genome sequence of novosphingobium malaysiense strain musc 273t, novel alpha-proteobacterium isolated from intertidal soil hooi-leng ser1,2,3, wai-fong yin4, kok-gan chan4,5, nurul-syakima ab mutalib6, learn-han lee1,2,3,7* 1novel bacteria and drug discovery (nbdd) research group, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2biofunctional molecule exploratory (bmex) research group, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 3biomedical research laboratory, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 4division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia 5vice chancellor office, jiangsu university, zhenjiang 212013, pr china 6ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia 7center of health outcomes research and therapeutic safety (cohorts), school of pharmaceutical sciences, university of phayao, phayao, thailand progress in microbes and molecular biology abstract : novosphingobium malaysiense strain musc 273t is a recently identified gram-negative, aerobic alpha-proteobacterium. the strain was isolated from intertidal soil with strong catalase activity. the genome sequence comprises 5,027,021 bp, with 50 trna and 3 rrna genes. further analysis identified presence of secondary metabolite gene clusters within genome of musc 273t. knowledge of the genomic features of the strain may allow further biotechnological exploitation, particularly for production of secondary metabolites as well as production of industrially important enzymes. keywords: proteobacteria; antioxidant, mangrove; secondary metabolite *correspondence to: learn-han lee, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. lee.learn.han@monash.edu; leelearnhan@yahoo.com received: 20th july 2018 accepted: 5th august 2018 published online: 15th august 2018 citation: ser hl, yin wf, chan kg, et al. genome sequence of novosphingobium malaysiense strain musc 273t, novel alpha-proteobacterium isolated from intertidal soi. prog micobes mol biol 2018; 1(1): a0000003. short introduction members of the genus novosphingobium can be found in wide range of habitats including soil, deep sea as well as contaminated environments[1-3]. their ability to degrade monoand polycyclic aromatic hydrocarbons (pahs) has attracted much biotechnological interest, notably for biomediation purposes. novosphingobium malaysiense strain musc 273t was isolated as novel species from intertidal soil obtained from pahang, malaysia[4]. based on 16s rrna gene analysis, the strain displayed highest similarities to novosphingobium indicum h25t, a type strain which has been shown to be able to degrade pahs. furthermore, musc 273t exhibited strong catalase activity, thus was selected for genome sequencing. data description extraction of genomic dna was using masterpuretm dna purification kit (epicentre, illumina inc., madison, wi, usa) genome report copyright 2018 by ser hl et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) treatment[5-7]. genomic dna quality was checked using nanodrop spectrophotometer (thermo scientific, waltham, ma, usa) and a qubit version 2.0 fluorometer (life technologies, carlsbad, ca, usa). following that, dna library was prepared with nexteratm dna sample preparation kit (nextera, usa). prior to sequencing, quality of dna library was examined with bioanalyzer 2100 high sensitivity dna kit (agilent technologies, palo alto, ca). whole genome shotgun project of musc 273t was carried out using paired sequencing in an illumina miseq platform with miseq reagent kit 2 (2 × 250 bp; illumina inc., madison, wi, usa), producing 4,376,924 pairedend reads. the assembly of trimmed seqeuence was done with clc genomic workbench version 5.1 (clc bio, denmark), resulting in 85 contigs and an n50 contig size of approximately 631,941 bp. the assembled genome size comprised 5,027,021 bp, with an average coverage of 100.0-fold and g + c content of 63.3%. the genome sequence of novosphingobium malaysiense musc 273t has been deposited at ddbj/embl/genbank under accession of jtdi00000000. the strain was deposited at three figure 1. subsystem category distribution of novosphingobium malaysiense musc 273t (based on rast annotation server). ser hl, ab mutalib ns, yin wf, et al. genome sequence of streptomyces antioxidans musc 164t isolated from mangrove forest. prog microbes mol biol 2018. url: http://www.journals.hh-publisher.com/index.php/ pmmb/article/view/1/14. aziz rk, bartels d, best aa, et al. the rast server: rapid annotations using subsystems technology. bmc genomics 2008; 9: 75. lowe tm, eddy sr. trnascan-se: a program for improved detection of transfer rna genes in genomic sequence. nuc acids res 1997; 25: 955–964. lagesen k, hallin p, rodland ea, et al. rnammer: consistent and rapid annotation of ribosomal rna genes. nuc acids res 2007; 35: 3100–3108. hyatt d, chen gl, locascio pf, et al. prodigal: prokaryotic gene recognition and translation initiation site identification. bmc bioinform 2010; 11: 119. blin k, medema mh, kazempour d, et al. antismash 2.0—a versatile platform for genome mining of secondary metabolite producers. nucleic acids res 2013; 41(w1): w204–212. 7. 8. 9. 10. 11. 12. gupta sk, lal d, lal r. novosphingobium panipatense sp. nov. and novosphingobium mathurense sp. nov., from oil-contaminated soil. int j syst evol microbiol 2009; 59(1): 156–161. yuan j, lai q, zheng t, et al. novosphingobium indicum sp. nov., a polycyclic aromatic hydrocarbon-degrading bacterium isolated from a deepsea environment. int j syst evol microbiol 2009; 59(8): 2084–2088. kämpfer p, young cc, busse hj, et al. novosphingobium soli sp. nov., isolated from soil. int j syst evol microbiol 2011; 61(2):259–263. lee lh, azman as, zainal n, et al. novosphingobium malaysiense sp. nov. isolated from mangrove sediment. int j syst evol microbiol 2014; 64(4): 1194–1201. ser hl, tan ws, cheng hj, et al. draft genome of amylolytic actinobacterium, sinomonas humi musc 117t solated from intertidal soil. mar gen 2015; 24: 209–210. ser hl, tan ws, ab mutalib ns, et al. genome sequence of streptomyces pluripotens musc 135t exhibiting antibacterial and antioxidant activity. mar gen 2015; 24: 281–283. 1. 2. 3. 4. 5. 6. references culture collection centers under accession of dsm 27798t = mccc 1a00645t = nbrc 109974t. table 1. general genomic features of novosphingobium malaysiense strain musc 273t. novosphingobium malaysiense musc 273t genome size (bp) 5,027,021 contigs 85 contigs n50 (bp) 631,941 g + c content % 63.3 protein coding genes 4,738 trna 50 rrna 3 putative secondary metabolite gene clusters 9 homoserine lactone 2 type 3 polyketides 1 bacteriocin 1 the assembled genome was annotated using rapid annotation using subsystem technology (rast)[8]. gene prediction was performed using prodigal version 2.6, while ribos omal rna (rrna) and transfer rna (trna) were pre dicted using rnammer and trnascan se version 1.21, respectively[9-11]. the analysis from rast revealed 4,738 protein-coding genes, along with 50 trna and 3 rrna genes. out of 4,738 protein-coding genes, 35 genes were identified to encode for enzymes involved in aromatic compounds metabolism. additionally, the genome also contained 3 genes responsible for production of catalase (e.c. 1.11.1.6) and/or peroxidase (e.c. 1.11.1.7). presence of these genes may suggest the genomic potential of strain musc 273t, most notably for industrial application and environmental bioremediation purposes. closer investigation of musc 273t genome with antibiotics & secondary metabolite analysis shell (antismash) unveiled several clusters of secondary metabolite genes[12]. total of 9 biosynthetic gene clusters were detected: 2 gene clusters associated with production for homoserine lactone; 1 each for type 3 polyketide and bacteriocin. these results indicate that strain musc 273t may produce natural bioactive compounds (including antibiotics), thus future studies involving characterization of these gene clusters and isolation of the secondary metabolite may assist in the discovery of these valuable compounds. acknowledgement this work was supported by the university of malaya for high impact research grant (um-mohe hir nature microbiome grant no. h-50001-a000027) awarded to k.-g. c. and external industry grant from biotek abadi sdn bhd (vote no. gba-808138 & gba-808813) awarded to l.-h. l. genome sequence of streptomyces antioxidans musc 164t isolated from mangrove forest hooi-leng ser1,2,3, nurul-syakima ab mutalib4, wai-fong yin5, bey-hing goh1,2,3,6, learn-han lee1,2,3,6*, kok-gan chan5,7* 1novel bacteria and drug discovery (nbdd) research group, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2biofunctional molecule exploratory (bmex) research group, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 3biomedical research laboratory, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 4ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia 5division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia 6center of health outcomes research and therapeutic safety (cohorts), school of pharmaceutical sciences, university of phayao, phayao, thailand 7international genome centre, jiangsu university, zhenjiang 212013, pr china progress in microbes and molecular biology abstract : members of streptomyces genus are well known to produce bioactive compounds of various structures[1-3]. having a complicated development life cycle with the ability to form spores, streptomyces species are ubiquitous in nature and can be found in interesting places like deep sea, hot springs and also mangrove forest[3-11]. streptomyces antioxidans musc 164t was originally isolated from mangrove forest in the east coast of peninsular malaysia and has been deposited at two culture collection centres (=dsm 101523t = mccc 1k01590t)[12,13]. after rounds of in vitro screening using human neuronal cell line (i.e. sh-sy5y), the extract of musc 164t was found to possess significant neuroprotective effect against hydrogen peroxide[13]. here, a high quality genome sequence of musc 164t is reported, while its genome potential to produce pharmaceutically important compounds is also discussed. keywords: streptomyces; antioxidant; genome; mangrove; actinobacteria; next generation sequencing (ngs) *correspondence to: learn-han lee, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; lee.learn.han@monash.edu; leelearnhan@yahoo.com; kok-gan chan, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia. kokgan@ um.edu.my received: 14th january 2018 accepted: 29th january 2018 published online: 16th april 2018 citation: ser hl, ab mutalib ns, yin wf, et al. genome sequence of streptomyces antioxidans musc 164t isolated from mangrove forest. prog microbes mol biol 2018; 1(1): a0000001. short introduction as a gram-positive bacterium, streptomyces antioxidans musc 164t forms yellowish white aerial mycelium and brilliant greenish yellow substrate mycelium on yeastmalt (isp 2) agar[13]. based on polyphasic study, this strain was concluded as a novel species belonging to the genus streptomyces, displaying with highest partial 16s rrna gene sequence similarity with type strain streptomyces javensis nbrc 100777t (99.6 % sequence similarity), streptomyces yogyakartensis nbrc 100779t (99.6 %) and streptomyces violaceusniger nbrc 13459t (99.6 %). the methanolic extract of musc 164t showed significant antioxidative and neuroprotective activities genome report copyright 2018 by ser hl et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) against hydrogen peroxide. as an attempt to further explore its bioactive potential, the strain was selected for genome sequencing. data description genomic dna of musc 164t was extracted using masterpure tm dna purification kit (epicentre, illumina inc., madison, wi, usa) before performing rnase (qiagen, usa) treatment[14,15]. dna quality was evaluated using nanodrop spectrophotometer (thermo scientific, waltham, ma, usa) and a qubit version 2.0 fluorometer (life technologies, carlsbad, ca, usa). whole-genome shotgun sequencing of strain musc 164t was carried out on illumina miseq platform with miseq reagent kit 2 (2 × 250 bp; illumina inc., madison, wi, usa), and it generated 8,806,977 raw reads. subsequently, ambiguous nucleotides, reads that are shorter than 50 bps and low quality reads were removed from the data. the draft genome was assembled with clc genomics workbench version 5.1 (clc bio, denmark). contigs with at least 200 bp and 30-fold coverage were selected for gene prediction and annotation. the bacteria identity was also checked by local blast against ncbi prokaryotic 16s rrna database. bacteria gene coding sequence (cds) was predicted from the draft genome using prodigal (version 2.6.1)[16]. gene annotation was performed by local blast of translated predicted cds against ncbi-nr database and also on rapid annotation using subsystem technology (rast) server[17]. presence of rrna and trna genes were detected using rnammer and trnascan se version 1.21[18,19]. a total of 282 contigs were generated with n50 size of 111,730 bp. the assembled genome size of musc 164t contains 9,118,065 bp, with an average genome coverage of 141-fold with a g + c content of 71.5 % (table 1). the whole genome project was deposited at ddbj/ embl/genbank under accession lakd00000000. the version described in this paper is the second version, lakd02000000. it is composed of 282 contigs and there were 7,214 protein coding genes (out of a total of 7,620 predicted genes) (figure 1). table 1. general features of streptomyces antioxidans musc 164t draft genome. attribute value genome size (bp) 9,118,065 dna g+c (bp) 6,516,852 dna scaffold 282 total genes 7,620 protein coding genes 7,214 rna genes (5s, 16s, 24s) 4, 4, 2 pseudo genes 331 crispr repeats 7 using antismash to detect biosynthetic gene clusters, the analysis revealed presence of 11 biosynthetic gene clusters which exhibited more than 70 % similarities with known gene clusters[20]. out of which, five clusters were associated with production of non-ribosomal peptides, including coelichelin, roseoflavin and paenibactin. interestingly, the genome of musc 164t revealed potential production of valuable siderophores like desferrioxamine b. in short, the availability of genome sequence for streptomyces antioxidans musc 164t has suggested its genome potential and prompted further improvement work (e.g. media optimization, strain improvement) to fully invoke the bioactive potential of the strain, particularly for production of valuable pharmaceutical compounds like desferrioxamine b. figure 1. subsystem category distribution of streptomyces antioxidans musc 164t (based on rast annotation server). acknowledgement this work was supported by mosti escience funds (project no. 06-02-10-sf0300) and external industry grant (biotek abadi vote no. genome sequence of streptomyces antixiodans... gba-808813 & gba-808138) awarded to l.-h.l., and k.g.c. was financially support by jbk grants (no. ga0012016, ga002-2016) and ppp grant (pg083-2015b). conflict of interest the authors declared that there is no conflict of interest. references 1. berdy j. bioactive microbial metabolites. j antibiotics 2005; 58: 1–26. 2. ser hl, tan lth, law jwf, et al. focused review: cytotoxic and antioxidant potentials of mangrovederived streptomyces. front microbiol 2017; 8: 2065. 3. subramani r, aalbersberg w. marine actinomycetes: an ongoing source of novel bioactive metabolites. microbiol res 2012; 167(10): 571–580. 4. kamjam m, sivalingam p, deng, z, et al. deep sea actinomycetes and their secondary metabolites. front microbiol 2017; 8: 760. 5. yang n, song f. bioprospecting of novel and bioactive compounds from marine actinomycetes isolated from south china sea sediments. curr microbiol 2017: 1–8. 6. al-dhabi na, esmail ga, duraipandiyan v, et al. isolation, identification and screening of antimicrobial thermophilic streptomyces sp. al-dhabi-1 isolated from tharban hot spring, saudi arabia. extremophiles 2016; 20(1):79–90. 7. duan yy, ming h, dong l, et al. streptomyces calidiresistens sp. nov., isolated from a hot spring sediment. antonie van leeuwenhoek 2014; 106(2): 189–196. 8. tan lth, ser hl, yin wf, et al. investigation of antioxidative and anticancer potentials of streptomyces sp. mum256 isolated from malaysia mangrove soil. front microbiol 2015; 6: 1316. 9. zainal n, ser hl, yin wf, et al. streptomyces 10. ser hl, palanisamy ud, yin wf, et al. streptomyces malaysiense sp. nov.: a novel malaysian mangrove soil actinobacterium with antioxidative activity and cytotoxic potential against human cancer cell lines. sci rep 2016; 6: 24247. 11. ser hl, zainal n, palanisamy ud, et al. streptomyces gilvigriseus sp. nov., a novel actinobacterium isolated from mangrove forest soil. antonie van leeuwenhoek 2015; 107(6): 1369–1378. 12. lee lh, zainal n, azman as, et al. diversity and antimicrobial activities of actinobacteria isolated from tropical mangrove sediments in malaysia.  scientific world j 2014; 2014: 1–14. 13. ser hl, tan lth, palanisamy ud, et al. streptomyces antioxidans sp. nov., a novel mangrove soil actinobacterium with antioxidative and neuroprotective potentials. front microbiol 2016; 7: 899. 14. ser hl, tan ws, ab mutalib ns, et al. draft genome sequence of mangrove-derived streptomyces sp. musc 125 with antioxidant potential. front microbiol 2016; 7: 1470. 15. ser hl, tan ws, ab mutalib ns, et al. genome sequence of streptomyces mangrovisoli musc 149t isolated from intertidal sediments. braz j microbiol braz j microbiol. 2018; 49(1): 13–15 16. hyatt d, chen gl, locascio pf, et al. prodigal: prokaryotic gene recognition and translation initiation site identification. bmc bioinform 2010; 11: 119. 17. aziz rk, bartels d, best aa, et al. the rast server: rapid annotations using subsystems technology. bmc genomics 2008; 9: 75. 18. lowe tm, eddy sr. trnascan-se: a program for improved detection of transfer rna genes in genomic sequence. nuc acids res 1997; 25: 955–964. 19. lagesen k, hallin p, rodland ea, et al. rnammer: consistent and rapid annotation of ribosomal rna genes. nuc acids res 2007; 35: 3100–3108. 20. blin k, medema mh, kottmann r, et al. the antismash database, a comprehensive database of microbial secondary metabolite biosynthetic gene clusters. nuc acids res 2016; 45: d555–d559. ser hl, et al. 3 humi sp. nov., an actinobacterium isolated from soil of a mangrove forest. antonie van leeuwenhoek 2016; 109(3): 467–474. progress in microbes and molecular biology genome report 1 genome sequence of vibrio sp. sall 6 isolated from shellfish vengadesh letchumanan1*, hooi-leng ser1,2, tan wen-si3,4, kok-gan chan4,5, nurul-syakima ab mutalib6 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2institute of biomedical and pharmaceutical sciences, guangdong university of technology, guangzhou, 510006, p. r. china 3illumina singapore pte ltd, woodlands industrial park e1, singapore 4division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia 5international genome centre, jiangsu university, zhenjiang 212013, pr china 6ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia abstract : members of the vibrionaceae family are well known as foodborne pathogen that cause hazard to human in many forms of clinical infection and also affecting aquaculture via infection to livestock. this pathogen has caused seafood associated gastroenteritis cases in many countries including united states, asian, and south east asian countries. antibiotics are usually used as prophylactic and therapeutic to manage the rising vibrio infections, however, this in turn led to emergence of antibiotic resistant strains in the environments. vibrio sp. sall 6 isolated from shellfish was selected for genome sequencing to further explore its antimicrobial traits. here, a high-quality genome sequence of vibrio sp. sall 6 is reported, while its genome reveals a potential for future antibiotic resistance managements. keywords: vibrionaceae; foodborne; gastroenteritis; antibiotics; genome received: 26th october 2019 accepted: 26th november 2019 published online: 06th december 2019 citation: letchumanan v, ser h-l, tan w-s, et al. genome sequence of vibrio sp. sall 6 isolated from shellfish. prog microbes mol biol, 2019; 2(1): a0000044 introduction vibrio species are natural inhabitant of aquatic environments and are the main cause of seafood-borne gastroenteritis[1,2]. this gram-negative halophilic bacteria belong to the vibrionaceae family[3–5] and many of them are linked with aquatic animals such as crustaceans, molluscs and fish[6]. of the 12 identified pathogenic vibrio sp., the three commonly reported are vibrio cholerae and vibrio parahaemolyticus — associated with seafood contamination, and vibrio vulnificus — related via wound infections[7,8]. the increase in seafood consumption worldwide lead to the global rise of seafood production from aquaculture. this causes the marine animals to be prone to bacterial infections[9]. the occurrence of vibrio sp. in our environments does raise a public concern on food safety due to the rising number of reported foodborne cases worldwide[10]. this situation has worsen by the emergence of antibiotic resistant bacteria which cause a delay in treatment, prolong hospitalization and even mortality. antibiotics are used in the aquaculture sector to treat bacterial infection, however, the misuse of them has resulted in the rising number of resistant foodborne pathogens such as vibrio sp.[11–18], listeria sp.[19,20], and salmonella sp.[21–24]. antibiotic resistance among foodborne pathogens is a major health issue and a great challenge to worldwide drug discovery programmes[25]. the clinical and environmental vibrio sp. strains are reported to exhibit antibiotic resistance traits[26]. hence, it is vital to continuously monitor and manage the occurrence of vibrio in seafood and environments. vibrio sp. sall 6 strain was isolated from shellfish originated from a wetmarket in selangor, malaysia. it formed green colony on thiosulphate citrate bile salt sucrose (tcbs) agar. the strain exhibited multidrug resistance profile towards 3/14 antibiotics tested. based on the antibiotic susceptibility phenotype, vibrio sp. sall copyright 2019 by letchumanan v et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) *correspondence: vengadesh letchumanan, novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; lvengadesh@yahoo.com. 2 6 strain was resistant to ampicillin, 3rd generation cephalosporin (cefotaxime) and aminoglycoside (amikacin). this is a distressing condition as the antibiotic resistant profile shown by this strain is among the recommended antimicrobials agents used in treatment of vibrio sp. infections[27,28]. as an attempt to further explore its antimicrobial resistance traits, this strain was selected for genome sequencing. data description genomic dna of vibrio sp. sall 6 was extracted using masterpuretm dna purification kit (epicentre, il lumina inc., madison, wi, usa) before performing rnase (qiagen, usa) treatment[29,30]. the dna quality was quantified using nanodrop spectrophotometer (thermo scientific, waltham, ma, usa) and a qubit version 2.0 fluorometer (life technologies, carlsbad, ca, usa). illumina sequencing library of genomic dna was prepared using nexteratm dna sample preparation kit (illumina, san diego, ca, usa) and library quality was validated by a bioanalyzer 2100 high sensitivity dna kit (agilent technologies, palo alto, ca) prior to sequencing. the genome of sall 6 strain was sequenced on miseq platform with miseq reagent kit 2 (2 x 250bp; illumina inc, san diego, ca, usa)[31]. the trimmed sequences were de novo assembled with clc genomic workbench version 5.1 (clc bio, denmark). contigs with at least 200 bp and 30-fold coverage were selected for gene prediction and annotation. the bacteria identity was also checked by local blast against ncbi prokaryotic 16s rrna database. bacteria gene coding sequence (cds) was predicted from the draft genome using prodigal (version 2.6.1)[32]. gene annotation was performed by local blast of translated predicted cds against ncbi-nr database and also on rapid annotation using subsystem technology (rast) server[33]. presence of rrna and trna genes were detected using rnammer and trnascan se version 1.21[34,35] a total of 81 contigs were generated with n50 size of 193,737 bp. the assembled genome size of vibrio sp. sall 6 contains 4,989,632 bp, with an average genome coverage of 54fold with a g + c content of 45.4 % (table 1). the whole genome project was deposited at ddbj/embl/genbank under accession mqvk00000000. the version described in this paper is the first version mqvk01000000. it is composed of 81 contigs and there were 4,500 protein coding genes (out of a total of 4,681 predicted gene) (table 1). table 1. general features of vibrio sp. sall 6 draft genome attribute value genome size (bp) 4,989,632 g + c content % 45.4 dna scaffold 81 total genes 4,681 protein coding genes 4,500 rna genes (5s, 16s, 24s) 2, 6, 1 pseudo genes 66 the analysis obtained from rast server revealed 544 subsystems (figure 1). the annotated genome has 74 genes responsible for resistance to antibiotic and toxic compounds including 28 genes for multidrug resistance efflux pumps, one gene for beta-lactamase, four genes for resistance to fluoroquinolones, and two genes for tetracycline resistance. the phenotypic resistance shown by vibrio sp. sall 6 toward ampicillin and cefotaxime is closely related to the gene coding beta-lactamase in the genome. multidrug resistance profile seen in the phenotype and genes of vibrio sp. sall 6 genome illustrates how extensive antibiotics have been utilized in agriculture and aquaculture settings. the efficacy of clinical antibiotics are declining, thus there is a need for non-antibiotic method such as bacteriophage application or natural plant antimicrobials to manage vibrio infections in the aquaculture[36,37]. genome squence of vibrio... figure 1. subsystem category distribution of vibrio sp. sall 6 (based on rast annotation server 3 conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. acknowledgement this work was supported by university of malaya for high impact research grant (um-mohe hir nature microbiome grant no. h-50001-a000027 and no. a00000150001) and ppp grant (pg090-2015b) awarded to k-gc. reference 1. elmahdi s, dasilva lv, and parveen s. antibiotic resistance of vibrio parahaemolyticus and vibrio vulnificus in various countries: a review. food microbiol, 2016; 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9: 102. letchumanan v et al. progress in microbes and molecular biology original research article 1 methicillin-resistant staphylococcus aureus (mrsa) on dispensing counters of community pharmacies in klang valley yao-li chow1, hong-wai tham1* 1biopharmaceutical research unit, faculty of pharmacy, segi university, petaling jaya, selangor, malaysia. abstract: staphylococcus aureus has been causing contamination and infection in the hospital and community settings. methicillin-resistant staphylococcus aureus (mrsa) was first discovered in the 1960s and epidemics of mrsa were reported soon after the usage of methicillin. the incidence rate of mrsa infections has been increasing for the past 50 years, and community-associated infection may be slowly replacing hospital-associated mrsa strains. this study aimed to investigate the prevalence of mrsa on the dispensing counters of community pharmacies under different settings community pharmacies in shopping malls and high streets in klang valley. with verbal consent, swab samples were collected from dispensing counters of 23 community pharmacies using sterile cotton buds moistened with sterile sodium chloride (nacl) solution. samples were spread on nutrient agars and brilliance mrsa 2 selection agars and incubated at 37ºc. the numbers of colony were documented and statistically analysed using microsoft excel and statistical package for the social sciences (spss) statistics. the results showed that the prevalence of mrsa on the dispensing counters was 22% (5 out of 23), and the difference in mrsa contamination between community pharmacies in shopping mall and high street setting was insignificant (p > 0.05). this study serves as the pioneer study of its kind in klang valley. all healthcare professionals and individuals are strongly advised to practise a good level of hygiene to avoid mrsa cross contamination. keywords: mrsa; pharmacy; dispensing counter; cross-contamination; klang valley received: 10th april 2020 accepted: 12th may 2020 published online: 18th may 2020 citation: chow y-l and tham h-w. methicillin-resistant staphylococcus aureus (mrsa) on dispensing counters of community pharmacies in klang valley. prog microbes mol biol 2020; 3(1): a0000084. https://doi.org/10.3687/pmmb. a0000084 introduction asia has one of the highest incidence rates of mrsa infections in the world[13]. besides mrsa, vancomycinintermediate s. aureus (visa) strains and vancomycinresistant s. aureus (vrsa) strains are also reported in several asia countries[4]. from 1986 to 2007, the prevalence rate of mrsa infections in malaysia has increased from 17% to 44.1%[5]. on average, mrsa patients in intensive care units are hospitalised three times longer and have five times greater risk of death compared to other patients in the intensive care units[6]. in addition, there have been reports in which patients infected with mrsa have a mortality rate of 34% within 30 days compared to 27% in patients infected with non-antibiotic resistant s. aureus[7]. researchers have been working persistently to search for potential compounds to inhibit mrsa, while some studies exhibited good potential but further works are needed before these discoveries could be developed as marketable drugs[8–11]. hospital-associated mrsa (ha-mrsa) and community-associated mrsa (ca-mrsa) differ in terms of their molecular and clinical epidemiology. hamrsa infection is linked to serious, invasive diseases, such as skin and soft tissue infections (sstis), pneumonia and bloodstream infection (bsi) in hospitalised patients[12]. on the other hand, ca-mrsa infection may slowly replace ha-mrsa[13] with cross contamination between hospital and community settings[14,15]. camrsa is defined as mrsa infection which happens without any recent hospital exposure, and is usually associated with sstis in healthy, young individuals[16]. in addition, uncommon cases of skin infections, for instance, necrotizing fasciitis has been reported to be associated with ca-mrsa[17]. furthermore, ca-mrsa strains also cause other invasive infections; for example, urinary tract infections, osteomyelitis, bacteremia, pneumonia and septic shock. however, each of these infections only accounts for less than 4% of all camrsa infections[18]. with the statistics and adverse effects of mrsa discussed above, this study aims to copyright @ 2020 by chow y-l, tham h-w and hh publisher. this work under licensed under the creative commons attributionnoncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: hong-wai tham, segi university, jalan teknologi, kota damansara, petaling jaya, selangor, 47810, malaysia; thamhongwai@outlook.my 2 study the prevalence of mrsa in selected community pharmacies in klang valley, malaysia. dispensing counters of these pharmacies will be the study site of this project. material and methods agar preparation and procurement nutrient agars (oxoid) were prepared according to the manufacturer’s instructions. brilliance mrsa 2 premade agar plates were purchased from thermo fisher scientific and stored in dark at 4°c until used. study sites twenty-three (23) community pharmacies were randomly identified for sample collection in klang valley. all pharmacies were either located in shopping complexes, or at high-street setting. sample inoculation all samples collected were inoculated onto nutrient agars and brilliance mrsa 2 agars and incubated at 37°c for 12-16 hours. the numbers of visible colony were documented and data were analyzed. the results were presented in colonies forming unit per ml (cfu/ml). all procedures were conducted under aseptic conditions. ethical clearance this study was conducted under the approval of segi ethics committee, with ethics approval number of segi/ rimc/fop/28/2018. results study sites figure 1 indicates the pharmacies visited over the course of the sample collection period in this study. the blue pointers indicate the absence of mrsa and the red pointers indicate the presence of mrsa. in kuala lumpur city, three community pharmacies were found to have mrsa on their dispensing counters. meanwhile, in damansara and petaling jaya area, one community pharmacy in each area was found to have mrsa on the dispensing counter. mrsa prevalence... microorganism prevalence were detected on all 23 dispensing counters of community pharmacies. mrsa was detected on five dispensing counters out of 23 community pharmacies in klang valley. prevalence of total microorganism and mrsa counts based on the graph in figure 2, different levels of figure 1. selected community pharmacists for this study. (blue pointers) community pharmacies without mrsa detected on dispensing counter; (red pointers) community pharmacies with mrsa detected on dispensing counter. 3 chow y-l and tham h-w mrsa contamination between community pharmacies in shopping mall and high street settings figure 3 shows mrsa was detected on two dispensing counters of community pharmacies in high street. three dispensing counters of community pharmacies in shopping mall setting were detected with mrsa. figure 3. no significant difference in mrsa contamination between community pharmacies in shopping mall and high street settings. table 1 shows that the nature of community pharmacy settings (shopping mall or high street) did not significantly correlate with the presence of mrsa contamination on dispensing counter (p > 0.05). table 1. independent t-test on difference in mrsa contamination between community pharmacies in shopping mall and high street settings. t-test for equality of means sig. (2-tailed) location equal variances assumed .708 discussion a comparable study was conducted at yamaguchi university hospital, japan in 2013. the study assessed the presence of mrsa contamination on the surfaces of bed side rails, overbed tables and curtains in the rooms of 24 inpatients that were infected with mrsa infections. the prevalence of mrsa contamination on the surfaces of bed side rails and overbed tables was 31.6% (6 out of 19) and 25% (6 out of 24) respectively. the total number of bedside rails examined was 19 instead of 24 because 5 of the patients were using beds without side rails. however, there was an absence of mrsa contamination on the surfaces of all 24 curtain samples[19]. three other similar studies that evaluated the prevalence of mrsa contamination on the surfaces of curtains have presented different results. the prevalence of mrsa contamination was reported to be 28% (14 out of 50)[20], 15.5% (31 out of 200)[21], and 92% (12 of 13 samples) [22]. the method used for mrsa detection may have been different for each study to which it leads to differences in results. therefore, further studies are needed to answer the inconsistencies in these results. in this study, the prevalence of mrsa on the dispensing counters of community pharmacies in klang valley was found to be 22% (5 out of 23). the number might be worrying since community pharmacies are easily accessible by the public. although the data collected could be improved, the cleanliness of dispensing counters should be prioritized since patients normally receive their medications from pharmacists over the dispensing counters. as a result, the transmission of mrsa to other healthy populations might occur through the platform of dispensing counters. in addition, we also found that the occurrence of mrsa contamination between different community pharmacy settings (shopping mall or high street) was not statistically significant. the location of pharmacies whether in a shopping mall or at high street, did not significantly reflect the hygiene of the dispensing counter. we would like to highlight other factors that may affect the cleanliness of pharmacy, for instance, the responsibility of pharmacist and staff members. in addition, increasing awareness of public to consult community pharmacists might also lead to cross figure 2. microorganism count on 25 cm2 of dispensing counters of selected community pharmacies. (a) total microorganism count on nutrient agars; (b) total mrsa count on brilliance mrsa 2 agars. 4 contamination of antibiotic-resistant microorganism[23], with several research studies reported evidence of microorganism transmissions between environmental surfaces and patients[24]. based on the good dispensing practices by world health organization (who), a clean, organized and safe working environment provides a basis for good practice. dispensing environments of a community pharmacy should be clean, hygienic and uncontaminated since most of the pharmaceutical products are for internal use. the dispensing environment of a community pharmacy includes work surfaces, staff members, physical surroundings, shelves, counters and so on. in addition, staff members should maintain good personal hygiene and wear clean clothing if they are involved in dispensing. the staff members should also avoid skin contact with pharmaceutical products during dispensing to prevent any contamination. maintaining a clean working environment in the community pharmacy requires daily cleaning of floors and working surfaces, daily removal of waste and a regular routine of shelves cleaning. it is also essential to clean the equipment used after handling any pharmaceutical products[25]. private healthcare sector such as community pharmacy is in growing need in developing countries. these pharmacies are always the primary source of healthcare for the public due to convenience, close in proximity, reasonable price, responsiveness and flexibility in operating hours[26]. therefore, community pharmacy should be kept at good level of hygiene to prevent cross contamination of multidrug-resistant microorganism. conclusion mrsa contamination has been detected on dispensing counters of community pharmacies in klang valley. the prevalence of mrsa on the dispensing counters was 22% (5 out of 23). in addition, this study also showed that the presence of mrsa contamination was independent from the location and setting of community pharmacies. authors contribution the research and manuscript writing were performed by y-lc. h-wt founded the research project. conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. acknowledgement authors would like to acknowledge faculty of pharmacy, segi university for supporting the research project through university research fund. reference 1. chuang y-y, huang y-c. molecular epidemiology of communityassociated meticillin-resistant staphylococcus aureus in asia. lancet infect dis 2013; 13(8): 698–708. 2. banerjee t, anupurba s. colonization with vancomycin-intermediate staphylococcus aureus strains containing the vana resistance gene in a tertiary-care center in north india. j clin microbiol 2012; 50(5): 1730–2. 3. khan tm, kok yl, bukhsh a, et al. incidence of methicillin resistant staphylococcus aureus (mrsa) in burn intensive care unit: a systematic review. germs 2018; 8(3): 113 4. wan mt, lauderdale tl, chou cc. characteristics and virulence factors of livestock associated st9 methicillin-resistant staphylococcus aureus with a novel recombinant staphylocoagulase type. vet microbiol 2013; 162(2–4): 779–84. 5. ahmad n, ruzan in, abd ghani mk, et al. characteristics of community and hospital-acquired meticillin-resistant staphylococcus aureus strains carrying sccmec type iv isolated in malaysia. j med microbiol 2009; 58(pt 9): 1213–8. 6. noskin ga, rubin rj, schentag jj, et al. the burden of staphylococcus aureus infections on hospitals in the united states: an analysis of the 2000 and 2001 nationwide inpatient sample database. arch intern med 2005; 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47(3): 703–5. 20. trillis f, eckstein ec, budavich r, et al. contamination of hospital curtains with healthcare-associated pathogens. infect control hosp epidemiol 2008; 29(11): 1074–6. 21. klakus j, vaughan nl, boswell tc. meticillin-resistant staphylococcus aureus contamination of hospital curtains. j hosp infect 2008; 68(2): 189–90. 22. ohl m, schweizer graham m et al. hospital privacy curtains are frequently and rapidly contaminated with potentially pathogenic bacteria. am j infect control 2012; 40(10): 904–6. 23. gebel j, exner m, french g, et al. the role of surface disinfection in infection prevention. gms hygiene infect control 2013; 8(1). 24. otter ja, yezli s, french gl. the role played by contaminated surfaces in the transmission of nosocomial pathogens. infect control hosp epidemiol 2011; 32(7): 687–99. 25. who. ensuring good dispensing practices. (mds-3: managing access to medicines and health technologies, chapter 30) [internet]. 2012 [cited 2018 dec 4]; available from: http://apps.who.int/ medicinedocs/en/d/js19607en/ 26. newton pn, amin aa, bird c, et al. the primacy of public health considerations in defining poor quality medicines. plos med 2011; 8(12). mrsa prevalence... progress in microbes and molecular biology original research article 1 coliform contamination on faucet surface of water vending machines in klang valley yi-mian ang1, hong-wai tham1* 1biopharmaceutical research unit, faculty of pharmacy, segi university, petaling jaya, selangor, malaysia. abstract: in malaysia, water vending machine serves as an alternative to drinking water supply. however, the quality of drinking water obtained from water vending machines may vary due to microorganism contamination which caused by inadequate hygienic practices and routine maintenance of the machines. in this study, 100 water vending machines were randomly selected from 10 districts of klang valley. sterile cotton swabs were used to collect swab samples from all selected subjects, with swab samples collected on the outer surface of water faucets. samples were sent to laboratory for culture analyses using nutrient agars (oxoid) and hicrome™ coliform agars (himedia laboratories). the results showed that none of the water vending machine was contaminated by faecal coliform, however, with close to 80% of the subjects were found contaminated by total coliform (eg. klebsiella, enterobacter or citrobacter species). although the presence of total coliform may not be deleterious to the health of end users, our findings highlights the need for authorities and water vending service providers to set an effective sanitation procedure in maintaining the hygienic level of water vending machines. keywords: vending machine; microorganism; faecal coliform; water; hygienic; klang valley malaysia received: 12th april 2020 accepted: 14th may 2020 published online: 20th may 2020 citation: ang ym and tham h-w. coliform contamination on faucet surface of water vending machines in klang valley. prog microbes mol biol 2020; 3(1): a0000086. https://doi.org/10.3687/pmmb.a0000086 introduction food and water have never been safer in terms of the incidence of infectious illness. a variety of bacteria, viruses and even parasite are transmitted via food and water, thus causing a rise of cases in recent years[1–13,]. a growing population with limited resources of clean water has resulted in 4 billion cases of diarrhoea every year[14]. waterborne diseases can be caused by various pathogenic agents, including escherichia, enterobacter, shigella, klebsiella, campylobacter, cryptosporidium, giardia, salmonella and enteric viruses[15]. of this, salmonella, vibrio, escherichia, campylobacter and citrobacter are among the causative agent of foodborne pathogens as well[16–31]. in malaysia, quality of drinking water remains as one of the main concerns of consumers. a survey conducted by aini et al. reported that odour, taste and colour were the major issues with water supply[32]in spite of these, quantity and quality of drinking water is still one of the main concerns of malaysian consumers today. an exploratory study was undertaken to determine the level of awareness of respondents on water issues, assess their perception on drinking water quality, and identify measures undertaken by households to improve drinking water quality and to determine sustainable water practices. a cross-sectional research design, utilizing a survey was conducted among urban residents of seremban town. data showed that each household had a mean of five members, with an average household income of rm3788.00 (us$1000). unaffordable water filtration systems have resulted in good sales of product water from vending machines[33], which serve as an alternative source of drinking water. several factors such as hygienic practice of users, routine maintenance by owners, quality control by authorities are crucial in maintaining the quality of drinking water[34]the vending machine has contributed to a revolution in how we buy food and drink. despite the very obvious benefits associated with this technology, vending machines have not always been welcome by the customers they are intended to serve. although occasionally blamed for various nonspecific illnesses, there have been very few studies about the microbiology of food and drink served from such machines. the few studies that have been reported have found high total viable counts (tvc). faecal coliform is a rod-shaped anaerobic bacterium with no sporulation. its members consist of various species of bacteria such as escherichia, enterobacter, klebsiella, salmonella and shigella. they exist in the faecal materials and intestinal tract of humans or warmblooded animals and enter the water bodies through copyright @ 2020 by ang ym and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: hong-wai tham, biopharmaceutical research unit, faculty of pharmacy, segi university, jalan teknologi, kota damansara, petaling jaya, selangor, 47810, malaysia; thamhongwai@outlook.my 2 the waste products. however, they are usually nonpathogenic and can be used as an indicator organisms to indicate the presence of faecal material in water[33,35]. e.coli o157:h7, a pathogenic strain of e. coli, is pathogenic and causes gastroenteritis, bloody diarrhoea, urinary tract infection (uti) and haemolytic-uremic syndrome (hus)[36]. according to united states environmental protection agency, the presence of e. coli serves as a good indicator of faecal coliform contamination to evaluate the microbiological quality of water. on the other hand, the presence of other coliforms such as klebsiella, salmonella, or shigella in consumable products also increases the risk of health conditions such as urinary/respiratory infections[37], salmonellosis[16,38–40], and shigellosis[41], respectively. according to national water quality standards, ministry of health malaysia, a class i water quality should contain total coliform at the maximum of 100 cfu/ml, with faecal coliform at the maximum of 10 cfu/ml. in view of the importance of drinking water quality in relation to the health of users in public, this study aimed to test the presence of faecal coliform and total coliform contamination on the faucets of water vending machines. the results were also correlated to different districts, brands, status of maintenance and licence. material and methods agar media nutrient agar powder (oxoid) and hicrome™ coliform agar (himedia laboratories) were purchased and prepared according to manufacturer’s instructions. all procedures in this section were conducted under aseptic conditions. sampling sites ten water vending machines were randomly chosen from commercial areas of each of the 10 districts of klang valley, including ampang, cheras, gombak, kajang, klang, kuala lumpur, puchong, petaling jaya, subang jaya, and shah alam. these areas were selected for their higher population density. in total, 100 sampling sites were identified. sampling sample sample collections were conducted in biological triplicates, using sterile cotton buds collecting microbiome on the outer surface of water faucets at 1 cycle anticlockwise. the cotton buds were then immediately kept in sterile 15 ml falcon tubes pre-filled with 1 ml of pre-sterilised 0.8% nacl solution. samples were sent to biology laboratories for downstream assays within 5 hours. detection of total microbial and coliform all samples were spread on either nutrient agar or hicrome™ coliform agar. all agars were incubated (37 °c, 18 hours) and the numbers of colony were documented (in cfu/ml) and analysed. meanwhile, the colour and morphology of colonies were also observed and recorded. some colonies were selected for gram staining for further characterisation. statistical analysis all results and data were analysed using statistical package for the social sciences (spss) software (version 23). kruskal-wallis tests and mann-whitney u tests were used in these analyses. statistical analyses were conducted based on total microbial counts, between brands, maintenance status, licence status of water vending machines. results sampling sites the sampling sites were presented in figure 1, which was generated using google my maps. coliform contamination... figure 1. sampling sites of this research project. red pointers indicate the location of water vending machines detected with enterobacter or citrobacter; black pointers indicate the presence of salmonella or shigella; purple pointers indicate the presence of klebsiella; blue pointers indicate the absence of coliform contamination. 3 ang ym et al. total microbial counts of various districts in klang valley total microbiome on the faucet surface of water vending machines were measured using nutrient agars and hicrome™ coliform agars (figure 2). figure 2. average of total microbiome (measured in cfu/ml) of various samples collected from 10 districts in klang valley. table 1 illustrates the outcome of kruskal wallis test between different districts. the difference between total cfu/ml detected on hicrome™ coliform agars were significant (p < 0.05), while results presented by nutrient agars showed the opposite (p > 0.05). table 1. correlation between different districts of klang valley and total microbial counts detected on nutrient agars and hicrome™ coliform agars. asymptotic significance pvalue hicrome coliform agar with sls (ha) .015 < 0.05 nutrient agar (na) .112 > 0.05 correlation between microbial counts and maintenance status of water vending machines table 2 shows that the status of licence during sampling does not contribute significantly to the hygienic level of water vending machines (p > 0.05). table 2. correlation between the status of licence during sampling and total microbial counts detected on nutrient agars and hicrome™ coliform agars. asymptotic significance pvalue hicrome coliform agar with sls (ha) .464 > 0.05 nutrient agar (na) .179 > 0.05 table 3. total number of vending machines with colonies presented in light pink, salmon red, or opaque white. district total number of vending machines with: light-pink colonies salmon-red colonies opaque-white colonies ampang 1 2 10 cheras 1 0 8 gombak 4 1 8 kajang 1 0 8 klang 2 0 8 kuala lumpur 1 0 6 petaling jaya 1 0 4 puchong 3 2 10 shah alam 1 0 10 subang jaya 1 3 5 total 16 8 77 discussion in 2018, the national water service commission (span) has confirmed the absence of e. coli from malaysia’s water supply, which was also claimed safe for direct consumption. nevertheless, for the supplier or owner of water vending machines located in various business centres in klang valley, bacterial colonies on hicrometm coliform agars the number of bacterial colonies tabulated in table 3 indicates the presence of klebsiella species (light-pink colonies), enterobacter or citrobacter species (salmonred colonies), or other gram negative coliform bacterial species (opaque-white colonies). 4 they should maintain a routine maintenance to prevent the filter membranes in ro system become overgrown with microorganisms. the quality control of water vending machines by authorities or service providers is crucial in maintaining the health of end users. our study suggested that the level of contamination was not significantly associated with different districts of klang valley (table 1). this conclusion was drawn from the p-value given using nutrient agar as a general growth medium. the low p-value from hicrome™ coliform agars was deemed less reliable since the agars were supplied with sodium lauryl sulfate (sls), which suppresses the growth of many microorganisms[42]. this finding was in accordance to the dynamic growth and strong population movement within klang valley[43,44]. during the study, we noticed that less than half (43%) of the 100 water vending machines received routine maintenance. nevertheless, the status of maintenance was not found strongly associated with the hygienic level on the surface of water vending machine faucets. our record shows that, in shah alam, 7 out of 10 selected water vending machines had undergone routine maintenance around a week before the day of sampling, whereas only 30–60% of water vending machines had clear indication of routine service from other areas. nevertheless, statistical analyses have shown that the status of routine maintenance was not strongly associated with the presence of total coliform on the water faucet surface. the same trend was observed for the status of licence issued by respective authorities (table 2), with only 9 out of 100 subjects were found labelled clearly with licence obtained from authorities. despite the standard operating procedure (sop) set by authorities or service providers, faucets of water vending machines can still be contaminated due to physical contact with hands of user or exposure to fomites on water containers. routine maintenance with effective sanitisation of all outer and inner surfaces is crucial[45]. we would like to highlight the stressing needs for proper sanitisation on the contact points between users and water vending machines for the persistent hygienic issues[46–48]. although the presence of faecal coliform in water supply may not be harmful, but it is an indication of the presence of faeces[49]. despite cases of water vending machines contaminated by faecal coliforms (e. coli o157:h7)[50], fortunately, none of the selected subjects in this study was found to harbour faecal coliform (figure 1, table 3). however, in accordance to other studies[51,52],the presence of other total coliform (eg. klebsiella, enterobacter or citrobacter species) was reported in our findings. conclusion in conclusion, our findings report the absence of faecal coliform from the faucet surface of all selected water vending machines in klang valley. however, the presence of other total coliforms highlights the importance of a proper and effective sanitisation by authorities and service providers — regardless of the area, licence and maintenance status of the machines. authors contribution the research and manuscript writing were performed by y-ma. h-wt founded the research project. conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. funding this project was fully funded by segi university research grant. acknowledgement authors would like to thank the faculty of pharmacy (segi university) and research & innovation management centre (rimc) of segi university for their full support in conducting the research project. reference 1. letchumanan v, ab mutalib n-s, wong sh, et al. determination of antibiotic resistance 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mangzira kemung1,2, loh teng-hern tan2, kooi yeong khaw1, yong sze ong1,3, chim kei chan4, darren yi sern low5, siah ying tang5,6, bey-hing goh1,6,7* 1biofunctional molecule exploratory research group (bmex), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength, jeffrey cheah school of medicine and health science, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 3health and well-being cluster, global asia in the 21st century (ga21) platform, monash university malaysia, bandar sunway 47500, malaysia 4de duve institute, avenue hippocrate 75, 1200 brussels, belgium 5chemical engineering discipline, school of engineering, monash university malaysia, jalan lagoon selatan, 47500 bandar sunway, selangor, malaysia 6advanced engineering platform, monash university malaysia, jalan lagoon selatan, 47500 bandar sunway, selangor, malaysia 7college of pharmaceutical sciences, zhejiang university, hangzhou, china abstract: biofilms form protective layers over bacteria that are associated with a majority of the hospital infections contributing to antibiotic resistance development in susceptible strains. nowadays, there is a pressing need for developing effective anti-biofilm agents to help address the growing problem of biofilm-producing bacteria associated with antibiotic resistance. in recent years, zinc oxide nanoparticles (zno-nps) has emerged as a prospective candidate for new anti-biofilm agents. the present method paper described an optimized anti-adherence and anti-biofilm assay using zno-nps. the antibiotic-resistant bacteria methicillin-resistant staphylococcus aureus (mrsa atcc4330) and vancomycin were used as the growth control and positive control, respectively. the result showed concentration-dependent anti-adherence and antibiofilm activity. the zno-nps effectively prevented attachment of bacterial cells onto walls of wells with 51.69 ± 2.55% at the highest concentration tested (65.4 µg/ml). zno-nps was also able to break-up 50% pre-formed mrsa biofilm at the lowest concentration of 13.5 µg/ml. interestingly, zno-nps at lower concentrations demonstrated significantly stronger antibiofilm activity than that of the positive control vancomycin, demonstrating that zno-nps is a promising anti-biofilm agent. this method could be used as a preliminary screening of transition metal oxide nanoparticles as potential anti-adherence and anti-biofilm agents followed by other specific anti-biofilm assays. keywords: methicillin-resistant staphylococcus aureus; mrsa; anti-biofilm; anti-adherence; zinc oxide nanoparticles; zno-nps received: 3rd may 2020 accepted: 3rd june 2020 published online: 12th june 2020 citation: kemung hm, tan lt-h, khaw ky, et al. an optimized anti-adherence and anti-biofilm assay: case study of zinc oxide nanoparticles versus mrsa biofilm. prog microbes mol biol 2020; 3(1): a0000091. https://doi.org/10.3687/ pmmb.a0000091. introduction over the course of history, nature through its subsidiary plants and microbes, has proven to be an essential player in driving development of future drugs by virtue of their potential in producing secondary metabolites with antibacterial, anti-cancer, anti-oxidant and neuroprotective activities[1–18]. even to this day, nature continues to instill its significance in society as a prominent resource for future antibiotics in treating antibiotic-resistant infections[19]. despite the use of current antibiotics, infectious diseases acquired either in hospitals or through consumption of foods contaminated by food-borne copyright @ 2020 by kemung hm and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: bey-hing goh, school of pharmacy, monash university malaysia, 47500, bandar sunway, selangor darul ehsan, malaysia; goh.bey.hing@monash.edu 2 pathogens[20–26] remain a major public health problem. additionally, the unscrupulous practice of antibiotics for various ailments has encouraged antibiotic-susceptible infectious bacteria to form natural defenses against them. one such defense established by these bacteria is the biofilm[19]. biofilm is a term that describes a community of microorganisms within a self-produced matrix of biopolymers attached on surfaces[27]. microbes tend to produce biofilm on surfaces evading harmful effects of antibiotics as well as detergents and persists in hospitals causing many internalized hospital-related infections. it was estimated that biofilm contributes to approximately 60 to 80% of hospital infections[28–30]. given that staphylococcus aureus normal flora is the skin, suggests that it is among the most common causative agent in hospital-acquired infections associated with medical implants[31,32]. moreover, it was shown that s. aureus was tolerant against higher doses of antibiotics and may thus contribute to development of antibiotic resistance in susceptible strains[32]. recent years has seen a growing interest in the study of biofilm inhibitors acting as adjuvant agents in reducing biofilm layer of pathogenic bacteria[33]. this has led to the use of anti-biofilm assays to identify alternative sources as potential inhibitors of microbial biofilm. previous studies have highlighted the antibacterial potential of transition metal oxides for crop protection[34] and disease eradication[35–37]. nanoparticles especially those of metallic nature are one of the newest emerging systems which have great potential in inhibiting the formation of biofilms accredited to their high anti-microbial and anti-bacterial properties. the use of nanoparticles as anti-biofilm agents have found its way in many different sectors such as in healthcare (drug delivery, therapeutics and dentistry) or even in the food industry with a plethora of tailored applications[38,39]. the potency of these metallic nanoparticles in resisting the production of biofilm is high due to its nanoscale size and active participation in most of the stages in biofilm production. if the nanoparticles can successfully prevent adherence of microbes, then cycle of biofilm production is halted from the start. sometimes, these nanoparticles disrupt the biofilm at the proliferation or even maturation stages, generally through the formation of radicals and reactive oxygen species (ros) which affects gene expressions and breaks dna strands[40]. in this context, the zno-nps were chemically synthesized using a zinc nitrate precursor and subsequently characterized to confirm its identity. this includes conducting elemental analysis, fourier-transform infrared spectroscopy and morphological analysis using electron microscopy. the zno-nps synthesized as an anti-adherence and antibiofilm agent have nanorice morphologies and have an average size of 250 nm. the aim of this methodology article is to present step-bystep and optimized anti-adherence and anti-biofilm assays to evaluate the efficacy of zno-nps as anti-adherence and anti-biofilm agents[41] (figure 1). to validate the test method, methicillin-resistant staphylococcus aureus (mrsa) atcc 43300 and vancomycin hydrochloride were used as the control bacteria and positive control, respectively. the experiment set-up consisted of a 96well plate with a flat bottom, crystal violet as a staining agent and a 96-well microplate reader for quantification of both the anti-adherence and anti-biofilm activities. the result obtained indicate zno-nps has anti-adherence and anti-biofilm properties against mrsa atcc 4330. given that crystal violet anti-adherence and anti-biofilm assay is an indirect measure of biofilm biomass, this study could be used as a preliminary screening to investigate the antibiofilm properties of transition metal oxide nanoparticles prior to studying the mechanism of action of anti-adherence and anti-biofilm properties. method details synthesis of zno-nps a weighted measurement of 1.90 g of zinc nitrate hexahydrate (zn(no3)2.6h2o) is first dissolved in 100 ml of ultrapure water under constant stirring. subsequently, the ph of the mixture was adjusted to ph 10 using 1m of sodium hydroxide (naoh) solution. next, the solution is heated for 1 hour at 85°c under continuous stirring. the white suspension was then centrifuged for 5 minutes at 7000 rpm. upon removing the supernatant, the residue is washed with distilled water and then subjected to another cycle of centrifugation before removing the supernatant again. the residue was then dried in an aerated oven at 60°c overnight, yielding a white powder. anti-adherence and anti-biofilm assay materials • biosafety level 2 cabinet, functional incubator, 96-wells microplate reader • sterile disposable consumables: 96-wells microtiter, 15 ml centrifuge tubes • bacterial cells (american type culture collection strainatcc) • bacteria nutrient-rich media. the use of tryptic soy broth (tsb) which contains glucose and stimulates biofilm formation especially with methicillin-resistant staphylococcus aureus. • aqueous crystal violet (0.1% w/v) • glacial acetic acid (30% v/v) • 1×phosphate buffer saline • multichannel pipette (preferable) • vancomycin hydrochloride drug as the positive control • zinc oxide nanoparticles (zno-nps) prepared in different test concentrations procedure bacterial culture preparation inoculate 3 to 5 pure colonies of mrsa atcc43300 from an optimized anti-adherence... 3 kemung hm et al. the culture plate into 15 ml tsb. revive the bacteria in the shaker incubator at 200 rpm and at 37°c for 18 to 24 hours prior to the experiment so that they are preferably in their log phase of growth. ensure sterile tsb is used by autoclaving tsb at 121°c for 15 minutes. anti-adherence assay 1. inoculate 50 μl of zno-nps and vancomycin into designated wells at a series of concentration. (vehicle control e.g. dmso is also needed to be aliquoted into appropriate wells if used as the diluent for the test substance) 2. prepare a bacterial suspension (~1 x 108 cfu/ml equivalent to uv absorbance reading of 0.08 to 0.1 with wavelength at 600 nm) in 15 ml from a 24-hour bacterial culture. alternatively, a 0.5 mcfarland standard can be used to determine the optimal bacterial suspension. make a 1:100 dilution in a separate centrifuge tube to obtain a 106 cfu/ml bacterial suspension. 3. add 50 μl diluted bacterial concentration in respective wells using an appropriate multichannel pipette. using a multichannel pipette is a faster and more efficient mean of adding the bacterial suspension into the wells. 4. add sterile distilled water to the 4 corners of the microplate to prevent evaporation of water from the test wells. evaporation of water in test wells can interfere with the results. alternatively, the microplate can be kept in a container placed with moist filter paper during the incubation. 5. cover the plate with the lid and place the plate in an incubator at 37°c for 18 to 24 hours. 6. take the 96-well plate out from incubator and slowly remove the tsb either by decanting or pipetting. rinse the plate thrice with sterile double distilled water and allow the plate to air dry under the biosafety cabinet. turn the plates upside down to hasten the process of drying. ensure it is dry before moving on to the next step. 7. dispense 100 µl of aqueous crystal violet (1% w/v) into the test wells and let it stain the bacterial cell walls for 10 to 15 minutes. decant the crystal violet either into a sink or onto clean disposable tissues. 8. rinse the test wells three times with sterile double distilled water and allow the wells to dry under the biosafety cabinet. alternatively, the plate can be bathed subsequently with 3 dishes of water. 9. add 30% (v/v) glacial acid in water to solubilize crystal violet and leave it standing for 15 minutes. ensure there is clear blue/violet solution with no visible residue in each of the test wells. 10. read the uv absorbance of all the wells at 570 nm (suggested range would be between 570 to 600 nm) 11. calculate the anti-adherence activity of test substance and vancomycin using the following formula: anti-biofilm assay 1. follow the steps stated in bacterial culture preparation and step 2 in anti-adherence assay to prepare 106 cfu/ ml bacterial suspension. inoculate 100 μl of the diluted bacterial suspension in tsb into respective well of a new 96 well microplate. 2. incubate the plate at 37oc in an incubator for 24 hours. 3. decant the tsb broth completely from the microplate, wash the well gently without disrupting the biomass formed attaching on the bottom and wall of the wells with sterile phosphate buffer saline (pbs) 3 times. 4. add in 100 µl of freshly prepared sterile tsb broth (control well), zno-nps suspended in tsb with test concentrations and tsb containing the vancomycin in test concentration. 5. repeat the steps 5 to 10 from the above anti-adherence assay protocol. 6. calculate the anti-biofilm activity of the test substance and vancomycin using the following formula: method validation determination of biofilm formation based on the protocol, biofilm formation was indicated by the violet stains. this show that tsb media was adequate for biofilm formation whilst 0.1% (w/v) concentration of crystal violet was sufficient for visible observation with the naked eye and quantification by the spectrophotometer. assessment of anti-adherence assay this protocol allows the determination of anti-adherence property of zno-nps versus vancomycin using the 96-well plate. the biomass of the bacterial cell was quantitatively analyzed on the microplate reader at absorbance of 570 nm showing a decreasing trend of biomass attachment with increasing concentrations of zno-nps tested. the result show that zno-nps at 65 μg/ml achieved a significant antiadherence activity of 51.69 ± 2.55%. however, vancomycin at 0.5 μg/ml did not exhibit significant anti-adherence activity when compared to negative control (tsb only) (figure 2). anti-adherence activity% = × 100% absorbance of control-absorbance of test sample absorbance of control antibiofilm activity% = × 100% absorbance of control-absorbance of test sample absorbance of control 4 figure 1. schematic diagram shows the step-by-step protocol of the optimized ant-adherence and anti-biofilm assays. more detailed protocol should refer to the text. an optimized anti-adherence... 5 figure 2. anti-adherence assay using vancomycin as positive control and mrsa atcc 43300 as growth control. experiment evaluated based on quadruplicate results with standard deviation. (n = 4, p < 0.05). *indicates signifcant difference when compared to negative control (tsb only). assessment of anti-biofilm assay this method allowed the determination of anti-biofilm property of zno-nps versus vancomycin using the 96-well plate. the biomass of bacterial cell was quantitatively analyzed on the microplate reader at absorbance of 570 nm showing significant reduction of biofilm when increasing concentration of zno-nps used when compared to the control (tsb only). the result shows that zno-nps at 54 µg/ml exhibited significant anti-biofilm activity of 77.35 ± 2.67%. meanwhile, the anti-biofilm activity of the positive control vancomycin was measured at 40 ± 8.39% at higher concentration of 100 µg/ml tested (figure 3). figure 3. anti-biofilm assay using vancomycin as positive control and mrsa atcc 43300 as growth control. experiment evaluated based on quadruplicate results with standard deviation. (n = 4, p < 0.05). * indicates significant difference when compared to negative control (tsb only). conclusion collectively, the present study shows step-by-step optimized protocol of anti-adherence and anti-biofilm assays which incorporate the crystal violet biofilm staining method of visualization and quantification of biofilm biomass in a 96-well microplate reader. the use of 96-well plates has allowed more samples that can be tested at any one time and is preferable to be carried out post-mic assessment. conflict of interest the authors declare no conflict of interest. acknowledgement this work was inspired by monash phd research training module which entitled “bioprospective of microbes with biopharmaceutical potential with bioinformatics and drug discovery platforms” and financially by external industry grants from biotek abadi sdn bhd (vote no. gba-81811a) and monash global asia in the 21st century (ga21) research grant (ga-hw-19-l-01 and ga-hw-19-s02) and fundamental research grant scheme (frgs/1/2019/ wab09/musm/02/1 and frgs/1/2019/skk08/ musm/02/7). reference 1. lee l-h, zainal n, azman a-s, et al. streptomyces pluripotens sp. nov.: a bacteriocin-producing streptomycete that inhibits meticillin-resistant staphylococcus aureus. int j syst evol microbiol 2014; 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choo715@uitm.edu.my (c-yc) 6laboratory of human pathologies biology, department of biology, faculty of sciences, mohammed v university in rabat, morocco; a.bouyahya@um5r.ac.ma (ab) 7school of pharmacy, taylor’s university, subang jaya, selangor, malaysia; ganeshsritheran.paneerselvam@taylors.edu.my (gsp) 8faculty of pharmacy, university of cyberjaya, cyberjaya, selangor, malaysia; liewkaibin@cyberjaya.edu.my (kbl) 9faculty of data science and information technology, inti international university, 71800 nilai, malaysia; khangwen.goh@newinti.edu.my (kwg) 10pap rashidah sa’adatul bolkiah institute of health sciences, universiti brunei darussalam, gadong, brunei darussalam 11school of medical and life sciences, sunway university, bandar sunway 47500, selangor, malaysia; longchiauming@gmail.com (lcm) *corresponding author: ching siang tan; school of pharmacy, kpj healthcare university, 71800 nilai, malaysia; tcsiang@kpjuc.edu.my (tcs); andi hermansyah; 3department of pharmacy practice, faculty of pharmacy, universitas airlangga, surabaya, 60115, indonesia; andi-h@ff.unair.ac.id (ah); yaser mohammed al-worafi; college of medical sciences, azal university for human development, sana’a, yemen; yworafi@yahoo.com (yma) received: 26 february 2023; received in revised form: 13 june 2023; accepted: 18 june 2023; available online: 21 june 2023 pmmb 2023, 6, 1; a0000337 2 of 13 abstract: this article explored the potential applications, benefits, and risks of using chatgpt in medical and health sciences research. the experimental study was performed with content analysis of the potential applications, benefits and risks of using chatgpt in medical and health sciences research. this study shows many potential applications, benefits, and risks of using chatgpt in medical and health sciences research. the average experts’ chatgpt appropriateness and accuracy rates in the eight research themes were between 60% and 95%. this concludes that chatgpt could help medical and health sciences researchers, especially new researchers, with caution in many aspects of research. the chatgpt is still in the early phase of use by researchers worldwide, and its ability to help in research will be better soon. attending training workshops about chatgpt and ai is very important and highly recommended. the practice of chatgpt in medical and health sciences research is important and recommended to explore the potential uses, benefits, risks and suggest recommendations for the best practice. keywords: artificial intelligence; chatgpt; medical; health sciences 1. introduction researching the various aspects of medical and health sciences education and practice is very important to evaluate the current practices, identify the challenges and suggest recommendations to address the identified challenges. the evolution of technology has contributed effectively to the medical and health sciences education, practice, and research during the last decades; one of the new technologies is artificial intelligence (ai) which has been implemented in many fields, including medical and health sciences education, practice and research [1-9]. ai can be defined as the “science and engineering of making intelligent machines”, especially intelligent computer programs. it is related to the similar task of using computers to understand human intelligence. still, ai does not have to confine itself to methods that are biologically observable” [9], or as “a field of science and engineering concerned with the computational understanding of what is commonly called intelligent behavior, and with the creation of artifacts that exhibit such behavior” [10]. one of the recent advances in ai development is the launch of a model called chatgpt, which interacts conversationally. it is an innovative kind of ai that can produce writing that seems to have been written by a person [11]. the dialogue format allows chatgpt to answer follow-up questions, admit mistakes, challenge incorrect premises, and reject inappropriate requests; chatgpt is a general large language model (llm) developed recently by openai. while the previous class of ai models has primarily been deep learning (dl) models, which are pmmb 2023, 6, 1; a0000337 3 of 13 designed to learn and recognize patterns in data, llms are a new type of ai algorithm trained to predict the likelihood of a given sequence of words based on the context of the words that come before it [12]. the chatgpt can be used to assist researchers and health professionals in generating real time information dissemination [13]. for instance, in a study done on the covid-19 pandemic among brunei darussalam patients [14], by using chatgpt, the researcher can quickly gather data and help in answering queries about the latest case numbers, government regulations and guidelines, testing centers, vaccination sites and trends in vaccination. in a systematic review, the potential benefits of chatgpt health care education, research, and practice have been highlighted in improving writing, research efficiency, streamlined workflow, and personalized learning [15]. moreover, it can assist in drug use pattern-related studies, where researchers conducting a comparative analysis of the factors that impact the pattern of procurement and usage of antithrombotic drugs can leverage the insights gained to inform their medical and health sciences research [16]. additionally, they can harness the capabilities of chatgpt, an ai-powered language model, to further enhance their studies. by synergistically combining the findings from their comparative analysis with the support of chatgpt, medical and health sciences researchers can effectively advance their understanding of the subject matter and contribute to evidence-based practices in this domain. furthermore, chatgpt can be considered a supportive tool to facilitate the literature review process. it can help the researcher access a wide range of literature sources and provide an overview of studies from various disciplines and publications. for instance, in gathering data related to mangrove antinobacterial diversity [17], use of ibrutinib in multiple myeloma [18], or in identifying the prevalence of listeria monocytogens [19] related studies, chatgpt can provide relevant summaries of the studies, helping to identify important findings, methodologies, and identifying the research gaps in existing research. besides that, by leveraging chatgpt, researchers can access up-to-date information on topics like mycobacterium ulcerans [20], facilitating data gathering and answering queries related to pathogenesis, diagnosis, and treatment. additionally, chatgpt can aid in exploring breakthroughs in actinobacteria drug discovery research [21] by assisting in data analysis, literature reviews, and generating insights. furthermore, in studying the chemistry of the gut microbiome [22], chatgpt can assist researchers in retrieving relevant studies, analyzing complex interactions, and generating hypotheses for further investigation. overall, chatgpt can be a valuable tool in medical research, supporting researchers with information retrieval, data analysis, and knowledge synthesis [23]. pmmb 2023, 6, 1; a0000337 4 of 13 as massive studies show that chatgpt has the potential to revolutionize medical research in various ways [24-27], many medical and health sciences researchers started using chatgpt at the end of 2022 for many purposes, including research and scientific writing. with its potential to revolutionize the clinical and translational medicine fields, chatgpt offers benefits like access to current information, enhanced patient engagement, and reduced healthcare provider workloads [28]. this article aimed to explore the potential applications, benefits, and risks of using chatgpt in medical and health sciences research. 2. materials and methods 2.1. study design the researchers will perform the content analysis of the potential applications, benefits, and risks of using chatgpt in medical and health sciences. 2.2 data collection the research was conducted between 01 january 2023 and 20 february 2023 to explore the potential applications, benefits, and risks of using chatgpt in medical and health sciences research. questions related to the medical and health sciences research were asked to explore the ability of chatgpt to answer them; the questions were divided into the following themes: 2.2.1. theme 1. questions related to the research ideas the questions were to suggest ideas for research projects; eight questions were related to pharmacy practice, public health, and patient care. 2.2.2. theme 2. questions related to the research proposal the questions were to write a proposal for the research projects. three projects were selected from what chatgpt suggested in the previous step (theme 1) related to pharmacy practice, public health, and patient care. 2.2.3. theme 3. questions related to the research background the questions were to write a background for the research projects. three projects were selected from what chatgpt suggested in the previous step (theme 2) related to pharmacy practice, public health, and patient care. pmmb 2023, 6, 1; a0000337 5 of 13 2.2.4. theme 4. questions related to the literature review the questions were to write a literature review for the selected three projects. 2.2.5. theme 5. questions related to the research questions and objectives the questions were to suggest research questions and objectives for the selected three projects. 2.2.6. theme 6. questions related to the research hypothesis the questions were to suggest null and alternative hypotheses for the selected three projects. 2.2.7. theme 7. questions related to the significance of the study and expected outcomes the questions were to write the significance of the study and expected outcomes for the three selected projects. 2.2.8. theme 8. questions related to the methodology the questions were to suggest different study designs, data collection, populations, sample size calculation, sampling methods, and data analysis for the three selected projects. 2.3. data analysis four professors with long experience in medical and health sciences research (two from pharmacy school and two from medicine school) independently evaluated the answers by chatgpt and rated the appropriateness and accuracy of each question as a percentage out of 100; the average of four professors’ evaluations was considered in this study. moreover, they will comment on the potential applications, benefits, and risks of using chatgpt in medical and health sciences research. 3. results 3.1. theme 1 questions related to the research ideas analysis of the expert’s opinion shows that chatgpt was able to suggest more than ten research ideas for each specialty. moreover, the ideas were good. the average of experts’ rates of appropriateness and accuracy was 95%. pmmb 2023, 6, 1; a0000337 6 of 13 3.1.1. potential benefits chatgpt can help medical and health sciences researchers, especially new researchers, select good ideas. 3.1.2. potential risks chatgpt can generate general ideas but cannot generate specific ideas. 3.1.3. recommendations medical and health sciences researchers, especially new researchers, can use chatgpt as a guide for generating research ideas. 3.2. theme 2 questions related to the research proposal analysis of the expert’s opinion shows that chatgpt could write brief research proposals for the three selected proposals. the average experts’ rates of appropriateness and accuracy were 60%. 3.2.1. potential benefits chatgpt can help medical and health sciences researchers, especially new researchers, as a guide in writing brief research proposals. 3.2.2. potential risks there are many steps in writing good proposals, which chatgpt will not be able to do. 3.2.3. recommendations medical and health sciences researchers cannot use chatgpt to write good research proposals. 3.3. theme 3 questions related to the research background analysis of the expert’s opinion shows that chatgpt could write research background with the rationality for the three selected projects. however, chatgpt was not able to cite what was written appropriately. the average of experts’ rates of appropriateness and accuracy was 75%. pmmb 2023, 6, 1; a0000337 7 of 13 3.3.1. potential benefits chatgpt can help medical and health sciences researchers, especially new researchers, as a guide in writing background about research projects. 3.3.2. potential risks the background should include the appropriate references, which chatgpt will not be able to do it. 3.3.3. recommendations chatgpt can help medical and health sciences researchers, especially new researchers, as a guide in writing the research background. 3.4 theme 4 questions related to the literature review analysis of the expert’s opinion shows that chatgpt was able to write a research literature review for the three selected projects. however, chatgpt could not cite what was written appropriately. moreover, the literature review was not up-to-date. the average experts’ rates of appropriateness and accuracy were 70%. 3.4.1. potential benefits chatgpt can help medical and health sciences researchers, especially new researchers, as a guide in writing literature reviews. this was in line with the finding by vaishya et al. (2023), which states that the current chatgpt version appears helpful to medical professionals [29]. 3.4.2. potential risks writing a literature review should include the appropriate references and update the literature, which chatgpt will not be able to do. 3.4.3. recommendations chatgpt can help medical and health sciences researchers, especially new researchers, as a guide in writing the literature review. pmmb 2023, 6, 1; a0000337 8 of 13 3.5. theme 5 questions related to the research questions and objectives analysis of the expert’s opinion shows that chatgpt was able to suggest good objectives and study questions. the average experts’ rates of appropriateness and accuracy were 90%. 3.5.1. potential benefits chatgpt can help medical and health sciences researchers, especially new researchers writing the research objectives and study questions. 3.5.2. potential risks chatgpt cannot generate specific objectives and research questions. 3.5.3. recommendations medical and health sciences researchers, especially new researchers, can use chatgpt as a guide for writing the study objectives and questions. 3.6. theme 6 questions related to the research hypothesis analysis of the expert’s opinion shows that chatgpt was able to suggest good null and alternative hypotheses for the research projects. the average of experts’ rates of appropriateness and accuracy was 90%. 3.6.1. potential benefits chatgpt can help medical and health sciences researchers, especially new researchers, write the null and alternative hypotheses for research projects. 3.6.2. potential risks chatgpt cannot generate specific null and alternative hypotheses for the research projects. 3.6.3. recommendations medical and health sciences researchers, especially new researchers, can use chatgpt as a guide for writing the null and alternative hypotheses for research projects. pmmb 2023, 6, 1; a0000337 9 of 13 3.7. theme 7 questions related to the significance of the study and expected outcomes analysis of the expert’s opinion shows that chatgpt was able to write the significance of the study and expected outcomes for the research projects. the average experts’ rates of appropriateness and accuracy were 80%. 3.7.1. potential benefits chatgpt can help medical and health sciences researchers, especially new researchers, write the study's significance and expected outcomes for the research projects. 3.7.2. potential risks chatgpt cannot generate specific significance of the study and expected outcomes for the research projects. 3.7.3. recommendations medical and health sciences researchers, especially new researchers, can use chatgpt as a guide for writing the significance of the study and expected outcomes for the research projects. 3.8. theme 8. questions related to the methodology analysis of the expert’s opinion shows that chatgpt could suggest different study designs, data collection, populations, sample size calculation, sampling methods, and data analysis for the selected projects. the average experts’ rating of appropriateness and accuracy was 60%. 3.8.1. potential benefits chatgpt can help medical and health sciences researchers, especially new researchers suggest different study designs, data collection, populations, sample size calculation, sampling methods, and data analysis for the selected projects. moreover, chatgpt can elaborate on each method, data collection, population, sample size calculation, sampling methods, and data analysis. pmmb 2023, 6, 1; a0000337 10 of 13 3.8.2. potential risks chatgpt cannot generate valid and reliable methods. there are many steps to design valid and reliable questionnaires or qualitative interviews, which chatgpt will not be able to do. 3.8.3. recommendations medical and health sciences researchers, especially new researchers, in many can use chatgpt as a guide for methodology. however, medical and health sciences researchers cannot use chatgpt to develop valid and reliable questionnaires and qualitative interviews. 4. discussion this study explored the potential applications, benefits, and risks of using chatgpt in medical and health sciences research. the findings of this study can be classified into the following themes. 4.1. theme 1 research ideas this study's findings show that chatgpt could suggest good research ideas. research ideas are critical to start any research project; this could help new researchers in their early careers as a guide. however, researchers should apply critical thinking and search for new research ideas to fill the gap in the literature. moreover, more information and research about this issue must be needed to compare and conclude that chatgpt or the new ai technologies positively impact generating ideas. in conclusion, many medical and health sciences researchers, especially new researchers, can use chatgpt as a guide for developing research ideas. for example, using a medical mobile phone application linked to chatgpt assists in collecting a wealth of data and analyzing and interpreting this data into meaningful patterns and insights to support medical and health sciences research [30]. 4.2. theme 2 research proposal this study's findings show that chatgpt could write brief research, which is unacceptable to depend on for acceptable research proposals. however, chatgpt is still in the early development phase, and it could be better soon and be able to generate better proposals. pmmb 2023, 6, 1; a0000337 11 of 13 4.3. theme 3 research background and literature review this study's findings show that chatgpt could write research background without the appropriate references and updated literature. this could be due to many reasons, such as the nature of chatgpt as a new language model and the need for access to scientific databases. 4.4. theme 4 research questions, objectives and hypothesis this study's findings show that chatgpt was able to suggest good research objectives, questions, and hypotheses. therefore, many medical and health sciences researchers, especially new researchers, can use chatgpt as a guide for generating research objectives, questions, and hypotheses. 4.5. theme 5 expected outcomes and significance of study this study's findings show that chatgpt could write the significance of the study and expected outcomes for the research projects but cannot generate specific significance of the study and expected outcomes for the research projects. this could be due to the nature of chatgpt as a new language model. chatgpt is still in the early development phase and could be better soon. 4.6. theme 6 research methodology this study's findings show that chatgpt cannot generate valid and reliable methods. however, chatgpt was able to suggest different study designs, data collection, populations, sample size calculation, sampling methods, and data analysis, which could help medical and health sciences researchers, especially new researchers. 5. conclusions in conclusion, chatgpt could help medical and health sciences researchers, especially new researchers, with caution in many aspects of research. chatgpt is still in the early phase of use by researchers worldwide, and its ability to help in research will be better soon. attending training workshops about chatgpt and ai is very important and highly recommended. practice chatgpt in medical and health sciences research is very important and highly recommended to explore the potential uses, benefits, and risks and suggest recommendations for the best practice. pmmb 2023, 6, 1; a0000337 12 of 13 author contributions: all authors contributed equally to this study. funding: no external funding was provided for this research. conflicts of interest: the authors declare no conflict of interest. references 1. petitti db, crooks vc, buckwalter jg, et al. blood pressure levels before dementia. arch neurol 2005; 62(1): 112– 116. 2. masters k. artificial intelligence in medical education. medical teacher. 2019; 41(9):976-80. 3. loeckx j. blurring boundaries in education: context and impact of moocs. international review of research in open and distributed learning. 2016;17(3):92-121. 4. ahuja as. the impact of artificial intelligence in medicine on the future role of the physician. peerj. 2019;7:e7702. 5. bohr a, memarzadeh k. the rise of artificial intelligence in healthcare applications. inartificial intelligence in healthcare. academic press. 2020; 1:25-60. 6. secinaro s, calandra d, secinaro a, et al. the role of artificial intelligence in healthcare: a structured literature review. bmc medical informatics and decision making. 2021;21:1-23. 7. davenport th, ronanki r. artificial intelligence for the real world. harvard business review. 2018 1;96(1):108-16. 8. hosny a, parmar c, quackenbush j, schwartz lh, aerts hj. artificial intelligence in radiology. nature reviews cancer. 2018;18(8):500-10. 9. roll i, wylie r. evolution and revolution in artificial intelligence in education. international journal of artificial intelligence in education. 2016;26:582-99. 10. mccarthy j. what is artificial intelligence. available from : https://cse.unl.edu/~choueiry/s09-476876/documents/whatisai.pdf . 2007 11. javaid m, haleem a, singh rp. chatgpt for healthcare services: an emerging stage for an innovative perspective. benchcouncil transactions on benchmarks, standards and evaluations 2023; 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a0000336. doi: 10.36877/pmmb.0000336 http://journals.hh-publisher.com/index.php/pmmb original research article quality assessment of hydroxychloroquine tablet: a comparative evaluation of drug produced by different pharmaceutical companies in bangladesh md. raihanur islam1, md. sakhawat hossain2*, md. sanower hossain3*, mohammad touhidul islam1, sharifa sultana1, nisarat nizhum2, kutub uddin ahamed4, chee-yan choo5,6, ching siang tan7*, khang wen goh8 article history 1department of pharmacy, daffodil international university, daffodil smart city, ashulia, dhaka, bangladesh; raihanur.islam.2374@gmail.com (mri); touhidul.ph@diu.edu.bd (mti); sharifa@daffodilvarsity.edu.bd (ss) 2pharmaceutical sciences research division, bcsir dhaka laboratories, bangladesh council of scientific and industrial research (bcsir), dr. qudrat-i-khuda road, dhanmondi, dhaka 1205, bangladesh; sakhawat.hossain@bcsir.gov.bd (msh), nisarat.nijhum@gmail.com (nn) 3centre for sustainability of ecosystem and earth resources (pusat alam), universiti malaysia pahang, kuantan 26300, malaysia 4bcsir rajshahi laboratories, bangladesh council of scientific and industrial research (bcsir), rajshahi 6206, bangladesh; kutubuddinjnu@gmail.com (kua) 5faculty of pharmacy, universiti teknologi mara, puncak alam, selangor, malaysia; choo715@uitm.edu.my (cyc) 6medchem herbal research group, faculty of pharmacy, universiti teknologi mara, puncak alam, selangor, malaysia 7school of pharmacy, kpj healthcare university college, 71800 nilai, malaysia 8faculty of data science and information technology, inti international university, 71800 nilai, malaysia; khangwen.goh@newinti.edu.my (kwg) *corresponding author: md. sakhawat hossain; pharmaceutical sciences research division, bcsir dhaka laboratories, bangladesh council of scientific and industrial research (bcsir), dr. qudrat-i-khuda road, dhanmondi, dhaka 1205, bangladesh; sakhawat.hossain@bcsir.gov.bd (msh); md. sanower hossain, centre for sustainability of ecosystem and earth resources (pusat alam), universiti malaysia pahang, kuantan 26300, malaysia; mshossainbge@gmail.com; mshossainbge@ump.edu.my (msh); ching siang tan; school of pharmacy, kpj healthcare university college, 71800 nilai, malaysia; tcsiang@kpjuc.edu.my (cst) received: 03 february 2023; received in revised form: 03 june 2023; accepted: 18 june 2023; available online: 05 july 2023 abstract: hydroxychloroquine is the most commonly prescribed antimalarial extensively used to treat rheumatoid arthritis. it is extensively utilized as a repurposing drug, as well, in many countries worldwide to treat covid-19. the pharmaceutical sector of bangladesh is much enriched, and different pharmaceutical companies in bangladesh produce this drug. since the drug quality might vary significantly among different brands, assuring the quality mailto:raihanur.islam.2374@gmail.com mailto:touhidul.ph@diu.edu.bd mailto:sharifa@daffodilvarsity.edu.bd mailto:sakhawat.hossain@bcsir.gov.bd mailto:nisarat.nijhum@gmail.com mailto:kutubuddinjnu@gmail.com mailto:choo715@uitm.edu.my mailto:sakhawat.hossain@bcsir.gov.bd mailto:tcsiang@kpjuc.edu.my pmmb 2023, 6, 1; a0000336 2 of 11 of medicine is absolutely necessary considering the health issues, particularly therapeutic efficacy and safety. therefore, this study examined the quality of hydroxychloroquine produced by bangladeshi pharmaceutical companies, concentrating on quality control parameters: the assay, dissolution, disintegration, hardness, friability, and weight fluctuation. all the brands of hydroxychloroquine tablets contained the stated amount of api between the range of 96.41±0.62 and 100.61±0.71 that met usp specification (100±5%). all brands met the pharmacopeial limit for the percentage of weight fluctuation, hardness test, friability, and disintegration time. weight variation was between 0.31±0.01% and 0.46±0.02%, hardness was between 4.31 ± 0.88 and 7.36 ± 0.74 kgf, friability was less than 1%, and disintegration time was 5.42± 0.11 and 5.42± 0.11 min. in the dissolution test, all the samples attained more than 70% dissolution after 30 minutes. the mean percentage of hydroxychloroquine released in phosphate buffer was between 95.44±0.55 (brand b) and 98.19±0.39 (brand c) after 60 min. no significant difference was among the tested drugs from different companies, and all quality assessment parameters were within usp specifications. therefore, hydroxychloroquine from the bangladesh market is safe and effective. keywords: hydroxychloroquine; dissolution; disintegration; hardness; bangladesh 1. introduction the pharmaceutical industry in bangladesh has grown tremendously over the past few decades and has become one of the most vital sectors of the country's economy. this is one of the largest sectors in south asia, with a market size of around usd 3 billion. according to industry experts, this sector is projected to continue its upward trajectory, and the market size is expected to exceed usd 6 billion by 2025[1]. there are 231 active and licensed pharmaceutical companies available in bangladesh[2]. these pharmaceutical companies meet about 97% of the country's total demand[3]. thus, ensuring the quality, therapeutic efficacy, and safety of drugs has become a fundamental issue in the postmarketing monitoring of the drug. this type of monitoring can play a leading role in ensuring quality products[4]. the support from the pharmaceutical industries was remarkable during covid-19 pandemic. various precautious programs and strategies were undertaken to stop the spread of sars-cov-02 throughout the world, e.g., in south africa, australia, malaysia, etc.[5–10], as well as a unique strategy like establishing artificial intelligence to provide advice and inquiries about covid-19, was done by spain[11]. an exception, although a lockdown has been implemented to decelerate the transmission of covid-19, the number of deaths in the united kingdom was still rising rapidly[12]. pmmb 2023, 6, 1; a0000336 3 of 11 repurposing a medicine refers to using existing pharmaceuticals for new therapeutic possibilities rather than their primary uses[13]. from the early phase of the covid-19 pandemic, several investigations towards repurposing drugs for covid-19 have been performed, and clinical trials on several drugs are still being conducted[14]. hydroxychloroquine has been widely used in different countries worldwide as a repurposed drug to overcome the covid-19 pandemic[13]. hydroxychloroquine has been recommended for the last 60 years as a safer analog of chloroquine and is also the most commonly prescribed antimalarial drug[15]. this drug is broadly used as a therapeutic agent against systemic lupus erythematosus and rheumatoid arthritis[16–17]. it has become a challenging issue for physicians to choose the right drug as these drugs are readily available in different brand names. so, assessing the quality of medicine has become more critical. since effective and safe treatment is vital for everyone, such research plays a significant role in this case. the primary goal of this research is to compare the efficacy of hydroxychloroquine (200 mg) generic drug (tablet) produced by different pharmaceutical companies in bangladesh and to decrease health risks by ensuring drug safety. 2. materials and methods 2.1. collection of standard api powder of hydroxychloroquine was supplied pharmaceutical sciences research division, bangladesh council of scientific and industrial research (bcsir), dhaka, bangladesh, as a gift for conducting the research. 2.2. collection of sample five different brands of hydroxychloroquine (200 mg) tablets respectively were bought from the resident drug shop of dhaka metropolitan city. all the samples were precisely examined during purchase for their physical appearance, manufacturing license numbers, batch numbers, manufacturer name, dar numbers, manufacturing date, expiry dates, and maximum retail price. the samples were arbitrarily coded as a, b, c, d, and e. samples were kept by maintaining standard storage conditions (humidity: 45–60% and temperature: 25±2°c). 2.3. solvents and reagents this study used purified water, sodium hydroxide, analytical grade of di-sodium hydrogen phosphate, and sodium dihydrogen phosphate. pmmb 2023, 6, 1; a0000336 4 of 11 2.4. weight variation test twenty-five tablets from each of the 5 (five) different brands of hydroxychloroquine were taken and weighed individually with the help of an analytical weighing balance (and ek-600i; electronic precision balance). then the mean weight for each brand was calculated, and the weight variation from the actual value was calculated using the following equation[18]. % weight variation = 𝐼𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 − 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑊𝑒𝑖𝑔ℎ𝑡 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑊𝑒𝑖𝑔ℎ𝑡 × 100 2.5. hardness test to resist friability and remove mechanical shocks during packaging and shipping, tablets need a definite amount of strength or hardness[19]. a monsanto-type (vinsyst technologies, india) hardness tester was used to perform this test. ten tablets were arbitrarily chosen from each brand of hydroxychloroquine. the tested tablet was placed vertically between the spindle and the anvil. each tablet was subjected to pressure in a clockwise direction, and the amount of pressure required to break each tablet was noted[20]. 2.6. friability test a friability test can be done to evaluate the ability of tablets to withstand abrasion, transporting, and handling. friabilator is made of a plastic chamber with two parts and revolves at 25 rpm. ten tablets from each brand of hydroxychloroquine were taken and weighed together. then the machine was fixed at 25 rpm for 5 minutes, and all the tablets (10 tablets) were placed on the roche friabilator. after 125 revolutions, the tablets were removed from the machine and weighted together again. the loss in weight indicated friability[21]. the following equation was used to determine the percentage of friability: % friability (f) = 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 −𝐹𝑖𝑛𝑎𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 × 100 2.7. disintegration test the process by which a tablet breaks down into smaller pieces is called disintegration[22]. at first, the vessel (1000 ml) of tablet disintegration tester (disintegration test apparatus, single unit; cat no. 2249) was filled with 900 ml distilled water, and the temperature was set to 37±0.5°c. six tablets from each brand of hydroxychloroquine were taken and placed in the disintegration chamber basket and the disk pmmb 2023, 6, 1; a0000336 5 of 11 appropriately, respectively. the machine was started, and the disintegration time (dt) was noted when no particles were in the system basket[23]. 2.8. assay preparation twenty tablets from each of the brands of hydroxychloroquine were taken and finely pulverized. the powdered samples were accurately weighted. powdered samples were taken in a 200 ml volumetric flask equivalent to 200 mg of hydroxychloroquine sulfate, and 150 ml phosphate buffer (ph 6.8) was added to make the solution cooled at room temperature and filtered. 5 ml of filtered solution were transferred to a 100 ml volumetric flask and diluted using phosphate buffer. absorbance was measured at 343 nm wavelength (λmax) using a uv-vis spectrophotometer (t60 u, pg instruments limited, uk). 2.9. preparation of standard curve in a 100 ml volumetric flask, 100 mg hydroxychloroquine sulfate was dissolved with the help of 100 ml phosphate buffer to make a concentration of 1000 μg/ml. from this standard solution, 10 ml of the solution was transferred to a 100 ml volumetric flask, and up to 100 ml of phosphate buffer was added to make the concentration 100 μg/ml. the serial dilution (2.5, 10, 10, 15, 20 μg/ml) was made using the above method. the absorbance of the following concentration was measured using a uv-vis spectrophotometer (t60 u, pg instruments limited, uk) at 254 nm wavelength. phosphate buffer was used as a blank. a standard curve was obtained by plotting the measured absorbance against the corresponding concentration (figure 1). 2.10. dissolution test dissolution tests are typically conducted to identify the drug release pattern over time. the dissolution study of five different brands of hydroxychloroquine was performed with usp apparatus type ii (paddle; rc-8; minhua pharmaceutical machinery co., ltd, china) at 50 rpm. as dissolving media, 900 ml of phosphate buffer (ph 6.8) was utilized, and the temperature was always maintained at 37±0.5°c. in the entire procedure, 10 ml of sample were taken out at 10, 20, 30, 40, 50, and 60 minutes, respectively, and substituted with phosphate buffers of equal volume. samples were kept dark and then examined by uv-vis spectrophotometer (t60 u, pg instruments limited, uk) at 343 nm. utilizing the standard curve of api (the data shown in the result section) of generic hydroxychloroquine drugs, the concentration of the sample was calculated from the equation y = mx + c. pmmb 2023, 6, 1; a0000336 6 of 11 3. result and discussion all the results were represented as mean ± sd, and the obtained data were then analyzed using nonlinear regression with the help of graphpad prism software (version 8.1). to evaluate quality parameters, various tests were performed for all the tablets of different brands of hydroxychloroquine obtained at dhaka city's local market. the weight variation test is an effective method for assessing the uniformity of the api present in the dosage form. keeping the weight variation accurate serves as an indicator of gmp compliance for every manufacturing organization. for tablets weighing 130 mg or less, the weight difference deviation range is ±10%, ±7.5% for tablets weighing more than 130 mg to 324 mg, and ±5% for tablets weighing more than 324 mg. if not more than two tablets cross the percentage limit, the sample passes the usp test, and if no tablet does not cross two times the percentage limit[24]. when the weight variation is within the pharmacopeial specifications, the active ingredient in the tablets is suspected to be uniform, providing the desired therapeutic response[22]. the weight variation of all the tablets of the same brand tested in this study met the usp specifications (table 1). the hardness test determines how resistant a tablet is to pressure or stress during manufacture, packaging, handling, and transportation[25]. it affects disintegration and dissolution, thus affecting bioavailability. a tablet may not disintegrate in the required amount of time if it is too hard, and if it is too soft, it may not resist handling. it has been observed that several factors may influence a tablet's hardness, including drug concentration, particle size and density, binder type, lubricant type and concentration, compression force, etc.[26–28]. the acceptable range of the hardness of an oral tablet is usually 4–8 kgf [25]. the hardness of five brands of hydroxychloroquine was between 4.31 ± 0.88 to 7.36 ± 0.74 kgf, which is within the usp specification (table 1). another mechanical property of a tablet is friability. friability is a surface deformation, whereas hardness is a bulk deformation[29]. according to the usp guidelines, the percentage of ideal friability should be more than 1%[30]. in this study, all of the evaluated brands of hydroxychloroquine tablets had a percentage of friability of less than 1%; hence they all met the specification of pharmacopeia. as a result, the five brands of hydroxychloroquine tablets available in bangladesh tested in this study had good strength and could withstand shocks during handling and transportation. disintegration is the initial stage of dissolution and involves breaking down a tablet into smaller pieces[31]. the fact that a drug has a fast disintegration time does not necessarily mean it will be quickly available for the body to absorb. other factors are also linked to the pmmb 2023, 6, 1; a0000336 7 of 11 drug's bioavailability[32]. a longer disintegration time indicates that the tablet is probably too tightly compressed. when the disintegration time is irregular, a lack of batch consistency will likely present[21]. the rate of dissolution is directly proportional to the disintegration rate. disintegration time also affects the absorption and efficacy of a drug[25]. according to usp guidelines, uncoated and film-coated tablets need 5–30 minutes to disintegrate[30]. the disintegration time of five brands of hydroxychloroquine tablets meets the usp specification (table 1). pharmaceutical product assay is a crucial quality characteristic needed to check that the stated quantity of api is present in a specific dosage form; failing to achieve the standard specifications will result in substandard quality medications. an insufficient amount of api may result in poor-quality treatment, whereas an excessive amount of api may create adverse drug reactions[21]. the assay result of all five brands of hydroxychloroquine met the usp standardization, which lies between 96.41±0.62 to 100.61±0.71 (table 1). the dissolution test determines the percentage of medications that dissolve in a specific time under controlled in vitro conditions. the dissolution rate determines the rate and extent of drug absorption and the subsequent therapeutic result of medicine. dissolution has been used to demonstrate bioequivalence and is considered the most essential tool for predicting in vivo bioavailability. the dissolution test of solid oral medicinal formulations has evolved into an important quality control test for ensuring product homogeneity and batch-to-batch consistency[25]. the percentage of drug release of five brands of hydroxychloroquine at different time intervals is shown in figure 2. the mean percentage of hydroxychloroquine released in the phosphate buffer was found to be between 95.44±0.55 (brand b) and 98.19±0.39 (brand c) after 60 min. following the usp specification, the percentage of hydroxychloroquine dissolved should not be less than 70% after 60 min[33]. the dissolution test results showed that each brand passed the usp-instructed dissolution standards that ensure the quality of this medication produced in bangladesh. table 1. weight variation, hardness, friability, disintegration time, and assay results of various brands of hydroxychloroquine (200 mg) tablets. brand code weight variation (%) hardness (kgf) friability % dt (min) drug content (%) a 0.45±0.01 7.36 ± 0.74 0.44 5.79 ± 0.46 98.28±0.89 b 0.46±0.02 4.31 ± 0.88 0.45 6.46 ± 0.29 96.70±0.45 c 0.31±0.01 6.39 ± 0.43 0.65 8.52 ± 0.05 99.82±0.91 d 0.32±0.01 5.62 ± 1.03 0.63 5.42± 0.11 96.41±0.62 e 0.39±0.01 5.71 ± 0.44 0.91 7.23 ± 0.14 100.61±0.71 usp specification ± 5% to ± 7.5% 4–8 kgf less than 1% 5–30 min 95–105% values are expressed as mean ± sd (n=5) pmmb 2023, 6, 1; a0000336 8 of 11 figure 1. standard curve of hydroxychloroquine. 0 20 40 60 80 0 50 100 time (min.) % o f d r u g r e le a se brand a brand b brand c brand d brand e figure 2. intra-brand dissolution profile of hydroxychloroquine. data represents as the mean±sd (n=5). 4. conclusion in vitro trials play an essential role in ongoing industrial practice because comparing different brands of the same generic drug makes it easy. at the same time, such exercises play a beneficial role in manufacturing an effective dosage form. hydroxychloroquine is a widely used antimalarial drug but has been used to repurpose therapeutics for treating covid-19. in this study, we evaluated in vitro comparison of five brands of hydroxychloroquine available in bangladesh. the results obtained from this research have met the pharmacopeial specification. these findings conclude that the hydroxychloroquine tablets of these five brands sold in bangladesh satisfy the therapeutic efficacy quality requirement. pmmb 2023, 6, 1; a0000336 9 of 11 every brand, including lower-ranked ones, has shown that their products meet official standards for quality. this research may help the drug regulatory authority and the general public overall idea regarding the quality of commercialized hydroxychloroquine tablets in bangladesh. since a small number of pharmaceutical companies were selected for this research, it is necessary to conduct further research involving many manufacturing companies to understand the overall scenario. high throughput analytical techniques like high-performance liquid chromatography (hplc) are highly recommended for quantifying hydroxychloroquine since it has high sensitivity and can separate and identify impurities or closely related compounds. author contributions: mri laboratory work, calculation, manuscript writing; msh concept design, manuscript writing, and review, data analysis; msh manuscript review and editing, supervision, data interpretation, and visualization; mti manuscript drafting and reviewing, data analysis; ss manuscript review and supervision; nn laboratory work, manuscript writing; kua laboratory work, data analysis; cyc manuscript review and editing; cst manuscript review and editing; kwg; manuscript review and editing. funding: this study did not receive any external funding. conflicts of interest: the authors declare no conflict of interest. references 1. bangladesh investment development authority (bida). the bangladesh pharmaceutical industry-the emerging asian hub for generic medicines [accessed on 25 nov 2022]; available from: https://bida.gov.bd/pharma-api. 2. allopathic [internet]. 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edition. j pharm technol 2010; 26(3):167–8. 32. jambhekar ss and breen pj. drug dissolution: significance of physicochemical properties and physiological conditions. drug discov today 2013; 18(23–24):1173–84. 33. usp monographs: hydroxychloroquine sulfate tablets; available from: http://www.pharmacopeia.cn/v29240/usp29nf24s0_m38910.html. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology review article 1 covid-19: are malaysians embracing or suffering the new normality? dinyadarshini johnson1, stanley eng chee ren2, hema darshinee johnson3, vengadesh letchumanan1* 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2hospital sultan ismail, jalan mutiara emas utama, taman mount austin, 81100 johor bahru, johor, malaysia 3hospital selayang, b21, lebuhraya selayang kepong, 68100 batu caves, selangor, malaysia abstract: the covid-19 pandemic has inevitably rendered a paradigm shift in peoples’ day-to-day normality. the pandemic has precipitated various reaction and responses from people across the globe especially with the enforcement of preventive measures initiated by their respective government forces. the movement control order (mco) was one of the drastic measures taken in malaysia adhering to the guideline released by world health organization (who) and has been made effective since 18th of march 2020. the execution of mco in a developing setting like malaysia certainly impacts its people on several fronts, especially those from low-socioeconomic background. it creates a domino effect from an economical to psychological aspects at both societal and individual levels. subsequently, a conditional mco (cmco) has been introduced during midway through phase 4 of mco with eased restrictions, particularly considering economic downturn. cmco is followed by recovery mco (rmco) phase. in this article, we aim to share some insights while highlighting the impacts of covid-19 with an emphasize on the psychosocial aspect, particularly during mco phases, which has thus imposed a new normality on malaysians. keywords: covid-19; malaysia; movement control order (mco); impacts; new normality received: 1st august 2020 accepted: 20th august 2020 published online: 31st august 2020 citation: johnson d, ren sec, johnson hd et al. covid-19: are malaysians embracing or suffering the new normality? prog microbes mol biol 2020; 3(1): a0000102. https://doi.org/10.3687/pmmb. a0000102 introduction a coronavirus disease, known as covid-19, is a deadly manifestation of a novel virus which came to light in december 2019 when pneumonia of unknown cause was first reported in china[1–4]. the outbreak took a downward spiral when world health organization (who) subsequently declared it as a pandemic in early march 2020 with death approaching nearly 1000 in the european region[5,6]. to date, a whopping total of approximately 770, 000 deaths and close to 22 million confirmed cases of covid-19 have been reported globally[7]. amongst southeast asia countries, malaysia records one of the highest numbers of confirmed cases with over 6000 cases and 103 total deaths by early may 2020, however this scenario gradually changed as the vigorous measures taken by the authorities through mco managed to reasonably arrest the rapid spread of covid-19 infection in local setting. since june 2020 until the present, the reported number of new cases remains within 2 digits which inevitably reflects the effective countermeasures executed by the local authorities[8,9]. as part of the federal government’s initiative to combat the rapid spread of covid-19 with an alarming surge in new cases amounting to 190 cases on 15th of march 2020, a movement control order (mco) has been issued effective from 18th of march 2020 till 12th of may 2020[10]. the initial order for a period of 2 weeks has been gradually extended to a total period of 8 weeks, adhering to the who’s guideline[8,11]. each phase of mco, lasting for 2 weeks respectively, sees a gradual modification on the restriction rules, depending on the reported number of new cases and recovery rate. the strictest rules were observed during phase 2 and phase 3 of mco starting from 1st of april 2020 till 28th of april 2020. during these phases, travel distance was limited to within 10km of radius from home, only one representative from each family allowed to travel at a time to buy essentials, and business hours and delivery copyright @ 2020 by johnson d and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: vengadesh letchumanan, novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia; vengadesh.letchumanan1@monash.edu. 2 time were limited to 8am till 8pm. there were some areas in major cities subjected to complete lockdown as well. almost no tolerance was given to mco violators where 9090 offenders were arrested while 4036 compounds were issued as of 13th of april 2020[12,13]. as soon after implementation of mco, the number of new cases peaked at most 235 cases per day in march end with a more vigorous screening process taking place across the nation. the trend has been generally decreasing since then, however, with some fluctuations in number of new cases reported on a daily basis. the recovery rate has been reported as high as 70%[8]. during midway through phase 4, from 29th of april 2020 till 12th of may 2020, a conditional mco has been introduced as phase 5 starting from 4th of may 2020 by easing some of the restrictions mainly catering to reopening of selected economic sectors involving essential services and manufacturing of critical products. the psychosocial well-being of general public has also been considered by allowing two family members to travel in a car, beyond 10km of radius from home for health needs, groceries and food, and some outdoor sports activities were also allowed while strictly exercising social distance and self-hygiene[11,14–16]. challenges and difficulties inevitably accompany the execution of this obligatory mandate at numerous aspects as far as the public is concerned. however, the same amusingly unfolds some fundamental values which hold the society together amid an unfathomable crisis. this article intends to explore some insights and impacts relating to the covid-19 situation in malaysia from a societal perspective during the mco phases particularly. measure of societal awareness and readiness the announcement of mco has inevitably provoked an alarming sense of fear and insecurity amongst the public, however, at varying concerns amongst the different strata within the society, largely influenced by individual socioeconomic status. panic-buying is one of the resulting occurrences which happened at early phase of mco where people flocked to the supermarkets, grocery stores and pharmacies, especially in major cities to stock up on provisions for their homes[17]. essentials were absurdly purchased in larger than usual amount with sole aim to survive the foreseen restriction due to mco. this is probably an immediate but fading gratification considering the mismatch between the needs and the wants in a long run and poorly understood terms of mco. the wearing of face mask is another aspect which becomes a highly critical measure of societal awareness and knowledge. mask has become one of the most sought-after essentials during this phase. while the usage of mask becomes a matter of debate, it has been made compulsory to wear a face mask upon entering any outside premises currently. although this was not one of the mandates by the government at the initial stage, starting from 1st of august 2020, it has been made compulsory for everyone to wear mask in public domains[18,19]. given the asymptomatic yet possibly contagious phase of covid-19 incubation period, the wearing of mask is merely recommended for everyone being outside[20,21]. however, the paranoia of wearing a mask to the extent of making own masks from random cloths or even tissue paper, and stealing masks from hospitals while neglecting other preventive measures only point to the lack of comprehensive understanding on the rationale behind proper usage of personal protective equipment (ppe) amongst the public[22–24]. while panic-buying and face mask obsession became the highlight during the initial phases of mco, the announcement of conditional mco with eased restrictions triggered an unexpected response from 130, 000 over malaysians who signed a petition to call it off and continue with the existing mco rules. a fear of repeating mistakes of other countries by easing restrictions only to see another wave of covid-19 has provoked such a response amongst the public[18]. resonating with the public’s fear, an increased number of new cases was seen during phase 4 after easing some movement restriction, with 94 cases reported on 29th of april 2020, followed by another peak at 105 cases on 2nd of may 2020[8]. chain of impacts and remedies from a societal perspective the mco period has created a new normality for the entire nation at a liability yet to be fully apprehended. the new normality in malaysian context became apparent with the enforcement of mco when strictest restrictions were introduced during critical phase 2 and phase 3. the glaring impacts from economical and psychosocial fronts remain a huge concern given the prolonged period of mco with uncertainties regarding its further extension. a recent survey conducted by the department of statistics, malaysia, participated by 168,182 respondents aged 15 years and above, revealed that almost 50% of the selfemployed workers have lost their job while 94.8% reported reduced monthly income. it further revealed that more than 50% of the working community is not financially prepared for a total lockdown[25]. another group detrimentally affected by the restriction is the daily wage workers, more so those with families to support due to complete loss of income[26]. malaysians residing in the southern-most state of johor who travel across the causeway for work in singapore were also impacted by the mco issued by both the countries. some barely had enough savings to support their prolonged stay in singapore while some were asked to go on an unpaid leave[27]. the psychosocial effect resulting from the restrictions imposed via mco differ from one stratum of the community to another to some large extent. these differences are probably attributable to the differences in economic capacity of a stratum. for an instance, panic-buying, is probably an incongruent description of the economically disadvantaged stratum of the society for buying capacity becomes a disheartening matter of concern. people who have been charged for violation of mco include joggers and golfers at one end of the spectrum and fishermen who were in dire need to feed their families at another[26,28,29]. the markedly differing concerns and priorities divide the psychosocial effects within the different strata of community. paradoxically, the virus does not have the capacity to be selective of its sufferer, thus a social dilemma covid-19: are malaysians embracing... 3 johnson d et al. fund of myr 1600 and myr 800 respectively[37]. other funding aids, including deferment of loan payment up to six months and 15–50% discount on electricity bills do provide a temporary relief [38]. the temporary diversion due to mco may possibly mask the actual extent of economic burden imposed on both individuals and nation. nevertheless, it certainly signals a forewarning on a long term and lasting detrimental consequences considering a recent estimate of malaysian ringgit (myr) of 2.4 billion loss per day owing to economic shutdown during mco[39]. in the face of countless issues and concerns, the forthcoming of several non-governmental organizations (ngos) and individuals to reach out to the needy including the homeless and refugees is commendable. free food distribution, shelter to the homeless and fund-raising via social media are all some of the noteworthy deeds[40]. the nation’s frontline healthcare workers could not have become more visible for their unremitting labor in fighting covid-19. the public took it to social media to express their unwavering gratitude for all these selfless heroes while ceaselessly reminding one another to stay home and obey the mco rules[41]. enduring eternal claustrophobic hours in ppe suit, heavily drenched in sweat, heading home with exhaustion and fear of possibly carrying and passing on the infection to their loved ones only to call it a day could not be described as nothing but a precious call to perform their clinical duties in this most needed time of crisis. the fellow countrymen are probably more aware than ever the value of a timeless noble service like this and that it deserves far a greater recognition and salutation in days to come. the four phases of mco were probably the strictest and tightest lockdown period malaysians have had endured to date. following mco, cmco was introduced in phase 5 till 9th of june and subsequently recovery mco (rmco) up to 31st of august 2020[42]. restrictions were eased and economic sectors were gradually allowed to operate at staged phases. standard operating procedure (sop) was administered across all the operating business outlets. a mobile app known as ‘mysejahtera’ has been introduced by the ministry of health (moh) malaysia as a mean to monitor covid-19 outbreak. this app provides its user with an updated information on covid-19 status in malaysia and health screening facilities, individual risk status and qr code reader to enable individual check-in at each visit to various locations. this app channels the information to the moh, thus paves the way for effective countermeasures which marks the importance of digital health application in the midst of public health crisis[43]. as of 15th of august 2020, malaysia has recorded a total number of 9175 positive cases with 96.25% or 8831 cases of discharges, and 1.36% or 125 number of total deaths (figure 1). since late may, not long after cmco was introduced, the number of active cases based on daily record has remained within 2 digits and this marks the successful execution of covid-19 measures in malaysian setting[44]. heavy penalties will be imposed on individuals who disobey the set sops, including a fine not exceeding rm1000 or imprisonment up to six months [16]. although the battle to combat covid-19 is still ongoing, malaysians continue to hope for brighter days ahead while recognizing the applaudable measures taken by the respective authorities. arises when it comes to the execution of the general order like mco in a developing setting. befrienders, a non-profit organization which provides a 24/7 helpline for emotional support, reported an increase of 13% of calls during the initial phase of mco with 9% expressing concerns and anxiety due to covid-19 and mco[28]. ‘talian kasih’, a 24/7 helpline by government body dedicated to providing support related to welfare and community, reported a 57% of rise in number of calls during mco. concerns were expressed regarding an increase in domestic violence cases and further predicts a possible rise in child abuse and incest during mco[30]. loss of job and income without a financial backup while being confined in a space with families to support would impact the psychological well-being of any affected individual even for a short period. on the other hand, those who have the privilege of continuing work from home requires a minimum of well-equipped networking facilities and an ideal environment free from distractions. this becomes a challenge in a rural setting, particularly in some parts of east malaysia, where there is no internet viability or basic amenities to support work-from-home alternative which depends on internet[31]. individuals who are committed to families, especially with younger children or family members who need extra care and attention, equally have to juggle between work and family, especially when interrupted. the closing of academic institutions and postponement of major public exams become another form of distress to parents, educators and students themselves. online and home-based learning has been highly encouraged and advocated, however, students from rural background with no access to internet and proper coaching would undoubtedly jeopardize students’ academic performance in a long run[32–34]. it would be an unrealistic expectation to have all the school children remain motivated to study throughout the mco period. boredom, agitation and stress could not only affect the children, but also the parents or caregivers who equally have the responsibility to ensure an adequate and healthy home environment for their children. from another perspective, in a common ground of psychosocial effects, the most difficult situation comes when one has to grapple with the loss of their loved ones, be it due to covid-19 or other causes. it becomes a painful bid-adieu to these souls for the sorrowful memory it creates[35]. there were people arrested and fined for attending funeral of loved ones during mco which makes the grieving even more painful[36]. perhaps no remedy is capable of recovering one from the loss of loved ones in such circumstances. as remedial measures tackling financial aspect, malaysians were given financial aid through the ‘prihatin economic stimulus package’ where a one-off amount was credited into accounts of eligible applicants based on net monthly household income of no more than myr 8000. the ‘b40 group’, with net monthly household income of less than myr 4000 for married individuals and less than myr 2000 for singles, was eligible for the highest aid 4 conclusion the gigantic wave of covid-19 has awakened the entire nation and calls for a bigger reformation in days to come at both individual and national fronts. economic downturn becomes a huge concern especially in developing countries like malaysia. psychosocial effect is influenced by economic capacity of an individual to a great extent; however, it becomes a borderless effect when emotional sufferings and trauma are considered. despite various limitations and concerns given the poorly anticipated circumstances, a responsible member of the nation should remain focused on working hand in hand to eradicate the egregious conquest of an invisible creature. the rapid spread of covid-19 leaves no space for any excuse to violate the governmental mandates. the miserable and dampening economical and psychosocial repercussions of covid-19 could only be revived by a robust and healthy citizen who survive this phase with deepest sense of conscience and responsibility. every individual would shoulder even a greater responsibility in curbing the pandemic as well as recovering from personal losses after cessation of mco. a continuous spread of awareness and reminders are necessary to ensure strict compliance to preventive measures. technology can never be more accommodating than now to keep the momentum going. all said, let this phase not be taken for granted, for a profound sense of humility has been awakened in the face a natural calamity. conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. author contributions the literature search and data collection were performed by dj. the manuscript was written by dj. technical supports and proofreading were contributed by sc-he, hdj and vl. vl set up the research project. funding this work was supported by seed funding from microbiome and bioresources research strength (mbrs), jeffrey cheah school of medicine and health sciences (jcsmhs) (vote number: mbrs/jcsmhs/01/2020). acknowledgement authors would like to acknowledge the support and guidance from professor shajahan yasin, head of school, jeffrey cheah school of medicine and health sciences (jcsmhs), monash university malaysia that enabled the success of this project. covid-19: are malaysians embracing... figure 1. the daily confirmed covid-19 cases reported in malaysia up to 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[cited 2020 august 16]. available: http://covid-19.moh.gov.my/ terkini. johnson d et al. progress in microbes and molecular biology review article 1 dietary glycaemic index and type 2 diabetes mellitus: potential modulation of gut microbiota hanusha durganaudu, thubasni kunasegaran, amutha ramadas* jeffrey cheah school of medicine & health sciences, monash university malaysia, jalan lagoon selatan, 47500 bandar sunway, selangor darul ehsan, malaysia abstract: diet therapy is often the first-line approach in prevention and management of type 2 diabetes mellitus (t2dm). adoption of low glycaemic index (gi) diet one of the recent dietary strategies to modulate glycaemic response in individuals with t2dm. generally, diet has strong influence on the gut microbiota, which recently have been found to be associated with insulin resistance and the inflammatory response in diabetes. the possible modulation of the gut microbiota with dietary intervention is a topic of emerging interest, with limited evidence among t2dm population. in this review, we have narrated the available evidence and discussed the current knowledge about diet manipulation associated with dietary gi in order to shape the gut microbiota. as a conclusion, we have pointed out several key research directions that may have helpful impact on diet interventions with modulation of gut microbiota on the pathogenesis and therapeutic implications in t2dm. keywords: microbiome; type 2 diabetes mellitus; carbohydrate-glycaemic index; nutrition received: 10th april 2020 accepted: 12th may 2020 published online: 19th may 2020 citation: durganaudu h, kunasegaran t and ramadas a. dietary glycaemic index and type 2 diabetes mellitus: potential modulation of gut microbiota. prog microbes mol biol 2020; 3(1): a0000082. https://doi.org/10.3687/pmmb.a0000082 introduction diabetes mellitus is defined as a “metabolic disorder of multiple aetiology characterised by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both”[1]. type 2 diabetes mellitus (t2dm) is the most common form of diabetes, accounting for about 80% of all diabetes cases mostly after the age of 30[2]. t2dm is characterised by three basic abnormalities — insulin resistance, impaired insulin secretion and increased hepatic glucose production, either of which may be the predominant feature[3,4]. this results in a disorder of carbohydrate metabolism, and fat, protein and mineral metabolism also can be affected. the world health organization has defined the diagnostic criteria for diabetes through a single raised glucose reading with symptoms, otherwise raised values on two occasions, of either fasting plasma glucose ≥ 7.0 mmol/l (126 mg/dl) or with a glucose tolerance test, two hours after the oral dose a plasma glucose ≥ 11.1 mmol/l (200 mg/dl)[1]. in diabetes management, nearly all t2dm related research outcomes are looking at reduction in glycosylated haemoglobin (hba1c) and report from the uk prospective diabetes study (ukpds) has recommended hba1c to be maintained below 7.1% to minimise t2dm-related complications[5]. it has been shown that individuals from primitive societies, where the incidence of diabetes is low who then move to societies where food is too readily available, often progress to develop diabetes[6]. although many people with t2dm could be managed by dietary modification alone, eventually they may require insulin therapy due to its progressive nature. although t2dm could be inherited, modifiable factors such as body composition and nutrition play important roles in the etiology of t2dm[7]. the goals of medical nutrition therapy (mnt) in people with diabetes are to achieve and maintain normal blood glucose, lipid and lipoprotein and blood pressure levels by addressing the nutritional needs and maintaining the pleasure of eating which eventually, prevent or slow the development of complication[8,9]. one of such dietary modifications that many mnts recommend will be adoption of low glycaemic index (gi) diet. gi ranks carbohydrate-containing foods according to their effect on postprandial glycaemia[10,11]. gi is a measure of the increase in blood glucose two hours after consumption of the food of interest, with reference to glucose or white bread. glycaemic load (gl), a product of gi and amount of carbohydrate in the food, is another measure of glycaemic response to carbohydrate-containing foods and could improve the copyright @ 2020 by durganaudu h and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: amutha ramadas, jeffrey cheah school of medicine & health sciences, monash university malaysia 47500, bandar sunway, selangor darul ehsan, malaysia; amutha.ramadas@monash.edu 2 evaluation of glycaemic response to a diet[12]. foods with low gi such as parboiled rice, barley, oats and legumes lower the postprandial hyperglycaemia, while high gi foods such as white bread, potatoes, white rice and commercial breakfast cereals will show the opposite effect[8,13]. additionally, increasing evidence has highlighted that the role of gut microbiota in a multitude of human illnesses, both inside and outside of gut, including irritable bowel syndrome[14], colorectal cancer[15,16], skin diseases[17,18], neurological-related diseases[19,20]. similarly, there are also emerging findings on the strong association between development of diabetes and gut microbiota[21,22]. gut microbiota is a dynamic identity that can result in various pathophysiological changes in human’s internal environment, though the composition changes gradually over the lifetime[23,24]. gut microbiota plays a critical role in several metabolic functions namely amino-acid synthesis, absorption of dietary fats and fatsoluble vitamins, production of short-chain fatty acids (scfas), activation of glucose homeostasis, lipid energy metabolism, calorie removal and regulating bile acid transformation among others[25]. this may predispose or protect an individual against diabetes. as gut microbiota can be easily influenced by environmental factors especially one’s dietary intake, there is a potential for gut microbiota to act as a modulator of dietary gi and t2dm relationship. in this review we will be summarizing the evidence associating dietary gi and t2dm, and subsequently discuss the potential role of gut microbiota in modulating this relationship. dietary glycaemic index and diabetes a cross-sectional study in south italy found a dosedependent relationship between dietary gi and gl with hba1c, where patients with diabetes with highest gi and gl had the highest hba1c levels[26]. gi-based dietary education has been suggested to be a useful tool in diabetes management[27,28]. miller and gutschall[29] reported a nine-week nutrition education regarding gi and gl which improved dietary intake, knowledge, outcome and efficacy expectations and empowerment for diabetes management. although gi could lead to a better dietary intake in people with diabetes, only few organisations recommended the use of low gi diets[30–32]. retrospective study by burani et al.[26] found a reduction in hba1c of 19% and body mass index (bmi) of 8% following inclusion of low gi diet in lifestyle intervention, which was well accepted by the participants. several trials have supported the role of gi in glycaemic control in patients with t2dm[34]. significant improvements have been seen in hba1c and/or fpg[34–37], insulin sensitivity[34] and serum fructosamine[36] with low gi diets. the role of gi diets in glycaemic control has been confirmed by several meta-analyses and reviews[13,38,39]. however, there is a lack of studies on the technology assisted low gi interventions, and only one feasibility nutritionist-delivered, pda-assisted low-gi dietary intervention by ma et al.[40] has been discovered. low gi diets also have been shown to have favourable impact on lipid profile and reduction in cardiovascular risk of patients with t2dm[35,41–44]. studies have reported significant decrease in total cholesterol and ldl-c with prescribed low gi diets[34]. longer educational programmes to improve diet quality by emphasizing low gi diets also have shown to lower the ldl-c[45,46] and increase the hdl-c[37]. besides the cholesterol levels, a low gi or gl diet may be preferred for the dietary management of t2dm because of sustained reductions in postprandial glucose and c-reactive protein[47] and the increase in the plasma adiponection concentrations[48]. there is also evidence showing the positive impact of low gi diet on other health outcomes in people with diabetes. low gi diet accompanied with exercise programme was found to improve cardiovascular health[49] and protect against-induced hypoglycaemia in t2dm patients[50]. low gi diet could assist with the weight management programme in patients with diabetes[43,46]. yusoff and colleagues[36] reported a significant reduction in waist circumference in asian patients after 4 months of following a low gi diet. low gi diet has also generally results in better cognitive performance in the postprandial period in adults with t2dm and reduce their dependency on diabetes medication[45,51]. interestingly, there were studies which did not support the role of gi in diabetes management[52]. in one of such studies, low gi diet with calorie restriction in overweight patients with t2dm did not find any significant reduction in hba1c[46]. data derived from the atherosclerosis risk in communities study suggest high gl intake to be a chd risk factor only among whites without diabetes and not in individuals with diabetes[53]. cheong et al.[54] concluded addition of a low-gi component to a walking did not improve anthropometric or metabolic outcomes in diabetic patients. a review by barojek and morello[55] has identified short coming in terms of power of the study and confounders, which could have affected otherwise positive findings in these studies. as low gi foods are generally rich in fiber and other nutrients, the consumption of this diet has been shown beneficial to diabetic patients[56]. incorporation of such foods in every day diet may be an effective approach for weight management, glycaemic control and favourable lipid profile. however, the concept of gi should not be used in isolation, but to be used as an adjunct treatment to existing lifestyle management of t2dm in fine-tuning the glycaemic control[45,57]. dietary glycaemic index and gut microbiota exploration of a potential association between dietary gi and changes in the proportion of certain gut microbiota is relatively a newer research area, with limited evidence among t2dm population. an experimental study among individuals at risk of metabolic syndrome showed largest increase in the bifidobacterium spp. (an established gut health biomarker) level in high carbohydrate/high glycaemic index (hc/ hgi) group compared to control group[58]. this change was dietary glycaemic index... 3 durganaudu h et al. barley-based bread in regulating gut microbiota. role of gut microbiota in type 2 diabetes several studies have investigated and showed some forms of association between gut microbiota and development of t2dm. the role of gut microbiota may be evaluated in several aspects. insulin resistance in t2dm patients may have resulted from increased production of hepatic triglyceride facilitated by firmicutes and bacteroidetes, as they enhance the monosaccharide uptake from the host gut[65]. aside from the impact on carbohydrate metabolism, high ratios of firmicutes to bacteroidetes is also found to alter the production of scfas with an increase in acetate production and decrease in butyrate production[66]. the increased levels of acetate in the blood is found to result in insulin resistance and heightened production of ghrelin (an-appetite stimulating hormone) in the stomach, as illustrated by a recent study among individuals with metabolic syndrome[67]. on the other hand, decrease in butyrate levels also encourage insulin resistance through promotion of low-level inflammation[68]. aside from the diversity of gut microbiota, another aspect that could be looked into is its role in facilitating immune response which lead towards development of t2dm. abundance of prevotella bacterial species was found particularly in obese t2dm individuals, and known to increase the levels of pro-inflammatory cytokines, besides encouraging low-grade inflammation and insulin resistance[69]. verrucomicrobia, on the other hand, which are known to contribute towards the maintenance of antiinflammatory state of gut and improve insulin sensitivity were found less in t2dm individuals in a study conducted in pakistan[70]. the same study also found an increase in the levels of gram-negative bacteria such as dialister and allisonella, which may have contributed to the rise in levels of lipopolysaccharide (lps), which eventually binds with cd14 and mediates inflammatory response. another class of bacteria which is found to be abundant among people with diabetes is fusobacteria, which plays critical role in inflammatory responses, mounting adhesiveness to host epithelial cells and energy generation[71,72]. furthermore, the link between gut microbiota and t2dm can also be explored by looking into their association with bile acids. it is well-known that one of the important symbiotic roles played by gut microbiota is in terms of bile acid transformation, as their composition has been found to affect the concentration and composition of circulating bile acids[73]. this in turn affects the individual’s risk in developing obesity and related disorders. most of the bile acid biotransformation occurs in the large intestine which is extremely rich in microbiota, through a complex process[74]. this is one process in which the role of gut bacteria can be directly observed, as it is catalysed by bile salt hydrolase (bsh), an enzyme which is present in several gut bacteria such as clostridium, bacteroides, lactobacillus, bifidobacterium and enterococcus[75]. bile acids in the blood circulation play a role in homeostasis of carbohydrates and lipids, further reinforcing their importance as regulatory molecules in that aspect[73]. bile acids also pose some direct associated with reduced fasting glucose, fasted insulin and cholesterol levels compared to baseline. furthermore, hc/hgi group was also associated with increased bacteroides numbers as well as reduction in body weight, bmi and waist circumference. both bacteroides and bifidobacterium spp. have been independently associated with reduction in risk factors for metabolic syndrome and improved body energy regulation in the past[59]. interestingly, the study found an increased abundance of faecalibacterium prausnitzii with both high saturated fat (hs) diet and high carbohydrate/low glycaemic index (hc/lgi) diets. hs group also experienced an increase in faecal scfa concentration. however, faecal acetate percentage may be inversely correlated with absorbed acetate percentage (after rectal infusion), as demonstrated by vogt and wolever[60]. in that case, higher scfa noted in the hs group may be due to decreased absorption, instead of higher colonic fermentation. a study exploring specific foods with lower gi responses in vitro, found minimally processed wholegrain cereals such as wholegrain oats and granola resulted in significant growth in the friendly bacteria namely bifidobacterium genus and lactobacillus-enterococcus group[61]. wholegrains with minimal processing also resulted in increase in atopobium cluster and bacteroidesprevotella group after 10 hours. increase in clostridium histolyticum after instant porridge fermentation (highly processed wholegrains) is also noted to be significantly higher compared to the decrease observed in minimally processed wholegrains. another low gi grains that were investigated in the past were barley grains. an in vivo study found barley intake lead to increased abundance of prevotella and lactobacillus, as well as candidatus homeothermaceae in obese and lean mice[62]. barley intake was also linked with lower levels of plasma insulin and resistin, a cysteine-rich peptide secreted by adipocytes, immune cells, and epithelial cells which are found in higher levels in metabolic syndrome cases[63]. another study utilised models such as static in vitro digestion and dynamic gastric model to simulate the normal digestion process in investigating relationship between different types of barley grains and microbiota[64]. during early stage of digestion in the study, it noted that the digesta of wild-type barley hordeum vulgare cv golden promise (hv) and amylose-only (ao) breads portrayed higher abundancy of firmicutes, whereas wheat and wild barley hordeum vulgare subsp. spontaneum (hs) grain bread digesta contained lower levels of it. hs grain bread digesta demonstrated increased abundance of actinobacteria (30-fold higher than control) to the detriment of bacteroidetes and firmicutes. however, samples representing later stages of digestion behaved differently as bacteroidetes and actinobacteria were found to be abundant in all digesta. specifically, increased actinobacteria was noted in ao fermentation (compared to early digestion stage samples) while bacteroidetes was more abundant in fermentation of hs grain digesta. aside from that, proteobacteria levels in wheat bread digesta fluctuated from early digestion to late digestion stage. therefore, these results portray the potential of low gi 4 antimicrobial action which subsequently impacts the survival and colonisation of certain gut microbiota[76]. as an individual’s food consumptions affect both gut microbiota and development of diabetes, further exploration can be made to investigate the changes in gut microbiota according to the type of prescribed dietary pattern, and its impact on diabetes development and/or severity. conclusion the search for the optimal nutritional strategy in t2dm patients remains an unresolved issue. it is important that the potential benefits of suitable gi diet and the t2dm patient’s microbiota, which in turn will impact on the progression of disease complication, are taken into consideration. in this review, we have provided evidence that different dietary patterns have different impacts on gut microbial composition. these findings suggest that the gut microbiota contribute to the pathophysiological regulation of glucose tolerance, insulin secretion and in inflammation. hence, future studies should define the features of the gut microbiome in diet consumption that contribute to the t2dm in defined populations. furthermore, with the emerging advances in technology, the relationship between important biomarkers to changes in gut microbiota and microbiota metabolites modulated by recommended diets are worth investigating in future studies to aid prevent or treat diabetes-related disorders in a strategic manner. in addition, it is also important to focus on bile stress and its effects on the gut microbiota in future direction in finding therapeutic strategies to reduce the aggregate metabolic burden in human populations. some bacteria can use bile as their host to regulate virulence determinants and produce secondary bile acids that regulate the normal homeostasis of tissues in human body which leads to unwanted complications. thus, further studies should investigate how bacteria sense bile and regulate it response it induces and reveal the effect of different bile acid profiles in host tissues by virulence factor production. lastly, approaches to modulate the microbiome-bile acid formation through diet may likely reduce the risk and/or treat metabolic diseases and this need further investigation. author contributions the review and manuscript writing were 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and the gut microbiome. curr opin gastroenterol 2014; 30(3): 332–338. 75. begley m, gahan cgm, hill c. the interaction between bacteria and bile. fems microbiol rev 2005; 29(4): 625–651. 76. dawson pa and karpen sj. intestinal transport and metabolism of bile acids. j lipid res 2015; 56(6): 1085–1099. progress in microbes and molecular biology original research article 1 landscape of hoxa genes methylation in colorectal cancer muhiddin ishak1, rashidah baharudin1, loh teng-hern tan2, learn-han lee2*, nurul-syakima ab mutalib1* 1ukm medical molecular biology institute (umbi), universiti kebangsaan malaysia, jalan yaacob latif, 56000 cheras, kuala lumpur, malaysia 2novel bacteria and drug discovery research group (ndbb), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia abstract: colorectal cancer (crc) is among the most common cancers worldwide and the second leading cause of cancerrelated death in malaysia. the hoxa gene cluster is a family of homeobox a genes encoding transcriptional regulators that play vital roles in cancer susceptibility and progression. dysregulated hoxa expression influences various aspects of carcinogenesis processes. therefore, this study aims to elucidate the methylation landscape of hoxa genes in crc. twelve pairs of crc — adjacent normal tissues were subjected to infinium dna methyepic array. differentially methylated regions were identified using the champ bioconductor and methylation levels of hoxa genes were manually curated. we identified 100 significantly differentially methylated probes annotated to hoxa genes. hoxa3 has the highest number of differentially methylated probes (n=27), followed by hoxa2 (n=20) and hoxa4 (n=14). the majority (43%) of the probes were located at the transcription start site (tss) 200, which is one of the gene promoters. in respect to cpg islands (cgi), the probes were equally located in the island and shore regions (47% each) while a minor percentage was in the shelf (6%). our work gave a comprehensive assessment of the dna methylation pattern of hoxa genes and provide the first evidence of hoxa2, hoxa3 and hoxa4 differential methylation in malaysian crc. the new knowledge from this study can be utilized to further increase our understanding of crc methylomics, particularly on the homeobox a genes. the prognostic and diagnostic roles of the differentially methylated hoxa genes warrant future investigations. keywords: homeobox a genes; colorectal cancer; dna methylation; hoxa2; hoxa-as3 received: 10th april 2020 accepted: 12th may 2020 published online: 18th may 2020 citation: ishak m, baharudin r, tan lt-h et al. landscape of hoxa genes methylation in colorectal cancer. prog microbes mol biol 2020; 3(1): a0000085. https://doi.org/10.3687/pmmb.a0000085 introduction cancer is a continuous global burden and colorectal cancer (crc) placed as the fourth most frequently diagnosed cancer worldwide[1] and second in malaysia[2,3]. cancer occurs through the accumulation of multiple genetics and epigenetics changes[4]. somatic mutation in apc, braf, kras, pik3ca and tp53[5–8] are identified in crc at varying frequencies and are perceived as the drivers of crc formation. although there are many efforts on investigating the molecular alterations involved in crc pathogenesis, the existing knowledge remains inadequate for an early diagnosis and prognosis assessment. therefore, further understanding of epigenetic components involved in crc carcinogenesis is highly sought after and will unravel new genes which can be utilized as the diagnostic, prognostic and predictive biomarkers to prevent crc-related mortality. epigenetics mechanism can be generally categorized into histone modification and dna methylation[9], with the latter being the most widely investigated. the clinical application of dna methylation markers to determine at-risk patient populations, improve diagnostic criteria, and provide prognostic factors to guide treatment decisions are becoming increasingly relevant. this copyright @ 2020 by ishak m and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: nurul-syakima ab mutalib, ukm medical molecular biology institute (umbi), universiti kebangsaan malaysia, jalan yaacob latif, 56000 cheras, kuala lumpur, malaysia; syakima@ppukm.ukm.edu.my. learn-han lee, novel bacteria and drug discovery research group (ndbb), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; lee.learn. han@monash.edu. 2 is because dna methylation can be reversed, thereby providing alternative treatment options for patients with methylation phenotype. clinically, dna methylation has also been demonstrated to have significant utility owing to its stability and relative ease of testing[9]. for instance, sept9 gene is the first us food and drug administration (fda)–approved diagnostic assay for crc screening (https://www.epiprocolon.com). however, the clinical utility of the methylated sept9 assay is still limited owing to patients’ heterogeneity factor which includes various demographic characteristics and pathological features[10]. the search for a good diagnostic, prognostic and predictive dna methylation markers in crc is an active area of research. homeobox genes play a role as the master regulators of morphogenesis and are aberrantly expressed in cancer[11]. these genes possess a highly conserved dna sequence that code for the homeodomain proteins, which act as transcription factors that bind specifically to the dna motifs and regulate the genes involved in cellular processes including adhesion, proliferation and differentiation[11]. over 200 homeobox genes have been discovered in the human genome and are divided into four hox gene clusters namely hoxa, hoxb, hoxc, and hoxd, positioned at the chromosome 7p15, 17q21.2, 12q13, and 2q31 loci, respectively. of these, there are 11 hoxa genes[12] (table 1). table 1. the list of 11 hoxa genes under hoxl subclass homeoboxes [11]. hgnc id (gene) approved symbol approved name hgnc:5099 hoxa1 homeobox a1 hgnc:5103 hoxa2 homeobox a2 hgnc:5104 hoxa3 homeobox a3 hgnc:5105 hoxa4 homeobox a4 hgnc:5106 hoxa5 homeobox a5 hgnc:5107 hoxa6 homeobox a6 hgnc:5108 hoxa7 homeobox a7 hgnc:5109 hoxa9 homeobox a9 hgnc:5100 hoxa10 homeobox a10 hgnc:5101 hoxa11 homeobox a11 hgnc:5102 hoxa13 homeobox a13 dysregulated homeobox gene expression is a frequent in cancer and one of the mechanisms causing such dysregulation is dna methylation. the hoxa cluster is often methylated in the non–small cell lung cancer (nsclc)[13,14], while hoxa5 and hoxa11 promoter methylation diminishes their tumour-suppressive function through transcriptional silencing[15,16]. hoxa11 is hypermethylated in gastric cancer tissues and is significantly associated with tnm iii and iv patients[17]. hypomethylation hoxa10 and hoxa11 able to discriminate ovarian cancer tissue from the normal tissue[18]. hoxa9 was reported to be hypermethylated in half of the ovarian cancer patients and is significantly associated with endometrioid histological subtype[19]. in bladder cancer, hoxa9 promoter methylation has been linked with cisplatin chemotherapy-resistant and metastatic bladder cancer, and reversing the dna methylation using decitabine sensitized the cancer cells to cisplatin[20]. yet, little is known about hoxa methylome in crc. only recently, li and colleagues reported the dna methylation status of three hoxa genes, which are hoxa2, hoxa5 and hoxa6 in crc using targeted bisulfite sequencing assay[21]. nevertheless, a comprehensive, unbiased methylome profile of hoxa genes has not been described. therefore, this study aims to investigate the methylation landscape of hoxa genes in crc context. material and methods clinical specimens the archived 12 pairs of tumour-adjacent normal fresh frozen colon tissues (n=24) from crc patients were retrieved from the umbi-hctm biobank. the tissues were collected according to the procedures approved by ukm research ethics committee. as a quality control procedure, all tissues were cryosectioned, followed by haematoxylin and eosin staining for the pathologist to determine the percentage of tumour cells and normal cells contents. only tumour samples with ≥ 80% cancerous cells and normal adjacent colon tissues with ≤ 20% necrosis were selected for dna extraction using allprep dna/ rna/mirna universal kit (qiagen, usa) according to the manufacturer’s instructions. then, the integrity of dna was assessed using agarose gel electrophoresis while the quantity and purity were evaluated using nanodrop 2000c spectrometer (thermo fisher scientific, usa). bisulfite conversion and methylation microarray five hundred nanogram (500 ng) of dna was subjected to bisulfite conversion to change all unmethylated cytosine to uracil using the ez dna methylation — gold kit (zymo research, usa) according to the manufacturer’s protocol. the effectiveness of bisulfite conversion was determined using universal methylated dna standard & control primers (zymo research, usa) according to the manufacturer’s protocol. the infinium dna methylationepic assay, covering 850,000 cpg dinucleotides spread over the whole genome, was performed according to the manufacturer’s specifications (illumina, inc.). methylation microarray data analysis the raw idat files obtained from methylation microarray were subsequently analyzed using genomestudio v1.9.0 and champ bioconductor packages[22]. filters were applied to all datasets where cpg sites with detection p-values ≥ 0.01 in one or more samples were omitted from further analysis. to reduce the technical biases intrinsic to the probe design, the raw intensities were swannormalized prior to statistical analysis[23]. β-values were then extracted and subjected to further statistical analysis. expression of hoxa genes from the cancer genome atlas (tcga) study the mrna expression data of selected hoxa genes in crc were retrieved using web-based firebrowse gene expression viewer tool (https://gdac.broadinstitute.org/) landscape of hoxa... 3 ishak m et al. from broad institute. this tool provides access to results of various omics analyses involving more than 14,000 cancer cases, from 38 types of cancers based on tcga data version 2016_01_28. statistical analysis a t statistic from the limma bioconductor package was used to determine the differentially methylated cpg sites[24,25]. the cpg sites were further filtered at an adjusted p-value < 0.05 to identify significant differentially methylated hoxa genes. to substantiate the specificity and accuracy of the differentially methylated probes, the discriminative performance of the probes was evaluated by receiver operating characteristic (roc) curves, and the area under the roc curve (auc), specificity, and sensitivity at the optimal cut-offs were determined using graphpad prism v8 (graphpad software, inc., usa). results locations of differentially methylated loci in hoxa genes we analysed the differential methylation status of 12 crc tissue samples with the 12 adjacent cancer-free colonic tissue samples and only differentially methylated regions with adjusted p-value < 0.05 were reported. from the list of differentially methylated probes, we further filtered for hoxa genes. here, we found that there are 100 crcassociated differentially methylated probes in 11 hoxa genes, noncoding hoxa-as3 and hoxa10-hoxa9 readthrough. hoxa3 has the highest number of differentially methylated probes (n=27), followed by hoxa2 (n=20) and hoxa4 (n=14) (figure 1a). the majority (43%) of the probes were located at the transcription start site (tss) 200 (figure 1b), which is one of the gene promoters. in respect to cpg islands (cgi), the probes were equally located in the island region and shore regions (47% each) while a minor percentage was in the shelf (6%) (figure 1c). figure 1. differentially methylated hoxa genes in crc. (a) the number of differentially methylated probes in each hoxa genes. (b) distribution of methylated loci in hoxa genes with respect to features. (c) distribution of methylated loci in hoxa genes with respect to cgi. the genomic and gene-related regions of the significant differentially methylated hoxa genes were distributed differently. generally, 53 probes (in seven genes) were hypermethylated compared to 47 loci (eight genes) that were hypomethylated. the largest portion of hypomethylated sites (55.3%) were in the shore and subsequently decreased in other categories (island 42.6% and shelf 2.1%). in contrast, more than half (50.9%) of the significantly hypermethylated loci of hoxa genes were on the island, followed by the shore (39.6%), and shelf (9.4%). none of the loci was identified in opensea region. meanwhile, most of the significantly hypomethylated loci were in the 5’utr (46.8%), followed by tss200 (14.9%), gene body (12.8%), and 8.5% in each 1st exon, 3’utr and tss1500. meanwhile, around a quarter (26.4%) of the significant hypermethylated loci were located in tss200 and tss1500, while the rest were mainly found in the gene body (20.8%), 1st exon (15.1%), and, to a lesser extent, in the 5’ and 3’ utr (7.5% and 3.8%, respectively). differentially methylated hoxa genes all of the 11 hoxa genes are significantly differentially methylated. due to the power of comprehensive contents in the microarray platform, we also identified significant hypomethylation of the noncoding hoxa-as3 and hoxa10hoxa9 readthrough. high resolution, probe-level analyses revealed hypomethylation of 20 loci in hoxa3, with the remaining seven loci were hypermethylated. in hoxa2 and hoxa9, all the probes were hypermethylated, while probes in hoxa4 were hypomethylated. hoxa6 exhibited eight hypermethylated loci and only one was hypomethylated. the summary of hypoand hypermethylated probes in each gene were summarized in table 2. 4 table 2. the number of hypermethylated and hypomethylated probes in the hoxa genes. genes number of hypermethylated probes genes number of hypomethylated probes hoxa1 2 hoxa-as3 4 hoxa2 20 hoxa10 4 hoxa3 7 hoxa10-hoxa9 1 hoxa5 5 hoxa11 1 hoxa6 7 hoxa13 2 hoxa7 2 hoxa3 20 hoxa9 10 hoxa4 14 hoxa6 1 the 100 significant probes with the methylation changes (δβ) are illustrated in table 3. it is worth mentioning that the loci in hoxa2 were the locations with the highest methylation changes; these probes were hypermethylated in crc as compared to the normal colon. on the other hand, the loci in hoxa3 were the most hypomethylated probes in crc as compared to the normal colon. table 3. the 100 significant differentially methylated probes in hoxa genes. genes probes adjusted p-value δβ feature cgi hoxa2 cg06055873 2.66e-05 0.368 1st exon shore hoxa2 cg24058604 1.35e-04 0.362 tss200 shore hoxa2 cg05921905 2.49e-04 0.362 tss200 shore hoxa2 cg04737131 3.75e-04 0.357 tss1500 shore hoxa2 cg17353412 6.30e-06 0.357 1st exon shore hoxa2 cg20747380 2.23e-05 0.356 1st exon shore hoxa2 cg02979457 3.64e-04 0.326 tss200 shore hoxa2 cg22943986 1.19e-03 0.322 tss1500 shore hoxa2 cg06786372 5.16e-03 0.319 body shore hoxa2 cg26069745 1.55e-04 0.317 1st exon shore hoxa2 cg06769202 8.92e-04 0.312 tss200 shore hoxa2 cg09871315 1.95e-03 0.293 tss1500 shore hoxa2 cg23979631 2.88e-04 0.291 tss200 shore hoxa5 cg03744763 7.31e-03 0.286 tss1500 island hoxa1 cg07450037 1.02e-03 0.282 body shore hoxa2 cg20087093 2.13e-03 0.276 tss1500 shore hoxa2 cg02803819 7.12e-03 0.258 body shelf hoxa9 cg12600174 1.19e-02 0.249 tss200 island hoxa3 cg27539480 4.52e-03 0.244 3’utr shore hoxa2 cg23206851 6.94e-03 0.243 tss1500 shore hoxa3 cg02627455 5.21e-03 0.240 5’utr shelf hoxa2 cg13661519 4.09e-03 0.240 body shelf hoxa3 cg07153966 2.74e-02 0.234 body island hoxa9 cg21001184 1.42e-02 0.228 tss200 island hoxa9 cg03217995 3.19e-02 0.226 body shore hoxa2 cg00188704 3.46e-02 0.222 body shelf hoxa1 cg03116258 4.38e-04 0.220 1st exon shore hoxa5 cg14882265 4.94e-02 0.211 tss1500 island hoxa2 cg02225599 1.51e-02 0.204 tss1500 island hoxa3 cg14216068 9.16e-03 0.194 3’utr island hoxa3 cg09591524 3.06e-02 0.186 5’utr island hoxa6 cg14044640 3.34e-02 0.184 tss200 island hoxa5 cg03368099 2.16e-02 0.183 tss1500 island hoxa7 cg20725013 2.91e-02 0.182 body shore hoxa3 cg02439266 1.46e-02 0.182 5’utr island hoxa5 cg01748892 3.58e-03 0.180 tss1500 island landscape of hoxa... 5 hoxa3 cg12305431 1.79e-02 0.175 5’utr shelf hoxa5 cg13694927 1.20e-02 0.168 tss1500 island hoxa9 cg26476852 4.68e-02 0.167 1st exon island hoxa9 cg20399871 2.10e-02 0.164 1st exon island hoxa6 cg03529432 4.98e-02 0.161 tss200 island hoxa9 cg16104915 1.81e-02 0.154 tss200 island hoxa6 cg22469274 4.68e-02 0.154 tss200 island hoxa6 cg09936824 3.98e-02 0.153 tss1500 island hoxa6 cg19183743 4.34e-02 0.150 tss1500 shore hoxa7 cg21778348 3.36e-02 0.149 body island hoxa9 cg16913789 4.62e-02 0.146 body island hoxa2 cg01217984 1.10e-02 0.134 tss1500 island hoxa9 cg05065989 1.07e-02 0.129 tss200 island hoxa9 cg07778029 3.18e-02 0.125 1st exon island hoxa6 cg04265576 2.56e-02 0.123 tss200 island hoxa9 cg03698009 4.16e-02 0.108 body island hoxa6 cg12810523 4.84e-02 0.103 tss200 island hoxa10 cg08938793 4.30e-02 -0.041 3’utr shore hoxa6 cg23590202 8.97e-04 -0.052 tss1500 shore hoxa13 cg01363170 4.33e-02 -0.056 3’utr shelf hoxa-as3 cg10374314 4.89e-02 -0.070 body shore hoxa10 cg05092861 5.66e-03 -0.078 tss200 shore hoxa4 cg23884241 2.58e-02 -0.078 1st exon island hoxa4 cg04317399 1.43e-02 -0.084 1st exon island hoxa13 cg02366798 4.96e-02 -0.089 3’utr shore hoxa4 cg03724423 2.08e-02 -0.096 tss1500 shore hoxa4 cg11410718 1.61e-02 -0.108 tss200 island hoxa4 cg07317062 2.15e-02 -0.114 5’utr island hoxa10 cg01078824 2.94e-02 -0.118 tss200 shore hoxa4 cg19142026 1.84e-02 -0.137 5’utr island hoxa4 cg17591595 4.32e-02 -0.151 tss1500 shore hoxa4 cg22997113 1.80e-02 -0.170 1st exon island hoxa3 cg16406967 1.99e-02 -0.196 5’utr island hoxa3 cg22798849 3.57e-02 -0.198 5’utr island hoxa3 cg18680977 1.76e-02 -0.209 5’utr island hoxa3 cg16748008 4.02e-02 -0.220 5’utr island hoxa4 cg11532431 2.10e-02 -0.231 body island hoxa11 cg05516617 8.55e-03 -0.231 3’utr shore hoxa3 cg23403004 2.42e-04 -0.244 5’utr shore hoxa3 cg04778178 2.48e-04 -0.254 5’utr island hoxa3 cg16644023 4.88e-02 -0.255 5’utr shore hoxa3 cg15982700 4.41e-02 -0.259 5’utr shore hoxa3 cg23806243 2.21e-03 -0.260 5’utr shore hoxa3 cg00318947 1.81e-02 -0.270 5’utr shore hoxa4 cg00562553 1.01e-02 -0.272 1st exon island hoxa4 cg20171892 5.24e-04 -0.273 body island hoxa3 cg04351734 2.10e-02 -0.274 5’utr island hoxa10 cg05517976 1.19e-02 -0.275 tss200 shore hoxa4 cg11227540 1.46e-02 -0.279 body shore hoxa4 cg09799676 3.54e-03 -0.286 body island hoxa3 cg21556281 1.04e-02 -0.291 5’utr shore hoxa-as3 cg14429861 7.94e-03 -0.299 tss200 shore hoxa3 cg18430152 7.29e-04 -0.305 5’utr island ishak m et al. 6 hoxa10-hoxa9 cg22274074 1.72e-03 -0.307 tss1500 shore hoxa3 cg14072564 6.32e-04 -0.311 5’utr island hoxa4 cg17132446 4.36e-03 -0.323 body shore hoxa3 cg01820751 2.39e-03 -0.328 5’utr shore hoxa-as3 cg06188746 3.12e-03 -0.331 tss200 shore hoxa-as3 cg18091117 3.05e-03 -0.331 tss200 shore hoxa3 cg03483713 4.12e-04 -0.346 5’utr shore hoxa3 cg26297005 1.81e-03 -0.361 5’utr island hoxa3 cg15725372 6.95e-04 -0.367 5’utr island hoxa3 cg00431187 7.17e-04 -0.381 5’utr shore hoxa3 cg09798023 2.84e-04 -0.390 5’utr shore tss: transcription start site utr: untranslated regions expression of hoxa genes and their correlation with methylation level using firebrowse, gene expression of the hoxa genes were retrieved from coad[5] and coadread[26] studies (figure 2). the data were presented as log2 fold change. the expression of seven hoxa genes was downregulated (hoxa1, hoxa2, hoxa4, hoxa5, hoxa6, hoxa7, and hoxa13) while four of genes (hoxa3, hoxa9, hoxa10, hoxa11) were upregulated. the expression profiles of hoxa1, hoxa2, hoxa5, hoxa6, hoxa7, hoxa10, and hoxa11 are inversely related to the methylation level as predicted, but not the hoxa4, hoxa9 and hoxa13. figure 2. gene expression of hoxa genes from tcga coad and coaread studies. landscape of hoxa... 7 receiver operating characteristics (roc) curve analysis lastly, the sensitivity and specificity of the methylation levels were further assessed using receiver-operator curve (roc) analysis. the methylation levels of 10 topmost hypermethylated cpg sites significantly differentiated the crcs from the normal colonic tissues (p-value 0.0032 to 0.0002) (table 4). the highest discriminative accuracy was demonstrated by hoxa2 cg06055873 (auc = 0.9514, confident interval = 0.8547 to 1.000, p-value = 0.0002). other candidate probes also reached high diagnostic accuracy (table 4; figure 3). table 4. receiver operating characteristics (roc) curve analysis of the top 10 differentially methylated probes in hoxa2 gene. gene_probe area std. error 95% confidence interval p value hoxa2 cg06055873 0.9514 0.04935 0.8547 to 1.000 0.0002 hoxa2 cg24058604 0.9444 0.04489 0.8565 to 1.032 0.0002 hoxa2 cg05921905 0.875 0.08441 0.7096 to 1.04 0.0018 hoxa2 cg04737131 0.8958 0.07498 0.7489 to 1.043 0.001 hoxa2 cg17353412 0.9444 0.05546 0.8357 to 1.053 0.0002 hoxa2 cg20747380 0.9375 0.06155 0.8169 to 1.058 0.0003 hoxa2 cg02979457 0.8681 0.07918 0.7129 to 1.023 0.0022 hoxa2 cg22943986 0.8611 0.09216 0.6805 to 1.042 0.0027 hoxa2 cg06786372 0.8542 0.08306 0.6914 to 1.017 0.0032 hoxa2 cg26069745 0.9167 0.0691 0.7812 to 1.052 0.0005 figure 3. box plot illustrating the comparison of β values and roc curve-based evaluation of the diagnostic accuracy for the top 10 hypermethylated hoxa2 probes in cancerous and normal tissues. ishak m et al. 8 discussion in this study, we analyzed in greater detail the genomewide methylation patterns of hoxa genes from 12 crcs compared with their adjacent normal tissues. interestingly, due to the high throughput nature of microarray platform, our analysis revealed that all of the 11 hoxa genes are significantly differentially methylated in crcs. we are the first to report this new finding and shed new light on the possible role of these genes in colorectal carcinogenesis. epigenetic alteration of hoxa genes has been widely studied in many cancers, especially non-small cell lung cancer (nsclc)[27–29], yet, the investigation about this gene cluster in crc is lacking. to date, there is only a handful of study which investigates the methylome of hoxa genes[21,30–32]. hoxa2 is the most significantly hypermethylated hox gene in our study, and the methylated loci were mostly located in the promoter regions (tss200 and tss1500). hypermethylation of this gene in crc has also been recently reported in concordance with our finding[21]. in addition, the significant association between hoxa2 methylation with age, node status (n), stage, metastasis (m), lymphovascular invasion, perineural invasion, as well as the number of lymph node was also demonstrated[21]. another study has shown that hoxa2 is hypermethylated in the rectal cancer mucosa compared to the nonmalignant rectal mucosa[30]. dna methylation is known to be inversely correlated with mrna expression; however, the published data on hoxa2 expression is lacking and the aforementioned two studies did not investigate the gene expression levels. therefore we attempted to investigate the mrna expression using the tcga crc dataset and the finding is in agreement with our hypothesis. the expression of hoxa2 is indeed downregulated in crc compared to the normal tissues by 0.68 fold, and thus, could be explained by its hypermethylation status. nevertheless, hoxa2 promoter is also found to be hypermethylated in nasopharyngeal cancer, whereby it associates with low mrna expression in the biopsies and cell lines[33]. moreover, in a study involving 101 patients from stage i–iii nsclc, methylation of hoxa2 was proposed to have prognostic significance in squamous cell carcinoma (scc) subtypes patients[34]. due to the limited number of patients and the lack of clinical information in our study, the association with clinical features were not established and warrant further investigation. in addition, roc curves for hoxa2 gene show exceptional diagnostic ability in differentiating crc from the normal healthy tissue, especially in stage i patients with the auc = 0.9979 [21]. we also observed a similar trend, whereby the loci in hoxa2 are the most significantly hypermethylated and exhibited high discriminative accuracy. hoxa5 and hoxa6 promoters were both hypermethylated in our study and this is supported by recent data by li and colleagues[21]. the authors went on to demonstrate a significant association between hoxa5 hypermethylation and age, tumour (t), metastasis (m), stage, and patients’ tumour status, while hoxa6 hypermethylation is correlated with age and presence of kras mutation. similarly, with hoxa2 genes, hoxa5 and hoxa6 have not been studied in detail in crc. in other malignancies, such as nsclc, hoxa5 is hypermethylated[35] and a separate study showed that low hoxa5 expression indicates unfavourable prognosis and reduces cell proliferation by via p21 expression[36]. our tcga analyses revealed downregulation of hoxa5 and hoxa6 by 0.58 and 0.62 fold, respectively. the relationship between downregulation of hoxa5 and hoxa6 with crcs patients prognosis is the subject for further research. hoxa5 also plays a role in haematopoietic differentiation, whereby hoxa5 is hypermethylated in the development of acute myeloid leukaemia (aml)[37]. additionally, hoxa6 hypermethylation was reported in oral cancer [38] and more recently in meningiomas[39]. we observed the hypomethylation of hoxa3 and hoxa4 among our patients, which is in disagreement with other crc studies [31, 32]. it is unclear what causes the discrepancies and it will be worthwhile to reconfirm this finding in a larger cohort. by looking at the gene expression of hoxa3 and hoxa4 in crc, several studies partly support our findings. for instance, zhang and colleagues[40] demonstrated that hoxa3 expression is increased in both crc tissues and cell lines. their analysis of the relationship between hoxa3 and tumour progression has revealed that elevated hoxa3 expression is linked with poor survival rates in crc. on the other hand, our finding on hoxa4 hypomethylation is also partially supported by bhatlekar and colleagues whereby hoxa4 is found to be overexpressed in crc[41]. they further demonstrated that overexpression of hoxa4 encourages self-renewal, leading to the overabundance of colon cancer stem cell[42], which play an essential role in the metastasis and relapse of this disease. taken together, hoxa3 and hoxa4 hypomethylation, as identified from our study, may play an important role in crc. to the best of our knowledge, hypomethylation of the long noncoding rna hoxa-as3 and hoxa10-hoxa9 readthrough has never been reported before. therefore we are the first to notice such observation in crc. hoxa10hoxa9 readthrough represents a naturally occurring read-through transcription between the hoxa10 and neighbouring hoxa9 and is a candidate for nonsensemediated mrna decay (nmd), which does not produce any protein product. published literature regarding this readthrough is severely lacking. on the other hand, hoxa-as3 has been gaining more attention from cancer researchers. in lung cancer, hoxa-as3 expression was significantly increased and inhibition of hoxa-as3 impairs cancer cell proliferation, migration, and invasion[43]. in vitro experiment further supported its oncogenic role, where the a549-derived xenografts with silenced hoxaas3 had significantly reduced tumour weights and volumes. these findings suggest the potential application of hoxa-as3 inhibition as an effective targeted therapy for lung cancer patients[43]. the authors also concluded that the upregulated hoxa-as3 expression was shown to be caused by histone acetylation, and the link between histone deacetylation and dna methylation has been established[44]. furthermore, hoxa-as3 confers resistance towards cisplatin treatment via interaction with hoxa3 landscape of hoxa... 9 in nsclc[45]. in glioma, hoxa-as3 upregulation promotes tumour progression and predicts poor prognosis[46]. it is probable that the hypomethylation of hoxa-as3 in our crc patients could lead to its increased expression. it will be interesting to validate this observation and investigate its function in crc. an interrogation hoxa-associated oncogenes or tumour suppressors as prospective mechanisms as predictive biomarkers may offer novel therapeutic strategies for treating cancers[47], including crc. yet, the obstacle lays in the fact that our knowledge and understanding of hoxa genes in the context of crc are still insufficient. in this study, we further extended the understanding of crc pathology by investigating the methylome landscape of hoxa genes. nevertheless, our study is not without limitation. while our sample size is rather small, the hypoand hypermethylation of the hoxa genes reported in this study are relevant to carcinogenesis as reported in several studies. for future study, validation of hoxa methylation changes in cancer tissues from a larger cohort is necessary, and the association with survival and other clinicopathological data is warranted. furthermore, an integrated analysis with gene expression data will be of importance to further establish the correlation between hoxa methylation and gene regulation. conclusion using the latest methylation microarray platform, we report a detailed, unbiased landscape of hoxa genes methylome and discovered epigenetically regulated candidate genes in crc carcinogenesis. specifically, our results provide the primary evidence that aberrant methylation of hoxa2, hoxa3 and hoxa4 in malaysian crc. the new knowledge from this study can be utilized to further increase our understanding of crc methylomics, particularly on the homeobox a genes. the prognostic and diagnostic roles of the differentially methylated hoxa genes warrant future investigations. author contributions mi and rb performed the lab experiments, data analysis and manuscript writing. ltht, l-hl and ns-am provided vital guidance for the project and improvement of the writing. the project was conceptualised by ns-am. conflict of interest the authors declare that there is no conflict of interest in this work. acknowledgement this work was fully supported by universiti kebangsaan malaysia under research university grant scheme (gup-2018-070 geran universiti penyelidikan). references 1. bray f, ferlay j, soerjomataram i, et al., global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin 2018; 68(6): 394– 424. 2. azizah am, nor saleha it, noor hashimah a, et al., malaysian national cancer registry report 2007-2011. 2016, putrajaya, malaysia: national cancer institute. 3. ishak m, baharudin r, rose im, et al., genome-wide open chromatin methylome profiles in colorectal cancer. biomol 2020; 10(5): 719– 719. 4. chow yp, yunos rim, rose im, et al., 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jemli1,2, abdelhakim bouyahya3*, khang wen goh4, ganesh sritheran paneerselvam5*, belatar bahia6, yahia cherrah1, katim alaoui1 article history 1laboratory of pharmacology and toxicology, faculty of medicine and pharmacy, mohammed v university, rabat, morocco; d.zouhra@yahoo.fr (dz); eljemli.meryem@gmail.com (mej); cherrahy@yahoo.fr (yc); alaouikma@yahoo.fr (ka) 2mohammed vi university of health sciences (um6ss), casablanca, morocco 3laboratory of human pathologies biology, faculty of sciences, department of biology, and genomic center of human pathologies, faculty of medicine and pharmacy, mohammed v university in rabat, morocco 4faculty of data science and information technology, inti international university, nilai 71800, malaysia; khangwen.goh@newinti.edu.my (kwg) 5school of pharmacy, taylor’s university, 47500 subang jaya, malaysia 6research unit of cerebral monitoring in neuro-reanimation, faculty of medicine and pharmacy, university mohamed v of rabat, morocco; belatar.belatar80@gmail.com (bb) *corresponding author: abdelhakim bouyahya, laboratory of human pathologies biology, faculty of sciences, department of biology, and genomic center of human pathologies, faculty of medicine and pharmacy, mohammed v university in rabat, morocco; a.bouyahya@um5r.ac.ma (ab); ganesh sritheran paneerselvam, school of pharmacy, taylor’s university, jln taylors, 47500 subang jaya, selangor; ganeshsritheran.paneerselvam@taylors.edu.my (gsp) received: 26 march 2023; received in revised form: 21 may 2023; accepted: 28 may 2023; available online: 07 june 2023 abstract: important moroccan medicinal plants known for their food and medicinal potential include urtica urens and mercurialis annua. we examined the antioxidant activity of the methanolic and aqueous extracts by several in vitro systems of the assay, namely the ferric reducing/antioxidant power (frap) assay, the dpph radical-scavenging activity, and the abts free radical-scavenging capacity. in addition, all active extracts were subjected to phytochemical screening to determine the key groups of chemicals implicated in their therapeutic activity. total phenolic and flavonoid concentrations were also measured to see how they affected the antioxidant properties of the plant extracts. the extracts of urtica urens and mercurialis annua were found to have different levels of antioxidant effect in the systems tested. based on the three different antioxidant assays, the mercurialis annua extracts showed the highest values of antioxidant ability, based on the three used assays. mailto:cherrahy@yahoo.fr mailto:alaouikma@yahoo.fr mailto:belatar.belatar80@gmail.com pmmb 2023, 6, 1; a0000335 2 of 14 phytochemical testing of studied extracts revealed the presence of flavonoids, anthocyanins, and tannins. total phenol and flavonoid contents were shown to provide the highest association with different antioxidant methods. the present study provides evidence that the extracts of urtica urens and mercurialis annua are a potential source of natural antioxidants, and this justifies their uses in moroccan traditional medicine. keywords: urtica urens; mercurialis annua; phytochemical screening; phenolic and flavonoid contents; antioxidant effects. 1. introduction free radicals produced in the body during normal physiologic functions or introduced from the entourage are highly unstable products capable of inducing several side health effects and diseases [1–5]. medicinal herbs are known to have the ability to reduce this damage and thus prevent oxidative stress-related diseases [6–9]. urtica urens and mercurialis annua are the most common species worldwide, extensively used for diverse medicinal purposes. urtica urens (urticacea), locally known as “hurriyqa”, is a perennial plant with stinging hairs frequently used as decoctions, infusions, or powders to treat anxiety, arthritis, rheumatism, cancer, toothache, scabies, and pruritus [10–13]. seeds soaked in milk are frequently used against cough, kidney stones, cystitis, and oliguria [10]. the aerial parts are known for diuretic, galactogenic, and aphrodisiac properties [10], and are used to treat cancer and sciatica [14], and as a styptic [15], the leaves are frequently used against burns, rheumatism, and urinary diseases [14–16]. several pharmacological and phytochemical works have confirmed their traditional uses in folk medicine [11,17,18]. the methanolic extract of the urtica urens aerial part has proved to have anxiolytic activity [11], free radical scavenging activity, and antimicrobial potential [19]. the ethanol and aqueous extracts of the leaves of urtica urens have been tested on staphylococcus aureus, pseudomonas aeruginosa, and escherichia coli and show considerable antibacterial activity [17]. many studies have shown that the biological properties of u. urens are especially related to its richness on various bioactive compounds such as polyphenols [20], caffeic acid [21] , and chlorogenic acid [22]. mercurialis annua (euphorbiaceae), popularly known in moroccan folk medicine as “hurriyqa l-melsâ”, is largely used in the treatment of a range of disorders [11,23,24]. in morocco, the plant is extensively used as a purgative, anxiolytic, and also in treating female infertility[10,11,25]. in portugal, the whole plant is known for its diuretic effect and is traditionally used to treat internal parasitosis and oral inflammation [26]. the italians widely use leaf infusions against diuresis and constipation [24]. one cup of a leaf decoction is taken pmmb 2023, 6, 1; a0000335 3 of 14 three times/day to treat diabetes and cancer in israeli folk medicine [27]. the plant has also been used as a laxative [28], anti-warts, and antibronchial [29]. the dried, powdered plant also promotes wound healing [30]. several studies have confirmed the various traditional reputations [18,23,31]. mercurialis annua is plenty of biological effects, such as anti-cancer [23], anti-inflammatory [23], anti-microbial [23], anti-anxiety [18], and anxiolytic effects [31]. many reports have demonstrated that pharmacological activities of m. annua are related to their rich content of phenolic compounds such as rutin, narcissin, flavonol glycosides [32–34]. despite the large flow of data of urtica urens and mercurialis annua uses, and to the authors’ knowledge, no works have so far been performed to evaluate the antioxidant effect of the moroccan urtica urens and mercurialis annua. accordingly, this study mainly tested the abts and dpph radical-scavenging abilities, the ferric reducing power (frap) effect, the phenolics contents of urtica urens and mercurialis annua methanolic extracts. 2. materials and methods 2.1. plant materials mercurialis annua and urtica urens aerial parts were manually collected from wazzan town (jaaouna el basra), morocco, and were identified by pr. m. ibn tatou and pr. h. khammar of the scientific institute of rabat, mohammed v university, rabat, morocco, where voucher specimens have been deposited at its herbarium (voucher number rab78984 and rab78983 respectively). the samples were thoroughly dried at laboratory ambient temperature, grounded into a fine powder, and kept at room temperature until further use. 2.2. extracts preparation methanolic extract of mercurialis annua (mam) and urtica urens (uum) were separately performed by cold maceration using 100 g of dried and powdered aerial parts in 1000 ml of methanol at room temperature 24 h. aqueous macerate of mercurialis annua (maa) and urtica urens (uua) were prepared by the same procedure. the macerates were filtered, and the filtrates obtained were evaporated under reduced pressure and at a temperature lower than 65 °c. 2.3. phytochemical screening mercurialis annua and urtica urens extracts were evaluated for the qualitative determination of major phytoconstituents, i.e., alkaloids, flavonoids, tannins, saponins, and cardiac glycosides following these methods [35,36]. pmmb 2023, 6, 1; a0000335 4 of 14 2.4. determination of total phenolic content total phenolic content (tpc) was spectrophotometrically determined according to the folin-ciocalteu method [37] . a total of 20𝜇l aliquot of the tested extract was mixed with 1.16ml of distilled water, 100𝜇l of folin ciocalteu reagent, and 300𝜇l of na2co3 solution (20%). the tubes were kept at 40∘c for 30 min, and the absorbance was measured at 760nm utilizing uv–visible spectrophotometer. the total phenolic content was obtained from the extrapolation of the calibration curve, which was made by various concentrations of gallic acid solution in methanol. tpc was expressed as µg of gallic acid equivalents (𝜇g gae)/mg dry extract. 2.5. determination of total flavonoid content the overall quantity of flavonoids (tfc) of mercurialis annua and urtica urens extracts was investigated using the method described by ordonez et al., 2006 [37] with slight modifications. briefly, 0.5ml of the extracts was mixed with 0.5ml of 2% alcl3. after 1 hour of incubation at room temperature, the absorbance was measured at 420 nm. tfc was expressed as microgram quercetin equivalent per milligram dry plant extract (µg qce/mg dry extract). 2.6. antioxidant activity the antioxidant effects of mercurialis annua and urtica urens extracts were tested using three complementary standard tests: dpph free radical scavenging, abts scavenging, and ferric-reducing antioxidant power (frap) assays. all samples were analyzed in triplicate. 2.7. dpph free radical-scavenging activity the dpph radical scavenging effect of mercurialis annua and urtica urens extracts was evaluated according to sahin method [38]. a mixture containing 0.50 ml of various extracts concentrations and 2 ml of the dpph methanolic solution (60𝜇m) was incubated in the dark for 20 min. the absorbance was then measured at 517 nm and the percentage of dpph radical scavenging performance was calculated as following eq. (1).: percentage (%) of dpph radical scavenging ability = [(a0-a1)/a0] x 100 where, a0 was the absorbance of blank, and a1 was the absorbance of the studied extract. quercetin (qc) and butyl hydroxytoulene (bht) were taken as standards. the extract pmmb 2023, 6, 1; a0000335 5 of 14 concentration providing 50% of inhibition (ic50) was calculated from the plotted inhibition curve (%) against concentrations. 2.8. abts free radical-scavenging assay the ability of water and methanolic extracts of mercurialis annua and urtica urens to scavenge the abts radical was estimated using the method described by pukalskas et al. [39]. a radical solution was produced by combining 10 ml of abts (7 mm), and 10 ml of potassium persulphate (70 mm) was kept in the dark at room temperature for 16 h. after the incubation period, the abts solution was diluted to obtain an absorbance of 0.700 ± 0.002 at 734 nm. a total of 100𝜇l of the diluted extract was added to 2ml of abts solution freshly prepared, and the absorbance was measured at 734 nm after 1 min. the percentage inhibition and the half-maximal inhibitory concentration (ic50) of the extract were calculated with the same procedure described above. 2.9. reducing power determination the ferric-reducing ability of mercurialis annua and urtica urens extracts was investigated using the method of tundis [40]. a mixture containing 0.2ml of each extract at various concentrations, 2.5 ml of potassium ferricyanide (1%), and 2.5 ml sodium phosphate buffers (0.2m, ph 6.6) was incubated at 50 °c for 20 min. after the incubation was completed, 2.5 ml of trichloroacetic acid (10%) was added, and the resulting mixture was centrifuged at 1000 r/min for 10 min; then, 2.5 ml of the supernatant was mixed with 2.5 ml of distilled water and 0.5 ml of fecl3 (0.1%). the absorbance was measured at 700 nm, and the ic50 was calculated by plotting the absorbances versus the sample concentrations. 2.10. statistical analysis all analyses were carried out in triplicate, and these values were then presented as mean values ± their standard derivations (sd). data were analyzed using the graph pad prism 6.0. statistical comparisons were performed with one-way analysis of variance (one-way anova), and (p < 0.05) were regarded as significant. the correlation coefficients (r) between dpph, abts, frap, tpc, and tfc were calculated to determine their relationship. pmmb 2023, 6, 1; a0000335 6 of 14 3. results and discussion 3.1. extraction yield the extraction yields of mercurialis annua and urtica urens aerial parts in methanol and water were determined and are shown in figure 1. depending on the species and the extraction solvent, the dry weight yield in the studied samples was remarkably affected. mercurialis annua aqueous extracts showed the highest extraction yield (32.7%), followed by the methanolic extract from the same plant (21.23%). while urtica urens samples extracted with water and methanol gave extract yields of 12.12% and 11.92%, respectively. the difference observed can be explained by the solvents' polarity and the solubility of bioactive compounds [41]. m a a m a m u u a u u m 0 10 20 30 40 y ie ld ( % ) figure 1. yield (%) of mercurialis annua and urtica urens extracts. 3.2. phytochemical screening phytochemical screening of the crude extracts of m. annua and u. urens revealed the major classes of compounds present to be polyphenols, flavonoids, and tannins (table 1). these constituents are known to possess various pharmacological activities including antioxidant, anticancer, antifungal, antibacterial, anti-inflammatory, and antidiabetic [42–46]. thus, the presence of these compounds may be responsible for the antioxidant abilities of the extracts. pmmb 2023, 6, 1; a0000335 7 of 14 table 1. phytochemical screening of crude extracts of mercurialis annua and urtica urens. maa mam uua uum total polyphenols ₊₊ ₊₊₊ ₊₊ ₊₊ flavonoids ₊₊ ₊₊ ₊ ₊ anthocyanins ₊ ₊ tannins ₊₊ ₊₊ ₊ ₊ alkaloids terpenes saponins ₊ ₊ ₊ ₊ quinones ₊ ₊ ₊ ₊ total phenolic content mercurialis annua and urtica urens extracts were characterized by a considerable amount of phenolic content (figure 2). the highest tpc was found in mam with (223.7 ± 1.59) 𝜇g gae/mg edw, followed by maa (168.274 ± 2.64) 𝜇g gae/mg dry plant extract (edw), uum (130.816 ± 2.82) 𝜇g gae/mg edw, and uua (109.768 ± 3.60) 𝜇g gae/mg edw. the richness of mercurialis annua and urtica urens on phenolic compounds agrees with several reports [33,47]. however, the variation in total phenolic content of the different studied extracts can be explained by various parameters and conditions such as genetic factors and type of extract. m a a m a m u u a u u m 0 50 100 150 200 250 𝜇 g g a e /m g e d w figure 2. total phenolic content expressed as gallic acid equivalents (𝜇ggae)/mg plant extracts of mercurialis annua and urtica urens. data are expressed as mean ± sd (n=3). pmmb 2023, 6, 1; a0000335 8 of 14 3.3. total flavonoid content flavonoid content in investigated extracts ranged from 10.98 to 80.71 𝜇g (qe)/ mg edw (figure 3). the high flavonoids concentration was determined in mam extract (80.71 ± 2.27𝜇g (qe)/mg edw) followed by uum (43.79 ± 2.10𝜇g (qe)/mg dry plant extract (edw)), maa (29.51 ± 0.51 𝜇g (qe)/mg edw). the lowest total flavonoid content was determined for uua extract (10.98 ± 0.39𝜇g (qe)/mg edw). the richness of mercurialis annua and urtica urens extracts on flavonoid compounds is in agreement with several studies [33,47]. however, the variation in total flavonoid content between the studied extracts can be explained by various parameters and conditions such as genetic factors and type of extract. m a a m a m u u a u u m 0 20 40 60 80 100 𝜇 g q e /m g e d w figure 3. total flavonoid content expressed as quercetin equivalents (𝜇gqe)/mg plant extracts of in mercurialis annua and urtica urens. data are expressed as mean ± sd (n=3). 3.4. antioxidant activity the antioxidant activity of the mercurialis annua and urtica urens, evaluated by the three assays varied significantly among the extracts (table 2). the mam and maa extracts presented the most substantial reducer effect in the frap assay (ic50 = 79.15 ± 0.14 and 91.52 ± 1. 30 𝜇g/ml, respectively) and the better antioxidant capacity in the dpph (ic50 = 51.16 ± 0.09 and 61.32 ± 0.65 𝜇g/ml respectively) as well as in the abts assays (ic50 = 59.00 ± 1.48 and 73.61 ± 0.88 𝜇g/ml respectively). the lowest capacities were recorded for the urtica urens extracts with an ic50 ≥ 227.11 𝜇g/ml. however, all the tested extracts exhibited low antioxidant capacities compared to quercetin, trolox, and bht with ic50 from 1.29 to 7.02 𝜇g/ml. the antioxidant potential of mercurialis annua and urtica urens might pmmb 2023, 6, 1; a0000335 9 of 14 be explained partly by their richness in polyphenols such as flavonoids previously revealed in our study (table 1, figure 1, figure 2). the antioxidant activities of these compounds have mainly been reported to have strong antioxidant power. various reports have been on the antioxidant effects of several mercurialis and urtica species [48,49]. urtica urens aerial parts extract exhibited great antioxidant activity which has been strongly associated with its richness in phenolic compound [49]. aerial parts of urtica membranacea also showed a potent antioxidant effect [49]. the whole plant of urtica dioica showing a strong antioxidant activity in the cupric reducing antioxidant capacity (cuprac) and the ferric reducing/antioxidant power (frap) assays which mainly due to its richness on phenolic compounds such as ursolic acid and quercetin [50]. stinging nettles provide high antioxidant activity and richness in phenolic compounds [51]. the antioxidant ability of the nettle (urtica dioica) has mainly been attributed to phenolic compounds such as rutin, quercetin 3-o-glucoside, chlorogenic acid, 2-o-caffeoylmalic acid, isorhamnetin 3-o-rutinoside, kaempferol 3-o-rutinoside, caffeic acid derivatives [26,52,53]. in the same sense, several studies have shown the richness of mercurialis spp. on phenolic compounds widely known for their potent antioxidant power, such as kaempferol, squalene, and cycloartenol [54–57]. table 2. ic50 values (𝜇g/ml) of mercurialis annua and urtica urens extracts and of quercetin, bht, and trolox. assays plant extracts positive controls maa mam uua uum qc bht trolox dpph 61.32 ± 0.65 51.16 ± 0.09 335.1 ± 2.18 282.58 ± 3.38 1.29 ± 0.01 4.20 ± 0.02 abts 73.61 ± 0.88 59.00 ± 1.48 376.53 ± 4.85 289.90 ± 1.13 1.93 ± 0.01 frap 91.52 ± 1.30 79.15 ± 0.14 372.35 ± 1.94 227.11 ± 0.89 2.06 ± 0.01 7.02 ± 0.02 3.5. relationship between antioxidant assays to evaluate the reliability and suitability of the three antioxidant assays used to determine the antioxidant abilities of mercurialis annua and urtica urens extracts, we performed a correlation analysis of the three used tests. as shown in table 3, the values of dpph were highly correlated with abts (r=0.997; p>0.05) and frap (r=0.955; p>0.05). the relation between abts and frap also showed a high and insignificant correlation pmmb 2023, 6, 1; a0000335 10 of 14 (r=0.976; p p>0.05). this suggests that dpph, abts, and frap can be predictive assays for each other. the relationship between the three used assays has been largely reported [58,59]. table 3. the correlation coefficient among antioxidant tests and total phenolic and flavonoid contents. dpph abts frap abts 0.997 frap 0.955 0.976 tpc -0.903 -0.908 -0.879 tfc -0.622 -0.656 -0.710 3.6. relationship between antioxidant effect and phenolic content the relationship between the antioxidant effect and phenolic content of mercurialis annua and urtica urens extracts is described in table 3. the tpc was negatively but highly correlated with dpph (r=−0.903; p<0.05), abts (r=−0.908; p<0.05), and frap (r=−0.879; p<0.05). the negative relationship is due to the antioxidant activity in terms of ic50 decreases, the tpc increases. the results show that high tpc indicates a better antioxidant effect in terms of dpph, abts, and frap assays of mercurialis annua and urtica urens extracts. the tfc was correlated with dpph (r=−0.622; p<0.05), abts (r=−0.656; p<0.05), and frap (r=−0.710; p<0.05). the results of flavonoid content were quite similar to those of phenolic content; thus, both of them can be a measure to assess the antioxidant effects of mercurialis annua and urtica urens extracts, but still, the role of other phenolic compounds and metabolites present in the plants. generally, individual flavonoid compounds cannot be as effective as in combination with other phenolic compounds. the high correlations between the phenolic content (tpc and tfc) and antioxidant potential have been reported for other medicinal plants such as hedychium spicatum, azadirachta indica, juniperus thurifera, j. oxycedrus, j. phoenicea and tetraclinis articulate [58,60,61]. therefore, total phenolic and flavonoid content can be used to assess medicinal plants' antioxidant ability. 4. conclusion the results obtained in this work support the therapeutic uses of mercurialis annua and urtica urens. their phenolic content contributed an increased antioxidant activity of those moroccan species in addition, the study suggests that the water and methanolic extracts pmmb 2023, 6, 1; a0000335 11 of 14 from the mercurialis annua and urtica urens constitute a valuable source of antioxidant metabolites. further investigations are required to identify the bioactive metabolites. the study also confirms that phenolic content can predict antioxidant assays like abts, dpph, and frap. author contributions: author contributions: conceptualization, d.z., a.b., l.c.m., k.a.; methodology, d.z., m.e.j., a.b., k.a.; software, d.z., k.w.g., k.a.; validation, d.z., m.e.j., k.a.; resources, a.b., k.w.g., l.c.m., k.a.; writing—original draft preparation, d.z., k.a.; writing—review and editingd.z., a.b., l.c.m., b.b., y.c., k.a.; visualization, d.z., k.w.g., k.a.; supervision, a.b., k.a,; project administration, d.z., k.a.; all authors have read and agreed to the published version of the manuscript. funding: all the materials and working tools for my thesis are available in the laboratory of pharmacology and toxicology, faculty of medicine and pharmacy, university mohammed v rabat. acknowledgments: we thank hayat aouari, for their technical assistance throughout the studies. we also thank professor amina zellou for his advice regarding the analysis of the experiments presented in this paper. competing interests: the authors declare that the research was conducted in the absence of 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https://onlinelibrary.wiley.com/toc/17454514/2017/41/2 progress in microbes and molecular biology review article 1 mycobacterium ulcerans and mycobacterium marinum: pathogenesis, diagnosis and treatment loh teng-hern tan1*, pendru raghunath2, long chiau ming3, jodi woan-fei law1* 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. 2department of microbiology, school of medicine, texila american university, georgetown, guyana. 3paprsb institute of health sciences, universiti brunei darussalam, jalan tungku link gadong be1410, brunei darussalam. abstract: skin and soft tissue infections are common presentations for non-tuberculous mycobacteria (ntm). the cutaneous infections caused by ntm may cause localized or diffuse lesions. m. ulcerans is one of the most identified pathogens that involves in the skin and soft tissue mycobacterial infections. meanwhile, m. marinum, as an ntm has also become important emerging causal agents of cutaneous disease in various geographical regions. although having common ancestry and highly similar in genetic makeup, m. ulcerans and m. marinum have differential impacts on the host innate immune system. in term pathogenesis, prolonged cell exposure to exotoxin mycolactone produced by m. ulcerans could lead to buruli ulcer. meanwhile, like most pathogenic mycobacteria, m. marinum evades the host immune responses by invading and replicating inside host cells and it is capable of modulating host immune responses. this article aims to provide a general overview and comparisons between the pathogenesis, diagnosis, prevention and therapeutic strategies for m. ulcerans and m. marinum. keywords: mycobacterium ulcerans; mycobacterium marinum; diagnosis; pathogenesis; treatment received: 17th august 2020 accepted: 18th september 2020 published online: 30th september 2020 citation: tan lth, raghunath p, ming lc et al. mycobacterium ulcerans and mycobacterium marinum: pathogenesis, diagnosis and treatment. prog microbes mol biol 2020; 3(1): a0000114. https://doi.org/10.36877/pmmb.a0000114 introduction mycobacteria are a group of aerobic, acid-fast bacteria which are slender, non-flagellated and rod in shape. they possess waxy cell wall composed of mycolic acid, enabling them to be resistant to decolorization even with the use of acidified alcohol. the acid-fastness property of mycobacteria can be demonstrated by the ziehlneelsen stain[1]. in general, the genus mycobacterium is broadly categorized into three main groups which include the mycobacterium tuberculosis complex (mtc), mycobacterium leprae and non-tuberculous mycobacteria (ntm). being the two major human pathogens, m. tuberculosis and m. leprae cause tuberculosis and leprosy, respectively. unlike the other two groups which are pathogenic to human, ntm, or atypical mycobacteria, encompasses a variety species which commonly inhabit the aquatic and terrestrial environments. more than 170 species of ntm has been discovered and the list keeps increasing. these mycobacteria form biofilm that contributes to their survival in diverse ecological niches[2], including soil, water (such as household water) plants, animals and food products. although ntm disease is not notifiable in most countries, the rise in the prevalence of ntm disease has become a growing health concern in the recent years. the reasons include the aging of the population, the increasing number of immunocompromised patients and the increased awareness of the disease. the ntm can cause pulmonary as well as extrapulmonary diseases (lymphadenitis, cutaneous disease, disseminated disease), that often inflicting immunocompromised individual and patients with pre-existing conditions. generally, ntm infections are acquired from environmental exposures via inhalation (e.g. aerosol) or inoculation (e.g. trauma, plastic surgery, acupuncture) [3,4]. skin and soft tissue infection is one of the common copyright @ 2020 by tan lth and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: loh teng-hern tan, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, bandar sunway, selangor darul ehsan, malaysia; loh. teng.hern@monash.edu. jodi woan-fei law, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, bandar sunway, selangor darul ehsan, malaysia; jodi.law1@monash.edu. 2 presentations for ntm. mycobacteria responsible for most skin disease include m. ulcerans, m. marinum, m. chelonae, m. fortuitum, m. avium-intracellulare, m. tuberculosis and m. leprae. the cutaneous diseases caused by mycobacteria usually manifest as nodules with characteristics of crusting, ulcers and hypoand hyperpigmentation. furthermore, cutaneous infections associated with these mycobacteria may cause localized or diffuse lesions. m. ulcerans is one of the most identified pathogens that involves in the skin and soft tissue mycobacterial infections. meanwhile, m. marinum, has also become the emerging pathogens causing cutaneous diseases in people from various countries. although having a common ancestry and they are highly similar in terms of genetic makeup, m. marinum and m. ulcerans exhibit differential impacts on the innate host immune system. the production of mycolactone plays a main role of m. ulcerans in the pathogenesis of buruli ulcer disease. meanwhile, m. marinum is similar to most pathogenic mycobacteria where the bacteria evade the host immune responses by invading and replicating inside host cells and are capable to modulate host immune responses (figure 1). mycobacterium ulcerans... figure 1. the differential clinical presentations and the pathogenesis between m. marinum and m. ulcerans despite they share high genomic similarity. the aim of this review is to provide an overview on both m. ulcerans and m. marinum as the two major human pathogenic mycobacteria species commonly implicated in cutaneous diseases. besides that, the pathogenesis and diagnosis of m. ulcerans and m. marinum infections are summarized and compared. the prevention and ideal strategies to control the diseases are also discussed in this review. mycobacterium marinum versus mycobacterium ulcerans both m. marinum and m. ulcerans are known to be the two opportunistic mycobacterial pathogens, also named as the mycolactone-producing mycobacteria (mpm), that secrete plasmid-encoded mycolactone exotoxins[5]. mycolactone is a cytotoxic polyketide metabolite produced by mpm, essential for bacterial virulence, to induce buruli ulcer-like lesions characterized by extensive necrosis and void of inflammation in intradermal administered animal models[6]. the mycolactone molecule is known to cause apoptosis of mammalian cells, especially more toxic toward the anchorage-dependent cells leading to cytoskeletal rearrangements and detachment in in vitro experiments. interestingly, studies also indicate that mycolactone alters the primary role of innate immunity, including immune cells trafficking and tlr-induced cytokine production. m. marinum was first isolated from salt water dead fish by aronson (1926)[7] and was considered as an opportunistic human pathogen after its retrieval from granulomatous skin lesions from swedish swimmers in the year of 1951[8]. m. marinum is categorized under the runyon’s group i photochromogenic ntm that are commonly found in nonchlorinated fresh or salt water[9]. being an opportunistic human pathogen, m. marinum causes zoonotic infection in individuals who had exposed through direct-contact with the bacterium from fishes, especially when handling the contaminated aquariums[10]. in general, m. marinum infections manifest as superficial skin infections that marked by granuloma and lymphangitis. thus, the infection of m. 3 tan lth et al. the conventional microbiological detection of mycobacteria can be done in specific solid or liquid medium to assist in devising successful antibiotic regimens. the cultivation of ntm is greatly varied depending on the type of pathogen. incubation of both solid and liquid media at both 30oc and 35oc are usually done to optimally recover the ntm. different media compositions and conditions according based on specific ntm metabolic needs are also required for their isolation, hence the suspected mycobacterial species could be suggested by the clinicians based on source of exposure and the clinical symptoms to the microbiologists[25]. being one of the most commonly used molecular-based method, polymerase chain reaction (pcr) has been used for rapid detection of pathogens based on specific target dna sequence[26-28]. specific polymerase chain reaction (pcr) assays are available and have been established as the gold standard for the identification and discrimination of ntm that representing public health hazard[29]. pcr can be performed on different clinical samples, including swabs, punch biopsies and fine needle aspirates from nodules, plaques as well as edematous lesions that have not ulcerated yet. several target genes or sequences are commonly used to differentiate mycobacterium sp., including the 16s rrna, internal transcribed spacer (its), 23s rrna, hsp-65, reca and rpob genes. among the different genes, pcr targeting the insertion element is2404 has shown to be highly sensitive and specific for m. ulcerans detection as it is present in high copy numbers (>200 copies) in the m. ulcerans genome[30] timothy. a highly specific real-time qpcr assay was demonstrated to confer enhanced sensitivity of 10-folds higher than the is2404 pcr and provide quantitative assessment of m. ulcerans dissemination in buruli ulcer lesions[31]. meanwhile, molecular detection technique for m. marinum is limited due to its high homology with m. ulcerans. typically, molecular identification of m. marinum was performed by analysing 16s rrna or other conserved gene related to a week-growth of photochromogenic colonies[29]. although pcr has high sensitivity, sophisticated laboratory infrastructure and well-trained personnel are required to obtain reliable pcr assays with strict quality control[32]. meanwhile, these criteria are not available in endemic communities. recently, loop-mediated amplification (lamp), an isothermal amplification technique, has been proposed as an efficient diagnostic tool for detection of m. ulcerans. being a promising alternative to pcr, this technique offers readily readable results within a short turnaround time and without the need of a thermocycler, hence extending molecular diagnosis in fieldwork and at the point of care[33]. pathogenesis of m. ulcerans and m. marinum m. ulcerans causes an ulcerative skin infection, namely buruli ulcer, which is a destructive infection of subcutaneous tissues that result in ulcerative lesions in skin, soft tissue and even the bone. m. ulcerans differs from many other mycobacteria in term of its implications for pathology and immune response in human. m. ulcerans is found to be distributed extracellularly around the coagulative necrosis regions which is different from other mycobacteria that are marinum is also termed as the ‘fish tank granuloma’ or ‘aquarium granuloma’. meanwhile, more severe infections that can spread in a sporotrichoid pattern[11] to deeper tissue inflicting tendinitis, arthritis and osteomyelitis may occur especially in immunocompromised host[12,13]. m. ulcerans is the causative pathogen of a neglected tropical disease, buruli disease, being one of the most common mycobacterial disease worldwide after tuberculosis and leprosy[14]. buruli disease is a chronic, necrotizing skin disease with cutaneous tissue destruction and large ulcerations. the buruli disease cases primarily occur in central africa, and other regions, including asia, south america, the western pacific and australasia[15]. m. ulcerans strains possess a large circular virulence plasmid named pmum which contains 3 genes encoding polyketide synthases (mlsa1, mlsa2 and mlsb) responsible for the synthesis of the lipid toxin mycolactone[16]. interestingly, m. ulcerans is genetically closely related to the m. marinum, thereby they share 99% dna identity in which m. ulcerans exhibit reduced genomes. the comparative whole genomic studies suggest that the emergence of m. ulcerans has recently evolved from a m. marinum progenitor via acquisition of the virulence plasmid pmum and subsequent reductive evolution[16]. acquisition of this plasmid has been considered to be the main contributor for buruli ulcer in humans[17]. the reduced genome of m. ulcerans was subjected to substantial gene loss due to dna deletion s, dna rearrangements mediated by insertion of is2404 and is2606 elements for niche adaptation[18,19]. both m. ulcerans and m. marinum strains have optimum growth temperature around 32°c[20], but they grow poorly at 37°c and above, thus reflecting the preference of both strains for the skin and their limited systemic dissemination[21]. considering both pathogens belonging to the group of slow-growing mycobacteria, m. marinum has longer doubling time than m. ulcerans when grown in microbial culture medium[22]. in australia, a mean incubation period of four and half months was identified for m. ulcerans infections[23]. meanwhile, the incubation period of m. marinum was approximately ∼3 weeks but can be prolonged up to 9 months prior to symptoms onset[24]. nevertheless, both m. marinum and m. ulcerans infections may resolve by host immune responses while long-term antibiotic therapy is required on an established infection. rapid identification and differentiation of mycobacterium species are crucial to determine the appropriate therapeutic regimens. however, a definitive diagnosis of cutaneous mycobacterial infections can be challenging to make in the clinical routine. there are several diagnostic methods available include histology, microbiological cultures and molecular detection. histologically, m. marinum infections manifest non-specific acute or chronic inflammation as well as positive for tuberculous granulomas and abscesses. however, the detection of acid-fast rod-like bacteria is unusual. meanwhile, m. ulcerans infections are associated with septal subcutaneous necrosis of adiposerich tissue and positive for acid-fast bacteria detection[25]. diagnosis of m. ulcerans and m. marinum 4 intracellular macrophage pathogens. this observation led to early proposal that m. ulcerans produces an exotoxin[34]. the cytotoxic molecule ‘mycolactone’ was successfully isolated and purified from the acetone soluble fraction of lipid extracts of m. ulcerans in 1999[35]kathleen. principally, the pathogenesis of m. ulcerans is mediated by the production of mycolactone which is uncommon among other bacterial exotoxins. mycolactones consist of a group poorly immunogenic polyketide-derived macrolides that have strong cytotoxic effects against most of the immune cells and skin cells. there are different variants of mycolactone molecules have been identified[36,37]. the typical mycolactone a/b occurs in africa while mycolactone c is found in australia. in in vitro, mycolactone a/b is more toxic than type c, the clinical significance of these differences remains elusive. the paramount role of mycolactone in the pathogenesis of m. ulcerans was first established via the administration of the purified mycolactone into the skin of experimental animals which resulted in cell death but devoid of acute inflammatory response[38]. the major role of mycolactone in buruli ulcer pathogenesis was further fortified by the infection of laboratory animals with m. ulcerans mutants which lack of mycolactone production. in contrary to the extracellular infection induced by the wild-type m. ulcerans, an intracellular inflammatory infection identical to that of m. marinum[39] was resulted by the mycolactone-negative mutants[40,41]. mycolactone has three major adverse implications on the host in mediating the pathogenesis of m. ulcerans infection. the chief destructive outcome of mycolactone is its apoptosis and necrosis inducing effects on an array of cells, including the immune cells. mve-obiang et al. (2003)[42] revealed the potent cytotoxic effect of mycolactone a/b, as low as 0.1 ng/ml was sufficient to induce cell death associated with apoptosis[38] and necrosis[43]. recently, mycolactone was demonstrated to induce bim-dependent cell apoptosis via the mtorc2akt-foxo3 axis [44]. secondly, a down-regulation of overall host immune defence due to the impairments on the production of tumor necrosis factor (tnf) and other secretory proteins via the blockade of sec61 by mycolactone. sec61 is a heterotrimeric complex responsible for the transport of all secretory and integral transmembrane proteins into the endoplasmic reticulum in eukaryotic cell. the blockade of sec61 activity affects the production of interferon-gamma (ifn-γ) and ifn-γ in activated lymphocytes as well as nitric oxide synthase production in macrophages[45]. thus, an effective immune response is failed to be activated by the host to act upon the underlying mycobacterial infection. thirdly, mycolactone also causes impairment of pain sensitivity by targeting the type 2 angiotensin ii receptors (at2r) to mediate its analgesic effect. the mycolactone was suggested to induce analgesia by direct cytotoxicity against sensory neurons and schwann cells, hence resulting in nerve damage[46,47]. as for m. marinum, this bacterium causes tuberculosislike infection in the ectotherms and induces caseating granulomas in zebrafish that are similar to those in humans[48]. m. marinum is an opportunistic intracellular pathogen that multiplies in non-acid phagosome of macrophages prior to phagolysosome fusion[49]. within the cells, m. marinum acquires the ability to escape from the phagosome into the cytoplasm to actively stimulate actinpolymerization, resulting to direct spread into adjacent cells via actin-based motility. the translocation of m. marinum into the host cell cytosol depends on an intact region-of-difference-1-locus (rd1) which encodes a type-vii secretion system (esx-1) that plays a role in mycobacterial virulence[50,51]. thus, this mechanism confers immune evasion for m. marinum by spreading from cell to cell, contributing to permanent infection. m. marinum was further found to employ the nonlytic spreading mechanism where the mycobacterium is ejected from the cell via the ejectosome, a f-actin based structure, enabling the transmission to naïve host macrophages[52]. then, the macrophages migrate into deeper tissue, where they start to form the pathological granuloma-like aggregates after phagocytosis of m. marinum. moreover, m. marinum was shown to harbour the esx-5 system of mycobacteria that responsible for the production of various prolineproline-glutamic acid (ppe) and proline-glutamic acid (pe)-polymorphic gc-rich repetitive sequence (pgrs) proteins. these proteins were demonstrated to interact with host immune system and evade the innate immune response via antigenic variation[53], thus contributing to persistent infection[54,55]. for instance, the expression of ppe38 protein on the cell wall of m. marinum was shown to involve in bacterial surface properties and proinflammatory effects on infected macrophages[56]. in the view of granulomas as host-beneficial protective structures that has long been a tenet of medical and immunology textbooks, studies employing the zebrafish embryo model of m. marinum infection have challenged the idea and provided evidence that the granulomas may be harnessed by mycobacteria for their dissemination and proliferation[57,58]. the study revealed that m. marinum employs the esx-1-dependent early macrophage aggregate to promote spread and growth[57]. on top of that, the mature and established granulomas are found to be porous, where newly infecting mycobacteria can infiltrate and persist within[59]. an utmost priority to curb buruli ulcer disease is to enhance our knowledge on the transmission pathway of m. ulcerans to human that could aid in the preventive measures focusing on early detection and administration of effective treatment. however, the greatest challenge in buruli ulcer control is that the reservoir and transmission of m. ulcerans are unclear. exposure to water sources near endemic villages has been shown to increase the risk for developing buruli ulcer, but it is a challenge to reduce the exposure, particularly in children, to such sources in rural west africa[60]. the development of an effective vaccine to confer protection has enormous significance in areas of high endemicity for buruli ulcer. however, there is no effective vaccine specifically targeting m. ulcerans is available clinically. the bacillus calmette-guérin (bcg) mycobacterium ulcerans... prevention and treatment strategies 5 vaccine is the only licensed vaccine against mycobacterial infections approved clinically that used to prevent tuberculosis. although the bcg is cross-protective against m. ulcerans, it has only been associated with delaying the onset of disease and short-lived protection in small trials. collectively, outreach programs to educate communities in endemic areas to recognize early stage of buruli ulcer is extremely crucial for prevention of severe forms of the disease. although there are no vaccine and effective protective strategies, antimicrobial therapy has showcased effective treatment of the disease and lowered the recurrence rate. given that single-drug treatment led to relapse of mycobacterial disease due to the emergence of drugresistant mycobacterial strains, multi-drug treatment regimens have been employed for mycobacterial infections. it is a common phenomenon that drug-resistant mutants repopulate the lesions following monotherapy, especially in both tuberculosis[61] and leprosy[62]. therefore, a second companion drug should be combined with the highly active core antimicrobial agent to prevent treatment failure and relapse. in 2004, who recommended a combination antibiotics alone for small early lesion or as an adjunct to surgical resection for large lesions[63]. a randomised controlled trial reported similar efficacy between the use of either rifampicin and streptomycin (8 weeks) or rifampicin and streptomycin (4 weeks) followed by rifampicin and clarithromycin (4 weeks) which resulted in high recovery rates of exceeding 90% for patients inflicted with early (<6 months) and small lesion (<10 cm)[64]. recently, a fully oral rifampicin and an extended release formulation of clarithromycin has shown comparable effectiveness for treatment of early and limited buruli ulcer[65]. according to center of disease control and prevention, the public water facilities such as swimming pools, spas and hot tub are advised to maintain adequate concentrations of free chlorine, ranging between 0.4 to 1 mg/liter in swimming pool and 2 to 5 mg/liter for spas and hot tub[66,67]. frequent sanitation and disinfection, and removal of infected fishes are the main control strategies of m. marinum infection in fishes. the maintenance personnel for the aquarium should use waterproof gloves to prevent any potential upper limb skin lesions exposure to the pathogen during fish tankrelated activities. proper training is also essential for high-risk populations, such as fishermen and marine-life handlers, to identify signs of m. marinum in fish or human in order to facilitate more prompt treatment[68]. to date, there is no clinical trial available which could suggest optimal management of m. marinum infections. furthermore, there is no standardized treatment for cutaneous infections due to m. marinum, the therapeutic choice is mainly based on the severity of the infection and the immunocompetency of the patient[9]. principally, rapid recovery from m. marinum in man requires the proper treatment and prevent further progression to deeper tissues. based on retrospective case studies, single agent antibiotic therapy was shown to successfully treat majority of the limited cutaneous m. marinum infections. these single antibiotic agents include minocycline, doxycycline, cotrimoxazole-trimethoprim and clarithromycin have demonstrated positive outcomes in the treatment of m. marinum infections[9,69]. besides that, the combination use of 2 active agents including ethambutol, clarithromycin/ azithromycin or rifampicin for 3 to 4 months has been reported to be effective adjunct therapy together with surgical debridement for invasive m. marinum infections[70]. other antimicrobials used for treatment of m. marinum infection include ciprofloxacin, moxifloxacin, isoniazid and protionamide[71]. nevertheless, there were reported cases that yielded negative therapeutic outcomes[72,73]. several reports also described the worsening of m. marinum infection in patients receiving anti-tnf-α therapy[74,75]. thus, it should be recommended to halt the use of tnf-α inhibitor or other immunosuppressive therapy in m. marinum infected patients who are under the course of antibiotics. surgical debridement remains a controversial therapy option for m. marinum infection and it should be limited to cases that fulfil certain criteria, including cases that associated with poor prognosis involving deep lesions, persistent drainage of sinus and chronic pain[76]. there are also other therapeutic modalities such as local hyperthermic therapy, photodynamic therapy, electrodessication, cryotherapy and x-ray therapy have been recommended to treat m. marinum infection[9]. bacteriophage therapy represents an interesting strategy to be developed for the management of m. marinum infection despite only phage therapy using mycobacteriophage d29 for treatment of buruli ulcer is available at the moment[77]. conclusion research on both m. ulcerans and m. marinum is vital for much needed advancement in the prevention and management of buruli ulcer and fish tank granuloma, which are both challenging diseases that have been largely neglected. a clearer view of the exact innate and adaptive immune mechanisms leading to protection from m. ulcerans and m. marinum infections will greatly propel the development of new strategies for effective vaccine design. moreover, future research focusing on clinical applications and epidemiology is essential to advance our knowledge of mycobacterial pathogens that cause cutaneous infection and improve our capability to control and treat these infections with optimal medical interventions. authors contributions the literature review and manuscript writing were performed by lt-ht and jw-fl. pr and lc-m provided vital insight and performed proof-reading. the research project is conceptualized by lt-ht and jw-fl. conflict of interest the authors declare that there is no conflict of interest in this work. tan lth et al. 6 mycobacterium ulcerans... reference 1. sethuraman g, dev t, and ramesh v. mycobacterial skin infections. harper’s textbook of pediatric dermatology 2019; 485–502. 2. esteban j and garcía-coca m. mycobacterium biofilms. front microbiol 2018; 8: 2651. 3. lyman mm, grigg c, kinsey cb, et 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bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2ukm medical molecular biology institute (umbi), ukm medical centre, universiti kebangsaan malaysia, kuala lumpur, malaysia abstract: using seven complete genomes of human sars-cov-2 (retrieved from gisaid) isolated in malaysia for phylogenetic tree construction, the current study showed that these strains formed four distinct clades when compared with other representative strains from asia, europe and us. in light of that, the genome sequences of these strains isolated in malaysia suggested that there is currently more than one “type””of strain within the country. complementing with epidemioogical and experimental studies, these findings allow better understanding the prevalence of certain types in malaysia and permits further in-depth studies on the virulence and pathogenic mechanisms of these strains which is particularly critical to speed up the development of effective treatment regime. keywords: sars-cov-2; malaysia; genome; phylogenetic analysis; snvs; mutations received: 4th may 2020 accepted: 4th june 2020 published online: 18th june 2020 †these authors contributed equally in the writing. citation: ser h-l, tan lt-h, law jw-f, et al. genomic analysis of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) strains isolated in malaysia. prog microbes mol biol 2020; 3(1): a0000093. https://doi.org/10.3687/pmmb. a0000093. introduction taking a closer look at coronavirus, this notorious bug has caused several global outbreaks throughout human history, with notable instances such as 1918 (h1n1) influenza pandemic, and more recently middle east respiratory syndrome (mers) caused by mers-cov in 2012. fast forward to the last month of 2019, the who china country office was informed of pneumonia cases with unknown etiology, which later on determined a new type of coronavirus known as sars-cov-2[1]. as of 30th april 2020, the world health organization reported that there are more than 3 million confirmed covid-19 cases globally and has impacted more than 210,000 covidrelated deaths[2–6]. at the time of writing, malaysia’s government announced to ease the “partial lockdown” of more than six weeks, allowing almost all economic sectors to reopen, in parallel with the decreasing trend of covid-19 confirmed cases during the end month of april 2020. in the south east asia region, malaysia was among the first few countries to implement the movement control order (mco) to curb the spread of the coronavirus on 18th march 2020 (figure 1). the first case of covid-19 in malaysia was detected on 24th january 2020[7]. the first case involved a chinese national from wuhan, who had travelled from singapore to johor bahru in a group of eight for holiday on 22nd january 2020. they were quarantined at a hotel on the following days after coming into contact with an infected patient in singapore. since then, there were around 22 positive covid-19 cases as the first wave of outbreak cases in the country, of which all the patients were discharged upon recovery[8]. however, a gradual increase in positive cases begun on 27th february before a sudden surge was observed on 15th march 2020 reaching as high as 428 cases after 11 days of zero reported case (i.e. from 16th to 26th february 2020). these cases were regarded as the second wave of outbreak and attributed to a religious gathering event which was attended by more than ten-thousands of people[8,9]. as a consequence, the shift and firm response from the government into the mco implementation has been critical to limiting the spread of covid-19. copyright © 2020 by ser h-l and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: hooi-leng ser, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; ser. hooileng@monash.edu. learn-han lee, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; lee. learn.han@monash.edu. 2 to gain further understanding on the molecular epidemiology of the covid-19 outbreak in malaysia, we used seven publicly available whole genome sequences sampled up 14th april 2020 to analyze the phylogenetic evolution of the sars-cov-2 strains isolated from the patients in malaysia with worldwide sars-cov-2 genome sequences. comparisons of the single nucleotide variants (snv) are often used for evolutionary studies as the virus genome is subjected to frequent mutations due to an error-prone rna-dependent rna polymerase responsible for the virus genome replication. these mutations might be associated with the changes in transmissibility and virulence of the virus. thus, we also performed the snv analysis to investigate the genotype changes during the transmission of sars-cov-2 in the country. sars-cov-2 in malaysia figure 1. the daily confirmed covid-19 cases and deaths reported in malaysia up to 28th april 2020 based on statistics obtained from ministry of health malaysia. the stacked bar graph shows the daily confirmed cases and deaths since the first case reported in malaysia while the line graph shows the cumulative confirmed cases. a heat map shows the distribution of confirmed cases from different states in malaysia. materials and methods phylogenetic and snv analysis using complete genome of sars-cov-2 complete genome sequences of sars-cov-2 were retrieved from gisaid (https://www.gisaid.org) and ncbi database[10,11]. nc_045512 genome sequence fetched from ncbi was used for reference and genomic coordinates in this study are based on this reference genome. the alignment of sequences were formed via clustalx software, verified manually and adjusted prior to the reconstruction of phylogenetic trees[12]. phylogenetic trees were constructed with the maximum-likelihood algorithm (figure 2) using molecular evolutionary genetics analysis across computing platforms (mega) version 7.0[13–15]. support for the tree topology was estimated with 1,000 bootstrap replicates. snv composition of the selected strains based on the phylogenetic tree was analyzed and compared with the reference sequence nc_045512 using bioedit and 2019 novel coronavirus resource (2019ncovr, https:// bigd.big.ac.cn/ncov)[16]. data availability statement all data are available in the main text and the supplementary materials. the genome sequences used in the current study is retrieved from gisaid (https:// www.gisaid.org/) and ncbi databases (https://www. ncbi.nlm.nih.gov/nuccore/nc_045512). results phylogenetic analysis using maximum likelihood algorithm in order to observe the genomic relationship between sars-cov-2 strains isolated in malaysia, a dataset of 7 publicly available complete genomes of sars-cov-2 from different countries was retrieved from gisaid (https://www.gisaid.org, accessed on 14th april 2020; supp table 1)[10,11]. a phylogenetic tree which was constructed using the maximum-likelihood algorithm revealed that the seven strains isolated in malaysia formed four major clades (figure 2). majority of the strains (n = 3) showed that they were closely related, forming clade iii with the closest related strain isolated from singapore (gisaid accession id: epi_isl_407987). on the other hand, another strain isolated from a 45-year old male patient in clade i (gisaid accession id: epi_isl_416886) displayed a close evolutionary relationship with strain from us (gisaid accession id: epi_isl_404895) and china (fujian, gisaid accession id: epi_isl_411060). the fourth clade (i.e. clade iv) which consist of strain isolated from a 67-year old female patient (gisaid accession id: epi_isl_416907) was closely related to another strains from asia region including china (gisaid accession id: epi_isl_408486, epi_isl_403930), japan (gisaid accession id: epi_isl_410532, epi_isl_413459, epi_ isl_412969) and singapore (gisaid accession id: epi_isl_406973). in addition, the other two strains epi_ isl_417917 and epi_isl_417418 which were isolated later in late march formed a separate clade (clade ii). 3 ser h-l et al. figure 2. phylogenetic analysis of forty-five sars-cov-2 complete genome sequences (-29880 nucleotides) showing relationship between seven malaysian strains with representatives’ complete sequences from different countries. it is a maximum-likelihood tree with bootstrap values (>50%) based on 1000 re-sampled datasets are shown at branch nodes. envelope (e) protein, matrix (m) protein, and nucleocapsid (n) protein[17,18]. along with these, there are a total of six accessory proteins coded by orf3a, orf6a, orf7a, orf7b, orf8 and orf10 genes located across the ~29kb genome of sars-cov-2. in the current analysis, a total of seven strains isolated in malaysia was used to perform in the comparison along with representative strains from asia, us and europe (note: all sequences of this study were retrieved from ncbi or gisaid database). most of these strains were closely related with strains isolated from asia region based on figure 2. however, some strains displayed more snvs than the rest of the strains isolated in malaysia (figure 3). for instance, epi_isl_417917 and epi_isl_417918 formed clade ii; analyses on snvs and amino acid variations reflected that these strains have more than 90% common nucleotide/ amino acid in several genes. for these strains, most of the variations occurred within the orf1ab genes including 6310c>a, 6312c>a, 11083g>t, 13730c>t, 19524c>t. these snvs then corresponded for four missense variants and one synonymous variant for polyproteins 1a/1ab. in addition, there are two more variations occurring only in epi_isl_417917 (but not epi_isl_417918) at position 2737t>a (synonymous variant) and 13975g>a (missense variant). in addition to that, there is a snv observed in gene n at 28311c>t in both strains which encodes for nucleocapsid protein (structural protein) that wraps the genomic rna (grna) into a helical structure. one of the important points to note is that these two strains were obtained from patients in march 2020. as suggested by previous literature, rna viruses like coronavirus can have high mutation rates and more often than not, these mutations are correlated with virulence modulation and single nucleotide variants (snvs) analysis the genome-wide snvs for selected strains are as reported in table 1 using a strain isolated from china in december 2019 (ncbi accession number: nc_045512) as reference strain. a total of 30 snvs was identified. epi_isl_417917 showed the highest number of snvs, with 9 positions showing variance compared to the reference strain. furthermore, one of the closely related strain isolated in malaysia, epi_isl_417418 (based on phylogenetic analysis) revealed 8 snvs; this strain shared 7 common snvs with epi_isl_417917 with addition to a snvs at 25473 nt (compared to nc_045512) where gene m is located. additionally, all members of clade iii showed 27147g>c and one missense variation in gene m which encodes for matrix protein. among the seven strains isolated in malaysia, epi_isl_416866 displayed the lowest number of snvs only one at 27147 nt (gene m). similar pattern was observed with amino acid variations as shown in table 2. discussion the availability and data-sharing of sars-cov-2 whole genome sequences allow researchers to study the evolutionary relationships and patterns of molecular divergence between coronaviruses around the globe. in fact, nearly two-thirds of the viral genome falls within the first orf (orf1a/b) which translate to (nonstructural) two polyproteins (pp1a and pp1ab), while the remaining genome encodes four essential structural proteins, including spike (s) glycoprotein, small 4 evolvability, thereby improving their adaptation ability[19– 21]. five of the seven strains, epi_isl_416886, epi_ isl_416866, epi_isl_416884, epi_isl_416829, epi_ isl_416907 (clade i, iii and iv) were isolated in late february in malaysia. interestingly, epi_isl_416886 reflected several snvs within two well-studied genomic regions including orf1ab (6025t>c, 8782c>t and 18060c>t) and orf8 (28144t>c). as a matter of fact, epi_isl_416886 is the only unique strain isolated from malaysia that exhibited co-variations at these two locations (i.e. 8782c>t and 28144t>c). as it has been noted that most of 8782c>t and 28144t>c variant sub-strains are found outside of wuhan, one of the two closely related strains of epi_isl_416886 was identified to be strain from fujian (epi_isl_411060) which also carried snvs at these two locations. a simple pairwise comparison showed that epi_isl_416886 exhibited ~99.7 % similarity to closely related strains epi_isl_411060 and epi_isl_404895 which isolated in us. there have been ongoing discussions on the topic of characterizing sars-cov-2 strains based on variation at 8782 (c or t) and 28144 (t or c), either (a) to trace the routes of infections by observing their network nodes, or (b) to study the virulence of strains (i.e. s or l subtypes) [22,23]. having said that, the actual function of orf8 in sars-cov-2 is yet to be discovered given that it lacks a known useful motif or region and seems to be highly divergent from orf8b in sars-cov which induce intracellular stress pathways[17,24,25]. in addition to this, epi_isl_416886 carried a snv at position 18060 nucleotide belonging to orf1ab (which encodes for nsp14). even though pachetti et al.[20] reported that this snv was mostly related to sars-cov-2 strain isolated from north america, further investigation into the travel history of the patient whom epi_isl_416886 was isolated from may be worthwhile to obtain a clearer understanding on how the strain acquired these snvs, whether it’s a spontaneous mutation and/or how fast before these snvs emerged. still and all, the changes at this position (i.e. 18060) from c>t resulted in a synonymous variant in amino acid sequence, thus the actual effect of this substitution is still pending to be discovered. s glycoprotein encoded by gene s is one of the crucial factors in determining host range and pathogenicity of sars-cov-2. a study in early february by zhou et al.[26] has confirmed that sars-cov-2 shares similarity in the target receptor for cellular entry, recognized as angiotensin converting enzyme ii (ace2) receptors. in other words, the s glycoprotein of sars-cov-2 can attach to ace2 receptors available on several types of human cells before hijacking the host machinery[26–30]. from table 1, a total of 9 snvs was found to be associated with s glycoprotein (or gene s). despite of that, three strains (epi_isl_416884, epi_isl_417917 and epi_isl_417918) isolated in malaysia carried one or more snvs within this gene. epi_ isl_416884 (clade iii) displayed highest number of snvs within gene s with 7 snvs, and subsequently resulted in 1 synonymous and 5 missense (amino acid) variants in the s glycoprotein, while epi_isl_417917 and epi_isl_417918 only reflected one snv (i.e. 23929c>t) without changes in the amino acid composition (i.e. synonymous variant). at the time of writing, these 7 snvs found in epi_isl_416884 (21767c>g, 21772c>t, 21773t>a, 21776g>t, 21779a>t, 21780c>t, 21782a>c) seems to be novel” and the true effect of these changes are still unknown. taking note that these snvs are mostly located near the n-terminal domain of the s1 subunit of s protein which is not directly involved with binding to ace2, hence the variations may not be influencing/altering the binding ability of the virus[31]. on the flip side, it is quite conservative or safe to mention that the actual functional impact of figure 3. single nucleotide variants detected from the seven malaysia strains of sars-cov-2 in comparison to the reference strain (nc_045512). sars-cov-2 in malaysia 5 ser h-l et al. n t g en e re f cl ad e i cl ad e ii cl ad e iii cl ad e iv pr op or ti on o f st ra in s w it h sn vs n c _0 45 51 2 ep i_ is l_ 41 68 86 ep i_ is l_ 40 48 95 ep i_ is l_ 41 10 60 ep i_ is l_ 41 79 17 ep i_ is l_ 41 79 18 ep i_ is l_ 41 68 84 ep i_ is l_ 41 68 29 ep i_ is l_ 41 68 66 ep i_ is l_ 40 79 87 ep i_ is l_ 41 05 32 ep i_ is l_ 40 39 30 ep i_ is l_ 41 69 07 ep i_ is l_ 40 84 86 ep i_ is l_ 40 69 73 ep i_ is l_ 41 34 59 ep i_ is l_ 41 29 69 39 8 o r f1 ab c c c c c c c t c c c c c c c c c 1/ 16 17 58 c c c c c c c c c c c c t c c c c 1/ 16 27 37 t t t t a t t t t t t t t t t t t 1/ 16 60 25 t c t t t t t t t t t t t t t t t 1/ 16 63 10 c c c c a a c c c c c c c c c c c 2/ 16 63 12 c c c c a a c c c c c c c c c c c 2/ 16 69 96 t t t t t t t t t t t c t t t t t 1/ 16 82 74 g g g g g g g g g g g g g a g g g 1/ 16 87 82 c t t t c c c c c c c c c c c c c 3/ 16 10 60 4 c c c c c c c c c c c c t c c c c 1/ 16 11 08 3 g g g g t t g g g g g g g g g t t 4/ 16 13 73 0 c c c c t t c c c c c c c c c c c 2/ 16 13 97 5 g g g g a g g g g g g g g g g g g 1/ 16 18 06 0 c t t t c c c c c c c c c c c c c 3/ 16 19 52 4 c c c c t t c c c c c c c c c c c 2/ 16 21 76 7 s c c c c c c g c c c c c c c c c c 1/ 16 21 77 2 c c c c c c t c c c c c c c c c c 1/ 16 21 77 3 t t t t t t a t t t t t t t t t t 1/ 16 21 77 6 g g g g g g t g g g g g g g g g g 1/ 16 21 77 9 a a a a a a t a a a a a a a a a a 1/ 16 21 78 0 c c c c c c t c c c c c c c c c c 1/ 16 21 78 2 a a a a a a c a a a a a a a a a a 1/ 16 23 92 9 c c c c t t c c c c c c c c c c c 2/ 16 25 06 0 a a a a a a a a a a a a a a g a a 1/ 16 25 47 3 o r f3 a t t t t t c t t t t t t t t t t t 1/ 16 27 13 1 m c c c c c c c t c c c c c c c c c 1/ 16 27 14 7 g g g g g g c c c c g g g g g g g 4/ 16 28 14 4 o r f8 t c c c t t t t t t t t t t t t t 3/ 16 28 31 1 n c c c c t t c c c c c c c c c c c 2/ 16 29 63 5 o r f1 0 c c c c c c c c c c c c c c c t t 2/ 16 pr op or ti on o f s n vs 4/ 30 3/ 30 3/ 30 9/ 30 8/ 30 8/ 30 3/ 30 1/ 30 1/ 30 0/ 30 1/ 30 2/ 30 1/ 30 1/ 30 2/ 30 2/ 30 ta bl e 1: si ng le n uc le ot id e va ri at io ns (s n v s) d ed uc ed b y co m pa ri so n of c om pl et e w ho le g en om e se qu en ce s of s a r sc ov -2 is ol at ed in m al ay si a an d se le ct ed c lo se ly -r el at ed s eq ue nc es (n = 1 6 se qu en ce s) u si ng n c _0 45 51 2 as re fe re nc e ge no m e (b lu e: v ar ia nt ). 6 a a g en e r ef c la de i c la de i i c la de i ii c la de i v pr op or ti on o f st ra in s w it h va ri an ts n c _0 44 51 2 e pi _ is l _4 16 88 6 e pi _ is l _4 04 89 5 e pi _ is l _4 11 06 0 e pi _ is l _4 17 91 7 e pi _ is l _4 17 91 8 e pi _ is l _4 16 88 4 e pi _ is l _4 16 82 9 e pi _ is l _4 16 86 6 e pi _ is l _4 07 98 7 e pi _ is l _4 10 53 2 e pi _ is l _4 03 93 0 e pi _ is l _4 16 90 7 e pi _ is l _4 08 48 6 e pi _ is l _4 06 97 3 e pi _ is l _4 13 45 9 e pi _ is l _4 12 96 9 45 o r f1 ab h h h h h h h y h h h h h h h h h 1/ 16 49 8 a a a a a a a a a a a a v a a a a 1/ 16 82 4 t t t t t t t t t t t t t t t t t 1/ 16 19 20 y y y y y y y y y y y y y y y y y 1/ 16 20 15 s s s s r r s s s s s s s s s s s 2/ 16 20 16 t t t t k k t t t t t t t t t t t 2/ 16 22 44 i i i i i i i i i i i t i i i i i 1/ 16 26 70 c c c c c c c c c c c c c y c c c 1/ 16 28 39 s s s s s s s s s s s s s s s s s 3/ 16 34 47 p p p p p p p p p p p p s p p p p 1/ 16 36 06 l l l l f f l l l l l l l l l f f 4/ 16 44 89 a a a a v v a a a a a a a a a a a 2/ 16 45 71 g g g g s g g g g g g g g g g g g 1/ 16 59 32 l l l l l l l l l l l l l l l l l 3/ 16 64 20 l l l l l l l l l l l l l l l l l 2/ 16 69 s h h h h h h d h h h h h h h h h h 1/ 16 70 v v v v v v v v v v v v v v v v v 1/ 16 71 s s s s s s t s s s s s s s s s s 1/ 16 72 g g g g g g w g g g g g g g g g g 1/ 16 73 t t t t t t s/ i t t t t t t t t t t 1/ 16 74 n n n n n n h n n n n n n n n n n 1/ 16 78 9 y y y y y y y y y y y y y y y y y 2/ 16 11 66 l l l l l l l l l l l l l l l l l 1/ 16 27 o r f3 a d d d d d d d d d d d d d d d d d 1/ 16 20 3 m n n n n n n n n n n n n n n n n n 1/ 16 20 9 d d d d d d h h h h d d d d d d d 4/ 16 84 o r f8 l s s s l l l l l l l l l l l l l 3/ 16 13 n p p p p l l p p p p p p p p p p p 2/ 16 26 o r f1 0 y y y y y y y y y y y y y y y y y 2/ 16 p ro po rt io n of va ri an ts 0/ 29 4/ 29 3/ 29 3/ 29 9/ 29 8/ 29 7/ 29 3/ 29 1/ 29 3/ 29 0/ 29 1/ 29 2/ 29 1/ 29 1/ 29 2/ 29 2/ 29 ta bl e 2: a m in o ac id v ar ia tio ns d ed uc ed b y co m pa ri so n of c om pl et e w ho le g en om e se qu en ce s of s a r sc ov -2 is ol at ed in m al ay si a an d se le ct ed c lo se ly -r el at ed s eq ue nc es (t ot al n = 1 6 se qu en ce s) u si ng n c _0 45 51 2 as re fe re nc e st ra in (b lu e: m is se ns e; y el lo w : s yn on ym ou s) . sars-cov-2 in malaysia 7 these variants remain unclear and further investigations are neccessary to assess its importance, particularly for the development of vaccines[25,32]. above all, these findings of this study were consistent with reports from other research teams, with most of the strains showing high genomic similarities between strains derived from the asia region. in the current study, regardless of sampling time (february or march), all the strains isolated in malaysia reflected highly similar “pattern” of snvs when compared to the other strains derived from asia including china (epi_isl_411060, epi_isl_403930 and epi_isl_408486), singapore (epi_isl_407987 and epi_isl_406973), and japan (epi_isl_410532, epi_isl_413459 and epi_isl_412969). when compared within the seven strains isolated in malaysia, members of clade ii epi_isl_417917 and epi_isl_417918 were shown to be more closely related to each other (<0.15% difference by pairwise comparison) compared to the other five strains (supp table 2). a recently published study by forster et al.[22] reconstructing evolutionary paths of sarscov-2 using 160 complete human sars-cov-2 and found three central variants distinguished by amino acid changes which was named a, b, and c, with a being the ancestral type according to the bat outgroup coronavirus. based on this finding, two members of clade i (epi_isl_404895 and epi_isl_411060) in this study were determined to be a type, while most of the members of clade iii and iv were determined as derived-b type which as suggested by its name derived from a type. forster et al.[22] mentioned that sarscov-2 would to first mutate into derived b type (from a type), before finally turning into b type which is the most common type in east asia. altogether, these findings may indicate that there are currently more than one subtype present in malaysia. nonetheless, there is still much more to be explored, particularly on the transmittance of sars-cov-2: how these variations/mutations emerged over time in malaysia, and then how these changes caused to the behavior of the virus (e.g. on their pathogenicity and virulence). given the recent emergence of sars-cov-2 around the world, researchers have witnessed the growing number of genome sequences deposited in publicly available database like gisaid and ncbi. the policy on data sharing can potentially hasten the identification of covid-19 infection sources while expediting the drug designing process and vaccine(s) development based on the availability of these complete viral genomes derived from different parts of the world[33]. all and all, by garnering a more thorough perspective on covid-19 infection sources, the current study could serve as the launching pad to inspect and understand the dynamics of the local transmission of sars-cov-2 in malaysia. complementing with epidemiological studies, these findings could essentially gather valuable information on the prevalence of certain strains in malaysia, whereas in-depth experimental research would then shed some light on the virulence and pathogenic mechanisms of these strains. conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. author contributions the experiment and data analysis were performed by h-ls, lt-ht, l-hl, the manuscript was written and proof by h-ls, lt-ht, jw-fl, vl, n-sam and l-hl. the project was founded by h-ls and l-hl. acknowledgments we sincerely appreciate the researchers worldwide who sequenced and shared the complete genome data of sars-cov-2 from gisaid (https://www.gisaid.org) and ncbi databases (https://www.ncbi.nlm.nih.gov/ nuccore/nc_045512). this research is dependent on these precious data. references 1. world health organization. who director-general’s opening remarks at the media briefing on covid-19 13 april 2020. 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hallmarks; dirty drug; natural product; metabolites received: 8th june 2020 accepted: 8th july 2020 published online: 18th july 2020 citation: tan lks, how cw, foo jb, et al. resveratrol as a potential broad-spectrum compound for cancer treatment. prog microbes mol biol 2020; 3(1): a0000098. https://doi.org/10.3687/pmmb.a0000098. introduction cancer, the most commonly diagnosed non-communicable disease, has imparted significant mortality and morbidity worldwide[1]. in 2018, the world health organisation ranked cancers as the leading cause of premature death with 9.6 million cases reported worldwide[2]. despite the advancement in cancer therapy, the overall survival rate and quality of life of cancer patients have not been improved. the development of personalised drug on the other hand, is slow, costly and may not guarantee good clinical outcomes. particular attention has been devoted to explore the potential of pleiotropic, “broadspectrum” compounds or “dirty drugs” which could simultaneously target multiple mechanisms to overcome the aforementioned issues in achieving effective cancer treatment[3]. the ten cancer hallmarks proposed by hanahan and weinberg in 2011, has become a general a guide to evaluate the potential of specific compound as a promising “dirty drug”[4]. this means compounds that could disrupt any of the processes responsible for the cancer hallmarks would almost certainly hinder cancer progression. in an ideal circumstance, a compound that could disrupt multiple pathways not only would produce superior effect, it would also minimise the risk of side effects that is otherwise introduced by multiple drug administration. natural products have been a rich and excellent source to search for multi-target bioactive compounds with improved therapeutic efficacy and safety[5,6]. these multi-target bioactive compounds are derived from natural sources such as plants[7,8], microorganisms[9–16] and animals[17]. in a work published by block et al. (2015)[3], they found that most of the compounds that targets all cancer hallmarks were the phytochemicals. phytochemicals are naturally produced as a result of evolution against pathogens with evident role playing in human health. these natural compounds have attracted much attention from community for their promising effects in treating diseases[18–22]. stilbenes are one of the examples of secondary metabolites that was produced in stressful condition to fight against fungal infection and uv radiation[23]. it is one of the nonflavonoids which consists of two aromatic rings linked by an ethylene copyright @ 2020 by tan lks and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: yong sze ong, school of pharmacy, monash university malaysia, 47500, bandar sunway, selangor darul ehsan, malaysia; ong.yong.sze@monash.edu. 2 or ethane bridge (c6-c2-c6 carbon skeleton) and usually found in plants. among the 400 natural stilbenes discovered, rsv is the most widely investigated compound due to its vast pharmacological activities[24]. resveratrol resveratrol (3,4’,5-trihydroxy-trans-stillbene) (rsv) (figure 1) can be found in the skin of grapes, red wines, peanuts, pineapple and mulberries. this compound could be synthesised from phenylalanine pathway through several enzyme reactions into para-coumaroyl-coa, which is condensed with malonyl coa to form rsv[25]. since the discovery of rsv by siemann and creasy in red wine in 1992[26], extensive studies have proven that rsv exhibits its biological functions such as antimicrobial, cardiovascular disease, anticancer, anti-inflammatory, antidiabetic and neurodegenerative diseases[27,28]. it is believed that most of the pharmacological activities are attributed to its anti-oxidant mechanisms involving the competition with coenzyme q, free radical scavenging and inhibition of lipid peroxidase in the mitochondria[29]. rsv has demonstrated anti-cancer properties in both in vitro and in vivo settings. the type of cancers tested are ovarian[30], lung[31], colon[32], prostate[33], breast[34] and cervical cancers[35]. as a pleiotropic compound which acts on different mechanisms, it also showed to play a possible role to counteract multidrug resistance[36]. as of june 2020, the keywords of “resveratrol and cancer” brings up 3660 results in pubmed database. correspondingly, rsv possesses great commercial value as evidence by a surge in patent filed in the lens patent database (http://lens. org) related to the application of rsv in cancer therapy in recent decades (figure 2). modulation of cancer hallmarks the surge of publications and patents undeniable reflect the increased interest of the scientific community toward the therapeutic effect of rsv. in an attempt to investigate the potential of rsv as a “broad spectrum” chemotherapeutic agent, this review summarised the cancer hallmarks and the target molecules that have been claimed to be modulated by rsv in various cancer cell lines and animal models (figure 3). resveratrol as a potential... figure 2. number of patents filed with keywords “resveratrol” and “cancer” from 1997 to 2020. data was obtained from the lens patent search engine. figure 1. chemical structure of resveratrol. 3 tan lks et al. of pten, the tumour suppressor gene, which then led to the downregulation pi3k/akt signalling evidenced by the phosphorylation of akt1/2[32]. activation of growth suppressors on top of sustaining the proliferation, the cancerous cells are also overpowered the growth suppression pathway. there are two commonly known mechanisms of tumour suppressors, the retinoblastoma-associated (rb) and tp53 proteins. these proteins prevent the progression of cancerous cells growth through cell cycle arrest and cell death via induction of apoptosis. evidence suggests that rsv induces cell death through the activation of p53 pathway, which further triggered apoptosis via mitogen-activated protein (map) kinases in t98g glioblastoma cells[41]. the results were in congruent to with another study in which the p53-dependent apoptosis was activated due to the binding of rsv to integrin αvβ3, more specifically the β3 monomer[42]. research found that the abruption and suppression in the transforming growth factor β (tgf-β) and “contact inhibition” pathway which involve e-cadherin and epidermal growth factor (egf) receptors has led to the proliferation of cancerous cells[36]. zhong et al. (2015)[43] postulated that rsv suppressed the proliferation of ovar3 ovarian cancer prevention of proliferative signalling most cancer cells have upregulated proliferative signalling pathways in order to maintain their survival. the signalling pathways are often associated to the alteration in the expression of proteins related to cell cycle, metabolism and cell proliferation[37–39]. rsv promoted the growth of ovarian cancer cell (ovcar-3) by blocking pi3k and akt signalling pathways, through reduction of phosphorylated extracellular signal-regulated kinase (erk)1/2, downregulation of cyclin d1 and protein kinase b[40]. cancer cells have prevented the proliferation via activation of autophagy-suppressing pathway such as phosphatidylinositol 3-kinase (pi3k)/mammalian target of rapamycin (mtor)[30]. autophagy has become a target that may play an important role in cancer treatment. studies have shown that rsv could induce autophagy cell death. zhang and the research team proved that the treatment with rsv caused accumulation of calcium ions which then led to the activation of phosphorylated ampk and phosphorylated raptor via ca2+/ampk-mtor signalling pathway in the a549 human lung adenocarcinoma cells[31]. the anti-proliferative effect of rsv has been determined in xenograft animal model (hct116 colon cancer cells in athymic nude mice). the rsv induced the gene expression figure 3. cancer hallmarks and molecular targets modulated by resveratrol. 4 cells by upregulated the protein expression of e-cadherin which subsequently increased the β-catenin on the cell membrane, leading to the inhibition of proliferation. promotion of cancerous cell death normal cells maintain the homeostasis by programmed cell death through apoptosis. however, this normal cellular homeostasis is abrogated during tumorigenesis. cancerous cells modulate the regulatory and effector components in the cell via increase the anti-apoptotic proteins such as bcl-2 and downregulate the proapoptotic proteins such as bax, puma, bim and caspases. induction of apoptosis is considered as one of the strategies in cancer treatment due to the minimal inflammatory response[18]. convincing evidences showed that rsv induced apoptosis in various cancer cells such as t acute lymphoblastic leukaemia cell line[44], hct116 colorectal carcinoma cell[45], caco-2 colorectal cancer cells[46], tramp murine prostate cancer cells[33] and mcf-7 breast cancer cell[34] through upregulation of bax and downregulation of bcl-2 expression. some studies have suggested that the changes in the expression on both bax and bcl-2 were due to the disruption in the mitochondria membrane potential (δψm) after treatment with rsv[33]. apart from apoptosis, autophagy plays an important role in maintaining the cellular homeostasis by clearing up the aggregated proteins, damaged organelles or exogenous components via a lysosomal degradation process[30]. studies suggested that rsv could be a natural autophagy agent. luyten and the research team found out that rsv induced autophagy via mtor-dependent pathway with the presence of ip3rs and extracellular ca 2+ in hela human cervix carcinoma cells[35]. the results is in compliance with park et al. (2016)[47] in which they suggested that rsv inhibited mtor by directly docking onto the atp-biding site of mtor[47]. disabling replicative immortality telomeres have long been regarded as the guardian of genome stability. the telomere theory of aging and longevity of cells is well-recognised through its ability to elongate the dna[48]. notwithstanding, the association of telomeres with cancer has been suggested in several studies that consistently demonstrated that the tumour cells contains higher percentage of telomerase activity as compared to normal cells. these observations strengthened the coherency that explains the immortality of cancerous cells. the understanding of the mechanisms underlying telomerase activity and telomere structures have offered another option for cancer therapy[49]. wang et al. (2011)[50] reported that rsv delayed the senescence of endothelial progenitor cells isolated from human peripheral blood via the downregulation of telomerase activity. the study also suggested that the compound has regulated the expression of human telomerase reverse transcriptase (htert), the rate limiting component of telomerase activity, via the pi3k/akt pathway[50]. the results were in accordance to mirzazadeh et al. (2017)[49] in which they pointed out the role of rsv in regulating the replication immortality by inhibition of the htert gene expression in a dose-dependent manner in human glioblastoma cell line u-87mg[49]. inhibition of angiogenesis one of the vital components for cancer development and progression is the formation of blood vessels (angiogenesis) in the tumour area. cancerous cells regulate the pro-angiogenic factors such as vascular endothelial growth factor (vegf) to stimulate vascular endothelial cells to enter the tumour hypoxic areas and anti-angiogenic factors such as thrombospondin-1 (tsp1) to inhibit the effect of vegf[51]. studies showed that treatment with rsv has decreased the stabilisation of hypoxia-inducible factor1 (hif-1α) which in turn downregulated the vegf protein expression and upregulated the tsp1 protein expression in spheroid a375 melanoma cells[52]. another study by mikuła-pietrasik et al. (2012)[53] suggested that rsv suppressed the angiogenesis by downregulating il-8/ cxcl-8, the proangiogenic chemokine which regulated the expression of vegf, in human peritoneal mesothelial cells (huvec, hmvec and hmec-1)[53] . a combination of rsv with 5-fluorouracil (5fu) has enhanced the in vitro anti-angiogenic effect in b16 melanoma cells, as compared to treatment with either drug alone. the results demonstrated that the combined treatment has synergistically decreased the vegf protein expression. as a proof of concept, further study was performed with b16 tumour-bearing balb/c nude mice in the effort to assess the in vivo anti-angiogenic effect. after subcutaneous injection for 10 days, the combined treatment of rsv and 5fu has significantly decreased the vegf protein expression with reduced microvessel density as compared to the negative control group[54]. rsv has shown its antiangiogenic effect by inhibiting the tube formation and cell migration in primary human vascular endothelial cells (huvecs). the authors reported that the process was achieved by the suppression of pkg-1 kinase and four inhibitors of apoptosis proteins (c-iap1, c-iap2, livin and xiap)[55]. deactivation of invasion and metastasis epithelial-mesenchymal transition (emt) is a process where the epithelial cells lose the cell-cell contact by exhibiting mesenchymal phenotype. this process potentially worsen the prognosis in cancer patients by enhancing the invasiveness and migration of cancerous cells[56]. several studies have confirmed that rsv inhibited metastasis and invasion in various cancer cells through emt signalling pathway[57–59]. yuan and team proved that rsv reversed emt via regulation of akt/gsk3β/snail signalling pathway both in vitro colon cancer cells (sw480 and sw620) and in vivo lung metastasis animal model (sw480 tumor bearing nude mice)[58]. the effect was verified using proteomic experiments, indicating that the treatment with rsv has upregulated the expression of e-cadherin and downregulated the expression of n-cadherin, p-akt, p-gsk-3β and snail in both models[58]. another key regulator, the metastasisassociated protein 1 (mta1), is also known to correlate with the aggressiveness of the tumour metastasis. li et al. (2013)[60] has first demonstrated that rsv modulated the expression of mta1-mediated proteins such as ac-p53 in resveratrol as a potential... 5 pyruvate kinase m2 (pkm2), a catalyst in converting the phosphoenol-pyruvate to pyruvate[73], in cervical cancer hela, liver carcinoma hepg2 and breast cancer mcf-7 cell lines[74]. promotion of immune destruction in normal cells, immune system acts as a surveillance to protect our body from against foreign microorganisms. in events where infection occurs, the immune system will initiate a series of mechanism to eliminate the source of infection. principally, the immune cells could mount a response in eliminating the cancer cells which are recognised as “non-self”[75]. however, the cancerous cells gain ability to alter the immune system through several mechanisms such as modulating the expression of major histocompatibility class (mhc1) or immunosuppressive products[76](print. studies have suggested that rsv could modulate the molecular modulators of the inflammatory response in vitro and in vivo, via the activation of macrophage, t cells and natural killer[77]. rsv suppressed the proliferation of cancerous cells via the secretion of ifn-γ, il-4, il2, cd4+ in lymphocytes and elevated the secretion il10 via downregulation of the cd80 on macrophages[78]. the compound was proven to enhance the expression of fcγriib, a receptor for igg that blocks the activation of b cells in mice[79]. as a result of the regulation of immune system, lee-chang et al. (2013)[80] showed that rsv could inhibit the lung metastasis in 4t1-tumor bearing balb/c mice via inhibition of phosphorylation of stat3 and hence inactivation of tbregs[80]. moving towards clinical trials in order to realise the clinical translation for cancer therapy, the toxicity of rsv have been investigated to gauge its safety and effectiveness in both the animals and human. a commercialised rsv (resvida) was found to have noobserved-adverse effect levels (noaels) of 750 mg/kg/ bw in rats via oral administration, which then converted to human equivalent dose of 450 mg/kg[81]. at higher dose of 3,000 mg/kg, oral administration of rsv does seem to caused renal and bladder toxicity after a daily administration for one month in rats[82]. in human, the oral consumption of 5 g of srt501, a proprietary micronised formulation of rsv by glaxosmithkline (gsk) for 14 days are well-tolerated in colorectal cancer patients[83]. however, the toxicity seems to be largely affected by the pre-existing conditions of patients. for instance, one clinical trial that recruited subjects with multiple myeloma was prematurely terminated due to high incidence of renal toxicity after given a dose of rsv similar to srt501. the patients had experienced an elevated serum creatinine level with more than 500 µmol/l with minority developed crystal nephropathy and acute tubular damage[84]. the toxic effect is believed to happen specifically to multiple myeloma patients which had developed pre-existing renal damage prior to the treatment. a number of clinical trials have been conducted in recent decades to investigate the anti-cancer effect of rsv. table 1 summarizes the clinical trials involved the use of resveratrol as a single therapeutic agent or combination therapy in cancer treatment: prostate cancer xenografts animal model, consolidating the ability of rsv in deactivation of metastasis. epigenetic modification epigenetic modification such as dna methylation and histone modification are the important key regulators in affecting tumorigenesis[61]. a number of research has been done to investigate the interaction of rsv with epigenetic targets such as dna methyltransferase (dnmt), histone deacetylase (hdac) and lysine-specific demethylase-1 (lsd-1) in regulating the histone proteins and dna molecules[62]. rsv showed its ability to reverse carcinogenesis by significantly reducing the dnmt mrna expression (dnmt1, dnmt3a and dnmt3b) in human breast cancer cell lines (mcf-7 and mdamb-231), evidenced by mirza et al. (2013)[63]. similarly, in combination of another plant-based pterostilbene, rsv has significantly decreased the activation of dnmt enzyme via restoration of oestrogen receptor-α (erα) expression in erα-negative breast cancer mda-mb-157 cell line[64]. ameliorating the tumour-promoting inflammation studies showed that 25% of the cancer occurred due to the chronic inflammation as a result of simultaneous destruction and healing of body tissues. tumour-associated inflammation is always linked with poor prognosis[65–67]. signal transducer and activators of transcription (stat) and nf-κb signalling pathways are the two promising targets for cancer therapy as both collaboratively play role in inflammatory response via inducing the expression of pro-tumorigenenic genes such as il-1β, cox-2 and cyclin d1. many studies have been focusing on the effect of polyphenols including rsv in tumour-associated inflammation. rsv has shown its efficacy by suppressing both stat3 and stat5 phosphorylation in 786-o renal cell carcinoma cells as evidenced by kim et al. (2016) [68]. it also co-regulated both stat3 and nf-κb pathways in medulloblastoma uw228-2 and uw228-3 cell lines, which subsequently lead to the up-regulation of bcl-2 protein expression[69]. interestingly, rsv also regulated jak-stat pathway in colon cancer ht-29 cell line with significantly reduced expression level of nitric oxide, prostaglandin e2, inducible nitric oxide synthase (inos) and cyclooxygenase-2 (cox-2), consolidating the effect of rsv in inflammatory process in various cancers. programming energy metabolism warburg effect was observed in cancer where the cells altered energy metabolisms, including increased glycolysis and lactate production, to cope with their growth requirements. therefore, the proteins that are involved in the metabolism become the key elements in tumour progression and therapeutic targets for cancer treatment[70–72]. saunier et al. (2017)[70] proved that rsv managed to reverse warburg effect via activation of pyruvate dehydrogenase in colon cancer caco2 cells. interestingly, the reversal effect could be observed even with low doses of rsv that mimicked the drug concentration in human patients[70]. another study proposed that rsv modulated the warburg effect via mtor signalling pathway by down-regulation of tan lks et al. 6 type of cancer phase and status treatment outcome ref. colon phase i (completed) oral administration of 20, 80, 160 mg/day of rsv two weeks treatment significant inhibition of the gene expression of wnt target in colonic mucosa [85] colon and rectal phase i (completed) treatment is given after surgical resection oral administration of rsv for 9 days continuously (dose is not mentioned) outcome is not provided multiple myeloma phase ii (terminated) oral administration of 5.0 g srt501 (rsv) for 20 days the trial was terminated due to reported renal toxicity [84] lymphangioleiomyomatosis phase ii (recruiting) escalating doses of rsv from 250 mg daily (for 8 weeks, 500 mg daily (8 weeks) and 500 mg twice a day (for 8 weeks) (route of administration is not reported) outcome is not yet reported polycystic ovary syndrome phase 4 (recruiting) a combination treatment of 500 mg of rsv and 20 mg simvastatin daily (route of administration is not reported) outcome is not yet reported unspecific solid tumour phase i (completed) oral administration of 0.5, 1.0, 2.5, or 5.0 g rsv daily for 29 days a decrease in level of igf-i and igfbp-3, suggesting rsv exhibits the chemopreventive property [86] table 1. clinical trials of resveratrol in cancer treatment, data retrieved from https://clinicaltrials.gov/. conclusion currently, the search and development of a more effective cancer therapeutic regimen with low toxicity and “broad spectrum” that could simultaneously target several mechanisms has been encouraged in response to the intolerable conventional chemotherapy. the cancer hallmarks have been adopted to provide a clear insight in understanding the potential of specific compounds as “dirty drugs” on the molecular basis. as can be seen, rsv was found to be interfering with all the cancer hallmarks and modulating the key signalling pathways in tumour development, hence suggesting that the compound hold a great promise for future anticancer drug development. on top of that, several studies have found that rsv exhibit synergistic anticancer effect when combined with other therapeutic agents. even though the preclinical studies showed promising results, the compound has been disappointing with its toxicity and poor pharmacokinetics in human. we believe that rsv should be considered favourably as a broad-spectrum chemotherapeutic agent that might overcome the limitations of conventional treatment. the drug design and delivery formulation of this compound should be further improved for clinical translation. acknowledgements the authors would like to acknowledge monash tropical 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malaysia (upm), serdang, selangor, malaysia 2department of pathology, faculty of medicine and health sciences, universiti putra malaysia (upm), serdang, selangor, malaysia 3department of medicine, faculty of medicine and health sciences, universiti putra malaysia (upm), serdang, selangor, malaysia 4allergy and immunology research centre, institute for medical research, jalan pahang, kuala lumpur, wilayah persekutuan kuala lumpur, malaysia abstract: systemic lupus erythematosus (sle) is a chronic autoimmune disease with a varying clinical phenotype. this disease occurs when the body’s tissues are attacked by its own immune system. the aetiology of sle is not fully understood, but both genetic predisposition and ecological triggers are thought to be involved in the disease manifestation. the study on the prevalence of sle allows us to identify potential risk factors associated with the disease for the disease and allows proper management and treatment in response to overall disease burden. hence, this review aims to discuss the prevalence, mortality, clinical manifestation and disease assessment of sle. keywords: systemic lupus erythematosus; autoimmune; aetiology; mortality; assessment received: 31st july 2020 accepted: 1st september 2020 published online: 05th september 2020 citation: selvaraja m, abdullah m, md shah a, et al. systematic lupus erythematosus (sle): a review on the prevalence, clinical manifestation, and disease assessment. prog microbes mol biol 2020; 3(1): a0000106. https://doi.org/10.3687/ pmmb.a0000106 introduction systemic lupus erythematosus (sle) is a multisystemic autoimmune disease which can cause chronic inflammation and damage to tissue and almost every organs in our human body[1,2]. the exact aetiology of sle remains unknown, however, it has been hypothesized that genetic, environmental, and other hormonal factors are likely to play a vital role in the occurrence of sle[3]. there are various immunological faults that have been described in the development of sle which include tand b-cell abnormalities, and the failure to clear autoantibodies that leads to generation of immune complexes[4]. in addition, an association between sle disease onset and age, sex, geography and race have been also reported. sle affects women more commonly than men with a ratio of 9:1[5]. however, male patients tend to have more severe disease manifestation than the female patients[6]. it is because the occurrence of lupus nephritis among men is more than women and more likely to develop to end-stage renal disease (esrd) than women[7]. sle could occur at any age, however, more prevalent in people between the ages of 10 and 50. in terms of ethnicity, the disease affects african american, asians, hispanic and native americans more frequently than other races in the world. this could be due to genetic and geographical influences which are thought to play a role in the development of sle. the overall survival rates for 5-year and 10-year was recorded as 82% and 70%, respectively, whereas survival for 1-year was 93%[8]. on the other hand, the overall mortality rate among sle patients was reported as 20.2%[9]. the cause of mortality among sle patients differs from one to another individual, due to demographical changes including country or region of patient’s origin, age, gender and ethnicities. the exact cause/aetiology of sle remains elusive, however, the fundamental defect in sle clearly elucidate the presence of various autoantibodies against self-constituent due to failure in mechanisms that maintain self-tolerance[10]. in recent years, the mortality rate of sle has been declining which possibly due to early detection and pharmacological advances in controlling and managing the disease progression. this review aims to discuss the prevalence, mortality, clinical manifestation and disease assessment of sle. copyright @ 2020 by selvaraja m and hh publisher. this work under licensed under the creative commons attributionnoncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: syafinaz amin nordin, department of medical microbiology & parasitology, faculty of medicine and health sciences, universiti putra malaysia (upm), serdang, selangor, malaysia; syafinaz@upm.edu.my 2 prevalence of systemic lupus erythematosus worldwide, the disease affects a minimum of 5 million people. the current prevalence of sle is 20 to150 cases per 100,000 people[11]. in the united states (us), the prevalence of sle among the caucasians is approximately 51 per 100,000[12]. the prevalence of sle amongst the african-american women is three times higher than the caucasian women[13]. based on a study in the us, the prevalence of definite sle within the us community is 54.3 per 100,000 people whereas the suspected cases of sle is 108.6 per100,000 people[14]. it is reported that the prevalence of sle among africanamerican women was 286 per 100,000 people, a figure nearly twice as high as the prevalence among white woman[15]. the incidence rates reported in north america and south america range from 2 to 8 cases per 100,000 people per year. one study by naleway and colleagues reported the incidence of sle amongst the us population is 5.1 per 100,000–1.9 per 100,000 in adult men and 8.2 per 100,00 in adult women[16]. previous study reported an overall incidence of sle amongst the us community was 5.56 per 100,000 people[17]. looking into the european continent, an epidemiological research reported an increase in sle prevalence from 64.99 per 100,000 people in 1999 to 97.04 per 100,000 people in 2012[18]. the incidence of sle during the same study period was 4.91 per 100,000 people with a yearly decline of 1.8%. approximately, 60% of sle populations were women. the highest incidence was reported between the ages of 50 and 59. in terms of ethnicity, africo-caribbean people had 2.3-fold higher rate of prevalence and incidence which could be due to regional variation[18]. in terms of gender, the females had 10-fold higher prevalence than the males. the same study also stated that the africo-caribbean sle population had a higher tendency to develop complication such as renal disease and end-stage renal disease (esrd) approximately 40.5% and 15.3% respectively as compared to caucasians in the same sle population whose proportions were only of 18.8% and 4.5% respectively [18]. in another study, it was reported that the caucasians of england origin had a lower prevalence compared to afro-caribbean, hispanics and asians[19]. in addition to the above, table 1 shows the prevalence rate for other countries in the european continent. table 1. summary of sle disease prevalence for european states. prevalence lithuania 16.2/100,000 (0.016%) [20] south ireland 21.7–39.5/100,000 (0.022%–0.039%) [21] norway 47.6–57.9/100,000 (0.048%–0.058%) [22] for oceanic continent, looking at australia, although there is a lack of published information on sle, one identified study has reported that two ethnic groups, namely aboriginal australian and caucasians were known to have sle. whereas, a study reported in 1998 stated that the prevalence of sle in queensland, australia was 45.3 per 100,000 people[23]. in another study conducted at the central part of australia from the period of 1996– 1997, the prevalence of sle was 92.8 per 100,000 people among the aborigines ethnic group[24]. besides that, one study carried out at the australia northern territory from the period of 1986–1990, reported sle prevalence of 19.3 per 100,000 and 52.6 per 100, 000 people amongst the australian caucasians and aborigines community respectively. another study reported the incidence of sle amongst aborigines people was 11 per 100,000 people at the northern territory of australia[25]. a study based in canada reported that the prevalence of sle in canada falls within the range of 22.1 and 51.0 cases per 100,000 people[26,27]. in addition to that, another study reported that the disease generally affects 0.05% of canadian adults and it is 10 times more prevalent amongst women than men[28]. one study from brazil population showed that the prevalence of sle is 3 per 100,000 people with predominantly women[29]. similarly, another study in brazil reported sle prevalence of 20 to150 cases per 100,000 people[30]. in saudi arabia, the prevalence of sle amongst its nationals was estimated at 19.28 per 100,000 people[31]. amongst asians, sle prevalence has been estimated to be between 30 and 50 per 100,000 individuals. it has been reported that sle is more common among the chinese population in asia[32]. another study reported three cases of sle out of 4192 adults registered for rheumatic disorder in the north of china (beijing), and one out of 5057in the south of china (shantou) from han population, to have sle disease[33]. similarly, the prevalence of sle amongst the han population in china was estimated at 37 to 70 cases per 100,000 people[34]. these data show the chinese population has a higher prevalence of rheumatic disease including sle in comparison to the caucasians. in taiwan, the prevalence of sle among paediatric population was reported at 6.3 per 100,000 people. the prevalence in girls were 11.2 cases per 100,000, which is 6.2 times higher than that in boys (1.8 cases per 100,000)[35]. in india, a study conducted in northern india demonstrated a prevalence of 3.2 per 100,000 people with sle, which is at lowest compared to other asian countries[36,37]. moreover, studies also reported that there were no sle cases found amongst adults from certain rural and urban areas of jammu, india[38]. amongst malaysian populations, consisting of three major ethnic groups; malays (55.1%), chinese (24.3%) and indians (7.4%) out of a total population of 22 millions of people, sle prevalence has been reported to be 43 per 100,000 people[37]. amongst the three communities, chinese population represents the highest prevalence rate of sle of 57 per 100,000 followed by malays 33 per 100,000 and indians with only 14 per 100,000 people. survival and mortality of sle generally, the survival rates of sle can be categorized into three groups. the categories are survival rates at 1 year, 5 years and 10 years. the overall survival rate at 1 year fall systematic lupus... country reference 3 selvaraja m et al. worsening multi-systemic disease with mild signs and lesser number of affected systems in the beginning stage. after some years or with a rapidly progressive condition, over a few weeks or months, it continues to involve and affect more systems severely. over time, there will be various clinical and laboratory manifestations. autoantibodies are the hallmark of laboratory manifestation for sle diagnosis. fatigue is the one of the most common complaints of sle patients. however, it has a broad association with many other diseases such as fibromyalgia, hypothyroidism, depression, anaemia, pulmonary or cardiovascular diseases. moreover, some other intrinsic signs of an active lupus that may commonly occur amongst the patients include fever, anorexia, lymphadenopathy and weight loss. however, all of the above symptoms cannot be attributed to lupus alone until infection and malignancy causes are ruled out. the stage of involvement and severity based on every organ system is further elucidated as below. mucocutaneous manifestations / dermatological features dermatological features amongst sle patients account for four out of 11 revised criteria based on the sle classification tool portrayed in the american college of rheumatology (acr). the skin involvement is claimed to be as high as 85% among the sle patients[44]. one of the most commonly identified signs amongst the sle patients are the photosensitivity rashes and the butterfly or malar rash on the face. other familiar mucocutaneous signs in sle patients include mouth ulcers and alopecia or hair loss. hair loss can be categorized into three types which include localized diffused alopecia, “frizz” or frontal hair loss and severe diffused alopecia with minimal sign of new hair growth[45]. discoid lesion (dl) and maculopapular lesion, splinter haemorrhages, dilated capillaries at the nail base, bullous lesions, angioneurotic oedema and livedo reticularis are also some of the identified dermatological manifestations[46]. the discoid lesion repeatedly leads to scarring and older lesions may result in pigmentary changes, either hypopigmentation or hyperpigmentation. alopecia scaring may appear in relation to discoid rash on the scalp region. ulcers in mouth, nasal and genital have also been associated with sle but they are less common. raynaud’s phenomenon is one of the mildly described signs amongst sle patients. it can be linked to severe digital ischaemia and maturation of gangrene. raynaud’s is a disease affecting the blood vessels at the fingers and toes. it will make blood vessels to be narrowed around the area in cold or stress conditions. this feature is difficult to be recognized amongst the dark skinned individuals unless there is sparing of some fingers[47]. vasculitis is usually identified at the nail folds and finger tips[44]. when it occurs, it may develop into a tender, deep, frank ulceration which can either take months to heal or lead to secondary infections. the other more frequently found lesion is shingles, which caused by herpes zoster and often occurs in patients taking immunosuppressant. maculopapular rash is a type of rash that appears flat and red on the skin and covered with small confluent bumps which can become infected in the sle patients. musculoskeletal involvement arthralgia is defined as an inflammatory joint pain which develops along with morning stiffness or gelling that within 93 to 98%, followed by at 5 years ranging from 60 to 97% and 70 to 94% at 10 years. moreover, with the advancement in pharmacological therapy and treatment, the overall survival rate of sle is expected to improve. the cause of mortality amongst sle patients varies from one individual to another partly due to demographical variances. the demographical differences include geographical origin of patients, age, gender and ethnicity. these factors are considered crucial as some studies have proven that they have major roles in the pathogenesis and survival of sle. socioeconomic status often determined by occupation and residing area of patients have also been found to effect survivability and management of sle disease[39,40]. besides that, the disease duration also determines the mortality rate amongst the patients. several studies have reported that generally, active disease and multiple kind of infections are the most common factors leading to early death among sle patients. during active phase of sle, patients receive numerous immunosuppressant which may have negative impact on their natural immune system, thus paving the way for opportunistic infections and even death in severe cases. on the other hand, death in later stage of sle is usually related to vascular events such as cardiovascular disease[41], thrombotic events[42], non-hodgkin lymphoma, lung cancer and renal disorders such as lupus nephritis[26]. however, these underlying causes of death vary according to geographical location and ethnicity. for instance, amongst the us populations, the african-american, american indians and the asians have higher mortality rate compared to the caucasians[41]. it was also found that the causes of death amongst the younger and elderly patients were mainly due to infections in the former and cardiovascular or renal complications in the latter. another study conducted in the us reported the sle mortality rate is higher among the african americans, american indians and asians compared to the americans[41]. it is noted that ethnicity and socioeconomic could be the possible cause of higher mortality rate among the african americans. based on medical intervention and clinicians, infections are the main cause of death among the younger patients, whereas cardiovascular or renal complications are the clinical manifestation among the elderly group. a study conducted in malaysia on the mortality patterns amongst malaysian sle patients, reported that 30% of patients died of infection, followed by 15% of renal disorders, 14% of pulmonary disorders, 7% of cardiovascular diseases, 5% of central nervous system, 1% of malignancy, 1% for acute anaphylaxis, 27% of unknown reason and remaining 19% died due to sle as a contributory factor of death. the study also mentioned that the chinese community represents the majority of those who live with sle at approximately 73%, followed by malays at 18% and indians the least at 9%. the average age at death reported was 28.6 years[43]. clinical manifestation of sle systemic lupus erythematosus is known to be a gradually 4 happens after a period of rest. it occurs approximately in 90% of sle patients[48]. synovial effusion is less common. however, only small volume is present even if it happens. in addition, non-erosive arthritis with joint tenderness and swelling may also form[49]. about 10% of sle patients have been found to develop jaccoud’s arthropathy, whereas among rheumatoid arthritis patients the deformities occur along with joint erosions. it has also been pointed out that sle patients may develop osteoarthritis as they age. bouchard’s nodes that occur at the proximal inter phalangeal joints and heberden’s nodes that occur at the distal interphalangeal joints caused by osteophytes in osteoarthritis is different from acute synovitis that occurs due to lupus flare. tenosynovitis is an early symptom of sle associated with tendon rupture found on patellar tendon, achilles tendon, the long head of the biceps, the triceps and the extensor tendons of the hands. it has been estimated that the, muscle involvement among sle patients is up to 30–50% of patients[50]. respiratory/ lung involvement lung is one of the vital organs in human body and it is highly susceptible to secondary infections caused by bacteria, viruses and fungi which may lead to pneumonia in sle patients. pain on deep inspiration is one of the frequent complains amongst sle patients which is caused by pleurisy. generally, the immunosuppressive agents taken by the sle patients may result in an immunosuppression which may trigger infections. pleural effusion is a sign found in approximately half of the sle patients specifically during disease flares. pulmonary fibrosis, pulmonary haemorrhage, oedema and pulmonary embolism caused by lupus pneumonitis are other rare manifestations[51]. cardiovascular features the most prevalent cardiovascular sign in sle is chest pain. a pericardial rub is more common than a significant pericardial effusion amongst sle patients. myocardial involvement in sle is less common than pericardial disorder. studies have reported that arthrosclerosis increases the risk of cardiovascular events amongst sle patients. systolic murmurs are also commonly found sign, affecting 30% of sle patients, whereas diastolic murmurs are less common[52,53]. neuropsychiatric features sle can affect both the central and peripheral nervous systems. some of the most frequently observed central nervous system features are headache (benign intracranial, hypertension), seizure disorders, psychosis, myelopathy, movement disorders, mood disorder, demyelinating syndrome, cognitive dysfunction, cerebrovascular disease, anxiety disorder, aseptic meningitis and delirium[54]. at some instance, drugs could possible precipitate some of the above-mentioned conditions. steroids are known to induce psychosis in some sle patients. acute inflammatory demyelinating polyradiculoneuropathy (guillain-barre-syndrome), autonomic disorder, mononeuropathy (single or multiplex), myasthesia gravis, neuropathy (cranial) and plexopathy are some of the manifestations involving peripheral nervous system[55]. severe neuropsychiatric condition of lupus is known as neuropsychiatric sle (np-sle), the third leading cause of death amongst sle patients[56]. gastrointestinal involvement various non-specific gastrointestinal manifestations have been reported in sle patients. nausea is one of the common symptoms followed by abdominal pain in sle patients. vomiting and diarrhoea are less common symptoms but may occur with active sle. when perforation occurs, necrotizing vasculitis can be seen pathologically. ascites, dysphagia and pancreatitis are other rare gastrointestinal manifestations[57]. ophthalmic involvement the recurrent factors that leads to red eye in lupus are episcleritis and sicca (dry eye). approximately 8% of sle patients develop inflammation of the retinal artery during the early days of disease course. retinal vasculitis is an active systemic disorder which can lead to visual loss besides optic neuritis. uveitis is less common and affects less than 1% of patients. vaso-occlusive disorder that affects the retina or choroidal vessels and causes anterior ischaemic optic neuropathy may also lead to a vision loss. myositis of eye muscles that lead to diplopia and / or proptosis is a rare occurrence amongst sle patients[58]. haematological abnormalities it is very common among sle patients to have haematological abnormalities such as anaemia, thrombocytopenia, and leukopenia during the course of disease[59]. autoimmune haemolytic anaemia in sle is another distinguishing symptom amongst sle patients reflecting decreased level of serum iron and iron binding capacity. multiple mechanisms lead to iron deficiency which include excessive usage of steroidal and non-steroidal anti-inflammatory drugs resulting in gastrointestinal bleeding[57]. renal insufficiency, blood loss, dietary insufficiency and infections may lead to anaemia. another persistent and typical feature of sle is leucopoenia (<4.0 x 109/l) which is found in over 90% of sle patients. autoantibody deposition that diminishes the function of immune cells and compliment activation is also partly involved in the development of haematological abnormalities amongst sle patients. moreover, immunosuppressive agents also contribute to severe leucopoenia[60]. thrombocytopenia (platelet count <100 x 109/l) is one of the common laboratory findings in sle patients. it can appear in either chronic or acute form. chronic form is related to mild disease, whereas acute form is similar to idiopathic autoimmune thrombocytopenic purpura. platelet dissociation is known to be directed by anti-platelet antibodies (apl). apl is also associated with thrombocytopenia and thrombosis[61]. renal involvement more than 70% of patients with sle have been estimated to have lupus nephritis (ln) at some stage of their disease progression. the world health organization systematic lupus... 5 histopathological biopsies by the international society of nephrology and the renal pathology society (table 2)[62]. (who) classification for lupus nephritis has been published to facilitate a more accurate description of renal selvaraja m et al. table 2. classification of lupus nephritis. classes description minimal mesangial lupus nephritis normal glomeruli at light microscopy mesangial immune deposits on immunofluorescence mesangial proliferative lupus nephritis mesangial hyper-cellularity or expansion with mesangial immune deposits in light microscopy some subepithelial or subendothelial deposits on immunofluorescence or electron microscopy focal lupus nephritis involves less than 50% glomeruli active or inactive lesions with subendothelial deposits diffuse lupus nephritis involves more than 50% glomeruli active or inactive diffuse, segmental, or global endoor extracapillary glomerulonephritis with subendothelial deposits. divided into diffuse segmental when <50% of involved glomeruli have segmental lesions and diffuse global when >50% of involved glomeruli have global lesions membranous lupus nephritis global or segmental subepithelial immune deposits by light microscopy and immunofluorescence or electron microscopy, with or without mesangial changes. class v lupus nephritis may occur in combination with class iii or class iv disease, in which case both are diagnosed. class v disease may show advanced sclerosis advanced sclerosis lupus >90% of glomeruli scleroses without residual activity obstetric-related issues although, sle is not directly associated with infertility, it has been associated with pregnancy, particularly in the second or third trimester which is caused by lupus flare. moreover, in sle patients who develop active lupus nephritis (ln) during conception, either as new onset or flare, increases the risk of preterm delivery, preeclampsia, maternal mortality, foetal / neonatal demise and intrauterine growth restrictions. other common conditions in sle pregnant women are cutaneous disease (25–90%), arthritis (20%) and haematological involvement which include thrombocytopenia (10–14%)[63]. laboratory criteria the evolution of autoantibodies in human body has been associated with the manifestation of sle disease. one of the classic, non-specific antibodies that are present in approximately 96% of sle patients is anti-nuclear antibodies (anas) (manson and isenberg, 2003). anas are antibodies or immunoglobulins that bind to one or more antigens expressed within the nucleus of human cells in relation to connective tissue disease or infections. anti-double stranded dna (adsdna) are more specific anti-nuclear antibodies that only target double stranded dna. studies have reported that anti-dsdna antibodies have a strong link with glomerulonephritis in sle patients. anti-dsdna is also used for diagnosis purpose and to monitor the disease progression and anti-smith (anti-sm) antibodies[4]. anti-smith (anti-sm) antibodies identify extractable nuclear antigens (anti-ena) which include anti-ro and anti-la antibodies. other antibodies that have been found to be associated with sle disease include antirnp antibodies, rheumatoid factor, igg anticardiolipin antibodies, igm anti cardiolipin antibodies and lupus anti-coagulant. moreover, complement activation plays a vital role in the deposition of immune complexes when autoantibodies bind to their target antigens. low level of complements, c3 and c4 are highly associated with lupus nephritis and vasculitis[64]. non-specific features lymphadenopathy is defined by changes in number, characteristics or size of the lymph node. in sle, this is a benign condition which can be diagnosed at any stage of the disease development. studies have l reported that this sign is commonly found in younger patients with cutaneous involvement[65]. other sign including spleen enlargement or known as splenomegaly is estimated to happen in 10% of sle patients. the disease assessment one of the challenging and difficult aspects in sle is measuring the disease activity due to the complexity of disease nature which affecting multiple organs and the clinical outcomes from one to another sle patient. number of studies attempted in order to find definition for disease activity in sle and how it should be measured. measuring the disease activity in sle is one of the three domains of sle assessments that include the measuring of damages caused by sle and the quality of life[66]. generally, corticosteroids and immunosuppressive that is used in controlling sle symptoms plays a vital role in disease activity. this is due to their pharmacological properties producing various side effects such as diabetes, 6 osteoporosis, arterial hypertension and neoplasia among sle patients. various indices or disease assessment tools were discovered with the objective standardizing sle activity assessment. the commonly used indices are as follows: lai (lupus activity index); slam (systemic lupus activity measure); eclam (european consensus lupus activity measurement); bilag (british isles lupus assessment), sledai (systemic lupus erythematosus disease activity index); selenasledai (safety of estrogen in lupus erythematosus national assessment-sledai) and sledai-2kg[67]. among the few listed instruments above, sledai that been introduced in 1985 was proven to be the most reliable and reproducible apart from being sensitive to change in a patient condition when used by various investigators. it is an index that measures disease activity by considering the organ affected and includes 24 clinical and laboratory parameters of nine organ systems in its evaluation. sledai assesses disease activity in the previous 10 days. it assesses 16 clinical features and 8 laboratory indices. the scores of the descriptors range from 1 to 8. active disease indicated by scores greater than 8 and the total possible score for all 24 descriptors is 105[68]. later sledai-2k was introduced in 2002, a modified version of original sledai but there was one limitation. this revised index does not account for severity within each descriptor[69]américa the systemic lupus activity measure-revised, the mexican systemic lupus erythematosus disease activity index (sledai. however, it was proven to have best discriminative validity in which able to differentiate active patients from inactive ones and lowest cost. later another novel tool was developed in order to measure lupus disease activity index known as sledai-2k gcs (sledai-2kg) which outlined disease activity while considering on glucocorticosteroid (gcs) dose and having same descriptors as sledai-2k but with different weight scores based on the dose of gcs[70]. slam (systemic lupus activity measure), was first reported in 1986 and later revised in 2001 based on consensus of the lupus council of the american college of rheumatology[71]. the difference between slam and sledai is illustrated in table 3. slam measured the disease severity in the domains of 9 organ systems and 7 laboratory measures as follows; constitutional, integument, eye, reticuloendothelial, gastrointestinal, cardiovascular, pulmonary, neuromotor, joints and laboratory parameters. the total slam score ranges from 0–84, with each organ items scored 0–3 points manifested within one month (30 days) period, in which severity having highest score by item[72]. table 3. difference between sledai and slam scoring system. slam · reliable, validated, sensitive and responsiveness to adapt with time · very practical and widely used for clinical and research purpose. · scored within last days if present with symptoms · none of the sledai version captures on improving or worsening and do not include severity within an organ system. thus, this is less sensitive to compared to other instruments [73] · laboratory information required. · responsiveness to patient care [69] · reliable, validated, sensitive and responsiveness change over time · measures disease severity within one month · laboratory studies needed · responsiveness to patient care: highly responsive · excellent sensitivity as compared to other instruments [74] conclusion systemic lupus erythematosus (sle) is an autoimmune disease that effects the quality of life for many people. this disease gradually worsen over the time and patients are on medications for life long. moreover, the disease manifestation and complications vary from one to another individual, taking into the consideration of their ethnicity and geographical region where they are origin. it is a daunting task to determine the cause of the disease in one population. hence, continuous monitoring and surveillance is required to manage the disease progression. author contribution the literature search and manuscript writing were performed by ms. ma, am-s and ma provided vital guidance and support as content expert and proofread of the writing. the project was founded by sa-n. conflict of interest all the authors declare that the research was conducted in the absence of any commercial or financial relationships that could 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1–10. 74. chang er, abrahamowicz m, ferland d, et al. organ manifestations influence differently the responsiveness of 2 lupus disease activity measures, according to patients’ or physicians’ evaluations of recent lupus activity. the journal of rheumatology 2002; 29(11): 2350–2358. systematic lupus... pmmb 2023, 6, 1; a0000332. doi: 10.36877/pmmb.0000332 http://journals.hh-publisher.com/index.php/pmmb original research article a study comparing immunohistochemistry, fluorescence in situ hybridization, genexpert® breast cancer strat4 assay for determining hormone receptor, human epidermal growth factor receptor 2, ki67 status in invasive breast carcinoma in moroccan women rajaa el aje1*, abdellah naya1, ayoub khoaja2, younes zaid1,3, mounia oudghiri1, jodi weidler4, mehdi karkouri2,5 article history 1immunology & biodiversity laboratory. biology, faculty of sciences ain chock, hassan ii university, casablanca, morocco; anaya@mabiotech.com (an); y.zaid@um5r.ac.ma (yz); mouniaoudghiri@gmail.com (mo) 2pathological laboratory, ibn rochd hospital. casablanca, morocco; akhoaja@gmail.com (ak); mehdi.karkouri@gmail.com (mk) 3faculty of sciences, mohammed v university in rabat, rabat, morocco 4medical and scientific affairs and strategy, oncology, cepheid, sunnyvale, ca, usa; jodi.weidler@cepheid.com (jw) 5laboratory of genetics and molecular pathology, faculty of medicine and pharmacy, hassan ii university, casablanca, morocco *corresponding author: rajaa el aje, immunology & biodiversity laboratory. biology, faculty of sciences ain chock, hassan ii university, casablanca, morocco; elaje.rajaa@gmail.com (re) received: 03 april 2023; received in revised form: 10 may 2023; accepted: 30 may 2023; available online: 02 june 2023 abstract: the accurate assessment of hormone receptors, her2, and ki-67 proliferative index provides meaningful information about breast cancer prognosis and prediction of therapy response. immunohistochemistry, the most common method for evaluating these prognostic biomarkers, can be impacted by numerous variabilities due to preanalytical/analytical factors and subjective interpretation by pathologists. the xpert® breast cancer strat4, an rt-qpcr based system, can be used to classify invasive breast carcinomas based on the assessment of these four biomarkers; methods: we evaluated esr1, pgr, erbb2, and mki67 mrna expression by xpert breast cancer strat4 and er, pr, her2 and ki67 by ihc (fish for her2 ihc 2+) in 200 formalin-fixed paraffin-embedded (ffpe) tissue blocks with invasive breast cancer, collected from the pathology department of casablanca ibn rochd university hospital; results: concordance between xpert ® breast cancer strat4 and ihc was 93.5% for er, 83.51% for pr, 95% for her2 (92% for ihc+fish), and 81.20% for ki67 (excluding intermediate ihc staining 10 ≤ %ihc <20). the simple kappa coefficient was, for er, 0.830 (p < 0.0001), 0.565 (p < 0.0001) for pr, mailto:mouniaoudghiri@gmail.com mailto:elaje.rajaa@gmail.com pmmb 2023, 6, 1; a0000332 2 of 16 0.838 (p < 0.0001) for her2-ihc, 0.771 (p < 0.0001) for her2 ihc+fish and, for, ki67, 0.458 (p < 0.0001); conclusions: we demonstrated globally a high concordance between centrally assessed ihc, ihc+fish and mrna measurements of er/esr1 and her2/erbb2, and a moderate agreement between pr/pgr and ki67/mki67. these findings provide an additional, objective, and quantitative assessment of tumor receptor status in breast cancer. keywords: breast cancer; hormone receptors; human epidermal growth factor receptor 2; ki-67; immunohistochemistry; xpert strat4 1. introduction cancer poses a significant global health challenge, as evidenced by the staggering numbers of new cases and deaths recorded in 2020, which stood at 19.3 million and 10.0 million, respectively [1-4]. individuals face a 20% chance of developing cancer at some point, with a 10% risk of dying. one in five people will develop cancer during their lifetime, and one in 10 will succumb to it [1]. in 2020, female breast cancer surpassed all other types of cancer as the most common cancer worldwide, with an estimated 2.26 million new cases, followed closely by lung cancer with 2.21 cases [1,3-4]. approximately 494,000 new cases are diagnosed annually in europe, and it is estimated that 143,000 women will succumb to the disease [5]. morocco saw around 11,000 new breast cancer diagnoses in 2020 [6], making it the most common malignancy in the country and the leading cause of cancer-related deaths among moroccan women [6]. improved treatment and early detection have led to a 34% decrease in breast cancer mortality over the past 30 years [7]. immunohistochemical assessment of estrogen and progesterone receptor protein expression has become a valuable tool for predicting patient outcomes and response to endocrine therapy in breast cancer [8-12]. another alteration that has received significant attention in the last two decades is amplifying the her-2/neu gene in human breast cancers. measuring protein overexpression or gene amplification of the human epidermal growth factor receptor 2 (her2 or erbb2) can serve as a useful prognostic marker and a predictor of response to trastuzumab or other her2-targeted therapies [13,14]. several retrospective studies of breast cancer patients have shown the marker of proliferation ki67 (mki67) to be an important prognostic factor. it has various potential uses, including prognosis, prediction of response to chemotherapy or endocrine therapy, estimation of residual risk in patients on standard therapy, and as a dynamic biomarker of treatment efficacy in samples taken before, during, and after neoadjuvant therapy [14,15]. the european society for medical oncology (esmo) recommends that all primary breast carcinomas be tested for estrogen receptor (er), progesterone receptor (pr), her2/erbb2, and ki-67 at the time of diagnosis, according to treatment guidelines [16-18]. pmmb 2023, 6, 1; a0000332 3 of 16 immunohistochemistry (ihc) on formalin-fixed paraffin-embedded (ffpe) tissues is the gold standard for assessing hormonal receptor status, her2, and ki67 in breast cancer [7,13,14]. when the her2 score is equivocal (2+), fluorescence in situ hybridization (fish) is typically used to clarify her2 immunohistochemical results, and some institutions use fish for initial her2 evaluation status in all patients [13,19]. despite being used for a long time, immunohistochemical assays have not been adequately standardized across laboratories. the accuracy and reproducibility of results for these four biomarkers can be impacted by pre-analytical or analytical limitations such as tissue fixation, choice of antibodies, use of manual versus computer-assisted scoring methods, and interpretation of results in assay performance, which can significantly affect ihc and fish results [14,16,20-22]. the xpert® breast cancer strat4 test is an in vitro diagnostic medical device (ceivd: in vitro diagnostic medical device. may not be available in all countries. not available in the u.s.) that utilizes a semi-quantitative assay with qualitative cut-off values to detect the mrna levels of estrogen receptor (esr1), progesterone receptor (pgr), her2/erbb2, and marker of proliferation ki-67 (mki67) in ffpe invasive breast cancer samples [21,22]. this test measures the target mrna levels of these four biomarkers and the mrna levels of the reference gene cyfip1 in ffpe breast cancer tissue using a self-contained cartridge. the genexpert® (gx) system is responsible for automating and integrating all aspects of the sample processing, including rna isolation, amplification, and detection of the target sequences in ffpe samples through real-time reverse transcriptase, polymerase chain reaction assays (rt-pcr) [21,22]. the main goal of this first retrospective study in the north africa region is to assess the clinical performance of the genexpert instrument systems compared to the currently used ihc and fish methods in evaluating routine biomarkers (er, pr, her2 and ki67) in moroccan women. the study aims to verify the efficacy of this new approach. according to earlier research conducted in various other countries, xpert® breast cancer strat4 is highly reproducible and shows a high level of agreement with ihc and her2 fish results [21-29]. 2. materials and methods our study included two hundred blocks of ffpe tissue corresponding to 200 patients, aged ≤5 years and archived in the pathology department of casablanca ibn rochd university hospital. the histopathology of all samples remaining in the blocks was reviewed, and only specimens still containing invasive breast carcinoma cells were included in the study. the ffpe tissue sections were obtained from core biopsies (55 cases) and surgical specimens (145 cases), from a selection of patients with invasive breast cancer whose tumor samples were collected and routinely evaluated for breast cancer biomarkers (er, pr, her2 and ki67) according to the standard of care (soc) ihc and/or fish assays at the pathology laboratory. the immunohistochemical status (ihc) was sought on 4 μm tissue sections pmmb 2023, 6, 1; a0000332 4 of 16 treated and incubated with the antibodies er: flex monoclonal rabbit anti-human estrogen receptor α clone ep1 ready-to-use; pr: flex monoclonal mouse anti-human progesterone receptor clone pr 636 ready-to-use and ki67: flex monoclonal mouse anti-human ki-67 antigen clone mib-1 ready-to-use, according to the dako protocol on the autostainer link 48 ihc platform. her2 status is first assessed by ihc, using the antibody ventana pathway anti-her-2/neu (4b5) rabbit monoclonal primary antibody on a ventana gx automated platform. tumors were classified as er positive or pr positive when ≥1 % of invasive tumor cells showed definite nuclear staining, irrespective of staining intensity. a tumor was considered her2 positive if an ihc score equal to 3+ was found and her2 negative if a score of 0 or 1+ was observed (asco/cap guidelines) [30]. her2 equivocal (ihc 2+) results were subsequently tested by fish with manual technique using the probes (her2 iqfish pharmdx) to confirm the final her2 status. the patient tumors selected for our study represent the various breast cancer subtypes as determined through surrogate ihc subtyping by the routine assays performed at our laboratory, as follows: 25 triple negatives (er negative, pr negative and her2 negative) 25 her2+ (hormone receptor (hr) negative / her2 +) 100 luminal a (hr positive / ki67 < 20 %) 25 luminal b (hr positive / ki67 ≥ 20 %) 25 her2 ihc 2+ (8 cases her2 fish positive and 17 her2 fish negative using current asco/cap her2 guidelines) [31]. the mrna levels of esr1, pgr, erbb2 (her2), and mki67 were assessed by quantitative gene expression readouts using the xpert® breast cancer strat4 assay. breast cancer ffpe tissue samples were prepared for the assay as tissue scrolls (10 µm thickness) and placed into a 1.5ml eppendorf tube. for some surgical specimens (ffpe section contained <30% invasive tumor), macrodissection (tumor area defined by the pathologist) was carried out, and the ffpe section was scraped off the slide and placed at the bottom of the tube. ffpe samples were first treated with the recommended volumes of ffpe lysis reagent (1.2 ml) and proteinase k (20 μl) provided by the xpert® ffpe lysis kit (ceivd*) before use in xpert® breast cancer strat4. the solution was then incubated in a heat block at 80 °c for 30 minutes. then 1.2 ml of ≥95% ethanol was mixed with the sample. once the tissue lysate was prepared, a 520 µl aliquot was placed into the appropriate sample chamber in the xpert® breast cancer strat4 cartridge. the testing cartridge was inserted into a module of a genexpert® system for processing, where the system fully automated and thoroughly integrated nucleic acid purification, amplification, and real-time pmmb 2023, 6, 1; a0000332 5 of 16 detection. the final results of strat4 testing are available approximately 70 minutes after starting the test. 2.1. statistical analysis statistical analysis was done in graphpad prism software. for each of the four biomarkers studied, agreement measurements between xpert® breast cancer strat4 and ihc and/or fish, which were considered as the reference methods, were based on contingency table analysis and included overall concordance (overall percent agreement), positive percent agreement (sensitivity) defined as the number of samples classified positive by both ihc and xpert® breast cancer strat4 divided by the number of positive samples using immunohistochemistry, negative percent agreement (specificity), and cohen's κ coefficient scores. the kappa (κ) statistic numeric values are categorized into the slight agreement (≤0.2), fair agreement (between 0.21 and 0.40), moderate agreement (between 0.21 and 0.40), substantial agreement (between 0.61 and 0.80), and almost perfect agreement (between 0.81 and 1.00). all measurements were associated with 95% confidence intervals (95% ci), compared using fisher’s exact test, and considered significant for p < 0.05 [32]. 3. results for each sample, we evaluated mrna results by xpert® breast cancer strat4 and compared them to the results obtained by the already routinely performed ihc+her2 fish. 3.1. concordance between ihc/fish and xpert strat4 3.1.1. estrogen receptor the overall concordance rate between xpert® breast cancer strat4 esr1 mrna results and er protein ihc results was 93.50% using either the ihc cut-off of ≥1% or ≥10% immunostaining level for positivity and using a pre-defined delta ct cut-off (dct ≥ −1) for esr1-positivity by xpert® breast cancer strat4 based on prior concordance studies [21,25]. the cohen’κ coefficient score was equal to 0.830 (95% confidence interval: from 0.741 to 0.919) (table 1). only 2% of immunohistochemistry-er-positive samples were classified as negative using strat4, whereas 4.5% of immunohistochemistry-er-negative samples showed a positive xpert® breast cancer strat4 er status (figure 1). these results demonstrate perfect agreement between strat4 and ihc for the esr1/er biomarker and suggest a low discordance level exists between the two methods. pmmb 2023, 6, 1; a0000332 6 of 16 table 1. comparison of protein status for er, pr, her2, and ki67 and mrna expression for esr1, pgr, erbb2, and mki67 between immunohistochemistry « ihc », fluorescence in situ hybridization « fish » and rt-qpcr « xpert® breast cancer strat4 test*». analyte reference total ihc+/ rtqpcr + ihc+/ rtqpcr ihc-/ rtqpcr ihc-/ rtqpcr + sensitivity (ppa) specificity (npa) concordance rate (opa) kappa statistic er/esr1 (ihc+ 1%) ihc 200 142 4 45 9 97.26% 83.33% 93.5% 0.830 er/esr1 (ihc+ 10%) ihc 200 142 4 45 9 97.26% 83.33% 93.5% 0.830 pr/pgr (ihc+ 1%) ihc 194 130 5 32 27 96.3% 54.24% 83.51% 0.565 pr/pgr (ihc+ 10%) ihc 194 114 1 36 43 99.13% 45.57% 77.32% 0.488 her2/erbb2 ihc 175 30 3 136 6 90.91% 95.77% 95% 0.838 her2/erbb2 fish 25 7 1 10 7 87.50% 58.82% 68% 0.387 her2/erbb2 ihc/ fish 200 37 3 147 13 92.5% 91.88% 92% 0.771 ki67/mki67 (ihc+ ≥20%) ihc 184 92 3 32 57 96.84% 35.96% 67.39% 0.334 ki67/mki67 (ihc+ >10%) ihc 184 94 6 29 55 94% 34.52% 66.85% 0.299 ki67/mki67 (excluding 10≤ihc%<20 range) ihc 133 92 3 16 22 96.84% 42.11% 81.20% 0.458 pmmb 2023, 6, 1; a0000332 7 of 16 -10 -5 0 5 10 esr1 dct vs. ihc% staining ihc (% erpositive cells) x p e rt ( e s r 1 d c t) dct cutoff (dct = -1) <1 % 1 % figure 1. comparison of estrogen receptor status determined by rt-qpcr and immunohistochemistry. graph of mrna expression esr1 dct determinated with xpert® breast cancer strat4 test* by er ihc result categorized as negative (<1%), or positive (≥1%). 3.1.2. progesterone receptor regarding the pr data, six cases with “indeterminate” xpert® breast cancer strat4 pgr results were excluded. taking into account all the remaining cases, concordance between xpert® breast cancer strat4 pgr and pr ihc results using an ihc cut-off of ≥1% as recommended by asco-cap [16] was 83.51% using pgr dct cutoff of 3.5. the statistical kappa value is around 0.565 (95% confidence interval: from 0.435 to 0.694) (table 1). only 2.5% cases of ihc pr-positive samples were classified negative by rt-qpcr. in contrast, 14% of ihc pr-negative cases showed a positive xpert® breast cancer strat4 pgr status based on current xpert® breast cancer strat4 cutoffs (figure 2). by using the ihc cut-off of ≥10% to determine pr-positive status, the overall concordance between both methods was 77.32%. in comparison, the cohen’s κ coefficient score was equal to 0.488 (95% confidence interval: from 0.372 to 0.603) (table 1). in this case, only 0.5% of the samples were classified pr-positive by ihc and negative by xpert® breast cancer strat4, whereas 22% of the samples of ihc prnegative became xpert® breast cancer strat4 pgr-positive (figure 2). based on this data, we note a moderate agreement between strat4 and ihc for the pgr/pr biomarker with an acceptable discordance percentage between the two methods (table 1). pmmb 2023, 6, 1; a0000332 8 of 16 -10 -5 0 5 10 pgr dct vs. ihc% staining ihc (% prpositive cells) x p e rt ( p g r d c t) < 1% 19% 1 0% dct cutoff (dct= -3.5) figure 2. comparison of progesterone receptor status determined by rt-qpcr and immunohistochemistry. graph of mrna expression pgr dct determined with xpert® breast cancer strat4 test by pr ihc result categorized as negative (0%), low positive (1–9%), or positive (≥10%). 3.1.3. her2 for her2 status determination, we studied the concordance on three levels, first of all between ihc and strat4 excluding equivocal cases (her2 score = 2+), secondly between fish and strat4 exclusively for equivocal cases, and lastly between strat4 and the two reference methods (ihc and fish), including all cases of the study. the overall concordance rate between xpert® breast cancer strat4 and ihc was approximately 95% (excluding equivocal cases) with a κ coefficient equal to 0.838 (95% confidence interval: from 0.735 to 0.940) (table 1). the percentage of discordant cases classified by ihc as her2positive and erbb2negative on xpert® breast cancer strat4 was only 1.7%, whereas 3.5% of her2-negative cases on ihc were erbb2 positive using xpert® breast cancer strat4 (figure 3). the concordance between xpert® breast cancer strat4 and fish for equivocal cases was 68%, and the cohen's coefficient was equal to 0.387 (95% confidence interval: from 0.073 to 0.700). solely 4% of fish positive samples were classified negative using xpert® breast cancer strat4, while 28% of fish negative samples were classified positive using xpert® breast cancer strat4 (figure 3). considering both reference methods (ihc and fish) as asco/cap guidelines recommended in evaluating the her2 status [33]. the concordance rate between xpert® breast cancer strat4 and reference methods (ihc and fish), including all samples, was 92%. the kappa coefficient equals 0.771 (95% confidence interval: 0.666 to 0.877) (table 1). in this case, the cohort was divided into her2negative (score 0, score1, score 2/fish non-amplified) and her2positive (score3, score2/fish amplified). therefore, the pmmb 2023, 6, 1; a0000332 9 of 16 percentage of discordant between her2negative cases on ihc/fish and her2-positive cases using xpert® breast cancer strat4 was 6.5%, while only 1.5% her2– positive cases on ihc/fish became her2negative using xpert® breast cancer strat4 (figure 3). in general, the cohen’s kappa value and the overall percent agreement (opa) showed that her2 results ranged from good to very good agreement; however, results suggest a minimal percentage of disagreement between methods (table 1). -10 -5 0 5 erbb2 dct vs. ihc (2+ with fish results) x p e rt ( e r b b 2 d c t) 0/ 1+ 2+ , f is h n eg at iv e 2+ , f is h p os iti ve 3+ dct cutoff (dct= -1) ihc (her2, 2+ with fish results) figure 3. comparison of her2/erbb2 determined by either rt-qpcr or by immunohistochemistry with or without fish assessment of ihc2+. graph of mrna expression erbb2 dct determined with xpert® breast cancer strat4 test by ihc/fish her2 results including all sample size. 3.1.4. ki67 last, we examined the xpert® breast cancer strat4 mki67 dct values and ki67 results by using ki67 ihc cutoff of 10% and 20% to discriminate “high proliferation rate” from “low proliferation rate”. in comparison, we used an intermediate zone (equivocal results) between 10 and 20% for the mki67 dct distribution. we excluded sixteen cases with « indeterminate » xpert® breast cancer strat4 mki67 status from this cohort. the overall concordance between xpert® breast cancer strat4 mki67 and ki67 ihc, considering a high proliferation rate of≥20%, was 67.39% (table 1). only 1.6% of samples with high ki67 ihc results were classified as low ki67 using xpert® breast cancer strat4, while 31% of cases with low ki 67 showed a high ki67 in xpert® breast cancer strat4 (figure 4). pmmb 2023, 6, 1; a0000332 10 of 16 -10 -5 0 5 x p e rt ( m k i6 7 d c t) < 10 % 10  ih c % < 2 0  2 0% ihc (%ki67positive cells) dct cutoff (dct= -4) mki67 dct vs. ihc% staining figure 4. comparison of ki67 proliferation rate determined by either rt-qpcr or immunohistochemistry. graph of xpert® breast cancer strat4 mki67 dct values by ki67 ihc % staining where the ihc low proliferation rate cutoff is defined as <10%, ihc high proliferation rate cutoff is defined as ≥20%, and the intermediate proliferation rate (equivocal range) is defined as 10 %≤ ihc% < 20%. when we considered >10% cutoff, the overall agreement was 66.85%. discordant cases consisted of ihc high ki67 and xpert® breast cancer strat4 low ki67 (3%) and ihc low ki67 / xpert® breast cancer strat4 high ki67 (≈ 30%) (table 1). when we excluded samples with ihc staining in the 10 %≤ ihc % < 20% range, the overall agreement was 81.2% (table 1). we noted only 2% of discordant cases with high ki67 on ihc and low ki67 using xpert® breast cancer strat4. however, 16.5% cases showed a low ki67 on ihc and a high ki67 by xpert® breast cancer strat4 (figure 4). the κ coefficient equals 0.334 (95% confidence interval: 0.224 to 0.445) using 20% ki67 ihc cutoff, kappa = 0.299 (95% confidence interval: 0.182 to 0.417) using 10% ki67 ihc cutoff. when we excluded equivocal cases, the kappa equals 0.458 (95% confidence interval: from 0.289 to 0.628) (table 1). comparison of mki67 strat4 results with ihc % immunostaining for ki67 demonstrates that ki67 results ranged from slight to moderate agreement between the methods and a significant discordance percentage. for er and pr, we used ihc cutoffs of 1% as recommended by the asco/cap 2010 er/pr testing guidelines [34] and 10% as described elsewhere [24,34]. central ihc and central fish resolve ihc her2 2+ to either fish-negative or fish-positive, as recommended by the asco-cap guidelines [33] for her2 testing. for ki67 we used ihc cutoff of ≥ 20% and >10% to discriminate “high proliferation rate” from “low proliferation rate”.“indeterminate” results for pgr or mki67 in the xpert breast cancer strat4 pmmb 2023, 6, 1; a0000332 11 of 16 indicate that the cyfip1 reference gene included in the assay cartridge had a ct that was not within the valid range or the endpoint was below the threshold setting required for pgr or mki67 status determination. for these cases, the assay needed to be redone using the concentrated lysate procedure provided by the test kit. 4. discussion immunohistochemical assays are the gold standard for evaluating er, pr, her2, and ki67 status in invasive breast carcinoma. the main advantages of ihc for assessing these markers are that it is rapid and simple, it can be performed in most pathology laboratories, and (compared with other assays) it is relatively inexpensive. however, ihc assay reliability has been questioned because alterations during tissue processing, manipulation, and fixation, as well as the antibody clone, internal controls, and scoring system, may affect the results' precision. in addition, inter-observer variability in interpretation may also play a role as ihc remains a semi-quantitative and non-standardized method [23-25]. the ambiguity encountered in interpreting her2 ihc results, especially in cases with her2 equivocal scores (her2 ihc =2+), may represent an issue. most laboratories in resource-constrained settings may be unable to overcome it, as the gold standard would be fish for quantifying her-2 gene amplification in these cases. indeed, fish has the advantage of being a quantitative method and is considered the gold standard method for confirming the her-2 status, not only to resolve ihc 2+ cases but also for all other cases where it has an excellent correlation with the her2 ihc results [36,37]. major disadvantages are that fish is technically complicated to execute, arduous to establish, has a long run time, and is costly, making it not routinely available in all pathology laboratories worldwide. moreover, another limitation of this method is that it does not necessarily reflect target protein expression, and counting fish spots is wearisome and can be biased by tumor heterogeneity [13,24,25]. nevertheless, these causes of assay variability may explain the differences in er, pr, her2, and ki67 ihc results in breast carcinomas reported previously. currently, treatment of invasive breast carcinoma relies essentially upon er, pr, her2, and ki67 status [38,39], and the accuracy of assays is critical. hence, to overcome the limitations of ihc and her2 fish, there have been efforts to establish alternative methods to assess the 4 biomarkers of interest as accurately as possible [40]. one of the options is to use rt-qpcr. reverse transcription quantitative pcr (rt-qpcr) represents a sensitive, efficient, and reliable approach for analyzing rna. the initial step in rt-pcr is the production of a single-strand complementary dna copy (cdna) of the rna through the action of the retroviral enzyme reverse transcriptase, to amplify that part of this cdna by pcr. rt-pcr is used to analyze differential gene expression or cloned cdnas. rt-pcr is more sensitive and easier to perform than other rna analysis techniques [41]. in this study, our statistical data demonstrated that the xpert® breast cancer strat4 closed-system rt-qpcr method shows a good concordance rate with ihc+her2 fish results. the concordance between xpert® breast cancer strat4 and her2 ihc+her2 pmmb 2023, 6, 1; a0000332 12 of 16 fish has been evaluated in other studies and varies between 91% and 98%. in the current analysis, our data demonstrate that the xpert® breast cancer strat4 assay shows greater than 91% concordance with her2 ihc+her2 fish, suggesting that our results are generally concordant with the previous studies [21-25,37,42,43]. for both esr1/er and erbb2/her2, data suggested almost perfect agreement between xpert® breast cancer strat4 and central ihc (κ "er"= 0.830; κ "her2"= 0.838), with nearly all of the discordant cases with quantitative dct values close to the esr1 and erbb2 dct cutoffs, respectively. our findings are in good agreement with previously reported results: we found overall concordance of 93.50% and 95% for er and her2, respectively, and previously published papers have reported values of 97% to 98% for er and of 93% to 97% for her2 [21-23,25]. the results showed a moderate kappa correlation agreement for pgr/pr (using pr ihc+ 1%) and mki67/ki67 (excluding equivocal cases) between both assays (κ "pr"= 0.565; κ "ki67"= 0.458). xpert® breast cancer strat4 demonstrated a significant overall concordance with ihc for pgr (83.5%) and mki67 (81%). the concordance rates observed in other studies vary from 81% to 92% for pr and from 78% to 89% for ki67, in accordance with agreement percentages obtained in our analysis [21-23,25,43]. discordance between assay methods can be attributed to several factors, including tissue fixation, antibody clones used in ihc, and scoring methods. preanalytical factors are essential to monitor, and a quality assessment scheme should be put in place in any laboratory routinely assessing the 4 biomarkers [44]. particular attention should be paid to the impact of sample handling, time of fixation, duration of tissue fixation, antibody selection, control samples and interpretation of assay on xpert® breast cancer strat4 results [11,14,15,18]. in spite of the systematic practice of immunohistochemistry methods, procedural inconsistency remains elevated in clinical settings, leading to interlaboratory and intralaboratory variations and high false-negative (for er and pr) and false-positive (for her2) [19]. this inconsistency emphasizes the importance of a standardized retrieval method in performing reliable ihc and/ or fish for the four markers routinely screened in breast cancer diagnosis. xpert® breast cancer strat4 has already shown good agreement with automated semi-quantitative ihc [21-23,26,27,29]. esr1 and erbb2 assessments have the highest pertinence in all studies. comparison between the xpert® breast cancer strat4 pgr status and the pr ihc status resulted in more discrepancies. this discrepancy between the two methods could be explained by the fact that total mrna does not necessarily reflect the total protein and vice versa [45]. denkert et al. have demonstrated that ki67 ihc results are greatly variable. significant variability in concordance rate has been noted for this biomarker, although this is not unforeseen given the challenges associated with ki67 ihc evaluation [23]. major gaps in diagnostic availability exist in many low-income and middle-income countries (lmic) [46]. ihc is not widely available and faces many challenges, such as lack of skilled human resources and adequate equipment, insufficient and unreliable funding to pmmb 2023, 6, 1; a0000332 13 of 16 run the facilities, and erratic supply chain management [47]. ihc would, therefore, either be unavailable at all or only available at a central level, in a centralized manner, resulting in very important delays in getting the results for breast cancer [13,15,21-25,43]. since genxpert technology is already widely available in lmic, especially in africa, as some countries have been largely equipped for the hiv and/or tb programs [48], existing instruments could be mutualised and used for breast cancer. since genxpert technology is very easy to use, implementing such technology would be very swift. in situations where delays exist in obtaining ihc results, such as heavy workloads or centralized ihc services, alternative diagnostic methods such as genxpert can be utilized for timely preliminary results. this enables oncologists to initiate treatment promptly while awaiting ihc results. after ihc results become available, treatment plans can be adapted if necessary. additionally, in cases where ihc is available but ineffective due to small specimen sizes or technical limitations, strat4 can be employed as a tiebreaker. considering our results and similar results found in other published papers, the xpert® breast cancer strat4 assay is highly reproducible. it shows a high level of concordance with ihc (and her2 fish) results. therefore, it may be a potential solution to overcome ihc/fish limitations and improve the management of invasive breast cancer, especially in low-resource countries [13,15,21-25,43]. 5. conclusions in conclusion, determining hormone receptors (er/pr), her2, and ki67 status by immunohistochemistry (and in situ hybridization for her2 ihc 2+ cases) is part of the standard management of invasive breast cancer. there are questions related to technical issues with the standard tests used, and all international recommendations insist on improving the quality of tissue samples analyzed (pre-analytical phase), analytical techniques, and interpretation of results to ensure quality immunohistochemistry and molecular biology tests. the new rt-qpcr assay « xpert® breast cancer strat4 » gave promising results and a remarkable agreement with the reference techniques (ihc and fish). molecular diagnostics have become more and more essential in diagnosing and managing cancer in general, and further studies of xpert® breast cancer strat4 are needed with a larger sample size to support and confirm the results already published. ethics approval and consent to participate: all experiments were performed retrospectively following the moroccan bioethics law 28-13, and after the casablanca biomedical research ethics committee (cerbc) approval. author contributions: re realised the technical part, analyzed and interpreted the data, and significantly contributed to the manuscript. an study monitored and corrected the manuscript. ak selected samples with the pathologist and contributed to the technical analysis. yz contributed to the statistical study. mo & jw corrected the manuscript. mk study supervisor, selected eligible cases for the study, reviewed the h&e stained slide, interpreted the data, and corrected the manuscript. all authors read and approved the final manuscript. funding: this study was funded by cepheid®. cepheid provided all xpert® breast cancer strat4 (ceivd) kits. cepheid was not involved in sample selection nor the final data analysis. acknowledgments: the authors would like to thank all the collaborators who ensured the excellent progress of this study. pmmb 2023, 6, 1; a0000332 14 of 16 conflicts of interest: j.w. was an employee of cepheid at the time of this study. the funders had no role in selecting samples used in the study, collecting data from participating sites, interpreting the final data analyses in the study, or deciding to publish the results. the other authors declare that they have no competing interests. references 1. ferlay j, colombet m, soerjomataram i et al. cancer statistics for the year 2020: an overview. int j cancer 2021; 149(4):778-89. 2. lye kl, tan lt, yap hm. insight of 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distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology 1 original research article microbial community diversity in the soil of barrientos island estimated by rapd and biolog ecoplate methods learn-han lee1, vengadesh letchumanan1, nurul-syakima ab mutalib2, yoke kqueen cheah3* 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia 2ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia 3department of biomedical sciences, faculty of medicine and health sciences, universiti putra malaysia, serdang 43300, malaysia abstract: the diversity of soil microbial communities at barrientos island with different soil characteristics were evaluated using pcr-based method random amplified polymorphic dna (rapd) and community level physiological profiles (clpp) of biolog ecoplate. the soils were selected from 17 different locations around barrientos island inhabited by different breeders. shannon-weaver index and multivariate analysis were performed to characterize variations of soil microbial communities. both rapd and clpp methods exhibited that most soils with different type of rookery and characteristics could possibly affect the dna sequence diversity and soil microbial diversity. the abandoned type of rookery had the highest shannon-weaver index as exhibited by soil sample 445 (3.4 for rapd) and 450 (3.09 for clpp). higher coefficients of dna sequence similarity were found in soil samples colonized by similar breeders, like soil 442 and 446 (both were active chinstrap rookery) shared highest similarity in dna sequences (73.53). the cluster analysis of rapd profiles by upgma and principle component analysis (pca) of biolog ecoplate exhibited similar influence of type of rookery and soil condition towards soil microbial community diversity. the results may suggest that the change in microbial community dna composition is accompanied with the change in microbial functional properties. keywords: microbial; diversity; random amplified polymorphic dna (rapd); biolog; soil; barrientos island received: 9th november 2019 accepted: 10th december 2019 published online: 18th january 2020 citation: lee l-h, letchumanan v, ab mutalib n-s, cheah y-k. microbial community diversity in the soil of barrientos island estimated by rapd and biolog ecoplate methods. prog microbes mol biol 2020; 2(1): a0000046. https:// doi.org/10.36877/ pmmb.a0000046 introduction the soil microbial community is the most important composition of the soil ecosystem[1]. they are involved in many ecosystem processes such as nutrient transformation, litter decomposition, plant health maintenance and soil organic matter formation[2]. soil microbiological parameters, such as microbial biomass carbon and basal respiration have been suggested and used as possible indicators of soil quality[3–5]. recently, soil microbial community structure is frequently used as indicators for soil quality and fertility[1]. however, the use of cultivation-based method to study soil microbial community are proven to be challenging as only a small fraction of microorganisms (less than 10%) are culturable[6,7]. the limitation of culturable methods resulted in difficulty to understand the shift in the complex microbial community of soils[8]. these limitations could be overcome by using biolog system and various molecular methods such as rapd, pcr-dgge, rflp and others[1,9–14]. the advancement of molecular methods has enabled various advancement and discovery in the study of microbial genomics[15–28]. the rapd analysis uses arbitrary short primers that amplify the intervening portion of the genome, and creating variably sized amplicons[29] that could be applied to study soil microbial community structure[9] and bacteria genome[30,31]. rapd has become a popular dna-based method as it is rapid, simple and able to provide meaningful information about soil microbial community than isolate-based methods[32]. the biolog system is used to assess community level physiological profiles (clpp) of a soil sample. it is a means of investigating the functional diversity of soils as they could reflect how the soil microbial communities could utilize a range of carbon substrates[33]. in this study, rapd and biolog ecoplate copyright 2020 by lee l-h et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) *correspondence: yoke kqueen cheah, department of biomedical sciences, faculty of medicine and health sciences, universiti putra malaysia, serdang 43300, malaysia. email: ykcheah@upm.edu.my 2 methods were used to investigate the influence of soil characteristics like type of rookery on soil microbial community diversity. material and methods environmental sampling in 2007, during the xi ecuadorian antarctic expedition to the research station “pedro vicente maldonado”, greenwich island, south shetland islands, sampling for analysis of microbial communities from the soil was carried out at barrientos island (coordinates: s 62° 24’ 18.7” to s 62° 24’ 32.4” and w 59° 44’ 13.2” to w 59° 45’ 39.3”). top soil samples of upper 20 cm layer (after removing the top 2–3 cm) were collected from 17 different sites within barrientos island. these sites have various interesting fauna and flora activities (table 1). soils were sampled into sterile plastic bags using an aseptic metal trowel, and kept in the dark for transport to malaysia. soils were subsequently stored at -20°c, with an aliquot stored at -80°c for molecular analysis like rapd. while a portion of each soil sample was stored in 4°c and analyzed by biolog ecoplate assay. table 1: soil samples characteristics. soil reference type of rookery / nest soil condition 442 active chinstrap penguin guano 443 abandoned gentoo penguin guano 444 abandoned gentoo penguin no guano 445 abandoned gentoo penguin no guano 446 active chinstrap penguin guano 447 active chinstrap penguin no guano 448 active penguin no guano 449 active chinstrap penguin guano 450 abandoned penguin guano 451 active gentoo penguin (resting-deleted) area guano 452 abandoned penguin guano 453 penguin resting area guano 455 active gentoo penguin guano 456 abandoned penguin no guano 457 active gentoo penguin no guano 458 seal colony guano 460 giant petrel nest no guano soil dna extraction and purification to minimize possible contaminants, all post-sampling manipulations were performed in a uv-sterilized laminar box hood, using sterile glass vials. total soil dna was extracted and purified from 1g dry weight of soil using gf-1 soil sample extraction kit (vivantis, selangor, malaysia). the kit uses a specially-treated silica-based material fixed into a column to efficiently bind dna in the presence of high salt. this kit applies the principle of a mini-column spin technology and the use of optimized buffers to ensure that only dna is isolated while cellular protein, humic acid and other low molecular weight impurities are removed during the subsequent washing stages. (cat. no: gf-sd-025). dna yield and quality were assessed by 0.8% (w/v) agarose gel electrophoresis following by dna quantification using a biophotometer (eppendorf, hamburg, germany) and ratio a260/a280 was measured. pure dna has an a260/a280 ratio of 1.7–1.9. pcr amplifications and fragment visualization rapd arbitrary primer opo 05 (5’-cccagtcact-3’), opo 06 (5’-ccacgggaag-3’) and opo 18 (5’-ctcgctatcc-3’) was used for amplification of soil dna by pcr using the eppendorf mastercycler (eppendorf, hamburg, germany). the pcr reaction mixture consisted of ~10 ng of soil bacteria dna, 2.0 ml of 10x optimized pcr buffer with 20 mm mgcl2, 2.0 ml of 10 mm dntps, 1 unit of taq polymerase (intron biotechnology, south korea) and 0.5 ml of 100 nm primer and sterile ultrapure water was added to final volume of 20 ml. the cycling parameters were 4 min at 94°c for pre-denaturation, 45 cycles each of 1 min at 94°c for denaturation, 1 min at 36°c for annealing, 1 min at 72°c for extension and 7 min at 72°c for final extension. the pcr amplification products were resolved by electrophoresis in 1.5% agarose gel (promega, madison, wi.), which was stained with ethidium bromide (0.5 mg ml–1) and viewed under a gel documentation system (alpha imager, alpha innotech, california). data analysis of rapd fingerprints three arbitrary primers were used to amplify microbial community dna from 17 soil samples. since primer sequences were random and non-selective to soil dna samples, amplification for one primer was equal to one random sampling from the whole microbial dna sequences[32]. the number of rapd fragments was considered to represent the rapd fragment richness (s) of the whole dna sequences. the calculations above rely on an assumption that each rapd fragment contributes equally to the microbial diversity[32]. since rapd fragments amplified in all 17 soil samples contributed to the diversity of dna sequences differently as compared to those fragments amplified in only one, two, and three samples, it was necessary to make a modification for another diversity calculation. a fragment amplified in all 17 soil samples had the smallest contribution to the diversity because of no polymorphism, and therefore scored 0 for the diversity. a fragment amplified in only one sample had the biggest contribution and scored 1. the other fragments, amplified in two or three samples, counted 2/3 or 1/3, respectively. this modification (modified richness, i.e., modified s), in fact, enlarged the contribution of the characteristic sequence to the dna sequence diversity. the richness and modified richness of soil microbial community dna sequences reflect to cermicrobial community diversity... 3 tain extent the diversity of soil microbial community dna sequences, but do not indicate the relative abundance of soil microbial community dna sequences. shannon-weaver index is developed to measure the species diversity of the community by integrating species richness and abundance. her, we used shannon-weaver index as a measure of soil microbial community dna sequence diversity by molecular marker. diversity of soil microbial community dna sequences was estimated using the equation below: s s dsh = -∑ pilnpi = ∑ (ni / n)ln(ni / n) i=1 i=1 where dsh is shannon-weaver index, pi is the percent of the ith rapd fragment gray degree to each dna sample, ni is the net gray degree quantity (subtracted by the background gray degree of a gel) of the ith rapd fragment in each dna sample, n is the total net gray degree quantity of all rapd fragments examined in each dna sample, and s is the number of rapd fragments in each dna sample. the range of dsh is between 0 and ln(s). by merging the data of rapd fragment net gray degrees from each primer into a single dataset, a cumulative diversity of shannonweaver index was calculated for each sample using the same equation above. rapd-based cluster analysis by using bionumerics version 6.0 gel analysis software (applied maths, kortrijk, belgium), the position of the markers in rapd gels were normalized from lane-tolane and gel-to-gel variation. this normalization enables comparison of banding patterns originating from different rapd gels, provided there was a high degree of gel reproducibility based on migration of standards. then a binary matrix was constructed for each microbial community based on the presence and absence of bands. jaccard’s coefficient (a similarity measurement) was used to calculate the matrix and the data were subjected to clustering based on the unweighted pair group method using arithmetic averages (upgma) to identify samples that generated patterns similar to each other [34]. results were displayed in dendrogram form to illustrate the relationship between microbial communities from different soil. biolog ecoplate inoculation and incubation one hundred and fifty ml of sediment slurry from each sample was placed in sterile 400 ml beakers. physiological saline solution was added to bring the volume to 200 ml. the resultant slurry was sonicated in a water bath for 5 min before 15 ml of the supernatant was extracted and centrifuged at 500 g for 3 min. a 150 ml aliquot of the centrifuged supernatant was then used to inoculate a microtitre biolog ecoplate. biolog ecoplates have 96 wells that contain 31 unique carbon compounds, in addition a control of distilled water repeated in triplicate. when bacteria use one of these carbon sources, tetrazolium dye is reduced by bacterial respiration and accumulates as insoluble formazin. these results in the well turning from clear to darker shades of purple depending on the amount of formazin produced[35]. each plate was cultivated at 25°c for 168 h, and the optical density at both 590 nm (color development plus turbidity) and 750 nm (turbidity only) was read every 24 h[36]. biolog data analysis the final values used to represent the activity in each well were the 590 nm values minus the 750 nm values after being corrected for readings in the control well at these wavelengths. well optical density values that were negative or under 0.06 were set to zero[36]. average well color development (awcd) was calculated as described by garland and colleagues, i.e. awcd (590–750 nm) = σ(c590–750)/31, where 31 represents the number of carbon sources used in biolog ecoplate. the final values of each well at 168 h were used to calculate the shannon’s diversity index (h), where h = − σ(pilnpi), pi is the proportional optical density value of each well, and pi=c590–750/σ (c590–750). normalized data were analyzed by principal component analysis. results rapd analysis in the present study, 3 arbitrary primers were used to amplify soil microbial dna. the rapd pattern generated by arbitrary primers opo 05, 06 and 18 for 17 soil samples were shown in figure 1 as gel photo. three primers generated a total of 416 rapd fragments with opo 05, opo 06 and opo 18 generated 130, 142 and 144 fragments, respectively. the number of fragments scored per primer ranged from 1 to 11, 5 to 11 and 3 to 12 for primer opo 05, 06 and 18, respectively. eight of the total fragments (1.9%) were polymorphic. the ratio of polymorphic fragments in each primer was 3.1% (opo 05), 1.4% (opo 06) and 1.4% (opo 18), respectively (table 2). lee l-h et al. table 2: three primer amplified outputs to microbial community dna from 17 soil samples. primers amplified fragment non-polymorphic amplified fragments polymorphic fragments ratio of polymorphic fragments to total fragments opo5 130 126 4 3.1% opo6 142 140 2 1.4% opo18 144 142 2 1.4% total 416 408 8 1.9% 4 microbial community diversity... figure 1: rapd fingerprint for 17 soil samples for opo 05 (a), opo 08 (b) and opo 18 (c). lane “m” contain dna molecular weight markers. the numbers indicate the soil sample number as in table 1. 5 rapd analysis — diversity of soil microbial communities dna sequence rapd fragment richness (s) in dna for 17 soils microbial communities in barrientos island are shown in table 3. average rapd fragments richness for sample 445 and 446 were both 10.7, respectively, which were the highest richness among all soils from barrientos island. soil sample 443, 456 and 444 were the subsequent samples with high average richness value of 10, 9.3 and 9, respectively. whereas soil 457 contain the lowest richness, with average only 4 rapd fragments per primer. three soils comprised of relatively low richness, which is 457, 458 and 455 with 4, 4.3 and 5.3 rapd fragments per primer, respectively. the modified richness (modified s) for 17 soils showed that sample 445 had the highest value for average of modified richness (7.09); as soil 457 had the lowest value (2.44) (table 4). by assigning a standard value of 1 for modified richness for the soil with the highest diversity (445), soils with high richness value were 445, 445 and 443 with 1, 0.95 and 0.91, respectively. the shannon-weaver indices (dsh) for microbial community at rapd are shown in table 5. the cumulative diversities of shannon-weaver indices for 445 were the highest (3.40), whereas 457 was the lowest (1.16). coefficient of dna sequence similarity indicated differences between soils at dna level (table 6). coefficient of dna sequence similarity was the highest (73.53) between sample 442 and 446. the similarity coefficient between sample 447 (active chinstrap penguin rookery) and sample 458 (seal colony rookery) was the lowest (6.07). lee l-h et al. table 3: richness (s) for soil microbial community in 17 soil samples at dna level. primer soil samples 442 443 444 445 446 447 448 449 450 451 452 453 455 456 457 458 460 opo5 7 10 6 9 11 7 9 9 9 10 5 7 5 6 1 4 9 opo6 7 9 9 11 11 9 9 8 8 6 9 6 7 11 8 5 9 opo18 12 11 12 12 10 7 7 7 8 5 7 10 4 11 3 4 8 total 26 30 27 32 32 23 25 24 25 21 21 23 16 28 12 13 26 average 8.7 10 9 10.7 10.7 7.7 8.3 8 8.3 7 7 7.7 5.3 9.3 4 4.3 8.7 table 4: modified richness (modified s) for soil microbial community in 17 soil samples at dna level. primer soil samples 442 443 444 445 446 447 448 449 450 451 452 453 455 456 457 458 460 opo5 3.50 5.88 3.31 5.69 6.44 3.31 6.56 5.19 6.38 6.56 2.88 4.13 3.63 4.06 0.69 2.63 6.5 opo6 3.56 5.81 4.56 6.88 7.00 5.00 6.06 4.44 4.63 3.56 6.06 2.94 4.75 7.69 5.19 3.13 5.88 opo18 7.88 7.69 8.25 8.69 6.81 4.50 4.56 3.56 4.88 3.13 5.00 6.69 2.50 7.25 1.44 2.13 4.94 average modified s 4.98 6.46 5.37 7.09 6.75 4.27 5.73 4.40 5.30 4.42 4.65 4.59 3.63 6.33 2.44 2.63 5.77 relative value 0.70 0.91 0.76 1 0.95 0.60 0.81 0.62 0.75 0.62 0.66 0.65 0.51 0.89 0.34 0.37 0.81 table 5: shannon-weaver diversity indices (dsh) of microbial community in 17 soil samples from random amplified polymorphic dna (rapd) and community level physiological profiles (clpp) analysis. methods soil samples 442 443 444 445 446 447 448 449 450 451 452 453 455 456 457 458 460 rapd 2.38 3.10 2.58 3.40 3.23 2.04 2.75 2.11 2.55 2.11 2.24 2.21 1.73 3.03 1.16 1.26 2.75 clpp 2.58 2.63 2.01 1.65 2.32 1.75 1.74 2.84 3.09 2.81 2.90 2.55 2.86 2.49 2.46 3.04 2.54 6 microbial community diversity... ta bl e 6: j ac ca rd ’s a ve ra ge s im ila ri ty c oe ffi ci en t o f 1 7 so il sa m pl es g en er at ed b y u pg m a a na ly si s. so il sa m pl es 44 2 44 3 44 4 44 5 44 6 44 7 44 8 44 9 45 0 45 1 45 2 45 3 45 5 45 6 45 7 45 8 46 0 44 2 10 0. 00 44 3 58 .2 9 10 0. 00 44 4 47 .1 1 49 .3 7 10 0. 00 44 5 58 .8 9 64 .3 3 48 .8 9 10 0. 00 44 6 73 .5 3 67 .7 1 61 .1 5 61 .6 2 10 0. 00 44 7 50 .3 8 56 .6 7 72 .7 5 40 .7 1 60 .8 8 10 0. 00 44 8 29 .0 2 35 .3 6 22 .9 3 18 .8 6 33 .9 0 23 .6 5 10 0. 00 44 9 46 .9 5 49 .0 3 51 .8 2 35 .3 3 53 .2 9 58 .1 9 33 .3 7 10 0. 00 45 0 43 .1 1 44 .0 2 47 .7 6 41 .6 1 38 .9 3 45 .3 8 18 .5 9 31 .5 0 10 0. 00 45 1 35 .1 0 36 .3 1 30 .6 3 23 .9 7 28 .2 6 24 .8 5 35 .4 5 33 .3 3 30 .8 0 10 0. 00 45 2 46 .6 7 47 .6 2 33 .0 6 35 .5 1 48 .1 1 55 .6 1 14 .6 5 46 .8 3 36 .4 5 23 .6 9 10 0. 00 45 3 32 .9 7 34 .1 7 21 .0 1 32 .2 9 31 .7 7 27 .1 5 37 .8 8 33 .3 3 35 .7 2 25 .9 7 23 .4 9 10 0. 00 45 5 24 .7 8 21 .7 2 33 .7 5 32 .9 2 26 .4 3 22 .0 9 14 .6 1 21 .3 7 20 .7 9 42 .0 3 23 .2 3 23 .7 1 10 0. 00 45 6 45 .8 7 28 .6 9 20 .6 4 37 .1 9 33 .9 9 27 .3 9 26 .1 5 44 .4 5 33 .7 9 27 .6 7 49 .7 5 25 .7 1 30 .4 7 10 0. 00 45 7 20 .4 5 22 .8 2 39 .9 7 35 .9 5 33 .4 3 22 .4 5 12 .9 7 17 .6 3 34 .2 9 34 .7 0 26 .3 9 23 .9 9 26 .6 7 22 .4 5 10 0. 00 45 8 15 .7 9 23 .2 3 17 .2 5 17 .5 0 24 .9 1 6. 07 ** 30 .9 1 6. 67 16 .2 4 13 .8 9 6. 67 29 .2 9 9. 53 19 .4 0 44 .4 5 10 0. 00 46 0 25 .0 0 29 .8 7 40 .2 7 38 .8 9 34 .7 6 40 .5 1 28 .2 9 13 .0 6 51 .3 6 21 .7 7 37 .7 8 26 .6 7 17 .9 9 30 .2 8 37 .6 8 37 .6 8 10 0. 00 7 rapd analysis — upgma analysis of rapd profiles rapd profiles were subjected to clustering based on unweighted pair group method using arithmetic averages (upgma) to identify soil samples that generated patterns similar to each other. upgma of 17 soil samples using composite analysis of opo 05, 06 and 18 revealed 8 clusters (figure 2) that clade together. within the 8 clusters produced, 5 clusters (i, iii, iv, vi, vii) comprised soil samples from similar type of rookery in each respective cluster. however, some soil samples from different type of rookery were clustered together, i.e cluster ii, v and viii. cluster i comprised of 2 abandoned lee l-h et al. penguin rookeries (452 and 456). cluster ii consisted of 1 abandoned penguin rookery (444) and 2 active chinstrap rookeries (447 and 449). cluster iii contained 2 active chinstrap rookeries (442 and 446) at high similarity level of 73.5% (table 6). cluster iv comprised 2 abandoned penguin rookeries (443 and 445) while cluster v contained 2 rookeries from abandoned penguin rookery (450) and petrel nest (460). cluster vi comprised of 2 penguin resting rookeries (448 and 453) whereas cluster vii consisted of 2 active gentoo rookeries (451 and 455). lastly cluster viii comprised of active gentoo (457) and seal colony (460) type of rookeries. figure 2. dendrogram derived from rapd composite analysis of arbitrary primer opo 05, 06 and 18. a total of 8 clusters were formed from 17 soil samples used in this study. biolog analysis — average well color development awcd of biolog ecoplates is an important index for elevated diversity of soil microbial biomass function. the values represent the changes of soil bacterial community activity in utilizing catabolic diversity in different type of rookery. the awcd generally followed the same pattern with incubation time, but the pattern varied for different soil samples (figure 3). in general, the awcd value was the highest in sample 458 (active seal rookery), and lowest in sample 444 (abandoned penguin rookery). the awcd values represent the metabolic activity of soil microbial community in using the carbon sources, thus proposed that the effect of rookery activities have influence on soil bacteria community metabolic function. biolog analysis — shannon’s diversity differences in the shannon-weaver index of the soil bacterial community of different rookery after incubating for 168 h in biolog ecoplate were observed (table 5). the shannon indices were significantly highest in sample 450, a soil with penguin abandoned type of rookery and soil surface covered by lots of guano. while sample 445 and 448 with low shannon indices of 1.65 and 1.74, respectively showed no guano at that area. biolog analysis — principle component analysis the principle component analysis was conducted to better understand differences in carbon utilization by soil microorganism. the pca plot shows that carbon substrate utilization profiles were able to clearly separate soil samples to group according to type of rookery (figure 4). five significant groups were formed (a, b, c, d, e), each according to certain type of rookeries. group a and b comprised of 3 (443, 444, 445) and 2 (452, 456) abandoned rookeries, respectively. group c consisted of soil samples from active chinstrap penguin rookeries (442, 446, 447, 448). group d comprised of penguin areas (453, 457) while group e consisted of gentoo penguin active rookeries (451, 455). 8 microbial community diversity... 0 0.2 0.4 0.6 0.8 1 1.2 24hrs 48hrs 72hrs 96hrs 120hrs 144hrs 168hrs 442 443 444 445 446 447 448 449 450 451 452 453 455 456 457 458 460 1 figure 3. difference in awcd of soil bacterial community over time for 17 different soil samples. figure 4. principle component analysis (pca) of biolog ecoplate data from different soil samples of barrientos island. discussion the potential microbial diversity as an indicator of soil quality is impeded due to the difficulties in measuring[9]. microbial population in soils are very large, with more than 109 organisms per gram of soil[37,38]. it is also very diverse with more than 104 species per gram of soil[39]. only 1–10% of these microbes can be isolated and studied in pure culture, therefore, various microbial methods are emerging as useful tools to study microbial communities in soils[9,40]. the difference among microbial species is fundamentally signified in their genetic diversity at dna sequence and their metabolic function. therefore, molecular method like rapd and substrate utilization pattern (biolog ecoplate) can be used to study soil microbial community diversity[9]. pcr-based technique like the rapd has become a popular method to assess soil microbial community as it is simple, rapid and sensitive means to identify small variations between similar genomes[41,42]. the biolog system was initially developed for bacterial identification. later the system has found to be useful to characterize soil microbial diversity from various different environments like soil, sediments, freshwater and seawater[35]. the substrate utilization patterns of the biolog system have been used to provide “fingerprints” of microbial community structure[43,44] and also as an indication of metabolic potentials[45,46]. the multivariate analysis of the utilization pattern of different carbon substrates generated from the 96-well biolog ecoplates enables the classification of microbial community functional diversity. the substrate utilization patterns in this study successfully differentiated most of the soil samples according to different type of rookery. 9 lee l-h et al. in this study, the rapd analysis demonstrated that different type of rookery activities could considerably affect soil microbial communities. as 80% (4/5) of highest rapd fragment rich samples were from abandoned penguin rookeries (445, 443, 444, 456) (table 1, table 3). compared to soil samples with active breeders’ type of rookeries (i.e. 457, 458, 455) that exhibited average richness range from 4–5.3, soil samples of abandoned penguin rookeries (443, 444, 445, 450, 452, 456) showed much higher average richness with value range from 7–10.7 (table 3). the shannon-weaver indices (dsh) for the microbial community at rapd in the 17 soils showed the average shannon indices for abandoned penguin rookeries (443, 444, 445, 450, 452, 456) were significantly higher than that of rookeries with active breeders (i.e. 457, 458, 455). this observation is indeed interesting and warrant further study to understand these patterns. for biolog ecoplate analysis of shannon-weaver indices, 77% (442, 443, 446, 449, 450, 451, 452, 453, 455, 458) of soil samples with high shannon index (>2.3) (table 5) were soil samples covered with guano, these could possibly infer that catabolic diversity of the soil bacterial community could be increased with the existence of organic manure like guano [1]. soil samples namely 445 (3.4 for rapd) and 450 (3.09 for clpp) which exhibited highest shannon index for rapd and clpp were both from abandoned type of rookeries. soil samples like 443, 450 and 452 all shared high shannon index in both rapd and biolog methods used. overall the correlation of shannon index between rapd and biolog is not really strong, this could be due to the variance of targeted microbes in both methods, as biolog method only reveals fast growth bacteria activity only. in regards to coefficient of dna sequence similarity, results indicated the possibility of different samples colonized by different breeders caused a shift in the species composition of soil microbial communities. as for most of the soil samples colonized by similar breeders, comparatively higher coefficients of dna sequence similarity were found, like sample 442 and 446 (both active chinstrap rookery) shared highest similarity in dna sequences (73.53) (table 6). the awcd analysis of biolog ecoplates revealed changes of soil bacterial community activity in utilizing catabolic diversity in different type of rookery. from 10 soil samples with highest awcd values, 7 were from active breeders’ rookeries and only 3 were from abandoned type of rookeries. the average awcd value for all the 9 active breeders rookeries (442, 446, 447, 449, 451, 455, 457, 458, 460) were 0.4203, which is significantly higher than average awcd of 6 abandoned rookeries (443, 444, 445, 450, 452, 456) with value of 0.2998. therefore, this suggested that the metabolic activity of soil bacteria was higher in active breeders’ rookeries and lower in abandoned type of rookeries. as a result, this observation showed that the effects of rookery activities have influence on soil bacteria community metabolic function. the cluster analysis of rapd profiles by upgma showed great similarity in dna profiles for microbial communities that shared similar type of rookery. total 63% (5/8) of the clusters formed were from similar type of rookery in each respective cluster (figure 2). these results suggest that there was a systematic change in the sequence diversity associated with different type of rookery at sampling sites. the pca results of ecoplate showed that carbon substrate utilization profiles were able to clearly separate most soil samples (76%) to group according to type of rookery (figure 4). sample 458 and 460, inhabited by seal and petrel respectively, both were evidently separated from the rest of the samples which were inhabited by penguins. this observation from clpp method revealed that there is positive influence of type of rookery and soil condition towards soil microbial community diversity. in generally, combination of molecular methods with other tools can be used to improve our understanding of the effect of different soil characteristics to soil microbial diversity. in this study, the genetic diversity of microbial populations by rapd genetic fingerprinting and metabolic diversity using biolog substrate utilization assays were used to investigate the effect of different type of rookery activities and characteristics on soil microbial populations. both rapd and clpp method revealed the similar influence of type of rookery and soil condition towards soil microbial community diversity. the results may suggest that the change in microbial community dna composition is accompanied with the change in microbial functional properties[9]. nonetheless, both the methods have limitations in determining soil microbial community. the rapd method may be affected by effects of pcr bias, like the size of random primer, sensitivity to reaction conditions and the possibility of co-migration[47,48]applications of random amplified polymorphic dna (rapd. the biolog system assesses the metabolic diversity of culturable bacteria only. it is a system that could indicate activity of the fast growth bacteria or eutrophic bacteria only. therefore, microorganisms like slow-growing bacteria, fungi and uncultured bacteria activity are expected to have minimal influence on the microbial metabolite profile[49,50]. so, only a part of soil microbial characteristics was discovered by the biolog ecoplate method. as a conclusion, it is necessary to incorporate comprehensive approaches at diverse level, including traditional, metabolic and molecular level to understand more precisely about the changes in the diversity of microbial communities[32,51]. author contributions 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microbiome in irritable bowel syndrome (ibs) shabnam mohajir selvaraj1, sunny hei wong2, hooi-leng ser1*, learn-han lee1* 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. 2li ka shing institute of health sciences, department of medicine and therapeutics, the chinese university of hong kong, shatin, hong kong. abstract: irritable bowel syndrome (ibs) is a chronic disease prevalent in today’s society and diet remains the most common aggravator of ibs symptoms. existing literature suggest that ibs patients are dysbiotic as evidence indicates decreased levels of bifidobacteria, bacteroidetes and faecalibacterium prausnitzii and increased levels of firmicutes in comparison to healthy individuals. studies suggest that changes in diet can modulate gut microbiota and therefore improve ibs symptoms. the two diets being investigated are the low fodmap diet and the use of probiotics. a low fodmap diet implements a reduction in the amount of poorly absorbed carbohydrates and probiotics are live microorganisms that have been proven beneficial when consumed appropriately. based on the literature acquired from pubmed, a positive correlation appears to exist between the low fodmap diet and ibs symptoms; 57% report symptom relief. there is also a notable effect on the gut microbiome after changing to low fodmap diet, noted with a significant decrease in levels of bifidobacterium, clostridium, f. prausnitzii and actinobacteria. this poses a concern as bacteria such as bifidobacteria and f. prausnitzii are beneficial for health. when probiotics are taken amongst ibs patients a reduction in symptoms is also observed. additionally, there is an increase in the abundance of bifidobacterium and lactobacilli. it is suggested that co-administration of probiotics with a low fodmap diet may ensure beneficial levels of bifidobacterium while ibs symptoms ameliorate. keywords: fodmap; gut microbiome; probiotics; irritable bowel syndrome (ibs). received: 10th march 2020 accepted: 12th april 2020 published online: 21th april 2020 citation: selvaraj sm, wong sh, ser h-l, et al. role of low fodmap diet and probiotics on gut microbiome in irritable bowel syndrome (ibs). prog microbes mol biol 2020; 3(1): a0000069. https://doi.org/10.3687/pmmb.a0000069 introduction irritable bowel syndrome (ibs) is a functional bowel disorder and it is more prevalent than one may think. in fact, ibs has a global prevalence of 11% and accounts for up to 60% of outpatient appointments in gastroenterology (figure 1)[1]. it is a disease that affects between 10–15% of individuals in western countries, while slightly lower rates are seen in asia, 2.3–11.5%[2]. while the prognosis for this condition is non-fatal, the impact on an individual’s quality of life remains significant; ibs patients are 7.93 times more likely to experience severe pain and discomfort, 2.83 times more likely to complain of issues relating to mobility and 2.39 times more likely to experience anxiety and depression[3]. ibs requires an effective and long-term treatment and existing literature has made it abundantly clear that food is a crucial factor correlating with the presentation and severity of gastrointestinal symptoms; 80% of all ibs patients report being able to identify a minimum of one food item that aggravates their symptoms[4]. furthermore, this disorder is also known to be influenced by a range of factors such as diet, anxiety, gastrointestinal inflammation and family history, thus may require a more holistic approach in its management. the current practice of management of ibs mainly focuses on relief of symptoms such as abdominal pain in combination with dietary modification including the introduction copyright © 2020 by selvaraj sm and hh publisher. this work under licensed under the creative commons attribution-noncommer cial 4.0 international lisence (cc-by-nc4.0) *correspondence: hooi-leng ser, novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; ser.hooileng@monash.edu; hooileng_ser@y7mail.com. learn-han lee, novel bacteria and drug disovery (nbdd) research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; lee.learn.han@monash.edu 2 of low fermentable oligosaccharides, disaccharides, monosaccharides and polyols (fodmap) diet. on the other hand, some studies have unveiled the relationship between gut microbiome and ibs, suggesting that the imbalance in microbial population may aggravate ibs symptoms[5]. therefore, these evidence then lead to the idea of probiotics intake as a means to reinstate balance in the microbial balance. probiotics is thought to prevent bacterial overgrowth by improving gut barrier function and receptor interactions, while at the same time producing a range of protective substance include short chain fatty acids (scfas)[6]. thus, the directive of the current study aims to provide an overview on the role of gut microbiome in ibs, while consolidating the data on how diet modifications, particularly low fodmap diet and/or intake of probiotics improve ibs via actions on the gut microbiome. ibs, fodmap, probiotics... what is irritable bowel syndrome (ibs)? as one of the most common functional bowel disorders (fbd), the pathogenesis of ibs remains convoluted. based on the rome iv criteria, ibs is defined as a fbd which is distinguished by recurrent abdominal pain accompanied with changes in bowel habits and defecation pattern, in addition to symptoms such as bloating[7]. the criteria classifies ibs into subtypes according to the bristol stool scale; ibs with diarrhea (ibs-d), ibs with constipation (ibs-c) and ibs with mixed bowel habits or cyclic pattern (ibs-m)[8]. ibs has been associated with a range of intestinal and extra-intestinal presentations; there is often an overlap between the following three conditions—functional dyspepsia, gastroesophageal reflux disease and ibs[9]. urological and gynaecological problems also arise in patients with ibs. on top of the associations made with physical health, mental health has been affected in ibs, with cases of depression and anxiety co-existing. the diagnosis of ibs is commonly suspected through extensive history taking; a pattern of intermittent or continuous abdominal pain, relief with defecation, bloating, flatulence and a lack of alarming features (loss of weight, loss of appetite or an abdominal mass) is suggestive of ibs. the aetiology of ibs is multifactorial and the pathogenesis of the disease is not fully understood. however, some evidence showed the potential role of gut-brain axis in development of ibs. known as the“second brain”, the huge network of neurons lining the gut forms the enteric nervous system which consists of more than 100 million neuronal cells[10]. evidence suggests that symptoms such as diarrhea and/or pain result from exaggerated responses of the intestine to food or stress and that abnormal mucosal secretions and serotonin uptake by enterocytes cause alterations in gastrointestinal motility[11,12]. visceral hypersensitivity or pain/discomfort occurs as a result of heightened perception of mechanical triggers applied to the bowel is commonly thought to be the conditions leading to ibs; up to 50% of patients have been found to have increased visceral perception[13,14]. as a matter of fact, gut-brain axis exists as a bidirectional interaction and psychological distress can cause ibs symptoms flareup and exaggeration[15]. besides causing activation in immune system, stress could further enhance dysbiosis or imbalance in the microbial population residing in the gut, which then lead to the development of vicious cycle of inflammation[16–20]. inflammation is another factor in the pathogenesis of ibs as there is increasing evidence of the association between enteric infection and the development of post infectious ibs (pi-ibs) along with studies demonstrating a greater abundance of pro-inflammatory cytokines like il-1β and il-8 in ibs patients[20, 21]. an eightyear follow up study conducted by marshall et al. showed that 15.4% of patients (n = 488) were diagnosed with ibs using the rome i criteria, suggesting an association between enteric infections and persistent ibs symptoms[20]. nevertheless, the study also noted other significant risk factors for the development of pi-ibs such as anxiety and depression, which is in-line with other evidences proposing participation of gut-brain axis in ibs[21, 22]. although the exact mechanism remains unclear, the authors suggested an association between enteric infections and persistent ibs symptoms, particularly caused by prolonged alteration in gut microbiota composition and chronic inflammation. above all, the current treatment for ibs patients is mainly to relieve symptoms, whereby the first line management primarily attempts to control diarrhea and constipation along with some over-the-counter medications (e.g. nonfigure 1: targeting gut microbiome for the treatment of ibs. 3 selvaraj sm et al. predominant bacterial phyla comprise of bacteroidetes and firmicutes, along with smaller populations of fusobacteria, actinobacteria and verrucomicrobia[37]. typically, the complex microbial community in gut stabilizes after two to three years of age[38]. nevertheless, it should be highlighted that variations exist between individuals as a multitude of factors contribute to the microbial composition such as age and diet to name a few[37,39,40]. dysbiosis in the gut microbiome has been suggested to be associated with a combination of both acute and chronic diseases, or, increase the risk of its development. the different potential mechanisms on how dysbiosis and/or certain metabolites can lead to mucosal leakiness in the gut and promote inflammation milleu in entire body has been actively conversed over the past ten years[41–46]. in the recent years, many researchers have witnessed the changes in gut microbiome and its relationship to the pathogenesis of illnesses, particularly in those with ibs[30,47–51]. in patients with pi-ibs, it is simply obvious that those with history of enteric infection have a higher risk of developing ibs, given that the abundance of microbial population in the gut changed with the colonization of pathogens [51,52]. these patients have been shown to have decreased levels of bifidobacteria, bacteroidetes and f. prausnitzii and an increase in the abundance of firmicutes. a 2-fold decrease in bifidobacteria is found in ibs patients compared to healthy controls; bifidobacteria is beneficial to the host as it produces acetic and lactic acid, thereby preventing the growth of pathogenic bacteria and maintaining the immune system[52–54]. apart from the differences in microbial taxa, studies have also suggested a reduction in microbial diversity, richness and temporal stability. tap et al. found that the severity of ibs symptoms is negatively associated with microbial richness and enterotypes enriched with clostridiales/prevotella species[55]. this was further emphasized by halkjaer et al. whereby ibs patients have a lower stool microbial biodiversity, suggesting an association between ibs and gut microbiome[56]. in conjunction with this, there are some studies suggest that microbial composition varies according to ibs subtypes. in a study conducted by jeffery and team, lactobacilli was found in greater abundance in ibs-d patients in comparison to ibs-c patients[52]. additionally, the luminal microbiota of patients with pi-ibs was distinguishable from non-pi-ibs patients, but like those with ibs-d. furthermore, kroguis-kirikk et al. found a reduction in microbial diversity in ibs-d patients and this can be related to the reduction in microbial composition identified in patients with acute diarrhea[57]. conversely, similar results were obtained in animal models of ibs, whereby fecal transplantation using stool from ibs patients induced visceral hypersensitivity in germ-free rats[58]. relationship between low fodmap diet and gut microbiome following the diagnosis of ibs, dietary changes are often crucial steps to control unwanted symptoms such as diarrhea and bloating. the traditional ibs diet focuses a great deal on when and how to eat. commonly, patients are told to eat 3 meals and 3 snacks a day and to reduce their intake of foods such as coffee, fatty foods, alcohol and spicy dishes[59]. along with that, since the knowledge of saccharides causes steroidal anti-inflammatory drugs, anti-flatulent, bulking agent for diarrhea)[8,23]. on top of that, patients are often advised to change their lifestyles, including stress management and dietary modifications such as high fiber intake, probiotics administration and/or adaptation of a special diet known as the low fodmap diet. fodmap refers to a group of carbohydrates, with 1–10 sugars, that are poorly absorbed[24,25]. when ingested, they have an osmotic effect; drawing water into the lumen of the small intestine. in the distal ileum and colon, fodmaps are fermented to produce short-chain fatty acids and gases such as methane, carbon dioxide and hydrogen. such products of fermentation result in gastrointestinal symptoms in individuals with existing issues of visceral hypersensitivity and gut motility[23,26]. another proposed mechanism through which fodmaps generate gastrointestinal symptoms is through immune activation. urinary metabolomic profiles of ibs patients were analysed after low and high fodmap diets. such an intervention indicated that patients who took a low fodmap diet demonstrated a slight decrease in urinary histamine levels. in addition, a decrease in inflammatory cytokines il-6 and il-8 have also been identified in patients undergoing a low fodmap diet[27,28]. nonetheless, fodmaps may act as prebiotics, inducing growth and/or activities of bacterial in the gastrointestinal tract thereby changing the composition of the gut microbiome[27,29]. so, what is the role of gut microbiome in ibs and more importantly can we tackle the symptoms of this chronic disease by targeting gut microbiome? gut health: ibs and microbiome out of the 38 trillion bacterial cells living in the human body, more than one-third (33%) of them are residing in the gastrointestinal tract[30,31]. gut microbiota has multiple functions and proves beneficial for a range of reasons. for instance, gut microbiome serves like a“physical barrier, preventing colonization by harmful pathogen[32]. in reality, gut microbiome was thought to be sterile before birth, but this dogma was challenged numerous times in the past 10 years with studies identifying bacterial dna and/ or their product present in the placenta, amniotic fluid or even meconium[33]. having said that, the gut microbiome also plays an indispensable role in the development of the immune system; the intestinal mucosa comes in contact with external antigens, presenting antigens and “educating” immune cells on when to attack via binding on receptors (e.g. toll-like receptor recognition) [32,34]. several observations in rodent models showed that germ free animals (i.e. absence of microbiota) displayed immunological defects with reduced number of immune cells and abnormal functioning[35]. the connection between the gut and health has been well established for centuries; in 400 b.c. hippocrates stated that death is in the bowels”and implied that indigestion would be the cause of all illnesses. thousands of years have passed since then and later in 1916, ali metchnikoff suggested that diseases originated from the gut, when bad”bacteria can no longer be controlled. the ideal state in gut microbiome or eubiosis is achieved when there is a balance in bacterial abundance, particularly relative abundance of pathogens that may pose serious infectious risk[36]. in the gastrointestinal (gi) tract, 4 gastrointestinal symptoms came to light, ibs patients are introduced to a meal plan”with low composition of fodmap content as these are considered potential triggers[25,30]. in a low fodmap diet, the daily intake of fodmaps in an ibs patient is reduced from 15–30 g/d to 5–18 g/d. the execution of this diet requires global restriction for 4–8 weeks followed by reintroduction of food according to a patient’s tolerance. as a consequence, this diet is not only “therapeutic” food avoidance plan but can also use as a diagnostic tool to test food intolerance. the low fodmap diet can be deemed clinically effective as a total of 50–70% of patients with ibs reported adequate symptom relief, marked by a lower ibs symptom severity score after implementing a low fodmap diet[1,27,60]. evidence suggests that gastrointestinal symptoms ameliorate, with reported reductions in bloating, borborygmi, stool frequency, urgency and improvement in consistency[1,59,61]. taking a closer look at the microbial composition, hustoft et al. indicated that reduction in fodmap intake (50%) produced a significant change in microbial composition; a 6-fold reduction was observed in those following low fodmap diet in the relative abundance of bifidobacteria, in comparison to individuals on normal diet (table 1)[28]. similarly, silk et al. found that a significant reduction in bifidobacteria abundance was seen following a 3-week implementation of a low fodmap diet. differing from histoft et al., this study also found a 47% overall reduction in total bacterial count and a reduction in abundance of other bacterial groups[62]. a significant reduction was found in clostridium, f. prausnitzii, bifidobacterium, megasphaera, pediococcus and actinobacteria and patients presented more dysbiotic gut microbiome. likewise, a study by mcintosh et al. highlighted an increase in the relative abundance of bacteria involved in gas consumption in patients on low fodmap diet[27]. members of genus adlercreutzia are described as hydrogen “consumers” for equol production, while reducing gas formation and eventually preventing symptoms of bloating and pain simultaneously[27,63]. table 1. effect of low fodmap diet on ibs symptoms and gut microbiota. study design methods impact of low fodmap diet on ibs symptoms references randomised controlled trial ibs symptom scoring system (ibs-sss) 57% had adequate pain relief vs control (38%) 73% achieved > 50 reduction of ibs-sss score vs control no significant difference for ibs-qol [61] randomised controlled trial ibs symptom scoring system (ibs-sss) mean decrease of 28% vs. control high fodmap group had a mean increase of 7% in symptoms 52% reduction in abdominal pain positive correlation between ibs symptom severity and level of fodmap consumption [27] randomised controlled trial ibs symptom scoring system (ibs-sss) symptom severity was reduced in both groups there was no significant difference between the ibs-sss scores in both groups compared to baseline. the number of bowel movements reduced in the low fodmap group compared to the traditional diet group. [59] study design methods impact of low fodmap diet on gut microbiota references randomised controlled trial 16s rrna increase in the number of dysbiotic patients after the low fodmap diet (60%) reduction in clostridium, faecalibacterium prausnizii, bifidobacterium, megasphaera, pediococcus and actinobacteria. [28] randomised cross over controlled trial fish no difference in the relative proportion of each bacterial group at baseline and post intervention. higher proportions of bifidobacterium spp. lower proportions of c. perfingens subgroup histolyticum and bacteroides/prevotella spp. [62] randomised controlled trial 16s rrna operational taxonomic units (otu) no difference between α-diversity or β-diversity. higher actinobacteria richness and diversity ibs-m and ibs-d had higher bacterial richness (firmicutes, clostridiales and actinobacteria). actinobacteria richness was increased [27] randomised controlled trial 16s rrna responders had high levels of: bacteroidaceae, clostridiales (ruminococcaceae, dorea and faecalibacterum prausnitzii) and erysipilotrichaceae [64] randomised controlled trial ga-maptm dysbiosis test responders has a high level of: bacteriodes fragilis, acinetobacter, ruminiclostridium, streptococcus and eubacterium. responders had lower levels of: clostridials, shigella/escherichia, unable to identify a significant change in dysbiosis. [65] ibs, fodmap, probiotics... 5 even so, some researchers argued that the effectiveness of the low fodmap diet may be dependent upon individual gut microbial composition. chumpitazi et al. conducted a study among children and concluded that 24% of patients who experienced symptom response had a high abundance of bacteroides, ruminococcaceae and f. prausnitzii[64]. these findings therefore proposed that the greatest benefit of a low fodmap diet can be seen in patients who have a high concentration of microbiota with saccharolytic potential. amongst adults, patients who respond significantly to the diet have been found to have higher levels of bacteroides fragilis, acinetobacter, ruminiclostridium, streptococcus and eubacterium[65]. administration of probiotics in ibs patients in ibs patients, several studies have indicated a drop in abundance of certain beneficial microbes or probiotics, particularly those under the phyla bifidobacterium and actinobacteria[51,52,66]. besides producing lactic selvaraj sm et al. acid from digested dietary sugars, some probiotics such as lactobacillus, lactococcus, bifidobacterium and streptococcus are capable of producing small chain fatty acids (scfas) like butyrate, propionate and acetate through fermentation of ingested food[67]. scfas, in particular butyrate is the primary energy source for colon cells and plays a central role in intestinal maintenance through antiinflammatory actions[68–71]. in addition to that, one of the most traditional theory on how probiotics confer protection to the host is by displacing pathogenic gi bacteria, preventing their colonization and subsequently re-establishing the balance in gut microbiome. in the light of current research on ibs, many researchers have reported the benefits of probiotics in ibs patients, particularly in the reduction of ibs symptoms such as abdominal pain and bloating[72–76]. a multicenter studies conducted in india by ducrotte and team showed that 78.1% of patients consider treatment with lactobacillus plantarum 299v to be effective (table 2)[73]. table 2. the effect of probiotics on ibs symptoms and gut microbiota. study design methods probiotics intervention (form: single/multi-strains; dose; duration) impact of probiotics on gut microbiota references monitoring ibs symptoms and quality of life monitoring of microbiome randomised controlled trial patient diary; rand-36 n.r. capsule: l. rhamnosus gg, l. rhamnosus lc705, b. breve bb99 and p. freudenreichii ssp. shermanii js (valio ltd, helsinki, finland. equal amount of strain: each 8–9×109 cfu/day duration: 6 months a. 42% reduction in symptom score b. probiotics appeared to be beneficial for all symptoms. c. significant reduction in scores for borborygmi [78] randomised controlled trial patient diary; rand-36 human intestinal tract chip (hitchip, agilent technologies) drink: l. rhamnosus gg (atcc 53103, lgg (valio ltd, helsinki, finland)), l. rhamnosus lc705 (dsm 7061), p. freudenreichii ssp. shermanii js (dsm 7067) and b. animalis ssp. lactis bb12 (dsm 15954) equal amount of strain: each 1×107 cfu/ml duration: 5 months a. higher mean reduction in ibs score in the probiotic group, recorded at 37%, compared to only 9% reduction in the placebo group b. health-related quality of life analysis showed improvement in the probiotic group for the domain describing bowel symptoms [72] randomised controlled trial visual analogue scale (vas) n.r. capsule: l. plantarum 299v (dsm 9843) amount: 1×1010 cfu/day duration: 4 weeks a. 45.2% reduction in mean severity of abdominal pain. b. 78.1% found the treatment good/excellent compared to placebo (8.1%) c. no significant side effects [73] randomised controlled trial n.r. qpcr (selected bacterial group only) capsule: l. rhamnosus gg (atcc 53103), l. rhamnosus lc705 (dsm 7061), p. freudenreichii ssp. shermanii js (dsm 7067) and b. breve bb99 (dsm 13692) equal amount of strain: each 8–9×109 cfu/day duration: 6 months a. intestinal microbiota remained stable except for bifidobacterium spp. b. number of bifidobacterium spp decreased [76] 6 randomised controlled trial clinical assessment at baseline and end of treatment telephone interview (1 month after end of treatment) qpcr vsl-3: 9.3 × 1010 cfu/g of bifidobacterium (b. longum y10, b. infantis y1 and b. breve y8), 2.7×109 cfu/g of lactobacillus (l. acidophilus, l. casei, l. delbrueckii subsp. bulgaricus and l. plantarum) and 2× 1011 cfu/g of streptococcus salivarius subsp. thermophilus. duration: 20 days a. significant increase in lactobacilli, bifidobacteria and streptococcus thermophilus b. no significant changes in enterococci, coliforms, bacteroides and clostridium perfingens. [82] randomised controlled trial n.a. qpcr (selected bacterial group only) capsule: lactobacillus rhamnosus gg, l. rhamnosus lc705, propionibacterium freudenreichii sp. shermanii js and bifidobacterium breve bb99 equal amount of strain: each 8–9×109 cfu/day duration: 6 months a. group receiving probiotics appeared to be display lesser monitored ibs-related gi symptoms. b. significant decrease in the amount of r. torques 94%, increase in c. thermosuccinogenes 85%. c. increased abundance of ruminococcus torques 93%. d. bifidobacterium decreased in the probiotic group [79] ibs patients reported overall reductions in stool frequency, bloating and feeling of incomplete emptying frequency after 4-weeks administration of probiotics. furthermore, there are studies emphasized that multi-strain probiotics have shown a more significant impact on improving gastrointestinal symptoms as compared to single strain probiotics. this is likely due to the fact that more niches are colonized, thus having a greater effect on gut motility[76,77]. a study using a probiotic mixture containing lactobacillus rhamnosus gg, l. rhamnosus lc705, bifidobacterium breve bb99 and propionibacterium freudenreichii sp. shermanii revealed that ibs patients experienced lesser symptoms, achieving a reduction rate as high as 42% in symptom score, whereas the placebo group only reported a 6% reduction[78]. also, the gut microbiome of ibs patient receiving probiotics was indeed different compared to the placebo as examined using similarity index which compared the stability of the microbiota composition at baseline and post-intervention. similar observation was obtained by lyra and team where they observed that probiotics intake reduced amount of ruminococcus torques 94%, which has been associated with gastrointestinal diseases like crohn’s disease as well as ibs[79–81]. after all these years, the use of probiotics in ibs remains as a compelling topic as some studies did not produce obvious long-term efficacy compared to placebo. for instance, using another commercially available probiotics capsule, vsl-3 which contains bifidobacterium, lactobacillus, streptococcus salivarius subsp. thermophiles, brigidi and team observed an increase in these probiotics, but not other bacterial population such as enterococci, coliforms, clostridium perfringens and bacteroides[82]. the same team also noted that the gut microbial composition returned to initial values once the probiotic had been suspended, indicating that the effects do not persist long term. however, bearing in mind that most of the previous studies investigated these using conventional qpcr targeting genes like 16s–23s ribosomal rna may not provide a full glimpse of which population changes after intervention. additionally, most studies have also highlighted that patients can continue with their normal diet, regardless of whether they are taking probiotics or placebo, which may explain for the inconsistencies in data. there are some concerns over the form of probiotics used such as capsule, liquid or powdered form, as studies have pointed out that the delivery systems vary greatly in effectiveness[83]. nonetheless, with the advancement in next generation sequencing and enhanced computational power, it may be easier to visualize how gut microbiome changes after probiotics intake in ibs patients, and at the same time answer the doubts on whether the observed effect is due to specific strains or a synergistic effect of these probiotics. consequently, this method would also assist the development of biomarkers to facilitate the detection and/or diagnosis of ibs on top of classic clinical assessment. conclusion and future recommendations it is evident that gut microbiome plays an important role in maintenance of gut health, including ibs. even though some medications may alleviate symptoms for ibs patients, there is still no one-for-all drug or treatment plan that would work for all of them[59,60]. the low fodmap diet has shown to be one of the clinically effective strategy by reducing overall symptom severity in multiple studies. by adhering to the low fodmap diet, patients may not experience symptoms flare-up but it is a challenging diet to adopt in certain regions of the world, particularly limitation of food choices as well as insufficient knowledge in delivery of the diet plan among medical practitioners[84,85]. some clinicians may doubt its implementation in the long run as a significant reduction in carbohydrates, iron and fiber can lead to other issues like calcium deficiency. while this may be concerning, concurrent use of probiotics along with ibs, fodmap, probiotics... 7 low fodmap diet may really help to ensure beneficial levels of bifidobacteria are maintained. theoretically, the gut microbiome patients receiving probiotics could make up for the reduced diversity following the restrictive diet, particularly by increasing the abundances of these probiotics[1,76,77]. in actual fact, there are still much to do before researchers could unravel the role of gut microbiome or even specific microbial population in ibs. amongst existing studies, there is a lack of consistency and clarity which may be attributed by the heterogeneity between them. this heterogeneity exists in variations in individual baseline microbiota, potential microbial differences according to the ibs subtypes, study designs, the methods of analyzing the microbiota composition, the definition of clinical response and the population of selected individuals for the study[4,52]. above and all, an innovative approach like combining probiotics and low fodmap diet in combating ibs is clinically applicable with no or minimal side effects. further investigations incorporating high throughput next generation sequencing technologies, analytical tools like chromatography and mass spectrometry techniques as well as improvement in delivery methods of probiotics may expedite the development of the next effective regime 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cancer is among the most predominant cancer in the world including malaysia. numerous factors could contribute towards colorectal carcinogenesis and one of the factors is genetic predisposition. mutations in the v-kiras2 (kras) oncogene have been implicated in 30-50% of the colorectal cancer patients and usually lead to poorer prognosis. the challenging ability for the early detection of colorectal cancer still poses an enormous challenge to oncologist as there are limited or no signs or symptoms in the early stage of colorectal cancer. many studies were conducted hoping to further understand colorectal cancer for a better diagnosis and prognosis. as early detection of colorectal cancer frequently leads to good prognosis. the gold standard for prognosis depends on the stage of the tumor at the time of diagnosis. lately a group of small, non-coding rnas termed micrornas (mirnas) exhibited capable outcomes in cancer research. numerous mirnas were discovered to play a key role in regulatory mechanism in numerous cancers. differential mirnas expression among tumors and non-tumor controls are highly valuable in recognizing mirnas that could have vital role in carcinogenesis. recently some mirnas were discovered to play a vital role in colorectal carcinogenesis. thus, mirnas have emerged as highly useful tool for scientists to comprehend carcinogenesis better. for example, mir-21 and mir-106a were highly expressed in colorectal cancer. while mirnas including mir-17-92 cluster, mir-21, mir-34, mir-135 and mir-196a also exhibited high association with colorectal cancer. therefore, this article aims to provide insight of mirnas role in colorectal cancer for a better understanding of this disease. keywords: microrna; mirna; colorectal cancer; malaysia; cancer received: 9th april 2020 accepted: 10th may 2020 published online: 15th may 2020 citation: lye kl, tan l-ht and yap h-m. insight of microrna role in colorectal cancer. prog microbes mol biol 2020; 3(1): a0000083. https://doi.org/10.3687/pmmb.a0000083 introduction colorectal cancer colorectal cancer is a kind of cancer that forms in the tissues of the colon and rectum. classically, most colorectal cancers are adenocarcinomas or cancer arises from cells making and releasing fluids such as mucus. alike other cancers[1], colorectal cancer is a multifactorial disease that exhibited complex interactions among inherited susceptibility and environmental factors leading towards the development of the disease[2,3]. prevalence of colorectal cancer a total of 115,238 new cancer cases were diagnosed in malaysia for the period of 2012-2016. the malaysian national cancer registry report 2012–2016 indicated that age-standardized incidence rates (asr) for all cancers were 86.1 for males and 101.6 for females per 100,000 populations[3]. the 5 most common cancers among malaysian were reported as breast, colorectal, trachea, bronchus and lung (tbl), lymphoma and nasopharynx cancer. the statistics indicated that colorectal cancer ranked 2nd most common cancer in malaysia. total asr was at 13.5 per 100000 population. colorectal cancer was somewhat higher in males (14.8 per 100000) as compared to females (11.1 per 100000)[3]. looking at different ethnicities, chinese documented the highest asr (21.4 per 100000), followed by indian (11.3 per 100000) and malay (9.5 per 100000)[4]. data obtained from the national cancer registry 2007 indicated that colorectal cancer ranked 2nd as one of the most common cancers in malaysia, with asr for male at 85.1 per 100000 and female at 94.4 per 100000 population[5]. diagnosis of colorectal cancer copyright @ 2020 by lye kl and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: kwan-liang lye, department of biomedical sciences faculty of medicine and health sciences, universiti putra malaysia, 43400 upm serdang, selangor darul ehsan, malaysia; kl_lye86@hotmail.com 2 diagnosis is extremely important in the management of colorectal cancer. currently there are few types of tests and procedures used to diagnose and detect colorectal cancer as listed here: • fecal occult blood test — detecting occult blood in the feces • double contrast barium enema — liquid containing barium is placed into the rectum and coats the lower gastrointestinal tract. x-rays are undertaken to detect abnormal regions of the colorectal • colonoscopy — a colonoscope is introduced via the rectum into the colon to investigate polyps, abnormal areas or cancer • sigmoidoscopy — a sigmoidoscope is introduced via the rectum into the sigmoid colon to view for abnormal areas, polyps or cancer. • virtual colonoscopy — using computed tomography to generate series of photos of the colon and allows the view of any abnormal areas such as polyps in the colon. staging of colorectal cancer staging is the important procedure of investigating how extensive a cancer has spread and is one of the most important factors in defining the prognosis and treatment choices for cancer patients. several staging systems are employed for colorectal cancer. the most common staging system is the tnm system established by american joint committee on cancer (ajcc). tnm system illustrates three key evidence: • t illustrates how extensive the main tumor has spread into the wall of the intestine. • n explains the extent of spread to nearby (regional) lymph nodes. • m specifies whether the cancer has metastases or spread to the other organs of the body. the wider staging of a cancer is usually represented by roman numbering for instance i, ii, iii and iv originated from the tnm values. specifics of this staging system are demonstrated in table 1. insight of micrrna... table 1. colorectal cancer staging system. ajcc stage tnm stage details 0 tis n0 m0 tumor has not expanded beyond the inner layer (mucosa) i t1 n0 m0 tumor has invaded submucosa i t2 n0 m0 tumor has invaded muscularis propria ii a t3 n0 m0 tumor has invaded subserosa ii b t4 n0 m0 tumor has invaded adjacent organs or perforated the visceral peritoneum iii a t1-2 n1 m0 tumor has extended to 1-3 regional lymph nodes. t1 or t2. iii b t3-4 n1 m0 tumor has extended to 1-3 regional lymph nodes. t3 or t4. iii c t1-4 n2 m0 tumor has extended to 4 or more regional lymph nodes. iv t1-4 n0-2 m1 tumor has extended to 1 or more distant organ(s) or set of lymph nodes. another staging system utilized is the dukes system, it is a less complex staging system. itemized here are the descriptions of the stages in the dukes system: • a denotes the tumor that is confined to the intestinal wall. • b denotes the tumor that begins invading over the intestinal wall. • c denotes the involvement of lymph node(s) • d denotes distant metastases present treatment for colorectal cancer different kinds of treatments are available for colorectal cancer and are administered according to the diagnosis of the colorectal cancer by oncologist. the available treatments for colorectal cancer are discussed below: • surgery — as one of the most frequent treatment for colorectal cancer. a clinician could do local excision if the cancer is still at an early stage. if the cancer is larger, the clinician could do colectomy to remove the cancer and nearby normal tissues. additional options consist of radiofrequency ablation and cryotherapy. • chemotherapy — to use of medications that destroys cancer cells or inhibiting cancer cells division. numerous medications could be given concurrently to increase treatment effectiveness. one of the regular medicines for treating colorectal cancer is 5-fluorouracil (5-fu), commonly apply concurrently with oxaliplatin and leucovorin in a combination recognized as folfox. • radiotherapy — the use of high doses of radiation to kill cancer cells via destructing the targeted cells genetic materials. there are 2 types of radiotherapy, the external radiation therapy that uses a beam of radiation to target on the cancer area and repeated over a few days. the other would be the internal radiation therapy that uses radioactive materials inserted into or near the tumor via small thin tubes or needle. • immunotherapy — the use of individual body’s immune system to combat cancer. this therapy primarily promotes the immune system to response and combat more effectively against cancer. monoclonal antibodies therapy is one of the immunotherapies using generated monoclonal antibodies to target the tumor in the body and to deliver drug or radioactive materials directly to tumor cells. other examples of immunotherapy are colony-stimulating factors, tumor vaccines and biological response modifiers. other than existing treatment options, scientists conducted many experiments in hope to search for useful metabolites 3 lye kl et al. treated with oxaliplatin chemotherapy. the increase of response rate associates with increased progression free survival in colorectal cancer. nevertheless, kras mutations have been connected to decreased response degree towards chemotherapeutic agents. lievre et al.[33] reported that kras mutation was decreasing the reaction of anti-epidermal growth factor receptor (egfr), therefore patients with mutant kras demonstrated lower survival rate as compared to patients with wild-type kras. hence, the prognosis of patients could be improved by verifying the mutation status of the kras gene. the micrornas micrornas (mirnas) are small, non-coding rna found in the genomes of vertebrates, invertebrates and plants[34]. the typical size of mature mirnas is 21–25 nucleotides. mirnas demonstrated important role in many vital processes for instance cell proliferation, differentiation and apoptosis[35]. mirnas controls gene expression by various ways for example mrna cleavage, deadenylation and translational repression. the exciting element is that mirna is able to control the expression level by partial complementary binding to the target mrna. this allows a single mirna to control and regulate more than one target mrna and perform several roles in our biological processes. discovery of mirnas the first mirna, the lin-4 was discovered in 1993 by the ambros’s and ruvkun’s research team[36,37]. the gene lin4 was detected by isolation of null mutation that triggers a breakdown in temporal growth in caenorhabditis elegans[36]. ambros et al.[34] reported a 700bp fragment that may well have lin-4 gene but unable to locate the conventional start and stop codons, hence indicated that lin-4 is not encoding protein. researchers also discovered two small transcripts of lin-4 of 61nt and 22nt in length that correspond to the common precursor mirna and mature mirna length[37]. the second mirna discovered was let-7, that is likewise a heterochronic gene of c. elegans. reinhart et al.[39] discovered that let-7 was a 21nt rna regulating the transition stage from l4 to adult in the larval development. dissimilar to the lin-4, let-7 sequence is conserved across species from invertebrates to complex organisms for instance humans. nevertheless, let-7 was not found in unicellular organisms and plants. curiously, the expression level of let-7 is dissimilar in different types of human tissue[40]. the finding that let-7 was conserved across numerous species initiated the surge of research in the small rna called microrna (mirna). till date, more than 38 thousand mirnas is listed in the mirbase database (http:// http://www.mirbase. org/) and keeps rising. mirna synthesis as depicted in figure 1, mirnas synthesis starts in the nucleus, with the primary transcripts (pri-mirnas) processed into mirna precursor (pre-mirna) facilitated by drosha and dicer, which are rnase iii enzymes. the pre-mirnas are next exported from the nucleus into the cytoplasm by exportin-5 and cut by dicer into a 22-nucleotide mature double stranded mirna[41]. this strand is then fused into the argonaute protein to create the effector rna-induced or compounds that could potentially inhibit the growth of cancer cells[6–12] especially colorectal cancer[13–19]. researches have gained some good findings, but more tests need to be done before these potential compounds could be ready for clinical application. the ras oncogene the ras is a family of related proteins known as small gtpase. they are vital for signal transmission between cells. the naming of “ras” derived from “rat sarcoma”, indicating the source from which the first member of the protein family was discovered. the ras family comprises of 3 members, nras, hras and kras[20]. members of the ras family are triggered after a nearby transmembrane receptor is bound by its corresponding ligand. the ras protein is activated by guanine nucleotide exchange factors (gefs) that leads to the development of gtpbound state, followed by inactivation by gtpase activating proteins (gaps) forming the gdp-bound state by gtp hydrolysis. consequently, these will be activated and regulating other genes participating in cell differentiation, growth and survival. hence, mutation in ras genes could lead to overexcited ras signaling, therefore triggering uncontrolled cell division and cell growth that would eventually become cancerous[21]. kras mutation in colorectal cancer kras gene is a vital gene in colorectal cancer, this gene expresses a protein involved in the epidermal growth factor receptor (egfr) signaling pathway. the kirsten rat sarcoma viral oncogene homolog or kras gene belongs to the ras family of oncogenes, and mutations are common notably in colorectal cancer, pancreatic cancer and lung cancer. kras gene mutation is among the earlier events in colorectal carcinogenesis, and the ability to detect kras gene mutation is very important for diagnosis. kras mutation was reported in the earlier stages of molecular alteration contributing to the development of colorectal adenoma to carcinoma. the kras protein has vital role in tumor growth via the regulation of downstream proteins involved in survival, proliferation, and metastasis[21,22]. the prevalence rate of kras mutation was reported to be 20% to 50%[23,24]. reported here are countries and their respective rate of kras mutation amongst colorectal cancer patients; italy (46.3%), usa (40%), iran (37.4%), turkey (34.2%), jordan (33.3%), taiwan (26.5%) and egypt (18.4%)[25,26,27]. one of the most common kras mutation occurs at codon 12 and codon 13 [28]. it was reported that codon 12 has higher mutation rate as compared to codon 13 [21,22], with mutation of codon 12 reported at ~35% based by bazan et al.[29]. researchers suggested the high mutation rate of codon 12 is because of the vulnerability of codon 12 to carcinogen binding, furthermore along with the poor repair mechanism of the resulting adduct[21,22]. also, other studies indicated that kras mutation normally implicated codon 12, 13, 59 or 61[30,31]. reports indicated that kras mutations contributed to higher probability of death and lower progression-free survival[22]. reinacher-schick et al.[32] also showed that kras mutations associated to lower progression-free survival in patients with advanced colorectal cancer 4 silencing complex (risc) after which the mirna and its mrna target interact. the mirna will only interact with mrnas containing anti-sense sequences. nevertheless, this interaction could occur even if they are partially complementary to each other[42]. mirna role in human diseases the discovery of mirnas had quickly led to key research conducted to investigate their roles in humans especially in disease progression. cancers were extensively studied, as one of the most common chronic disease affecting human. furthermore, mirnas were as well correlated to a multitude of other diseases. van rooij et al.[43] firstly reported the association of mirnas with cardiac hypertrophy and heart failure. the study utilized mirna microarray analysis demonstrated 12 mirnas were deregulated during cardiac hypertrophy and heart failure[43]. furthermore tijsen et al.[44] reported another mirna, mir-423-5p was elevated in heart failure patients. while for acute myocardial infarction patients, mir-208b and mir-499 was discovered to be greatly elevated and associated with the plasma troponin level[45]. `moreover, mirnas have demonstrated important role in autoimmune diseases. stanczyk et al.[46] demonstrated mirnas role in autoimmune diseases with the discovery of mir-146 and mir-155 overexpressed in rheumatoid arthritis synovial fibroblast and synovial tissue. the finding agreed with another study by nakasa et al.[47] that validated the expression of mir-146 in rheumatoid arthritis synovial tissue. in 2007, dai et al.[48] described a total of 16 mirnas were differentially expressed in patients of systemic lupus erythematosus (sle). both the mir-21 and mir-148a were overexpressed in cd4+ t cells of sle patients and contributing to down-regulation of the dnmt1 gene, triggering dna hypomethylation[49]. a couple of mirnas were correlated to neurodegenerative diseases for instance parkinson, alzheimer and huntington’s disease. lukiw (2007)[50] reported that in alzheimer’s patients, mir-9, mir-25b and mir128 were up-regulated, whereas mir-124a was downregulated. an increase in mir-9, mir-128 and mir-125b were observed on cultured human fetal brain-derived primary neural cells, that was treated with metal salts to create reactive oxygen species (ros)[51]. while mir133b was absent in brain tissue of parkinson’s disease patients, indicating mir-133b vital roles for maturation and function of dopamigernic neurons[52]. mirna in cancer in 2002, mirnas were first reported to be involved in cancer, with mir-15 and mir-16 discovered to be downregulated or deleted in chronic lymphocytic leukemia (cll)[53]. cimmino et al.[54] demonstrated that mir-15 and mir-16 have a role in apoptosis by targeting bcl2 mrna. various studies then reported different mirna expression in almost all types of cancer. mirnas can behave as tumor suppressors or oncomirs in carcinogenesis[55,56,57,58]. mirnas that were up-regulated act as oncomirs, while down-regulated mirnas in cancer usually operate as tumor suppressor. the overexpressed mirnas leading to cancerous growth operate as oncogenes, whereas underexpressed mirnas leading to cancerous growth act as tumor suppressor. there were numerous mirnas identified as tumor suppressor. johnson et al.[59] demonstrated that let-7 expression was down-regulated in lung cancer tissue as compared to normal tissue. furthermore, it associates with the increased of ras protein in the lung cancer samples. while study by garzon et al.[60] indicated that mir-29 was down-regulated in acute myeloid leukemia. while mir-34 was reported to be down-regulated in colon, pancreatic and breast cancers[61]. the mir-155 was among the first mirna to be linked as oncomirs. it was up-regulated in various cancers such as acute myeloid leukemia[60], hodgkin disease[62], burkitt lymphoma[63], and lung cancer[64]. mirnas in colorectal cancer micrornas (mirnas) have exhibited vital role in colorectal cancer genesis, progression and response to figure 1. the mirna synthesis pathway. insight of micrrna... 5 treatments. researchers demonstrated that in colorectal cancer samples, more mirnas were up-regulated than down-regulated[65,66,67]. the mir-21 is one of the properly studied mirna and discovered to be associated in many types of cancers. it was reported among the first mirna discovered as oncomir and was associated to a multitude of tumor suppressor genes for instance pdcd4, pten and bcl-2. faltejskova et al.[68] indicated higher expression of mir-21 was associated with shorter general survival of colorectal cancer patients. they performed silencing of mir-21 expression in dld1 cell lines and observed 30% suppression of the cancer cells migration ability, thus leading to lower cancer cells viability. this finding demonstrated the role of mir-21 in cancer cells migration in tumorigenesis. likewise, link et al.[69] demonstrated that mir-21 was highly expressed in patients with adenomas and colorectal cancer as compared to healthy individuals. another study of stage ii colon cancer patient exhibited increased mir-21 expression levels were associated to decrease recurrence-free cancer-specific survival[70]. furthermore, the plasma mir-21 was able to differentiate colorectal cancer patients from normal controls with 90% sensitivity and specificity[71]. these findings demonstrated that mir-21 could be a reliable and non-invasive marker for colorectal cancer. link et al.[69] reported that mir-106a was up-regulated in colorectal cancer patients when compared with normal patients. furthermore mir-106a was overexpressed in colorectal cancer and regulates the retinoblastoma 1 (rb1) gene in sporadic colorectal cancer patients[72]. lately, feng et al.[73] demonstrated that mir-106a was highly expressed in metastatic colorectal cancer cells and regulating the migration and invasion both in vitro and in vivo. the mir-106a prevents the expression of transforming growth factor-b receptor 2 (tgfbr2), leading to increase tumor cells migration and invasion. while diaz et al.[74] stated the downregulation of mir-106a contributes to lower disease-free survival and overall survival of colon cancer patients, regardless of the tumor stage. the mir-135b was regularly found up-regulated in cancer samples. faltejskova et al.[75], reported the increased of mir-135b expression in crc tumor tissues. furthermore, mir-135b was correlated with higher serum levels of cea and ca19-9. moreover, xu et al.[76] indicated the elevated expression levels of mir-135b in crc tissues compared to normal tissues, and the positive association of mir-135b with the clinical stage. the mir-135b targets the adenomatous polyposis coli (apc) gene, an important gene in colorectal carcinogenesis[77]. they discovered that mir-135b was up-regulated in colorectal carcinomas and associates with the low apc mrna levels leading to colorectal cancer. the high-throughput sequencing was used to compare between paired tumor and normal tissue, and results identified that 37 mirnas were dysregulated, with mir1 among the down-regulated mirnas[77,78]. furthermore, mir-1 down-regulation was correlated to colorectal cancer progression, hence attributes that mir-1 can be a potential tumor suppressor via down-regulating met oncogene at rna and protein level[79]. for mir-504, researchers have reported its association in cancer in various studies[80,81,82]. the mir-504 was upregulated in oral cancer, by increasing the invasion and migration capabilities of oral cancer cells[80]. while hu et al.[81] indicated that mir-504 down-regulates the p53 protein via binding to the 3’-utr of p53 gene, hence stimulating tumorigenesis. in 2011, another study also demonstrated that mir-504 down-regulates p53 protein levels and damages its function particularly in p53-mediated apoptosis and g1 cell cycle arrest[82]. bauer and hummon (2012)[83] indicated that mir-145 was down-regulated in colon cancer and created distinctive molecular alterations. earlier studies have indicated that these mirnas were also down-regulated in more types of cancers. kent et al.[84] established that ras activation leads to the down-regulation of mir-145 that propels tumorigenesis. the down-regulation of mir-145 was reported to increase risk of esophageal cancer[85]. whereas in bladder cancer, both mir-133a and mir-145 were discovered to be downregulated and targeted the oncogenic fscn1 mrna[86]. hence, mir-133a and mir-145 could act as a probable tumor suppressor by regulating the fscn1 gene. three mirnas from the mir-182/183 cluster, namely mir-182, mir-183 and mir-96 were up-regulated various studies[78,87,88,89]. sarver et al.[78] indicated that these mirnas (mir-182, mir-183 and mir-96) were up-regulated in colon cancer tissues. the mir-183 was overexpressed in rhabdomyosarcoma and colon cancer[90], and the mir-183 demonstrated a function as oncomir via controlling the tumor suppressor egr1 and pten and advocating tumor cell migration[90]. cekaite et al.[87] demonstrated that mir182 was overexpressed by more than 2 fold in colon cancers throughout all clinical stages. likewise, mir-96 was among the mirnas reported to be up-regulated in colorectal cancer sample as compared to adjacent normal tissue[78,86]. intriguingly, yu et al.[89] reported differing finding of mir96 down-regulated in pancreatic cancer and act as a tumor suppressor gene via inhibiting kras oncogenic gene. the mir-224 expression was elevated in colorectal cancer cell lines and in wild type kras and braf colorectal tumors. arndt et al.[91] indicated that mir-224 was overexpressed and correlated to colorectal cancer tumor progression. yet, mencia et al.[92] suggested that mir-224 was underexpressed in colorectal cancer cell lines and leading to the cells exhibiting rise in resistance towards methotrexate. likewise, previous studies have suggested that mir-203 was up-regulated in colorectal cancer, but down-regulated in another study[93]. chiang et al.[94] reported that mir-203 has really low expression in colorectal cancer tissue and cell lines. mir-31, a mirna discovered to be highly expressed in colorectal cancer tissues and correlated with advance tumor stage and poor differentiation[95]. furthermore wang et al.[96] also reported mir-31 to be up-regulated in crc samples and positively related to advanced tnm stage. while reports indicated that mir-17 was up-regulated in colorectal cancer and promotes tumor cell proliferation, growth and cell cycle progression[97]. mirnas in cancer pathways mirnas participation in cancer pathways have been lye kl et al. 6 highlighted in many studies. many proteins in key signaling pathways of colorectal cancer are changed and controlled by mirnas. the wnt pathway is one of the important pathways in early colorectal cancer development. in the wnt pathway, inactivation of the apc gene is one of the key beginning steps for colorectal carcinogenesis[98]. nagel et al.[77] suggested that mir135a and mir-135b reduce the translation of apc gene in vitro. also, the expression of these mirnas increases in colorectal cancer and associated with low expression of apc gene. egfr and kras signaling pathways lead to the initiation of numerous signal transduction molecules which started a cascade of downstream effectors regulating tumor growth, angiogenesis and metastasis[99]. the upregulation of kras will start a cascade of downstream activation of mek gene, raf gene and mapk gene, therefore increasing the proliferation of tumor cells[100]. the mir-1 and mir-106a were reported for presumed targets of the mapk gene family, therefore play a part in the kras signaling pathway. the pi3k pathway is an important signaling pathway downstream of the egfr pathway. researchers reported that mir-135b and mir21 targets the genes involved in pi3k pathway. with mir-21 clearly repressing the tumor suppressor, pten gene leading to lower survival rate of cancer patients[101]. another renowned tumor suppressor gene, p53 was mutated in estimated 50–75% of all colorectal cancer and other tumors. the p53 protein responds to dna damage and deregulation of oncogenes through the initiation of cell cycle checkpoints, cellular senescence or apoptosis[102,103]. the mir-504 exhibited putative target of bcl-2 gene, one of the gene that regulates the p53 pathway. researchers indicated that mir-504 down-regulates the p53 protein, thus promoting cancer progression[81]. these results was also in agreement by feng et al.[82] that indicated that mir-504 down-regulates p53 protein and damages its function in p53-mediated apoptosis and g1 cell cycle arrest. conclusion this article provided vital insight into the roles of mirnas in colorectal cancer. in colorectal cancer, studies indicated that many mirnas are involved in the pathogenesis of the disease. they control the known oncogenes or tumor suppressor pathways by targeting proteins for instance p53, kras and phophatidylinositol3-kinase (pi3k). nagel et al.[77] demonstrated that mir-135a and mir-135b reduce the translation of the apc transcript in vitro. the inactivation of the apc gene an important stage in colorectal carcinogenesis. furthermore, let-7 and mir-143 were described to target kras oncogene. the kras signaling leads to the initiation of numerous signal transduction molecules that starts a cascade of downstream effectors regulating angiogenesis, tumorigenesis and metastasis. some mirnas were discovered to be correlated with colorectal cancer for instance the mir-17-92 cluster, mir-21, mir-34, mir135 and mir-196a[104,105,106,107]. in conclusion, this article shed light of mirnas role in colorectal cancer that enabled a much better understanding of the disease. author contributions the literature review and manuscript writing were performed by k-ll, lt-ht and 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bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 3division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia 4international genome centre, jiangsu university, zhenjiang 212013, pr china 5college of pharmaceutical sciences, zhejiang university, 866 yuhangtang road, hangzhou 310058, china 6health and well-being cluster, global asia in the 21st century (ga21) platform, monash university malaysia, bandar sunway 47500, malaysia. abstract: the present study explored the antioxidant potential of a streptomyces sp. strain musc 5 from the mangrove forest soil in the pahang state, peninsular of malaysia and determined the presence of biologically active chemical constituents contained in the methanolic extract. the 16s rrna genomic dna extraction, phylogenetic analysis and phenotyping methods were used to confirm identity of strain. the antioxidant potential of methanolic extract from streptomyces sp. strain musc 5 was assessed using a number of antioxidants assays which included free radical scavenging assays, metal chelation and ferric reduction antioxidant power (frap) assay. furthermore, gas chromatography-mass spectrometry (gc-ms) was used to determine the presence of biologically active chemical compounds in methanolic extract of streptomyces sp. strain musc 5. the strain was confirmed as belonging to streptomyces genus. antioxidant studies of the methanolic extract from streptomyces sp. strain musc 5 revealed antioxidant activity of 24.97 ± 0.99 %, 22.95 ± 3.21% and 26.81 ± 1.05% against free radicals abts, dpph and metal chelation, respectively. the result of frap assay was expressed in dose of 1-2 mg which was equivalent to 1.732.15 microgram (µg) of ascorbic acid. gc-ms analysis carried out on the methanolic extract of streptomyces sp. strain musc 5 detected the presence of 11 known compounds belonging to pyrrolopyrazine, esters, fatty acid esters, triterpene and an alkane group of compounds. the study supports the notion that streptomyces from underexplored mangrove forest offer promising streptomyces with antioxidant activity and could serve as important sources for new antioxidant agents. keywords: streptomyces; antioxidative; mangrove; radical scavenging received: 18th april 2020 accepted: 20th may 2020 published online: 28th may 2020 citation: kemung hm, tan lt-h, chan k-g, et al. streptomyces sp. strain musc 5 from mangrove forest in malaysia: identification, antioxidant potential and chemical profiling of its methanolic extract. prog microbes mol biol 2020; 3(1): a0000087. https://doi.org/10.3687/pmmb.a0000087 introduction oxidative stress is a pathological condition caused by presence of high levels of reactive oxygen species (ros) with an insufficient amount of defensive antioxidants in the body originating from mitochondria, in some instances, can be acquired as air pollutant such as carbon monoxide[1]. to date, there is growing evidence linking oxidative stress caused by mitochondrial dysfunction with the progression of degenerative diseases in the copyright @ 2020 by kemung hm and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: kok gan chan, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia. kokgan@um.edu.my; bey-hing goh, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. goh.bey.hing@monash.edu 2 adult population[2]. the role of antioxidant is to supply the body with optimal antioxidants, thereby reducing unwanted effects of circulating ros. previously, a study that investigated natural antioxidants as dietary supplements was shown to be effective in regressing cancer[3]. over the years, microbes have risen on the stage of prominence as producers of useful drugs against debilitating diseases[4–10]. the bacteria genus of streptomyces is characterized as a gram-positive, filamentous, soil-dwelling and saprophytic in nature[11, 12]. the soil consists of a diverse microbial community which includes bacteria such as streptomyces[13] and competition for nutrients can be extremely intense[14]. nevertheless, streptomyces adapted well to the surrounding environment and this may be justified by the fact that they carry a large genome size with high g-c content[15–21] that enable the production of vast array of enzymes, proteins and secondary metabolites to process variety of materials needed for growth and survival[22–26]. the genus of streptomyces under the actinobacteria phylum are by far the most recognized producers of current drugs and secondary metabolites with diverse biological activities[4,27–31]. there is renewed interest in the streptomyces strains and species lurking in understudied ecological niches and awaiting discoveries which can aid development of new and needed drugs[32–34]. additionally, there is growing evidence suggesting streptomyces from the understudied mangrove habitat as potential sources of antioxidants in the pharmaceutical industries[35–50]. the mangrove forest is a unique ecosystem which consists of stretch of forests at the confluence of land and the marine ecosystems. concentrated mostly in 15 countries with an estimated coverage of 75% of their coastline margin[51], mangrove forests are home to a rich microbial community[52] which remain relatively understudied for their biologically active properties[53–56]. herein, we report a streptomyces sp. strain musc 5 with antioxidant potential that was previously isolated from the soil in the mangrove forest of malaysia. in addition, gcms analysis detected 11 known compounds present in the methanolic extract of streptomyces sp. strain musc 5. overall, this study supports the notion of mangrove forest in malaysia harbouring promising streptomyces that have yet to be investigated for important biologically active compounds. materials and methods sampling and maintenance of streptomyces sp. strain musc 5 the soil sample for the present study, was collected from mangrove forest in tanjung lumpur, malaysia (musctls4 3°48’21.3” n 103°20’3.3”e) in december 2012[43,57,58]. soil sample collected consisted of portion of the soil layer just beneath 2–3 mm of the surface with a depth up to 30 cm and was achieved by a sterile trowel. soil samples were aseptically packed into a plastic bag and delivered safely to be stored at 20 ºc prior to air drying. air-dried samples were then ground and processed by wet heat sterilization. pre-treated sample was suspended in previously autoclaved water, diluted and plated uniformly across isp 2 media supplemented with antifungal drugs which selectively promoted growth of streptomyces. growth was monitored by continuous subculture onto freshly made isp 2 media until pure isolates was achieved. pure isolates were then kept on isp2 agar slant and 20 % glycerol at 20 ºc as stocks for future work. phylogenetic analysis of streptomyces sp. strain musc 5 the isolation of genomic dna (gdna) was for the purpose of amplifying the 16s rrna region in the genome[59, 60]. the sequenced 16s rrna gene of streptomyces sp. strain musc 5 was then entered into genbank/ embl/ ddbj database to retrieve member type strains with the closest match. multiple alignment for all the member type strains retrieved from genbank/embl/ddbj database was achieved using clustal-x software[61]. the stability of the generated phylogenetic tree was verified by following felsenstein method[66]. phenotypic characterization of streptomyces sp. strain musc 5 phenotypic characterization of streptomyces sp. strain musc 5 consisted of growth characteristics, physiological tolerance levels and production of extracellular enzymes. growth characteristics of streptomyces sp. strain musc 5 cultured at 28 °c for 7–14 days, was visually inspected on conventional nutrient media — international streptomyces project (isp) 2, isp3, isp4, isp5, isp6, isp7[67], streptomyces agar (sa)[68], nutrient agar (na) [69], actinomycete isolation agar (aia)[70] and starch casein agar (sca)[71]. soluble pigment and colony colour on each growth media were monitored[72]. physiological tolerance assessment of streptomyces sp. strain musc 5 was evaluated by growing at temperature range of 4–50 °c, salinity levels of 0–10 % w/v and ph 2–10. biochemical properties of streptomyces sp. strain musc 5 evaluated the production of extracellular enzymes. presence of catalase was investigated by observing bubble formation following the dropping of 3 % (v/v) hydrogen peroxide onto a culture of streptomyces sp. musc 5[73]. test to detect hemolysis activity was carried out on a 5-day old culture grown on blood agar media[74]. production of extracellular enzymes were determined on isp 2 media[75]. fermentation process and extract preparation a 10-day culture of streptomyces sp. strain musc 5 in 10 ml of tryptone soya broth (tsb) media and afterwards inoculated in 200 ml sterile han’s fermentation media 1 (biomerge, malaysia) shaken in incubator at 28 °c, 220 rpm for 10 days. secondary metabolites in supernatant was collected by initial centrifuging followed by filtration and freeze-drying[60]. extraction of secondary metabolites from freeze-dried supernatant was performed using organic solvent methanol. crude methanolic extract was collected after evaporation of methanol using of rotary evaporator and stored conveniently at – 20 °c for future use[43]. antioxidant assays scavenging of abts radical streptomyces sp.strain... 3 kemung hm et al. scavenging of 2,2’-azino-bis(3-ethylbenzothiazoline6-sulfonic acid) (abts) radical by the methanolic extract was examined according to tan et al. (2017) [41]. in short, abts radical (abts•+) was obtained from the reaction of 7 mm of abts with 2.45 mm of potassium persulfate (k2s2o8). the six concentrations (0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml and 4 mg/ml) of methanolic extract were prepared by 2-fold dilution in 96 well plate. the abts radical was then introduced into the 96 well plate. the plate was then kept in dark for 20 minutes prior to reading of the uv absorbance at 734 nm. gallic acid served as the standard for this experiment. the following formula was used to calculate the radical scavenging activity of methanolic extract in percentage (%): scavenging of dpph radical scavenging of 2, 2-diphenyl-1-picrylhydrazyl (dpph) radical by the methanolic extract was performed according to tan et al. (2017)[41]. the test was run in a 96-well microplate. a series of concentration of methanolic extract was prepared in the 96-well plate by 2-fold dilution ranging from 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml and 4 mg/ml. a solution of dpph ethanol (0.016 % w/v) was then added into 96 well plates containing the methanolic extract and left standing in the dark for 20 minutes at room temperature. the uv absorbance of the mixture was read at a wavelength of 515 nm. gallic acid was used as the control for this test. the following formula was used to calculate the radical scavenging activity of methanolic extract in percentage (%): chelation of metal ions the metal chelating assay was assessed according to adjimani and asare[76] and performed in a 96 well plate. methanolic extract were prepared in the concentration of 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ ml, 1 mg/ml, 2 mg/ml and 4 mg/ml and placed in individual wells. a 2mm ferrous sulfate (feso4) was afterwards added into the wells followed by the addition of 5 mm ferrozine. in this reaction mixture, both methanolic extract and ferrozine compete with each other for ferrous ion. ethylenediaminetetraacetic acid (edta) was the standard used in this experiment. the following formula was used to calculate the metal chelating activity of methanolic extract in percentage (%): ferric reduction antioxidant power (frap) assay reduction of ferric ion was evaluated according to adjimani and asare[76] with few alterations made. series of concentration of methanolic extract ranging from 5 mg/ ml, 10 mg/ml, 20mg/ml, 40 mg/ml and 80 mg/ml, were prepared in 25 µl in a 1.5 ml microcentrifuge tubes. a 25 µl from phosphate buffer (0.2 m) and 25 µl from potassium ferricyanide (1 %) were subsequently added into sterile 1.5 ml. the reaction mixtures were then heated to 50 °c and temperature kept constant for 20 minutes before cooling down to room temperature. a 25 µl of 10 % trichloroacetic acid (tca) was afterwards added to cease the reaction. an 80 µl was transferred to wells in 96 well plate with a further addition of 20 µl of ferric chloride (fecl3). the uv absorbance reading was taken at wavelength of 700 nm and results expressed in equivalent dose of ascorbic acid. detection of compounds in methanolic extract with gc-ms profiling of individual constituents in the methanolic extract was achieved by using agilent technologies 6980n with a 5979 mass selective detector [42]. a hp-5 ms (5 % phenyl methyl siloxane) capillary column was used as helium gas carrier at 1 ml every 1 minute. heat was gradually applied until 40 °c was reached whilst keeping it constant for 10 minutes; then, increased by 3 °c every minute until peak temperature of 250 °c keeping it constant for 5 another minutes. ms was functioning at 70 ev. individual compounds detected by gc-ms were matched with nist 05 reference library. statistical analysis antioxidant tests were repeated three times and the result expressed in means ± standard deviation (sd). statistical analysis of antioxidant result was computed using statistical package for the social sciences software (spss) and included using one-way analysis of variance (anova) whilst tukey’s post hoc determined the statistical significance at p-value < 0.05. in order to correlate the antioxidant activity to phenolic content in methanolic extract, a pearson’s correlation in spss software was performed. results genomic and phylogenetic analysis of streptomyces sp. strain musc 5 the 1489 bp 16 s rrna sequence of streptomyces sp. strain musc 5 (genbank accession number kp998433) from its gdna enabled retrieving representative of closely related taxa and manually aligned. the phylogenetic tree of streptomyces sp. strain musc 5 is depicted in figure 1. the phylogenetic tree constructed showed strain musc 5 forming sister clade with streptomyces drozdowiczii nbrc 101007t at bootstrap value of 63 %. closest representation of taxa was established between streptomyces drozdowiczii nbrc101007t (99.52 %) followed by streptomyces laculatispora bk166t (99.37%) and streptomyces brevispora bk160t (99.30 %). % abts scavenging activity = absorbance of control absorbance of sample absorbance of control ×100% % dpph radical scavenging activity = absorbance of control absorbance of sample absorbance of control ×100% % metal chelating activity = absorbance of control absorbance of sample absorbance of control ×100% 4 cultural characterization, physiological tolerance and biochemical characterization the growth of streptomyces sp. strain musc 5 on various media is shown in table 1. streptomyces sp. strain musc 5 grew abundantly on isp 2, isp5, isp6, isp7 and sca and sa after 7–14 days at 28 °c. this is in agreement by gottlieb and shirling who recommend isp media for the growth of streptomyces[67]. colour of figure 1. neighbour-joining phylogenetic tree based on 1489 nucleotides of 16s rrna gene sequence showing the relationship between strain musc 5 and closely related member strains. numbers and nodes indicate percentages (> 50 %) of 1000 bootstrap re-sampling. bar, 0.002 substitutions per site. colony of mycelia were noted on all media grown, except isp4. a light greyish olive and dark yellow pigments were visible on isp 6 and na, respectively (table 1). optimal conditions for growth were temperature of 26 °c, ph of 6–7 and salinity concentration of 2 % w/v. streptomyces sp. strain musc 5 was tested positive for catalase. results of the biochemical analysis suggested the production of catalase, xylanase and cellulase and amylase by streptomyces sp. strain musc 5 (table 2). table 1. cultural characteristics of streptomyces sp. strain musc 5. media growth colony colour soluble pigments aerial mycelia substrate mycelia isp 2 well brilliant greenish yellow vivid yellow isp 3 poor light olive grey moderate olive isp 4 isp 5 well dark greyish yellow light yellowish brown isp 6 well pale yellow moderate yellow light greyish olive isp 7 well light olive grey light greyish olive aia moderate pale greenish yellow pale greenish yellow sca well medium grey light greyish olive sa well light orange yellow brilliant yellow na moderate yellowish grey light greyish yellow brown dark yellow no growth on isp 4 and no production of soluble pigment streptomyces sp.strain... 5 table 2. biochemical and physiological characteristics of streptomyces sp. strain musc 5. catalase + haemolytic enzymatic test chitinase activity (2.5 % chitin) xylanase activity (0.5 % xylan) + amylolytic activity (0.2 % starch) (+) protease activity (2 % casein) lipase activity (1 % tributyrin) cellulase activity (0.5 % cmc) + temperature (oc) growth 26-37 optimum 26 nacl (%) tolerance growth 0-6 optimum 2 ph tolerance growth 6-8 optimum 6-7 no activity; + activity; (+) weak activity abts radical scavenging assay scavenging of abts radical by the methanolic extract was assessed by reacting abts radical cation with methanolic extract and thereafter observing visible colour change from blue-green to colourless. the colour change is suggestive of abts scavenging activity. the uv absorbance of free abts radical was taken at 743 nm with result showing a concentration dependent scavenging of abts radical (p < 0.05) with 24.97 ± 0.99 % as the highest activity measured at 4 mg/ml (table 3). dpph radical scavenging assay scavenging of dpph radical by methanolic extract was assessed based on the visible colour change from purple (dpph radical) to yellow (diphenylpicrylhydrazine) in the reaction mixture. quantitative analysis of this antioxidant activity was based on the uv absorbance reading taken at 515 nm which is the wavelength that detects free dpph radical. the result of this experiment demonstrated the dpph radical scavenging potential of methanolic extract musc 5 with an activity (p < 0.05) of 22.95 ± 3.21 % at its highest concentration of 4 mg/ml (table 3). metal chelating assay in this experiment, the ferrozine reagent was used to assess the ferrous ion (fe2+) chelating ability of methanolic extract. the metal chelating potential of the methanolic extract was thereafter evaluated by taking the absorbance of complex of fe2+-ferrozine at 562 nm. a low absorbance reading normally suggests that most of the ferrous iron have been prevented to form complex with ferrozine by the metabolites within the methanolic extract. the result of this study indicated that methanolic extract had a metal chelating activity (p < 0.05) of 26.81 ± 1.05 % at 4 mg/ml (table 3). table 3. scavenging of free radicals and chelation of metal ion by methanolic extract. concentration (mg/ml) antioxidant activities (%) abts radical scavenging activity dpph radical scavenging activity metal chelating activity 0.125 3.04 ± 0.54* 2.11 ± 4.92 3.05 ± 2.43 0.25 4.65 ± 0.95* 2.08 ± 4.73 4.13 ± 1.81* 0.5 4.61 ± 1.14* 4.35 ± 6.12 4.97 ± 0.65* 1 6.00 ± 1.15* 6.15 ± 6.73 8.60 ± 1.78* 2 14.55 ± 0.68* 16.39 ± 5.69* 12.94 ± 2.13* 4 24.97 ± 0.99* 22.95 ± 3.21* 26.81 ± 1.05* gallic acida 42.50 ± 0.60* gallic acidb 53.99 ± 4.06* edtac 68.49 ± 7.68* *statistically significant at p < 0.05; aactivity of gallic acid at 12.5 μg/ml; bactivity of gallic acid at 10 μg/ml; cactivity of edta at 0.125 mg/ml kemung hm et al. 6 figure 3. the molecular structures of the chemical compounds detected by gc-ms in the methanolic extract of streptomyces sp. strain musc 5. frap assay reduction potential of ferric ion to fe2+ form by methanolic extract was assessed through frap assay. the amount of fe2+ fe3+ complex formed was measured with uv wavelength light absorbance of 700 nm. given there is activity, the colour of the reaction mixture changes to prussian blue and indicates the methanolic extract has reducing power. the result showed that methanolic extract absorbance was 0.77–0.85, in the dose range of 1–2 mg (figure 2) which was equivalent to 1.73–2.15 µg of ascorbic acid. gc-ms chemical profiling of methanolic extract of streptomyces sp. musc 5 chemical profiling of various constituents was achieved by the use of the gcms together with the mass spectral streptomyces sp.strain... figure 2. frap of the methanolic extract of streptomyces sp. musc 5. the 6 doses (0.0625 mg, 0.125mg. 0.25 mg, 0.5 mg, 1 mg and 2 mg) represents the 6 test concentrations employed (2.5 mg/ml, 5 mg/ml, 10mg/ml, 20 mg/ml, 40 mg/ml and 80 mg/ml). values are based on experiment run in triplicates ± standard deviation. data provided by the nist library. from this, 11 compounds identified belonged to the class of pyrrolopyrazine, esters, fatty acid esters, triterpene and an alkane. further information regarding individual compounds are provided in table 4 and figure 3. table 4. compounds detected by gc-ms. no. constituents retention time (min) molecular formula molecular weight similarity (%) 1 pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro53.752 c7h10n2o2 154 94 2 pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)55.048 c11h18n2o2 210 81 3 tricosanoic acid, methyl ester 58.797 c24h48o2 368 80 4 triacontanoic acid, methyl ester 63.184 c31h62o2 466 86 5 tetracosanoic acid, methyl ester 65.669 c25h50o2 382 93 6 pentacosanoic acid, methyl ester 77.07 c26h52o2 396 93 7 hexacosanoic acid, methyl ester 81.538 c27h54o2 410 93 8 squalene 76.578 c30h50 410 95 9 propanoic acid, 2-hydroxy-, methyl ester 3.852 c4h8o3 104 99 10 dl-proline, 5-oxo-, methyl ester 38.665 c6h9no3 143 96 11 hentriacontane 79.802 c31h64 436 96 7 discussion the 16s rrna is widely recognised as the golden standard for identification of bacteria at the genus level[77] and was therefore applied to validate that the strain belonged to the streptomyces genus[12]. having acquired the 16s rrna gene sequence of streptomyces sp. strain musc 5 with a 1489 bp, assisted with the construction of phylogenetic tree (figure 1). analysis of the tree placed the strain musc 5 within the streptomyces genus. apart from conferring the status of streptomyces to the strain musc 5, close relations were also investigated revealing streptomyces drozdowiczii nrbc 101007t at bootstrap value of 63 %. closest relation was established with streptomyces drozdowiczii nbrc101007t (99.52 %) followed by streptomyces laculatispora bk166t (99.37 %) and streptomyces brevispora bk160t (99.3 %). further information regarding the physical, physiological and biochemical characteristics of streptomyces sp. strain musc 5 was also conducted in the current study to provide phenotypic characterization and could be also useful for other research purposes. the strain was able to utilize a wide range of nutrients, produce soluble pigment and coloured colony, as shown by the result presented in table 1. the strain showed potential in producing extracellular enzymes as well as tolerating different temperatures, salinity and ph (table 1). the methanolic extract was evaluated for antioxidant potential utilized a combination of radical scavenging assays and reduction power of antioxidants. the radical scavenging assays was chosen on the basis of its sensitivity and ease of performance and thus the use of abts and dpph[78]. herein, we report the radical scavenging activity of methanolic extract as having an abts and dpph activity of 24.97 ± 0.99 % and 22.95 ± 3.21 %, respectively, at 4 mg/ml (table 3). the metal chelation antioxidant assay was carried out to examine the potential of the methanolic extract to interfere with the formation of coloured complex between ferrozine with ferrous ion[41]. in the biological system, fe2+ is involved as a catalyst in the formation of hydroxyl radical (oh•-) through the fenton reaction[79]. the oh•are the most destructive of all ros, and removing it from the system is critical for maintaining homeostasis. the use of antioxidants that are capable to terminate hydroxyl radicals by chelating with ferrous ion would proof worthwhile. the result of the metal chelating test show that methanolic extract had a moderate activity (p < 0.05) of 26.81 ± 1.05 % at 4 mg/ml (table 3). frap is an antioxidant assay that measures a different aspect of ros by process known as dismutation. in this experiment, both the oxidation and reduction happen concurrently involving an exchange of electrons between a reductant and an oxidant[80]. here, the methanolic extract and ferrocyanide undergo dismutation. given that methanolic extract possess antioxidant potential, it will exchange its electron with ferrocyanide and become oxidised. the ferricyanide is transformed to ferrocyanide and later reacted with ferric chloride forming fe2+-fe3+ complex which has an absorb uv-vis light at 700nm. in this experiment, the frap value was found within the dose range of 1–2 mg (figure 2). this was equivalent of 1.73–2.15 µg of ascorbic acid. given that methanolic extract demonstrated antioxidant activity, these findings have prompted further investigation into identification of the antioxidative compounds. the gcms has become an common tool for chemical profiling of bioactive compounds after determining their biological activities[81]. for this reason, gc-ms was used in the present study to detect chemical constituents in the methanolic extract musc 5 (table 4 and figure 3). the gc-ms analysis led to the detection of 11 known compounds, including pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro (1), pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3(2-methylpropyl)(2), tricosanoic acid, methyl ester (3), triacontanoic acid, methyl ester (4), tetracosanoic acid, methyl ester (5), pentacosanoic acid, methyl ester (6), hexacosanoic acid, methyl ester (7), squalene (8), propanoic acid, 2-hydroxy-, methyl ester (9), dl-proline, 5-oxo-, methyl ester (10) and hentriacontane (11). pyrrolopyrazines exist as a class of heterocyclic compounds frequently recovered from microbial extracts. for example, compound (1) isolated from a bacillus strain was identified as the active compound with strong dpph radical scavenging activity as well as exhibiting activity against multi-drug resistant staphylococcus aureus[82].compound (2) among the 3 constituents present in ethyl acetate extract was identified in a marine streptomyces sp. s2a as the major constituent responsible for the antioxidant activities (dpph, abts, metal chelating and frap), antibacterial, enzyme inhibitory and cytotoxic effects[83]. in addition to the two pyrrolopyrazine compounds, several of the fatty acid esters were also detected by gc-ms in the current study. in the current study, gc-ms detected 5 fatty acid methyl esters (3–7) in the methanolic extract of streptomyces sp. strain musc 5. compound (3) has been previously detected by gc-ms in microbes[84]. triacontanoic acid, methyl ester (4) present in a plant extract showed anticancer property[85]. another plant extract displaying antidiabetic activity was found to contain tetracosanoic acid, methyl ester, (5)[86]. both the ethanolic and water extract of propolis demonstrated antioxidant and antimicrobial activities and pentacosanoic acid, methyl ester (6) was confirmed in both extracts[87]. hexacosanoic acid, methyl ester (7) was reported in a plant extract demonstrating anticancer properties[88]. interestingly, gc-ms detected squalene (8), a biologically active triterpene, which was first reported from a shark liver oil by tsujimoto (1916)[89]. to date, shark liver oil has been the major source of squalene, although this compound is also naturally produced in minute quantity by streptomyces. for instance, streptomyces sp. qc45b was shown to synthesize squalene via the methylerythritol phosphate pathway (mep) [90]. other miscellaneous compounds detected by gc-ms in the current study include propanoic acid, 2-hydroxy-, methyl ester (9) commonly known as methyl lactate. it is a volatile oil produced by plants and was detected by gc-ms in studies investigating the chemical composition of coffee kemung hm et al. 8 beans[91]. compound (10) was detected in methanolic extract of bacillus sp. strain sb1 and halobacillus sp. strain sb2 having demonstrated antioxidant activity[92]. a long chain alkane hentriacontane (11) in a plant extract displayed biological activities including, antimicrobial and antioxidant activity[93]. conclusion the methanolic extract of streptomyces sp. strain musc 5 has demonstrated antioxidant activity by radical scavenging and frap assays. furthermore, gc-ms analysis detected the presence of 11 compounds in the methanolic extract having a variety of known biological activity. this study adds support to the notion that understudied mangrove forest in malaysia hold promising streptomyces with antioxidant metabolites that could be resourceful in the development of safer antioxidant agents in addressing oxidative stress which has been associated with several medical conditions. author contributions k-hm, lt-ht and b-hg executed the experiments, analysed data and participated in the writing of the manuscript. additional technical supports and proofreading were provided by lt-ht, h-ls, jw-fl. 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calamintha baborensis batt. pharm chem j 2018; 52(4): 347– 356. kemung hm et al. progress in microbes and molecular biology review article 1 cancer, natural products and nanodrug delivery systems yong sze ong1,2*, loh teng-hern tan3 1biofunctional molecule exploratory research group (bmex), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2health and well-being cluster, global asia in the 21st century (ga21) platform, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 3novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia abstract: as an offshoot of nanotechnology, nanomedicine has made great impact in the field of pharmaceutical and biomedical sciences by achieving breakthroughs in therapeutics and diagnostics of diseases in living organisms. one of the promising breakthroughs is the application of natural product-based nanoformulations for the treatment of various human diseases, such as cancer. principally, the nanoparticle-based drug delivery system (ndds) aims to overcome the limitations of conventional drug delivery system. ndds improves the in vivo pharmacological and therapeutic properties of the poorly soluble drugs by dissolving, encapsulating, absorbing and/or attaching the drugs with the matrices of the nanoparticles. the nanoparticles that act as drug reservoirs also aim to control the drug release, enhance the drug uptake by targeted delivery and protect the drug against enzymatic degradation. this review presents a summary of the integration of nanotechnology and phytotherapy to achieve an improved pharmacological response and better clinical outcome in patients undergoing chemotherapy. keywords: nanomedicine; chemotherapy; natural product; drug delivery; nanoparticles received: 27th april 2020 accepted: 28th may 2020 published online: 5th june 2020 citation: ong yz, and tan lt-h. cancer, natural products and nanodrug delivery systems. prog microbes mol biol 2020; 3(1): a0000089. https://doi.org/10.3687/pmmb.a00000089 introduction cancer remains as one of the leading causes of mortality globally, irrespective of the advent of current available forms of cancer treatment. in 2018, cancer has accounted for an estimated of 9.6 million deaths worldwide[1]. the current therapeutic modalities, such as chemotherapy and radiotherapy, are associated with limitations, including damaging to proliferating healthy tissues, systemic toxicity, chronic side effects and emergence of drug resistance within tumor cells. hence, there is always a continuous need of more effective strategies for the treatment of different human malignancies. throughout the history of mankind, natural products have played a pivotal role in the treatment of various diseases. natural products derived from plants, animals and microorganisms represent the prolific bioresources for pharmacologically active compounds, including as chemotherapeutic agents[2–12]. these natural products have been extensively studied for the prevention and treatment of cancer, such as paclitaxel, vincristine, camptothecin and resveratrol[13]. although they have been demonstrated to possess strong therapeutic value, the clinical application of these promising natural product derived compounds is severely hampered by their poor solubility and bioavailability[14]. as an offshoot of nanotechnology, nanomedicine has made great impact in the field of pharmaceutical and biomedical sciences by achieving breakthroughs in therapeutics and diagnostics of diseases in living organisms. one of the promising breakthroughs is the use of nanoparticles in the development of drug delivery system to improve the treatment of various diseases, including cancer[15]. principally, the nanoparticle-based drug delivery system (ndds) aims to improve the in vivo pharmacological and therapeutic properties of the poorly soluble drugs by dissolving, encapsulating, absorbing and/or attaching the drugs with the matrices of the nanoparticles. the nanoparticles that act as drug reservoirs also aim to control the drug release, enhance the drug uptake by targeted delivery and protect the drug against enzymatic degradation. in 2005, the national cancer institute (nci) has launched the alliance for nanotechnology in cancer due to the emerging of nanomedicine and their potential applications in copyright @ 2020 by ong yz and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: yong sze ong, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. ong.yongsze@monash.edu 2 cancer research[16]. under this approach, nanoparticlebased drug delivery system has been approved as one of the effective strategies to overcome the limitations of conventional chemotherapy with numerous nanoparticlebased drugs and delivery system that are in clinical use. in this review, we aim to critically consolidate the advent of nanotechnology in the context of chemotherapeutic drug delivery. this review also presents the examples of the extensively studied natural product-derived compounds using nanotechnology, including doxorubicin, paclitaxel and vincristine. brief overview of cancer cancer is a collection of genetic diseases characterized by the uncontrolled cell growth as a result of dysregulated processes of cell division and cell death. cancer cells are abnormal cells which lose their ability to undergo apoptosis (the programmed cell death) and uncontrollably proliferate, subsequently leading to formation of malignant tumour that invades to adjacent tissues[16]. the formation of tumour (tumourigenesis) involves a complex multistep process (tumour initiation, tumour promotion and tumour progression) that transforms a normal cell into a malignant one due to dna mutation. in 2011, “the hallmarks of cancer” have been revised, explaining the defect mechanisms of malignant cells that deviate from normal cellular functions (figure 1)[17]. figure 1. the ten hallmarks of cancer[17]. these cancer hallmarks are the common traits of tumour cells with their capabilities to alternate the cell growth (sustaining proliferative signalling and evading growth suppressors), to evade apoptosis and cell cycle (resisting cell death and enabling replicative immortality), to modify cellular metabolism (deregulating cellular energetic), to destruct immunological function, to induce angiogenesis, invasion and metastasis. additionally, the genomic instability[18-22] and inflammation by immune cells facilitate the tumourigenesis[17]. apoptosis and cancer one of the prominent hallmarks of cancer is the ability to resist cell death[17]. cell death is a fundamental programme that happens for two purposes; under defence mechanism when the cells received stressful stimuli or under homeostasis mechanism to remove damaged or aged cells for maintaining cell population[23, 24]. the cell death could occur by either necrosis or apoptosis, depending on the response of the cell to the stimuli[25]. necrosis is an “accident” or premature cell death that occurs when the cells encountered irreversible stimuli such as infectious agents, hypoxia, heat and radiation[26]. the necrotic cells experience rupture in the plasma membrane and organelles due to the swelling of cytoplasm. the cytoplasmic contents are then released to the extracellular space and triggered an inflammatory response[27]. in contrast, apoptosis is a “suicide” or programmed cell death that occurs under some physiological conditions to maintain homeostasis[28]. aged and damaged cells that may interfere with body function are removed from the system[29]. apoptotic cells experience the blebbing of plasma membrane, cell shrinkage, dna fragmentation and activation of specific proteases (caspases). as different from necrosis, the apoptotic process does not trigger the inflammatory response as the apoptotic cell externalises the phosphatidylserine at the outer leaflet of plasma membrane to be recognised and engulfed by .phagocytes[30,31]. tumour initiation (hyperproliferation of cell) happens when apoptosis fails to eliminate the genetic mutated cells[32]ryia-illani mohd. these mutated cells proliferate uncontrollably by evading apoptosis via several mechanisms. one of the common mechanisms is the loss of function of tp53 tumour suppressor gene[17]. tp53 tumour suppressor gene encodes the p53 protein which is responsible to transcript more than 125 genes coordinating the repairing of dna, cell cycle and apoptosis. the mutation of tp53 gene has been identified as a common molecular characteristic in human cancer[33]. therefore, tp53 has become a potential target in cancer treatment with the aim to restore the transcription function[34]. apoptosis is executed via two downstream signalling pathways: intrinsic and extrinsic pathways, depending on the source of the stimuli (figure 2)[35]. the intrinsic pathway receives the intracellular death signals from non-receptor-mediated stimuli and initiates the events in mitochondria[24]. the up-regulation of pro-apoptotic proteins (bax, bad and bim) or/and down-regulation of anti-apoptotic proteins (bcl-2, bcl-xl and bag) stimulate the release of cytochrome c from mitochondria, leading to the formation of apoptosome in the cytosol. the apoptosome then initiates a cascade of proteolysis with the effector caspases (caspase-3 and -7) that lead to the execution of apoptosis[36]. on the other hand, extrinsic pathway receives extracellular death signals from transmembrane death receptors such as tumour necrosis factor (tnf) receptor and fas receptors after binding with their homologous ligands (tnf-α and fasl). activation of these death receptors results in the activation of fas-associated death domain protein (fadd) and caspase-8 that further initiates the effector caspases (caspase-3 and -7) and triggers apoptosis[37, 38]. cancer, natural products... 3 ong yz et al. figure 2. the intrinsic and extrinsic pathways of apoptosis[35]. angiogenesis and cancer another hallmark of cancer is the induction of angiogenesis[17]. angiogenesis is the formation of new blood capillaries from existing blood vessels[39]. formation of new capillaries in the tumour enables the tumour cells to obtain nutrients and oxygen and to remove the metabolic waste and carbon dioxide. this has promoted the tumour growth and metastasis of tumour cells to other organs[17,40]. vascular endothelial growth factor (vegf) family members such as vegf a and b proteins are the key components to induce angiogenesis. in response to hypoxia, the level of hypoxia inducible factor-1 α (hif-1α) promotes the expression of these pro-angiogenic vegfs in tumour[41]. the vegfs then bind to their respective receptors on the endothelial cells in the extracellular matrix, causing the differentiation of endothelial cells to form new capillaries[42]. therefore, vegf has become the therapeutic target to control the tumour growth, angiogenesis and metastasis[41,43]. metastasis and cancer activation of invasion and metastasis is one of the characteristics of malignant cancer[17]. metastasis is defined as the spread or development of cancer in other distinct organs from the primary tumour through blood and/or lymphatic vessels[44]. it is always related to poor prognosis as it is the leading cause of cancer death[45]. for tumour cells to pass through the tissue barriers, they have to develop the ability to break through the extracellular matrix (ecm)[46]. one of the proteolytic enzymes that are responsible to degrade ecm is the matrix metalloproteinases (mmps). ecm metalloprotease inducers (emmprin) are produced on the membrane of the tumour cells to activate mmps including the mmp-2 (72-kda gelatinase a) and mmp-9 (92-kda gelatinase a)[47]. natural products as a source of chemotherapeutic agents natural products are defined as the compounds that are produced from nature such as plants, microorganisms and animals as a result of nutritional needs and evolution to adapt the environmental challenges[48–62]. historically, plants have been extensively documented in traditional medical systems such as traditional chinese medicine, indian ayurvedic system and egyptian “ebers papyrus” to promote health and cure diseases[63]. nowadays, plants have become an important source for discovery of bioactive compounds (phytochemicals) with promising therapeutic activities such as anti-cancer, anti-inflammatory and anti-bacterial effects[13,64–75]. majority of the marketed chemotherapeutic agents were isolated and derived from medicinal plants due to the advantages of readily available and cost effective[76]. the well-known examples of plant-derived chemotherapeutic compounds are paclitaxel from taxus brevifolia[77], camptothecin from camptotheca acuminata[78], vinblastin and vincristine from catharanthus roseus[79]. these phytochemicals can be further classified into alkaloids, flavonoids, taxanes, lignans, stilbenes and more[76]. table 1 summarises some of the potential and clinically approved plant-derived chemotherapeutic compounds with their mechanisms of action. table 1. potential and clinically approved plant-derived chemotherapeutic compounds with their mechanisms of action phytochemical plant name and part mechanism of action and type of cancer reference camptothecin synthetic derivatives: topotecan and irinotecan camptotheca acuminata (bark and stem) dna topoisomerase i inhibitor in glioblastoma, ovarian, lung and colorectal cancers [78] vinca alkaloids (vincristine and vinblastine) synthetic derivatives: vinorelbine, vindesine, and vinovelbine catharanthus roseus (leaves and bark) microtubules acting agent that arrests cell cycle in leukemia, breast, lung and liver cancers [79,80] 4 colchicine synthetic derivatives: colchicinamide, deacetylcolchicine colchicum autumnale (leaves) arrest cell cycle by binding to tubulin in melanoma, colorectal and breast cancers [81,82] pomiferin maclura pomifera (fruits and flowers) inhibit histone deacetylase and cause oxidative dna damage in colorectal cancer [83] paclitaxel synthetic derivative: docetaxel taxus brevifolia (barks and leaves) microtubules acting agent that binds to β-tubulin and arrests cell cycle in breast, ovarian and prostate cancers [77,84] podophyllotoxin synthetic derivatives: etoposide and teniposide podophyllum peltatum (leaves) inhibit microtubule polymerization and arrest cell cycle at g2/m phase induce apoptosis via modulation of intrinsic and extrinsic pathways in non-small cell lung carcinoma [85,86] resveratrol veratrum grandiflorum (leaves) inhibit angiogenesis and induce apoptosis via modulation of vegf, p53, bax, bcl-2, cytokines, and cox-2 proteins in breast, liver, prostate, lung and colorectal cancers [87,88] thymoquinone nigella sativa (seed oil) induce apoptosis via modulation of intrinsic pathway in breast cancer [89] geniposide gardenia jasminoides (fruit) antioxidant and anti-inflammatory via induction of nrf2 and gpx [90] nanotechnology in drug delivery: nanomedicine the word “nano” comes from the greek “nannos” (dwarf or a very short man) that refers to the prefix of one-billionth of a meter (a factor of 10-9)[91]. according to the national nanotechnology initiative (nni), nanotechnology is defined as “the knowledge and manipulation of matter within nanometer scale (1–100 nm) that comprises multidisciplinary fields of nanoscale science, engineering and technology”. owing to the abilities to shape, process and create things at nanoscale, nanotechnology has been addressed as the next “industrial revolution” that offers tremendous advances to human being in the application of communications, chemistry, engineering, medicine and robotics[91,92]. one of the most active applications of nanotechnology is nanomedicine[93]. nanomedicine is defined as “the application of nanotechnology in the field of medicine”[94]. with the utilisation of nanoparticles, nanotechnology has brought positive impact to human health in diagnosis, prevention and treatment of diseases[95]. it involves the development of nanomaterials and nanodevices for the applications of drug delivery, in vivo diagnosis, implants and nanotheranostics[96]. nowadays, various types of material have emerged as useful nanomaterials for the development of drug delivery system. the nanoparticles can take various shapes and sizes with distinct properties depending on the types of nanomaterials used. generally, nanoparticle-based drug delivery systems can be categorized based on biological-origin materials (such as phospholipids, dextran, lipids, chitosan, and lactic acid) or inorganic materials (such as polymers, carbon, silica and metals)[97,98]. the family of nanoparticles, including polymeric nanoparticles, lipid nanoparticles and inorganic nanoparticles, is illustrated in figure 3[99]. cancer, natural products... figure 3. family of nanoparticles in drug delivery systems. 5 drug delivery is defined as “a process of delivering a pharmaceutical agent for pharmacological reactions”[100]. an ideal drug carrier used in the drug delivery system should possess the ability to transport the optimum dose of pharmaceutical agent to the desired site without causing adverse side effects in other tissues due to unwanted accumulation[101]. besides, the intentions of drug carriers are to protect the therapeutic agents from degradation by gastrointestinal enzymes, to improve the bioavailability of hydrophobic or lipophilic therapeutic agents and to facilitate controlled release of drug[102–106].on top of these criteria, nanoparticles have been developed as the novel drug delivery system that provide extra advantages of low possibility of rapid clearance from the body through extravasation or prevention from phagocytosis by macrophage due to their nanometer size[107,108]. limitations of conventional delivery of chemotherapeutic agents chemotherapy is the most common therapeutic approach for breast cancer. the highly-cytotoxic chemotherapeutic agents are directly administered into the blood circulating system by intravenous or oral route to kill rapidly-dividing cells[110]. however, this conventional drug delivery system encounters some drawbacks as follows: a non-specificity of chemotherapeutic agents: the random distribution and non-specific targeting of chemotherapeutic agents in the body system have caused unwanted accumulation and toxicity to other normal cells that divide rapidly such as bone marrow, hair follicle and digestive tract cells[111]. the most common adverse effects of chemotherapy are anaemia, alopecia, nausea, vomiting and acute cholinergic gastrointestinal effects[112]. b poor pharmacokinetics via oral administration: cancer is considered as chronic disease that requires long term frequent treatment. therefore, oral route of drug administration has been the most preferred choice due to the reasons of patient’s convenience, compliance, lower cost and painless[113]. nevertheless, the desired therapeutic dose needed for maximum therapeutic effect is hard to achieve via oral administration of active drugs due to the enzymatic and hydrolytic degradation in the gastro-intestinal fluids, low cell uptake in the gastro-intestinal tract, firstpass hepatic metabolism, susceptibility to efflux transport and short biological half-life[114, 115]. c poor aqueous solubility: conventional delivery of chemotherapeutic agents remains a challenge as more than 40% of the potential anti-cancer compounds are hydrophobic or lipophilic. the insolubility of these drugs in water becomes an issue as they could not achieve the desired concentration in the systemic circulation. therefore, various methods have been applied to improve the drug solubility such as drug carrier, chemical/physical modification of drugs, use of surfactant and salt formation[14]. application of novel drug delivery system in cancer therapy nanotechnology has appeared as an attractive approach for solving the drawbacks of conventional drug delivery system of chemotherapy. in principle, the potential or existing chemotherapeutic agents are conjugated or encapsulated in the nanoparticles in order to improve their pharmacokinetics and bioavailability[116,117]. as compared to discovery of new chemotherapeutic agents, the development of this novel drug delivery system only utilized half of the time (6–8 years) and 20% of the cost ($50–$60 million) to be clinically approved and marketed[111]. in 2005, the national cancer institute (nci) has launched the alliance for nanotechnology in cancer due to the emerging of nanomedicine and their potential applications in cancer research. under this approach, nanoparticle-based drug delivery system has been approved as one of the promising strategies to overcome the limitations of conventional chemotherapy with numerous nanoparticle-based drugs and delivery system that are in clinical use. an effective chemotherapeutic drug delivery to a solid tumour involves five steps which are circulation in blood, accumulation and penetration in the tumour, internalization by cancerous cell and drug release (capir)[118] (figure 4). figure 4. the capir cascade of chemotherapeutic drug delivery[118]. the overall therapeutic efficiency of chemotherapeutic drugs is improved by nanoparticle-based drug delivery system by its abilities of accumulation and penetration in tumour, which could be achieved by either passive or active targeting (figure 5)[119]. figure 5. targeted delivery system by nanoparticles[15]. ong yz et al. 6 in passive targeting, the circulating nanoparticles could passively extravasate from blood vessels and accumulate in the tumour via enhanced permeability and retention (epr) effect. this epr effect could only be achieved in tumour due to its unique characteristics of leaky blood vasculature and poor lymphatic drainage[120]. the normal blood vessels are endowed with tight junctions that could allow molecules less than 10 nm to permeate; meanwhile, the fast growing blood vessels that surround the tumour are highly disorganized and dilated due to the defective smooth muscle and enlarged gap junctions, allowing molecules less than 600 nm to permeate[121,122]. furthermore, the tumour possesses poor lymphatic system that further enhances the entrapment of nanoparticles in the tumour[123]. on the other hand, active targeting can be achieved by attaching the ligands (proteins or antibodies) on the surface of nanoparticles that can interact with specific over-expressed receptors on the tumour[123,124]. in principle, this mechanism allows the nanoparticles to identify and recognise cancerous cells, thus minimising the unwanted systemic exposure of chemotherapeutics to other tissue[123]. table 2 shows some examples of clinically approved nanomedicine for cancer chemotherapy[109,125]. conclusion natural products have been showing impressive potentials as chemotherapeutic agents for cancer treatment, but their success in clinical trial has been limited as they have low solubility and bioavailability. the era of nanomedicine in cancer therapeutics has promisingly overcome the challenges hampering conventional therapy regimes hurdled by barriers of low solubility and physiological stability, poor bioavailability and specificity and high toxicity. nanotechnology has revolutionized current cancer therapeutic modalities with the introduction of nano-drug delivery systems which are more efficient and less toxic as well as enhanced specificity to target tumor cells. the present in vitro and in vivo results pose a promising picture, but much more efforts are required from basic molecular aspects to preclinical and clinical trials before advancing more clinical use of nanoformulations in cancer chemotherapy. nanoparticle trade name description type of cancer manufacturer liposome doxil® doxorubicin encapsulated in pegylated liposomes reduced toxicity of doxorubicin ovarian and breast cancer orthobiotech, schering-plough myocet® doxorubicin citrate encapsulated in liposomes lower clearance and higher half life of drug breast cancer elan/sopherion therapeutics marqibo® vincristine sulfate encapsulated in liposomes lower clearance acute lymphoblastic leukemia talon therapeutics, inc. daunoxome® doxorubicin encapsulated in non-pegylated liposomes prolonged circulation time and higher accumulation in tumour site hiv-associated kaposi’s sarcoma galen pharmaceutics marqibo® vincristine encapsulated in non-pegylated liposomes reduced toxicity and higher accumulation in tumour site philadelphia chromosomenegative acute lymphoblastic leukemia spectrum pharmaceuticals onivyde® irinotecan encapsulated in non-pegylated liposomes prolonged circulation time, and higher accumulation in tumour site pancreatic cancer merrimack pharmaceutics table 2. clinically approved nanomedicine for cancer treatment[109,125]. polymerbased nanoparticle genexol® paclitaxel in peg-pla copolymer micelles increased solubility of paclitaxel metastatic breast cancer and pancreatic cancer samyang biopharmaceuticals opaxio® soluble ester conjugate of paclitaxel and α-poly(l)-glutamic acid prolonged tumour exposure of paclitaxel reduced neutropenia and alopecia ovarian cancer cell therapeutics eligard® lueprolide acetate and polymer prolonged drug circulation time and controlled drug release prostate cancer tolmar pharmaceutics cancer, natural products... 7 abbreviation: peg-pla, poly(ethylene glycol)-poly(d,l-lactide). author contributions y-so, lt-ht participated in the writing of the manuscript. y-so conceptualized the project. conflict of interest the authors hereby declare no competing 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for tumor targeting. drug discov today 2006; 11(17–18): 812–818. 122. cho k, wang x, nie s, et al. therapeutic nanoparticles for drug delivery in cancer. clin. cancer res 2008; 14(5): 1310–1316. 123. bazak r, houri m, el achy s, et al. cancer active targeting by nanoparticles: a comprehensive review of literature. j cancer res clin oncol 2014; 141(5): 769–784. 124. tan lt-h, chan k-g, pusparajah p, et al. targeting membrane lipid a potential cancer cure? front pharmacol 2017; 8: 12. 125. ragelle h, danhier f, préat v, et al. nanoparticle-based drug delivery systems: a commercial and regulatory outlook as the field matures. expert opin drug deliv 2017; 14(7): 851–864. ong yz et al. progress in microbes and molecular biology review article 1 updates on the development of vaccines and therapeutic options against rabies roshan arjun ananda1,2†, hooi-leng ser1†, vengadesh letchumanan1* 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia. †these authors contributed equally in the writing. abstract: even though rabies has been claiming more than 50,000 deaths annually worldwide, it is considered as a vaccinepreventable viral disease. more than 95 % of the total human rabies cases are caused by dogs. during the initial stage of infection, affected individuals usually show weakess at the bitten extremities and the virus can ultimately travel to the brain causing neurological signs. in attenuated (inactivated) form, the currently in use vaccines have been recommended by who for the prevention (i.e. pre-exposure prophylaxis, prep) and treatment (i.e. post exposure prophylaxis, pep) regime against the rabies virus (rabv). however, given that they normally require refrigeration and are costly, there have been discussions revolving around potential development of newer, safer and cheaper alternative that can perform better and more convenient than the ones that are currently in use. the current review aims to explore general characteristics of rabv before looking into potential candidates of vaccines that have been studied. further studies on the pathogenic mechanism of rabv and therapeutic approaches are still required to prevent the deathly infection following clinical manifestation. in sum, integrated interventional strategy emphasizing human health and animal health is essential and requires collaboration between health authorities and the public. keywords: rabies; vaccines; treatment; development; therapeutic received: 27th june 2020 accepted: 28th july 2020 published online: 7th august 2020 citation: ananda ra, ser h-l, letchumanan v. updates on the development of vaccines and therapeutic options against rabies. prog microbes mol biol 2020; 3(1): a0000100. https://doi.org/10.36877/pmmb.a0000100 introduction rabies is one of the dangerous zoonotic diseases, claiming more than 50,000 deaths per year worldwide[1]. the “culprit” behind rabies is known as rabies virus (rabv) which can be transmitted by animals including bats, raccoons and foxes[1–3]. still, dog-mediated rabies infection accounts for more than 95% of the total human rabies cases as the virus can replicate in salivary glands of infected dogs. as a results, rabv can be easily transmitted from affected dogs through bite wounds, licking of damaged skin, or direct mucosal contact[1,4]. the virus attaches itself to its cellular targets by its surface protein (i.e. rabv-g), rapidly gaining access to peripheral nerves. via retrograde axonal transport and trans-synaptic spread, rabv ultimately enters the brain[5]. if the disease is not treated in a prompt manner, death can occur within 5–7 days upon onset of symptoms[6]. the incubation time before clinical manifestation is influenced by several variables including distance of injection site from the central nervous system (cns) and virus load at the wound site[7]. shorter incubation period was observed in victims with a wound on the head/neck or category iii exposure[8].commonly, initial presentation of rabies-infected victims is the weakness at the bitten extremities, which subsequently progresses into acute neurological signs[6,9]. however, there are circumstances in which the victims presented unusual symptoms including severe abdominal pain and abnormal sexual behaviours[10,11]. rabies infection can manifest as furious or paralytic form. limbic signs are predominant in furious rabies while paralysis of lower motor neuron is hallmark for paralytic rabies[10,12]. even after recovery, most rabies survivors suffer from neurological impairment[6,13]. as a consequence, an “ideal” effective immunological defense against rabies would be the interception of virus before productive neuronal infection, considering copyright @ 2020 by ananda ra and hh publisher. this work under licensed under the creative commons attributionnoncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: vengadesh letchumanan, novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. vengadesh.letchumanan1@monash.edu. 2 there is still established, effective therapy for those who developed rabies encephalomyelitis[14]. canine rabies remains endemic in most developing countries, it is a huge global burden with estimated 3.7 million disability-adjusted life years and 8.6 billion usd economic losses every year[15]. the world health organization (who) has issued a notice in the past to discontinue the usage of nerve tissue vaccine and replace the vaccination program with newer vaccine produced from cell-culture or embryonated eggs[1,16]. these newer vaccines typically consist of purified inactivated virus that can be used as prevention (i.e. pre-exposure prophylaxis, prep) and treatment (i.e. post exposure prophylaxis, pep). thus, the current review aims to provide an overview on the characteristics of rabv before exploring available vaccines and those which are in development. indeed, rabies may not seem like a disease that can be eradicated completely worldwide, thus it is imperative to continuously seek for safer vaccines or drugs with higher efficacy to curb the spread of such harmful pathogens. discovery and characteristics of rabv as one of the oldest communicable disease known to man, rabies was documented several times in historical records, as early as 4,000 years ago in the pre-mosaic eshnunna code[17–19]. in the code, it was stated that the owner of a rabid dog that bit a person who later died due to rabies must pay a fine. rabies is an acute, lethal disease marked by encephalomyelitis in which the causative agents are identified to be viruses belong to the genus lyssavirus. rabv or taxonomically known as rabies lyssavirus (under family: rhabdoviridae, genus: lyssavirus) is a negative-strand rna virus. in general, those within the genus lyssavirus are enveloped rna virus that are viewed as bullet-shaped when cut tangentially or “bulls-eye” in cross sectional view under transmission electron microscope[20,21]. the genome of lyssaviruses is approximately 11–12kb in size, encoding five proteins including glycoprotein (g), phosphoprotein (p), nucleoprotein (n), matrix protein (m), rna-dependent rna polymerase (l) (figure 1) [22,23]. belonging to phylogroup i, rabv seems to be far more “adaptable” compared to other strain in the same phylogroup — circulates in both chiroptera (i.e. bats) and carnivora (i.e carnivores) including wolves, foxes, shunks and dogs[24]. several strains of rabv have been previously described and it has been discussed that certain polymorphisms within the genome can alter virulence and transmission[25]. for rabv, its genome encodes viral proteins in the sequence of 3’-n-p-m-g-l-5’[26,27]. the viral structure consists of m protein encoded by gene m and transmembrane g protein encoded by gene g. g protein plays critical roles in the pathogenesis of rabies by binding to neural receptors and cellular entry via fusion with the cellular membrane[28–30]. as the only surface proteins, g protein is the only protein that is capable of inducing production of virus neutralizing antibodies (vnas) by the host, therefore essential in determining the evasiveness of rabv against the host immune system[30,31]. a study in 2019 compared laboratory-adapted rabv strain (b2c) and a wild type (wt) rabv isolated from rabid dog in mexico in 1990s (drv); the team discovered that lesser g molecules were incorporated into mature virions by wt rabvs when compared to laboratory-adapted rabvs[30]. while recombinant virus with additional g protein (i.e. triple g expression) showed higher expression and incorporation of g protein, the virus activated more dendritic cells (dc) compared to its corresponding wild type form. conversely, wild type rabvs that were treated with subtilisin or dithiothreitol (dtt)/nonidet p-40 (np40) to remove g protein failed to activate any dc and/or vnas expression. without g protein, these g protein-depleted virus evaded the host immune response and caused lethal infection in mice. furthermore, another study showed that single amino acid change(s) at position of 255 or 349 in g protein decreased the viral pathogenicity of rabv[31,32]. for instance, after introducing the amino acid change at position 349 nucleotide substituting glycine with glutamine (gly349→glu349), the mutant strain (known as rgdsh-g349) exhibited decreased rabv pathogenicity without affecting its propagation rate[32]. on top of that, the same strain was able to induce higher immunogenicity in mice with higher level of vna observed compared to its parent strain. altogether, these important findings greatly benefited the scientific community by providing crucial insights into “behaviour changes” of the virus while at the same time enabling researchers to exploit these mutation points for development of therapeutic drugs and vaccines against rabies. rabies vaccine development... figure 1. illustration of rabies virus genome and its common animal reservoir. 3 ananda ra et al. tissue-based vaccines were developed and used as rabies vaccine but they are being phased out in most countries in the 21st century since the introduction of non-neural tissuebased vaccines. in addition, the crude neural vaccines made from sheep or mouse brains caused severe adverse effects and neurological sequalae including acute demyelinating encephalitis[46,47]. even so, a few countries including ethiopia are still using neural vaccines due to their affordability and high cost of newer vaccine[46,48]. as for preventive measures, who recommends rabies prep for individuals who are at high risk of exposure to rabies including veterinarians, laboratory workers, travellers or residents of rabiesendemic nations[1,49]. prep obviates rabies immunoglobulins administration and reduces the number of vaccine doses required when an individual is exposed to rabies[1,49-51]. according to who guidelines, a complete prep consists of single-dose intramuscular (im) or two-site intradermal (id) vaccination on both day 0 and day 7. at the same time, who has also published a detailed guidelines and recommendation on pep to assist physicians in making decision on treatment: (i) category i involves touching of animals or licks on intact skin; (ii) category ii involves nibbling of uncovered skin or non-bleeding minor abrasions; and (iii) category iii involves transdermal bites, direct contact with bats, licks on broken skins or mucous membranes[1,51]. no pep is indicated for category i exposure, but only active immunization (i.e. vaccine) will be given for those with category ii exposure. for category iii exposed individuals, they will be given both active and passive immunization (i.e. vaccine and monoclonal antibodies administration). the recommended dose of passive immunization is given at 20 iu/kg body weight for hrig and 40 iu/kg body weight for erig and f(ab’)2 products. full dose of rabies immunoglobulins (rig) can be given into or around wound site, but it can be diluted with physiological buffered saline to ensure better wound coverage in severe cases. instead, purified cell-cultureor embryonated-eggbased rabies vaccines can be administered intramuscularly or intradermally[1,36]. pep regimens recommended by who include two-sites id rabies immunization (2-2-2-0-0) on day 0, 3 and 7; two-weeks im rabies immunization (1-1-1-1-0) on day 0, 3, 7 and 14; three-weeks im rabies immunization (2-0-1-0-1) on day 0, 7 and 21–28[1,50,51]. im and id immunization were commonly recommended because subcutaneous injections of rabies vaccines failed to induce sufficient antibody response after 1 month completing immunization protocol[52]. receiving rabies vaccination and rig within first 7 days and 48 hours respectively is considered as timely pep response[53]. though, people who received at least two doses of rabies pre-exposure vaccines do not require rig infusion [1,51]. in events of re-exposure to animal bites, a previously immunised patient only require booster injections on day 0 and 3[49-51]. so the next important question would be — what are these vaccines make of? as discussed earlier, the host immune system must first recognize the pathogens before initiating “attacks” on the intruders. having that said, it may seem to be unwise to inject someone with live virus to activate someone’s immune system; nevertheless, looking at the history, one of the earlier version of “vaccination program” was done in small pox known as variolation, whereby they on the other hand, the n protein is thought to be a preferred target for phylogenetic studies given that it’s highly conserved and expressed while accountable for activating immunogenic response from the host[33–35]. as a matter of fact, n protein which forms the major component of helicoidal nucleocapsid that encapsidates the genomic rna plays a determining role in viral replication; it facilitates the temporal transition between transcription and replication of the viral genome during the replicative cycle[35]. in contrast, the phosphoprotein encoded by gene p serves as a cofactor for l protein, connecting it to n protein and finally leading to the formation of ribonucleoprotein complex in viral rna synthesis[36,37]. besides polymorphism, the rearrangement of viral genes such as p and n has been shown to affect its pathogenicity and immunogenicity. a team led by mei et al. in 2019 found that the rearrangement of gene p in rabv led to its low gene expression which then suppressed n gene and attenuated the pathogenicity of the virus [32,33]. similar results were reported by morimoto and team whereby the p-gene deficient (def-p) virus was apathogenic in adult and suckling mice. it was also described that even though the def-p virus can perform the primary rna transcription, no further progeny virus was produced by the infected host (with def-p virus)[37]. located on the third position in rabv genome, the m protein encoded by gene m is an important component during viral assembly and budding, covering the rnp coil and maintaining the viral bullet-shaped form [38,39]. on top of that, some studies have highlighted the role of m protein in viral transcription, whereby genetic manipulation on gene m via codon deoptimization led to inhibition of rabv replication at the initial stage of infection but increased viral titre at later stages[40,41]. likewise, the codon deoptimization strain caused higher level of apoptosis in neuronal cell compared to its parental strain[42]. besides shedding light on the transmission and replication mechanisms of rabv, the understanding on its genomic content allows researchers to identify and exploit these “weak points” in designing treatments or vaccines against this deadly virus, while monitoring rabv outbreaks and evolution. treatment and vaccines development against rabies lyssavirus for human use in order to fend off infections, the infected host needs to have sufficient and/or adequate immune response to first recognize the infectious agent(s) before eliminating it from the body. before discussing in-depth about each vaccines that are in-use or in development (i.e. novel), it is important to note that currently in-use vaccines for rabies can be used as prevention (i.e. pre-exposure prophylaxis, prep) and treatment (i.e. post exposure prophylaxis, pep); however, the only difference between these two lies in immunization schedule[43,44]. moreover, there are two forms of immunizations: (a) passive immunization — by administration of monoclonal antibodies (e.g. human rabies immunoglobulins (hrig), equine rabies immunoglobulin (erig)) and (b) active immunization which involves the use of cell cultureor embryonated egg-based inactivated virus[44,45]. in 1888, crude nerve 4 inoculate a boy with materials from cowpox pustule and observed protective effect against matter from smallpox lesion[54]. for rabv, there have been many studies looking into potentially more effective vaccines over the years, apart from the attenuated rabv vaccines that are currently in use. nucleic acid-based vaccines are getting more popular these days, as researchers are working around the clock to develop them given that this approach combines the positive attributes of both live-attenuated and subunit vaccines[55]. a research team in germany successfully developed a synthetic messenger rna (mrna) based vaccine which consists of an optimized non-replicating rabies virus glycoprotein (rabv-g) mrna sequence in 2016. when compared with licensed rabies vaccines, this mrna vaccine managed to induce comparable cd4+ t cells and cd8+ t cells responses upon two injections[56]. subsequently in 2017, stitz and team described another attractive feature for their mrna vaccine — thermostability; they showed that the mrna vaccine that retained its immunogenicity and protective effects against rabv even after exposure to temperatures as high as 70°c[57]. the development of thermostable vaccines provides extended shelf life in challenging conditions especially in tropical countries and economical vaccine stockpiling in preparation for epidemic threats. in fact, a phase i clinical trial carried out in 2016 using the same mrna vaccine technology (rnactive®), studying the safety of and immunogenicity of this vaccine in healthy volunteers (nct02241135)[58]. a total of 101 participants were enrolled and vaccinated with 306 doses of mrna (80–640 μg) by needle-syringe or needle-free devices (via intradermal or intramuscular route). as the first drug substance of mrna vaccine against rabv, cv7201 or nadorameran (as named by who) was described as generally safe with a reasonable tolerability profile. the same study reported the observation on vna titres of 0·5 iu/ml or more across dose levels and schedules in 71% of participants given 80 μg or 160 μg cv7201 doses intradermally and 46% of participants given 200 μg or 400 μg cv7201 doses intramuscularly. nonetheless, 57% of them (i.e. 8 out of 14 participants) achieved titres of 0·5 iu/ml or more after receiving needle-free booster shot of cv7201 at 80 μg intradermally, while those underwent intradermal or intramuscular needlesyringe injection failed to respond (i.e. no immune response) except one participant who received 320 μg of cv7201 intradermally. additionally, another recent study highlighted that the co-administration of rnabased adjuvant cv8102 with licensed vaccine for rabies, rabipur® in phase i clinical trial (eudract no. 2013-004514-18, nct02238756) indicated that cv8102 was safe up to 50 μg and enhanced immunogenicity of the licensed rabies vaccine significantly[59]. even so, there is still much to do to determine the appropriate vaccination dose and schedule of mrna vaccine alone or as adjuvant for prep and/or pep regime. besides that, there are several groups discussing the use of viral vector-based vaccines against rabv, such as via the incorporation of rabv glycoprotein genes into west nile virus backbone to induce protective effect [7,60–62]. in the study by giel-moloney and team, the rabv g protein expression remained stable after multiple in vitro passages and the vaccine exhibited durable protective immunity with high titres of complementing t helper cells[60]. the vaccine which uses replivax® technology is a highly promising vector delivery system, given that immunized dogs displayed durable protective immunity when tested at oneand two-year post immunization. in addition to that, there are other recombinant rabies vaccines generated using different viral vector systems, such as poxvirus[7,63,64], newcastle disease virus[65], parainfluenza virus[66], adenovirus[67,68], or baculovirus[69,70]. despite of that, these vaccines may not be available for clinical use at the moment, particularly regarding efficacy restricted to certain species but not human, safety considerations, and similar to the concern with mrna vaccines—usage as pep and/or prep vaccination. looking on the bright side, there are two ongoing phase i clinical trial studying the safety and immunogenicity of novel recombinant rabies vaccines, chad155-rg (nct04019444) and chadox2 rabg (nct04162600)[71,72]. in reality, another critical point to consider in designing recombinant vaccine is that the viral vector used must not be pathogenic while being able to trigger protection against certain pathogens (including rabv)[73,74]. decades have passed since the first vaccine for smallpox and an increasing number of researchers are considering the possibility of immunization against multiple pathogens with the use of single viral vector carrying fragments of another virus (e.g. multivalent vaccine)[74,75]. for instance, a novel vaccine consisting of inactivated rabv that expressed protein fragments of middle east respiratory syndrome coronavirus (mers-cov) was proven to be effective in producing antibodies against rabies and mers-cov infection[74]. developed by wirblich and team, bnsp333-s1 is an inactivated rabv-mers s-based vaccine and the team observed increased antigen-specific igg responses over time after each immunization. besides that, there is another genetically modified rabv vectorbased rift valley fever virus (rvfv) vaccine which induced significant rabies vna level but it is still unsure whether it can protect against rvfv as it failed to induce rvfv vna (despite high titres of anti-rvfv igg antibodies)[75]. therefore, multivalent vaccines against rabies and other infectious diseases can be developed, but further validation tests should be conducted thoroughly in clinical studies to confirm its efficacy and safety. current measures in place to control the spread of rabv from animals to humans animal mass vaccination while the development of rabv vaccine for human use is essential to combat against rabv, the preventive measures and management of wild life including carrier of rabv are equally important. who recommended that mass vaccination of at least 70% rabies-susceptible dog population is essential to achieve herd immunity and contain the virus as 95% of human rabies cases were caused by dog bites[1]. although mass vaccination of dogs is the most cost-effective method for significant decrease rabies vaccine development... 5 neutralization tests when other samples collected are of low quality[96]. therefore, bi-annual and annual bait distribution schedules are sufficient for rabies control in wildlife reservoirs[92]. outbreak prevention appropriate health promotion measures, coordinated rabies surveillance and mass vaccination program can prevent rabies outbreaks[97]. introduction of rabies-infected subject to a community could be an imminent threat and trigger an outbreak, especially for a previously rabies-free region[98]. in developing countries, significant stray dog population and inevitable dog movement are recognized as public health risk and could result in rabies outbreak with subsequent bites to other animals by an infected animal[99,100]. while stray dog population in rural regions is correlated with carcass availability, economic implications of dog bites and rabies infections are significant following decline in vultures population. local government should implement strategies for carcass disposal including incinerations. as significant stray dog population and rabies infection are major concerns, bhutan had implemented catch-neuter-vaccinaterelease (cnvr) program[99]. moreover, rabies outbreak in wildlife is of huge concern as animals like fox and wild dogs are highly mobile and travel over a long distance between habitats in different countries, further enhancing the spread of rabies infection[101]. health promotion measures including domestic dog control regulation and mass vaccination program had been implemented in the early 19th century during the japanese colonial period[102]. however, the japanese colonial government was widely criticized in korea for brutality and poor understanding of traditional dog-human relationship. in the 21st century, cultural obligations to dog population remains significant in the rural communities, especially indigenous community, as harming dogs will result in sickness[98]. during an outbreak in india, most people only received rabies preventive measures from friends and consumed traditional herbal medicines[100]. poor knowledge and practices of preventive measures reflected on the health-seeking behaviour of rural communities following an outbreak. as a previously rabies-free region, rabies remains endemic in bali since the introduction of the virus by a sub-clinically infected dog in 2008[103,104]. poor surveillances, diagnostic facilities and treatment policy in 2008 had resulted in circulation of rabies virus across the island following the outbreak[104]. however, local authority in bali has implemented program dharma to improve dog care practices and facilitate mass vaccination program, in addition to reducing roaming dog density. hence, public health officials should organise education awareness campaign to emphasize the significance of dog ownerships and public cooperation as preventive strategies of outbreak[103]. poor handling of outbreak in wildlife or local community could facilitate transmission of zoonotic diseases to human. despite its rabies-free status, australia has identified potential areas of rabies incursion and implemented community-based health promotion approach to increase preparedness of the local community[98]. rabies surveillance including strict monitoring programs is important for prompt control measures as potential cases are identified to halt spreading[97]. in human rabies cases and mortality, local government particularly in endemic countries often neglect these preventive efforts [76,77]. according to world organization for animal health (oie), animals are considered to have protective immunity against rabies infection if they have minimum post-vaccination rabies vna of 0.5 iu/ ml[78]. several canine rabies control strategies including immunization, movement restriction and culling of stray dogs were carried out in several asian and african countries over many decades but were not effective in eliminating rabies from the population[79,80]. introduction of a simple centralised canine rabies vaccination campaign to a rural area in africa increased vaccination coverage from initial estimated 9.5% to between 60 and 70%[79]. there was a significant decline in incidence of dog rabies by 97% after the second vaccination programme with more than 60% coverage of the dog population. however, in korea, canine rabies was successfully controlled with low vaccination coverage, which ranged between 30% and 50%[81]. genetic, temporal and spatial heterogeneities that influence contact and transmission rate can have significant impact on the design of immunization program[82]. besides vaccination coverage, relative success of large-scale vaccination program is also determined by frequency of vaccination campaign and dog density in the area[83]. satisfactory rabies knowledge and awareness in the population will increase rabies immunization coverage[84]. in countries with high birth and death rate of dogs, there is substantial risk of outbreaks occurrence between vaccination campaigns due to rapid decline in overall population coverage following a campaign[79]. oral rabies vaccination (orv) is a cost-effective and socially acceptable technique that can be incorporated into large scale rabies control programmes for canine or wildlife reservoirs[85]. international researchers have generated several effective vaccines over the years. in the late 20th century, a mass vaccination programme using live attenuated rabv vaccine (era-bhk21) successfully eliminated arctic rabies virus variant from red fox population in eastern ontario[86]. similar result was observed in europe where the spread of rabies infection was prevented by vaccinating approximately 60% of fox population with a different live vaccine (sad)[2]. despite the successful results and cost-effectiveness, the use of live-attenuated vaccines in orv programmes remains controversial due to residual pathogenicity, vaccine-induced rabies infection, thermal instability and ineffectiveness of oral immunization in rabies reservoirs including skunks and raccoons[2,85–91]. alternatively, recombinant vaccines were constructed from heterologous virus vectors expressing rabv glycoprotein and were proven to have improved safety profile and thermal stability[92,93]. onrab® is a recombinant oral rabv vaccine generated using human adenovirus vector that expresses rabv glycoprotein and often distributed as bait to animals[92]. onrab® induced sufficient immune response in wildlife reservoirs including red foxes, raccoons and skunks with high survival rate after rabies challenge test 1 year post-vaccination[92,94,95]. during oral vaccination campaign, muscle extracts and thoracic liquid are considered potential samples for virus ananda ra et al. 6 conclusion and future recommendation since the description of rabies by the historical records, humans have made a long way in the discovery and development of vaccines for rabv. in actual, thorough research into molecular virology, immunology and epidemiology have provided remarkable understanding of the circulation of rabies virus. even though the current inactivated vaccine may seem to be working well, it is still far from “perfect” with some studies found inadequate antibodies titre among veterinary students at 2 years after pre-exposure rabies vaccination; the levels of antibodies was independently influenced by several variables including gender, vaccine type or manufacturer, bmi and interval between first and third vaccine doses[105,106]. furthermore, the currently approved vaccine for rabies needs to be refrigerated, complicating the logistics problem which can lead to delay in treatment time[107]. global burden of rabies infection is notable, but the zoonotic diseases as such is preventable. collaborative efforts from several countries have played an important role in improving public health and relieving the economic burden. as part of the drug discovery process, researchers have been studying the potential of small molecules or even peptides expressed by microorganisms and plants to be used to prevent and/or treat infectious diseases including rabv[108–117]. among these studies, tobacco mosaic virus (tmv) isolated from chimeric plants expressed spherical particles (i.e. coat protein of alfalfa mosaic virus fused with antigenic peptides of rabv) that can improve rabies vaccine protective properties due to the presence of rabv antigenic peptides[108,109]. yusibov and team showed that using plants as tool to produce antigens to be used in vaccines provide several advantages including lack of contamination of other human pathogens, reasonable ease of genetic manipulation and economical production[108]. after purification steps, spherical particles formed from the recombinant aimv cp was used to immunize mice and subsequently resulted in an antigenspecific humoral immune response, accompanied with vna. apart from that, nucleoside analogs are potential antiviral compounds because they can act as competitive inhibitors to interfere nucleic acid biosynthesis during replication of viral genome. for example, small molecule drugs such as ribavirin (which is an antiviral drug against respiratory syncytial virus) and favipiravir (i.e. antiviral drug against influenza) have been shown to be effective against rabv as well by acting as competitive inhibitors that interfere with nucleic acid biosynthesis during replication of viral genome[110–113]. along with these, there is also potential use of these molecules in combination and/or as adjuvant (booster) to increase the efficacy or performance of the vaccine. however, further studies on the pathogenic mechanism of rabies virus and therapeutic approaches are still required to prevent the deathly infection following clinical manifestation. having that said, integrated interventional strategy emphasizing human health and animal health is essential and via the collaboration between health authorities and the public, it is highly possible to control and prevent further spread of zoonotic disease like rabies. author contribution the literature review and manuscript writing were performed by r-aa and h-ls. h-ls and vl provided vital guidance and support as content expert and proofread of the 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vitro inhibitory effects toward rabies virus. antiviral res 2018; 154:1–9. 117. banyard ac, mansfield kl, wu g, et al. re-evaluating the effect of favipiravir treatment on rabies virus infection. vaccine 2019; 37(33): 4686–4693. rabies vaccine development... pmmb 2023, 6, 1; a0000338. doi: 10.36877/pmmb.0000338 http://journals.hh-publisher.com/index.php/pmmb original research article evaluation of antioxidant and antimicrobial activity of saponin extracts from different parts of argania spinosa l. skeels yousra el idrissi1, youssef elouafy1, hamza el moudden2, najoua mghazli3, chakir el guezzane1, adil el yadini1, hicham harhar1, abdelkader zarrouk1, khang wen goh4, long chiau ming5,*, abdelhakim bouyahya6,*, mohammed tabyaoui1 article history 1laboratory of materials, nanotechnology and environment lmne, faculty of sciences, mohammed v university in rabat, rabat bp 1014, morocco; yousra.elidrissi93@gmail.com (yei); youssef.elouafy@um5r.ac.ma (ye); chakir.elguezzane@gmail.com (ceg); a.elyadini@um5r.ac.ma (aey); h.harhar@um5r.ac.ma (hh); azarrouk@gmail.com (za); h.tabyaoui@um5r.ac.ma (mt) 2higher school of technology of el kelaa des sraghna, cadi ayyad university, el kelaa des sraghna bp 104, morocco; hamzaelm802@gmail.com (hem) 3team of microbiology and molecular biology, faculty of sciences, mohammed v university in rabat, bp 1014rabat, morocco; najoua_mghazli@um5.ac.ma (nm). 4faculty of data science and information technology, inti international university, 71800 nilai, malaysia; khangwen.goh@newinti.edu.my (kwg) 5school of medical and life sciences, sunway university, 47500 sunway city, malaysia 6laboratory of human pathologies biology, faculty of sciences, mohammed v university in rabat, bp 1014, rabat, morocco *corresponding author: long chiau ming; school of medical and life sciences, sunway university, 47500 sunway city, malaysia; longchiauming@gmail.com (lcm); abdelhakim bouyahya; laboratory of human pathologies biology, faculty of sciences, mohammed v university in rabat, bp 1014, rabat, morocco; a.bouyahya@um5r.ac.ma (ab) received: 14 february 2023; received in revised form: 11 june 2023; accepted: 22 june 2023; available online: 5 july 2023 abstract: the argan tree is a versatile forest tree (silviculture-fruit-forestry) of great importance for the country both in biological, phytogenetic and ecological biodiversity as well as in economic and social aspects. it has significant medicinal and therapeutic potential. the present study concentrated on saccharide-containing molecules, particularly saponins, for their medicinal properties. butanol was used to fractionate phytochemical groups from different parts of argania spinosa (branch, leaf, pulp, shell and seed), which were subsequently precipitated in ether. it was found that solvent fractionation increased the total saponin amount according to the colorimetric test using vanillin and sulfuric acid. the butanol fraction of the argan shell and its precipitate showed the highest levels of triterpene saponins compared to the rest of the plant parts studied. the samples showed antioxidant capacity by dpph versus ascorbic acid (ic50 = 1.92 µg/ml) and by abts using the similar pmmb 2023, 6, 1; a0000338 2 of 22 standard (ic50 = 11.31 µg/ml). the antioxidant activity of the samples was significantly improved from the crude shell extract (ic50 = 5.85 µg/ml) to its butanolic fraction (ic50 = 2.94 µg/ml) and its precipitate (ic50 = 1.57 µg/ml) using 2,2-diphenyl-picrylhydrazyl. indeed, the abts test, showed similarity to the core results. also, the solid disc diffusion method was used to highlight the antimicrobial activity of extracts from different parts of the argania spinosa plant. the etoh 70% and aqueous leaf extracts exhibited the highest antibacterial activity, resulting in an inhibition zone of approximately 13 mm against the growth of staphylococcus saprophyticus. on the other hand, the extract of saponin filtrate from the pulp part of the plant displayed the lowest antibacterial activity, with an inhibition zone of 9 mm. all studied types of saponin extract from different parts of the plant do not contribute to any antifungal activity against botrytis cinerea. keywords: argania spinosa; antioxidant activity; saponins; antibacterial activity; antifungal activity 1. introduction throughout the world, natural resources are facing a concerning degradation due to the excessive exploitation and mismanagement practices[1]. this not only poses a threat to the overall ecosystem but also has the potential to lead to the loss of valuable natural resources that possess significant pharmacological and biological properties. many natural resources harbor compounds and substances utilized for centuries in traditional medicine and have shown promising therapeutic potential[2–6]. morocco is well-known for its diverse and rich forestry, with several species of trees that possess significant medicinal properties[7–14]. among these trees, the argan tree (argania spinosa l. skeels) stands out as a highly endemic species, ranking as the second most abundant forest species in the country, following the holm oak (quercus ilex) and preceding the cedar (cedrus atlantica)[15]. it is estimated that the tree will live between 150 and 200 years. drought and heat are not a problem for it. the argan tree grows wild and abundant in southwest morocco's arid and semi-arid areas. in this geographical area, the argan tree plays an irreplaceable role in the ecological balance and in preserving biodiversity. in addition to this environmental role, the argan tree has a very important economic interest; the argan tree produces argan oil as its main product[16]. there are two categories of argan oil, cosmetic oil from unroasted almonds and edible oil extracted from roasted almonds. the physicochemical properties of these two types of argan oil have been well studied[17,18], as well as their production in a controlled and reproducible manner[19,20], and their pharmacological properties[21–23]. indeed, the nutritional properties of argan oil are due to its chemical composition. according to the moroccan standards institute (imanor), argan oil is composed of 80% unsaturated fatty acids[24], and is particularly rich in tocopherols, molecules with powerful antioxidant properties. the high content of tocopherols and the low content of linolenic acid are responsible for its oxidation resistance. the phospholipids present in argan oil contribute to oil preservation[25]. pmmb 2023, 6, 1; a0000338 3 of 22 unfortunately, the current worldwide fame of the argan oil has partially overshadowed the first intensive work carried out on the triterpenic saponins of the argan tree. for some time, this family of molecules has represented the most likely result to successfully save the argan tree from various threats it faced. in fact, saponins possess many pharmacological and biological properties[26], and even if these compounds are no longer studied as intensively as they were a few years ago, the need to diversify the a. spinosa resource is sufficient to warrant further research. literature reports indicate that other sapotaceae species contain glucuronidic triterpenes[27–35]. originally, pure ethanol was believed insufficient as a solvent to extract saponins from different parts of the a. spinosa plant. this paper investigated the total saponin content in the crude extract of the different parts of a. spinosa (branch, leaf, pulp, shell, and seed) as well as its fraction and precipitate. due to the complexity of phytochemicals contained in plant extracts, liquid-liquid extraction was used to separate the target compounds, utilizing butanol as an organic solvent. the organic fraction was then precipitated in diethyl ether to further recover the chemicals. saponins can be precipitated by utilizing ether to reduce the medium's dielectric constant. therefore, this paper aimed to compare the total saponins estimated in the extracts of different parts of the a. spinosa plant. in this work, we investigated the antioxidant power of dpph (2,2-diphenylpicrylhydrazyl) and abts (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) of the saponin extracts from the plant. however, the antimicrobial activity of the different extracts was carried out using four bacterial strains for the antibacterial activity, and two fungi for the antifungal activity. in addition, principal component analysis (pca) was employed to evaluate the correlation between antioxidant activity (dpph, abts), secondary metabolites (polyphenols, flavonoids, tannins), total sugars, and triterpene saponins in all extracts derived from the a. spinosa saponin extraction process. 2. materials and methods 2.1. preparation of argania spinosa extracts the characterization of the extracts of a. spinosa focused on the different parts of the tree, namely: branch, leaf, pulp, shell, and seed. the different parts of a. spinosa from lakhssas (29° 24′ 43″ north, 9° 43′ 34″ west), guelmim-oued noun region were harvested in the summer of 2020 for the study of saponins. the different parts of a. spinosa, freshly harvested, were washed with tap water and left to dry in the shade for two weeks to preserve as much integrity of their chemical composition as possible. after drying, the different parts of a. spinosa were finely crushed with a professional grinder and then sieved on sieves. 2.2. extraction of saponins samples from different parts of the plant were delipidated by soxhlet for 6 hours using hexane as an apolar solvent. the residue obtained was left in the oven at a temperature of 25 overnight to completely eliminate hexane. the residue was extracted with a 70:30 pmmb 2023, 6, 1; a0000338 4 of 22 etoh/water hydroalcoholic solution in a two-hour heat-reflux system. after extraction, a rotary evaporator was used to chill and filter the solution until it was completely dry[36–38]. secondly, the liquid-liquid extraction technique was employed. the crude 70% ethanol extract was reconstituted in water and extracted vigorously with butanol in a separatory funnel. for phase separation, the organic phase was removed, and more butanol was added to the residual aqueous solution for further liquid-liquid extraction. this fractionation process was further repeated in triplicate [36–38]. the organic butanol fraction was merged and dried by a rotary evaporator. the aqueous phase was coated and concentrated to dryness by a freeze-dryer. the butanol fractions of the five parts were reconstituted in absolute methanol. moreover, adding diethyl ether to the organic fraction was done in a very slow manner and a precipitate was formed at the end[39]. the phytochemicals that were not very soluble in diethyl ether were precipitated. the precipitate was filtered using sintered glass and separated from the solvent using a rotary evaporator (rotavapor r-100, buchi). 2.3. determination of secondary metabolites of argania spinosa extracts 2.3.1. determination of polyphenols the content of phenolic compounds in the different extracts of argania spinosa was estimated by the folin-ciocalteu method. briefly, 2.5 ml of folin reagent (10-fold diluted) is added to 500 μl of sample or standard (prepared in ethanol) with suitable dilutions, 2 ml of na2co3 sodium carbonate solution (7.5%). after 15 min of incubation in a water bath at 45 °c, the absorbance was measured at 765 nm against a blank without extract, and the calibration range obtained with gallic acid (1-200 g/ml) was used to calculate the quantification of total polyphenols under the identical circumstances as the sample. results are given as milligrams of gallic acid equivalent per gram of extract (mg gae/g)[40]. 2.3.2. determination of flavonoids the determination of total flavonoids was performed by the aluminum chloride colorimetric method. 6.4 ml of distilled water and 1 ml of an extract at a specific concentration were combined in a tube. then 0.3 ml of sodium nitrite (nano2) solution (5% concentration) was added. 0.3 ml of 10% aluminum chloride (alcl3) was added to the mixture after 5 minutes. it was left for six minutes. the tubes were then filled with 2 ml of sodium hydroxide (1m), thoroughly mixed, and agitated before being permitted to stand for 30 minutes. at 510 nm, the absorbances were measured. flavonoid concentration was deduced from a calibration range established with quercetin (2–200 μg/ml) and was expressed as milligram quercetin equivalent per gram of extract (mg qe/g)[41]. 2.3.3. determination of condensed tannins we adopted the vanillin method with hydrochloric acid. this method depends on the property of tannins to transform into red-colored anthocyanidols by reaction with vanillin[42]. pmmb 2023, 6, 1; a0000338 5 of 22 3 ml of an ethanolic solution of 4% vanillin is added to 0.5 ml of the sample. the final step is to add 1.5 ml of concentrated hydrochloric acid. the resulting combination is given 15 minutes to react at room temperature. at 500 nm, the absorbance is calculated in comparison to a blank. the calibration curve was generated using various concentrations between 1 and 800 g/ml made from a stock solution of catechin. while the final result was expressed as mg catechin equivalent (ce) per gram of extract (mg ce/g)[43]. 2.3.4. analysis of triterpene saponins by uv spectrophotometer 2.5 ml of sulfuric acid (72% concentration) was added to a 250 µl sample that included 1 mg/ml of sulfuric acid and 250 µl of vanillin (8 g/100 ml ethanol). the combination was heated for 10 minutes at 60 degrees celsius, then cooled for 5 minutes in an ice-water bath. the mixture's absorbance was measured using a uv/vis spectrophotometer. (llg-unispec 2) at 544 nm. different concentrations between 5 and 350 µg/ml prepared from a stock solution of oleanolic acid were used to plot the calibration curve. while the results were expressed as mg oleanolic acid equivalent per gram of extract (mg oae/g)[36]. 2.4. quantification of sugar in summary, 5 ml of concentrated sulfuric acid and 1 ml of phenol (5%) were combined with 1 ml of sample. stir and allow it to stand at room temperature for 10 minutes. after 20 minutes, the mixture was incubated at 30 °c in a water bath to measure the yelloworange color at 488 nm. quantification of the total score was calculated from the regression equation of the calibration range established with glucose (5–100 μg/ml) under the same conditions as the sample. results are expressed via mg glucose equivalent per gram dry matter (mg de-glu/g dm)[44]. 2.5. study of biological activity 2.5.1. antioxidant potentials of argan extracts 2.5.1.1. antioxidant test by dpph the dpph solution is solubilized in absolute ethanol (0.2 mm). 2.5 ml of the test extract was added to 0.5 ml of the dpph solution. after vortexing the mixture, the tubes were left at room temperature and dark for 30 minutes. the reading is made using a spectrophotometer to measure the absorbance at 517 nm[45]. the negative control (nc) contains only the dpph solution. the dpph concentration is deduced from a calibration range established with ascorbic acid (0.5–20 µg/ml)[45]. 2.5.1.2. cation-radical reduction test abts°+ the 7 mm abts stock solution was mixed with 2.45 mm potassium persulfate (1:1, v/v), and the mixture was allowed to stand for 4 to 8 hours to allow the reaction to finish, and pmmb 2023, 6, 1; a0000338 6 of 22 the absorbance was stable. the abts°+ solution was diluted in ethanol to an absorbance of (0.70 ± 0.05) at 734 nm for measurements. the photometric assay was performed on 0.9 ml of abts°+ solution and 0.1 ml of extracts dissolved in ethanol solution and mixed for 45s; measurements were made at 734 nm after 30 min in the dark[46]. 2.5.2. evaluation of antimicrobial properties from the saponin extraction, extracts of different parts of the argan tree of mass 10 mg were solubilized in 5 μl of dimethyl sulfoxide (dmso) and 995 μl of distilled water. they were filtered before any further use on millipore filters (0.22 μm). the antifungal activity was evaluated on the following fungi: fusarium oxysporum and botrytis cinerea. these fungi were provided by the team of microbiology and molecular biology (embm-fsr). the bacteria used are the following: raoultella ornithinolytica, (mz853484.2), microbacterium resistens (mz853466.2), and staphylococcus saprophyticus (op750262.1), all isolated from a mine highly contaminated with metals and posing major health problems [47]. escherichia coli (dh5α) was also tested. these bacteria were provided by the team of microbiology and molecular biology (embm-fsr). 2.5.2.1. antifungal test the fungi were grown on pda (potato dextrose agar) medium. after seven days of growth, these fungi were used to test the antifungal activity of the saponin extracts. to study the effect of the extracts on the growth of the fungi, petri dishes of 55 mm diameter were used. one hundred microliters of each extract were well spread on the plates containing pda medium, the plates were left open under a laminar flow hood for a maximum of 30 minutes allowing the extracts to dry on agar. finally, 0.6 cm discs of fungi were placed in the center of each plate and incubated at 25 °c for 4–5 days in the dark. a plate containing only the fungus was used as a control[47]. to evaluate the effect of the extracts on the growth of the fungi, the percentage of growth inhibition was calculated according to the following formula: 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % = (dc−𝑑0)−(𝑑𝑔−𝑑0) (𝑑𝑐−𝑑0) × 100 (1) where dg is the diameter of the fungus growth in the presence of the extract; d0 is the diameter of the fungus disc deposited at t0; and dc is the diameter of the fungus growth in the control box. pmmb 2023, 6, 1; a0000338 7 of 22 2.5.2.2. antibacterial test muller-hinton agar was prepared and sterilized by autoclaving for 20 minutes at 120 °c. then the medium was poured onto 90 mm petri dishes and left for 24 hours at room temperature to ensure no contamination. ten milliliters of nutrient broth were introduced into each test tube and then sterilized for 20 min at 120 °c by autoclaving. after 24h, the bacterial strains were plated on a liquid medium and left to grow for 24h. to prepare the working suspensions, the bacterial cultures were adjusted by nutrient broth to have an optical density of 0.1. the solid disk diffusion method demonstrated antimicrobial activity[4]. one hundred microliters of bacterial suspension with od of 0.1 of each microbial strain were well spread on a solid medium. then discs were placed on top of the dried bacterial layer, each disc was soaked with 10 μl of extract in such a way as to have different concentrations (a total of 5) of the same extract per petri dish. the control petri dish contained tetracycline (tet 30 µg) antibiotic disc (sigma). all tests were repeated three times. finally, the plates were incubated at 37 °c for 18 to 24 hours, depending on the bacterial growth. the diameter of the bacterial growth inhibition zone (a transparent halo around the discs) was measured (mm)[48], to evaluate the antibacterial activity. 2.6. statistical analysis 2.6.1. principal component analysis (pca) the pca is intended to establish the correlation between antioxidant activity, using dpph and abts as much as free radicals and the chemical composition in polyphenols, flavonoids, and tannins for the crude extracts of different parts of the plant as well as the quantification of total sugars and tritepenic saponins of the set of extracts from the process of extraction of saponins from a. spinosa. pca was performed on the results of (tpt, tft, ttt, tsut, tsapt, and ttc) and antioxidant capacity by two tests, abts and dpph, which expressed the 8 variables for the five parts of a. spinosa. 2.6.2. correlation matrix pearson correlations between tpt, tft, ttt, tsut, tsapt, ttc, abts, and dpph were performed by pca. which pca expressed the 5 samples according to their response values in a graph to facilitate the understanding of the variations of the data according to the nature of the argan parts[49]. 2.6.3. analysis of the data pearson correlation was used to investigate the correlation between all parameters used for the argan parts in this paper. pca was performed by xlstat 2014 software. data pmmb 2023, 6, 1; a0000338 8 of 22 were represented as mean ± standard error of the mean and were performed using ibm spss statistics 21 software. 3. results and discussions 3.1. yield of extracts of different parts of argania spinosa the yields obtained from the different extracts of each plant part of a. spinosa are shown in figure 1. in the current study, water-ethanol was selected as the extraction solvent for its low toxicity and great solubility for both terpenes and saponins[50]. most of the plant extract's saponins had low solubility, as evidenced by the fact that decreasing the solvent's polarity by adding more ethanol[36]. indeed, a hydroalcoholic solution (ethanol/water 70/30) was used to extract different parts of the plant. branch leaf pulp shell seed 0 5 10 15 20 25 y ie ld ( % ) different parts of a. spinosa etoh (70%) buoh precipitate filtrate aqueous figure 1. histogram of yields of the different extracts of the parts of the a. spinosa. the yields of the argan tree plant differ from one part to another. however, the leaves of the argan tree present very high yields, followed by the pulp, the shell, the branch, and finally the seed. generally, the ethanolic extracts of the different parts of a. spinosa showed a significantly high percentage, followed by that of the aqueous phase, then the organic phase (butanol) then the precipitate, and finally the filtrate. the saponin content of fresh argan grains is 0.5%[51], while the wood of the argan tree is also particularly rich in saponins, these being found at a concentration of 5% to 6%[51,52], or a concentration twelve times higher than that of saponins in the cake. the crude content of saponin in the shell of the fruits of argan can be 0.01% to 1%[51,52]. however, the pulp of the fruits of argan is poor in saponins, whose concentration is only 0.02%[51]. pmmb 2023, 6, 1; a0000338 9 of 22 3.2. quantitative evaluation of total secondary metabolites 3.2.1. quantitative evaluation of total polyphenols, flavonoids, and condensed tannins in this work, we were interested in analyzing these results, focusing on the composition of polyphenols of 70% ethanolic extracts of different plant parts. the content of phenolic compounds of different parts of the argan tree was evaluated by the colorimetric method of folin-ciocalteau. these results show that the contents of total polyphenols, are higher at the level of the raw extract of the shell’s value of 406.94 mg gae/g extract followed by that of branch of 207.52 mg gae/g extract, then leaf of 145.34 mg gae/g extract then pulp 73.58 mg gae/g extract, and finally the seed of 16.68 mg gae/g extract (table 1). the results show that the flavonoid dosage follows the same logic as those of polyphenols. the dosage of total flavonoids was carried out again on the 70% ethanolic extracts of different parts of a. spinosa. as for the polyphenols, the content of total flavonoids differs from one part to another of the argania plant. the shell is the part of the plant that has the highest content of total flavonoids compared to other parts of the plant value of 50.67 mg qe/g extract, followed by a branch of 32.36 mg qe/g extract, then a leaf of 13.27 mg qe/g extract then pulp 7.74 mg qe/g extract, and finally, the seed of 2.03 mg qe/g extract (table 1) the aerial part of the argan tree is particularly rich in flavonoids. this metabolic fraction can reach 17% of the mixed leaves and branches[53,54]. the flavonoid content of the fruit pulp varies according to the degree of maturity of the fruit and according to more complex criteria (genotypic) whose impact would also be reflected in the shape of the fruit[55]. tannins represent an exceptional class of polyphenols. condensed tannins were determined only on the 70% ethanolic extracts of different parts of a. spinosa. the tannin molecules showed their presence only in two ethanolic extracts of the a. spinosa plant. however, the leaves present a significant value in condensed tannins at 182.66 mg ce/g of extract, compared to the shell, a value of 61.55 mg ce/g of extract (table 1). table 1. secondary metabolite content of 70% ethanolic extracts from different parts of a. spinosa. parts tpt (mg gae/g) tft (mg qe/g) ttt (mg ce/g) branch 207.52 ± 0.79a 32.36 ± 0.26a nr leaf 145.34 ± 0.66b 13.27 ± 0.65b 182.66 ± 0.55a pulp 73.58 ± 0.72c 7.74 ± 0.58c nr shell 406.94 ± 1.05d 50.67 ± 0.26d 61.55 ± 0.55b seed 16.68 ± 0.26e 2.03 ± 0.065e nr data are presented as the mean of three individual triplicates (n = 3 ± sem); means followed by similar letters exposed in the same column are not different (p < 0.05). nr: no results. pmmb 2023, 6, 1; a0000338 10 of 22 3.2.2. evaluation of triterpene saponins present in extracts of different parts of the argan tree saponin assay showed that the total triterpene saponins of argan parts increased proportionally from the 70% ethanolic extract, followed by the butanolic extract, and finally, the precipitate (table 2). total saponin concentration in the organic fraction appears to increase modestly with cold ether precipitation. again, the precipitate provided the highest total saponin content. ether, thus contributing to higher saponin content. also, since highly polar molecules like organic acids, polysaccharides, and proteins can stay in the aqueous phase, their higher solubility in ethanol than in water can be explained. while other relatively less polar substances, such as terpenoids and saponins, are dispersed in the organic phase, the butanolic phase has a higher total saponin content as a result. table 2. tritepene saponin content (mg oae/g) of extracts from different parts of a. spinosa. etoh70% buoh precipitate filtrate aqueous branch 99.74±0.71a 117.42±0.53a 137.42±0.53a 54.74±0.35a 65.46±0.36a leaf 98.14±0.89a 104.56±0.53b 160.10±0.26b 50.81±0.71b 64.92±0.53a pulp 78.85±0.53b 84.21±0.54c 130.81±0.71a 9.21±0.54c 24.93±0.18b shell 246.28±0.39c 297.47±0.26d 342.60±0.39c 121.17±0.35d 126.89±0.36c seed 43.31±0.35d 53.31±0.35e 61.71±0.54d 9.21±0.54c 14.21±0.54d data are presented as the mean of three individual triplicates (n = 3 ± sem), means followed by similar letters exposed in the same column are not different (p < 0.05). we were also interested in analyzing these results, focusing on the composition of triterpenic saponins of different parts of argania spinosa. for any type of extract, the triterpene saponin content is best in the shell, followed by the leaf, branch, pulp, and the seed. the shell precipitate presented the best content of triterpenic saponins with a value of 342.60 mg oae/g of extract compared to the other precipitates of the plant. on the other hand, the seed precipitate of a. spinosa presented the lowest triterpene saponin value of 61.71 mg oae/g of extract. 3.3. quantification of total sugars sugars constitute the hydrophilic part of saponins. they can comprise one or more osidic chains (linear or branched) at different positions on the aglycone[51]. the contents of soluble sugars vary according to the plant species presented in table 3. generally, the amount of sugar in the 70% ethanolic extract is remarkably high in the pulp part of 477.50 mg dglue/g extract, followed by the branch part of 447.28 mg d-glue/g extract, the leaf part of 379.42 mg d-glue/g extract, the shell part of 291.86 mg d-glue/g extract, and finally the seed part of 216.97 mg d-glue/g extract. pmmb 2023, 6, 1; a0000338 11 of 22 table 3. total sugar content (mg d-glue/g) of extracts from different parts of a. spinosa. etoh70% buoh precipitate filtrate aqueous branch 447.28±1.77a 254.75±0.44a 300.97±0.89a 58.51±0.16a 160.08±0.44a leaf 379.42±1.11b 268.97±0.44b 340.97±0.89b 70.24±0.11b 193.48±0.11b pulp 477.50±1.77c 261.95±0.8c 270.31±0.89c 44.35±0.22c 147.86±0.66c shell 291.86±1.55d 143.59±0.44d 408.31±1.11d 119.70±0.55d 127.26±0.33d seed 216.97±0.44e 175.86±0.66e 317.86±0.44e 22.79±0.22e 336.30±0.22e data are presented as the mean of three individual triplicates (n = 3 ± sem); means followed by similar letters exposed in the same column are not different (p < 0.05). the part of the branch, leaf, and pulp have kept the total sugar classification of extracts as: ethanol 70% ; precipitate ; butanol ; aqueous ; and filtrate. however, the seed and shell precipitate extract showed better total sugar contents than the ethanolic extract with values of 408.31 mg d-glue/g extract and 317.86 mg d-glue/g extract, respectively. also, the aqueous phase of argan seed presented a total sugar content of 336.30 mg d-glue/g extract higher than its precipitate. the sugar content is strongly influenced by the plant parts and the choice of solvent. 3.4. evaluation of the antioxidant activity 3.4.1. determination of the scavenger effect of the dpph radical the data on the antioxidant activity of the extracts of different parts of argania towards the free radical dpph are summarized in table 4. the anti-free radical activity by dpph was measurably better in the precipitate of each part of the argan tree compared to the other extracts studied. the precipitate of the shell shows the best ic50 value of 1.57 µg/ml, followed by the leaves (2.63 µg/ml), the branch (11.23 µg/ml), the pulp (90.74 µg/ml), and finally, the seed (372.87 µg/ml). the ic50 values of the shell as well as leaf and branch are very close to that of ascorbic acid (1.92 µg/ml) compared to those of pulp and seed. the shell, leaf, and branch present better antioxidant activity than the pulp and seed. with the exception of the seed of the argan tree, the other extracts demonstrated significant antioxidant power as assessed by dpph. according to the results obtained in table 4, we noticed a chronological order in ic50 of the dpph of the extracts of the argan tree as follows: precipitate ; butanol ; ethanolic 70% ; aqueous, and filtrate. while the argan tree seeds showed no antioxidant activity by dpph in the butanolic, ethanolic, filtrate, and aqueous extracts for a concentration range of up to 2000 µg/ml. this result of antioxidant activity can be translated by the content of secondary metabolites. the free radical scavenging activity of the saponins in the cake was determined against dpph, and ic25 = 85 mm was observed, while an ic25 = 560 mm was established against hydroxy radicals [56]. pmmb 2023, 6, 1; a0000338 12 of 22 table 4. evaluation of the antioxidant activity of extracts of different parts of a. spinosa by dpph. ic50 (µg/ml) etoh70% buoh precipitate filtrate aqueous branch 15.77±0.11a 12.57±0.04a 11.23±0.16a 25.27±0.37a 17.03±0.03a leaf 7.52±0.17b 3.00±0.01b 2.63±0.02b 9.74±0.13b 8.51±0.065b pulp 164.27±0.8c 149.12±1.7c 90.74±0.79c 180.35±0.70c 177.34±0.85c shell 5.85±0.16b 2.94±0.02b 1.57±0.2b 8.20±0.02b 6.48±0.085b seed na na 372.87±0.05d na na ascorbic acid 1.92 ± 0.04d data are presented as the mean of three individual triplicates (n = 3 ± sem); means followed by similar letters exposed in the same column are not different (p < 0.05). na: no activity. 3.4.2. determination of the scavenger effect of the abts radical. in our work, we opted for abts to test extracts of different parts of a. spinosa. the results obtained illustrated in table 5 showed that the inhibitory action of the samples was improved from the crude extract of 70% ethanol to the butanolic fraction as well as its precipitate for the different parts of the plant except the seeds. the precipitate of the latter part showed low antioxidant activity in a concentration range of up to 2000 µg/ml. table 5. evaluating the antioxidant activity of extracts of different parts of a. spinosa by abts. ic50 (µg/ml) etoh70% buoh precipitate filtrate aqueous branch 61.25±0.49a 59.4±0.33a 56.53±0.57a 73.82±0.06a 68.68±0.39a leaf 33.85±0.28b 29.6±0.19b 24.07±0.12b 46.05±0.22b 41.01±0.06b pulp 210.64±1.55c 179.13±1.03c 174.24±0.78c 270.16±2.91c 255.34±1.26c shell 31.48±0.17b 27.25±0.47b 21.77±0.02b 44.16±0.41b 39.37±0.24b seed na na 516.86±4.03d na na ascorbic acid 11.31±0.05e data are presented as the mean of three individual triplicates (n = 3 ± sem); means followed by similar letters exposed in the same column are not different (p < 0.05). na: no activity. however, the other extracts of a. spinosa seed showed no activity against the abts radical, which means that a high concentration of the samples is necessary to present its antioxidant power. as for the dpph test, the precipitate of the shell has the best ic50 value (21.77 µg/ml), followed by leaves (24.07 µg/ml), the branch (56.53 µg/ml), the pulp (174.24 µg/ml), and finally the seed (516.86 µg/ml). the precipitate of the shell and leaf was very close to the value of ascorbic acid (11.31 µg/ml). saponins were associated with various biological activities, such as antioxidant activity[57]. it was shown that the total flavonoid extract of the leaves has antiradical and antioxidant activity. the antioxidant effect was confirmed by observing a reduction in the effects of oxidative stress produced on uvpmmb 2023, 6, 1; a0000338 13 of 22 irradiated human skin cells[58]. the extract of flavonoids from the argan tree could thus be used in cosmetology as a skin protector[58]. the confirmation of these effects in vivo would allow great valorization of the leaves of the argan tree. 3.5. evaluation of the antimicrobial activity of argan extracts the study of the antimicrobial activity of the extracts of different parts of argania prepared at a concentration of 10 mg/ml was examined on microorganisms by the disc diffusion method. 3.5.1. antibacterial activity the antibacterial activity of the extracts of different parts of argania spinosa was evaluated on different bacterial strains: escherichia coli, microbacterium resistens, raoultella ornithinolytica, and staphylococcus saprophyticus. the inhibition results generated by the extracts from different parts of argania spinosa after 24 hours at 37 °c incubation are recorded in table 6. based on the results in table 6, the extracts of different parts of the argania spinosa plant showed no bactericidal activity on escherichia coli and raoultella ornithinolytica strains. however, saponin precipitates obtained from the leaf and shell of argania spinosa had inhibitory effects on the growth of microbacterium resistens, with inhibition zone measurements of 10 mm and 11 mm, respectively. as for the extracts etoh 70%, aqueous and filtrate of the leaf of the argan tree showed an inhibition of the growth of the bacterium staphylococcus saprophyticus, yielding inhibitory zones of 13 mm, 13 mm, and 12 mm, respectively. the aqueous phase of the leaves was the only one that showed antibacterial activity against both microbacterium resistens and staphylococcus saprophyticus. the filtrate of the argania spinosa pulp exhibited sensitivity against microbacterium resistens bacteria, resulting in an inhibition zone of 9 mm. generally, the leaves of argania spinosa were the only ones that exhibited stronger antibacterial activity against microbacterium resistens and staphylococcus saprophyticus compared to other parts of the plant studied. saponin extract from argan oilcake showed antibacterial activity at a concentration of 500 μg/ml on cutibacterium acne bacteria, while no inhibition of bacterial growth was observed against prevotella intermedia up to the highest concentration (12500 μg/ml)[59]. table 6. inhibition zone diameter (mm) of different extracts from different parts of argania spinosa compared to tetracycline (tet) as reference. inhibition zone diameter (mm) escherichia coli microbacterium resistens raoultella ornithinolytica staphylococcus saprophyticus branch etoh 70% buoh aqueous precipitate filtrate leaf etoh 70% 13 ± 0.05a buoh aqueous 15 ± 0.02a 13 ± 0.07a pmmb 2023, 6, 1; a0000338 14 of 22 inhibition zone diameter (mm) escherichia coli microbacterium resistens raoultella ornithinolytica staphylococcus saprophyticus precipitate 10 ± 0.03a filtrate 12 ± 0.03a pulp etoh 70% buoh aqueous precipitate filtrate 9 ± 0.03a shell etoh 70% buoh aqueous precipitate 11 ± 0.05a filtrate seed etoh 70% buoh aqueous precipitate filtrate tet 30 µg 23 33 15 25 data are presented as the mean of three individual triplicates (n = 3 ± sem), means followed by similar letters exposed in the same column are not different (p < 0.05). 3.5.2. antifungal activity the antifungal activity of different parts of argania spinosa extracts was investigated against two fungi botrytis cinerea and fusarium oxysporum. the results obtained on botrytis cinerea, after incubation in the dark at 25 °c for five days, show that our prepared samples do not contribute to any antifungal activity (table 7). unlike fusarium oxysporum which showed inhibitions ranging from 1.92% to 32.69% for some extracts from the five parts of a. spinosa. the 70% of leaf, pulp, shell, and seed ethanolic extracts showed various inhibitions of 1.92%, 13.46%, 26.92%, and 1.92 %, respectively. while the butanolic fraction showed antifungal activity only at the branch, shell, and seed parts. the latter part of the organic fraction showed good antifungal activity with a value of 32.69% compared to the branch and shell of 5.77% and 17.31%, respectively. as for the saponin precipitates, the different parts of the plant revealed their ineffectiveness towards the two studied fungi, which means that a high concentration of the saponin precipitates samples is necessary to present its antifungal power and/or the range of chosen fungi or yeasts to prove this effect. also, the aqueous phase of the branch, pulp, and seed showed inhibitions towards fusarium oxysporum fungus of a significant value of 32.69% for seed and pulp, while the branch presented a low value of 1.92%. in addition, the seed is the only part of the argan tree that showed antifungal activity in its different extracts except for its saponin precipitate. this result confirms then that the seed part of the argan tree fruit has antifungal potential rather than antibacterial activity. saponins are natural substances with a wide spectrum of biological activity; some are already used in therapeutics as hepatoprotective, anti-inflammatory, and anticancer activities[37]. antifungal studies have revealed an action against cladosporium cucumerinum (phytopathogenic fungus of the cucurbitaceae family), polysticus versicolor, and a fungus pathogenic to humans: candida pmmb 2023, 6, 1; a0000338 15 of 22 albicans. the minimum inhibitory concentrations are 12.5 and 50 μg/ml for c. cucumerinum and p. versicolor, respectively. on the other hand, the mixture of saponins does not show any activity against candida albicans[60]. table 7. evaluation of the antifungal power of extracts of different parts of argania spinosa by the disc diffusion method. inhibition % botrytis cinerea fusarium oxysporum branch etoh 70% buoh 5.77 ± 0.05a aqueous 1.92 ± 0.06b precipitate filtrate leaf etoh 70% 1.92 ± 0.02b buoh aqueous precipitate filtrate pulp etoh 70% 13.46 ± 0.08c buoh aqueous 32.69 ± 0.08d precipitate filtrate shell etoh 70% 26.92 ± 0.07e buoh 17.32 ± 0.04f aqueous precipitate filtrate seed etoh70% 1.92 ± 0.02b buoh 32.69 ± 0.07d aqueous 32.69 ± 0.09d precipitate filtrate 9.62 ± 0.04g data are presented as the mean of three individual triplicates (n = 3 ± sem); means followed by similar letters exposed in the same column are not different (p < 0.05). 3.6. statistical analysis 3.6.1. correlation matrix the correlation coefficients for tpt, tft, ttt, tsut, tsapt, abts, and dpph are presented in table 8. also, dpph (1/dpph ic50) and abts (1/abts ic50) represent the free radical inhibition power, dpph, and abts, respectively. however, table 9 shows the p-values of the correlation matrix coefficient between all variables. table 8. coefficient of the pearson correlation matrix between the antioxidant capacity and antioxidant compounds in the different parts of the argan tree. variables tpt tft ttt tsut tsapt 1/dpph 1/abts tpt 1 tft 0.980 1 ttt 0.210 0.057 1 tsut -0.042 -0.003 -0.037 1 pmmb 2023, 6, 1; a0000338 16 of 22 variables tpt tft ttt tsut tsapt 1/dpph 1/abts tsapt 0.963 0.925 0.369 -0.226 1 1/dpph 0.861 0.769 0.673 -0.113 0.930 1 1/abts 0.801 0.717 0.730 0.059 0.863 0.977 1 values in bold differ from 0 at a significance level of alpha = 0.05. table 9. p-values of the correlation matrix coefficient between all variables. variables tpt tft ttt tsut tsapt 1/dpph 1/abts tpt 0 tft 0.004 0 ttt 0.734 0.928 0 tsut 0.946 0.996 0.952 0 tsapt 0.009 0.025 0.541 0.714 0 1/dpph 0.061 0.128 0.213 0.857 0.022 0 1/abts 0.104 0.173 0.161 0.925 0.060 0.004 0 values in bold differ from 0 at a significance level of alpha = 0.05. according to table 8 and table 9, pearson's correlation analysis between antioxidant activity and phytochemical amounts revealed that tsapt had a strong positive correlation (pvalue < 0.05) with antioxidant activity. the correlation data of tsapt was 0.930 with the scavenger effect of dpph radical. the linear positive correlation between polyphenols (tpt) and flavonoids (tft) was also observed (r2 = 0.980). however, tannins (ttt) did not contribute much to antioxidant activities. in addition, the correlation matrix showed a strong correlation between abts and dpph (r2 = 0.977). 3.6.2. analysis of the data by pca the results of the testing on phytochemicals are regarded as variables. the pca in figure 2 (f1-f2) factorial design projects them. 67.84% of the information is presented in the first component (f1), and 17.11% is explained in the second component (f2). 84.94% is the combined proportion of the first two elements. however, because their linear combination is more than 50%, it already adequately represents the variables. as a result, the first two axes can adequately explain all of the data. the plane created by the axes f1 and f2 in figure 2 depicts the correlation between the variables. the positive correlation between the dpph and abts tests and the tpc, tfc, and tsapt tests substantially determines the shape of the f1 axis (figure 2). pmmb 2023, 6, 1; a0000338 17 of 22 figure 2. factorial plan of principal component analysis carried out on the values (tpt, tfc, ttc, tsapt, tsut, dpph and abts) of different parts of the argan tree. figure 3. projection on the factorial plane (f1×f2) of the variables of different parts of a. spinosa. gi: group i; gii: group ii dpph(1/dpph ic50;abts(1/abts ic50). the five individuals are depicted in figure 3 as split into two groups (extracts). group i contain 8 extracts (shell p, leaf b, leaf p, shell e, leaf f, shell a, shell f, shell b), according to the dpph and abts assays, this group's antioxidant capacity is noticeably stronger than that of group ii. this group's antioxidant activity is also distinguished by extremely high tsapt levels. tpt tft ttt tsut tsapt dpph abts -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 f 2 ( 1 7 .1 1 % ) f1 (67.84 %) variables (axes f1 et f2 : 84.94 %) pmmb 2023, 6, 1; a0000338 18 of 22 the 17 remaining extracts comprise group ii, distinguished by its high tsut level and lower antioxidant activity when compared to extracts from group i by the dpph and abts assays. this low antioxidant activity is reflected in the low content of tsapt. 4. conclusions the technique of butanol fractionation followed by ether precipitation appears to be important for recovering saccharide-containing compounds from the crude extract of a. spinosa. given the calorimetric results of the colorimetric test using vanillin and sulfuric acid, it turned out that the precipitates of the different parts of a. spinosa are rich in saponin molecules. this family of molecules seems to be the secondary metabolites with the best potential for industrial use. for this purpose, the biological activities of the extracts of different parts of a. spinosa was evaluated. the study of the antimicrobial activity of the extracts of the argan tree prepared at a concentration of 10 mg/ml was examined on microorganisms by the disc diffusion method. the extracts of saponin etoh 70% and aqueous from leaves of a. spinosa plant showed significant antibacterial activity against microbacterium resistens, with a remarkable inhibition zone of 13 mm for both extracts. while the saponin extracts did not exhibit any antifungal activity against botrytis cinerea, many of them presented an antifungal activity ranging from 1.92% to 32.69% inhibition of the growth of fusarium oxysporum fungi. in addition, the antioxidant activity of the extracts of different parts of the studied plant was also evaluated by two methods: dpph free radical test and abts free radical test. the different tests showed that the samples' inhibitory action was improved from the crude extract of 70% ethanol to the butanol fraction as well as it’s precipitate for the different parts of the plant except the unroasted shells of a. spinosa. in addition, the pca data analysis study demonstrated a positive correlation between the content of saponin compounds and antioxidant activity. author contributions: conceptualization nm, ceg, aey. hh, ab; 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from argania spinosa (l.) skeels. pharmacognosy j 2019; 11(1): 26–31. 60. charrouf z. valorisation d’argania spinosa (l.) sapotaceae: etude de la composition chimique et de l’activité biologique du tourteau et de l’extrait lipidique de la pulpe, mohammed v university: rabat, 1991. pmmb 2023, 6, 1; a0000338 22 of 22 author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology review article 1 bioprospecting of microbes for valuable compounds to mankind nurul-syakima ab mutalib1, sunny hei wong2, hooi-leng ser3, acharaporn duangjai4,5, jodi woanfei law3, shanti ratnakomala6, loh teng-hern tan3, vengadesh letchumanan3* 1ukm medical molecular biology institute (umbi), ukm medical centre, universiti kebangsaan malaysia, kuala lumpur, malaysia 2li ka shing institute of health sciences, department of medicine and therapeutics, the chinese university of hong kong, shatin, hong kong 3novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 4division of physiology, school of medical sciences, university of phayao, phayao, thailand 5center of health outcomes research and therapeutic safety (cohorts), school of pharmaceutical sciences, university of phayao, phayao, thailand 6research center for biotechnology, indonesia institute of sciences (lipi), cibinong 16911, indonesia abstract: the most biological multiplicity on this planet is almost certainly concealed in soils. many valuable bacteria had been extensively dispersed in soils worldwide, with soils from terrestrial, desserts and antarctic. hence, soils become an intensively utilized ecological niche for the inhabitants to generate various useful biologically active natural products such as antibiotics, antifungal, antiviral, antioxidant, neuroprotection, anticancer and other important compounds. bacteria including actinobacteria have been exceptionally valuable for the pharmaceutical industry due to their limitless capability to generate secondary metabolites with various biological activities and chemical structure. therefore, this article aims to provide critical insight of bioprospecting of microbes for valuable compounds to mankind. keywords: bioprospecting; microbes; compounds; metabolites; actinobacteria; soil received: 29th april 2020 accepted: 29th may 2020 published online: 7th june 2020 citation: ab mutalib ns, wong sh, ser hl, et al. bioprospecting of microbes for valuable compounds to mankind. prog microbes mol biol 2020; 3(1): a0000088. https://doi.org/10.3687/pmmb.a0000088. introduction biotechnology is an illustration of biodiversity as new products via the utilization of living organisms and bioprocesses in medicine, engineering, technology, and other fields that required bioproducts. the greater the biodiversity offered, the greater probabilities of discoveries that could be transformed into vital technologies[1]. the estimation of the environmental and economic gains that are a direct or indirect result of microbial diversity were approximate to be in the range of 16-54 trillion us dollars per year, with an average of 33 trillion us dollars per year[2]. the primary and secondary metabolism of prokaryotes has been utilized by industrial for the creation of diverse products such as antibiotics[3–6], amino acids[7,8], nucleotides[7], organic acids[9] and vitamins[10]. bacteria like actinobacteria are a particularly rich source of compounds with activities such as antimicrobial[6,11–22], anticancer[23–29], antioxidants[30–35], neuroprotective[36,37], enzymes[38–41] and immunosuppressive[29] as illustrated by figure 1. bérdy (2005)[42] reported that in 2002, over 10,000 bioactive compounds (45% of all microbial metabolites) were obtained from filamentous actinobacteria, out of which 7600 (75%) were obtained from streptomyces and 2500 (25%) from rare actinomycetes for instance actinomadura, streptoverticillium and micromonospora. despite the tremendous success of the past in obtaining copyright @ 2020 by ab mutalib and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: vengadesh letchumanan, novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; vengadesh. letchumanan1@monash.edu. 2 useful secondary metabolite, the probabilities of discovery novel biologically active molecules from bacteria such as actinobacteria was reduced and appears to be reaching a saturation curve. recently, isolating well known actinobacteria such as streptomyces from diverse environments were reported to obtain similar compound, potentially due to regular genetic exchange between species[43]. these challenges had led to intensely amplified in serious demand for new structures in pharmacology, hence propelled the investigation of new habitats with poorly explored areas and uncommon environments to become vital for the discovery of novel bacteria (e.g. actinobacteria) and useful metabolites[44–55]. reports from poorly explored areas from these regions (e.g. antarctic, australia, china, malaysia and jordan) suggested that the investigation of new habitats remain to be valuable in discovering novel microorganisms and useful metabolites[47,56–61]. moreover, the progression of new selective methods allows the screening and isolation of ‘rare’ actinobacteria that can lead to finding useful bioactive compounds[62–64]. the finding of “rare” actinobacteria has increased the array and diversity of genetic resources available for biotechnological utilization[62–66]. it is apparent that the findings of novel bacteria such as actinobacteria could increase the discovery novel bioactive metabolites[62,66–68]. bioprospecting of microbes... the genome sequencing of streptomyces coelicolor a(3)2t[69] and streptomyces avermitilis ma-4689t[70,71] discovered that these bacteria comprise more than 20 natural product gene clusters. this number of gene clusters is much more as compared to genomes of bacteria from another phylum[72,73]. for instance, bacillus subtilis strain 168t with three, ralstonia solanacearum strain gmi 1000t with two[74], and pseudomonas aeruginosa strain pa01t with four[75] pseudomonas aeruginosa strain. while most other bacteria genomes lacking any detected natural product gene clusters[69]. these reports indicated the capability to produce secondary metabolites are not evenly distributed among microbes. moreover, multiple gene clusters encoding for alike classes of secondary metabolites have been discovered in the genomes of other actinobacteria[76,77]. thus, explaining actinobacteria are highly prolific sources of bioactive metabolites[78] with high capacity to utilize a extensive range of compounds and create secondary metabolites with diverse chemical structures and biological activities[79,80]. unexplored environment — the antarctic the antarctic is the area at the earth’s south pole, contrary the arctic region at the north pole. the antarctic includes the continent of antarctica and the ice shelves, waters and island territories in the southern ocean situated south of the antarctic convergence. the area covers approximately 20% of the southern hemisphere, of which 5.5% (14 million km2) is the surface area of the continent itself. the antarctic is the coldest and windiest continent, it is a hostile, remote, and uninhabited area with its surrounding marine sites, provides an appropriate chance to investigate a still unexplored microbial biodiversity[81–87]. the uneven mixture of selection pressures has led to the evolution of novel biochemical adaptations and the likelihood of native species[88,89]. the production of metabolites such as antibiotics and toxins could confer a competitive survival benefit in this environment. therefore, the investigation of poorly explored areas such as the antarctic seemed as important region for discovering of potential novel bacteria and useful biological active metabolites[59,82,85,90,91]. bacteria from antarctic territories the information of prokaryotic biodiversity remains very sparse across antarctica[82,92,93]. nevertheless, in recent decades, the improvement in both culture dependent and culture-independent methodologies allow some studies focused on signy island were done[94,95,96,97,98,99]. this area figure 1. actinobacteria are prolific producers for metabolites with diverse activities. 3 ab mutalib et al. act as a benchmark site within the maritime antarctic, whose terrestrial ecosystems are demonstrative of the region[100]. furthermore, more studies are also emerging from other sites along the antarctic peninsula, such as the study of the prokaryotic communities of a series of antarctic terrestrial habitats along a latitudinal gradient as part of a larger regional microbial diversity study covering between the falkland islands (~500s) and mars oasis, alexander island (~720s)[101–103]. based on the restricted habitats studied, a fairly large bacterial diversity has been reported[96–99,104,105]. there is an agreement that spatial distinction between soil organisms is not random but displays expectable patterns over dissimilar spatial scales. the small-scale difference is found to exhibit superior diversity than large scale difference[106–108]. small-scale difference might be more vulnerable to local environmental effects such as areas of increased substrate availability[109]. scientists indicated that water content, organic content (loss on ignition) and total n showed substantial direct correlations with microbial counts from soil at 6 different sites on signy island, whereas ph exhibited an inverse association[94]. some recent culture-independent reports have demonstrated that soil prokaryote biodiversity on signy island have high association with elements such as conductivity, ph, lead and copper content. moreover, significant overlap was reported across sites evidently affected by penguins, seals, and the existence of vegetation[99]. the direct effect of soil properties for instance soil ph, nutrients and moisture on bacterial diversity were demonstrated[110–113], and remarkably these parameters also exhibited close connection to specific functional genes for instance glutamate dehydrogenase and nitrate reductase[102]. studies of the bacterial ecology of antarctic soils by means of culturing dependent methods demonstrated that bacterial abundance and diversity can differ with soil factors for instance moisture, ph, available nutrients, salinity, elevation, slope, solar radiation, and drainage[114]. suzuki et al. (1997)[115] isolated an obligate psychrophilic actinobacteria, cryobacterium psychrophilum from the antarctica soil. this bacterium grew best at 9–12oc and did not grow at temperatures higher than 18oc. while psychrophilic strains of modestobacter multiseptatus with optimum growth temperatures of 11-13oc have also been isolated from transantarctic mountain soils[116]. normally, the early studies on the bacterial diversity of antarctic soils were disadvantaged by the readiness of appropriate approaches. with the accessibility of dna-based culture-independent assays, analysis of mineral soils of the antarctic area has discovered that the soil bacterial communities have low diversity compared with temperate soils and may be dominated by a few bacterial phylotypes. bacteria reported from the soils typically group with the phyla actinobacteria, acidobacteria, bacteroidetes, deinococcus-thermus, firmicutes, cyanobacteria and proteobacteria[117-119]. apart from deinococcus and cyanobacteria, they are among the phyla normally described from non-antarctic soils[120]. the phyla actinobacteria and bacteroidetes appear to be prevalent in antarctica while other phyla less broadly spread (e.g. acidobacteria). remarkably some bacteria have no close relatives demonstrating soils of the antarctic (e.g. ross sea region) are extremely potential as a natural reserve of novel and cold-adapted bacteria[118]. the closest relatives include members of the genera arthrobacter, brevundimonas, leptolyngbya, hymenobacter, nocardioides, sphingomonas and sporosarcina[117–119] all of which have been isolated from antarctic soil. the barrientos island of antarctic is situated at 62˚24’s, 59˚47’w, north entrance to english strait between robert and greenwich islands. the north coast of the 1.5km island is dominated by steep cliffs, reaching a height of nearly 70 metres, with a gentle slope down to the south coast. the eastern and western ends of the island are black sand and cobbled beaches. the western end has columnar basalt outcrops as a notable feature. the whole center of the island is covered by widespread moss carpet. lichens xanthoria spp., caloplaca spp. and other crustose lichen species are present. moreover, the green alga prasiola crispa is prevalent. soil samples were collected from this island and molecular identification, which was based on 16s rdna sequences analysis, discovered eight genera of actinobacteria namely actinomyces, actinobacterium, an uncultured actinomycete, streptomyces, leifsonia, frankinea, rhodococcus and mycobacterium. the uncultured actinomyces sp. and rhodococcus sp. appear to be the prominent genera of actinobacteria in barrientos island soil[121]. molecular methods were applied to investigate correlations between actinobacteria abundance and environmental features, for instance vegetation and type of rookery. there was a substantial positive association between type of rookery and the abundance of actinobacteria; soil samples collected from active chinstrap penguin rookeries had the highest actinobacteria abundance. vegetation type, for instance moss, which could provide a microhabitat for bacteria did not associate significantly with actinobacteria abundance[121]. in barrientos island, the selective isolation of culturable bacteria using 12 different isolation media were performed and total 96 bacteria isolates were isolated with 39 and 57 isolates belonged to phylum actinobacteria and proteobacteria, respectively. through 16s rrna gene analysis, 13 (arthrobacter, brevibacterium, demetria, gordonia, rhodococcus, janibacter, leifsonia, dermacoccus, kocuria, lapillicoccus, micromonospora, microbacterium, nocardioides) and 8 (bradyrhizobium, caulobacter, sphingomonas, methylobacterium, paracoccus, ralstonia, rhizobium, staphylococcus) different genera of actinobacteria and proteobacteria, respectively were discovered[122,123]. comparatively actinobacteria (13 genera) had substantial higher diversity than proteobacteria (8 genera)[122,123], hence showed that actinobacteria are proficient to prosper in an extensive range of diverse soil environments, and they could resist the pressure of harsh environment as they could persist in the viable but inactive state for a extended time with form of spore[124]. their extensive disseminations in antarctic suggest that their dispersals are extremely endemic, 4 predominantly in soil and sediment[112,125]. therefore, allowing the bio-prospecting of bacteria from sampling soil from widespread array of geographic sites, such as the antarctic areas to be benefitted. results showed that streptomyces agar (sa) was the most suitable medium for isolating actinobacteria from soil of barrientos island with 54% isolation rate, while starch casein agar (sca) was the most suitable medium to isolate proteobacteria with 19% isolation rate[122,123]. furthermore, researchers studied actinobacteria and proteobacteria isolates from barrientos island for ability of producing antibacterial and antifungal secondary metabolites[122,123]. by means of high-throughput screening models, about 23%, 9%, 6% and 1% of isolates inhibited growth of candida albicans atcc 10231t, staphyloccoccus aurues atcc 51650t, methicillinresistant s. aurues (mrsa) atcc baa-44t and pseudomonas aeruginosa atcc 10145t, respectively. a total 34 bioactive isolates were isolated and categorized into 13 genera, particularly 9 genera were actinobacteria. the high bioactivities of actinobacteria isolates (38%) as compared to proteobacteria isolates (25%) in this study[122,123] showed that actinobacteria still remain as the better source for bioprospecting of novel bioactive metabolites owing to their tremendous capability to produce secondary metabolites with varied chemical structure and biological activities[79,80,126]. these findings provided vital baseline data that barrientos island is a good source of isolation for bioactive actinobacteria and proteobacteria with good antibacterial and antifungal metabolites[122,123]. in barrientos island, the application of the polyphasic taxonomic such as on the basis of phylogenetic, chemotaxonomic, phenotypic and signature nucleotide pattern of the 16s rrna gene, these results indicated that strain 39t is unlike all the genera in the family dermacoccaceae. hence, it is recommended that strain 39t to be categorized in a novel genus in the family dermacoccaceae, as barrientosiimonas gen. nov., the type species of which is barrientosiimonas humi gen. nov., sp. nov. the strain was named after barrientos island, the origin of the sampling site[127]. bacteria as source of new natural products the natural products have been demonstrated to be the richest source for discovery of novel bioactive compounds[128]. previously, the majority bioactive products of microbial origin obtained from few taxonomic groups and mainly terrestrial environments[42,48]. in these decades, microbial natural products research inspired the progress of integrated methods merging specific isolation methods and the access to geographically diverse sources and to different ecological niches[128]. lately the advancement of technologies enables other initiatives like targeting the exploitation of the metabolic potential of environmental gene libraries without undertaking the need of culturing microbes[129–131]. the microbial secondary metabolites comprise of antitumor agents, antibiotics, pesticides, enzyme inhibitors, toxins, and pigments. the biosynthesis of these metabolites is usually coded by genes clusters on chromosomal dna and irregularly on plasmid dna[132]. the discovery of new classes of antibiotics are vital to fight the increased occurrence of multiple resistances among pathogens to the available drugs presently in clinical use[133]. the utmost producers of natural product antibiotics are actinobacteria as nearly two thirds of natural products have been derived from actinobacteria[20], with streptomycetes accountable for more than 80% of them. the phylum actinobacteria signify a significant constituent of the microbial population in most soils[134–138]; such as the antarctic region[117-119,139]. also, actinobacteria present in rhizosphere soil were reported for discovery of antimicrobial agents and other useful metabolites[140–151]. the genus streptomyces exhibited potential as bio-control agent of commercial crops against fungal pathogens[17,152]. moreover, streptomyces spp. derived from grapes exhibited antifungal activity that is pathogenic to fungi and yeast from the same habitat[153]. while the genus arthrobacter, a pervasive genus repeatedly discovered in antarctic and arctic areas is recognized for secondary bioactive metabolite production and for bioconversions[154,155]. rojas et al. (2009)[128] examined antarctic bacteria for creation of novel metabolites discovered a novel molecules associated to cyclic thiazolyl peptides active on gram positive pathogens produced by arthrobacter agilis derived from lake hoare and lake fryxell from the mcmurdo dry valley area in antartic[128]. the antarctic γand β-proteobacteria strains r-12535 and r-7687 derived from lake reid in the larsemann hills and lake hoare in the mcmurdo dry valleys produced bioactive metabolites that inhibited the growth of gram positive and negative pathogens such as e. coli and s. aureus[128]. moreover, the ms spectra of bioactive metabolites obtained from the γand β-proteobacteria strains r-12535 and r-7687 indicated no relatedness with any known compounds, suggesting a chemical novelty related to the bioactivity of these antarctic bacteria. these studies demonstrated the high occurrences of antimicrobial activities discovered from antarctic bacteria, which exhibited them as a prolific source of antimicrobial agents[42,62,156]. these findings support the notion that bacteria from antarctic habitats comprise a rich metabolic diversity and the production of antimicrobial agents could provide a competitive benefit in this situation[157]. other than antimicrobial agents, bacteria such as actinobacteria produced enzymes that are vital and extensively used in medical therapy, bio-organic chemistry, molecular biology, detergent manufacturing, food processing, the textile and pharmaceutical industries[158]. for instance, thermophilic thermoactinomyces candidus could yield extracellular enzyme keratinase that could degrade wool[159]. the antimicrobial agents and keratindegrading producing actinobacteria (streptomyces, nocardioides, saccharomonospora, nonomuraea and nocardiopsis) have been utilized to transformed poultry farm feather waste by composting into pathogen-free bioprospecting of microbes... 5 and odourless bio-fertilizer with complete biological degradation[160]. crawford (1978)[161] reported that streptomycetes can decay lignin by producing the enzyme lignin peroxidase. the extracellular lignin peroxidase derived from streptomyces viridosporus has been studied[162] and it was the first report of a lignin peroxidase from a bacterium. in nature, lignin physically covers cellulose to form lignocellulose (65% cellulose, 25% lignin, and small quantities of hemicellulose glucans), and is resilient to degradation by most microorganisms. streptomyces viridosporus t7a could depolymerizes lignin while degrading cellulose[161] and generates a modified water-soluble, acid-precipitable polymeric lignin (appl) as a key lignin degradation product[163]. pasti et al. (1990)[164] revealed novel streptomyces strains, the s. rochei and s. chromofuscus that were discovered to be superior or equivalent in lignocellulose-degrading capability to streptomyces viridosporus t7a. the enzyme chitinase were discovered from the culture filtrate of streptomyces cinereorube[165]. the enzyme was inhibited by ag+, hg+, hg2+ and r-chloromercuribenzoate. this enzyme is stable in ph range 4.0-10.0 and the optimum ph and temperature for chitinase activity were 4.5 and 50oc, respectively. gomes et al. (2000)[166] reported that streptomyces spp. obtained from a brazilian forest soil exhibited exceptional endochitinase activity and very active against three phytopathogenic fungi, namely fusarium solani, magnaphorte grisea and aspergillus parasiticus. streptomyces ipomoea cect3341 and s. scabies cect3340 in liquid culture produces great levels of enzyme mannanase[167]. the potential of mannanase enzyme in refining the bleachability of pine kraft pulp was demonstrated. with bio-bleaching examinations by means of treatment of the enzyme to result in the release of chromophoric and color material from the pine kraft pulp, together with an increase in pulp brightness and an absence of differences in the viscosity values. berens et al. (1996)[168] effectively obtained the enzyme endoxylanases from the thermophilic actinobacteria microtetraspora flexuosa siix. these thermostable enzymes reported to have optimal activities at ph 6.0 and 80oc. the hydrolysis of hemicellulose generated mostly xylobiose and xylotriose, the latter will be hydrolysed to xylobiose and xylose. researchers demonstrated the production of endoxylanase from streptomyces noboritoensis[169]. moreover, a cellulasefree and endoxylanase-producing streptomycete, streptomyces thermocoprophilus sp. nov. was discovered by kim et al. (2000)[170]. busch and stutzenberger (1997)[171] discovered the thermomonospora fusca, a facultative thermoalkalophilic actinobacteria that produces an extracellular α-amylase which generates maltotriose as the key product. the optimum ph and temperature for the amylase activity were 6.0 and 65oc, respectively. the enzyme activity was not blocked by the addition of glucose due to the preference of the actinobacteria for maltotriose. pasti and belli (1985)[172] reported isolation of streptomyces sp. and micromonospora sp. from termite gut whereby these strains produce enzyme cellulose that contributed to their cellulolytic activity. a total of 4 different termites were reported for the isolation of cellulolytic actinobacteria, namely armitermes, macrotermes, odontotermes and microcerotermes spp. all actinobacteria strains effectively degraded both soluble and insoluble cellulose with some shown persistent activity up to a week. waldron et al. (1986)[173] reported the isolation of microbispora bispora from soil samples of hot springs, geysers and composts was found to grow at 55oc and create thermo-stable extracellular endoglucanase in good concentration with broad ph range of 5.5–7.2. all these reports indicated the practicality of various enzymes produced by various bacteria such as actinobacteria. the value of bacteria in the production of enzymes is heightened by their comparatively high produces, cost efficiency and susceptibility to genetic manipulation. these enzymes enabled bacteria to have a key role in numerous areas for instance the biodegradation of plant litter especially the recalcitrant lignocellulose component [174] and the decomposition of soil organic matter[175]. conclusion as a conclusion, the research of microbial diversity and the isolation of novel microorganisms signify a key chance for developments in biology[67,176–181]. the search and discovery of novel microbes that produce new useful secondary metabolites remains important in the fight against antibiotic resistant pathogens[182], and new emerging diseases[183–185]. author contributions n-sam, shw, h-ls, lt-ht and jw-fl performed the literature search, critical review and performed the writing of this review. guidance, support, and proofreading were contributed by ad, sr and vl. n-sam and vl founded the review writing project. references 1. cowan da. the marine biosphere: a global resource for biotechnology. trends biotechnol 1997; 15: 129–131. 2. costanza r, d’arge r, de groot r, et al. the value of the world’s ecosystem services and natural capital. nature 1997; 387: 253–260. 3. bérdy j. thoughts and facts about antibiotics: where we are now and where we are heading. j antibiot 2012; 65(8): 385. 4. goodfellow m, williams st and mordarski m. 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universiti putra malaysia, 43400 upm serdang, selangor darul ehsan, malaysia 2novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), abstract: the increased concentration of extracellular glutamate has been reported to play a key role in most of the neurodegenerative diseases, such as parkinson’s disease and alzheimer’s disease, even though its importance as an amino acid neurotransmitter in mammalian. glutamate toxicity, which can be caused by excessive intake of monosodium glutamate (msg), is the major contributor to pathological neuronal cell death. it causes neuronal dysfunction and degeneration in the central nervous system (cns). glutamate neurotoxicity can be categorized into two forms, which are receptor-mediated glutamate excitotoxicity and non-receptor mediated glutamate oxidative toxicity. the receptor-mediated glutamate excitotoxicity involved excessive stimulation of glutamate receptors (glurs) which lead to excessive ion calcium (ca2+) influx and activates a cell death cascade involving the accumulation of mitochondrially generated reactive oxygen species (ros). studies showed excessive extracellular glutamate leads to nerve cell death via the activation of n-methyl-daspartate (nmda) receptors in the cases of trauma or stroke. whereas non-receptor mediated oxidative toxicity involved the breakdown of the cystine/glutamate antiporter (xc -) mechanism, which leads to the depletion of glutathione (gsh) and causes oxidative stress and cell death. the cystine/glutamate antiporter couples the import of cystine to the export of glutamate. the increased concentration of extracellular glutamate could inhibit the uptake of cystine, which is required for the synthesis of the intracellular antioxidant gsh. gsh plays an important role in the disposal of peroxides by brain cells and in the protection against ros. depletion of gsh renders the cell to oxidative stress and ultimately leading to cell death. this article aims to provide a comprehensive review of neurodegenerative diseases and the role of neurotoxin agents, glutamate in these diseases. keywords: neurodegenerative diseases; alzheimer’s disease; parkinson’s disease; neurotoxin agents; glutamate received: 5th march 2020 accepted: 19th april 2020 published online: 22nd april 2020 citation: yap, h.m, lye k-l and tan lt-h. comprehensive insight of neurodegenerative diseases and the role of neurotoxin agents –– glutamate. prog mircobes mol bio1 2020; 3(1): a0000070. https://doi.org/10.3687/pddbs.a0000070 introduction nervous system nervous system is the major controlling, regulatory and communicating system in the body responsible for receiving and transmitting stimuli throughout the body[1]. nervous system also monitors and coordinates internal organ function and initiates response to the changes in the external environment. nervous system can be divided into central nervous system (cns) and peripheral nervous system (pns). cns is composed of the brain and spinal cord, which serves as the main processing center for the entire nervous system[2]. it receives information from and sends information to the pns. pns is made up of all the nerves connecting the body to the brain and spinal cord. sensory neuron is responsible in sending information from internal organ or from external stimuli to cns, whereas motor neuron transmits information from cns to organs, muscles and glands[2]. the motor nervous system can be further divided into two systems; autonomic nervous system and somatic nervous system. autonomic nervous system is responsible for involuntary or visceral bodily function while somatic nervous system is mediating voluntary reflexes for instance skeletal muscle. in general, activity of the nervous system depends on the connectivity among two cell types, which are neurons and glia[3–5]. copyright © 2020 by yap and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: hui-min yap, department of biomedical sciences faculty of medicine and health sciences, universiti putra malaysia, 43400 upm serdang, selangor darul ehsan, malaysia; huimin050686@gmail.com. jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2 neuronal cell neuronal cell is an excitable nerve cell that processes and transmits information by electrochemical signaling. most neurons cannot divide; thus, neurons cannot be replaced if lost due to injury or disease[1]. there are 3 types of neurons based on functions, which are sensory neurons, motor neurons, and interneuron. interneurons carry information between sensory neurons and motor neurons and can be found in cns. generally, neuronal cell is comprised of neuronal cell body (soma), axon and dendrite. the axon has a terminal end that constitutes the pre-synaptic site of the synapse while the dendrite has an apical site and multiple branches. neuron communicates with each other at synaptic cleft (synapse), which involved release of neurotransmitter by pre-synaptic cell and binding of neurotransmitter to the receptor on post-synaptic cell. neurotransmitter will trigger an electrical response or a secondary messenger pathway that may either excite or inhibit postsynaptic neuron[4]. excitatory neurotransmitter causes changes that generate action potential in the responding neuron while inhibitory neurotransmitter will block the changes[6,7]. major neurotransmitters found in the body include glutamate, dopamine, glycine, gamma-aminobutyric acid (gaba) and serotonin. glial cell the glial cells provide vital support for the proper functioning of neurons. glial cells are also known as nerve glue, are non-neuronal cells of the nervous system. it does not transmit electrical impulses, but it appears to be the most abundant cell types in the cns. glial cell can be divided into macroglia (astrocytes, oligodendroglia and schwann cells) and microglia. different types of glial cell provide different support to neurons. microglia cells in cns protect neurons from bacteria invasion. oligodendrocytes and schwann cells provide myelination to axons in cns and pns respectively. myelin sheath provides insulation to the axon that allows electrical signals to be transmitted efficiently[8]. astrocytes not only provide physical support to neurons, but also support neurons by providing antioxidant protection, substrates for neuronal metabolism via neurovascular coupling, digest dead neurons and glutamate clearances[4,9]. also, astrocytes have interaction with the cerebral endothelium in determining blood brain barrier function, morphology and protein expression[10]. neuron-astrocyte interaction in the brain astrocytes are the most numerous and diverse glial cells in the cns. many studies demonstrating astrocyte dysfunction throughout the neurodegenerative process as a prominent determinant for survival for both neuronal cell and the entire organism. neuronal activity leads to increase of potassium ion (k+) and glutamate levels in extracellular space. as a supportive cell, astrocyte plays important role in maintaining the extracellular k+ homeostasis through potassium channels expressed by astrocytes[11]. moreover, astrocytes are known to express high level of glutamate transporter to remove glutamate from extracellular space[12]. the increased concentration of extracellular k+ and glutamate will trigger astrocyte glycolysis and further enhance lactate and pyruvate production to fuel neuronal tricarboxylic acid cycle (tca cycle)[13,14]. astrocyte and neuronal cell also coupled by glutamate-glutamine cycle. astrocytes take up glutamate and convert it to glutamine. this glutamine is then shuttled back to presynaptic terminals (neuronal cells) and used by neuronal cell to replenish the neurotransmitter glutamate[15]. in addition, neurotransmitters released by neuronal cells induce transient elevations of internal ca2+ levels in astrocytes due to the activation of metabotropic glutamate receptors on astrocytes[16,17]. the increased level of astrocytic ca2+ triggers the release of chemical transmitters from astrocytes, which can cause sustained modulatory actions on neighbouring neuronal cells[17]. neurodegenerative diseases neurodegenerative diseases are becoming huge public health challenge due to their increasing medical and social impact. it is one of the major global health burdens in the western world and is often found in aging population[18]. these diseases can be life-threatening and have great impacts on both the patients and caregivers. neurodegenerative diseases can be illustrated by progressive nervous system dysfunction that causes deterioration of many of human body’s activities, including speech, movement, balance, heart function and breathing[19]. the causes of neurodegenerative diseases comprise genetic factors, infections or autoimmune diseases, brain trauma or injury and environmental toxins[20–23]. most of the neurodegenerative disorders are incurable; thus, treatments are applied with the hope to relieve pain, improve symptoms, and increase mobility. neurodegenerative diseases are categorized based on primary brain region or type of affected brain cell, clinical symptoms, and type of protein aggregated in brain[24]. parkinson’s disease (pd), alzheimer’s disease (ad), and amyotrophic lateral sclerosis (als) are the most common age-related neurodegenerative diseases. some forms of these diseases could be inherited. moreover, morphological, biochemical, genetic, cell and animal model studies reveal that mitochondria play roles in the neurodegeneration through rendering vulnerable neurons susceptible to stress, cellular aging and genetic variations[25]. alzheimer’s disease (ad) alzheimer’s disease (ad) can be categorized by progressive degenerative disorders of the brain which causes irreversible loss of neurons and memory. it is one of the common causes leading to dementia. late-onset of alzheimer’s disease (load) with age 65 years or older is representing the majority of ad. senile plaques, intracellular neurofibrillary tangles (nfts), extracellular degenerating neurons, dystrophic neuritis, and activated comprehensive insight of... 3 yap et al. astrocytes or microglial found in the brain underlie the pathogenesis of ad[26]. senile plaque is mainly composed of β-amyloid (aβ) while nfts consist of hyperphosphorylated and aggregated forms of the tau protein. tau is a microtubule-binding protein that acts as neuronal cytoskeleton stabilizer and participates in vesicular transport and axonal polarity. the deposition of hyperphosphorylated tau protein impairs axonal transport therefore affecting the nutrition of dendrites and axon terminals. while the accumulation of aβ activates cellular stress responses and disrupts signal transduction pathways[27]. studies indicated that aβ also induces inflammatory reaction in the brain and causes release of various cytokine such as tnf-α (tumor necrosis factoralpha)[28]. tnf-α could induce infiltration of leukocytes and possible t-cells into the central nervous system through its capacity in enhancing blood brain barrier permeability[29]. the increased permeability of blood brain barrier causes influx of neurotoxin, such as glutamate and arachidonic acid, which could lead to substantial damage to the brain. cholinergic and glutamatergic neurotransmission systems, which play role in cognition, were affecting in patients with alzheimer’s disease[30]. parkinson’s disease (pd) parkinson’s disease (pd) is the second most common latelife neurodegenerative disease after alzheimer’s disease. it can be characterized by progressive dopaminergic cell loss in the substantia nigra pars compacta which leads to dysfunction of the basal ganglia system and motor symptoms and subsequently causing tremors, slow movement, rigidity and gait impairment[31,32]. another pathological feature of pd is the accumulation of lewy bodies in neurons of the brainstem nuclei, substantia nigra, hippocampus, cerebral cortex, myenteric plexus, and olfactory bulb. lewy bodies are accumulations of microscopic protein deposits in the brain, mainly alpha-synuclein. the pathology of pd is not yet fully understood. some genetic and toxin-based animal models suggest that inflammation, oxidative stress, mitochondrial dysfunction, aberrant processing of proteins by the ubiquitin-proteasome system, and activation of apoptotic pathways are playing roles in dopaminergic cell death[33]. furthermore, some studies indicated the increased glutamatergic transmission may contribute an excitotoxic component to the cellular insults that lead to degeneration in the substantia nigra pars compacta as well as the glial response that arises in the striatum and the substantia nigra[34]. other neurodegenerative diseases other neurodegenerative disorders include huntington’s disease (hd) and amyotropic lateral sclerosis (als). hd can be categorized by cognitive decline, psychiatric disturbances and motor dysfunction, which will lead to dementia and death approximately 15–20 years after the onset of the disease[35]. hd is an autosomal-dominant disorder caused by the expansion of a cag trinucleotide repeat encoding an abnormally long polyglutamine track in the n terminus of the huntingtin protein[36]. this protein may inhibit mitochondrial function and proteasome activity[37]. hd has a prevalence of 5–10 cases per 100,000. thus, hd is known as one the most common inherited neurodegenerative disease[35]. while amyotrophic lateral sclerosis seemed to be a progressive and usually fatal disorder caused by motorneuron degeneration in the brain and spinal cord. motorneuron degeneration in the brain and spinal cord will lead to paralysis of voluntary muscles. the mutation in superoxide dismutase 1 (sod1) seems to be associated with amyotrophic lateral sclerosis. this mutation will form aggregates in mitochondria which could interfere anti-apoptotic function of b-cell lymphoma 2 (bcl-2), mitochondrial import and generate free radicals. reactive oxygen species produced will inhibit the function of glial glutamate transporter (eaat2) and lead to the increase of extracellular glutamate, next triggering motor neuron death[38]. factors involved in neurodegenerative diseases neurodegeneration is due to the death of neuronal cells through the delayed process of apoptosis or necrosis. among the known risk factors for neurodegenerative diseases, such as genetic polymorphism, infection and head trauma, the role of neurotoxin agents such as glutamate and an oxidative stress received huge attention recently. this is because most of the pathologies of neurodegenerative diseases involve oxidative stress triggered by neurotoxin agents or imbalance in pro-oxidant/antioxidant homeostasis[39,40]. astrocytes in neurodegenerative diseases astrocytes have many housekeeping functions, such as the maintenance of the extracellular environment and synaptic function in the cns. also, astrocytes play a vital role in the maintenance of neurotransmitter synthesis and neuronal metabolism. astrocytes become activated (reactive) in response to a variety of brain insults, such as trauma, stroke and neurodegenerative diseases[41]. reactive gliosis can be characterized by the increase expression of vimentin (vim), glial fibrillary acidic protein (gfap), s100β and proliferation that could probable occur in neurodegenerative diseases[42]. astrocytes will be activated under stress and injury, leading to an upregulation of proinflammatory cytokines and chemokines, which are related to the pathogenesis of ad[43]. recent studies suggested that activated astrocytes play a role in the clearance of the aβ-peptide and thus preventing the plaque formation in ad. furthermore, astrocytosis (abnormal increase number of astrocytes) was discovered in affected brain region of hd patients. the huntingtin protein was also observed to be co-localized with reactive astrocytes[15]. glutamate l-glutamate is a non-essential amino acid which can be gained from alimentary protein, endogenous protein and 4 also monosodium glutamate (msg) that is used as food additive[44]. it is one of the most abundant neurotransmitter in mammalian central nervous system and it is involved in numerous normal brain functions, such as memory, cognition and learning[45]. furthermore, glutamate has an essential role in regulating bio-energetic processes such as reactions of glycolysis, gluconeogenesis, citric acid cycle and synthesis of ketone bodies. it is the precursor for γ-aminobutyric acid (gaba) and glutathione (gsh) [46]. glutamate is one of the key factors in central pain transduction mechanisms and excitotoxic neuronal cell injury[45]. glutamate receptors neurotransmission occurs at synapses and it involved processes of neurotransmitters carrying signals released from presynaptic neuron terminals to synaptic cleft followed by binding of neurotransmitter to the receptors on the postsynaptic neuron. binding of excitatory neurotransmitters, such as glutamate, to the receptor proteins on the postsynaptic neuron will cause sodium ion (na+) channels to open and allow influx of na+ to neuronal cell, and causes the neuron membrane to depolarize due to an action potential. the excitation of glutamate can be mediated via activation of metabotropic glutamate receptors and ionotropic glutamate receptors[47]. there are 3 families of ionotropic glutamate receptors; n-methyl-d-aspartate (nmda), α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (ampa) and kainate receptors. the ionotropic glutamate receptors are coupled directly to membrane ion channels which conducting only na+ or both na+ and ca2+. metabotropic glutamate receptors comprise of g-protein coupled receptors (mglur1–8) that are important in regulating ion channels and enzymes producing second messenger[48]. it is categorized into 3 groups based on the sequence, function and pharmacology. group i mglurs (mglur1 and 5) activates phospholipase c via gq proteins and initiate an inositol triphosphate/ diacylglycerol (ip3/dag) second messenger cascade. whereas group ii mglurs (mglur2 and 3) and group iii mglurs (mglur4, 6, 7 and 8) inhibit adenylyl cyclase via gi/o proteins. reports showed that group ii and iii were mainly localized on presynaptic neuron while group iii was localized on postsynaptic neuron[49]. metabotropic and ionotropic glutamate receptors present on both neuronal and glial cells[50,51]. nevertheless, the involvement of glutamate receptors of glial cells in neurotransmission remains unknown. while the binding of inhibitory neurotransmitters, such as serotonin or gaba, to the receptor proteins on the postsynaptic neuron will lead to hyperpolarization and efflux of potassium ion (k+) from neuronal cell[52]. glutamate transporters the level of extracellular glutamate must remain low to prevent excessive excitation that can injure or kill neuronal cells due to the deficiency of enzyme at extracellular space to metabolize glutamate. the excessive glutamate can only be removed by cellular uptake through glutamate transporters on the plasma membrane of astrocytes and neuronal cells. glutamate transporter can be discovered in the plasma membranes and intracellular membrane[53]. glutamate transporters in the plasma membranes can be categorized into high affinity glutamate transporters and low affinity glutamate transporters. there are 5 high affinity glutamate transporters, also known as sodium-dependent glutamate transporters, have been cloned, namely excitatory-amino acid transporter 1-5 (eaat1-5). these 5 glutamate transporters catalyze na+and k+-coupled transport of l-glutamate and also land d-aspartate[54]. each glutamate is co-transported with two or three na+ and counter-transport of one k+[55]. eaat3, eaat4 and eaat5 are known as neuronal glutamate transporters. however, there were studies that showed eaat4 was expressed in astrocytes as well[56]. eaat1 and eaat2, also known as glutamate aspartate transporter (glast) and glutamate transporter-1 (glt1) respectively, are expressed in astrocytes[57]. these astrocytic glutamate transporters are responsible for the majority of glutamate uptake near excitatory synapse[58,59]. cystine/glutamate antiporter (system xc-) is a low affinity glutamate transporter which also known as sodiumindependent glutamate transporter. system xcis composed of two subunits, which are xct light chain and 4f2 heavy chain. it is also known as cystine transporter which carries cystine into the cell in exchange for internal glutamate by using the transmembrane gradient of glutamate as driving force[54,60]. this system is suggested to mediate the cellular cystine uptake for the synthesis of gsh and maintenance of cystine—cysteine redox balance in the extracellular compartment, which is vital for the cellular protection from oxidative stress and cell death[61]. glutamate metabolism when glutamate taken up by cells, it is not only used as transmitter, but also used for metabolic purposes such as protein synthesis, energy metabolism and ammonia fixation[62]. in the nerve terminal, cytoplasmic glutamate is transported into synaptic vesicles by vesicular glutamate transporters (vgluts) and then released to the synaptic cleft by exocytosis. as neurotransmitter, the binding of a transmitter to postsynaptic ionotropic glutamate receptors (ampa, nmda and kainate receptors) will cause an ionic influx that depolarizes the neuron (excitation)[63]. whereas, a slower second messenger-mediated affects occur following the activation of the metabotropic glutamate receptors by glutamate[64]. to avoid excessive excitation that could injure and damage neurons, glutamate in the synapse should be removed rapidly. glutamate can be removed via the uptake into the postsynaptic compartment, uptake into nonneuronal compartment (astrocyte) and reuptake into the presynaptic compartment. glutamate uptake into presynaptic and postsynaptic neurons seems to be less important than astrocytic transport due to the membrane potential of astrocytes lower than neurons[65]. in astrocytes, glutamate taken up from extracellular fluid is metabolized by 2 different pathways, which are comprehensive insight of... 5 tricarboxylic acid cycle (tca cycle) and glutamateglutamine cycle. astrocytes convert glutamate and ammonia to glutamine (non-neuroactive species) via atpdependent glutamine synthetase. this pathway further supports the buffering of ammonia, which is a potential neurotoxic species. glutamine synthetase in the brain is located mostly in astrocytes[66]. glutamine produced is then released to the extracellular fluid and taken up by neuronal cell. glutamine taken up into neuronal cell could be hydrolyzed to glutamate and ammonia via the action of phosphate-dependent glutaminase. the regenerated glutamate will be reused as neurotransmitter or potential fuel by being oxidized to α-ketoglutarate for neuronal cells[67,68]. meanwhile, glutamate taken up by astrocyte could be converted to α-ketoglutarate and ammonia via reversible dehydrogenation by glutamate dehydrogenase (gdh) with either nad+ or nadp+ as a cofactor[69]. alpha-ketoglutarate is metabolized through tca cycle to succinate, fumarate and malate. malate is further decarboxylated to pyruvate and reduced to lactate. both pyruvate and lactate are exported from astrocytes to the extracellular fluid and taken up by neuronal cell. furthermore, glutamate uptake into astrocytes will stimulate glycolysis, that is glucose utilization and lactate production. glutamate activates astrocytic na+/k+ atpase by cotransport na+ into the cell via na+-dependent glutamate uptake by glutamate transporters. the resulted lactate will provide the fuel for neuronal tca cycle[70]. figure 1 denotes the metabolism of glutamate in neuronal cell and astrocyte. figure 1. glutamate metabolism in neuronal cell and astrocyte. glutamate neurotoxicity the maintenance of a low level (1–3 µm) of extracellular glutamate is vital for normal brain function to prevent excessive stimulation of receptors, excessive formation of ammonia by glutamate dehydrogenase and breakdown of glutamate/cystine antiporter. the excessive accumulation of extracellular glutamate is the main contributor to neuronal cell death under pathological conditions for instance ischemic insults and traumatic brain damage which could be associated to the disruption of glutamate metabolism[71,72]. the disruptions of glutamate metabolism underlie many neurodegenerative pathologies, for instance alzheimer’s disease, parkinson’s disease, huntington’s disease, brain ischemia and amyotrophic lateral sclerosis. these may be associated to variation of glutamate metabolism enzyme activities, disruption in mitochondrial homeostasis, and oxidation or redox imbalance in cells[73,74]. glutamate neurotoxicity can be classified into receptor-mediated glutamate excitotoxicity and non-receptor mediated oxidative glutamate toxicity. receptor-mediated glutamate excitotoxicity hayashi was the first to discover the excitatory action of glutamate in 1952 by injecting glutamate into mammalian brain able to produce convulsion[75]. toxicity of glutamate was then observed when the injection of monosodium glutamate destroyed the neurons in the inner layers of the retina and induced acute neuronal necrosis in brain and term as excitotoxicity[76–78]. elevated concentration of extracellular glutamate will cause excessive stimulation of glutamate receptors at postsynaptic membrane, which will further induce excessive influx of ca2+ and na+. ionotropic glutamate receptors directly activate channels that allow influx of extracellular ca2+ and na+ while metabotropic glutamate receptor stimulates calcium released from internal store (endoplasmic reticulum and mitochondria) [79,80]. the influx of ca2+ and na+ through glutamate receptor activation will result in membrane depolarizations which further activate voltage-dependent calcium channels. plasma membrane calcium transporters regulate the calcium homeostasis by maintaining low concentration of yap et al. 6 free intracellular calcium relative to free extracellular. basically, there are 3 types of membrane transporters, namely antiporters, symporters and atp-coupled active transporters. calcium is controlled by the antiporters and ca2+-atpase. antiporters and ca2+-atpase move ca2+ out of the neuron and restore ca2+ to mitochondria and endoplasmic reticulum[81]. antiporter transport ca2+ by a sodium-calcium exchange mechanism while the ca2+atpase transports one ca2+ for each atp hydrolyzed with a resultant increase in atp demand. in addition, the direct uptake of ca2+ into the matrix utilizes the proton circuit for oxidation phosphorylation and competes with mitochondrial atp synthesis[82]. therefore, mitochondrial atp synthesis in mitochondria may cease as the ca2+ flood from cytoplasm to the matrix[83]. moreover, there are studies that showed ca2+ influx from extracellular space activates cytosolic phospholipase a2 and lead to production of arachidonic acid[84,85]. ros will be created during arachidonic acid oxidative metabolism[86]. ca2+ excess of mitochondrial matrix triggers a mitochondrial permeability transition pore opens in the mitochondrial membrane, which will lead to collapse of mitochondrial membrane potential (mmp) [87]. furthermore, reactive oxygen species formed from oxidation phosphorylation also contribute to the opening of the mitochondrial permeability transition pore[88]. the maintenance of mmp is vital for atp synthesis. the collapse of mmp is related to a secondary increase in cytosolic ca2+ concentration and the release of cytochrome c followed by caspase activation and apoptosis[89]. moreover, caspase-independent death can also be induced by mmp through release of caspaseindependent death effectors such as apoptosis-inducing factor and endonuclease g[90]. non-receptor mediated glutamate oxidative toxicity the increased concentration of extracellular glutamate will lead to a prolonged cell death by oxidative stress known as glutamate oxidative toxicity. in glutamate oxidative toxicity, increased concentration of extracellular glutamate interferes with cystine uptake by blocking the glutamate gradient driven cystine/ glutamate antiporter (system xc-)[91]. in facilitating the uptake of cystine, it is vital to exchange with glutamate at an equimolar ratio. the breakdown of system xcwill lead to the depletion of cystine and glutathione (gsh). in normal situation, imported cystine (oxidized form of 2 cysteines) is reduced to cysteine, which primarily acts as a precursor for gsh synthesis. the depletion of gsh renders the cells to oxidative stress and cell death[92]. studies indicated that gsh depletion lead to activation of 12-lipoxygenase (12-lox) and 15-lipoxygenase (15-lox) and also causes inactivation of gsh peroxidase 4 (gpx4), which damage mitochondria and resulted to cytochrome c and apoptosis-inducing factor (aif) release, followed by reactive oxygen species accumulation and cell death[93–96]. oxidative stress oxidative stress built up caused by an imbalance of free radicals (pro-oxidant) and antioxidants in favor of the former. free radicals are atoms or groups of atoms that have one or more unpaired electrons which make it extremely reactive[97]. these extremely reactive molecules tend to react quickly with adjacent molecules by donating or capturing an electron in order to gain stability and start the free radical reaction chain. it usually arises during body normal metabolism and increase with age. also, free radicals can be generated by the immune system to destroy bacteria and viruses during infection. environmental factors for instance pollution and radiation can further increase the production of free radicals. the most common cellular free radicals include hydrogen peroxide (h2o2), hydroxyl (oh·), superoxide (o2-) and nitric monoxide (no·). high level of free radicals leads to redox imbalance and oxidative damage to biomolecules, such as protein, lipids and dna, then lead to many chronic diseases in human such as cancer, diabetics, atherosclerosis, postischemic perfusion injury, cardiovascular diseases, stroke, chronic inflammation and other degenerative diseases[98]. reactive oxygen species (ros) and the reactive nitrogen species (rns) are extremely reactive among free radicals, which acts as mediators in biological processes for instance respiration, cellular metabolism, gene translation and transcription, neurotransmission and inflammatorytype reactions[99]. mitochondria are the main contributor to ros production, and the electron transport chain that is found in the inner mitochondrial membrane uses oxygen to produce adenosine triphosphate (atp)[100]. cytochrome oxidase functions as the terminal enzyme of the chain by accepting an electron from cytochrome c and transferring it to oxygen (the final electron acceptor in the chain). the reduction of oxygen by one electron at a time by cytochrome oxidase make it becomes highly reactive. superoxide (o2-), the product of one-electron reduction of oxygen, occurs mainly within the mitochondria of a cell[101]. it also functions as the precursor of ros (such as hydroxyl radical) and as a mediator in oxidative chain reactions[102]. ros is known to initiate lipid peroxidation by targeting the carbon-carbon double bond of polyunsaturated fatty acids (pufas) which are ample in cellular membranes and lowdensity lipoproteins. high level of oxygen requirement and unsaturated lipids in brain make it particularly susceptible to oxidative damage and lipid peroxidation[103,104]. neuronal cells are most vulnerable to oxidative damage due to their low antioxidant activity[105]. furthermore, the metabolism of excitatory amino acids and neurotransmitters in neuronal cells further increase the production of ros and oxidative stress. therefore, oxidative stress plays vital role in the pathogenesis of neurodegenerative diseases[106]. mode of cell death in neuronal cells, calcium ions overload caused by glutamate excitotoxicity activates damaging enzymes for instance phospholipases that produce ros. ros induces neuronal injury such as cell membrane disruption, damage to intracellular proteins and dna, and induction of mitochondrial release of cytochrome c, and caspase comprehensive insight of... 7 activation. the release of cytochrome c, caspase activation and dna fragmentation are the molecular hallmarks of apoptosis[107–110]. furthermore, cystein protease or calpains are also activated by the elevation of intracellular ca2+. calpains cleave various intracellular proteins, such as regulatory protein, membrane channels and cytoskeletal that lead to neuronal cell death by necrosis[111,112]. apoptosis and necrosis signify 2 significant different cell death mechanisms. apoptosis, also well-known as programmed cell death or cell suicide, happens under normal physiological conditions. it is an important mechanism in maintaining the homeostasis and regulation of tissue size during postnatal life. apoptosis is signified by the activation of endogenous endonucleases that are activated directly or indirectly by a cascade of biochemical reactions, which involve cutting of dna into fragments and consequently causing cell death. apparently, cells continue to produce proteins and atp during apoptosis. as a result, each apoptotic body is surrounded by cell membrane which contains intact and functional cell components. apoptosis causes morphological changes that can be characterized by a deep chromatin condensation and formation of apoptotic bodies, followed by nuclear fragmentation and secondary necrosis that occur at the final stage. in contrast to apoptosis, necrosis occurs when cells are exposed to extreme variance from physiological condition. it starts with an impairment of the cell’s ability to maintain homeostasis. necrosis can be signified by the loss of metabolic functions and the loss of cell membrane integrity. the cells terminate their proteins and atp production during necrosis. the morphological changes of necrosis are signified by cell membrane rupture starts with swelling of cytoplasm and mitochondria. cell membrane ruptures results in the release of the cell’s components into the surrounding tissue. this process is recognized as cytolysis. the released cell’s components will induce an intense inflammatory response[113–115]. conclusion the excessive glutamate-induced neurotoxicity had been associated to many chronic neurodegenerative disorders in cns. under normal situation, the astrocyte is responsible in clearing up glutamate from synaptic cleft. hence, minimizes the neuronal injury from glutamate toxicity. however, excessive glutamate will induce oxidative stress in both astrocyte and neuronal cell and lead to cell death. studies demonstrated that radical scavengers like vitamin e, in the form of tocopherol and tocotrienol, can protect the glutamate-injured cells from oxidative stress. other than vitamin e, other sources such as microbial resources were also demonstrating strong antioxidants[116–126] and neuroprotective properties[127–132] such as metal chelating and radical scavenging potentials[133–140]. in conclusion, neuronal cell seemed to be the most vulnerable brain cells toward glutamate toxicity. in the absence of astrocyte, neuronal cells are 100-fold more vulnerable to glutamate toxicity. thus, more studies should be conducted to explore the protective effect of astrocyte on neuronal cell against glutamate toxicity. conflict of interest the authors declare that there is no conflict of interest in this work. author contributions the literature review and manuscript writing were performed by h-my, k-ll and 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novel actinobacterium isolated from malaysia mangrove soil exhibiting antioxidative activity and cytotoxic potential against human colon cancer cell lines. front microbiol, 2017; 8: 877. 140. tan, lt-h, chan, k-g, pusparajah, p, et al. mangrove derived streptomyces sp. mum265 as a potential source of antioxidant and anticolon-cancer agents. bmc microbiol, 2019; 19(1): 38. yap et al. progress in microbes and molecular biology review article 1 molecular profiling and detection methods of microrna in cancer research nurul-syakima ab mutalib1*, imilia ismail2, hooi-leng ser3 1ukm medical molecular biology institute (umbi), ukm medical centre, universiti kebangsaan malaysia, kuala lumpur, malaysia 2school of biomedicine, faculty of health sciences, universiti sultan zainal abidin, gong badak campus, 21300 kuala terengganu, terengganu, malaysia 3novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia abstract: a large portion of human genome was believed to be “useless” and termed as “junk dna” in the past, given that these sequences did not have any protein coding role. however, with more researchers dwelling into the world of these mysterious genetic codes, a group of non-protein coding rna (ncrna) known as micrornas (mirnas) is now being recognized to play important roles than they were thought to be. in truth, the first discovery of mirna was in a simple organism nematode (scientific name: caenorhabditis elegans), whereby a mutant displayed aberrant morphological changes. years after that, researchers then realized that these mirnas are actually important regulatory molecules — controlling cell division signaling, apoptosis and so on. in fact, the unusual expression of mirnas has also been associated in etiology of various cancers. acting like a “double-edge” sword, mirnas can control and/or act as tumor suppressor genes and oncogenes, thus any unwanted alterations in their expression would bring upon disastrous effects on the host. therefore, the current review aims to summarize the molecular detection tools that are available for mirna profiling in cancer research. keywords: mirna; noncoding rna; detection; profiling; cancer received: 22nd june 2020 accepted: 22nd july 2020 published online: 1st august 2020 citation: ab mutalib n-s, ismail i and ser h-l. molecular profiling and detection methods of microrna in cancer research. prog microbes mol biol 2020; 3(1): a0000099. https://doi.org/10.3687/pmmb.a0000099. introduction in the 1972, a geneticist susumu ohno coined the concept of “junk dna”, which was used to describe all the non-protein coding regions within the human genome[1]. since then, scientists around the world have been trying to decode the human genetic code or dna, particularly to illuminate and understand the genetics of gene regulation and function. less than twenty years later, the human genome project (hgp) commenced in the early 1990s as an international, collaborative research which is known to be an important milestone in understanding the human genome[2,3]. more importantly, apart from identifying the genes, one of the eight major goals proposed by hgp in 1998 was to elucidate functions of these genes, including non-protein coding sequences. one of the most gamechanging discovery was a type of small rna called micrornas (mirnas) in 1993 by lee and colleagues, in which they cloned the lin-4 locus almost two decades after its first description and this locus was proven to have exceptional characteristics compared with other normal coding genes[4]. rather than coding a protein, the lin-4 gene presents as a small rna molecule. two small lin-4 transcripts with approximately length of 22 and 61 nucleotides were discovered in caenorhabditis elegans; these were significantly smaller in size compared with other genes. also, lin-4 mrna transcripts displayed antisense complementarity to multiple sites in the 3’ untranslated region (utr) of another gene known as lin-14, implying that lin-4 regulates the translation of latter gene via an antisense rna-rna interaction. these remarkable breakthroughs subsequently instigated the concept of a unique class of small ncrna regulatory molecules acting via an antisense-like interaction[5,6]. accumulating evidence is pointing directly at the role of mirna in human chronic diseases, including diabetes, copyright © 2020 by ab mutalib n-s and hh publisher. this work under licensed under the creative commons attributionnoncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: nurul-syakima ab mutalib, ukm medical molecular biology institute (umbi), ukm medical centre, universiti kebangsaan malaysia, kuala lumpur, malaysia; syakima@ppukm.ukm.edu.my. 2 alzheimer’s disease, cardiovascular diseases as well as cancer[7,8]. carcinogenesis is a very complex, multi-step process which involves at least three stages: initiation, promotion and progression/metastasis[9]. the mutated cells somewhat managed to overcome fate of cell death or apoptosis and subsequently divide and expand itself; some scenarios would bring about life threating events particularly when these mutated cells gain the ability to promote blood vessel formation in order to gain access to nutrients and oxygen supply as well as ability to spread via moving or metastasize to nearby local tissue[9,10]. with the increasing interest in human genome, it was then noted that mirna expression patterns were certainly tissuespecific, with cancer cells demonstrating significantly different profile than their normal counterpart[11,12]. furthermore, additional studies also demonstrated that mirna expression in peripheral blood could be used as an alternative way to “gauge” mirna expression in the tumor biopsy. therefore, the current study is to summarize and discuss conventional molecular detection tools as well as some newer, innovative methods that are available currently for mirna profiling in cancer research. what are mirnas and their role in cancer? mirnas are recognized as a group of small non-protein coding rna (ncrna) sequences with regulatory potential. the biogenesis of mirna begins with the processing of rna polymerase ii/iii transcripts postor co-transcriptionally, with most of the mirna being intragenic and processed largely from introns[13]. two main pathways have been described for mirna biogenesis: (a) canonical pathway which involves processing by a microprocessor complex an rna binding protein digeorge syndrome critical region 8 (dgcr8) and a ribonuclease iii enzyme, drosha and (b) noncanonical pathway that can be either drosha/dgcr8independent and dicer-independent pathways[13,14]. nonetheless, it has been shown that animals cannot survive or reproduce normally without mirnas expression[15]. using c. elegans as an in vivo model in genetic study, sulston and brenner were able to screen for 300 mutants that gave rise to c. elegans displaying diverse developmental defects and behavioral changes in the early 1970s[16]. one of the mutants, designated as lin4, exhibited elongated and flaccid body morphology with diverse reiterations for particular cell lineages. little did the scientists noticed that this discovery became the founding member of the mirna family, whereby it was shown to be involved in the early development of c. elegans larval by influencing timing of developmental events across various cell types[17,18]. nonetheless, the atypical expression of mirnas has also been associated in etiology of various cancers, given that it can regulate and/or act as tumor suppressor genes and oncogenes[19–21]. for example, calin and team failed to notice any protein coding genes within chromosome 13q14 and later found that the loss of two mirnas, mir-15a and mir-16-1 in more than 65 % of b cell chronic lymphocytic leukemia patients[22]. later on, another study by bandi and team also revealed that these mir-15a and mir-16 are often deleted or downregulated in non-small cell lung cancer (nsclc) tissues[23]. even though the target for these two mirna seems to be unknown, some studies showed that these mirna may possess a negative regulatory role for antiapoptotic b cell lymphoma 2 (bcl2) protein and eventually induces apoptosis in leukemic cell model[20,24,25]. as such, alterations in mirna may indeed sound like “total chaos” for the human body, contributing to the hallmark features of human cancer including supporting cell division signaling, dodging growth suppressors, avoiding cell death, activating replicative immortality, increasing invasion and metastasis ability as well as promoting angiogenesis[26–29]. rather than a loss of function, oncogenic mirnas or “oncomir” grant cancer cells survival/growth advantages by evading apoptosis and tend to cleave target mrnas more frequently than those mirnas with tumor suppressors function[30–32]. one of the classic example for oncogenic mirnas would be mir-17-92 which exists as a mirna polycistron at chromosome 13q31 and is highly expressed in a range of human cancers including lymphoma, lung cancer, breast cancer, colon cancer as well as head and neck cancers[33–38]. all in all, extensive dysregulation of mirnas can have serious impact on the development of various human cancers, which in turn explains for its importance as diagnostic and prognostic markers as well as targets for new therapeutic agents[39]. detection and profiling of mirna currently, there are several methods to capture and quantify expression of these small non-coding mirna. in terms of mirna detection, there are three main techniques: (a) hybridization-based techniques (e.g. northern blots, microarrays), (b) amplification-based (e.g. rt-qpcr) and (c) cloning-based (e.g. mirage) (figure 1)[40]. the decision on which techniques to be used for detection is greatly dependent on number of specimens, running time and cost. for examples, norther blots developed by alwine and team in the late 1970s remained as the gold standard for gene expression changes and mirna studies[41]. however, in terms of expression throughput, multiplex rt-pcr and mirage can produce results in shorter time, but the newer techniques like microarrays are generally better choices if the study involves large number samples or requires in-depth analysis[40]. having said that, the quantitative power of these techniques varies; rt-qpcr produces quantitative data, whereas results from northern blots are semi-quantitative. hybridization-based technique like in-situ hybridization is normally non-quantitative. each of these techniques have got their advantages as well as limitations, therefore researchers may opt to combine these techniques in a study to improve and/or strengthen their findings. molecular profiling... 3 ab mutalib n-s et al. explained that the oligonucleotide probes with lna was designed in a way that every third nucleotide position was substituted by a lna monomer. this design subsequently resulted in an increase in its sensitivity yet retained its high specificity, which enables detection of both mature and precursor mirnas[46]. similarly, edc chemically mediates cross linking of rna to (nylon) membrane which has been shown to improve mirna detection by up to 25 to 50-fold, depending on plants or mammalian samples[47,48]. therefore, by combining the goodness of these three components, kim and team highlighted that led probes offer multiple advantages; it’s more environmentally friendly compared to conventional probes which can be stored for at least six months [42]. having said that, these “upgrades” seem to incur higher cost compared with the conventional old method, especially when lna probes can be more expensive than dna probe and dig-labeling which will also add up to the final cost. microarray-based method microarrays are amazing tools in cancer research. based on nucleic acid hybridizations between target molecules and corresponding complementary probes, it allows genotyping at multiple loci or targets at once. in reality, majority of the published studies reporting mirna profiling used a variety of microarray technologies. its high utilization in mirna profiling studies is fairly selfexplanatory, as most of the experimental (and analysis) steps can be done at any molecular biology laboratory and it also allows customization including probes design, chemistry of probe immobilization, labeling of samples and detection of signal[49,50]. at some point, some researchers have also implicated its potential to be used as a standard tool in the near future[51]. with the availability of mirna databases (e.g. mirbase, mirwaydb, omcd), these efforts in turn speeds up the northern blotting even though close of half a decade has passed since the first introduction of northern blots, this technique is still the gold standard for mirna expression profiling. in spite of that, there are several technical considerations which have been raised up by researchers when using northern blot as a routine mirna expression profiling tool. as much as the technique stands as a high sensitivity assay, northern blotting requires huge running time and large amounts (5–25 mg) of total rna from each sample, on top of potentially dangerous radioactive probes[40]. as a result, researchers have come up with alternatives in probe design, specifically to allow the detection of mirna without compromising its sensitivity and accuracy. one of which was a technique designed based on the northernblot principle by kim and team, known as led which consists of three “key players” – digoxigenin-labelled oligonucleotide probes containing locked nucleic acids (lna) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (edc) for cross-linkage with membrane[42]. the authors claimed that led is able to generate clearly visible signals for rna amounts as low as 0.05 fmol and requires only few seconds of membrane exposure, equivalent to ∼1000-fold improvement in exposure-time (kim et al., 2010). as a non-radioactive label, dig assay is safer than radioactive methods, while offering the same/ similar sensitivity as isotope labeling-based methods[43]. along with this, locked nucleic acid (lna)-modified oligonucleotides greatly improve the sensitivity of northern blotting technique, as high as ten-fold compared to conventional dna probes[44,45]. lnas were described as a new class of bicyclic high-affinity rna analogues, containing a furanose ring in the sugar-phosphate backbone that is chemically locked in an n-type (c3’endo) conformation by the introduction of a 2’-o,4’-c methylene bridge[45]. furthermore, válóczi and team figure 1. examples of mirna profiling detection methods in cancer research. 4 development of commercially available mirna microarrays. in general, a ready-to-use mirna microarray contains mirna oligonucleotide probes that have a aminemodified 5’-end which are immobilized onto glass slides by covalent crosslinking[52]. the detection of mirna type is based on the binding location of fluorescent-dye labelled mirna on the slides, while its expression level can be quantitated based on the levels of fluorescence emission. along with this, lna can also be used in mirna microarray due to its exceptional affinity and specificity to the complementary rna. on top of the efficient visualization fostered by the extraordinary thermal stability between lnas and their target rna molecules, lna seems to improve the mismatch discrimination which then lead to multiple attempts to incorporate lnabased probes in mirna microarrays design for profiling purposes[53–55]. exiqon (now under qiagen) used to manufacture microarray platform with probes containing lna bases, providing higher annealing affinities[49]. as a result, several studies attempted to compare the performance of commercial mirna expression array platforms[49,56]. git and team included six commercially mirna expression array platforms which consist of both singleand dual-channel fluorescence technologies in their studies[49]. based on their studies, all six platforms tested were equally applicable to cell line and tissue, but factors such as input sample amount may need to be considered when selecting a platform to be used. on the other side, another study compared four platforms including agilent, illumina (platform withdrawn since 2010), exiqon and miltenyi[56]. it was noted that higher modulated mirnas were identified via agilent and illumina platforms for class comparison analysis between tumor and normal samples. even though exiqon did not produce the same number of modulated mirnas as the two platforms, most of mirnas modulated in agilent and illumina were detectable, although they did not reach statistical significance. on the contrary, the same mirnas were mostly undetected on the miltenyi platform which can be explained by its sensitivity to gc content. sah and colleagues found similar results with ambion and agilent which exhibite better accuracy while illumina and exiqon displayed higher specificity[51]. another company, invitrogen also developed a series of microarray for mirna profiling, known as ncode™ multi-species mirna microarray. tchernitsa and team used the invitrogen ncode(™) multi-species mirna microarray probe set containing 857 mammalian probes to study six primary gastric cancers (compared with normal/ non-cancerous tissue); out of which three of them presented lymph node metastases, while the other three did not[57]. comparing gastric carcinoma with non-cancerous tissue, twenty mirnas were differentially regulated and six of these mirnas showed distinct expression which separated node-positive from node-negative gastric cancers, including mir-103, mir-21, mir-145, mir-106b, mir-146a, and mir-148a. these results indeed emphasized the importance of microarray as high throughput method in mirna studies. despite of that, researchers have also pointed out that the periodic changes to mirna database like mirbase then imposes a reannotation of microarray and qpcr probes prior to analysis. nevertheless, this would also provide researchers the flexibility in selecting types of microarrays to be used, be it pre-designed microarrays or customized microarrays. pcr-based technique: reverse transcription quantitative pcr (rt-qpcr) even though there are several options which allow measurement of multiple target mirnas simultaneously, rt-qpcr remains one of the popular choice for mirna profiling as it’s an important technique for validating expression data obtained from high-throughput screening (e.g. with microarray).the first step of rt-qpcr begins from precise and absolute conversion of rna into complementary dna (cdna) via reverse transcription. however, given its nature of mirna which has a limited length (~22 nucleotides), reverse transcription can be difficult to perform. apart from that, mature mirna sequence can exist in two forms – preand the primirnas, along with little or no common sequence feature to be used for their enrichment and amplification[58]. before quantification, there are two approaches for reverse transcription of mirnas: (a) by using mirnas-specific reverse transcription primers or (b) using a universal primer that targets the mirnas that are tailed with a common sequence (e.g. poly-a tail). while the use of mirnaspecific primers (msps) reduces background “noise”, using a universal reverse transcription method is useful when there are several different mirnas needed to be studied from a small amount of input. additionally, there is another alternative which enables multiple mirnas to be reverse transcribed by pooling stem-loop primers[59,60]. step-loop primers consist of a short single stranded sequence at their 3’-ends that anneals to the 3’-end of the mirna of interest, a double-stranded segment (the stem) and a loop. due to its structure, the primer will not be able to bind to priand pre-mirnas and to any dsdna that may be present. rt-qpcr possesses numerous benefits compared to microarray as it presents higher speed and sensitivity as well as larger dynamic range[40,61]. furthermore, rtqpcr also requires low amounts of starting specimen which makes it more user-friendly. at the time of writing, there is currently over 1,900 human mirna curated in mirbase (http://www.mirbase.org/). as single target rtqpcr can be both time and reagent consuming, several strategies can be used to perform parallel reverse transcription when there is a need to detect a large number of mirnas in a single sample. a study by tang and colleagues incorporated 220 individual stem-loop primers in their study to develop a multiplex reverse transcription assay[62]. a pre-pcr process was included to reduce significant loss of detection sensitivity using low amounts of rt-qpcr primers (pre-pcr) and subsequently the cdna product was diluted before multi-well plates containing msps, universal primers and taqman probes for rt-qpcr. apart from that, there are also mirna arrays available for molecular profiling... 5 spearman r > 0.7) between results generated from this technique and their previous microarray experiment (after data normalization). in 2017, androvic and team introduced a novel and costeffective strategy to quantify mirna expression known as the two-tailed rt-qpcr[67]. as the primers are made up of two hemiprobes (which are complementary to different regions of target mirna) with a oligonucleotide tether folded into a hairpin structure, this design then subsequently increase the binding strength to template, increasing its sensitivity while detecting all terminal variants of any mirna (isomirs). in another words, this assay enables a “true” reflection or measurement of total mirna content (of interests) in a sample. having the advantage of allowing multiplex during the reverse transcription step, the mirna profiles generated by two-tailed rt-qpcr displayed excellent correlation with the standard taqman mirna assays (r2 = 0.985). following the success of developing the rapid technique for mirna expression with a total analysis time of less than 2.5 hours, the team led by androvic subsequently published another work last year, highlighting the use of this technique as a quality control test for circulating mirna studies[68]. as a matter of fact, even though rt-qpcr is described as a “gold standard” for typical gene expression studies, its application in mirna profiling may be limited by the constantly increase number of mirna on a genomic scale, thus suggesting its utilization as a validation test for other high-throughput techniques such as microarray and next generation sequencing (ngs) which might be a more affordable plan (depending on factors such specimen and mirna target numbers). next generation sequencing (ngs) in biomedical science, sanger sequencing has always been the principal approach and gold standard for dna sequencing[40]. two decades ago, a team from lynx therapeutics (usa) (which was later acquired by illumina) launched the first ngs technologies known as massively parallel signature sequencing (mpss)[72]. the technological feature of this method is that clonally amplified or single dna molecules in mpss are spatially separated in a flow cell, making it different from the sanger sequencing which works based on the electrophoretic separation of chain-termination products produced in individual sequencing reactions[40]. as a general rule, ngs technologies involve repeated cycles of nucleotide extensions mediated by polymerase or by iterative cycles of oligonucleotide ligation in one format[73]. ngs platforms available today are roche 454 gs flx sequencing, illumina/solexa genome analyzer sequencing, applied biosystems/solid as well as helicos biosciences and single-molecule, real-time (smrt) sequencing by pacific biosciences (pacbio). in truth, quite a number of known mirnas were discovered by conventional cloning and sanger sequencing approach[74]. however, the sequencing power of ngs opens up a new window for researchers to discover novel mirnas as it is not hindered by variability in melting temperatures, neither co-expression of almost identical mirna family members nor post-transcriptional modifications as in other molecular techniques like microarray and rt-qpcr. at earlier times, several groups have attempted to use different ngs the use of mirna profiling. zhang and team measured differential expression of 95 mirnas in pancreatic cancer tissues and cell lines by rt-qpcr using the quantimir system (sbi system biosciences)[63]. as a consequence, unique mirna profiles were seen in pancreatic cancer tissues and cell lines which reflected individual diversity, compared to adjacent normal pancreatic tissue or cells. after another validation using rt-qpcr, a total of eight mirna were upregulated in most of pancreatic cancer tissues and cell types. on the other hand, as a mediumthroughput method for rt-qpcr, taqman array microrna cards (by applied biosystems) combine microfluidics technique and classic/advance chemistries which can measure up to 384 mirnas. based on project’s needs and directions, user can also select to use either pre-designed (standard) or custom-made cards. mees and colleagues used taqman low density microrna arrays (tlda) to investigate mirna expression in ductal adenocarcinomas of the pancreas[64]. the main advantage is that this system allows as little as 1 ng of rna in circumstances whereby pre-amplification pcr step is performed[65]. for this approach, each tlda card contains pre-loaded primers and taqman probes to amplify a single mirna[64,65]. after cdna synthesis using predefined pools of reverse transcription primers, cdnas are loaded into the micro fluidic card where amplification of individual mirna occurs[64]. this method provides a simple and convenient way for parallel monitoring of a large number of mirnas by using rt-qpcr. conversely, there are several innovative methods for quantification of mirna such as lna-enhanced primers with droplet digital pcr (ddpcr) and the two-tailed rt-apcr method which uses primers composed of two hemiprobes (and connected by a hairpin structure)[66-69]. the inclusion of lna in the forward pcr primers keeps its sequence to be short but highly specific and detects most of the mirna sequences, while in the reverse primer binds to the 3′ end of the mirna sequences[66]. andreasen and team concluded that this unique combination confers specificity plus extreme sensitivity thus making it useful especially for quantification of mirnas in difficult samples like ffpe tissues. complementing to the strategy of using lna-enhanced primer, a relatively new technology that serves the same purpose as qpcr machines comes into the picture, known as ddpcr[69]. this technique eludes several problems with conventional qpcr such as the need for reference gene or replicate samples as the system detects fluorescence signals in the nanoliter-sized water-inoil droplets containing target molecule[70,71]. using ffpe specimens, laprovitera and team used a customized, prespotted 96-well plates to study the expression of 92 mirna with different mircury lna mirna primers (qiagen, former exiqon)[69]. the customized plate was designed to cover most cancer-specific mirnas (including 89 cancer-specific mirna and 3 reference genes). in their study, the team managed to detect mirnas expression in 14 ffpe specimens representing different type of tumors comprising of liver, skin, breast, gastric, colon, ovary, prostate, gastrointestinal-neuroendocrine and so on. even though there wasn’t a direct comparison between this technique and microarray in the same study, the team observed a highly significant correlation (p < 0.0001, ab mutalib n-s et al. 6 technologies to uncover novel mirna or even generate mirnaome. in 2010, ramsingh and team used 454-based sequencing to study mirnaome in a patient suffering from acute myeloid leukemia[75]. a total of 472 mirna (including 7 of them being novel) was identified from leukemic myeloblasts; some of them showed differential expression compared to normal (healthy) cd34+ cells. besides that, creighton and team took advantage of another platform — illumina/solexa genome analyzer sequencing to discover of novel mirnas in female reproductive tract[76]. in the same study, they have added nearly 100 putative novel mirna (with mid-high confidence) derived from various organs of the female reproductive system (in both diseased and normal states), representing diseases such as ovarian cancer, endometriosis, and uterine tumors (benign and malignant). comparing two different systems, the illumina platform (e.g. hiseq) which uses typically generates more reads at lower cost compared to roche 454 pyrosequencing system[53,77]. furthermore, one of the main problems with the latter system is that it has got relatively high error rate (for poly-bases longer than 6 bp), even though the system does offer an automated process for library construction. even so, applied biosystems purchased solid or sequencing by oligo ligation detection sequencing in 2006, which stands as a twobase sequencing technology based on ligation. schulte and colleagues have adopted solid ngs in their study analysing small rna transcriptomes of neuroblastoma cases. the team subsequently successfully revealed the differential expression of mirnas in favourable versus unfavourable neuroblastoma[78]. the third generation of ngs was firstly introduced in 2008 and defined as single molecule sequencing which was entirely different from the clonal based second-generation sequencing methods[79,80]. the short running time of smrt sequencing by pacbio makes it particularly attractive for diagnostic use. a research team in india performed isoseq analysis on infratentorial ependymoma tumor tissue using smrt technology, pacbio rsii[81]. a total of 2952 unique transcripts were identified to be involved in 307 kegg pathways, with 22 transcripts coding 18 genes related with mirna biosynthesis/processes (ko0520622). with the fast development in ngs technology, the fourth generation of ngs technology such as the minion, a commercially available device from oxford nanopore technologies (ont) has also begun to gain attention as a potential tool for mirna profiling work[82,83]. even though it comes at a lower pricing compared to the third generation ngs, it is rather unfortunate to mention that more work/ modification may need to be done to overcome the compatibility issue of the technology for short nucleic acid strands or mirna due to their short length[83,84]. as much as ngs offers as somewhat powerful tool to identify and detect mirna in various samples, some researchers pointed out that there are possibilities of bias resulting from rna ligation and amplification steps[49,85]. furthermore, the increasing amount of data generated from these ngs technologies require high computation power for analysis. also, for high-throughput methods like ngs, additional data validation step is still required using other techniques like qrt-pcr[86,87]. bead-based method apart from techniques mentioned above, some researchers developed another exciting method for the profiling of mirna expression which works based on “beads” that capture different types of mirna. luminex corporation developed the bead-based array known as xmap™ system, which is a multiplexed microsphere-based suspension array (http://www.luminexcorp.com/technology/ index.html). for this method, the oligonucleotide-capturing probes complementary to mirnas of interest are linked to carboxilated 5-micron polystirene beads impregnated with mixture of two fluorescent dyes, each coded for a single mirna[88]. in 2005, lu and colleagues were able to differentiate tumors that were inaccurately classified by mrna profiles[89], while another study in united kingdom discovered new markers of human breast cancer subtype[90]. the bead-based mirna arrays offers several benefits compared to glass-slide microarrays: (a) user-friendly and easy to use, (b) relatively low cost with advanced statistical performance, (c) faster hybridization kinetics and (d) higher flexibility in preparation of the array[40]. this technique remains as a popular tool in cancer research studying mirna expression at present day. wang and team studied the expression of mirna in nsclc tissues using luminex xmap bead-based suspension array and highlighted that the system requires as little as 2 μl of sample volume without the need of running reverse transcription or amplification step[91]. though, this method demands for specialized equipment, thus limiting its usage. others: cloning based assay, mirage and rna primed–array-based klenow enzyme assay (rake) before witnessing the major breakthrough in developing ngs techniques, researchers have heavily relied on traditional methods like amplification and cloning methods to increase the efficiency of small rna species discovery including mirna. mirna serial analysis of gene expression or mirage was developed by cummins and colleagues that merged the aspects of direct mirna cloning and sage[92,93]. while allowing identification of new mirna, mirage is similar with conventional cloning approaches: it starts with the isolation of 18-26 base rna molecules and then ligation with specialized linkers and adapters before reverse-transcription step into cdna[93,94]. the cdna then will be subjected to another pcr reaction with the help of biotinylated primers before purification step with column of streptavidin-coated beads (to remove biotin tagged linkers). the eluded product will be purified mirnas and these mirnas are then concatenated, cloned, and sequenced for analysis. the main advantage of this approach is that it is able to generate large concatemers, enabling as many as 35 tags to be identified in a single sequencing reaction, whereas existing cloning protocols analyse approximately five mirnas per reaction[92]. molecular profiling... 7 aside from that, there is another method available for mirna detection known as rnaprimed, array-based klenow enzyme (rake) assay developed by nelson and colleagues in 2004[95]. as it doesn’t require any sample rna manipulation prior to hybridization, the unmodified mirna is hybridized to immobilized dna probes. biotinylated datp is incorporated to dna probe with hybridized mirnas acting as primers by klenow enzyme and subsequently a streptavidin-conjugated fluorophore is applied to visualize and analyze the expression of mirnas. rake offers a high throughput protocol with unique advantages for specificity over northern blots or other microarray-based expression profiling platforms[96]. moreover, the team demonstrated that mirnas can be isolated and profiled from ffpe tissue, which provides another alternative for analyses of small rnas from archival human tissue. on top of that, berezikov and colleagues proved that novel mammalian mirna candidates can be identified by extensive cloning and rake analysis[97]. conclusion and future recommendations the continuous expansion of mirna knowledge has deemed the importance of these small non-coding nucleic acid in the area of cancer research, particularly as biomarkers in diagnostic and therapeutic targets in drug discovery field[98]. even though newer methods are constantly being developed/introduced for mirna profiling use, there is still much room for improvement, given that these tools are still unable to replace the conventional method that “sits on the throne”. one of the bottlenecks would be experimental confirmation of mirna targets behind these phenotypes computed via data generated from high-throughput technology. upon obtaining a comprehensive understanding on their roles, selected mirna could be used in rna interference strategy to achieve therapeutic effects against different types of human cancers[99]. in fact, a quick search on clinical trials registry website (e.g. clinicaltrial.gov) revealed that there is a total of 327 studies related to mirna, out of which 98 studies were interventional studies (as of 8th june 2020). however, no completed results reported results related to the use of mirna as therapeutic agent(s) on the database yet. just like any other drugs, it is impossible to observe success with every mirna tested, but the “magic bullet” would pledge a promising future in the war against cancer. authors contribution the literature review and manuscript writing were performed by nsam and h-ls. nsam and ii provided vital guidance of the research and proof of the writing. conflict of interest the authors declare that there is no conflict of interest in this work. reference 1. ohno s. so much ‘junk’ dna in our genome. in evolution of genetic systems, brookhaven symp. biol 1972; 366–370. 2. collins fs and mckusick va. implications of the 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(jw-fl) received: 15 march 2023; received in revised form: 10 may 2023; accepted: 26 may 2023; available online: 02 june 2023 abstract: covid-19 bulletins from various countries have been published as the pandemic hit. this focused review provides an overview of the published covid-19 cases, including the first case detected in respective countries, preventive measures implemented, and vaccinations available for some countries, depending on the time it was written. the countries that will be covered in this review are the united kingdom, spain, malaysia, singapore, thailand, india, australia, south africa, and brunei. this review also discusses the variants of concern (vocs) that emerged since the start of the pandemic, which include variants: alpha (b.1.1.7), beta (b.1.351), delta (b.1.617.2), gamma (p.1), and omicron (b.1.1.529). covid-19-associated gastrointestinal manifestations and mental health implications and the application of probiotics as a potential adjunct therapy to psychotropic medications will be addressed in this review. additional information on covid-19 will also be reviewed, such as risk factors affecting covid-19 case fatality rate, positive impacts of public health measures, the cost-effectiveness of public health strategies on covid-19 control, and the covid-19 pandemic and diet. keywords: covid-19; sars-cov-2; variants of concern (vocs); vaccines; probiotics 1. introduction covid-19 is a global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this is the third zoonotic human coronavirus that emerged within the past century following the severe acute respiratory syndrome coronavirus (sarscov) and the middle east respiratory syndrome coronavirus (mers-cov)[1–3]. covid-19 started in early december 2019 when the news reported a pneumonia outbreak by an unknown aetiology in hubei province of china. this incident consequently led to closing of pmmb 2023, 6, 1; a0000333 2 of 22 a seafood market on 1st january 2020, and countries with travel links to wuhan were on high alert for travellers with respiratory diseases potentially linked to the pneumonia outbreak[4,5]. on the 30th january 2020, sars-cov-2 was declared a public health emergency by the world health organization (who)[6]. the sequenced sars-cov-2 genome was postulated to be of bat origin[7], but the intermediate host is uncertain[6], and the transmission of covid19 had progressed to human-to-human contact. sars-cov-2 mainly affects the respiratory system, with the most common clinical symptoms of covid-19 infection, including fever, cough, dyspnea, fatigue/myalgia, lymphopenia, and pneumonia accompanied by abnormal findings on chest computed tomography (ct) scans were reported in the initial cases from wuhan, china. other less common symptoms were sputum production, headache, haemoptysis, and diarrhoea[8]. by early april 2020, sars-cov-2 had spread worldwide, spawning several mutations in which spike protein amino acid d614g mutation was the dominant form of sars-cov-2 variant[9]. simultaneous mutations on the spike proteins consequently led to the emergence of variants of concern (vocs) which are more transmissible and have immune evasion potentials[10]. although there was a rapid development of vaccines against covid19, their efficacy could be affected by the vocs[10]. despite covid-19 vaccines being made available and the national vaccination program has been implemented worldwide, the issue on long covid-19 emerged as the health of many individuals were affected by post-covid19 infections [1, 3]. as of february 2023, there had been a total of 671,852,527 cases and 6,845,598 deaths recorded worldwide[11]. this focused review will consolidate and integrate all pertinent information obtained from covid-19 related articles published by pmmb, which include covid-19 bulletins, genomic analysis of sars-cov-2 strains isolated in malaysia, the variants of concern (vocs) of covid-19, the available covid-19 vaccines, covid-19 gastrointestinal manifestations and complications, and long-covid-19. 2. the onset of the covid-19 pandemic in various countries 2.1. united kingdom (uk) the outbreak started at the end of january 2020, and by the middle of march 2020, europe became one of the countries with the highest number of reported cases and deaths globally, following china. compared to other european countries excluding france, italy, and spain, the united kingdom (uk) was hit hard in february due to the lack of isolation, quarantine and control tracing, resulting in surging covid-19 cases as well as critically ill patients, consequently overwhelming the healthcare system. the first confirmed case was on 31st january 2020, involving two members of a family of chinese nationals staying in a hotel in york[12]. these cases were identified without any clear case definition and were purely due to information about the spread of covid-19 and clinical suspicion[13]. in terms of the actions in preparations for and response to covid-19, the uk government faced heavy criticism. although the uk government launched the “contain-delay-mitigate-research” on the 3rd march 2020, it had failed due to the lack of testing in the uk, which led the pmmb 2023, 6, 1; a0000333 3 of 22 government to shift to “suppress-shield-treat-palliate”[12,14]. on 23rd march 2020, the uk was officially placed under lockdown[15]. 2.2. spain spain was once hit by the 1918 influenza pandemic, known as “spanish flu”, caused by the h1n1 coronavirus[16–18]. in 2020, along with the rest of the world, spain was again hit by a pandemic ⸺ covid-19 and had the highest record of confirmed covid-19 cases in europe. the first covid-19 case in spain was reported on the 31st january 2020, which involved a german tourist in the canary islands who had contact with people who had travelled to china. although the spanish government took many counter-measures to reduce the spread of covid-19, including closure of schools, flight controls and gatherings (>1000 people) at closed venues at hardest-hit hit areas, but by the second week of march, spain has ranked second badly affected country among other european countries[18,19]. with that, a 15day national emergency was declared by the government using a royal decree (463/2020) starting 15th march[20]. it was extended to midnight of 12th april as cases continued to increase[21], and subsequent extension to stop the spread of the pandemic. additionally, the spanish government also incorporated artificial intelligence as an official channel to provide advice and enquiries about covid-19[22]. furthermore, they also set up research grants to expedite drug and vaccine development[18,23]. 2.3. malaysia malaysia is one of the countries with the highest number of recorded confirmed cases and deaths in southeast asia by early may 2020[24]. the first covid-19-positive case in malaysia was reported on the 25th january 2020, and it was an imported case from wuhan, china[25]. the first covid-19-positive case involving a malaysian was reported on the 3rd february 2020. this individual had just returned from a business meeting in singapore, which was also attended by a delegation from china[26]. two days later, his younger sister tested positive for covid-19, making it the first local sars-cov-2 transmission case in malaysia[27]. in march 2020, the covid-19 cases surged as a result of a mass gathering linked to a religious event held in sri petaling, kuala lumpur, which involved approximately 16,000 participants with roughly 1,500 foreign participants from other southeast asian countries[27,28]. subsequently, the government implemented the closure of international borders and started taking strict preventive measures[27]. the initiative taken by the federal government to combat the spread of covid-19 was the implementation of a movement control order (mco) for 2 weeks, effective from 18th march 2020. it gradually extended to a total of 8 weeks until 12th may 2020, encompassing a total of 4 phases. rules were strictest during phase 2 and phase 3 of mco; for instance, travel distance was limited to within a 10 km radius from home, business hours and delivery times were limited to 8 a.m.–8 p.m., and only one representative per household is allowed at a time to purchase essentials. following mco, midway through phase 4, a conditional mco (cmco) was introduced with eased restrictions followed through during phase 5 from 4th pmmb 2023, 6, 1; a0000333 4 of 22 may 2020 up until 9th june 2020. subsequently, a recovery mco (rmco) was introduced until 31st august 2020, restrictions were eased, and economic sectors resumed gradually in stages. nonetheless, strict social distancing and self-hygiene are to be practised[29]. notably, a standard operating procedure with the use of a mobile app known as “mysejahtera” introduced by the ministry of health (moh) was implemented to monitor covid-19 outbreaks. additionally, wearing a face mask in public domains was made mandatory starting 1st august 2020[29]. covid-19 has affected the psychosocial aspects of malaysians, especially during mco phases, subsequently imposing a new normality[29]. meanwhile, ser et al.[24] analysed the phylogenetic evolution of sars-cov-2 strains from malaysia to compare with worldwide sars-cov-2 genomes. based on seven publicly available whole genome sequences of sars-cov-2 isolated from patients in malaysia, they performed the single nucleotide variants (snv) analysis to investigate the genotype changes during sars-cov-2 transmission in malaysia[24]. the study concluded that the sars-cov2 strains from malaysia were highly similar to other strains derived from asia, and more than one subtype was detected. 2.3.1. vaccination in malaysia (up until december 2021) malaysia started ordering vaccines developed by sinovac (coronavac), pfizer (comirnaty), and astrazeneca (vaxzevria) since november 2020. in 2021 additional orders were placed to speed up malaysia’s vaccination process so an 80% immunity can be achieved by february 2022 for its 32 million population. the first batch of sinovac, pfizer, and astrazeneca vaccines arrived on the 27th february 2021, 21st february 2021, and 23rd april 2021 respectively, and more vaccine shipments arrived in the months following the first batch. the covid-19 vaccines were distributed in three phases via the covid-19 immunization program. the first phase was completed in april 2021 and prioritised front liners. on the other hand, the second phase was scheduled to complete in august 2021. it prioritised individuals in high-risk groups, including those with chronic disease and senior citizens. lastly, phase 3 prioritised individuals >18 years old[27]. later, the government approved the use and launched janssen, cansino, sinopharm, and moderna for the vaccination program. in december 2021, malaysia has achieved over 60% vaccinated individuals and aims to have nearly 80% of the population fully vaccinated by 4th august 2022[30]. 2.4. singapore with a population of 5.7 million, singapore has often been used as an exemplary for other countries in handling covid-19[31,32]. its first covid-19 case was an imported case that was reported on the 23rd january 2020. not too long after that, in early february local transmission was reported resulting in nationwide alert. consequently, the national assessment level, known as the disease outbreak response system condition (dorscon) turned from “yellow” to “orange”[33]. besides that, tracetogether, a mobile phone-based tracing application developed by the government technology of singapore (govtech) was pmmb 2023, 6, 1; a0000333 5 of 22 launched in march 2020 to track interactions between individuals diagnosed with sarscov-2 with their close contacts[33,34]. however, a surge in cases in march 2020 led to the implementation of a “circuit breaker” with a stringent set of lockdown measures from 7th april 2020 to 1st june 2020[33]. additionally, a national cloud-based registration system called safeentry was introduced by the government for contact tracing purposes[33,34]. furthermore, in may 2021, locally developed breathalysers were provisionally approved as a screening method to detect covid-19[33]. in terms of the reopening of singapore, after 1st june 2020 was phase 1 of reopening, followed by phase 2 on 17th june 2020 and later phase 3 on the 28th december 2020. the different phases of reopening were followed with rules that exhibited a slightly reduced level of strictness compared to the previous guidelines[33,35]. the governance, transparency, good cooperation between the public and the local government, the enforcement of social responsibility, utilisation of technological advancement, and high vaccination rates have all contributed to the proper control of covid-19 in singapore[33]. 2.4.1. vaccination in singapore (up until november 2021) in march 2021, singapore’s mass vaccination began with an estimated 800,000 individuals already receiving at least one dose of the covid-19 vaccine and the program was set to roll out for younger age group[33,36]. at that time, the covid-19 vaccines authorised for use by the health sciences authority in singapore under the pandemic special access route (psar) were vaccines developed by pfizer (comirnaty) and moderna[33]. in order to achieve herd immunity, on 18th may 2021, covid-19 vaccine by pfizer (comirnaty) was then authorised for individuals 12 to 15 years of age[33,37]. 2.5. thailand the first covid-19 case reported in thailand was on the 13th january 2020. prior to mid-march 2020, thailand’s covid-19 cases remained relatively low. still, it surged due to a boxing event on the 6th march 2020 as the people did not comply with the government’s ban on large gatherings implemented just a few days earlier. nonetheless, the government was able to keep covid-19 under control for most of the year 2020[38]. given that the government took measures to control the spread of covid-19, including comprehensive infection prevention and control measures, quarantine, travel restrictions, site closures, entry point screening, and contact tracking, nevertheless, the second wave still hit in middecember 2020[38]. the second wave of covid-19 hit thailand in december 2020 as covid-19 cases surged up to 7,284 daily new cases due to the incident in a wholesale shrimp market[38,39]. later, the third wave in thailand started in april 2021. the superspreading incidents were identified at entertainment establishments, karaoke lounges, bars, pubs, and gambling venues in different regions. the surge in cases during the third wave was greater than the first and second waves combined[38,39]. this could be a result of eased restrictions to prevent further economic downturns. furthermore, the government also permitted interprovincial travel during the thai new year holiday in april 2021[38]. pmmb 2023, 6, 1; a0000333 6 of 22 thailand aimed to achieve 70% herd immunity by the end of 2021, and in july 2021, it opened its borders for phuket’s tourism industry. then starting 1st november 2021, it opened its borders to fully-vaccinated individuals. however, on the 6th december 2021, thailand’s ministry of public health reported its first omicron case, which involved a citizen from the united states (visiting from spain). despite the emergence of the new omicron variant in thailand, its plan for border reopening continues[38]. 2.6. india india was one of the countries badly affected by covid-19[40,41]. the first confirmed covid-19 case was on the 30th january 2020, involving a patient returning to kerala from wuhan[41,42]. on the 22nd march 2020, the prime minister of india announced “janata curfew” for 14 hours. on the other hand, the ministry of health and family welfare took precautionary measures to ensure sufficient workers, personal protective equipment, isolation wards, medicines, and test kits. however, with the continuous surge of thousands of covid19 cases a day, a nationwide lockdown was implemented starting 25th march 2020, which was extended thrice until 31st may 2020[41,43-45]. among some of the measures taken by the indian government were improvements in the healthcare system and infrastructures, an increase in rapid antigen test, an increase in labs and the launch of a mobile application called “aarogya setu” to aid in contact tracing and surveillance as well as a video consultation program called e-icu. later, on the 8th october 2020, “jan andolan” was launched to observe behaviours, including wearing of face mask, hand washing and 6-feet social distancing. despite the decrease in daily new cases and deaths in november 2020, a sudden surge in cases occurred in february 2021, leading to the lockdown in new delhi. on the 1st may 2021, india became the first country to record >400,000 new covid-19 cases within 24 hours since the pandemic[41]. 2.6.1. vaccination in india (up until june 2021) the national vaccination program in india begins on the 16th january 2021, and intends to vaccinate 300 million individuals by july 2021. the national covid-19 strategy was divided into 3 phases. phase i targets healthcare and frontline workers, while phase ii’s registration and booking started on the 1st march 2021 and was meant for individuals ≥60 years old and individuals 45–69 years old with comorbidities, lastly, phase iii’s vaccination which begun after 1st may 2021 for individuals ≥18 years old[41,46,47]. at that time, the covid-19 vaccines granted emergency use authorization were covishield® (astrazeneca's vaccine), manufactured by serum institute of india (sii) and covaxin, manufactured by icmr in collaboration with bharat biotech international limited (bbil). later in mid-april, russia’s sputnik v was approved as the third covid-19 vaccine in india[41,46]. as of 1st may 2021, india had administered a total of 156,816,031 doses of covid-19 vaccines[48]. pmmb 2023, 6, 1; a0000333 7 of 22 2.7. australia the first covid-19 positive case in australia was reported in melbourne at the end of january 2020[49]. the first 12 covid-19 positive cases in australia were all acquired from china. the first local case was reported in queensland, whereby the patient had contact with a chinese tourist in the first week of february 2020[50]. covid-19 cases surged to over 4,000 by the end of march 2020[51]. then in late april 2020, australia launched its contact tracing app, covidsafe, which is part of the three key requirements (test, trace, responds) for easing restrictions[52]. in july 2020, due to a surge in cases in victoria resulted in a lockdown implemented in victoria, mitchell shire, and metropolitan melbourne. despite implementing mitigation measures by the australian government, cases continued to increase in august 2020. therefore, a nightly curfew was implanted together with compulsory face coverings in public, and both businesses and schools were told to close. in october 2020, as covid-19 cases started to decrease, restrictions were eased, and finally, the 112-day lockdown in victoria ended, and various sectors were allowed to reopen starting 28th october 2020 [51]. however, the lockdown was again implemented on the 19th november 2020 in response to cluster cases in south australia. it was revoked on the 22nd november 2020, as the outbreak was successfully controlled, leading to eased restrictions[51,53]. the covid-19 cases in australia showed a decreasing trend continuing into the year 2021, nevertheless, on the 30th november 2021 as announced by the local government, a total of six omicron variants of the covid-19 cases were reported in australia. the government began implementing travel restrictions, home isolation requirements, and revised quarantine[51]. 2.7.1. vaccination in australia (up until december 2021) vaccinations started in late february 2021and priority was given to frontline healthcare workers, quarantined and border workers, aged and disability care residents, and staff with significantly higher risk of covid-19. after that, the vaccines were distributed to the mass public. vaccines approved for use in australia are comirnaty (pfizer), vaxzevria (astrazeneca), and spikevax (moderna). by june 2021, >6.5 million vaccine doses had been administered in australia, but in july 2021, covid-19 cases started increasing. there are four phases in the national plan to transition australia’s covid-19 response. on 1st july 2021, australia entered phase one, known as “vaccinate, prepare, and pilot” which aimed to vaccinate as many individuals as possible and implement early, stringent, and short lockdowns in outbreak-occurring areas. on 20th october 2021, the phase two vaccination transition phase began. on 3rd december, 87.9% of the population was fully vaccinated. hence, it has achieved the target (80%) of fully vaccinated individuals, which was part of phase three, the vaccination consolidation phase. lastly, phase four, the post-vaccination phase was at that time put on hold due to the emergence of the omicron variant. additionally, the only approved and preferred covid-19 booster dose in australia at that time was comirnaty[51]. pmmb 2023, 6, 1; a0000333 8 of 22 2.8. south africa the south african government had prepared to face sars-cov-2 even before the country's first covid-19 case was detected by having planned an overlapping eight-stage program[54]. the first stage of the national covid-19 response program focuses on covid19 preparation which includes surveillance, education on sars-cov-2 and methods to prevent the spread[55]. the first covid-19 case in south africa involved a citizen who returned from italy on 1st march 2020 and was diagnosed on 5th march 2020[54,56]. ten days after the first covid-19 case was diagnosed, stage two of the program begins, and its focus is on covid-19’s primary prevention. the government announced a national state of disaster, promoting social distancing and hand hygiene, prohibiting international travel, closing schools, and restricting gatherings[54,55]. nonetheless, covid-19 cases continue surging. the first local transmission case documented in south africa was on the 18th march 2020; by the end of march, covid-19 had spread to all nine provinces in south africa[55,57]. then stage three of the national covid-19 response program was a strict level five (l5) national lockdown that took place to control the spread of covid-19[55,58]. in april 2020, stage four was commenced, and it involved active case-finding through contact tracing. however, in may 2020, the lockdown measures were eased to level four then in june 2020, it was further eased to level three[55]. later in july 2020, the covid-19 cases reached the peak and stage five to eight of the program commenced as hotspots were rapidly identified and medical care were provided promptly. in august 2020, the curve flattened, allowing the lockdown measures to drop to level two, but the national state of disaster was extended for another month [55]. additionally, in the same month, the covid alert sa app was launched to trace covid-19 positive individuals[55,59]. as covid-19 in south africa stabilised, the restrictions were further eased to level one[55,60,61]. the second wave of covid-19 occurred at the end of the year 2020, which caused a surge in cases, and subsequently, the hotspots for sars-cov-2 were put under stricter restrictions[54,55]. furthermore, the voc-beta variant (b.1.351) was detected in south africa, which resulted in >1 million covid-19 cases recorded on the 27th december 2020. hence, a partial level three lockdown was implemented for two weeks[55,62]. then in may 2021, the detection of vocalpha (b.1.1.7) and delta (b.1.617.2) in south africa led to a surge in covid-19 cases, resulting in the third wave of covid-19 and consequently, heightened lockdown restrictions at level two starting 31st may 2021[55]. however, in june 2021, the lockdown restrictions went up to level four, and as the cases decreases, it went back down to level one in october 2021. although in december 2021, the voc-omicron (b.1.1529) resulted in the fourth wave in south africa, it subsided quickly by the end of december 2021[55]. 2.8.1. vaccinations in south africa (up until february 2022) the first vaccines delivered to south africa were pfizer (comirnaty), and by the end of may 2021, 960,000 individuals had received one dose of the vaccine[63]. up until february pmmb 2023, 6, 1; a0000333 9 of 22 2022, only two vaccines were used in their national vaccination program, namely pfizer (comirnaty) and janssen (johnson & johnson)[55]. 2.9. brunei on the 9th march 2020, brunei reported its first covid-19 case involving a citizen returning from a tablighi jama’at in kuala lumpur, malaysia, on 3rd march 2020. by the first week of april, 71 covid-19 cases were reported and acquired in malaysia[64,65]. hence, the start of the first wave of covid-19 in brunei. on the 16th may 2020, the government implemented a four-stage de-escalation plan to control the outbreak. the guidelines laid down four operational readiness levels with specific restrictions: level 0very high restriction (closure), level 1high restriction, level 2-(medium restriction), level 3(low restriction), level 4normal operation (pre-normal)[64,66,67]. for most of 2020, the covid-19 outbreak was well-controlled in brunei until the arrival of the delta variant[64,68,69]. the second wave of covid-19 in brunei started on the 7th august 2021, involving a local transmission of the covid-19 delta variant. as the delta variant is a voc with a high transmission rate, it escalated covid-19 cases, thereby overwhelming brunei’s healthcare system[70]. with the increasing covid-19 cases and vaccine shortage, starting 9th august 2021, brunei went into a two-month partial lockdown[64,71]. restrictions imposed include “operasi pulih”, closure of several non-essential businesses and public facilities, work from home policies, restrictions on gathering, suspension of on-site activities for educational institutions, and dine-in were prohibited[64]. these measures successfully flattened the curve, but they had mental health impact on the people[72,73]. thus, there is a need for a plan to ensure a safe transition and stable situation with minimal disruption to the daily activities of the community. on 25th october 2021, the government announced a national covid-19 recovery plan framework consisting of the containment, preparation, transition, and endemic phases. the commencement of each stage depends on the vaccination coverage status and critical cases. the curve was successfully flattened in november, and on december 2021, the early endemic phase began[64,71]. however, in late december 2021, the emergence of omicron variant in brunei marked the third wave of covid-19, which peaked in march 2022 [64,74]. on 1st june 2022, brunei entered its endemic phase. as of february 2023, brunei had entered the “new normal” phase of its de-escalation plan[64,75]. brunei effectively controlled the covid-19 pandemic with the help of the whole nation. like other countries, brunei’s ministry of health launched a mobile app in may 2020 called “bruhealth” focused on contact tracing[76]. then another app called “premisescan” was launched to further ensure better accessibility of “bruhealth” by recording customers’ entry and exit of business premises[64,77]. 2.9.1. vaccination in brunei (up until february 2023) brunei’s national vaccination program commenced on the 3rd april 2021, in three phases. the first phase was targeted towards frontliners, senior citizens, and students studying abroad; the second phase targeted adults with comorbidities, child care centre staffs, and pmmb 2023, 6, 1; a0000333 10 of 22 teachers; and lastly, the third phase targeted individuals ≥18 years old, which began 5th july 2021. to achieve herd immunity, five covid-19 vaccines have been authorised by the brunei darussalam medicines control authority (bdmca) under emergency use authorisation, namely pfizer (comirnaty), vaxzevria (astrazeneca), spike vax (moderna), covilo (sinopharm), and janssen (johnson and johnson)[64,78]. on the 19th october 2021, vaccination for children aged 12 to 17 was set to roll out[79]. then on 3rd april 2022, kids 5–11 years old were administered the first dose of the pfizer vaccine[80]. around november 2021, the first booster doses were administered to frontliners, followed by senior citizens. for those who are 12–17 years old, they can receive their booster dose starting april 2022. later in june 2022, the second booster was offered to healthcare workers and frontliners with high-risk exposure, elderly ≥80 years old, adults ≥60 years old with chronic disease, and immunocompromised individuals ≥18 years old. lastly, the fourth dose is voluntary and given only to eligible individuals. the government encourages people to receive a booster dose even though it is not mandatory[81–83]. recently, in late november 2022, the bdmca authorised and rolled out a bivalent vaccine from moderna, and soon another bivalent vaccine from pfizer will be expected to be available in early 2023[84,85] . as of 11th november 2022, 78.9% of brunei’s population had received a third covid-19 vaccine dose[64]. 3. variants of concern (vocs) variants of concern (vocs) are variants with evolutionary advantages which are favoured[10,86,87]. since the start of the covid-19 pandemic, there have been a total of five vocs since the covid-19 pandemic started which are variants alpha (b.1.1.7), beta (b.1.351), gamma (p.1), delta (b.1.617.2), and omicron (b.1.1.529). however, it is to note that as of writing, the only voc is the omicron variant. these vocs have similarities in terms of the presence of multiple mutations on their spike protein, contributing to their increased transmissibility and immune evasion potentials. therefore, this leads to increased virulence. this review will briefly discuss the vocs in the following sections[10,30,88,89]. 3.1. alpha (b.1.1.7) the alpha variant was first detected in england on the 20th september 2020, and by february 2021, it had accounted for almost 95 % of england’s covid-19 transmission[90]. the alpha variant has higher transmissibility (43–82% more transmissible), higher viral load, longer duration of infection, higher hospitalisation rate, and higher mortality rate[10, 90–95]. in terms of vaccine efficacy, the impact from the susceptibility of neutralising antibody is minimal, and reinfection was found to be not higher than other earlier strains[10, 96–98]. there are nine mutations on the spike protein (two deletions and seven amino acid substitutions): δ69–70hv and δ144y deletions; n501y, d614g, a570d, p681h, t716i, s982a, and d1118h. additionally, e484k, s494p, and k1191n may be present in some sequences. nonetheless, mutations n501y, p681h, a570d, d614g, s982a, δ144y, and δ69–70hv deletions have been associated with this variant’s increase in binding affinity, cell entry, pmmb 2023, 6, 1; a0000333 11 of 22 infectivity, and neutralisation. details about the mutation affecting the different characteristics of the alpha variant have been discussed by thye et al.[10]. 3.2. beta (b.1.351) the emergence of the beta variant is likely from south africa’s first covid-19 wave that occurred in nelson mandela bay metropolitan area in the eastern cape province in october 2020. b.1.351 has higher transmissibility, viral load, reinfection, vaccine escape, hospitalisation rate, and mortality than the progenitor strain[10,94,99–102]. the beta variant seemed to have a lower vaccine efficacy rate and a higher risk of reinfection[10,101,103–106]. the beta variant has three amino acid deletions in the spike protein and eighteen amino acid mutations (seven in the spike protein). compared to the progenitor strain, the beta variant has ten changes: 3 receptor binding domain (rbd) mutations at e484k, k417n, n501y; replacements of a701v, d215g, d80a; d614g spike mutation; amino acid l242–244 deletions and r246i and l18f[10,99,100]. in this variant, mutations that are of great concern are at rbd: e484k, k417n, n501y and have been associated with increased binding affinity, cell entry, infectivity, resistance to neutralisation, and immune evasion ability that have been discussed in detail by thye et al.[10, 99]. 3.3. delta (b.1.617.2) delta variant was first detected in india in december 2020. based on the articles by thye et al., at that time of writing, this variant was relatively new, and information was limited[10,107]. nonetheless, this variant appeared to have higher transmissibility (60% more than the alpha variant), higher risk of hospitalisation and potential to escape the vaccine[10,107,108]. the delta variant has been reported to possess rbd mutations: d614g, l452r, t19r, t478k, p681r, r158g, g142d, a222v substitution along with 156–157 deletion in the ntd, s2 substitution in d950n[10,107,109]. however, the mutation g142d is found only in some delta strains[10]. mutations of particular interest, including l452r and p681r, have been further discussed by thye et al. by associating them with cell infectivity, binding affinity, immune evasion, and resistance to neutralisation[10,107]. 3.4. gamma (p.1) the gamma variant was first detected on the 6th december 2020 in manaus amazonas state, northern brazil. its emergence marks the start of manaus’s second covid-19 wave, and in january 2021, it was associated with 87% of all infections[10,110]. this variant consisted of mutations k417t, e484k, n501y in the rbd; l18f, t20n, p26s, d138y, r190s in the ntd; d614g and h655y at c terminus in s1; and v1176f and t1027i in s2. this variant potentially has higher transmissibility and result in increased hospitalisation. additionally, this variant seems less resistant than the beta variant to vaccine-induced or naturally-acquired antibody responses [10, 111]. more information on the associations of key mutations n501y, e484k, and k417t with this variant’s characteristics have been discussed by thye et al.[10]. pmmb 2023, 6, 1; a0000333 12 of 22 3.5. omicron (b.1.1.529) omicron is the latest addition and the only mutant of sars-cov-2 that remained a voc. this variant was detected in botswana on the 11th november 2021, but it was reported to the who in south africa on the 24th november 2021. later, b.1.1.529 was designated as a voc named omicron by the who on the 26th november 2021[88,112]. there have been three hypotheses on the origin of omicron: it could be circulated and evolved from hidden populations, evolved from immunocompromised patients or originated from animal reservoirs[30,88,113]. compared to previous vocs, omicron had the most mutations, with over fifty mutations [114]. the mutations include a67v, δ69–70, t95i, g142d/δ143–145, δ211/l212i, ins214epe, g339d, s371l, s373p, s375f, k417n, n440k, g446s, s477n, t478k, e484a, q493r, g496s, q498r, n501y, y505h, t547k, d614g, h655y, n679k, p681h, n764k, d796y, n856k, q954h, n969 k, l981f[115]. importantly, fifteen out of the thirty over mutations occur on the rbd, which has significant implications as the rbd is the key binding site for sars-cov-2 entry and a key target for neutralising antibodies[116]. additionally, thye et al.[88] showed that compared to previous vocs, the omicron variant seemed to have lower severity, lower hospital admission but higher transmissibility and infectivity, and have immune evasion potential. 4. vaccines and their concerns after the emergence of covid-19, vaccines were developed rapidly. the different types of vaccines include inactivated viral vaccines, mrna vaccines, recombinant viral vector vaccines, and protein subunit vaccines. as of january 2021, a few vaccines have been authorised for emergency use and have been administered to countries worldwide. review by loo et al.[117] (published march 2021) provided an overview into the vaccines approved for emergency use, vaccine candidates in phase iii trial (as of 30th january 2021), the different types of vaccines and their respective advantages and disadvantages, and strategies used to develop these vaccines. nonetheless, there have been concerns regarding covid-19 vaccines[118,119]. pregnant women are among the most vulnerable populations at risk of being infected with severe covid-19 and consequently experiencing poor pregnancy and neonatal outcomes. vaccination appeared to be the safest way of stopping the pandemic, and there is reassuring evidence supporting vaccination in pregnant women, but data is limited. hence, kwan et al. [118] reviewed the latest evidence on covid-19 vaccine’s safety, immunogenicity, and reactogenicity of these vaccines in pregnant women, the recommendations and guidelines provided by the public health authorities in southeast asia countries. they found that most countries in southeast asia generally recommend vaccination in pregnant women [118]. on the other hand, a scoping review by vairavan et al.[119] addresses the concern of autoimmune patients by exploring the safety, outcomes, and effects of covid-19 vaccines in autoimmune patients, mainly focusing on autoimmune diseases: myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. in conclusion, most autoimmune patients presented with good antibody response to vaccination, particularly after the second dose. pmmb 2023, 6, 1; a0000333 13 of 22 furthermore, the overall risk of flares and development of severe side effects was low after immunisation[119]. the development of booster vaccines was mainly due to the waning protection of covid-19 vaccines, the reduced protection against covid-19’s vocs and the inadequate protection of the primary vaccination for some risk groups. thus, the aim of booster vaccines is to improve and prolong the protection against covid-19. the review by thye et al.[120] discussed the various booster vaccines being authorised and administered in different countries, the eligibility criteria for the different booster vaccines, and the extent of protection it provides in several countries: uk, united states (us), israel, singapore, and chile. 5. covid-19 manifestations although the common symptoms upon contracting covid-19 are respiratory symptoms and fever, it can also involve the gastrointestinal tract, presenting symptoms such as diarrhoea, abdominal pain, nausea and/or vomiting[121]. many studies reported the presence of sars-cov-2 rna in stool samples of covid-19 infected patients [122-126]. besides, the angiotensin-converting enzyme-2 (ace2) is highly expressed in the gastrointestinal epithelial cells, especially the ileum, duodenum, jejunum, caecum, and colon [127]. a review by thye et al.[121] showed the prevalence of sars-cov-2 rna in covid19 patients’ stool samples and the gastrointestinal manifestations upon being infected with sars-cov-2. additionally, thye et al.[121] also discussed the possible pathophysiology of covid-19 associated gastrointestinal manifestations, which includes associations with the human-host receptor ace-2, the intestinal microflora, the use of antibiotics and antiviral medicines, and the direct and indirect inflammatory response as sars-cov-2 damages the digestive system. all in all, sars-cov-2 could infect and replicate in the gastrointestinal tract, suggesting possible sars-cov-2 tissue tropism in the intestinal cells, and it possibly transmits via the faecal-oral route in addition to droplet transmission[121]. another concern would be the issue of long covid-19. many studies have shown covid-19 survivors experienced increased mental health consequences than the general population, and some of the mental health implications include depression, anxiety, and posttraumatic stress disorder (ptsd) as presented in reviews by thye et al.[1,3]. the cause may be directly related to the infection via direct and indirect mechanisms, but the underlying aetiology is complex and multifactorial, involving biological, environmental, and psychological factors. risk factors and the prevalence rate seemed to vary across different studies, however, having a history of mental disorders and the female gender seems to be more consistent risk factors. on the other hand, the possible mechanisms by which sarscov-2 enters the brain may affect the central nervous system, resulting in these mental health implications include via the ace-2 receptors and through the implications of immune inflammatory signaling on neuropsychiatric disorders[1,3]. several reviews published in pmmb also discussed using probiotics as a potential adjunct treatment in addition to conventional psychotropic medication to alleviate and prevent covid-19 mental health implications[1,3,128,129]. pmmb 2023, 6, 1; a0000333 14 of 22 6. public health related various articles associating covid-19 with public health have also been published in pmmb. a review by goh et al.[130] examined the relationship between various prevailing population-based risk factors in comparison with the mortality rate and fatality rate (cfr) of covid-19. they found that countries having more older people aged >65 years old have a high risk of cfr due to covid-19. additionally, differences in gender and smoking prevalence did not prove a significant association with covid-19 mortality rate and cfr[130]. covid-19 pandemic has impacted various aspects, including the development of mobile applications to control the pandemic, the implementation of public health measures and public health strategies, and even promoted diet change. a review by loy et al.[131] assessed the features and content of covid-19 mobile applications accessible in the apple appstore. they found that the most popular mobile applications provided knowledge and guidance, contact tracing and hotspot information, and 50% of these evaluated mobile applications were maintained by respective countries’ health authorities, allowing the government to disseminate information to the public and collect data for covid-19 control[131]. in a review by hoo et al.[132], the author discussed the unforeseen positive impacts on healthcare as a result of the implementation of public health measures. the positive impacts includes positive environmental effects; increased health consciousness; decreased hospital admissions; more individuals quitting smoking; in some ways, mental health, sexual health, and family relationship were positively impacted; and the global movement towards healthcare innovation and technology development[132]. another systematic review by rayanakorn et al.[133] provided a critical summary of complete economic evaluations to inform decisions regarding adopting public health interventions. they concluded that tight and timely implementation of public health interventions is vital to flatten the covid-19 curve and that stringency, enforcement, timing, and adherence to interventions are key to the cost-effectiveness of epidemiological control measures. additionally, compared to nonselective widescale measures or single intervention, the application of a combination approach is more cost-effective. furthermore, the epidemiological characteristics of the virus, the healthcare capacity, and the local context need to be considered in the adoption of public health interventions[133]. lastly, a review by loh et al.[134] suggested that plant-based diets gained tremendous popularity globally during the covid-19 pandemic. the drop in meat and seafood sales could be associated with a few reasons; namely, the fear of meat contamination by sarscov-2, rise in price, and ethical reasons. furthermore, people believe that a plant-based diet can improve immunity, have more health benefits, and be more cost-effective than other diets. during the pandemic, there were more vegan venues and eateries with vegan options. their review concluded that there is a need for changes regarding the use of livestock and pmmb 2023, 6, 1; a0000333 15 of 22 wild animals to prevent future zoonotic pandemics. hence, promoting plant-based diets while decreasing animal-based diets are steps needed to prevent future zoonotic pandemics[134]. 7. conclusions pmmb had published many articles on covid-19, such as bulletins from various countries, emergence of vocs, covid-19 vaccines, covid-19 related public health measures and even genomic analysis of isolated sars-cov-2 strain from malaysia. this focused review compiles all covid-19 articles published by pmmb, providing a general overview of these articles. based on the various bulletins, it is clear that the cooperation between the government and people is essential to curb the covid-19 pandemic successfully and that the development of mobile applications for contact tracing plays a significant role in stopping the spread of the virus. key strategies used include the national vaccination program, contact tracing applications, and public health measures such as social distancing, wearing of face masks, hand hygiene etc. despite the pandemic, there are positive impacts from public health measures. in terms of vaccination, even though protection from primary vaccination may be waning, booster vaccines are available, and vaccination remains a key component in curbing the covid-19 pandemic. moreover, it is clear that sars-cov2 affects not just the respiratory system but could also affect the gastrointestinal system and have implications on mental health, in which probiotics have shown to be a potential adjunct treatment in addition to psychotropic medications. author contributions: 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control: a systematic review. prog microbes mol biol 2022; 5(1): a0000268. 134. loh hc, seah yk, and looi i. the covid-19 pandemic and diet change. prog microbes mol biol 2021; 4(1): a0000203. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology review article 1 critical review of fermentation and extraction of anti-vibrio compounds from streptomyces loh teng-hern tan1, learn-han lee1,3*, bey-hing goh2,3,4* 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. 2biofunctional molecule exploratory (bmex) research group, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. 3health and well-being cluster, global asia in the 21st century (ga21) platform, monash university malaysia, bandar sunway 47500, selangor, malaysia 4college of pharmaceutical sciences, zhejiang university, hangzhou, china abstract: a single streptomyces strain often have the potential to produce more than one bioactive compound. fermentation parameters include media compositions, temperature and ph, have great impact on the secondary metabolism of streptomyces and subsequently on production of different microbial products. this review aims to consolidate the studies on the cultivation parameters used to enhance the production of secondary metabolite with anti-vibrio activity from a single streptomyces strain. in turn, this review sheds light on the possible alterations of the cultivation parameters to obtain desired anti-vibrio compounds from streptomyces sp. furthermore, the bioactive compounds with anti-vibrio activity identified from streptomyces sp. were demonstrated to exhibit immense values for future antibacterial agent developments. keywords: streptomyces; vibrio; fermentation; extraction; secondary metabolites received: 26th november 2019 accepted: 7th january 2020 published online: 20th january 2020 citation: tan lt-h, lee lh, goh b-h. critical review of fermentation and extraction of anti-vibrio compounds from streptomyces. prog mircobes mol bio1 2020; 3(1): a0000051. https://doi.org/10.36877/pmmb.a0000051 introduction fermentation is an important process for the production of various structurally-diverse bioactive substances from microorganisms, including antibiotics, anticancer, antiviral and immunosuppressants[1,2]. given that the limited quantity of bioactive substances is usually produced by these microorganisms, fermentation is one of the feasible processes to continuously supply majority of these clinically useful drugs in the market currently. this is because the total chemical synthesis is way too complicated and costly than fermentation. for instance, antimicrobial peptides such as a novel class of antibiotics, which recently have received much attention, is not economically feasible to be synthesized chemically if involve larger or more complex peptides[3]. furthermore, medium optimization remains one of most critical steps in fermentation technology to enhance the production of valuable bioactive compounds. to achieve maximum production of desirable compounds, the production medium containing appropriate components (e.g., carbon, nitrogen, nacl, etc.) coupled with optimal fermentation conditions are required to be identified and optimized accordingly[1]. actinobacteria have been regarded as the most prolific producers in the microbial world[4–8], especially from the genus streptomyces[9,10]. the genus streptomyces has responsible for the production of more than 70% of commercially important antibiotics[2,11], as well as many bioactive compounds of pharmacological and agricultural interest[12–22]. the discovery of antibiotic from actinobacteria is highly dependent on the effect of growth conditions on the production of secondary metabolites[23–28]. these soil bacteria are known to have complex life cycle which is composing of different stages. secondary metabolites are usually produced by streptomyces sp. at the end of the active vegetative growth and during the dormant or reproduction stage[29]. the secondary metabolism of streptomyces is based on its unique genetic make-up but the expression can be copyright @ 2020 by tan lt-h and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: bey-hing goh, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; goh.bey.hing@monash. edu. learn-han lee, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; lee.learn.han@ monash.edu; leelearnhan@yahoo.com. 2 influenced by the surrounding manipulations[30]. therefore, the productions of secondary metabolites are often associated with the limitation of nutrients, presence of inducer or reduction of growth rate in streptomyces[23]. it is well known that secondary metabolite production can be repressed by readily available carbon source, high levels of nitrogen and phosphorus, all of which keeping the bacteria at active proliferative stage. this indicated that the production of secondary metabolites can be influenced significantly by various fermentation parameters including the nutrient availability, ph, temperature, mineral salts, inducers and inhibitors[31]. small modifications in the composition of growth media can result variation of the quantity of specific compounds, also these modifications could result in the production of a completely distinct pattern of molecules[32]. vibrio spp. is autochthonous to various aquatic environments, including estuarine, coastal waters and sediments[33–36]. vibrio spp. was known to be susceptible virtually to most of the antimicrobial agents[37,38]. however, antimicrobial resistance has emerged and evolved in many bacterial genera[39–41], including vibrio spp. as a result of excessive use of antimicrobial agents in various settings[42]. for instance, applications of antibiotics in aquaculture water as prophylactics to control infectious diseases in fish and aquatic organisms. furthermore, certain vibrio species, in particular v. parahaemolyticus and v. vulnificus are significant foodborne human pathogens[43–47]. hence, the increase in emergence of antibiotic-resistant bacterial pathogens, including vibrio spp. is a major public health concern[39,40,42]. this issue not only has immense impact on human health, it is also a concern on the future ability to treat the diseases as antibiotic resistance has developed over time, from single classes of antibiotics to multidrug resistance and eventually emergence of superbug with extreme drug resistance[48,49]. therefore, it has increased the interest of research on the search for more effective alternatives to cope with the issue of antibiotic resistant bacteria, including vibrio pathogens[50,51]. the exploration of bioactive compounds sourcing from natural resources, including plant[52–56], animal[57] or microbial origins[58–64] constitute an attractive bioprospection strategy among the drug discovery scientists. in fact, numerous efforts have demonstrated that the genus strepto-myces capable to synthesize various bioactive compounds against vibrio pathogens, representing a valuable source for antibacterial agents with anti-vibrio activities[65–67]. in the light of the promising potential of streptomyces as the bioresource of anti-vibrio compounds, this review provides the rationale for the designing and optimizing of fermentation medium to facilitate the process of antivibrio metabolite production in streptomyces sp. furthermore, the importance of extraction techniques for optimum yield of the desired bioactive compounds is discussed in this review (figure 1). critical review of fermentation... figure 1. fermentation and medium optimization for the production of anti-vibrio compounds by streptomyces sp. fermentation is conducted to induce the production of antivibrio active metabolites by streptomyces. starch and glycerol are both good carbon sources for the production of metabolites with better anti-vibrio activity (percentage shows the changes in the anti-vibrio activity when added with the indicated component in the fermentation medium). likewise, yeast extract and nitrate are the preferable nitrogen source as compared to casein and ammonium for the production of metabolites with better anti-vibrio activity by the streptomyces. the fermentation parameters presented are the optimum conditions for the production of anti-vibrio active metabolites. 3 fermentation process for production of anti-vibrio compounds by streptomyces sp. within the 64 studies analyzed in this review, a total of 38 studies conducted secondary screening of the metabolites produced by the anti-vibrio streptomyces via submerged fermentation process. this implies that 59.4% of the studies showed the anti-vibrio streptomyces strains displayed the antagonistic activities against different vibrio sp. through the production of bioactive secondary metabolites. thus, more study should perform fermentation in order to fully unravel the potential of the anti-vibrio streptomyces strains in the production of bioactive compounds against vibrio sp. solid state fermentation was reported as an alternative fermentation process to facilitate the secondary metabolites production from the anti-vibrio streptomyces[65]. the solid state fermentation involves the use of solid particles free of water or with little moisture for microbial growth and secondary metabolites production[68]. mohana and radhakrishnan (2014)[65] indicated that solid state fermentation process was more suitable for streptomyces ma7, a strain derived from mangrove rhizosphere sediment in producing antivibrio bioactive metabolites against vibrio pathogens such as v. cholerae o1, v. cholerae o139, v. parahaemolyticus and v. mimicus. however, there is limited information on studies comparing the two different fermentation techniques in the production of secondary metabolites with anti-vibrio activities. more study could be performed to investigate the optimal fermentation techniques for the production of anti-vibrio compounds from streptomyces at a higher yield. nevertheless, there was study suggested that solid-state fermentation is better for antibiotic production by streptomyces in the aspects of its stability and quantity[69]. for instance, solid-state fermentation tan lt-h et al. of streptomyces species resulted in higher yield and stability of well-known antibiotics including tetracycline[70], neomycin[71], cephamycin c[72] and oxytetracycline[73]. fermentation parameters affecting anti-vibrio compounds production media composition media composition plays an important role in determining the microbial secondary metabolites as it comprises of components that may act as activators of certain signaling pathway in the production of secondary metabolites[31]. thus, a single strain, grown under different condition may result in production of substantially different compounds. a study reported that by using a defined medium resulted in production of new metabolites which were not found in other media used to cultivate streptomyces sp. c34, and exhibited antibacterial activity towards v. parahaemolyticus[74]. the defined medium (table 1) containing 2 mm fluoride employed by the study was previously developed for the production of fluorinated secondary metabolites by streptomyces[75]. the mechanism for the production of the novel metabolites by streptomyces c34 has yet to be elucidated. nevertheless, it was suggested that the addition of fluoride salts could have activated the unique biosynthetic genes which responsible for the production of those new compounds[75]. therefore, other than depending on the biosynthetic potential of the microbes which determines the types of bioactive compounds, the composition of the media also plays a substantial role on the success of screening programs based on culturedependent bioprospecting strategy. according to the 38 studies that performed submerged fermentation, different types of fermentation broths were used, including starch casein broth, soybean meal broth, potato dextrose broth, arginine glycerol broth, actinomycetes isolation broth and glycerol asparagine. besides that, examples of fermentation broth with defined compositions used for the production of secondary metabolites from the anti-vibrio streptomyces can refer to table 1. parameters composition (% w/v)* studies that utilized mixture of complex and simple carbon and nitrogen sources# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 glucose 1 0.4 2 0.5 0.2 soluble starch 2 0.5 0.5 1 1 1 0.5 1 0.1 2 glycerol 1 1 1 1 myo-inositol 0.04 malt extract 0.4 soybean 2 0.5 0.5 0.1 1.5 casein 0.03 0.03 0.03 cornsteep powder 1 polypeptone 0.5 peptone 0.2 0.2 yeast extract 0.2 0.4 0.4 0.2 0.25 0.3 l-tyrosine 0.05 l-asparagine 0.1 msg 0.5 caco3 0.002 0.002 0.32 0.025 0.002 0.1 0.004 table 1. the composition of selected production media and fermentation conditions used for secondary metabolites production in the streptomyces sp. displaying anti-vibrio activity. 4 the influence of complex and simple carbon source on anti-vibrio activity the carbon source has significant effect on the production of antibiotic and the morphological development of streptomyces sp. several mechanisms have been described in the genus streptomyces to illustrate the carbon catabolite repression effects on secondary metabolites production[93,94]. as for aim of this review, it is to consolidate and rationalize the information available on the effect of different media composition on streptomyces toward the production of metabolites against vibrio sp. furthermore, major emphasis will be given towards the efficacy of the anti-vibrio metabolites produced by streptomyces in response to the presence of specific carbon source in the fermentation media. based on the data of media composition presented in the reviewed studies, carbon sources such as starch, glycerol and glucose are commonly used as growth substrate in the fermentation media used to produce secondary metabolites. majority of the studies incorporated starch (45.2%), a complex carbohydrate in the fermentation medium for the production of secondary metabolites with anti-vibrio activity (table 2). literatures demonstrated that the optimal production of secondary metabolites is generally achieved by culturing the microorganisms in media containing slowly assimilated nutrient sources while the readily utilized carbon source is often known to repress antibiotic production. for instance, the use of glucose as a carbon source had a negative influence on the production of nystatin as well as their morphology to a certain extent that resulted in termination of cell growth and nystatin production[95]. this is also commonly seen in other streptomyces sp., such as in the production of streptomycin, chloramphenicol and cephamycin by s. griseus[96], s. venezuelae[97] and s. clavuligerus[97] respectively. however, previous study indicated novobiocin production by s. niveus is subjected to catabolite repression by citrate assimilation and not caused by glucose assimilation[98]. streptomyces avermitilis was shown to assimilate glucose slowly and become the best carbon source in determining the production rate of avermec-tin[99]. ikeda et al. (1988)[99] suggested that the activity of 6-phosphogluconate dehydrogenase of the pentose phosphate pathway is associated with avermectin production, in which the nadph generated by the enzyme could be used as the intermediate for the biosynthesis of avermectin. previous study also indicated that glucose is important for the biosynthesis of ε-rhodomycinone, an important aglycone precursor to anthracycline antibiotic in streptomyces[100]. to identify the best carbon source for the production of anti-vibrio metabolites by streptomyces, the anti-vibrio activities of the streptomyces strains with or without the specific carbon source were compared based on the inhibition zones (table 3). in table 3, the anti-vibrio activity of metabolites produced by streptomyces strains increased by 33.1% in the presence of starch as carbon source. furthermore, a ten folds increment of anti-vibrio activity is demonstrated by streptomyces metabolites produced in the presence starch when compared to the use of glycerol as carbon source. in contrast, the use of glucose as carbon source is shown to repress the antivibrio activity of the streptomyces metabolites by 8.1% based on the median inhibition zones. this information is in line with other studies, indicating starch is a good carbon source for anti-vibrio metabolite production. the starch-based a1bfe medium (table 1) resulted in production of twice the amount of anti-vibrio compounds by streptomyces atrovirens pk288-21 compare to culture in glucose-based tcg medium[80]. the study suggested that streptomyces atrovirens pk288-21 utilized starch as the main carbon source that could increase the production of antibacterial compounds[80]. the continuous and gradual hydrolysis of starch could critical review of fermentation... nacl 0.2 0.05 0.2 0.1 0.05 0.2 0.05 0.08 naf 0.0084 nh4cl 0.15 0.1 kbr 0.01 kcl 0.01 k2hpo4 0.05 0.2 0.2 0.05 0.2 0.05 0.001 kno3 0.1 0.2 0.2 0.2 0.005 feso4 0.001 0.001 0.004 0.0025 0.001 mgso4 0.005 0.05 0.005 0.05 0.05 0.005 0.05 0.02 cocl2 0.001 znso4 0.001 seawater + + + + + + + + ph 7.5 ns ns 7.2 7.4 8 7 7.2 7.4 7.0±0.2 ns 7.5 7 7 temperature (oc) 27 28 28 29 30 ns 28 28 28 28 27 28 28 28 * the percentage of each composition was calculated using: w/v% = (weight of solute (g)/volume of media(ml)) × 100 # 1 soybean medium[65], 2 starch casein broth[76], 3 gsb broth medium[77], 4 casein glycerol/ starch medium[78], 5 production broth[79], 6 a1bfe media[80], 7 defined medium[74], 8 fermentation broth[81], 9 r2a medium[82], 10 starch casein broth[83], 11 soybean meal broth[84] 12 fermentation broth[85] 13 melanin production medium[86, 87],14 fermentation broth[88–92] 5 tan lt-h et al. avoid the carbon catabolite repression mechanisms that usually triggered by carbon sources that are more easily metabolized by the microorganism such as glucose[101]. in addition, the antibacterial compounds present were consisted of two benzaldehydes compounds identified from the fermented broth of s. atrovirens pk288-21. both of the benzaldehyde derivatives demonstrated antibacterial activity against both v. anguillarum and v. harveyi, particularly against v. harveyi with lower mic values reported as compared to ciprofloxacin (58 µg/ml). the work showed that the compound, 2-hydroxy-5-(3-methylbut-2-enyl)benzaldehyde (9) was as a new derivative while 2-hepta-1,5-dienyl-3,6dihydroxy-5-(3-methylbut-2-enyl)benzaldehyde (10) was previously reported from fungus eurothium rubrum. similarly, another 4 studies (table 2) also demonstrated the use of starch with concentrations ranging from 0.1 to 1% (w/v), as the sole carbon source in the fermentation medium for the production of secondary metabolites by streptomyces strains, and exhibited diverse strength of antibacterial activity against vibrio sp.[76,77,83,84]. overall, starch is recommended to be a good carbon source for the production of anti-vibrio metabolites from streptomyces. table 2. the compositions of fermentation medium and the fermentation conditions extracted from the reviewed studies on streptomyces with anti-vibrio activity. parameters compositions concentration % (w/v)/ units number of studies performed fermentation (n = 31) percentage (%) carbon sources complex carbon source only starch 0.1 1 0.5 2 1 2 sugarcane 1 1 • yeast extract 5 1 total = 7 22.6 • glucose 0.2 5 • glycerol 1 2 • glycerol, myoinositol 1, 0.04 1 total = 8 25.8 mixture of both complex and readily utilizable • starch, glucose 2, 1 1 • starch & glycerol 1 1 • glycerol, starch, glucose 1,1,1 2 • malt extract, glucose 0.4, 0.4 1 total = 5 16.1 media used by studies w/o specify the composition • starch casein broth 5 • potato dextrose broth 1 • arginine glycerol broth 1 • glycerol asparagine broth 1 • isp2 1 • soybean meal medium 1 • actinomycetes isolation medium 1 total = 11 35.5 nitrogen sources complex nitrogen source only • soybean 0.2 1 0.5 1 • tryptone, yeast extract 1, 5 1 • peptone, yeast extract 0.2, 0.4 1 • soybean, yeast extract 1.5, 0.25 1 • polypepton, yeast extract, corn steep liqour 0.5, 0.2, 0.1 1 • soybean meal, peptone, yeast extract 0.5, 0.2, 0.2 1 • malt extract, yeast extract 0.4, 0.4 1 6 • l-tyrosine, l-asparagine 0.05, 0.1 2 total = 10 32.3 mixture of both complex and readily utilizable • soybean, kno3 0.1, 0.005 1 • casein, kno3 0.03, 0.2 3 • yeast extract, nh4cl 0.3, 0.1 5 • msg, nh4cl 0.5, 0.15 1 total = 10 32.3 media used by studies w/o specify the composition • starch casein broth 5 • potato dextrose broth 1 • arginine glycerol broth 1 • glycerol asparagine broth 1 • isp2 1 • soybean meal medium 1 • actinomycetes isolation medium 1 total = 11 35.5 phosphate k2hpo4 0.001 5 0.05 4 0.2 3 total = 12 38.7 salt nacl 0.05 3 0.08 5 0.1 1 0.2 3 1 1 total = 13 41.9 ph 7 9 7.2 1 7.4 1 7.5 2 8 1 not specified 17 total = 31 temperature (oc) 23 1 25 1 27 2 28 13 29 1 30 5 32 1 35 1 26-30 1 28-32 1 not specified 4 total = 31 critical review of fermentation... 7 tan lt-h et al. table 3. the effect of carbon, nitrogen and nacl on the anti-vibrio activity of streptomyces metabolites. media composition (concentration range, w/v %) median of inhibition zone (mm) percentage of changes in anti-vibrio activity (%) absence presence carbon sources starch (0.32 – 2) 15.03 (n = 16) 20 (n = 10) increased by 33.07 glucose (0.2 – 2) 17.40 (n = 18) 16 (n = 9) decreased by 8.05 glycerol (0.12 – 1) 17.40 (n = 20) 18 (n = 8) increased by 3.45 nitrogen sources yeast extract (0.3 1) 16 (n = 16) 19 (n = 8) increased by 18.75 casein (0.03 1) 16 (n = 15) 17.4 (n = 10) increased by 8.75 ammonium salts, nh4 + (0.0001 0.12) 16.4 (n = 18) 18 (n = 5) increased by 9.75 nitrate salts, no3 (0.2) 16 (n = 16) 18 (n = 7) increased by 12.50 others nacl (0.05 1.2) 17 (n = 8) 18 (n = 16) increased by 5.88 influence of organic and inorganic nitrogen source on anti-vibrio activity nitrogen sources such as nitrate and ammonium salts which favorable for growth were shown to affect negatively on the production of secondary metabolites in streptomyces. the readily utilized nitrogen sources were demonstrated to cause repression of enzymes responsible for tylosin in streptomyces fradiae[102]. complex protein source such as soybean meal and the slowly assimilated amino acid such as proline are good nitrogen source to promote high secondary metabolites production. therefore, slow-metabolizing nitrogen sources are preferable to supply the essential nutrients to the antibiotic-producing strains. yeast extract, corn steep liquor and soybean flour are commonly used complex organic nitrogen sources[31]. based on the reviewed studies, soybean meal (0.2 and 0.5% w/v) was evidenced in studies[77,103] as a sole nitrogen source for the production of metabolites that exhibited anti-vibrio activities by the streptomyces strains (table 2). furthermore, the anti-vibrio activity of the streptomyces strains cultivated in different nitrogen sources were compared based on the median inhibition zone (table 3). the usage of yeast extract as a complex organic nitrogen source is found to enhance the anti-vibrio activity of the streptomyces metabolites by 18.75%, when compared to the only 8.75% increment in the presence of casein as an organic nitrogen source. besides that, nitrate is a more favorable inorganic nitrogen source when compared to the use of nh4 + in the fermentation media of the anti-vibrio streptomyces. none of the studies utilized ammonium or nitrate salts as the sole nitrogen source for the fermentation process. a total of 19 studies demonstrated the use of a mixture of readily and slowly utilizable nitrogen sources in the optimization of medium composition for the improvement of the yield of secondary metabolites (table 1). as the readily utilizable sources such as ammonium salts and nitrate salts serve to support the exponential growth of the bacteria while the slowly used sources such as yeast extract and casein serve to sustain the production of metabolites during the stationary phase, as the rapidly assimilated sources are depleted[31]. thus, the combination of yeast extract and nitrate salts could be used to serve as a good nitrogen sources in the production of anti-vibrio metabolites in the genus streptomyces. inorganic phosphate inorganic phosphorus is the common major growth-limiting nutrient in natural environments[31]. literatures showed that high concentration of inorganic phosphate in culture media causes negative regulation on the synthesis of secondary metabolites in different streptomyces sp.[104,105]. a total of 12 studies (38.7%) indicated the supplementation of dipotassium phosphate as a source of inorganic phosphate, with wide range of concentrations from 0.001 to 0.2% (w/ v) (~ 0.5–115mm) in the fermentation medium for the production of anti-vibrio secondary metabolites by streptomyces (table 2). none of the studies indicated the potential of inorganic phosphate that could resulted in lower production of anti-vibrio compounds. although some literatures demonstrated the supply of inorganic phosphate more than 3–5 mm are frequently inhibitory to antibiotic biosynthe-sis[105,106]. liras et al. (1990)[106] indicated phosphate stimulates the expression of genes involved in the biosynthesis of macromolecules and house-keeping genes essential for growth whereas it often inhibits expression of genes encoding for biosynthesis of secondary metabolites. the p-ami-nobenzoic acid synthase (paba synthase), that catalyzes the conversion of chorismic acid to paminobenzoic acid which is a precursor for candicidin (macrolide antibiotic) was found to be inhibited by potassium phosphate at 5 to 10 mm resulting in repression of candicidin biosynthesis in streptomyces griseus[107]. studies showed that the biosynthesis of several groups of antibiotic are 8 particularly sensitive to phosphate repression such as aminoglycosides[108], tetracyclines[109], macrolides[110] and polyenes[104]. meanwhile, the biosynthesis of beta-lactam antibiotic and peptide secondary metabolites were poorly sensitive to high concentration of inorganic phosphate. for example, the production of cephalosporin is optimal at 25 mm phosphate but higher concentrations of phosphate resulted in 85% reduction of cephalosporin production in s. clavuligerus[111]. these evidences suggested that the genes encoding the enzyme for the secondary metabolites produced by the anti-vibrio streptomyces may have lower sensitivity toward phosphate repression. however, the concentration of inorganic phosphate to be used in fermentation media should be optimized to ensure maximum production of anti-vibrio metabolites by the strep-tomyces. by comparing the anti-vibrio activity of the streptomyces metabolites under different concentration of k2hpo4 (table 4), based on the median of inhibition zone, it is observed that the antivibrio activity reduced by 33.3% when the concentration of k2hpo4 used is increased from 0.001% to 0.2% (w/ v). these data suggest that inorganic phosphate is recommended to be maintained at lower concentration such as at 0.001% (w/v) as a source of phosphorus in the fermentation media for optimal production of anti-vibrio metabolites from streptomyces. table 4. the effect of different compositions and the fermentation conditions on the anti-vibrio activity of streptomyces metabolite sodium chloride the supplementation of sodium chloride in the fermentation medium is one of the non-nutritional stress factors influencing the secondary metabolites production[112,113]. based on the reviewed studies, a total of 13 studies supplemented sodium chloride in the fermentation medium for the production of anti-vibrio secondary metabolites from streptomyces (table 2). the concentration of sodium chloride used was ranging from 0.05 to 1% (w/ v), showing production of anti-vibrio metabolites from streptomyces. in line with the literatures, the anti-vibrio activity of streptomyces metabolites is enhanced by 5.88% when cultivated in the presence of sodium chloride as compared to the metabolites produced in the absence of sodium chloride (table 3). barakat and beltagy (2015) [114] indicated the streptomyces ruber erh2 supplemented with 1% sodium chloride (w/v) produced metabolites against v. ordalii fish pathogen, with high inhibition zone measured at 15mm. as indicated in table 4, a small increase of sodium chloride concentration, such as from 0.05 to 0.08% (w/v) resulted in 99.7% increment in the anti-vibrio activity, thus indicated the optimum concentration of sodium chloride for the production of anti-vib-rio metabolites is at 0.08% (w/v) for streptomyces. at the meantime, the further increase of sodium chloride in the fermentation media from 0.08% (w/v) to more than 0.2% (w/v) may reduce the anti-vibrio activity from streptomyces metabolites by 50%. similarly, syvitski et al. (2006)[115] demonstrated that the presence of salt in the growth medium could result in differential production of antibiotic by streptomyces. furthermore, this study indicated the addition of 2.5% of sodium chloride inhibited the production of actinorhodin, but activated the production of undecylprodigiosin[115]. the study also reported high salt conditions that resulted in differential expression of these genes, actii-orf4 and redd encoding corresponding pathway specific transcriptional regulators for both actinorhodin and undecylprodigiosin biosynthesis in streptomyces coelicolor a3(2)[115]. temperature an optimal temperature is often required for the production of secondary metabolites. based on the reviewed studies, 28oc (41.9%) is the most common incubation temperature used for the secondary metabolite production. slightly higher incubation temperature at 30oc is also reported in several studies (16.1%) (table 2). there are also studies employed a lower incubation temperature ranging from 23-25oc[116,117]. the studies indicated that the optimal temperature for production of secondary metabolites can be varying considerably between the similar genera of actinobacteria. furthermore, some studies indicated that optimal temperature for production of secondary metabolites is generally lower, when compared to growth of streptomyces sp. thakur et al. (2009)[118] reported streptomyces sp. 201 showed narrow range of incubation temperature for growth and antibiotic production, maximum mycelial growth was measured at 35oc while highest antibacterial activity was observed at 30oc. thirumurugan and vijayakumar (2015)[119] also reported a strain, streptomyces ecr77 that produced antivibrio secondary metabolites after cultivated at 28-30oc although this strain showed optimal growth at 35oc. costa and badino (2012)[120] also recommended that the reduction of temperature could be useful in increasing the production of clavulanic acid by streptomyces clavuligerus. according to table 4, repression effect could occur via increase of fermentation temperature from the optimum 28oc to 30oc, resulting in 25% reduction of anti-vibrio activity (based on the median of inhibition zone) from the streptomyces metabolites. hence, these data suggest that lower incubation temperature results in lower cellular growth and substrate consumption which could minimize critical review of fermentation... parameters concentration (w/v %) / range median of inhibition zone (mm) nacl 0.05 15.02 (n = 2) 0.08 30.00 (n =3) 0.20 19.00 (n = 8) > 0.20 15.00 (n = 3) k2hpo4 0.001 30.00 (n = 3) 0.01 0.05 16.52 (n = 4) 0.2 20.00 (n = 1) ph 7 15.03 (n = 7) 7.1 – 7.3 18 (n = 5) > 7.3 22.5 (n = 2) temperature (oc) < 28 16.4 (n = 2) 28 20 (n = 12) 30 15 (n = 7) 9 the metabolite repression effects and also reduces end-product degradation, eventually increasing the yield of secondary metabolites production[120]. ph of fermentation media the ph of the cultivation media has substantial effect on the growth of streptomyces sp. and their antibiotic production ability[121,122]. based on the reviewed studies, a narrow range of initial ph (7–8) of the fermentation media were used in cultivation of streptomyces sp. for secondary metabolites production (table 2). kontro et al. (2005)[122] reported that the ph ranges for the optimal growth of streptomyces sp. were species specific and strongly influenced by the nutrient compositions of the media. the use of neutral to slightly alkaline ph as described by majority studies suggested that these ph range are more preferable for developing a fermentation medium for antibiotic producing-streptomyces. in agreement with others, anti-vibrio activity from streptomyces metabolites could be enhanced by performing the fermentation under slightly alkaline ph. as depicted in table 4, the anti-vibrio activity could be increased by 49.7% with a small increase of the fermentation media from ph 7 to 7.3. according to guimaraes et al. (2004)[121] findings, the low ph level of the cultivation media (at the end of the shake flask fermentation) resulted in no detection of retamycin although the final cell concentrations of s. olindensis icb20 reached 4 g/liter, indicating that the ph negatively affected the activity of the biosyn-thetic enzymes that involved in the secondary metabolism. meanwhile, at higher ph of 8.0, it was reported to have negative effect on the excretion of the antibiotic, demonstrated by the higher intracellular content of retamycin was produced, rather than the yield of extracellular retamycin[121]. extraction of secondary metabolites the extraction is a critical step to isolate the desirable secondary metabolites from the complex fermented products[123]. solvent extraction is one of the most common extraction methods due to the high selectivity and solubility of target compositions. it has been widely utilized to extract fermentation-derived microbial products prior to the final purification of bioactive compounds by chromatography[74,123,124]. there are a wide range of approaches available for the recovery of microbial metabolites. primarily, the types of extraction method employed is chosen depending on the compounds of interest residing whether it is excreted into the medium or produced intracellularly. generally, direct solvent extraction is conducted if the desired product is present in the cell and the medium. however, the common practice in extraction of microbial product from the cultivation media involves the separation of the microorganism biomass by centrifugation or filtration prior to solvent extraction of the cell free medium[125,126]. among the 31 studies that performed fermentation, 18 studies (58.1%) conducted solvent extraction method to extract and determine the antibacterial activity of the bioactive compounds present in the fermented product. the selection of most appropriate solvent is critical in determining the successfulness of yielding the desired product. nonpolar solvents (petroleum ether, chloroform and hexane) are useful in extracting lipophilic compounds such as alkanes, sterols, alkaloids, fatty acids, coumarins and some terpenoids. some alkaloids and flavonoids are compounds with medium polarity can be extracted with medium polarity solvents such as ethyl acetate. meanwhile the more polar compounds such as flavonoid glycosides, tannins and some alkaloids are extracted with the carbon-bonded oxygen-bearing extractants include alcohols, esters and ketones [123]. table 5 shows the examples of bioactive compounds isolated from anti-vibrio streptomyces using different organic solvents. tan lt-h et al. source compounds antibacterial activity references ethyl acetate of streptomyces rosa var. notoensis nanaomycin a (1) mic: 6.3 μg/ml against v. alginolyticus 138-2 mic: 3.1 μg/ml against v. parahaemolyticus k-1 [127] nanaomycin d (2) mic: <0.05 μg/ml against v. alginolyticus 138-2 mic: <0.05 μg/ml against v. parahaemolyticus k-1 methylene chloride extract of endophytic streptomyces sp. nrrl30562 derived from plant, kennedia nigriscans munumbicin b (3) 16 mm against v. fischeri pic345 [116] munumbicin c (4) 9 mm against v. fischeri pic345 munumbicin d (5) 12 mm against v. fischeri pic345 methanol extract of desert soil-derived streptomyces sp. c34 chaxalactin a (6) mic: 12.5 µg/ml against v. parahaemolyticus [74] chaxalactin b (7) mic: 20 µg/ml against v. parahaemolyticus chaxalactin c (8) mic: 12.5 µg/ml against v. parahaemolyticus acetone extract of streptomyces atrovirens pk28821 derived from marine seaweeds 2-hydroxy-5-(3-methylbut2-enyl)benzaldehyde (9) mic: 20 µg/ml against v. harveyi mic: 65 µg/ml against v. anguillarum [80] table 5. the bioactive compounds identified from the streptomyces sp. displaying anti-vibrio activities. 10 2 h e p t a 1 , 5 d i e n y l 3 , 6 dihydroxy-5-(3-methylbut2-enyl)benzaldehyde (10) mic: 32 µg/ml against v. harveyi mic: 65 µg/ml against v. anguillarum acetone extract of streptomyces sp. k01-0509 guadinomine b (11) ic50: 14 nm potent type iii secretion system (ttss) inhibitor [128] ethyl acetate extract of streptomyces sp. scsio 01689 derived from submarine sediment pyranosesquiterpene compound (12) mic: >100 µg/ml against v. anguillarum [82] cyclo(d)-pro-(d)-ile (13) mic: 0.05 µg/ml against v. anguillarum cyclo(d)-pro-(d)-leu (14) mic: 0.04 µg/ml against v. anguillarum cyclo(d)-trans-4-oh-pro(d)-phe (15) mic: 0.07 µg/ml against v. anguillarum critical review of fermentation... based on the data, the commonly used solvents for the extraction of bioactive compounds include, methanol, acetone, chloroform, ethyl acetate, n-butanol, n-hexane and petroleum ether. from these studies, ethyl acetate (83.3%) was the most commonly used solvent. this may be due to the property of ethyl acetate which is only partially miscible with water, hence allowing easier recovery of the metabolites from the fermentation broth by liquidliquid extraction methods. besides that, methanol was the second (27.8%) most commonly utilized solvent among the reviewed studies. usually, methanol is preferable for the extraction of unknown metabolites from new strains of bacteria. this is because methanol has been known to be efficient in extracting a wide range of metabolites from bacteria[129]. eventually, the resulting extract is filtered, concentrated by vacuum evaporation before being used for bioactivity analysis. it is imperative to remove the solvent or extractant completely from the resulting extracts as their presence in the final product is undesirable and might affect the results of the bioactivity screening. gas chromatography is a useful tool for the detection of residual solvents. this is because of the low figure 2. the chemical structures of anti-vibrio secondary metabolites isolated from streptomyces sp. detection limits allowing for the detection of trace organic compounds[130]. furthermore, supercritical carbon dioxide at 200 atm and 35oc was shown to be effective in removing organic solvents from antibiotic without affecting the antibiotic activities[131]. moreover, it is common to find that interesting compounds can be overlooked due to the presence of other molecules in a crude extract, or simply because of its low titers in an extract resulted overall low activity observed. fractionation step after the extraction could be a way to overcome these issues. for instance, the fractionation of streptomyces sp. c34 methanolic extracts with three other different solvents, n-hexane, dichloromethane and ethyl acetate and eventually identified the three novel macrolactones from the dichloromethane fraction with the most diverse metabolic profile[74]. once a bioactive extract is identified, a more detailed analysis is performed, normally involving chromatography-based separation of the individual constituents, to identify the specific bioactive molecules and also structure elucidation with nmr analysis. 11 subsequently, the bioactive compounds from these screening activities are tested in an in vivo model to examine efficacy and safety. most of current clinically used antibiotics have been discovered using this approach. for instance, barakat and beltagy (2015)[114] demonstrated that the phthalic acid isolated from s. ruber ekh2 with antagonistic activity against v. ordalii is non-toxic toward artemia salina (brine shrimp) up to 2800 µg/ml, suggesting that the compound is natural and minimum side effects. furthermore, the conventional screening process also provides valuable information such as the potency of the antibiotic by determining the minimum inhibitory concentration (mic) of the antibiotic toward specific pathogens, the spectrum of activity. cho and kim (2012)[80] determined the potency of benzaldehyde compounds isolated from s. atrovirens pk288-12, revealing a lower mic displayed by both compounds as compared to ciprofloxacin against v. harveyi. by referring to the studies which reported the isolation of streptomyces with anti-vibrio activity, most of them have focused on the preliminary screening and optimization of the various culture conditions. however, there is only limited number of the study that further analyzed and identified the bioactive compounds that displayed potent antibacterial activity against vibrio sp. hence, there is a need to improve the isolation and screening strategies, as the conventional methods of cultivation, extraction and bioac-tivity testing of anti-vibrio streptomyces are time consuming and prone to rediscovery of known compounds. new research strategies such as genome mining, which reveals the silence biosynthetic gene cluster, coupling with the advanced chemical separation and characterization techniques[132] have been developed to enhance the antibiotic production and discovery of new compounds in streptomyces. furthermore, more advanced extraction method could be employed to replace the conventional organic solvent extract method. for example, supercritical fluid extraction, pressurized solvent extraction and ultrasoundassisted extraction have been discussed as some of the better alternative extraction techniques to isolate bioactive natural products[133]. these advanced extraction methods are known for their higher selectivity, shorter extraction time, nontoxic organic solvents and more environmental friendly as compared to the conventional solvent extraction method[133]. majority of these advanced extraction methods have been widely used to extract biologically active compounds with antioxidant and antimicrobial activity from plants[53,134]. despite that, only a small portion of studies have utilized the advanced extraction methods to extract the bioactive compounds from the fermentation broth of microorganism. for instance, griseofulvin, which is one of the few examples of microbial antifungal antibiotic, was extracted with supercritical carbon dioxide extraction method[135]. although the supercritical carbon dioxide is less effective in extracting highly polar compounds, this extraction method offers a better alternative to organic solvents because of its nontoxic property, inexpensive and most importantly can be easily removed from the final products[133]. this is because the residual organic solvent presents a major concern over the safety of food and pharmaceutical products over the years[136]. therefore, future studies are encouraged to utilize one of these advanced extraction methods to improve the yield and purification of the biologically active compounds from streptomyces. conclusion given the ever-increasing reports of antibiotic resistant vibrio pathogens, there is a critical need to search for alternatives of major antibiotics. numerous studies demonstrated the production of promising bioactive compounds with anti-vibrio activity by streptomyces sp. fermentation parameters can have great impact on the secondary metabolism of streptomyces and subsequently on production of different microbial products. the information and knowledge obtained in this review could help in the optimizing of suitable fermentation medium is important for better yield and antimicrobial activity from streptomyces sp. we suggest that starch and yeast extract are both good carbon and nitrogen source for the secondary metabolites production by the anti-vibrio streptomyces. the temperature, concentrations of phosphate and sodium chloride are also important criteria should be taken into consideration when designing the fermentation medium and condition for the anti-vibrio metabolite production in the genus streptomyces. the limited findings on the bioactive compounds with antivibrio activity from streptomyces sp. suggesting that more studies should focus on identifying the potential bioactive compounds that specifically against vibrio sp. taken together, with optimal fermentation conditions and appropriate extraction techniques, future development of clinically important drugs is warranted from these streptomyces sp. to treat infections inflicted by vibrio pathogens. author contributions the literature review and manuscript writing were performed by lt-ht, l-hl and b-hg. l-hl and bhg founded the research project. conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. acknowledgments this work was supported by the monash university malaysia ecr grant (5140077-000-00), mosti escience fund (02-02-10-sf0215 and 06-02-10sf0300), monash global asia in the 21st century (ga21) research grant (ga-hw-19-l01 & ga-hw-19s01) and fundamental research grant scheme (frgs/ 1/2019/wab09/ musm/02/1 & frgs/1/2019/skk08/ musm/02/7). tan lt-h et al. 12 critical review of fermentation... references 1. singh v, haque s, niwas r, et al., strategies for fermentation medium optimization: an in-depth review. front microbiol 2017; 7(2087). 2. kemung hm, tan lth, 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739256 singapore 3department of pathology, faculty of medicine, universiti kebangsaan malaysia, 56000, kuala lumpur, malaysia 4department of surgery, faculty of medicine, universiti kebangsaan malaysia, 56000, kuala lumpur, malaysia abstract: majority of colorectal cancer (crc) patients are presented with advanced disease at diagnosis, particularly in cases of proximal crcs. little is known about the relationship between the genetic landscape and the anatomical location of the tumour; as well as the prognostication in crc patients. the objectives of this study were to determine the somatic single nucleotide variants (snv) and the cellular pathways between the proximal and distal crcs. whole exome sequencing was performed on the ion proton platform on 10 pairs of normal and crc samples. the sequencing results were analysed using the torrent suite software and the variants were annotated using annovar; followed by validation with sanger sequencing. apc is the most frequently altered gene in both proximal and distal crcs. kras and atm genes were particularly altered in the proximal crcs with a frequency of 60% and 40%, respectively. on the other hand, tp53 mutations did not show any crc anatomical predominance. there were five recurrent novel variants in proximal crcs and no recurrent variants identified in distal crc. wnt signalling pathway was the most frequently altered pathway in both proximal and distal crcs whereas tgf-β and pi3k signalling pathways were predominantly altered in the proximal crcs. we found that proximal crcs presented with more variants and different altered pathways as compared to distal crcs. however, further study in a larger series of samples coupled with functional studies will be required to confirm the identified variants and determine their roles in the pathogenesis of proximal and distal crcs. keywords: colorectal; proximal; distal; whole-exome sequencing; insertion; deletion received: 12th august 2019 accepted: 13th september 2019 publish online: 30th september 2019 *correspondence: nurul-syakima ab mutalib, ukm medical molecular biology institute (umbi), universiti kebangsaan malaysia, 56000, kuala lumpur, malaysia; syakima@ppukm.ukm.edu.my. rahman jamal, ukm medical molecular biology institute (umbi), universiti kebangsaan malaysia, 56000, kuala lumpur, malaysia; rahmanj@ppukm.ukm.edu.my. citation: mohd yunos r-i, ab mutalib n-s, khor ss, et al. whole exome sequencing identifies genomic alterations in proximal and distal colorectal cancer. prog microb mol biol, 2019; 1(1): a0000036 introduction colorectal cancer (crc) is the third most commonly diagnosed cancer worldwide[1] and is ranked as the second most common cancer in malaysia[2]. according to the national cancer registry report, crc is the most common cancer among men and the third most common among women in malaysia[3]. in the year 2018, it was estimated that there were 1.8 million cases and 881,000 deaths from crc worldwide[1]. anatomically, crcs are classified into three subsets named, proximal, distal and rectum. proximal crc is located on the right side of the colon, which includes cecum, ascending colon, hepatic flexure, transverse colon and splenic flexure; while distal crc is located on the left side of the colon, including sigmoid and descending colon[4]. it is postulated that both the proximal and distal crcs are anatomically different and arose from different biological pathways, suggesting different molecular mechanisms involved[5]. biological differences between the normal proximal and distal colon suggest that the carcinogenesis in these locations may be mediated via different molecular pathways[6,7]. this may have profound prognostic and therapeutic implications. for instance, it has been reported that the gene expression profile between adenocarcinoma of the proximal and distal crc is different, and therefore, the information copyright 2019 by mohd yunos r-i et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) 2 should be taken into consideration when investigating new predictive and prognostic biomarkers[8]. among the molecular characteristics that distinguish between proximal and distal crcs is the low frequency of tp53 and kras gene mutations, lower c-myc expression and dna mismatch repair (mmr) deficiency in proximal crc. distal crc, on the other hand, shows a higher frequency of tp53 and kras gene mutations and c-myc expression[9]. several large cohort studies in the last 20 years have demonstrated that the proximal and distal crcs differed in their susceptibility to screening tests, the stage at which they were diagnosed, patient characteristics, pathology and prognosis[10,11]. a few studies have discovered that the distribution of distal and proximal crcs varies according to ethnicity[12]. individuals with african ancestry are more likely to develop proximal crcs rather than distal crcs[13,14], whereas asians and pacific islanders are more prone to distal colon and rectal cancers[13]. a study by goh and colleagues (2005) supported this fact, whereby 77% of crc cases in malaysia were diagnosed as distal crcs[15]. however, despite the different distributions, the actual causative molecular events that lead to the different prognosis between proximal and distal crcs remains poorly understood. next-generation sequencing technologies have revolutionized cancer research and management, particularly in diagnostic and treatment strategies[16,17]. using wholeexome sequencing approach, we examined the exomes of malaysian patients diagnosed with proximal and distal crcs in order to identify the differences and the distinct signatures based on their anatomical distribution. we hope that this can contribute to better management and treatment of crc patients in the future. materials and methods clinical material a total of ten paired colorectal carcinoma and their corresponding adjacent normal tissues (five proximal and five distal), were collected from patients at universiti kebangsaan malaysia medical centre, kuala lumpur. the written informed consents were provided by patients to participate in the study. this study was approved by the ukm research ethics committee (reference no: ukm 1.5.3.5/244/umbi-004-2012). the tissues were subjected to cryosectioning; followed by haematoxylin and eosin (h&e) staining. our pathologist confirmed the presence of at least 80% of cancer cells in the tumour specimens and less than 20% necrosis in paired unaffected colorectal tissue adjacent to the tumour site. the dna extraction was performed using the qiaamp dna mini kit (qiagen, valencia, ca) according to the manufacturer’s protocol. quality and quantity of the extracted dna were assessed using qubit fluorometer (invitrogen, carlsbad, ca, usa), nanodrop 2000 spectrophotometer (nanodrop technologies, wilmington, de); as well as agarose gel electrophoresis. to confirm the identity of each tumour and normal paired samples, genotyping was performed by multiplex pcr based on microsatellite polymorphisms using coriell identity mapping kit (coriell institute, new jersey, usa). exome capture, library construction and next-generation sequencing one microgram (1 μg) of genomic dna from each pair of tumour and normal sample was mechanically sheared by ultrasonic fragmentation using the covaris® system to achieve fragments of about 50–500 bp. the fragmentation profile was assessed by the bioanalyzer high sensitivity dna analysis kit (agilent technologies, carlsbad, ca, usa). the fragmented dna was used to construct a fragment library using the ion plus fragment library kit (life technologies, guilford, ct) according to the manufacturer’s instructions for ligation, end repair, purification, size selection and final amplification prior to exome capture. for multiplexing the samples, adapters with short stretches of index sequences from ion xpress™ barcode adapters 1–16 kit was used and thus allowing the sequencing of two samples in a single ion pi™ chip run. five hundred nanograms (500 ng) of the amplified, size-selected library (~285 bp) from each sample was subjected to exome capture procedure using the ion targetseq™ exome kit (life technologies, guilford, ct) according to manufacturer’s protocol. exomes were captured using ~2 million targetseq™ capture probes with biotinylated oligos that range from ~50 bases to ~120 bases. the captured dna fragments were isolated using streptavidin-coated dynabeads paramagnetic beads and they were amplified and purified. finally, the samples were quantitated and qualitatively assessed on the bioanalyzer high sensitivity dna chip (agilent technologies, carlsbad, ca, usa). the purified, 10 pm of exome-enriched libraries were used for template preparation on ion pi™ ion sphere™ particles (isps) for sequencing on an ion pi™ chip using ion proton sequencer (life technologies, guilford, ct). bioinformatics analyses the data was processed using the torrent suite software v4.2.1. the torrent suite software automated the generation of sequence reads, trimming of adapter sequences, removal of poor quality reads; as well as sequence alignment to the hg19 human genome reference. variants were called using the torrent variant caller plugin, configured for somatic mutation detection with low stringency setting. variant filtering and calculation of transition to transversion ratio (ti/tv) were performed using snpsift tool in snpeff (version 4.0, 2014-11)[18]. the variants were then subjected to annotation using annovar, version 2013may09[19]. gene-based annotation was performed against refseq gene[20], ucsc known gene[21] and ensembl gene[22]. the variants were further annotated against the conserved region (phastconselements46way) [23], alternative allele frequency in all subjects in the national heart lung and blood institute exome sequencing project (nhlbi-esp) project with 6500 exomes (esp6500si_all)[24], alternative allele frequency data in 1000 genomes project (1000g2014oct_all) (the 1000 genomes project consortium, 2015), the exome aggregation consortium (exac 01)[25], dbsnp version 138 (snp138)[26], clinvar (clinvar_20140929)[27] and coswhole exome sequencing... 3 mic version 70 (cosmic70)[28]. protein impact prediction was also performed on annovar (version 2013may09) using command ljb26_all[19] which include sift[29] and polyphen2[30]. in order to identify potentially druggable variants, the drug-gene interaction database, dgidb 2.0 was utilized to annotate the variants against drugs genes interaction dataset (http://dgidb.genome.wustl.edu/)[31]. variants prioritization variants exclusion criteria included those with base quality less than q30, not resulting in amino acid changes, identified in unannotated genes (unknown), called in both tumour and normal exomes, representing probable mapping ambiguities, and have minimal allele frequency (maf) of more than 5% in the 1000 genomes project[32], exac and esp6500 database[25]. a variant in a tumour was considered to be a novel true candidate somatic mutation if the corresponding normal sample has at least 10 reads covering this position, zero variant reads and has not been reported in dbsnp138[26] or the 1000 genomes data set (october 2014)[32]. for the resulting candidate of somatic mutations, the alignment of each sample was manually examined to check for sequencing artefacts and alignment errors using the integrated genomic viewer (igv)[33,34]. we were then assessed for potential protein impact prediction of each genetic variant identified in both proximal and distal cohorts based on variant prediction algorithms, sift[29,35] and polyphen2[30]. statistical analysis we utilized the fisher’s exact test to define significance values in a number of protein-altering mutations and number of affected pathways between proximal and distal crc using the 2 × 2 contingency tables and the graphpad quickcalcs online calculator for scientists (http://www. graphpad.com/quickcalcs/index.cfm). all p values are twosided and statistical significance is denoted by p < 0.05. gene pathway analysis altered genes identified in both proximal and distal cohorts were pooled and run through the ingenuity pathway analysis (ipa) (qiagen, valencia, ca) software at the same time to identify the affected canonical pathways. variant confirmation variants were validated using the sanger sequencing method on tumour and normal samples. primers corresponding to the selected locations were designed using idt-dna primer quest (coralville, ia). pcr products were generated and cycle sequencing was performed using the big dye terminator v3.1 reagent (life technologies, guilford, ct). the cycle sequencing products were then processed using ethanol precipitation and sequencing was carried out using the abi 3130xl capillary electrophoresis (life technologies, guilford, ct). the results were analysed using the basic local alignment system tool (blast)[36] and sequence scanner (applied biosystem, foster city, ca). results clinicopathological characteristic of patients the characteristics of all ten patients were listed in table 1. with regards to cancer stage, 20% (n=2) of the patients were of dukes’ a, 40% (n=4) were dukes’ b and the remaining 40% (n=4) were dukes’ c. the average age of patients was approximately 69 years old (range 58–75 years). the samples comprised of three welldifferentiated adenocarcinomas, three moderate differentiated adenocarcinomas and four poorly differentiated adenocarcinomas. from these ten samples, four patients were positives for lymph nodes metastasis and six were negative. exome sequencing analysis and coverage the capture regions covered by ion targetseq was about 37.3 mb and we managed to obtain an average of 39.6 million reads. average coverage of about 70x for each sample was obtained and the coverage of the target region at 20x was more than 85%. this was comparatively higher than what was obtained by a study on pancreatic cancer[37] and a study on colon and rectal cancers[38]. on average, the number of variants detected at q30 for each sample was 35, 713 (32, 804 37,487 variants) (table 2). to assess the quality of variants, the ratio of the number of transitions to the number of transversions was determined. the expected ti/tv ratio for exome target regions is 2.8[39]. however, the target regions of exome capture kits often covered more than just exons. for snps resided in these target regions, ti/ tv ratios between 2.0 and 3.0 were observed[40]. the target regions of ion targetseq kit covered both exonic and non-exonic regions, such as 3’ utr and 5’ utr, therefore, we obtained an average ti/tv ratio of 2.7. upon variants prioritizations, we obtained a total of 4,835 and 4,177 variants in proximal and distal crc, respectively. among all the variants found in proximal crc, 539 were protein-altering variants in 508 genes located in the conserved regions. on the other hand, the distal crcs had 245 protein-altering mutations in 180 genes located in the conserved regions. the proximal crc showed significantly more protein-altering variants as compared to distal crc (p-value = 0.0001) (table 3). based on mutation rates, we stratified the cases into two groups: hypermutated (>30 per 106 bases) and non-hypermutated (<20 per 106 bases). the average mutation rate for proximal and distal crc is 22 per 106 bases and 26 per 106 bases respectively (median mutation rate in both proximal and distal is 15 per 106 bases). one case of each proximal (sample r4t, 72 per 106 bases) and distal (sample l2t, 58 per 106 bases) were classified as hypermutated. we then analysed the mmr gene mutations (msh2, msh3, msh6 and mlh1) in all samples. interestingly, we identified at least one somatic missense mutation in mmr gene presence in sample r4t (msh2 g.47656969c>t) and l2t (epcam g.47604176c>t), that possibly explain the hypermutation status of the samples. on the other hand, none of the rest of the samples was having a mutation in mmr genes. mohd yunos r-i et al. 4 whole exome sequencing... ta bl e 1. c lin ic op at ho lo gi ca l c ha ra ct er is tic s of c r c p at ie nt s sa m pl e id g en de r/ a ge e th ni c h is to lo gi ca l s ub ty pe st ag e d uk es ’ t n m s ta gi ng l 1 m /7 1 c hi ne se w el l d if fe re nt ia te d, a de no ca rc in om a b 2 t 3 n 0 m x l 2 m /6 6 c hi ne se m od er at el y d if fe re nt ia te d, a de no ca rc in om a a t 2 n 0 m x l 3 m /7 5 m al ay po or ly d if fe re nt ia te d, a de no ca rc in om a c t 3 n 1a m x l 4 f/ 73 m al ay w el l d if fe re nt ia te d, a de no ca rc in om a c 1 t 2 n 1b m x l 5 f/ 75 m al ay w el l d if fe re nt ia te d, a de no ca rc in om a a t 2 n 0 m x r 1 f/ 65 c hi ne se po or ly d if fe re nt ia te d, m uc in ou s a de no ca rc in om a b t 3 n 0 m x r 2 m /7 0 c hi ne se po or ly d if fe re nt ia te d, a de no ca rc in om a b 2 t 3 n 0 m x r 3 m /5 8 m al ay m od er at el y d if fe re nt ia te d, a de no ca rc in om a c t 3 n 1a m x r 4 m 72 c hi ne se po or ly d if fe re nt ia te d, a de no ca rc in om a b t 3 n 0 m x r 5 m /6 4 c hi ne se m od er at el y d if fe re nt ia te d, a de no ca rc in om a c t 2 n 1 m x l = l ef t, r = r ig ht , m = m al e, f = fe m al e, t n m = tu m or -n od em et as ta si s ta bl e 2. e xo m e se qu en ci ng c ov er ag e fo r n or m al a nd tu m ou r s am pl es sa m pl e to ta l a lig ne d o ut pu t ( g ) # of m ap pe d re ad s % r ea ds o n ta rg et a ve ra ge c ov er ag e u ni fo rm it y (% ) ta rg et b as e c ov er ag e at 1 x (% ) ta rg et b as e c ov er ag e at 2 0x (% ) ta rg et b as e c ov er ag e at 1 00 x (% ) ta rg et b as e c ov er ag e at 5 00 x (% ) v ar ia nt s l 1n 13 .7 38 ,3 27 ,1 22 80 .7 5 71 .3 9 93 .4 8 98 .0 3 90 .7 2 20 .6 6 0. 11 34 ,7 06 l 1t 13 .7 43 ,5 81 ,6 66 84 .7 9 84 .6 8 93 .7 8 98 .1 7 92 .6 4 29 .7 9 0. 21 35 ,6 75 l 2n 13 .6 37 ,2 48 ,5 04 79 .6 67 .2 1 94 .0 7 98 .5 90 .2 8 16 .8 4 0. 14 37 ,4 72 l 2t 13 .6 50 ,5 27 ,4 25 76 .2 9 88 .3 9 94 .4 0 98 .7 1 93 .5 5 30 .6 6 0. 35 37 ,4 87 l 3n 14 .1 45 ,5 86 ,5 46 83 .1 4 86 .3 5 93 .3 3 98 .0 8 92 .2 9 31 .8 6 0. 21 36 ,0 77 l 3t 14 .1 41 ,2 17 ,8 44 82 .6 6 77 .7 1 93 .2 1 98 .0 1 91 .1 2 25 .1 7 0. 19 35 ,3 58 l 4n 10 .7 38 ,2 73 ,5 07 77 .9 6 62 .2 4 92 .9 7 97 .8 5 88 .1 5 14 .3 8 0. 1 34 ,1 97 l 4t 10 .7 33 ,6 04 ,5 20 78 .6 1 55 .4 92 .7 7 97 .7 9 85 .5 3 10 .6 5 0. 07 33 ,5 19 l 5n 12 .3 41 ,5 24 ,9 01 78 .4 9 71 .8 1 93 .4 8 98 .0 4 90 .6 7 20 .4 8 0. 16 35 ,1 13 l 5t 12 .3 37 ,6 47 ,7 63 79 .9 9 66 .2 2 93 .0 4 97 .9 8 88 .9 0 17 .1 2 0. 13 34 ,5 63 r 1n 12 .5 40 ,7 60 ,4 56 81 .9 2 79 .2 6 93 .6 5 97 .9 5 92 .0 3 26 .0 1 0. 17 35 ,2 13 r 1t 12 .5 32 ,4 07 ,5 82 82 .1 4 62 .6 7 92 .8 5 97 .7 6 87 .6 9 15 .1 0 0. 12 32 ,8 04 r 2n 12 .7 36 ,5 90 ,3 33 86 .1 1 73 .4 7 93 .8 3 98 .1 2 91 .4 3 21 .7 0 0. 15 34 ,7 52 r 2t 12 .7 40 ,2 62 ,0 39 86 .9 9 80 .7 4 93 .8 7 98 .2 2 92 .3 4 26 .1 7 0. 21 34 ,8 62 r 3n 11 .9 38 ,2 79 ,1 57 73 .0 1 65 .6 9 93 .7 6 98 .0 6 90 .1 8 16 .2 4 0. 09 34 ,8 70 r 3t 11 .9 31 ,5 89 ,2 14 79 .7 8 58 .7 1 93 .5 1 97 .9 1 88 .0 9 11 .9 3 0. 07 35 ,2 74 r 4n 14 .6 40 ,8 55 ,5 11 77 .9 7 72 .7 9 93 .0 1 98 .0 2 90 .2 5 22 .2 6 0. 12 34 ,5 16 r 4t 14 .6 47 ,7 48 ,6 66 78 .0 7 85 .3 5 93 .3 5 98 .1 1 92 .2 7 31 .5 1 0. 18 39 ,3 37 r 5n 11 .6 37 ,2 43 ,8 95 75 .2 7 60 .6 4 93 .5 4 98 .0 4 88 .5 3 13 .4 8 0. 06 34 ,4 97 r 5t 11 .6 37 ,4 55 ,3 62 75 .7 9 62 .1 1 92 .5 9 97 .8 2 87 .4 8 15 .1 1 0. 07 33 ,1 75 m ea n 71 .6 4 93 .4 2 98 .0 6 90 .2 1 20 .8 6 0. 15 35 ,1 73 l = l ef t ( d is ta l) , r = r ig ht (p ro xi m al ), n = n or m al , t = tu m ou r mohd yunos r-i et al. 6 table 3. number of protein altering mutation and number of affected pathway proximal crc distal crc p value number of protein altering mutations 539 245 0.0001 number of affected pathways 5 3 0.66 profile of mutated crc-related genes and altered pathways we profiled mutated genes in our set of discovery patients based on thirty-three (33) genes that have been reported in the tumorigenesis of crcs, which is a compilation from ten publications[41–50]. we plotted each altered gene which was detected in our patients in figure 1a. altered genes were defined as any gene that has at least one or more proteinaltering mutations. fifteen (15) out of the 33 crc-related genes were altered in proximal cancers (29 mutations) and five crc-related genes were altered in distal cancers (eight mutations) as listed in table 4. we discovered that some of the nucleotide changes may lead to multiple protein amino acid changes. this is corresponding to different transcript isoforms. to explore the affected pathways and the differences between the two subsites of crc, we focused on major pathways involved in crc tumorigenesis[42,43,46–50]. this approach was adapted from ashktorab et al.[51]. an affected pathway is defined when at least one or more genes are altered in any pathway (figure 1b). we observed that 90% (n=9) of the crc patients shared an affected wnt signalling pathway with five genes being altered (12 mutations). rtk-ras and tp53 signalling pathways were also found to be altered in both proximal and distal crcs with six mutations in both pathways. we also discovered that the tgf-beta signalling (four mutations) and pi3k signalling (two mutations) pathways were exclusively altered in proximal crcs. overall, there were more altered pathways in proximal as compared to distal crcs. however, the number of different affected pathways between these two groups were not significant (p=0.66) (table 3). since this is discovery research, the lack of significance could be due to our small sample size. analysis using ingenuity pathway analysis (ipa) software identified the wnt signalling and growth factor signalling pathways as the most commonly affected (figure 3). figure 1. altered genes and pathway implicated in ten crc patients (five patients of each proximal and distal crcs). (a) the genes associated with existing treatment option or novel targeted therapies currently being investigated in clinical trials. (b) major signalling pathways altered in crcs. whole exome sequencing... 7 table 4. novel and known mutations in crc-related genes in proximal and distal crcs. sample id gene start end ref alt protein change exonic function dbsnp id cosmic id known / novel r1t apc 112175255 112175255 g t e1304x e1322x stop gain na cosm18702 known tp53 7578550 7578550 g a s88f s127f nonsynonymous na cosm216414 cosm3378368 cosm44226 cosm216412 cosm216413 cosm1637542 known r2t tp53 7577022 7577022 g a r174x r267x r306x stop gain rs121913344 cosm3388168 cosm10663 cosm1640820 cosm99947 known kras 25398284 25398284 c t g12d nonsynonymous rs121913529 cosm521 cosm1135366 known kras 25398284 25398284 c t g12d nonsynonymous rs121913529 cosm521 cosm1135366 known acvr2a 148683686 148683686 a k327fs k435fs frameshift deletion na cosm252949 known apc 112175639 112175639 c t r1432x r1450x stop gain rs121913332 cosm13127 known r3t atm 108141828 108141828 a g y959c nonsynonymous na na novel ctnnb1 41266136 41266136 t c s45p nonsynonymous rs121913407 cosm5663 known ctnnb1 41277302 41277302 a g r591g nonsynonymous na na novel igf2-as 2167618 2167618 a c t150p nonsynonymous na na novel kras 25380283 25380283 c t a59t nonsynonymous rs121913528 cosm546 cosm1562187 known r4t acvr1b 52387841 52387841 c t r489c r437c r530c nonsynonymous na na novel acvr1b 52379132 52379132 g a r379q r327q r420q nonsynonymous na na novel mohd yunos r-i et al. 8 r4t acvr2a 148683718 148683718 g a w337x w445x stop gain na na novel erbb2 37879658 37879658 g a r678 r648q r663q nonsynonymous na cosm436498 known erbb2 37864598 37864598 g a v84m v54m v69m nonsynonymous rs376524324 na known erbb3 56495023 56495023 g a r1127h nonsynonymous rs2271188 cosm1363018 cosm1363017 known erbb3 56478854 56478854 g a v104m nonsynonymous na cosm172423 cosm20710 cosm1152549 known fbxw7 153249456 153249456 c t r323q r361q r441q nonsynonymous na cosm1052092 cosm1052093 cosm1052091 cosm1052094 known lrp5 68201247 68201247 g a r733h r1314h nonsynonymous na na novel pten 89720812 89720812 a t321fs frameshift deletion na cosm5823 known tcf7l2 114925436 114925436 g a r482q r505q r499q nonsynonymous na na known tp53 7577138 7577138 c t r135q r108q r228q r267q nonsynonymous na cosm43923 cosm3691863 cosm1290766 cosm3691864 known tp53 7578458 7578458 g a r26c r119c r158c nonsynonymous na cosm1750371 cosm984954 cosm984957 cosm3932746 cosm984958 cosm984956 cosm43848 known r5t apc 112176559 112176559 tt gc 5214_5215gc 5268_5269gc 5268_5269gc nonframeshift substitution na na novel atm 108106477 108106477 g t g138x stop gain na na novel smad4 48573529 48573536 gagcaatt 38_40del frameshift deletion na na novel l1t apc 112162896 112162896 t g y482x y500x stop gain na na novel l2t apc 112175212 112175216 aaaag 1289_1291del 1307_1309del frameshift deletion na cosm18764 known erbb3 56478854 56478854 g c v104l nonsynonymous na cosm160824 known whole exome sequencing... 9 l3t tp53 7577121 7577121 g a r141c r114c r234c r273c nonsynonymous rs121913343 cosm3355991 cosm10659 cosm1645518 cosm99933 known l4t apc 112175322 112175322 c g s1326x s1344x stop gain na na novel fbxw7 153249385 153249385 g a r347c r385c r465c nonsynonymous na cosm1154293 cosm170727 cosm170726 cosm170725 cosm22932 known l5t ctnnb1 41266071 41266100 gtcactggcagcaa23_33del nonframeshift deletion na na novel tp53 7578263 7578263 g a r64x r37x r157x r196x stop gain na cosm1640847 cosm99667 cosm99666 cosm99668 cosm3378446 cosm99665 cosm10705 known figure 2. diagram illustrating the number of patients with alteration in the crc-associated genes. proximal distal mohd yunos r-i et al. 10 most frequently altered genes in proximal and distal crcs we discovered that the apc gene (six mutations) was the most frequently mutated gene with a frequency of 60% (n=3) in both proximal and distal crcs. the second most frequently mutated gene in both proximal and distal crc was tp53 gene (six mutations) with the frequency of 40% (n=2). this suggested that apc and tp53 did not have predominance for either side of the colon. interestingly, we found that the kras gene (three mutations) and atm gene (two mutations) were uniquely altered in proximal crcs with the frequency of 60% and 40% respectively (figure 2). among all of the frequently altered genes in this set of analysis, we identified three novel mutations in apc gene, namely, a non-frameshift substitution (apc g.112176559tt>gc) in tumour r5t, stop-gain (apc g.112162896t>g) in tumour l1t, stop-gain (apc g. 112175322c>g) in tumour l4t and two novel mutations in atm gene of tumour r3t (atm g.108141828a>g) and r5t (atm g.108106477g>t) (table 5). sanger sequencing successfully confirmed each of the 17 somatic mutations we had detected in these four genes. recurrent variants and mutated genes in this discovery set of ten malaysian crc patients, five genes with one mutation were demonstrated in at least two individuals of proximal crc patients. on the other hand, there is no recurrent mutation identified in individuals of distal crcs (table 6). detailed analysis revealed that the minimum reads covering the mutated allele were 35 reads with a minimum of 1% of the total reads containing the alternate base. this was found in the variant presented in c9orf50 gene. the variant in the kras gene (kras g.25398284c>t) had the highest variant coverage with 121 reads and 52 reads (63%) contained the variant in sample r2t. the same variant was detected in sample r5t with 96 reads and 48 of the reads (50%) contained the alternate base. figure 3. the most commonly mutated canonical pathway. the key mutated genes detected in proximal crc are highlighted in pink while mutated genes detected in distal crcs are highlighted in blue. whole exome sequencing... actionable alterations in proximal and distal crc we looked at the drug-gene interaction by annotating against the drug gene interaction database (dgidb) and identified ten genes implicated in crc tumorigenesis that are clinically relevant, including targets of new and existing therapies and genes. twenty-one (21) variants in ten genes and eight variants in three genes were identified in proximal and distal crcs, respectively. notably, 80% (8/10) of crc patients harboured at least one actionable alteration (range one to seven alterations) that has been linked to a clinical treatment option or is currently being investigated in clinical trials for novel targeted therapies. for example, in the present study, 5fu-based chemotherapy is considered to target patients with wild type tp53, which includes patients r2, r3, r5, l1, l2 and l4 (figure 1a). validation against tcga data we performed external validation using somatic mutation data from 618 patients (consisting of proximal and distal crc patients) in the tcga data set[38]. we successfully validate that znf337 and c9orf50 genes, both are recurrently mutated genes identified from our discovery data set, were exclusively mutated in proximal patients in tcga patients. while gpr6 gene was found to be less frequently mutated in tcga patients (about 5%), we discovered that two out of five patients in our proximal patients (40%) harboured one known gpr6 mutations (gpr6 g. 110301081g>a). we performed a comparison between mutation frequency in proximal versus distal crc of tcga patients. subsequently, we profiled our patients based on the significantly mutated genes from the tcga data set. we discovered that at least eight genes were found to be predominantly mutated in proximal crc (p=0.0032) (table 7). 11 tp53/ chr17 r1t 7578550 7578550 g a s127f s88f nonsynonymous na cosm216414 known r1t 7577022 7577022 g a r174x r147x r306x r267x stop gain rs121913344 known r4t 7577138 7577138 c t r135q g323a r108q r267q r228q nonsynonymous na cosm43923 known r4t 7578458 7578458 g a r26c r158c r119c nonsynonymous na known l3t 7577121 7577121 g a r141c r273c r234c nonsynonymous rs121913343 known l5t 7578263 7578263 g a r64x r37x r196x r157x stop gain na known kras/ chr 12 r1t 25398284 25398284 c t g12d non synonymous rs121913529 cosm521 known r2t 25398284 25398284 c t g12d non synonymous rs121913529 cosm521 known r3t 25380283 25380283 c t a59t non synonymous rs121913528 cosm546 known atm/ chr11 r3t 108141828 108141828 a g y959c nonsynonymous na na novel r5t 108106477 108106477 g t g138x stop gain na na novel na = not available mohd yunos r-i et al. table 6. recurrent variants and mutated genes in proximal crc sample id gene/ chr start end ref alt protein change exonic function dbsnp id cosmic id known / novel r2t, r5t c9orf50/ chr9 132377900 132377900 c r248fs frameshift deletion na na novel r1t, r4t gpr6/ chr6 110301081 110301081 g a a256t, a271t nonsynonymous na cosm3429854 cosm3429853 known r1t, r2t kras/ chr12 25398284 25398284 c t g12d nonsynonymous rs121913529 cosm521 cosm1135366 known r3t, r4t znf337/ chr20 25657029 25657029 g a r299x stop gain na na novel r3t, r4t znf783/ chr7 148963763 148963763 g a r121h nonsynonymous na na novel na = not available gene/ chr sample id start end ref alt protein change exonic function dbsnp id cosmic id known / novel apc/ chr5 r1t 112175255 112175255 g t r1432x r1450x stop gain na cosm18702 known r2t 112175639 112175639 c t 5214_5215gc 5268_5269gc stop gain rs121913332 cosm13127 known r5t 112176559 112176560 tt gc e1304x e1322x nonframeshift substitution na na novel l1t 112162896 112162896 t g y482x y500x stop gain na na novel l2t 112175212 112175216 aaaag 1289_1291del 1307_1309del frameshift deletion na cosm18764 known l4t 112175322 112175322 c g s1326x s1344x stop gain na na novel table 5. novel and known mutations in most frequently mutated genes in proximal and distal 12 discussion crc is a heterogeneous disease with the genetic landscape and clinical outcomes depend on the anatomic location of cancer. many efforts have been made to unveil the genetic alterations and molecular features of colorectal cancer[38,51]. studies show that these alterations would determine the prognosis and response to treatment[8,52,53]. nevertheless, how the anatomical location could have an impact on the molecular features and more importantly, the prognosis, is unknown. in this study, we analyzed somatic alterations between proximal and distal crc in malaysian patients. it has provided an insight into the identification of known and novel somatic mutations that suggest a relationship between the genomic alterations, cellular pathways, actionable genes and anatomical location of the tumour. overall, we discovered that proximal crcs exhibited a higher number of somatic mutations and altered pathways as opposed to distal crcs. proximal crcs has been proven to have increased mutational burden, with higher rates of microsatellite instability as compared to distal colon and rectal cancers[54]. these results may account for the poor prognosis of proximal crc patients. based on crc tcga data published in 2012 (accessed through cbioportal), 16.1% of distal crc and 47.1% proximal crc were microsatellite instable (msi), while 83.2% of distal and 52.7% of proximal crc were microsatellite stable (mss)[55]. even though generally, msi crc accounted for approximately 15% of sporadic crcs, the frequency of msi was higher in proximal crcs[55]. msi cancers were shown to have eight times more somatic nonsynonymous variants than mss cancers[56]. however, we are unable to perform msi determination status due to the lack of tissue for staining. the wnt signalling and egfr pathways are among the commonly affected pathways in crc tumorigenesis[57,58]. to identify possible pathway differences in proximal and distal, we performed pathway analysis using ipa. we discovered that the most enriched pathways are the wnt and the growth factor signalling pathways. ninety per cent (90%) of our patients in this discovery set, irrespective of their anatomical location, had a mutation in one or more members of the wnt signalling pathway, predominantly in apc. the frequency of mutated apc gene across proximal and distal crc patients in our study is 60% (figure 2). this is in concordance with recent findings where they discovered over 50% of the recruited patients in the study exhibited altered wnt signalling pathway with apc being the most significantly mutated gene[38,59]. wnt signalling pathway activation is required for maintenance of colorectal tumours harbouring apc mutations[60]. inactivating apc mutations occur in about 85% of crcs, resulting in β-catenin stabilization and increased signalling through the tcf/lef transcription factors. mutant β-catenin is free to enter the nucleus and constitutively activates transcription through tcfs[61]. β-catenin inhibition in vivo strongly inhibited the growth of established apc-mutant colorectal tumour xenografts[60]. in our small set of patients, those with wild type apc gene (r3t and l5t) harboured at least one mutation in the ctnnb1 gene (figure 1a), resulting in altered wnt signalling pathway. it has been shown that up to 50% of crc with wild type apc gene were found to have ctnnb1 mutations[62]. from our analyses, we also found that the tp53 signalling pathway was altered in both proximal and distal crcs. with a frequency of 40%, tp53 is the second commonest altered gene in our study. similarly, tp53 gene mutations in an iranian cohort of crc patients occurred as frequent as in other studies with equal distribution, suggesting no differences across the anatomic location[63,64]. however, our findings were in contrast with another study which different mutation spectra of tp53 was observed depending on proximal or distally located tumour[65]. alteration of tp53 may have different prognostic significance depending on the ethnic group[66], an anatomic subsite of the colon[65,66] and stage of disease[67,68]. there is convincing evidence that patients with wild-type tp53 gained survival benefit from the use of 5-fluorouracil (5fu)-based chemotherapy[69]. conversely, patients with mutant p53 do not gain this survival benefit[70]. we identified six patients with wild type tp53 and these patients could potentially benefit table 7. validation of mutation frequency in proximal and distal crc of tcga patients this study tcga crc gene proximal crc (%) distal crc (%) p-value proximal crc (%) distal crc (%) p-value kras 60 0 0.0001 29 26 n.s atm 40 0 0.0001 10 4 n.s znf337 40 0 0.0001 9 0 0.003 c9orf50 40 0 0.0001 3 0 n.s gpr6 40 0 0.0001 5 2 n.s znf783 40 0 0.0001 4 1 n.s pik3ca 20 0 0.0001 28 14 0.02 acvr2a 40 20 0.0032 15 3 0.005 tp53 40 60 0.0071 40 61 0.004 n.s = not significant whole exome sequencing... 13 from 5fu based chemotherapy. however, we postulated that four tp53 mutated patients, in our study, might not gain a survival benefit from 5fu treatment. in addition, the atm gene which plays a significant role in the tp53 signalling pathway was found to be mutated in 40% of the proximal crc patients. for the patients with mutated atm and wild type tp53 (r3 and r5) they have the additional option of the atm/atr kinase inhibitor as the alternative treatment. a study by batey and colleague demonstrated that atm is a valid target for the development of drugs designed to improve the activity of certain cytotoxic anticancer therapies[71]. small molecule inhibitors of atm are currently in preclinical and clinical development. ku59403 is the first atm inhibitor to show good tissue distribution and significant chemosensitization in in vivo models of human cancer, without major toxicity. this preclinical data of the atm inhibitor was utilised to support the future clinical development of atm inhibitors[71]. the tumour progression and response towards treatment are believed to be dependent on both atm and tp53 status[72]. for instance, atm signalling is necessary for the survival of tp53-deficient cells after dna damage; whereas in cancers with wild type tp53, inactivation of atm allows the survival of genomically unstable cells and induces chemoresistance[73]. in tp53-deficient settings, inhibition of atm dramatically sensitizes tumours to dna-damaging chemotherapy, whereas, conversely, in the presence of functional tp53, inhibition of atm actually promotes resistance effect towards chemotherapy[73]. thus, the specific set of alterations induced during tumour development play an important role in determining both the tumour response towards chemotherapy and specific susceptibilities to targeted therapies in a given cancer type. we highlighted here five genes which were of particular interest due to being recurrently mutated exclusively in proximal crcs. out of these six recurrently mutated genes, kras and gpr6 are known to be involved in cancer, as reported in cosmic. in our small set of data, we found three patients of proximal crc (r1, r2 and r3) harbouring at least one of kras druggable targeted variants in kras exon 2 (g12d) and kras exon 3 (a59t) and none were found in distal crc patients. previous studies also identified that kras-mutated carcinomas are more frequently found in the proximal colon[74,75]. activating kras mutations have been proven to predict a lack of response to anti-egfr therapy in patients with metastatic colorectal cancer (mcrc). the combination regimen of panitumumab and oxaliplatin have no value in metastatic colorectal cancer patients with mutated kras[76]. a substantial group of mcrc patients with mutated kras acquired resistance to anti-egfr treatment cetuximab and a study has suggested an early initiation of mek inhibitor to delay or reverse the drug resistance[77]. testing for kras exon 2 mutation is currently recommended to guide decisions regarding the eligibility for anti-egfr therapy in mcrc. profiling of tumour-specific genetic marker will help to guide the selection of patients who are likely to have a response to a particular treatment and prevent adverse effects on those who are unlikely to benefit. the gpr6 gene was mutated uniquely in two patients with proximal crc. gpr6 protein plays an important role in signal transmission and regulates many cellular functions. there are pieces of evidence implicating the gpr6 protein and its downstream signalling targets are involved in cancer initiation and progression, where it can influence cell growth and survival through the activation of akt/ mtor and mapk pathways[78]. cancer cells may exploit this pathway which can result in the promotion of tumour growth, angiogenesis and metastasis to distant sites[79]. findings of recurrently altered gpr6 gene unique to the proximal crc patients may explain the poor prognosis and low survival rate in these patients. by directly targeting gpr6 or its downstream signalling components, it may help to identify novel therapeutic opportunities for cancer prevention and treatment. however, further investigations will be warranted to examine the potential impact of this mutation. exome sequencing may lead to the discovery of novel targets, driver mutations as well as novel colorectal cancer-predisposing mutations. this application is getting more common in clinical practice and represents a costeffective approach to characterize somatic mutations. this discovery study in our own local crc patients provides a number of insights into the differences in genetic landscape of proximal and distal and identifies potential therapeutic targets in particular to the anatomic subsites of crc, specifically in malaysian patients. nevertheless, further study in a larger series of samples coupled with functional studies will be needed to confirm the identified variants and determine their role in the genesis of proximal and distal crcs. conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. author contributions rimy, nsam and rj conceived and designed the experiments. rimy, ss, nsmn, mrar and zar performed the experiments. rimy, nsam and jkss analysed the data. is and lm provide the tissue samples. imr is the pathologist who assessed the tumour percentage of the samples. rimy and nsam wrote the manuscript and prepare the figures and tables. na, nsam and rj provide critical reviews. funding the study was funded by a grant from the ministry of science, technology and innovation (07-05-mgigmb016). acknowledgements the authors thanked ms chia chiu lim for her extensive help in the validation process of the identified variants. this manuscript has been released as a pre-print at[80]. mohd yunos r-i et al. 14 reference 1. bray f, ferlay j, soerjomataram i, et al. globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin, 2018; 68(6): 394–424. 2. zainal ariffin o. and nor saleha it. national cancer registry report: malaysia cancer statisticsdata and figure 2007. 2011. ministry of health, malaysia. 3. lim gcc, rampal s, and halimah y (eds). cancer incidence in peninsular malaysia 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material the aligned whole-exome sequencing data in .bam format were deposited at the ncbi sequence read archive (sra) at http://www.ncbi.nlm.nih.gov/sra with accession number prjna382764. mohd yunos r-i et al. pmmb 2023, 6, 1; a0000340. doi: 10.36877/pmmb.0000340 http://journals.hh-publisher.com/index.php/pmmb review article the burden of vibrio sp. infections – a scoping review ke-yan loo1,9, jodi woan-fei law1,2, loh teng-hern tan1,3, priyia pusparajah4, sunny hei wong5,6, kok-gan chan1,7, learn-han lee1,8*, and vengadesh letchumanan1,9,* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia; ke.loo@monash.edu (k-yl) 2next-generation precision medicine and therapeutics research group (nmet), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia; jodi.law1@monash.edu (jw-fl) 3innovative bioprospection development research group (inbiod), clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) 4medical health and translational research group (mhtr), microbiome and bioresource research, strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, 47500, selangor darul ehsan, malaysia; priyia.pusparajah@monash.edu (pp) 5lee kong chian school of medicine, nanyang technological university, singapore 308232, singapore; sunny.wong@ntu.edu.sg (shw) 6state key laboratory of digestive disease, department of medicine and therapeutics, the chinese university of hong kong, hong kong sar, china 7division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, kuala lumpur 50603, malaysia; kokgan@um.edu.my (k-gc) 8sunway microbiomics centre, school of medical and life sciences, sunway university, sunway city 47500, malaysia 9pathogen resistome virulome and diagnostic research group (pathrid), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia *corresponding author: vengadesh letchumanan; pathogen resistome virulome and diagnostic research group (pathrid), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor, malaysia; vengadesh.letchumanan1@monash.edu (vl); learn-han lee; sunway microbiomics centre, school of medical and life sciences, sunway university, sunway city 47500, malaysia; learnhanl@sunway.edu.my (l-hl) received: 13 june 2023; received in revised form: 21 july 2023; accepted: 26 july 2023; available online: 28 july 2023 abstract: vibrios are a group of gram-negative bacteria ubiquitous in our surrounding environments and responsible for various clinically significant human infections. the three species responsible for human illness are vibrio cholerae, vibrio parahaemolyticus, and pmmb 2023, 6, 1; a0000340 2 of 25 vibrio vulnificus. v. cholerae causes cholera which may result in severe dehydration and death without timely treatment, v. parahaemolyticus infection causes gastroenteritis while v. vulnificus infections typically manifest as wound or soft tissue infections with poor prognosis, including amputation or death. available data on the epidemiology and clinical burden of vibrio infections were compiled systematically following a literature review, and 149 relevant studies published between 2010 to 2022 were identified. cholera represents the majority of vibrio infections, affecting individuals of all ages and gender, while v. parahaemolyticus infections were found to affect mostly adult males. v. vulnificus infections were mostly reported in males over 50 years old with pre-existing co-morbidities. this review's findings may guide planning and implementing preventative measures in affected regions to prevent future vibrio infections, disease transmission, and major outbreaks. keywords: vibrio infections; cholera; vibrio cholerae; vibrio parahaemolyticus; vibrio vulnificus; pathogens; sdg 3 good health and well-being 1. introduction vibrios are a group of gram-negative bacteria that can potentially cause infections in various species from aquatic animals to humans. these bacteria are transmitted to humans by environmental exposure or by consuming contaminated water or seafood [1]. the most common species within vibrio sp., which are pathogenic to humans are vibrio cholerae, vibrio parahaemolyticus, and vibrio vulnificus. these three vibrio species represent those with the most significant impact on human health based on statistics from reports of outbreaks and infections caused by these bacteria [2-8]. these vibrios are autochthonous to aquatic environments, and the warming of the oceans due to climate change has significantly increased their populations and distribution worldwide [9]. as shown by vezzulli et al., when there is an increase in sea surface temperatures, vibrio concentrations in the waters increase, and subsequently, vibrio infections outbreaks also increase globally [9]. cholera is a deadly disease caused by v. cholerae with serogroup o1 and o139; it presents as an acute diarrheal infection that can lead to potentially fatal dehydration [10]. symptoms of cholera include watery stool, nausea and vomiting, dehydration, abdominal pain, and leg cramps [2]. it is estimated that there are 1.3 to 4 million cases of cholera, with 21,000 to 143,000 deaths worldwide due to cholera annually [11]. the world health organization reported a surge in cholera cases globally since 2021 following years of decline; this increase can be attributed to poor health systems and climate change. in addition, when the covid-19 pandemic hit in 2021, healthcare systems worldwide were strained dealing with the novel coronavirus, resulting in a lack of resources and medical personnel to care for other disease outbreaks such as cholera [12]. v. cholerae possesses several features which optimize its success as a pathogen – it utilizes biofilms for survival in the environment and provides protection against host gastric acid barriers during infection. this organism is also adapted to maximize its chances of pmmb 2023, 6, 1; a0000340 3 of 25 colonizing the gut. during the early stages of infection, where v. cholerae cell density is low, virulence gene expression is active for virulence factors such as cholera toxin to cause diarrhea and toxin coregulated pilus for colonization in the gut. still, as v. cholerae begins to proliferate, diarrhea and environmental changes in the gut lead to the clearance of competitor microbes, disrupting the host gut microbiome. the profuse watery diarrhoea at this stage can lead to lethal dehydration. as cell density of v. cholerae and quorum sensing molecules increase during late infection, the bacteria detach from the intestinal mucosa, entering the luminal contents to be excreted in the stool and disseminated into the environment, causing further spread of cholera [13]. rehydration therapy and hydration maintenance are the primary treatment for cholera patients. antibiotics are recommended for severely ill patients or vulnerable groups such as pregnant patients; antibiotic therapy will be given based on the local antimicrobial susceptibility profile of the pathogen [14]. cholera vaccines have also been made available, and they are administered via the oral route. the vaccination is recommended for individuals traveling or residing in areas where local transmission of cholera occurs and during cholera outbreaks [15]. vibriosis is commonly caused by v. parahaemolyticus, and v. vulnificus, and can manifest as gastroenteritis or wound and soft tissue infections where necrotizing fasciitis and sepsis are widely reported [1, 16-19]. in the united states, statistics from the centers for disease control and prevention report 80,000 illnesses and 10 deaths caused by vibriosis annually, with the highest incidence of infection reported during the warmer months from may to october [20]. v. parahaemolyticus is often identified as the etiological agent in gastroenteritis outbreaks [17, 21, 22]. typical symptoms are watery diarrhea, abdominal cramps, nausea and vomiting, fever, and headache. during infection, v. parahaemolyticus utilizes multivalent adhesion molecule 7 to adhere to host cells, leading to up-regulation of other virulence factors. this is followed by upregulation of thermostable direct hemolysin (tdh) and tdhrelated hemolysin (trh) gene expression resulting in hemolysis of red blood cells, formation of pores in the cell membrane as well as alterations in ion flux thus causing gastroenteritis [23]. gastroenteritis caused by v. parahaemolyticus is often self-limiting and resolves within 72 days from onset of symptoms. medical intervention in the form of antimicrobial treatment commences only when the infection does not resolve or has progressed to systemic infection [14]. v. vulnificus infections include wound or soft tissue infections caused by wound exposure to contaminated water. wound infections typically present with symptoms such as fever, tenderness, swelling, warmth, pain, and discharge in the affected areas [20]. in addition, ingesting seafood contaminated with v. vulnificus can also result in severe systemic infection. the commonly reported clinical characteristics of infection are fever, chills, nausea, hypotensive septic shock, and formation of secondary lesions on the patient's extremities [24]. this opportunistic pathogen can evade destruction by the host’s gastric acid through the upregulation of lysine decarboxylase and manganese superoxide dismutase. these processes ultimately result in acid neutralization and reduction of oxidative stress, thus enabling v. vulnificus to evade host defenses in the upper gastrointestinal tract. the bacteria can then pmmb 2023, 6, 1; a0000340 4 of 25 penetrate the host intestinal wall and enter the bloodstream. v. vulnificus also expresses capsular polysaccharide (cps) on its cell surface to evade the host immune response. in addition, the exotoxin, vibrio vulnificus hemolysin a (vvha) expressed by v. vulnificus causes iron release from hemoglobin, causing a hemolytic effect. vvha also elicits cytotoxic effects on the host cells by causing cell death via pore formation in the cellular membrane. vibrio vulnificus metalloprotease (vvpee) also plays a role in causing tissue necrosis, particularly during systemic infections where edema and bullous lesions form [25]. furthermore, lipopolysaccharide (lps) of v. vulnificus that are released during cell lysis mediates endotoxic shock in severe disease which can result in anaphylactic shock and death [25, 26]. the rapid development of v. vulnificus infections requires prompt diagnosis and delivery of antibiotic therapy to prevent worsening of the infection that can lead to necrosis, resulting in amputation or fatality [14]. an additional challenge is the increased antibiotic resistance of these vibrio species, which has decreased the efficacy of the current antimicrobial treatments [27-30]. thus, exploring alternative treatments and preventative methods to manage vibrio infections and disease outbreaks is crucial. in addition, as reports of vibrio populations and spread continue to increase worldwide, an increase in infections or disease outbreaks due to these vibrio species is expected in the foreseeable future. thus, this review aims to study the clinical burden of infections caused by v. cholerae, v. parahaemolyticus, and v. vulnificus including the incidence of cases, mortality rates, symptoms, and risk factors. the insight provided by this study can serve as a guideline to implement more effective preventative measures and methods for disease or outbreak management based on the current trends of vibrio infections. 2. methods this scoping review was done in accordance with the preferred reporting items for systematic reviews (prisma) guidelines [31]. the search was conducted across three databases: ovid medline, embase, and pubmed by using the mesh terms “vibrio or vibriosis” and “clinical outbreak or population surveillance or incidence or burden or epidemiology”. the inclusion criteria for the search were original articles in english on vibrio infections, including cholera and vibriosis, with no time or country restrictions. since the vibrio species with the most significant impact on human health are v. cholerae, v. parahaemolyticus, and v. vulnificus, publications reporting on infections or disease outbreaks pertaining to these species were included in this scoping review. publications that report on the incidence of the relevant vibrio infections or outbreaks, attack rates, mortality rates, and risk factors for the disease were included. for example, national surveillance reports, epidemiology studies, and case-control studies were included in reporting on the significance of certain risk factors on disease occurrences. prevalence studies of vibrios in animals, seafood, environment, and humans without clinical data were excluded. in addition, publications related to vibrio infections that did not contain any inclusion criteria, such as studies on temporospatial effects on vibrio infections, estimation or projection of vibrio diseases, estimation of disease transmission, and vibrio vaccine studies were excluded. pmmb 2023, 6, 1; a0000340 5 of 25 from the search, the publications identified for further assessment were downloaded and imported into covidence, a primary screening and data extraction tool [32]. in covidence, duplicates were removed, and the remaining were screened for relevance based on the titles and abstracts. the full-text of the studies that passed the screening process were then assessed for inclusion, and data from included studies were extracted. the search and data extraction were done by one author and cross-checked by another author. 3. results 3.1. study selection a total of 3908 studies were identified from the search and imported into covidence for duplicate removal and primary screening, with 1416 duplicates removed, leaving 2492 studies for screening based on the article titles and abstracts. a total of 1240 studies were found to be irrelevant, the full-text for the remaining 1252 studies was retrieved and assessed for eligibility for inclusion. a total of 1103 studies were excluded: prevalence studies in humans without clinical data (248), prevalence studies in animals or seafood (165), prevalence studies in the environment (114), studies with irrelevant content for the review (184), publications with wrong study designs (347), articles not in english (32), studies which did not specify vibrio species (9), and articles with no full text (4) (figure 1). figure 1. study selection presented in prisma flow diagram. the results included data from various countries and their respective references where published data were obtained. the countries are: bangladesh [33-54], cameroon [55-58], canada [21], china [3, 5, 16, 19, 59-74], democratic republic of congo [75-78], ethiopia [79, 80], ghana [81-85], guinea [86], guinea-bissau [87], haiti [88-94], india [6, 95-105], iran [106-109], iraq [110], japan [4, 111113], kenya [114-119], korea [17, 18, 120-122], malawi [123], mozambique [124, 125], nepal [126], niger pmmb 2023, 6, 1; a0000340 6 of 25 [127], nigeria [128, 129], pakistan [130], papua new guinea [131, 132], philippines [133], sierra leone [134], singapore [135], south sudan [136], sweden [137], tanzania [138], thailand [139], togo [140], uganda [141-152], united states of america (usa) [7, 153-161], vietnam [162, 163], yemen [164-166]. additional data were obtained from two studies that published data from countries within africa [2, 167]. the data collected from the included studies were published from 2010-2022. among the studies included were epidemiological investigation and surveillance reports (100), case-control studies (28), cross-sectional studies (7), prospective studies (3), a prospective cohort study, a randomized controlled trial, retrospective case-control studies (3), a retrospective cohort study, a retrospective cross-sectional study, and retrospective studies (6) were also included. 3.2. incidence of vibrio infections 3.2.1 cholera of all the studies included in this review, 101/149 (67.8%) reported the incidence and deaths of v. cholerae infections (table 1). cholera was mainly reported in africa, asia, north america, and oceania. the democratic republic of congo reported the highest number of cholera cases, with one major study reporting recurrent outbreaks from 2008 to 2017, recording 270,852 cholera cases within that period [78]. two studies investigating the incidence of cholera in various african countries reported a total of 251,210 cases; 47% (8/17) of the countries included in this review had two or more studies reporting the incidence of cholera, with the highest reporting rates in uganda, followed by kenya. in addition, 82.4% (14/17) of the included studies from africa reported deaths due to cholera. the number of reported deaths is directly proportionate to the number of total reported cases, i.e., the higher the number of total reported cases, the higher the number of total deaths. in asia, the highest number of studies on the incidence of cholera cases came from bangladesh, with 19 studies, followed by india, with 12 studies. in yemen, a significant research analyzing surveillance data from 2016-2018 related to the cholera epidemic in yemen reported over 1.1 million cholera cases [166]. out of the 12 countries which reported cholera cases in asia, only 50% (6/12) of the studies reported cholera deaths, indicating that fatality rates due to cholera is lower in asia when compared to africa. data from north america came from three countries: the dominican republic, haiti, and the united states. haiti reported the highest number of cholera cases and deaths, with six studies reporting 1 089 923 cases and 14 112 deaths from 2013 to 2019. in oceania, two studies from papua new guinea reported 61 cholera cases with no deaths recorded. pmmb 2023, 6, 1; a0000340 7 of 25 table 1. reports of cholera and deaths caused by cholera by country. region/country number of studies total reported cases total reported deaths africa cameroon 3 [55, 57, 167] 27946 657 democratic republic of the congo 3 [75, 76, 78] 290729 5666 ethiopia 2 [79, 80] 4329 48 ghana 5 [81-85] 3002 4 guinea 1 [86] 2712 n/a guinea-bissau 1 [87] 14222 225 kenya 6 [114-119] 80144 2917 malawi 1 [123] 1171 21 mozambique 2 [124, 125] 27294 220 niger 1 [127] 16328 578 nigeria 2 [128, 129] 1649 54 sierra-leone 1 [134] 49 n/a south sudan 1 [136] 1451 44 tanzania 1 [138] 11921 n/a togo 1 [140] 12676 554 uganda 11 [141-151] 30297 674 asia bangladesh 19 [33, 34, 36-48, 51-54] 24475 13 china 1 [74] 46 n/a india 12 [6, 95-105] 41374 34 iran 3 [106, 107, 109] 2022 n/a iraq 1 [110] 136 3 korea 1 [121] 3 n/a nepal 1 [126] 111 2 pakistan 1 [130] 83 n/a philippines 1 [133] 55 2 singapore 1 [135] 210 n/a vietnam 2 [162, 163] 67 n/a yemen 3 [164-166] 1118929 2461 north america dominican republic 1 [77] 42 n/a haiti 6 [88, 90-94] 1082923 14112 united sates 2 [153, 156] 161 n/a oceania papua new guinea 2 [131, 132] 61 n/a pmmb 2023, 6, 1; a0000340 8 of 25 data from the included studies indicate no significant difference in the occurrence of cholera between males and females. the incidence of cholera was relatively equally distributed among both genders. the median age for cholera infections ranged between 15 to 44 years. cholera was most often reported in young adults, followed by adults and young children. results from this review show that cholera is an important disease in africa, as many cases and deaths have been reported in this region. countries in africa which only have one study reporting on cholera have also reported significantly higher number of cholera cases [87, 123, 127, 140] and deaths than countries in other regions with only one study reporting on cholera [74, 110, 135]. 3.2.2 vibriosis data on vibriosis represents a smaller fraction of the included studies, with 25/149 (16.8%) reporting on the incidence of v. parahaemolyticus infections (table 2), and 18/149 (12%) reporting on the incidence and deaths of v. vulnificus infections (table 3). v. parahaemolyticus infections were most commonly reported in asia, with the majority (63.2%, 12/19) of studies from china. countries in east asia had the highest number of v. parahaemolyticus infections, followed by south asia and southeast asia. however, the united states had five studies presenting 6169 cases of v. parahaemolyticus infections in north america. v. parahaemolyticus infections are typically non-fatal, thus, zero deaths have been reported. the included studies show that v. parahaemolyticus infections occurred more frequently in males than females, and most of these infections were reported in adults. interestingly, one study showed that the prevalence of v. parahaemolyticus infections was higher in males when the age group was less than 15. still, the prevalence of v. parahaemolyticus infections was higher in middle-aged and older women [111]. v. vulnificus infections are most commonly reported in asia, specifically in east asia, with the majority (75%, 9/12) of reports coming from china. despite high numbers of v. vulnificus infections in china, only 11 deaths were reported. in north america, six studies on v. vulnificus were from the united states, reporting 3400 cases and 76 deaths. data from the included studies show that males make up most of v. vulnificus infections, and the infections typically occur in older adults over 50 years. the severity of v. vulnificus infections is apparent as there are fewer reported cases than v. parahaemolyticus infections. still, the reported deaths for v. vulnificus infections are significantly higher throughout affected regions. pmmb 2023, 6, 1; a0000340 9 of 25 table 2. incidences of v. parahaemolyticus infections by country. region/country number of studies total reported cases asia china 12 [3, 5, 16, 60-64, 66, 67, 70, 74] 16501 india 1 [104] 137 japan 2 [111, 112] 1223 korea 3 [17, 120, 122] 1014 thailand 1 [139] 32 north america canada 1 [21] 82 united states 5 [7, 153, 159-161] 6169 table 3. incidences of v. vulnificus infections and deaths due to v. vulnificus infections by country. region/country number of studies total reported cases total reported deaths asia china 9 [19, 59, 65, 68-70, 72-74] 530 11 japan 2 [4, 113] 49 7 korea 1 [18] 5 n/a north america united states 6 [7, 153, 157, 159-161] 3400 76 *n/a: data is unavailable. 3.3 risk factors among the included studies in this review, 55/149 reported vibrio infections' risk factors (table 4). the risk factors identified for cholera among the studies were the source of drinking water, source of water for domestic activities, sanitation or hygiene practices, age, education level, eating out, exposure to cholera patients, household income, consumption of pmmb 2023, 6, 1; a0000340 10 of 25 raw food, residing in slums, consumption of food contaminated with v. cholerae, and accessibility to healthcare. risk factors associated with v. parahaemolyticus were consuming raw or undercooked seafood, shellfish, and eating outside the home. as for v. vulnificus infections, the identified risk factors are underlying co-morbidities, male gender, consumption of raw or undercooked seafood, age, exposure to contaminated seawaters, extensive skin lesions, and low platelet count. it is also vital for local governments to strengthen food safety measures by targeting food handlers, vendors, and eating places. also, education on this disease should be given to all food handlers and the community. table 1. risk factors for vibrio infections were identified from all studies. vibrio species/risk factor number of times reported vibrio cholerae source of drinking water 22 source of water for domestic activities 15 sanitation/hygiene practices 15 age 12 education 9 eating out 8 exposure to person with cholera 7 household income 4 consuming raw food 3 residence in slum 1 consumption of contaminated food 1 vibrio parahaemolyticus consumption of raw seafood 2 shellfish consumption 2 eating out 1 vibrio vulnificus underlying chronic disease 5 male 2 consumption of raw or undercooked seafood 2 age 2 exposure to contaminated seawater 1 4. discussion vibrio infections are without doubt a global public health concern, causing significant numbers of diseases and deaths over recent years. data from this review shows that v. cholera remains the most significant vibrio infection based on the incidence of cholera cases, followed by v. parahaemolyticus, and v. vulnificus. pmmb 2023, 6, 1; a0000340 11 of 25 in terms of risk factor analysis which may assist with targeted preventive methods for cholera, many studies which report high incidences of cholera also reported that there was a lack of access to clean water sources for drinking or domestic activities and poor sanitation and hygiene practices were also identified as the contributing factors for disease outbreaks [51, 103, 116, 129, 133, 142, 149]. the primary water sources of the residents who reported cholera infections were rivers or lakes. the waters were often contaminated by fecal matter due to open defecation or being near the latrines of the residents [51, 83, 147, 151]. the bacterial flora from fecal matter can potentially alter the bacterial flora in the nutrient-rich environments of rivers and lakes, resulting in plankton blooms often associated with cholera outbreaks [168]. water retrieved from wells or public taps for drinking or domestic use was contaminated with v. cholerae in several instances [34, 134, 144]. thus, it is essential to educate the residents in the local area on the importance of proper sanitation of latrines and to refrain from open defecation in rivers or lakes to prevent the spread of v. cholerae. it was found that cholera affects all age groups, with the highest occurrence in young adults, followed by adults and young children. the lack of information and knowledge on the disease and its risk factors among affected populations has also contributed to the prevalence of cholera cases [41, 42, 56]. for instance, exposure or contact with persons infected with cholera increases the chances of disease spreading; hence, it is important to practice hand washing with soap and water when caring for a cholera patient. it is also essential to sanitize the toilets used by cholera patients to prevent the rampant spread of the disease [15]. therefore, educating individuals of all ages and demographics on cholera and the importance of practicing good hygiene, such as hand washing with soap and water, is crucial in preventing infections. consumption of contaminated foods and eating out are also risk factors for cholera. thus, local authorities need to implement better food safety measures to monitor the cleanliness and safety of the food sold by restaurants and street vendors to prevent outbreaks. for v. parahaemolyticus infections, it has been well-documented that consuming raw or undercooked seafood is the primary infection source. this can be seen in data from asia, where v. parahaemolyticus infections are most common. eating seafood raw in several asian cuisines contributes to food-poisoning cases caused by v. parahaemolyticus [169]. the consumption of shellfish is also commonly reported to be the source of v. parahaemolyticus infections; which is perhaps expected given that bioaccumulation of v. parahaemolyticus often occurs in shellfish due to filter feeding. one study found that oysters may contain up to 100-fold higher concentration of v. parahaemolyticus than their surrounding environment due to filter feeding, which is further exacerbated during the warmer months in summer [170]. in order to prevent v. parahaemolyticus infections, it is vital to avoid consuming raw or undercooked seafood and to ensure that the seafood consumed is cooked thoroughly before consumption. moreover, when storing raw seafood for future use, keeping them separate from other fresh produce or food is vital to prevent cross-contamination [17]. although v. vulnificus infections have the lowest incidence among the three, the severity and prognosis of the infection still raise concerns [14, 24, 25]. infections of v. vulnificus often occur when the individual is exposed to the pathogen via seafood consumption or when pmmb 2023, 6, 1; a0000340 12 of 25 a wound is exposed to waters contaminated with the bacteria [18, 19, 69]. v. vulnificus infections can manifest as gastroenteritis which is usually self-limiting, and by ensuring the seafood is cooked thoroughly before consumption, the risks of v. vulnificus gastroenteritis can be reduced. however, wound infections involving v. vulnificus are severe and can potentially cause death. this review described data that shows males over the age of 50 contribute to the majority of v. vulnificus infections, and a number of those infected succumb to the disease [4, 59, 113]. pre-existing medical conditions are a significant risk factor for v. vulnificus infections, especially chronic illnesses, including hepatic dysfunctions, renal disorders, diabetes, heart diseases, and hematological disorders [4, 19, 59, 65, 113, 157]. the majority of v. vulnificus infections affect wounds and cause soft-tissue infections such as cellulitis and necrotizing fasciitis [25, 69, 72, 157]. these infections typically require hospitalization, debridement of the affected areas, and possibly even amputation of the limbs should the infection continue to spread [4, 69, 72, 73]. the poor prognosis of v. vulnificus infections renders it a critical vibrio infection as it can result in severe health consequences such as amputation, which will negatively impact the quality of life and reduce life expectancy. to prevent exposure to v. vulnificus in the environment, persons handling raw seafood should wear protective clothing, such as gloves, to prevent injuries to minimize exposure to the opportunistic pathogen. persons with open wounds should avoid exposing the wounds or broken skin to open waters inhabited by v. vulnificus. furthermore, protective footwear should be worn when participating in water-related recreational activities to prevent v. vulnificus from entering any open wounds [25, 171]. it is imperative to prevent these vibrio infections to minimize fatalities and to improve global public health. as these vibrio species are ubiquitous in our surrounding aquatic environments, pathogenic strains of the bacteria can easily be transmitted to humans, making primary prevention very challenging. an alternative strategy to improve outcomes is through early detection of these vibrio species in the early stages of infection using rapid detection methods to identify the specific causative agent, allowing the initiation of appropriate treatment promptly. for example, lateral flow immunoassays in dipsticks or immunochromatographic strips can detect the presence of v. cholerae from clinical samples in a few minutes. a positive test result can then prompt early treatment of infections and prevent significant outbreaks in the area. this can be very useful in low-resource settings where access to laboratories and expensive equipment is limited [172, 173]. cholera vaccines have also been developed and distributed to countries frequently plagued by cholera outbreaks. the vaccines have effectively protected against cholera and prevented the development of severe disease in individuals who have received the immunization [174-176]. vibrio infections may require antimicrobial treatment, but the rapid emergence of antibiotic resistance in these bacteria dramatically reduces the efficacy of antibiotics currently available [27, 177-181]. therefore, alternative antibiotics such as probiotics should be explored to manage vibrio infections effectively. the use of antibiotics to prevent vibriosis in aquaculture, which produces seafood for human consumption, is a significant contributor to antibiotic resistance in these vibrio species [27, 182]. probiotics are known for modulating pmmb 2023, 6, 1; a0000340 13 of 25 host gastrointestinal microflora by enhancing beneficial strains' growth and suppressing pathogenic bacteria such as vibrio species. thus, probiotics are an excellent alternative to control vibriosis and maintain a safe bacterial load in these marine animals to reduce transmission of pathogenic bacteria to humans [183, 184]. streptomyces sp., a filamentous, gram-positive bacteria, has been the focal point of many researchers in developing a probiotic to treat vibrio infections in the aquaculture industry [180, 185-187]. the bacteria produce a variety of secondary metabolites, which have been useful in drug discovery [188194]. these compounds possess many properties, such as antibacterial, antifungal, antiviral, antioxidant, and cytotoxic properties [195, 196]. for this context, their anti-vibrio properties can be beneficial in developing probiotics to fight against vibrio infections [197-199]. studies on the effects of streptomyces sp., on animal models have produced positive results, as it was demonstrated that the administration of streptomyces sp. enhanced immunity and provided protective effects from vibrio infections [200-202]. as positive data from animal models continue to increase, clinical trials on the impact of streptomyces sp. in humans can be done in the near future, providing an optimistic outlook for public health. the potential application of streptomyces sp. as a probiotic in aquatic life and humans can prevent vibrio infections and the further spread of antibiotic resistance in the environment. in addition, using probiotics in humans can help maintain a healthy gut microbiome [203]. the gut microbiome acts as a protective barrier to prevent colonization of pathogenic bacterium such as vibrio sp. in the git to maintain balance in the gut microbiome [204-206]. furthermore, when exposed to pathogens, the highly diverse gut microflora defends the host from infections via immunoregulatory responses [207]. interestingly, research has shown an association between the human gut microbiome and various diseases other than gastrointestinal diseases, such as infectious diseases, metabolic disorders, and cognitive disorders [208-210]. therefore, enhancing the gut microbiome via probiotics reduces the disease burden of vibrio infections on the immune system and lowers the risk of developing medical conditions attributed to microbiome disruption. 5. conclusions this review of 149 studies demonstrated that vibrio infections remain a persistent and significant public health threat globally. a broad spectrum of the population is at risk, with some variations between the key pathogenic species: for cholera, persons of all ages and gender can be infected, whereas in v. parahaemolyticus infections, mainly adult males are affected, and v. vulnificus infections primarily affected males over the age of 50 years old. the collected data can prompt continuous surveillance and monitoring within nations that are greatly affected by vibrio infections. the data can also be used to plan, implement and strengthen preventative measures in the relevant regions to protect the local communities. for example, quality assessment of drinking water and water used for daily activities should be done diligently and periodically to ensure the waters are free from contamination. local government authorities should also implement more stringent food safety measures to ensure the public's food source is safe to consume. in addition, a focus should also be put on providing education regarding the importance of maintaining good personal hygiene, pmmb 2023, 6, 1; a0000340 14 of 25 sanitation of living spaces, risks of consuming raw or undercooked food, and the risk of exposing wounds to open waters. empowering individuals with such knowledge may be the critical link to the primary prevention of vibrio infections in vulnerable communities. author contributions: k-yl conducted the literature search, critical data analysis, and manuscript writing. shw, and k-gc provided vital technical support, proofreading, and comprehensive editing. lt-ht, jw-fl, pp, l-hl and vl provided resources, supervision, and proofreading. vl and l-hl conceptualized and founded this review writing project. all authors have read and agreed to the published version of the manuscript. funding: this work is supported by the jeffrey cheah school of medicine and 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gut microbiota modulation. prog microbes mol biol 2022; 5(1). 207. thye ay-k, pusparajah p, tan lt-h, et al. covid-19: gastrointestinal manifestations and complications. prog microbes mol biol 2021; 4(1). 208. lau awy, tan lt-h, ab mutalib n-s, et al. the chemistry of gut microbiome in health and diseases. prog microbes mol biol 2021; 4(1). 209. ong ij, loo k-y, law ln-s, et al. exploring the impact of helicobacter pylori and potential gut microbiome modulation. prog microbes mol biol 2023; 6(1). 210. kong gy-e, letchumanan v, tan lt-h, et al. gut microbiome in obsessive compulsive disorder: potential of probiotics as an adjuvant therapy. prog microbes mol biol 2022; 5(1). pmmb 2023, 6, 1; a0000340 25 of 25 author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2022, 5, 1; a0000285. doi: a10.36877/pmmb.0000285 http://journals.hh-publisher.com/index.php/pmmb original research article features and functionalities of medical mobile applications for the endemic phase of covid‐19: review and content analysis mei jun loy1, khang wen goh2, norliza osili3, long chiau ming3,4,5*, jagjit singh dhaliwal3,6, andi hermansyah4*, yaser mohammed al-worafi7,8, kah seng lee9 article history 1faculty of engineering, universiti teknologi malaysia, johor bahru, malaysia; mei.jun.loy@gmail.com (mjl) 2faculty of data science and information technology, inti international university, 71800 nilai, malaysia; khangwen.goh@newinti.edu.my (kwg) 3pengiran anak puteri rashidah sa’adatul bolkiah institute of health sciences, universiti brunei darussalam, gadong, brunei darussalam; norlizaosili@gmail.com (nao); jagjit.dhaliwal@ubd.edu.bn (jsd) 4department of pharmacy practice, faculty of pharmacy, universitas airlangga, surabaya, indonesia 5school of medical and life sciences, sunway university, sunway city, malaysia 6the university of health sciences, khayaban-e-jamia punjab, pakistan 7college of pharmacy, university of science and technology of fujairah, fujairah, united arab emirates; yworafi@yahoo.com (ymaw) 8college of medical sciences, azal university for human development, amran, yemen 9faculty of pharmacy, university of cyberjaya, cyberjaya, malaysia; ksl.pharm@gmail.com (ksl) *corresponding author: department of pharmacy practice, faculty of pharmacy, universitas airlangga, surabaya 60115, indonesia; e-mail: andi-h@ff.unair.ac.id (ah) pengiran anak puteri rashidah sa’adatul bolkiah institute of health sciences, universiti brunei darussalam, be1410 gadong, brunei darussalam/ school of medical and life sciences, sunway university, sunway city 47500, selangor, malaysia e-mail: long.ming@ubd.edu.bn/ chiaumingl@sunway.edu.my (lcm) received: 30 november 2022; received in revised form: 21 december 2022; accepted: 26 december 2022; available online: 28 december 2022 abstract: the study's objective was to assess the features and content of the covid-19 mobile applications accessible in the apple appstore. a content analysis, comparison, and functionality evaluation of a few covid-19 related mobile applications was performed. the search for covid -19 related apps in the ios appstore took place between february 1 and march 31, 2022. the mobile applications received a maximum of 7 points (basic feature assessment) and 8 points overall (functionality assessment). the requirements were fully met mailto:mei.jun.loy@gmail.com mailto:khangwen.goh@newinti.edu.my mailto:yworafi@yahoo.com mailto:longchiauming@gmail.com/ mailto:chiaumingl@sunway.edu.my pmmb 2022, 5, 1; a0000285 2 of 17 by receiving one point. frequencies from descriptive statistics were used to allude to the applications' features according to the app's basic purpose. a total of 234 applications were recognized using the keywords to explorecovid-19 related mobile applications in apple appstore. however, 58 mobile applications (24.8%) relevant to covid-19 were evaluated. according to the findings of an evaluation of basic aspects of mobile applications, 89.7% require an internet connection, 70.7% have a size of less than 50 mb, 96.6% require no funding, 58.6% include educational content, and 60.3% offer advice from the applications. in terms of score, 41.4% scored three or below, whereas 58.6% scored four or above. functionality assessment wise, 79.3% included information regarding covid-19, 12.1% included covid-19 contact tracing, 17.2% had vaccination status, a health authority maintained 50%, 31.0% included covid-19 statistics, and 25.8% were able to report art/pcr test. in terms of score, 91.4% scored three points or less, and 8.6% scored four points or more. this study has discovered several applications that could effectively prevent covid-19 pandemic spread. based on the findings, mobile applications that would be recommended are the ones supported by the government health administration of the respective country. app development companies’ applications show that competent healthcare personnel was not involved in developing the applications. online consultation with healthcare professionals might help the public who do not have access to the nearest hospital. keywords: health education; mhealth; coronavirus; health system access; infectious disease; essential healthcare services; health emergency preparedness; disaster management 1. introduction sars-cov-2, also known as coronavirus disease 2019 (covid-19), has been infecting humans for more than two years, causing extraordinary levels of morbidity and mortality around the world [1-3]. according to the world health organization (who), covid-19 has caused around 585 million confirmed illnesses and over 6 million deaths worldwide as of august 14th, 2022 [4]. as the covid-19 pandemic spreads, increasing public health attempts to evaluate the viral epidemiology and determine its short and long-term repercussions become increasingly important. new solutions to meet unmet needs are required as the disease progresses and infects people worldwide [5]. during the spread of covid-19, urban lockdowns were established in numerous regions of the world, compelling people to stay at home and limit their activities [6]. while its impact on society has evolved regarding behavioural and psychological changes, the trend of using digital media is the most significant change [6]. applications (apps) for mobile health (mhealth) are software that can be described as “integrated into smartphones to enhance health outcomes, health research, and health care services” [7]. they are health applications that both patients and healthcare professionals may use and are available on mobile devices [8]. pmmb 2022, 5, 1; a0000285 3 of 17 according to a who’s 2011 study, the healthcare industry could take advantage of these benefits by developing mhealth applications to improve patient care [8]. in most nations mhealth is quickly becoming one of the most essential instruments for providing emergency medical assistance and patient monitoring [9]. the ability for healthcare workers to update patient records at the point of care using mobile devices has reduced the frequency of clerical errors. healthcare personnel can access these patient records immediately from any location with an internet connection. in addition, mobile technology enables healthcare workers to communicate with one another and their patients at any time, resulting in a reduction in patient load [10]. it is critical to collect adequate exposure data, characterize disease burden, and disseminate information that aids in prevention, containment, and contact tracing to support public health initiatives [5]. in order to quarantine possibly infected individuals and stop the spread of disease, contact tracing is the process of finding whom the sick individual may have contacted, as well as determining where the disease may have started or who might be the "patient zero" in a given location [11]. since who declared covid-19 a global pandemic, the demand for digital tools to enhance public health measures has surged, and it now provides information to assist various stages of the pandemic. early detection, public notifications, sharing of case data, rapid screening, contact tracing, patient monitoring, information exchange, education, and treatment management were all done using mobile health technologies in response to the outbreak [12, 13]. applications can help control covid-19 pandemic in various ways, from remote monitoring by healthcare workers to tracking the number of infections [14]. mhealth is presently being used to track people who have covid-19 symptoms. it can be utilized to detect virus exacerbations and, if necessary, clinical interventions. users' real-time, longitudinal, and dynamic viral experiences are also tracked using mhealth. fear of infection in the clinic has resulted in fewer on-site referrals and more use of mobile health services [9]. despite the growing use of mhealth applications by both health professionals and the general public, quality of content, such as significant knowledge gaps about their utility and efficacy, software functioning, as well as data privacy and security, are all areas of concern in mhealth applications [7, 15]. the success of an app is determined by the context in which it is used and the design’s suitability [14]. different countries used different strategies to stop covid-19 from spreading, with some being more effective than others [10]. although the use of mobile applications is growing, research flows remain irregular and fragmented [16]. it is vital to study covid-19 mobile applications to aid users in choosing a suitable app based on their needs and assist developers in refining the designs of their current or future mobile applications to improve quality. therefore, this study aimed to evaluate the contents and features of covid-19 mobile applications available in apple’s appstore. after analyzing the functions of each application, recommendations for future health applications could be synthesised to improve the applications’ practicality. pmmb 2022, 5, 1; a0000285 4 of 17 2. materials and methods a content analysis, comparison, and functionality assessment of selected mobile applications for covid-19 were performed. a search for covid-19 mobile applications was conducted via the appstore on the apple ipad 2017. the search was conducted from 1st february 2022 until 31st march 2022 using ios-based appstore to obtain mhealth applications that were related to covid-19 that had no language restrictions. the exclusion criteria included applications that supplied quarantined users with shopping or pharmacy items to contain the virus, paid applications, and game applications. the keywords “covid19,” “coronavirus,” and “covid” were used to search for covid-19 mobile applications in the appstore. an online search on google using the key terms “mobile app,” “mhealth,” “covid19,” “coronavirus,” “corona,” and “covid-19” was also conducted to ensure that all relevant mobile applications were included. boolean operator or was used to ensuring full expansion of the search. the relevance of covid-19 was then used to screen all mobile applications, which were further filtered using the inclusion and exclusion criteria. only applications with an english language interface were evaluated and rated, as the author is mainly proficient in english. figure 1 depicts the summary of the steps involved in picking appropriate mobile applications from the app store. figure 1. selection process of mobile health applications. pmmb 2022, 5, 1; a0000285 5 of 17 2.1 data collection in the study, the basic features and functionalities of the included mobile applications were carefully assessed by three researchers. we modified the classification of mobile health applications assessment requirement reported by nouri et al [17] and in the previous studies [11,16,21] to streamline the seven basic features. the seven basic features were (1) no internet requirement, (2) size of the app less than 50 mb, (3) no subscription needed (ie, free), (4) educational content (covid-19 teaching), (5) export data (sharing of user’s data with other platforms), (6) automated data entry (automatic update of data without user interference), and (7) advisory function. inter-rater agreements were achieved through the consensus of the three participating researchers. after completing the basic feature evaluation, the applications were categorized into distinct groups based on their purpose and functionality from reading the application’s developer overview and explanation provided [5]. the categorized five functionalities of mobile applications were (1) knowledge (information on covid-19), (2) tracing or mapping of covid-19 cases, (3) home monitoring surveillance, (4) online consultation with a health authority, and (5) official mobile applications run by a health authority [17, 18]. we repeated our study conducted in 2020 [18] during the early pandemic phase to examine the latest mhealth applications on covid-19 and incorporate updated features that can help in diagnosis, symptom reporting, and contact tracing of the disease. upon scrutiny of the content of the mobile applications of the researchers involved, two separate senior researchers provided advisory roles to finalize the functional classification of all selected mobile applications. we gave either one or zero points for the features that were fully or not/partially fulfilled by the said mobile application. a maximum score of 7 and 5 points could be given for the basic features and functionalities, respectively. statistically wise, descriptive statistics (frequencies) were utilised to represent the characteristics of the mobile applications. 3. results after using the keywords to explore covid-19 related mobile applications in apple appstore, 234 applications were discovered and identified. applications were ranked based on the inclusion and exclusion criteria, with a final number of 58 applications. as shown in supplementary appendix s1, the accessible mobile applications were divided into three categories i.e., universal covid-19, country-specific, and non-english applications. only 58 out of the total 234 (24.8%) mobile applications relevant to covid-19 were evaluated; others were omitted because they failed the inclusion criteria. the selected mobile applications were evaluated based on their main features (table 1 & 2) and functionality (table 3 & 4). the assessment’s conclusions were based on a scoring methodology in which the app was granted a score of one for each condition it met. pmmb 2022, 5, 1; a0000285 6 of 17 the main features were assessed by the prerequisite of internet connectivity to use the app, storage capacity, subscription requirement, educational content, ability to export data, automated data entry support, and an advisory feature. as per the findings of an evaluation of major aspects of mobile applications, as shown in figure 2, majority of the applications (52/58, 89.7%) requires an internet connection, have the size of less than 50 mb (41/58, 70.7%), requires no funding (56/58, 96.6%), included educational content (34/58, 58.6%), does not export data (35/58, 60.3%), allow automated data entry (31/58, 53.4%) and offers advice (35/58, 60.3%). figure 2. assessment of ios-based mobile applications (a: basic features; b: functionality assessment). covid-19: coronavirus disease. figure 3. total assessment score of ios and android-based mobile applications (a: basic features; b: functionality assessment). pmmb 2022, 5, 1; a0000285 7 of 17 as shown in figure 3, none of the applications scored a total of 0, 1, and 7. out of the 58 applications, 6 applications (10.3%) scored two, 18 applications (31.0%) scored three, 19 applications (32.7%) scored four, 11 applications (18.9%) scored five, and 4 applications (6.9%) scored six. apart from the basic features, the applications are also assessed based on the functionality assessment, as shown in figure 2. the criteria evaluated under functionality included information regarding covid-19 (46/58, 79.3%), covid-19 contact tracing (7/58, 12.1%), home monitoring surveillance (1/58, 1.7%), vaccination status (10/58, 17.2%), online consultation with a health authority (0/58), the application is maintained by a health authority (29/58, 50%), covid-19 statistics (18/58, 31.0%) and being able to report art/pcr test (15/58, 25.8%). from figure 3, none of the applications scored 6 and 8. out of the 58 applications, 3 applications (5.2%) scored zero, 24 applications (41.4%) scored one, 18 applications (31.0%) scored two, 8 applications (13.8%) scored three, 2 applications (3.4%) scored four and five and 1 application (1.7%) scored seven. pmmb 2022, 5, 1; a0000285. doi: a10.36877/pmmb.0000285 http://journals.hh-publisher.com/index.php/pmmb table 1. assessment of basic features of medical applications in appstore for general use. no. name no internet requirement size of application <50 mb no subscription requirement (i.e., free) educational content export data automated data entry advisory total score 1 covid-19! x x 1 1 x 1 1 4 2 coronavirus – covid19 x 1 1 1 x 1 1 5 3 who academy: covid-19 learning x x 1 1 x x 1 3 4 covid-19 ichart 1 x 1 1 x x x 3 5 healthlynked covid19 tracker x 1 1 x x 1 x 4 6 covid-19 advisor x x 1 x x 1 1 3 7 relief central | covid-19 x 1 1 1 x 1 1 5 8 disinfection checklist covid19 x 1 1 1 x x 1 4 9 covid-19: response x 1 1 1 x x 1 4 10 covid-19 resources for midwives x 1 1 1 x x 1 4 11 covid19 sounds x 1 1 x 1 x 1 4 12 covid-19 severity calculator 1 1 1 x x x x 2 13 ra & covid-19 x 1 1 1 x x 1 4 14 covid symptom x 1 1 x 1 x x 3 15 howwefeel x 1 1 x 1 x x 3 16 covid coach 1 x 1 1 x x 1 4 17 managing your stress & anxiety 1 1 1 1 x x 1 5 18 coronasurveys x 1 1 x x 1 x 3 19 corona expert x 1 1 1 x x 1 4 20 virusassist x 1 1 1 x 1 1 5 21 coronavirus support app (uk) x 1 1 1 x x 1 4 22 corona check screening x 1 1 1 x 1 1 5 23 coronareport x 1 1 1 x 1 x 4 24 best in covid: stats & updates x 1 1 x x 1 x 3 25 responsum for long covid x x 1 1 x 1 1 4 26 covidwatcher x 1 1 x 1 x x 2 27 covid protocols 1 1 1 1 x 1 1 6 28 be+ against covid x 1 1 1 x x 1 4 pmmb 2022, 5, 1; a0000285 2 of 17 table 2. assessment of basic features of medical applications in appstore with country-specific functionality. no. name no internet requirement size of application <50 mb no subcription requirement (i.e., free) educational content export data automated data entry advisory total score 1 coronavirus australia x 1 1 1 x 1 1 5 2 bruhealth x 1 1 1 1 1 1 6 3 myaus covid-19 x 1 1 1 x 1 1 5 4 covid19 – dxb smart app x x 1 1 1 1 1 4 5 infected – covid-19 nl x 1 1 x x 1 x 3 6 tracvirus covid19 test ecert x 1 1 x x 1 x 3 7 coronavírus sus x x 1 1 x x 1 3 8 my covid-19 tracker x x 1 x x 1 1 2 9 tousanticovid x x 1 x 1 1 x 2 10 zanzibar covid-19 testing x 1 1 x 1 1 x 3 11 covid-19 ehs x x 1 1 1 1 1 5 12 bc covid-19 support x 1 1 1 1 x 1 5 13 grey bruce covid19 vaccine app x 1 1 1 x x 1 4 14 aman aman.jo jordan covid-19 x 1 1 x 1 1 x 4 15 covid-19 virginia resources 1 1 1 1 x x 1 5 16 jamcovid19 x 1 1 1 1 1 1 6 17 nhsggc clinical guidelines x x 1 1 x x 1 3 18 apollo covid-19 x 1 1 x x x 1 3 19 uclh covid19 x 1 1 1 x x 1 4 20 путешествую без covid-19 (israel) release without covid-19 x x 1 x 1 1 x 3 21 hough home test kit x x 1 x 1 1 x 3 22 covid result verifier x x 1 1 1 1 1 5 23 wlnss x 1 x x 1 x x 2 x 1 1 x 1 1 x 4 האפלקציה למלחמה בקורונה 2המגן 24 25 nz covid tracer x 1 1 1 1 1 1 6 26 covid safepass x x x 1 1 1 x 3 27 covid secure x x 1 x 1 x x 2 28 nyc covid safe x 1 1 x 1 x x 3 29 covidpasseu x 1 1 x 1 1 x 4 30 uc covid check x 1 1 x 1 x x 3 pmmb 2022, 5, 1; a0000285 3 of 17 table 3. quality assessment of medical applications in appstore for general use. no. name knowledge covid19 contact tracing home monitoring surveillance vaccination status online consultation with health authority official mobile application maintained by health authority covid19 statistics report art1pcr result total score 1 covid-19! 1 x x x x x 1 x 2 2 coronavirus – covid19 1 x x x x x 1 x 2 3 who academy: covid-19 learning 1 x x x x 1 x x 2 4 covid-19 ichart 1 x x x x x x x 1 5 healthlynked covid-19 tracker x x x x x x 1 x 1 6 covid-19 advisor x x x x x x 1 x 1 7 relief central | covid-19 1 x x x x x x x 1 8 disinfection checklist covid-19 1 x x x x x x x 1 9 covid-19: response 1 x x x x 1 x x 2 10 covid-19 resources for midwives 1 x x x x 1 x x 2 11 covid19 sounds 1 x x x x x x x 1 12 covid-19 severity calculator x x x x x x x x 0 13 ra & covid-19 1 x x x x x x x 1 14 covid symptom x x x x x x x 1 1 15 howwefeel x x x x x x x x 0 16 covid coach 1 x x x x x x x 1 17 managing your stress & anxiety 1 x x x x 1 x x 2 18 coronasurveys x x x x x x 1 x 1 19 corona expert 1 x x x x 1 x x 2 20 virusassist 1 x x x x 1 1 x 2 21 coronavirus support app (uk) 1 x x x x x x x 1 22 corona check screening 1 x x x x x x x 1 23 coronareport 1 x x x x x x x 1 24 best in covid: stats & updates x x x x x x 1 x 1 25 responsum for long covid 1 x x x x x x x 1 pmmb 2022, 5, 1; a0000285 4 of 17 26 covidwatcher x x x x x x x x 0 27 covid protocols 1 x x x x 1 x x 1 28 be+ against covid 1 x x x x 1 x x 2 table 4. quality assessment of medical applications in appstore with country-specific functionality. no. name knowledge covid19 contact tracing home monitoring surveillance vaccination status online consultation with health authority official mobile application maintained by health authority covid-19 statistics report art/pcr result total score 1 coronavirus australia 1 x x x x 1 1 x 3 2 bruhealth 1 1 1 1 x 1 1 1 7 3 myaus covid-19 1 x x x x 1 1 x 3 4 covid19 – dxb smart app 1 x x x x 1 1 1 4 5 infected – covid-19 nl x x x x x x 1 x 1 6 tracvirus covid19 test ecert 1 x x 1 x x 1 1 3 7 coronavírus sus 1 x x x x 1 x x 2 8 my covid-19 tracker x x x x x x 1 x 1 9 tousanticovid x 1 x 1 x 1 x x 3 10 zanzibar covid-19 testing x x x 1 x 1 x 1 3 11 covid-19 ehs 1 x x 1 x 1 1 1 5 12 bc covid-19 support 1 x x x x 1 1 x 3 13 grey bruce covid19 vaccine app 1 x x x x 1 1 x 3 14 aman aman.jo jordan covid-19 x 1 n1a n1a x 1 x x 2 15 covid-19 virginia resources 1 x x x x 1 x x 2 16 jamcovid19 1 x x n1a x 1 1 n1a 3 17 nhsggc clinical guidelines 1 x x x x 1 x x 2 18 apollo covid-19 x 1 x x x x x x 1 19 uclh covid19 1 x x x x 1 x x 2 20 traveling without covid19 (russian путешествую без) x 1 x 1 x 1 x 1 4 pmmb 2022, 5, 1; a0000285 5 of 17 21 hough home test kit x x x x x x x 1 1 22 covid result verifier 1 x x x x 1 x 1 2 23 wlnss x x x x x x x 1 1 24 האפלקציה למלחמה 2המגן בקורונה defender 2 the corona war application (israel) x 1 x x x 1 x x 2 25 nz covid tracer 1 1 n1a 1 x 1 x 1 5 26 covid safepass 1 x x x x x x 1 2 27 covid secure x x x 1 x 1 x 1 2 28 nyc covid safe x x x 1 x 1 x 1 2 29 covidpasseu x x x x x x x 1 1 30 uc covid check x x x 1 x x x x 1 4. discussion this study assessed the fundamental characteristics and functionalities of covid-19 applications. this study could contribute to developing a top-notch covid-19 mobile application or aid in enhancing the current covid-19 mobile applications. only ‘bruhealth’ has the capability of home monitoring surveillance. in circumstances of insufficient inpatient conditions or medical resources, who emergency guidelines advocate adopting alternative quarantine techniques, such as homecare and isolation, for patients with covid-19 who have mild symptoms. identifying people with disease conditions that are likely to worsen or become serious may be challenging. therefore, the patients who are quarantined at home may require ongoing medical observation, which could save their lives [19]. only seven covid-19 contact tracing applications are available as per our criteria. controlling the spread of communicable diseases can be done by tracing and isolating those who have had contact with an unwell person. according to the classic definition of contact tracing, healthcare professionals will interview known disease carriers and then identify the type of close contact with the disease-positive person. subsequently, the close contact would be identified and quarantined. this method is used for illnesses that are not known to spread quickly and where patients can be identified without difficulty [20]. the results show that only 15 applications have the function to report art/pcr test. this feature helps with contact tracing of covid-19 cases. according to recent research, covid-19 cases might be contained using quick and accurate digital contact tracing (dct). ferretti et al. [21], argued that dct might dramatically minimize tracing delays, allowing the outbreak to be managed with a tracing probability much lower than 70%. as a result, it is critical that anyone even experiencing the mildest symptoms gain easy access to a covid-19 investigation [20]. four covid-19 mobile applications from the appstore scored the maximum score 6 in the calculation of key features, which are ‘bruhealth’, ‘jamcovid19’, ‘nz covid tracer’ and pmmb 2022, 5, 1; a0000285 6 of 17 ‘covid protocols’. meanwhile, the lowest score ‘two’ was obtained by 6 applications: ' my covid-19 tracker’, ‘tousanticovid’, ‘covid-19 severity calculator’, ‘wlnss’, ‘covidwatcher’ and ‘covid secure’. when evaluating the functionality assessment, only 1 app obtained the highest score of 7, which is ‘bruhealth’. meanwhile, 3 applications received zero scores for the evaluation, namely ‘covid-19 severity calculator’, ‘howwefeel, and covidwatcher’. overall, ‘bruhealth’ obtained the highest score (6 and 7) for both basic feature and functionality. it lacks the ‘no requirement of internet and online consultation’ with a healthcare person. the app was given a rating of 3.8 out of 5 (n=10) from the appstore. both ‘covid-19 severity calculator’ and ‘covidwatcher’ obtained the lowest score (2 and 0) for both basic features and functionality. ‘covid-19 severity calculator’ calculates the severity of covid-19 based on demographic, clinical, and laboratory values. meanwhile, ‘covidwatcher’ by columbia university is used as a research tool where the user needs to answer a survey regarding their symptoms and medical history. ‘healthlynked covid-19 tracker’ was given a ranking of 4.3 (n=2.8k) from the appstore, but after both basic feature and functionality assessment, the app received a score of 4 and 1, respectively. this is because the application does not have a reliable source of information regarding covid-19. the consequences of false or misleading health information can be devastating. misinformation can undermine healthcare professionals’ credibility and lead to a lack of trust in medicines and vaccines [22]. the only functionality assessment score that it received is the information regarding statistics of covid-19 cases globally. ‘wlnss’ and ‘covid safepass’ are the only two applications that did not meet the requirement for no subscription as both applications require the user to buy the companyspecific art kits. ‘covid-19 ichart’ is an app for healthcare professionals to cross reference for covid-19 drug interactions. 4.1. recommendations for covid-19 mobile applications based on the findings, mobile applications that would be recommended are the ones monitored by the health ministry of the respective nation. resources regarding covid-19 were updated, and protocols for each country would differ. information regarding the disease should be from an eligible source to not mislead the public. app development companies’ applications show that competent healthcare personnel were not involved in developing the applications [23]. online consultation with healthcare professionals might help the public when the country is in lockdown, and they do not have access to the nearest hospital. digital platforms could turn out to be patients’ number one source of information, or at the very least, an alternative to in-person communication when unavailable. as a result, mobile applications may bridge the gap and provide healthcare professionals with a new way of disseminating public health messages [24]. vaccination status is also an essential feature of the covid-19 application. securing covid-19 vaccination digital certificates or also namely passports are required to overcome pmmb 2022, 5, 1; a0000285 7 of 17 the constraints of old-fashioned immunization cards. old-style immunization cards or vaccination certificates are susceptible to falsification, manipulation, and modifications, and being hard to interpret by non-health professionals, easily forgotten, and susceptible to environmental conditions such as rain [25]. furthermore, the availability and appointment booking of covid-19 booster vaccines should be added to facilitate the update of immunization [26]. the booster dose info, along with the news update of the prevalence and spread of variants of concern and their main symptoms, is vital information update in such mobile applications [27,28,29] 4.2. limitations there are some limitations acknowledged in the current study. this study examined just one digital platform (apple appstore); therefore, we could not compare the results with another digital platform (android). the scoring system placed equal weight on each of the included eight criteria. additionally, several increasingly important aspects have been overlooked in this study i.e., privacy, security, and breach protection, including the use of information by third-party applications. these may imply a significant gap in the characteristics and functionality of the reviewed applications. lastly, applications other than the english language interface were also not studied. 5. conclusions a significant portion of the global smartphone user population can facilitate and carry out mobile interventions to distribute information and support the covid-19 pandemic response. the onslaught of the covid-19 pandemic has created a profound positive impact of using these mobile applications in promoting better information collection and dissemination, which is vital in saving lives during a pandemic. digital platforms could become patients’ favourite sources of information and substitute for face-to-face communication when unavailable. as a result, mobile applications may bridge the gap and provide healthcare professionals with a new way of disseminating public health messages. this study discovered mobile applications that may be useful in checking the spread of covid-19. the most popular applications provided knowledge and guidance, as well as information on contact tracing and hot spots, and half of the evaluated applications were maintained by respective country health authorities. governments could use these mobile applications to disseminate information to the general public and collect full data for disease control. given the current covid-19 situation, continuous development and optimization of these applications are critical in curbing the spread of covid-19. further studies on the applications are encouraged to expand the research scope to applications that have not been evaluated. pmmb 2022, 5, 1; a0000285 8 of 17 author contributions: conceptualization, lcm, ah; methodology, mjl, nao, lcm, ah; formal analysis, mjl, nao, jsd, ymw, ksl; data curation, mjl, kwg, nao, lcm; writing—original draft preparation, mjl, kwg, nao, lcm; writing—review and editing, jsd, ah, ymw, ksl. funding: no external funding was provided for this research. conflicts of interest: the authors declare no conflict of interest. references 1. goh, h. p., mahari, w. i., ahad, n. i., chaw, l. l., kifli, n., goh, b. h., . . . ming, l. c. (2020). risk factors affecting covid-19 case fatality rate: a quantitative analysis of top 50 affected countries. progress in microbes and molecular biology, 3(1). doi:10.36877/pmmb.a0000171 2. loo, k. y., & letchumanan, v. (2022). covid-19: understanding the new variants of concern. progress in microbes and molecular biology, 5(1). doi:10.36877/pmmb.a0000282 3. thye, a. y. k., tan, l. t. h., law, j. w. f., pusparajah, p., & letchumanan, v. (2022). long covid19: psychological symptoms in covid-19 and probiotics as an adjunct therapy. progress in microbes and molecular biology, 5(1). doi:10.36877/pmmb.a0000267 4. loo, k. y., & letchumanan, v. 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(2021). covid-19 booster vaccines administration in different countries. progress in microbes and molecular biology, 4(1). doi:10.36877/pmmb.a0000256 27. law, l. n. s., loo, k. y., goh, j. x. h., & pusparajah, p. (2021). omicron: the rising fear for another wave in malaysia. progress in microbes and molecular biology, 4(1). doi:10.36877/pmmb.a0000261 28. loo, k. y., & letchumanan, v. (2022). covid-19: understanding the new variants of concern. progress in microbes and molecular biology, 5(1). doi:10.36877/pmmb.a0000282 29. thye, a. y. k., loo, k. y., tan, k. b. c., lau, j. m. s., & letchumanan, v. (2021). insights into covid-19 delta variant (b.1.617.2). progress in microbes and molecular biology, 4(1). doi:10.36877/pmmb.a0000243 pmmb 2022, 5, 1; a0000285 10 of 17 author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2022, 5, 1; a0000284. doi: a10.36877/pmmb.0000284 http://journals.hh-publisher.com/index.php/pmmb original research article evaluation of antibiofilm activity of thymus syriacus essential oil against clinically isolated mdr bacteria basem battah1*, anas rajab1, lama shbibe2, jagjit singh dhaliwal3, khang wen goh4, long chiau ming5, yaman kassab1, abdelhakim bouyahya6, mahibub mahahamadsa kanakal7, chadi soukkarieh1,2 article history 1faculty of pharmacy, syrian private university (spu), damascus, syria, anasrajab@gmail.com, (ar) dryamankassab@yahoo.com, (yk) 2faculty of science, damascus university, damascus, syria, menaayoub1990@gmail.com, (ls), soukkarieh@gmail.com, (cs) 3pengiran anak puteri rashidah sa'adatul bolkiah institute of health sciences, universiti brunei darussalam, brunei darussalam, jagjit.dhaliwal@ubd.edu.bn, (jsd) 4faculty of data science and information technology, inti international university, 71800 nilai, malaysia, khangwen.goh@newinti.edu.my, (kwg) 5school of medical and life sciences, sunway university, sunway city 47500, malaysia, chiaumingl@sunway.edu.my (lcm) 6laboratory of human pathologies biology, faculty of sciences, mohammed v university in rabat, morocco, a.bouyahya@um5r.ac.ma, (ab) 7faculty of pharmacy, quest international university, ipoh, perak, malaysia; mehboobct@gmail.com *corresponding author: basem battah, department of biochemistry and microbiology, faculty of pharmacy, syrian private university (spu), daraa international highway, syria, basem.battah.sc@gmail.com, (bb) received: 29 november 2022; received in revised form: 21 december 2022; accepted: 24 december 2022; available online: 26 december 2022 abstract: finding alternative strategies to confront bacterial resistance is an urgent need. biofilm-forming bacteria have become a serious problem in medicine and industry. bacteria can use biofilm as a mechanism of resistance against antibacterial drugs and avoid the immune system. the aim of this study was to evaluate the antibacterial activity of thymus syriacus (t. syriacus) essential oil in a solid and liquid medium and to study its antibiofilm formation activity. the t. syriacus essential oil was extracted from the aerial parts of the plants. the minimum inhibitory concentrations (mic) measurements were used to identify the antibacterial activity of the essential oil against multidrug-resistant strains pseudomonas aeruginosa (p. aeruginosa), klebsiella pneumonia (k. pneumonia), streptococcus pneumonia (s. pneumonia) isolated clinically from blood infections. the microtiter plate was used in order to quantify biofilm formation by bacteria. the minimum inhibitory concentration (mic) against the three clinically isolated strains (p. aeruginosa, k. pneumonia, s. pneumonia) were (3.12, 1.56, 3.12 µl/ml) respectively. the formation of biofilm by (p. aeruginosa, k. pneumonia, s. pneumonia) was reduced up to (43%, 50%, 60%) respectively, when the essential oil was applied at mic concentrations for each strain. the observed antibacterial activity of t. syriacus essential oil was significant against pmmb 2022, 5, 1; a0000284 2 of 13 antibacterial-resistant strains and antibiofilm formation activity was identified. the novelty of this study is we confirmed that the essential oil of t. syriacus exhibited not just antibacterial properties but also antibiofilm formation effect. more studies are needed in order to continue studying this oil and evaluate its other medicinal properties and toxicity. keywords: antibacterial, antibiofilm, natural product, medicinal plants, essential oil, t. syriacus 1. introduction the abuse of antimicrobial agents in poultry farming is one of the main causative factors of bacterial resistance [1, 2]. likewise, irrational use of antibiotics in healthcare [3] renders antibacterial agents ineffective against bacteria such as legionella pneumophila [4] and mycobacterium tuberculosis [5, 6]. multidrug-resistant (mdr) acinetobacter baumannii strains are the main cause of urinary tract infections in the elderly with a biofilm-forming ability which is more difficult to treat [7]. furthermore, the resistance problem can be caused by other bacteria like methicillin-resistant staphylococcus aureus (mrsa) and s. pneumoniae, and gram-negative bacteria like k. pneumoniae, p. aeruginosa, escherichia coli (e. coli), and mycobacterium tuberculosis (mtb) [8, 9]. biofilm is a self-enclosed population of bacterial cells [10] biofilm producing organisms have a different pattern of growth rates, gene expression and metabolism with a far more resistant to antibacterial agents than not biofilm-forming bacteria [10, 11]. bacterial biofilms are a serious problem in clinical practice and industries, and numerous efforts have been made to eradicate the bacterial biofilm [12]. many studies have been conducted in order to understand the mechanism of biofilm formation and how the resistant genes and exopolysaccharides matrix expressed during biofilm formation, which can increase the biofilm antibiotic resistance and become a serious threat making treating such infections more difficult [13]. even though infectious control using nanomaterials and nanotechnology has been well researched, their application is still in the developmental stage [14-16]. therefore, finding alternative strategy in order to face this serious emerging problem and increase the activity of other antibacterial agents is an urgent need [17]. natural products like essential oils have been studied for a long time to verify their antibacterial effect [18-20]. it was suggested that essential oils affect bacterial proliferation and contain many compounds with different mechanisms like damaging cell membrane, increasing its permeability, damaging cytoplasmic membrane, cell lysis, leakage of intracellular material, inhibiting efflux pump mechanism of antibiotics rendering them more efficient [21-24]. the genus thymus is also known locally in syria as zattar [25]. it has numerous species and varieties native to southern europe and asia [26]. thymus genus has been claimed as a source of primary material for pharmaceutical industry used in medicine and cosmetics [25]. research and studies about thymus syriacus (t. syriacus) are scarce and more studies are pmmb 2022, 5, 1; a0000284 3 of 13 needed to verify its medical application and characteristics. the chemical composition of essential oil could be variable. thymus spp. essential oil is characterized by the presence of high concentration of isomeric phenolic monoterpenes thymol and or carvacrol [27]. the concentrations of thymol and carvacrol, the main components of the thyme essential oil range from (3-60%) of the total essential oil [28] and the other component are in trace amount. the antimicrobial activity of the essential oil and its mechanism of action may vary depending on the percentage of composition of its ingredients [29]. it was reported that the antibacterial activity of thymus essential oil has been widely used in food and cosmetics. moreover, many studies have suggested the application of essential oil in antibacterial therapy and found a role for essential oil against biofilm-forming properties of some microorganisms [30]. despite some reports about the antibacterial activity of thymus essential oil [31, 32], further evidence is desired to verify the antimicrobial activity of t. syriacus essential oils formulations. our study aimed to evaluate the bactericidal, bacteriostatic, antibiofim activity of t. syriacus essential oil against various clinically isolated drug-resistant bacteria. 2. materials and methods 2.1. preparation of essential oil t. syriacus were prepared after harvesting the plant from the rural parts of damascus during the month of may. the leaves of the plant were dried at 25°c. after drying, steam distillation was used to extract the essential oil. chloroform was used to separate the oil and water from the isotropic mixture. then the chloroform was evaporated, and we collected the essential oil. finally, a yield of 1.5% from the t. syriacus essential oil was obtained. 2.2. bacterial strain isolation and culturing the blood samples were isolated from university children's hospital in damascus. in order to identify the bacterial species, bacterial dna was directly extracted from the blood using tmbosis molysis immediately after extracting dna genomic from 2-3 ml blood taken with edta (molzym gmbh & co kg-germany). the pcr was based on 16s rdna (eurofins genomics ebersberg) . in addition, conventional microbiological methods was also used for the same isolates. three bacterial strains were isolated and identified s. pneumoniae, p. aeruginosa, k. pneumoniae. 2.3. diffusion disc bacterial antibiotic sensitivity we performed the antimicrobial susceptibility for each strain (s. pneumoniae, p. aeruginosa, k. pneumoniae) by using three commonly used antibiotics (vancomycin, ampicilin, amikacin, gentamicin) using kirby-bauer disk (bioanalyse) diffusion methods. the inhibition zone of each antibiotic for each strain was measured and bacterial strains were determined as susceptible or resistant strains [2, 33]. pmmb 2022, 5, 1; a0000284 4 of 13 2.4. evaluation of the antibacterial effect of t. syriacus essential oils on solid medium agar-well diffusion method was used to determine the antibacterial activity. the bacteria were cultured on mueller–hinton agar medium (biomerieuxfrance). then, 6 mm of wells were cut through the agar using a sterile cork borer. the agar was then removed, leaving empty wells that were filled with 20 µl of t. syriacus essential oil. the plates were incubated at 37°c for 24h. 2.5. determination of the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) of t. syriacus essential oils in order to evaluate the mic and mbc of t.syriacus essential oils, the three bacterial strains (p. aeruginosa, k. pneumoniae, s. pneumoniae) were cultured on lb broth medium (tmmedia, india) with dmso 10% (sigma aldrich) at 37c ͦ. a series of decreasing concentrations of the essential oil (100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0 µl/ml) were prepared and inoculated with approximately 5x103 cfu/ml and incubated at 37 c ͦ for 24h. microplate alamar blue assay was used to identify the t. syriacus essential oil mic [34]. in addition, in order to identify the mic and mbc concentration, cfus counting was used and aliquots were cultured on lb agar medium (tmmedia, india) and incubated for 24h at 37 c ͦ. each essay was assayed in triplicate. 2.6. measuring and quantifying biofilms the quantification of biofilm mass was performed by a microtiter plate [35]. the three strains (p. aeruginosa, k. pneumoniae, s. pneumoniae) were cultured in lb broth at 37°c to reach od600=1, then diluted 1:100 in lb broth . 1 ml of the bacterial culture was incubated in 24 microtiter plate at 37°c. in order to evaluate the antibiofilm activity of t. syriacus essential oil. the essential oil was added at two concentration mic and mbc concentrations at different time points (24h, 48h, 72h). the medium was removed and each well was left to dry at room temperature. then, 500µl of crystal violet 1% ( sigma aldrich) was added to each well and incubated for 10 minutes. in the next step, each well was washed with distilled water three times then let to dry at room temperature. finally, 1 ml of ethanol 95% was added to each well and 100µl from each condition was transferred to 96 well microtiter plate. in order to quantify the biofilm mass, od570 was measured to quantify the amount of crystal violet-stained biofilm. each condition was assayed in triplicate. 2.7. statistical analysis microsoft excel (2010) and graphpad prism software (graphpad prism 6 software, graphpad software inc, ca, usa) were used to analyze the data. all data were conducted as the mean ± standard deviation and analyzed by one-way or two-way anova comparison tests followed by appropriate correction, as specified in the caption under each figure. all experiments were performed in triplicates. pmmb 2022, 5, 1; a0000284 5 of 13 3. results 3.1. t. syriacus essential oil antibacterial activity on solid medium cultured with clinically isolated bacteria the studying of antibacterial activity of the t. syriacus on solid medium was verified by using three clinically isolated multidrug-resistant (mdr) bacteria (p. aeruginosa, k. pneumoniae, s. pneumoniae). the three strains involved in the study have different antibiotic sensitivity profiles. p. aeruginosa showed resistance against (gentamicin and ampicillin ) and sensitivity against (amikacin and vancomycin ), k. pneumoniae showed resistance against (ampicillin, vancomycin and gentamycin) and sensitivity against amikacin, while s. pneumoniae exert resistance against (gentamicin, ampicillin and amikacin) and sensitivity against vancomycin. in order to verify the antibacterial activity on solid medium, the inhibiting zone on the solid medium was measured at different concentration of the essential oils as described in material and methods and the observed results are reported in table 1. the obtained results showed good activity of t. syriacus essential oil at concentrations (25% 50% 100%) and moderate or intermediate activity at 10% concentration against the studied bacterial strains. table 1. t. syriacus essential oils antibacterial activity on solid medium (mueller-hinton agar). the three different multidrug-resistant (mdr) bacterial strains (p. aeruginosa, klebsiella pneumoniae, s. pneumoniae) were cultured on solid medium and different concentrations of t. syriacus essential oil were applied (100%, 50%, 25%, 10%, 5%). different results were obtained by measuring zone inhibition in mm after 24 h incubation at 37 c ͦ for each strain. zone inhibition against s. pneumonia in mm zone inhibition against p. aeruginosa in mm zone inhibition against k. pneumonia in mm concentration of t. syriacus essential oil (v̸v) 5.5 8 no growth 100% 3 3.5 5 50% 4.5 3 4 25% 2 0 1.5 10% 0 0 0 5% 3.2. the antibacterial activity of t. syriacus essential oil in liquid medium culture with clinically isolated bacteria the antimicrobial activity of t.syriacus essential oil was verified using three resistant bacterial strains clinically isolated (s. pneumoniae, k. pneumoniae and p. aeruginosa). the minimum inhibitory concentration (mic) and the minimum bactericidal concentration (mbc) in axenic culture were identified, and the results are reported in table 2. the observed results showed antibacterial activity of the t. syriacus essential oil against all three strains pmmb 2022, 5, 1; a0000284 6 of 13 and the mic results were arranged between (1.56 µl/ml) against k. pneumoniae and (3.12µl/ml) against s. pneumonie and p. aeruginosa. table 2. t. syriacus essential oil minimum inhibitory concentration of (mic) and minimum bactericidal concentration (mbc) against multidrug-resistant (mdr) strains of s pneumoniae, k. pneumoniae and p. aeruginosa. essential oil mic µl/ml s .pneumoniae mbc µl/ml s .pneumoniae mic µl/ml k.pneumoniae mbc µl/ml k .pneumoniae mic µl/ml p.aeruginosa mbc µl/ml p.aeruginosa t. syriacus 3.12 6.25 1.56 3.12 3.12 6.25 3.3. antibiofilm formation activity of the t.syriacus essential oils against clinically isolated bacteria three different strains (p. aeruginosa, k. pneumoniae, s. pneumoniae) were used to quantify the antibiofilm activity of t. syriacus essential oil by incubating the three different strains in contact with different concentrations of the t. syriacus essential oil representing a different mic concentration against different bacterial strains (3.12 µl/ml for s. pneumoniae, 3.12 µl/ml for p. aeruginosa, and 1.56 µl/ml for k. pneumonia). furthermore, in order to verify if the mbc concentrations have different effects, we incubated the three different strains with different mbc concentrations of the t. syriacus essential oil (6.2 5µl/ml for s. pneumonia, 6.25 µl/ml for p. aeruginosa and 3.12 µl/ml for k. pneumoniae). in order to quantify biofilm masses, a microtiter plate, as described in the material and methods, was used at different time points (24h, 48h, 72h) and compared with the control group (untreated bacteria). as shown in figure 1 (a), biofilm formation of s. pneumoniae (gram-positive bacteria) was significantly reduced by t. syriacus essential oil at mic and mbc concentration at (24h, 48h and 72h). the reduction was between (60 %) and (68%) at 72h post-treatment. the results in figure 1 (b) showed that biofilm formation of p. aeruginosa was significantly reduced by t.syriacus essential oil at (24h, 48h and 72h) but the reducing effect was less than the effect against s. pneumoniae, especially at 24h and 48h. the reducing effect was increased from (43%) to (75%) when the mbc concentration was used at 72h post-treatment. similarly, figure 1 (c) showed that biofilm formation was significantly reduced by t.syriacus essential oil against k. pneumoniae at different time points( 24h, 48h and 72h), but the reducing effect at 24h post-treatment was less than the effect against s. pneumoniae. the reducing effect was increased to (66%) after 48h posttreatment and from (50%) to (67%) when the mbc concentration was used at 72h posttreatment. pmmb 2022, 5, 1; a0000284 7 of 13 figure 1. (a) quantification of biofilm mass of s. pneumoniae after application of t. syriacus essential oils with different concentrations at different time points. the essential oil was added at mic (minimum inhibitory concentration) concentration (3.12µl/ml) and mbc (minimum bactericidal concentration ) concentration (6.25µl/ml). crystal violet staining was used to quantify the biofilm mass at different time points (24, 48, 72h). the obtained results showed a significant antibiofilm activity and a significant decrease in biomass after adding essential oil. ****p<0.00001 for t. syriacus with mic and mbc concentration at the three different time points (24, 48, 72h) versus control. (b) quantification of biofilm mass of p. aeruginosa after treatment with mic and mbc concentration of t. syriacus essential oils at different time points. the essential oil was added at mic concentration (3.12µl/ml) and mbc concentration (6.25µl/ml). the obtained results exhibited a significant antibiofilm activity and a significant decrease in biomass after adding essential oil. **p<0.001for t. syriacus with mic concentration and ***p<0.0001 with mbc at 24h post-treatment, ***p<0.0001 for t. syriacus with mic concentration and ****p<0.00001 with mbc at 48h post-treatment versus control , ****p<0.00001 for t. syriacus with two concentration mic and mbc at 72 post-treatment and****p<0.00001 for t. syriacus with mic concentration versus mbc concentration after 72 h of treatment. (c) quantification of biofilm mass of k. pneumoniae after treatment with mic and mbc concentrations of t. syriacus essential oils at different time points. the essential oil was added at mic concentration (1.56µl/ml) and mbc concentration (3.12µl/ml). the obtained results showed a significant antibiofilm activity and a significant decrease in biomass after adding essential oil. ****p<0.00001for t. syriacus with mic and mbc concentration versus control, ****p<0.00001for t. syriacus with mic concentration versus mbc concentration after 72 h of treatment. pmmb 2022, 5, 1; a0000284 8 of 13 4. discussions thymus vulgaris has been used for centuries in traditional medicine [36, 37]. the emergence of resistant and biofilm-forming bacteria makes finding new antibacterial strategies an urgent need [38, 39]. essential oils contain many compounds mixed together and could be a promising alternative strategy in front of emerging resistance bacterial infection [40-42]. in this study, we verified the antibacterial activity of t. syriacus essential oil extracted from the peripheral region of damascus against resistant bacteria clinically isolated. the yield of extraction was 1.5% lower than the 2.8% yield obtained in other studies conducted in syria [25]. the obtained results showed that t. syriacus essential oil have significant antibacterial activity against p. aeruginosa with mic (3.12 µl/ml), which is in agreement with another study conducted on t. syriacus essential oil. this study verified the activity of t. syriacus essential oil against gram-negative isolated bacteria and found that the mic was (3.12 µl/ml) against p. aeruginosa [25]. the minimum inhibitory concentration of the t. syriacus essential oil against k. pneumoniae was (1.56 µl/ml) in contrast with another study that showed thymus essential oil showed no antibacterial activity against a. baumanni and k. pneumonia [43]. it was also demonstrated in a study developed to evaluate the antibacterial effect and antibiofilm forming of thymus vulgaris essential oil against clinically isolated p. aeruginosa that the mic and mbc values were between 0.062 and 2% (v/v) of isolates [44]. in another study, the inhibitory effect of essential oil was also explained, and the results showed that the zone of inhibition of thymus vulgaris essential oil was less than (10 mm) for k. pneumonia, p. aeruginosa and s. pneumoniae. the calculated mic was (0.312 mg/ml) for s. pneumoniae, (0.625 mg/ml) for p. aeruginosa and (1.25 mg/ml) for k. pneumoniae which are less than our obtained results. previous and current findings confirmed that thymus essential oil is effective as an antibacterial for both gram-negative bacteria and grampositive like s. pneumoniae. furthermore, the antibacterial activity of thymus vulgaris against other microorganism gram-positive bacteria staphylococcus aureus (staph. aureus) with mic (0.625 mg/ml) and gram-negative bacteria e. coli with mic (2.5 mg/ml) was reported [45]. in addition, the minimum inhibitory concentration of thymus vulgaris essential oil against clinically isolated bacterial strains (mrsa, s. pneumoniae, p. aeruginosa, e. coli and k. pneumoniae ) was (0.625, 0.312, 0.625, 2.5 and 1.5 mg/ml) respectively [45]. it is important to note that thymus vulgaris essential oil with its major chemical compounds (thymol, linalool, carvacrol, 1,8-cineole, eugenol, camphor, camphene, 𝛼pinene, borneol, 𝛽-pinene) [46] have inhibiting activity against different microorganisms (clostridium perfringens, listeria monocytogenes, e. coli, salmonella typhimurium, staphylococcus aureus, salmonella typhi) with mic values (1.25, 0.04, 0.1, 0.2, 0.08 and 0.2 mg/ml) respectively [47]. the antimicrobial activity of thymus vulgaris essential oil was not restricted to bacteria but also to fungi. a study showed that thymus vulgaris essential oil with its major chemical compounds (thymol, p-cymene, carvacrol, carvacrol acetate, and linalool) have antifungal activity against candida albicans, candida glabrata, candida parapsilosis, aspergillus fumigates with mic (0.031, 0.031, 0.0625, 0.016µg/ml) respectively [31]. in addition, it was documented by other studies that the antimicrobial activity of the thymus pmmb 2022, 5, 1; a0000284 9 of 13 vulgaris essential oil was associated with its phenolic compound thymol and carvacrol [48]. the hydroxyl group of these compounds interacts with the cytoplasmic membrane, changes its permeability and affects the stability of the lipid bilayer, leading to disruption of the cytoplasmic membrane and leakage of intracellular components [49, 50]. in this study, we verified the activity of thymus syriacus essential oils in inhibiting the biofilm formation and found a significant reduction in biofilm mass after treatment with mic and mbc concentrations of essential oil against p. aeruginosa, k. pneumoniae and s. pneumoniae at different hours of incubation. the essential oil was more effective in reducing the biofilm mass by using mic and mbc concentration and at three different time points with more activity against s. pneumoniae biofilm formed. moreover, the antibiofilm activity of t. syriacus essential oil increased by augmenting the essential oil concentration at 72h post-treatment. another study that tested against k. pneumoniae determined the minimum biofilm inhibitory concentration was 5 mg/ml and the biofilm reduced by 70% after 24h of treatment. at the same time, the reducing effects were 20% and 85%, respectively, by using the same concentration of p. aeruginosa and s. pneumoniae, which aligns with the current findings that the antibiofilm effect of the thymus syriacus was more efficient against s. pneumoniae than p. aeruginosa and k. pneumoniae. the antibiofilm formation of thymus vulgaris essential oil was also established against other bacteria. it was demonstrated that using 0.01% of the essential was able to reduce the biofilm formation by 53%, and this effect was increased to 76% by increasing the concentration to 0.05% [51]. finally, our study confirmed with other studies the antibacterial and antibiofilm activity of the thymus essential oil. the different origin of essential oil with different chemical compositions leads to different obtained results of mic, and the reason that the essential oil was tested on different strains of bacteria. 5. conclusion in this study, we found a significant activity of t. syriacus essential oil against a group of multidrug-resistant bacteria. moreover, the results of our study showed a significant effect of t. syriacus essential oil against bacterial biofilm formation. the findings point out that t. syriacus essential could be an alternative armamentarium targeting bacterial infection and effective inhibitor of biofilm formation. its synergistic use with the existing range of antimicrobial agents could be explored too. although this study utilised only conventional testing methodology, the findings verified the activities of t. syriacus essential oil against other bacterial strains and other pathogenic microorganisms like viruses and fungi. furthermore, determining the cytotoxic effects of t. syriacus essential oil using different concentrations and verifying its effect using in vivo model are steps to be taken before further commercialization consideration. author contributions: conceptualization, bb, ar and yk; 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19(7): 681-687. pmmb 2022, 5, 1; a0000284 13 of 13 author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2022, 5, 1; a0000283. doi: a10.36877/pmmb.0000283 http://journals.hh-publisher.com/index.php/pmmb original research article comparison of the factors that influence the pattern of procurement and usage of antithrombotic drugs mohamed mansor manan1, aswani yanti baharuddin1, qi ying lean1, jagjit singh dhaliwal2, sachinjeet kaur sodhi dhaliwal2, muhammad junaid farrukh3, ching siang tan4, long chiau ming5* article history 1faculty of pharmacy, universiti teknologi mara, puncak alam, malaysia; mansormanan@uitm.edu.my (mmm), clinpharms@gmail.com (ayb); leanqiying@yahoo.com (qyl) 2pengiran anak puteri rashidah sa’adatul bolkiah institute of health sciences, universiti brunei darussalam, brunei darussalam; jagjit.dhaliwal@ubd.edu.bn (jsd); molars14@hotmail.com (sksd) 3faculty of pharmaceutical sciences, ucsi university, 56000 cheras, malaysia; junaid@ucsiuniversity.edu.my (mjf) 4school of pharmacy, kpj healthcare university college, nilai 71800, malaysia; tcsiang@kpjuc.edu.my (cst) 5school of medical and life sciences, sunway university, 47500 sunway city, malaysia *corresponding author: long chiau ming, school of medical and life sciences, sunway university, sunway city 47500, selangor, malaysia; chiaumingl@sunway.edu.my/ longchiauming@gmail.com received: 28 november 2022; received in revised form: 18 december 2022; accepted: 20 december 2022; available online: 22 december 2022 abstract: antithrombotics are used for many indications in prophylaxis and therapeutic treatment. it is often associated with a higher risk of adverse effects; hence a drug utilization evaluation is essential. the main aim was to describe the utilization of antithrombotic drugs. the specific objectives are to compare the pattern of procurement and usage of antithrombotic drugs, estimate the defined daily dose (ddd), and to compare the usage of antithrombotic drugs with the who recommended standard. this was a retrospective study evaluating the utilization of antithrombotics at a public tertiary care hospital in the urban city of klang valley, malaysia. a defined daily dose (ddd) per 1000 inhabitants/day was calculated to estimate patients with thromboembolic disorders receiving standard treatment on a daily basis. expenditures on all antithrombotic drugs were also assessed. the ddd was examined for correlation with gender, race, and age group. malaysian clinical practice guideline on the prevention and treatment of venous thromboembolism was used as a tool to evaluate the quality of prescribing. during the study period, among anticoagulants prescribed, warfarin was the most commonly utilized for treatment and prophylaxis in various disciplines, with 1.82 ddd. enoxaparin was the second most utilized anticoagulant, with 1.05 ddd, followed by tinzaparin, with 0.39 ddd. for antiplatelet drugs, acetylsalicylic acid was the most utilized antiplatelet with 8.5 ddd followed by clopidogrel pmmb 2022, 5, 1; a0000283 2 of 19 (1.42 ddd) and ticlopidine (0.16 ddd). the utilization pattern for all the antithrombotic drugs was in accordance with the who ddd guidelines. the pattern of antithrombotic drug procured was based on the demand for the drug supplies. age, gender, and race were determinant factors that influenced the choice of antithrombotic drugs and further affected the antithrombotic drug utilization pattern. still, additional information regarding patient’s status and concomitant disease condition is needed to establish the impact of utilization of antithrombotics fully. ddd analysis is an excellent practice to be conducted at planned intervals. it is a tool to update the changing or even new pattern of drug usage, which will assist in the justification of rational use of drugs. the mean difference in terms of ddds of the studied antithrombotic drugs impacts patient demographic characteristics, smoking history, presence of atherosclerotic diseases and peripheral artery diseases, comorbidity, patient compliance, and bleeding history. it is useful to evaluate trends and expenditures, which will ultimately avoid untoward adverse effects and unnecessary costs. keywords: drug utilization review; health outcomes; anticoagulant; antiplatelet; heart disease; myocardial infarction; healthcare delivery 1. introduction drug utilization, one of the areas within pharmacoepidemiology has been given more emphasis in the current economic climate, to recognize the variation among prescribing practices that could optimise healthcare expenditure. such utilization acts as indicators where these indicators provide information and give much input in strategic planning to healthcare managers to improve the quality of drug use, improvement of treatment guidelines and prescribing patterns, and development of national drug policy. they reflect the status of a vital characteristic of healthcare services [1,2]. according to a fact sheet by world health organization, more than 50% of all medicines are not prescribed, dispensed, and sold correctly and more than 50% of patients are not taking their drugs correctly. the situation is worse in developing countries, with 6070% of patients not being treated according to clinical guidelines. the approach to rationalizing the use of medicines should address all the factors that contribute to the irrational use of medicines, including patient-centred factors, prescriber-related factors, social and economic factors, healthcare system factors, and disease factors [3]. antithrombotic therapy is fundamental in treating arterial and venous thrombotic diseases and in percutaneous coronary intervention. it has had an enormous impact in several significant ways where anticoagulant becomes the cornerstone of treatment for thromboembolic disorders, either as prophylaxis or for treatment purposes, whilst antiplatelet therapy becomes a cornerstone in coronary artery disease management. the landscape of anticoagulant therapy for atrial fibrillation has changed significantly with the availability of targeted new oral anticoagulants that are safer than warfarin. antiplatelet therapy is essential to treating acute coronary syndrome and preventing ischemic complications of percutaneous pmmb 2022, 5, 1; a0000283 3 of 19 coronary intervention. both antiplatelets and anticoagulants work to prevent clots in the blood vessels, but they work in different mechanisms of action [4]. anticoagulant use is often associated with risks and complications such as venous thromboembolism (vte) and pulmonary embolism (pe). patients with acute coronary syndrome remain at risk of recurrent cardiovascular events despite the advances in medical therapy like antiplatelet therapy [5]. the findings would inform healthcare administration on the current status of anticoagulant drugs and antiplatelet drug utilization and the appropriateness of those drugs prescribed in the tertiary hospital setting. the study's outcome would be beneficial in updating the hospital drug formulary. it can also improve current policy on the utilization of anticoagulants and antiplatelet drugs in the hospital. despite the availability of comprehensive and evidence-based guidelines such as the malaysian clinical practice guidelines (cpg) on the prevention and treatment of venous thromboembolism 2013 [6], much of the care provided is not in line with cpg. published literature has shown that in a study performed to look at and monitor the appropriateness of care (care in line with evidence-based or consensus-based guidelines) at healthcare delivery in australia, it was found out that the percentage of vte compliance in healthcare delivery in australia was reported at only 58% [7]. with the new entry of medications to treat and prevent thromboembolism to the hospital formulary list, little is known about its utilization pattern. looking at the importance of anticoagulant and antiplatelet use and the associated risks of this therapy together with the shortage of budgetary resources and the increased cost of today's complex treatment of thrombosis, drug utilization evaluation of anticoagulant and antiplatelet medications is highly warranted. this study aimed to describe the utilization of anticoagulant and antiplatelet medications supplied to patients from various disciplines with a variety of underlying conditions and to compare the defined daily dose (ddd) of anticoagulant and antiplatelet medications prescribed with the who’s standard ddd to examine the appropriateness of the prescription. 2. materials and methods 2.1. study design this study was conducted at a tertiary public hospital located in the state of selangor, malaysia. this was a retrospective study on the utilization of selected anticoagulant medications. the drugs selected were warfarin, fondaparinux, tinzaparin, or enoxaparin) and selected antiplatelet medications such as acetylsalicylic acid, acetylsalicylic acid & glycine, clopidogrel, dipyridamole or ticlopidine. the data were obtained from dispensed prescriptions from the outpatient pharmacy department. the prescriptions were from several disciplines with various underlying conditions from january 2017 to december 2019. the selected hospital is a referral centre for haematology. it is a fully information technology (it) hospital using an established online healthcare application known as electronic hospital information system (ehis). the total number of patients in the outpatient pharmacy is pmmb 2022, 5, 1; a0000283 4 of 19 roughly 1000 patients per day from various disciplines and departments. record of all outpatient patients from all disciplines with a variety of underlying conditions who received at least one anticoagulant medication (i.e. warfarin, fondaparinux, tinzaparin or enoxaparin) or one antiplatelet (i.e. acetylsalicylic acid, acetylsalicylic acid & glycine, clopidogrel, dipyridamole, and ticlopidine) from the outpatient pharmacy during the study period was obtained from ehis. relevant data including patients' clinical and demographic data such as age, gender, race, medications use, anticoagulants use, antiplatelets use, diagnosis, prescribed drugs, dosing and indication, duration of therapy and cost of the drug were collected. the drug price was obtained from the medication purchase system. patients' identities remained anonymous, and only relevant information was retrieved from the records. 2.2. data collection the electronic hospital information system (ehis) was utilized to generate the list of all outpatient patients with various underlying conditions from all disciplines from january 2017 to december 2019 that was prescribed with at least one of the anticoagulants or antiplatelets studied. patients were then sorted and listed according to their registration numbers. randomization was conducted by employing the method by edward fury, 2011 [32]. the final list was computer generated with each patient's identification number only. the identification number was used to retrieve each patient’s dispensing records, such as the prescribed medicine, dose, indication, and frequency, and transcribed into the data collection form. additional information, such as demographic information and procurement and supply data (2017 2019), was used to quantify the cost of drug usage and to see the pattern in drug procurement. the data collection form was designed so that all appropriate information is not missed for further data analysis. these data were utilized to calculate each drug's defined daily dose (ddd) per 1000 inhabitants per day. malaysian cpg was referred to evaluate the quality of prescribing. the ddd was compared between drugs with respect to the patient's demographic characteristics. 2.3. patient selection criteria 2.3.1. inclusion criteria outpatient patients aged 18 years and above from all disciplines with various underlying conditions received at least one antithrombotic medication from an outpatient pharmacy from january 2017 to december 2019. patients with at least three (3) consecutive visits dose including during inpatient and outpatient visits to ensure the consistency and applicability of ddd except for tinzaparin and enoxaparin which 1 visit dose was sufficient. pmmb 2022, 5, 1; a0000283 5 of 19 2.3.2. exclusion criteria rivaroxaban (non-formulary item). heparin is given to patients for a short term during their stay in the ward. patients on dental prescriptions, haemodialysis, patients referred from other facilities, incomplete data. 2.4. sample size and sampling method the sample size was determined using the sample size formula [33]. the significance level was set at α =0.05 (two-tailed), and confidence interval (ci) was set as 95% for the calculation of sample size by using the formula above. the calculated sample size was based on the proportion of patients who received antithrombotic agents in the chosen hospital. a total of 176 patients on antithrombotic agents for the year 2019, 135 patients for the year 2018, and 156 patients for the year 2017 were sampled from ehis after considering approximately 20% for the drop-out patients following the inclusion and exclusion criteria. table 1. characteristics of patients receiving antithrombotic agents. variables all (n=673) anticoagulants (n=262) antiplatelets (n=402) both (n=9) x2 (df) pvalue* n % n % n % n % gender male 309 45.9 60 22.9 243 60.4 6 66.7 91.63 (2) <0.001 female 364 54.1 202 77.1 159 39.6 3 33.3 age (years) 18-30 52 7.7 46 17.6 4 1.0 2 22.2 201.46 (6) <0.001 31-45 146 21.7 111 42.4 34 8.5 1 11.1 46-60 150 22.3 31 11.8 118 29.4 1 11.1 >61 325 48.3 74 28.2 246 61.2 5 55.6 mean age (sd) 57.03±17.41 47.32±18.55 63.36±13.24 56.33±18.74 ethnicity malay 375 55.7 174 66.4 197 49.0 4 44.4 29.10 (6) <0.001 chinese 219 32.5 64 24.4 152 37.8 3 33.3 indian 51 7.6 10 3.8 39 9.7 2 22.2 others 28 4.2 14 5.3 0 0 0 0 diagnosis cvs conditions 521 77.4 114 43 .5 398 99. 0 9 100 288.38 (4) <0.001 non-cvs conditions: o&g 142 21.1 142 54 .2 0 0 0 0 non-cvs conditions: others 10 1.5 0 2. 3 4 1.0 0 0 *chi-square test pmmb 2022, 5, 1; a0000283 6 of 19 2.5. medicine list anticoagulants available in the ministry of health formulary are coumarins and indandiones group such as warfarin 1mg tablet, warfarin 2mg tablet, warfarin 3mg tablet, and warfarin 5mg tablet. another available anticoagulant medication is the factor xa inhibitors group i.e. fondaparinux 2.5mg/0.5ml injection and rivaroxaban 10mg, 15mg, and 20mg tablets. rivaroxaban is a control item purchased under the haematology discipline in this hospital, but patients are required to buy the medication out-of-pocket. 2.6. outcome parameter the data was expressed in defined daily dose (ddd) per 1,000 inhabitants per day. the ddd result was then compared with the current who classification of atc/ddd for the ddd analysis, obtained from the atc/ddd index 2020 [8]. for this study, the quantity of use of medicine was expressed as the number of ddds per 1000 inhabitants per day. 2.7. statistical analysis and interpretation data were analysed using ibm statistical package for social science (spss) programme version 25 and microsoft excel version 2016. the patient's prescribing record and demographic data were descriptively presented, such as the frequency, percentage, mean and standard deviation. the variables evaluated were patients' age, gender, and race. the variables' distribution was assessed with the kolmogorov-smirnov test. results would be statistically significant if the p-value were < 0.05. 2.8. ethical considerations the ethical approval application was obtained from uitm's ethics committee and medical research & ethics committee (mrec) before commencing this study (approval rec/04/2021), dated 30th april 2021. all the principles of the declaration of helsinki and the malaysian good clinical practice guidelines were followed. all the data obtained from ehis, such as patients' profiles and medical records, was restricted only to the investigators and kept confidential. 3. results the ehis database showed that the participating pharmacy unit in the hospital dispensed anticoagulant and antiplatelet agents to 37,314 patients from january 2017 to december 2019. based on the general inclusion and exclusion criteria, 27,471 patients were eligible for further investigation of their medication dispensing records. this resulted in 73.8% of patients receiving antithrombotic drugs throughout the study. out of 9843 patients excluded, some had incomplete dispensed prescription information and not meeting the criteria. the final randomization resulted in the screening are 673 patient’s records that constitute 2.4% of the total population who received antithrombotic drugs in the hospital which this study was conducted (table 2). pmmb 2022, 5, 1; a0000283 7 of 19 table 2. use of antithrombotic in ddd/1000 inhabitants/day and total expenditure. 3.1. patients’ socio-demographic characteristics the selected demographic data of patients who received antithrombotic agents (anticoagulant and/or antiplatelet agents) is summarised in table 3. anti-thrombotic drugs atc code year ddd/1000 inhabitants/day warfarin tablet b01aa03 2017 1.3153 2018 2.0447 2019 3.1305 enoxaparin injection b01ab05 2017 1.3699 2018 0.8173 2019 1.5732 fondaparinux injection b01ax05 2017 0.0023 2018 0.0009 2019 0.0000 tinzaparin injection b01ab10 2017 0.4820 2018 0.3304 2019 0.3012 acetylsalicylic acid tablet b01ac06 2017 7.9451 2018 7.4489 2019 6.4526 acetylsalicylic acid and glycine b01ac06 2017 7.9451 2018 7.4489 2019 6.4526 clopidogrel tablet b01ac04 2017 0.8418 2018 1.7177 2019 0.6883 dipyridamole tablet b01ac07 2017 0 2018 0 2019 0 ticlopidine tablet b01ac05 2017 0.3752 2018 0.0463 2019 0.0755 total 2017 2018 2019 pmmb 2022, 5, 1; a0000283 8 of 19 table 3. summary of the mean difference in ddd in relation to age group, gender, and race. antithrombotic drugs variables number of samples (n) mean ddd (sd) p value warfarin male 57 0.0518(0.2736) t=1.185, p=0.239 no significant difference female 47 0.0215(0.9056) 18-30 2 0.0518(0.2736) f=0.405, p=0.75 no significant difference 31-45 10 0.0215(0.9056) 46-60 24 0.0356(0.6316) >61 68 0.0355(0.6023) chinese 48 0.0393(0.5689) f=1.753, p=0.161 no significant difference indian 6 0.0984(0.4930) malay 49 0.0260(0.6913) enoxaparin male 2 0.0453(0.5027) t=-1.130, p=0.260 no significant difference female 152 0.0188(0.4748) 18-30 46 0.0186(0.4256) f=10.739, p<0.001 31-45 100 0.0168(0.4173) 46-60 4 0.0430(0.9244) >61 4 0.2697(0.4144) chinese 15 0.0294(0.6728) f=2.247, p=0.085 no significant difference indian 4 0.0279(0.6712) malay 122 0.0190(0.4510) others 13 0.0106(0.2575) acetylsalicylic acid male 237 0.0583(0.4737) t=0.524, p=0.601 no significant difference female 146 0.0620(0.4975) 18-30 5 0.0256(0.3911) f=3.103, p=0.027 31-45 35 0.0411(0.4826) 46-60 113 0.0564(0.4836) >61 230 0.0662(0.4767) chinese 144 0.0736(0.4423) f=8.426, p<0.001 indian 36 0.0611(0.5117) malay 190 0.0554(0.4779) others 13 0.0162(0.5028) clopidogrel male 74 0.0325(0.5713) t=2.152, p=0.034 female 42 0.0570(0.6094) 18-30 3 0.0421(0.5278) f=1.290, p=0.281 no significant difference 31-45 9 0.0169(0.4679) 46-60 31 0.0412(0.1316) >61 73 0.0435(0.5385) chinese 37 0.0649(0.6368) f=4.384, p=0.006 indian 13 0.0412(0.7087) malay 58 0.0345(0.5047) others 8 0.0114(0.4178) pmmb 2022, 5, 1; a0000283 9 of 19 3.2. defined daily dose (ddd) of antithrombotic drugs to estimate the ddd of antithrombotic drugs prescribed, 673 prescriptions sampled after randomization according to years from 2017 to 2019 were screened for all anticoagulant and antiplatelet drugs. ddds values were calculated from the information on dosage per day. it was found that the top three most utilized anticoagulant drugs from the year 2017 to 2019 were warfarin with 1.82 ddd, followed by enoxaparin (1.05) and tinzaparin (0.39). this implied that warfarin was the most utilized anticoagulant drugs in the population about two times more than the second most utilized drug i.e. enoxaparin followed by tinzaparin. fondaparinux was the least utilized anticoagulant drug in this study. the results for ddd of warfarin, enoxaparin, acetylsalicylic acid, clopidogrel according to gender, age and race were presented in table 4. comparison of ddd/1000 inhabitants’/day anticoagulant and antiplatelet drugs with msom 2015 – 2016 tabulated in table 5. table 4. association between antithrombotic drugs uses and demographic variables based on who defined daily dose (ddd) and malaysian cpg on the prevention and treatment of venous thromboembolism 2013. antithrombotic drugs variables accordance to who ddd’s accordance to cpg n % n % warfarin male 57 100 57 100 female 47 100 47 100 18-30 2 100 2 100 31-45 10 100 10 100 46-60 24 100 24 100 >61 68 100 68 100 chinese 48 100 48 100 indian 6 100 6 100 malay 49 100 49 100 enoxaparin male 2 100 2 100 female 152 100 152 100 18-30 46 100 46 100 31-45 100 100 100 100 46-60 4 100 4 100 >61 4 100 4 100 chinese 15 100 15 100 pmmb 2022, 5, 1; a0000283 10 of 19 indian 4 100 4 100 malay 122 100 122 100 others 13 100 13 100 acetylsalicylic acid male 237 100 237 100 female 146 100 146 100 18-30 5 100 5 100 31-45 35 100 35 100 46-60 113 100 113 100 >61 230 100 230 100 chinese 144 100 144 100 indian 36 100 36 100 malay 190 100 190 100 others 13 100 13 100 clopidogrel male 74 100 74 100 female 42 100 42 100 18-30 3 100 3 100 31-45 9 100 9 100 46-60 31 100 31 100 >61 73 100 73 100 chinese 37 100 37 100 indian 13 100 13 100 malay 58 100 58 100 others 8 100 8 100 pmmb 2022, 5, 1; a0000283 11 of 19 table 5. comparison of ddd/1000 inhabitants’/day anticoagulant and antiplatelet drugs with msom 2015 – 2016. drugs (atc code) ddd in msom 2016 (public sector) msom ranking 2016 (top 50) ddd/1000 inhabitants/day 2017 2018 2019 warfarin (b01aa03) 0.2852 1.3153 2.0447 3.1305 enoxaparin (b01ab05) 0.0986 1.3699 0.8173 1.5732 fondaparinux (b01ax05) 0.0259 0.0023 0.0009 0 tinzaparin (b01ab10) 0.0184 0.4820 0.3304 0.3012 acetylsalicylic acid (b01ac06) 8.5408 6 7.9451 7.4489 6.4526 clopidogrel (b01ac04) 0.6873 42 0.8418 1.7177 0.6883 dipyridamole (b01ac07) 0.0219 ticlopidine (b01ac05) 0.4728 0.3752 0.0463 0.0755 3.3. factors/determinants influence anticoagulant and antiplatelet drugs pattern it was found that both males and females in all age groups received antithrombotic drugs, and females were found to receive more antithrombotic agents than males for each year from the year 2017 to 2019. in terms of age, the patients’ age ranged >61 years old, which were the highest number of patients who received the antithrombotic drug for both genders. the highest anticoagulant received by the male population for the three years was warfarin which was almost two times more than females in 2017. as for 2018 and 2019, the prescription frequency of males to females receiving warfarin was similar. this was followed by tinzaparin, fondaparinux, and enoxaparin. the highest anticoagulant received by females for those three years was enoxaparin. females in the age group 31-45 years old received enoxaparin the most, followed by the age group 18-30 years old for the years 2017 to 2019. this was followed by warfarin, tinzaparin, and fondaparinux. the males received the highest antiplatelet drug for three years. they were in the age group >61, followed by 46-60. the highest antiplatelet drug received by the male population for the three years was aspirin followed by cardiprin and clopidogrel. the pattern of receiving antiplatelet drugs in the female group was similar to the male patients. ticlopidine and dipyridamole was the least antiplatelet drug that both groups received. pmmb 2022, 5, 1; a0000283 12 of 19 4. discussions 4.1. patients' socio-demographic characteristics the distribution of patients receiving antithrombotic agents was mainly from the medical department, o&g department and haematology department of this hospital which is also a referral hospital for haematology. the number of patients load is about 1000 patients per day. the percentage of patients who received antithrombotic drugs showed an increasing trend for antiplatelets, of which 5.5% for 2017 and 7.7% for 2019. for anticoagulants, the percentage also increased from 2.2% in 2018 to 2.46% in 2019. the patients were predominantly malay and chinese; most were >61 years old, followed by the age group between 45-60 and 31-45 years old. the distribution of patients resembled the demographics of patients reported by the department of statistics, malaysia, 2019 under the statistics on causes of death, 2019 [21]. the report showed that ischaemic heart diseases contributed to the highest principal causes of death in 41-59 years old and >60 years old in 2018. 4.2. appropriateness and rational use of antithrombotics in this study, the ddd for all antithrombotic drugs studied were in accordance with who ddd’s, i.e. lower than who ddd’s. all antithrombotic drugs were prescribed in compliance with the malaysian cpg on the prevention and treatment of venous thromboembolism 2013. this finding was inconsistent with a previous study done in canada by lloyd et al. where the management of deep vein thrombosis (dvt) despite the recommendation to support deep vein thrombosis (dvt) risk assessment and appropriate use of prophylaxis in medical inpatients. the authors further stated it was either neglected or prescribed unnecessarily by the clinicians [9]. the proportion of outpatient patients who received anticoagulants was 2.46% in 2019, which increased from 2018 to 2.2%. however, in 2017, the proportion of outpatient patients who received anticoagulants was 3.36%. the proportion of outpatient patients who received antiplatelets are 7.7% in 2019, which also showed an increasing trend from 2018, which was 5.4%, and 5.5% in 2017. among all the antithrombotic drugs, enoxaparin injection was the highest item purchased in 2019 (rm755, 992.49) where the total purchasing value had increased by about 32% from 2018. it was found that rm1, 528,234.14 had been allocated for antithrombotic drugs purchased in 2019 (about 2% of the total expenditure). this was fair in terms of budgetary implications since there were no significant changes in the drug price from 2017 to 2019 for most of the antithrombotic drugs studied. this showed that the pattern of drugs procured was based on demand. the evaluation of purchasing of anticoagulant drugs from 2017 to 2019 found that the total expenditure spent on the purchase of warfarin was in accordance with the usage pattern. pmmb 2022, 5, 1; a0000283 13 of 19 the total cost of warfarin usage was also in line with the ddd/1000population/day of warfarin from 2017 to 2019. this was consistent with a study by kirley et al., and theodorou et al. that stated although prescribing of noacs is increasing, recent data available suggested that warfarin remains the most commonly utilized oral anticoagulant in outpatient settings [10.11]. for enoxaparin, the second highest utilized anticoagulant drug, the total expenditure spent on purchasing this drug was appropriate with the total cost usage. from the total cost of enoxaparin usage, the decreasing pattern from 2017 to 2018 later rebounded from 2018 to 2019. this was in line with the ddd/1000population/day of enoxaparin from 2017 to 2019. the decreasing pattern of the usage of enoxaparin from 2017 to 2018 was due to the purchase of enoxaparin in 2017 (rm1, 267,153.20) constituted a 2.1% budget spent on drugs for that year. this has made it a controlled item and is strictly monitored by the pharmacy department. this control was with the agreement with the o&g department, where prescribing and supplying enoxaparin must be according to the scoring and criteria of post-natal patients upon discharge after delivery. this approach impacted the reduced budget spent on enoxaparin in 2018 (rm 512,909.20) i.e. only 0.8% of the budget was spent on drugs for that year. however, the total expenditure on enoxaparin increased from 2018 to 2019 due to the increase in deliveries in this hospital. tinzaparin, the second costliest anticoagulant drug, the total cost of usage for the outpatient setting was more than the total purchase for the total number of patients. this was contrary to fondaparinux, where total expenditure spent on it was high, but the actual total usage was very low. both tinzaparin and fondaparinux total cost usage were not in line with the ddd from 2017 to 2019. this highlighted the amount of money that could be saved to purchase other drugs, such as enoxaparin. as for antiplatelet drugs, from 2017 to 2019, the total expenditure spent on the purchase of aspirin and cardiprin was in accordance with the usage pattern. however, the total cost of aspirin and cardiprin was not in line with the ddd/1000 population/day of acetylsalicylic acid from 2017 to 2019. this finding was consistent with a study in the united states by ansa et al. that showed that not all groups of individuals are leveraging the benefits from the recommended use of aspirin and may consequently be at higher risks for cardiovascular events and premature death [12]. the total cost usage was in line with the ddd calculated for clopidogrel from 2017 to 2018 and not from 2018 to 2019. however, the total expenditure spent on the purchase of this drug was appropriate with the total cost usage. the drop in the ddd of clopidogrel from 2018 to 2019 might be contributed by the lack of awareness at the prescriber side on the availability of the generic clopidogrel which allows the prescriber to start prescribing clopidogrel for lifelong treatment from march 2010 onwards. this was due to the high budget implications since the cost of original clopidogrel for lifelong treatment would be rm2007.50 per patient/year. this is about eighteen times higher as compared to rm110 per patient per year for generic clopidogrel [13]. the usage of generic clopidogrel led to total pmmb 2022, 5, 1; a0000283 14 of 19 expenditure for the whole year 2019 for generic clopidogrel was only rm58, 344 or 0.1% of the budget spent on drugs in 2019. 4.3. defined daily dose (ddd) of antithrombotic drugs the ddds found in this study were based on the population sample at the outpatient pharmacy of the chosen hospital. the national reports (msom) include the utilization of the whole population data for public health institutions but sample population data for private health institutions. therefore, the comparison of the ddd from the study with the national reports was not comparable. overall, from 2017 to 2019, there was a fluctuation in the usage of antithrombotic agents, except for the use of warfarin from 2017 to 2019, which showed an increasing pattern. this was due to warfarin remaining the gold standard oral anticoagulant since the availability of other oral anticoagulants (doac), such as rivaroxaban, was purchased for the haematology patient only. hence warfarin is the preferred choice for the prescriber. the findings on warfarin being the highest ddd utilized, were in accordance with the current recommendations. warfarin was recommended for use as an initial treatment for venous thromboembolism (vte), a common cardiovascular disease after myocardial infarction and stroke [14]. enoxaparin, a low molecular weight heparin, was the second most utilized anticoagulant drug from 2017 to 2019. lmwh was proven in a few studies to be more effective than unfractionated heparin in preventing thrombosis without increasing the risk of bleeding [15-18]. these findings were consistent to another study that found enoxaparin was among commonly used anticoagulant drugs for various diagnoses such as ischemic heart disease (ihd), deep vein thrombosis (dvt), pulmonary embolism (pe), cardiovascular accident and others [18]. however, the high usage of enoxaparin was mainly utilized by females aged 31-45 and 18-30 years old for the indication for pregnancy and post-partum. this was consistent with current recommendations suggesting lmwh as the preferred agent during pregnancy for prophylaxis and vte treatment [19]. the least utilized antithrombotic agents at the outpatient pharmacy from 2017 to 2019 were tinzaparin and fondaparinux. this was due to the high budget implications of these drugs. its low utilization was monitored and presented during drug therapeutic committee meetings to the hospital departments, which created heightened awareness to the prescriber in prioritizing its use on patients. acetylsalicylic acid (aspirin) remained the most widely used agent for antiplatelet drugs since 2017. this was followed by cardiprin, clopidogrel, ticlopidine and dipyridamole. the usage pattern of antiplatelet drugs from 2017 to 2019 was consistent with the usage pattern of antiplatelet drugs from 2015 to 2016 that was reported in the malaysian statistics on medicines (msom) 2015 to 2016 [20]. pmmb 2022, 5, 1; a0000283 15 of 19 4.4. factors and determinants influence the antithrombotic drugs pattern regarding the finding on patient characteristics, it was found that the treatment intensity increased with age. patients in the age range >61 years old had the highest number of patients who received antithrombotic followed by patients in the age group 46-60 and 3145. the lowest range of age received antithrombotic was 18-30 years old. this was consistent with the study performed by man-son-hing et al. where among all age groups, elderly persons received the most significant absolute benefit from warfarin or aspirin prophylaxis [23]. man-son-hing et al., in the study, also highlighted a recommendation from an expert panel that for long-term warfarin treatment, all elderly people with atrial fibrillation should be treated, unless there was a contraindication. the choice of antithrombotic drugs was influenced by the patient's factors that can affect the antithrombotic drug utilization pattern as well as the outcome of the treatment. determinant factors include age, gender, race, smoking history, presence of atherosclerotic diseases & peripheral artery diseases, comorbidity, patient compliance, and bleeding history. patients in the presence of atherosclerotic diseases like angina and peripheral artery disease constitute the major determinants of the selection of antithrombotic monotherapy. comorbidity of cardiovascular risk factors (heart failure, hypertension, stroke, transient ischaemic attack (tia), systematic thromboembolism, pulmonary thromboembolism, deep vein thrombosis, peripheral artery diseases, myocardial infarction (mi) and angina were among the major determinants on the selection of antiplatelet and anticoagulant drugs (rauch, 2012). prescribing of anticoagulants was mentioned to be influenced to a greater extent by bleeding risk than it was by the risk of stroke [24]. another study also showed that changes in anticoagulant regimens were influenced by a history of bleeding [25]. for the anticoagulants studied, warfarin was the highest utilized anticoagulant for the three years. the mean difference in ddd was not statistically significant in relation to gender, age group, and race. this was supported by two studies in patients with atrial fibrillation (af) that demonstrated the factors for the usage of oral anticoagulants and their consistent use in these af patients. evidence suggested that oral anticoagulants, such as vitamin k antagonists (vka) or noac, were influenced by old age, race, and patientrelated and provider-related factors [26,27]. enoxaparin, the second most utilized antithrombotic drug from 2017 to 2019, the mean difference of ddd for enoxaparin was statistically significant only with age group but not statistically significant with gender and race. enoxaparin was mainly used for the indication of pregnancy and post-partum in women. this was supported by a study by ellison et.al that highlighted the use of enoxaparin in pregnancy was associated with a low incidence of complications and a dose of 40 mg once daily throughout pregnancy provides satisfactory anti‐factor xa levels and appears effective in preventing venous thromboembolism [28]. enoxaparin's highest female population to receive was in the age group of 31-45 followed by those in the age group between 18-30 years old. this was because 18-45 years pmmb 2022, 5, 1; a0000283 16 of 19 old was the child-bearing age that supported the high utilization of enoxaparin in that age range. this was consistent with the study highlighting the average female age of first-time pregnancy in the united states of america increased from 21 to 25 years in the 40 years after 1970, with a decline in the number of mothers under the age of 20 and a substantial rise in those over the age of 35 [29]. the highest utilized antiplatelet for the three years was acetylsalicylic acid. the mean difference in ddd was statistically significant in relation to age group and race but not statistically significant in gender. for clopidogrel, the second highest utilized antiplatelet for the three years of 2017, 2018, and 2019 the mean difference in ddd was statistically significant in relation to gender and race, but the mean difference of ddd was not statistically significant in relation to age group. this was consistent with the findings in the study by ahmed et al. that found there were differences in prescribing antiplatelet agents between different gender and different ages. the study reported that clopidogrel and aspirin were prescribed mainly for patients over 50 years old, and generally, majority of the male patients received antiplatelet agents [30]. this is the first study conducted in the region assessing the usage of antithrombotic agents, and the ddd values could serve as the standard for comparison with other studies. even though this study was only conducted in one particular hospital, we collected data for consecutive three years’ data to examine the continuous usage pattern. the study's outcome would be beneficial in developing a cost-effective hospital drug formulary and improving current policy on the utilization of anticoagulants and antiplatelets. this research could highlight the utilization pattern of anticoagulants and antiplatelets in a tertiary hospital in malaysia and its appropriateness in terms of ddd. 5. conclusions the findings on the trend of high warfarin utilization, a high risk medication that requires close monitoring to prescribe this medication through the effective collaboration among prescribers and pharmacists in managing thrombotic diseases. warfarin was also an efficient and cost-effective alternative due to favorable tolerability with fewer side effects than doac. aspirin and cardiprin were the highest utilized antiplatelet drugs, followed by clopidogrel and ticlopidine. this study showed that the mean difference in terms of ddds of the studied antithrombotic drugs have an impact of patient demographic characteristics in terms of age, gender, race, smoking history, presence of atherosclerotic diseases and peripheral artery diseases, comorbidity, patient compliance and bleeding history that affected the choice and utilization of antithrombotic drugs. future drug use evaluation should also have prospectively assessed the appropriateness of antithrombotic drug indications, drug selection, duration of treatment, drug interactions, and outcomes of treatment. pmmb 2022, 5, 1; a0000283 17 of 19 author contributions: conceptualization, mmm, ayb, lcm ; methodology, mmm, qyl, ksl, lcm ; software, ayb, qyl; validation, mmm, ayb, qyl, lcm; formal analysis, mmm, ayb, lcm ; investigation, mmm, ayb, qyl, lcm ; resources, qyl, ksl, lcm ; data curation, mmm, ayb, qyl, ksl, lcm ; writing-original draft preparation, mmm, ayb; writing-review and editing, qyl, jsd, sksd, mjf, ksl, lcm funding: no external funding was provided for this research. conflicts of interest: the authors declare no conflict of interest. references 1. hennessy s, bilker wb, zhou l, et al. retrospective drug utilization review, prescribing errors, and clinical outcomes. jama 2003;290(11):1494-9. 2. nasir hha, dhaliwal js, goh hp, et al. patient’s own medication use during hospitalization. progress in microbes & molecular biology 2022; 5(1). 3. world health organization. promoting rational use of medicines: core components. world health organization; 2002. available at https://apps.who.int/iris/bitstream/handle/10665/67438/who_edm_2002.3.pdf 4. goette a, vranckx p. atrial fibrillation patients undergoing percutaneous coronary intervention: dual or triple antithrombotic therapy with non-vitamin k antagonist oral anticoagulants. european heart journal supplements 2020; 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4(3):120-123. 12. unit kejururawatan, hospital ampang. buku daftar kemasukan hospital/institusi series per-pd 101 pin. 2/2009. available at https://www.moh.gov.my/moh/modules_resources/database_stores/87/195.pdf 13. ansa be, hoffman z, lewis n, et al. aspirin use among adults with cardiovascular disease in the united states: implications for an intervention approach. journal of clinical medicine 2019; 8(2):264. 14. næss ia, christiansen sc, romundstad p, et al. incidence and mortality of venous thrombosis: a population‐based study. journal of thrombosis and haemostasis 2007; 5(4):692-9. 15. garcia da, baglin tp, weitz ji, et al. parenteral anticoagulants: antithrombotic therapy and prevention of thrombosis: american college of chest physicians evidence-based clinical practice guidelines. chest 2012; 141(2): e24s-43s. 16. geerts wh, pineo gf, heit ja, et al. prevention of venous thromboembolism: the seventh accp conference on antithrombotic and thrombolytic therapy. chest 2004;126(3):338s-400s. pmmb 2022, 5, 1; a0000283 18 of 19 17. gould mk, garcia da, wren sm, et al. prevention of vte in nonorthopedic surgical patients: antithrombotic therapy and prevention of thrombosis: american college of chest physicians evidencebased clinical practice guidelines. chest 2012;141(2): e227s-77s. 18. jørgensen ln, wille‐jørgensen p, hauch o. prophylaxis of postoperative thromboembolism with low molecular weight heparins. british journal of surgery 1993; 80(6):689-704. 19. singh v, gopinath k, behzadpour a, et al. anticoagulant utilization evaluation in a tertiary care teaching hospital: an observational prospective study in medical in patients. indian journal of pharmacy practice 2015;8(2). 20. malaysian statistics on medicines 2015-2016; pharmaceutical services programme, ministry of health malaysia: kuala lumpur, 2020. available at https://www.pharmacy.gov.my/v2/sites/default/files/document-upload/malaysian-statistics-medicines2015-2016.pdf 21. department of statistics, malaysia (2019). statistics on causes of death, malaysia 2019. 22. master list contract registry, pharmaceutical services division dated 28 august 2020. 23. man-son-hing m, nichol g, lau a, et al. choosing antithrombotic therapy for elderly patients with atrial fibrillation who are at risk for falls. archives of internal medicine 1999;159(7):677-85. 24. kusakawa k, harada kh, kagimura t, et al. major determinants for the selecting antithrombotic therapies in patients with nonvalvular atrial fibrillation in japan (japaf study). journal of arrhythmia 2017;33(2):99-106. 25. frain b, castelino r, bereznicki lr. the utilization of antithrombotic therapy in older patients in aged care facilities with atrial fibrillation. clinical and applied thrombosis/hemostasis 2018;24(3):519-24. 26. tulner lr, van campen jp, kuper im, et al. reasons for undertreatment with oral anticoagulants in frail geriatric outpatients with atrial fibrillation. drugs & aging 2010; 27(1):39-50. 27. waldo al, becker rc, tapson vf, et al. hospitalized patients with atrial fibrillation and a high risk of stroke are not being provided with adequate anticoagulation. journal of the american college of cardiology 2005; 46(9):1729-36. 28. ellison j, walker id, greer ia. antenatal use of enoxaparin for prevention and treatment of thromboembolism in pregnancy. bjog: an international journal of obstetrics & gynaecology 2000 ; 107(9):1116-21. 29. mathews tj. delayed childbearing: more women are having their first child later in life. us department of health and human services, centers for disease control and prevention, national center for health statistics; 2009. 30. ahmed, n. j. age and gender differences in the pattern of antiplatelet agents prescribing. journal of pharmaceutical research international 2020; 87-91. 31. allemann ss, van mil jw, botermann l, et al. pharmaceutical care: the pcne definition 2013. international journal of clinical pharmacy 2014; 36(3):544-55. 32. edward furey, random number generator. 2011, retrieved from https://www. calculatorsoup.com/calculators/statistics/random-number-generator.php 33. naing l, winn t, rusli b. practical issues in calculating the sample size for prevalence studies. archives of orofacial sciences 2006; 1: 9-14. pmmb 2022, 5, 1; a0000283 19 of 19 author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology editorial note 1 efficacy of chlorine dioxide as a disinfectant shermaine yee1, ya chee lim2, choon fu goh3*, vijay kotra4, long chiau ming2* 1faculty of medicine, quest international university perak, ipoh, perak, malaysia 2pap rashidah sa’adatul bolkiah institute of health sciences, universiti brunei darussalam, gadong, brunei darussalam 3discipline of pharmaceutical technology, school of pharmaceutical sciences, universiti sains malaysia, penang, malaysia 4faculty of pharmacy, quest international university perak, ipoh, perak, malaysia abstract: chlorine dioxide plays a significant role in the industrial settings as disinfectants due to its broad antimicrobial property. despite commonly use as germicide, chlorine dioxide demonstrates a good safety profile, rendering its suitability for use at water treatment and food preparation zones. protein denaturation including envelope proteins is the major mechanism of chlorine dioxide to inactivate microorganisms even at low concentrations. adverse reactions are not widely reported due to the typical use at a low concentration. the effectiveness of chlorine dioxide against various microorganisms, in both liquid and gaseous forms, over a wide range of ph and at an extremely low concentration has confirmed chlorine dioxide as a vital and versatile disinfectant. keywords: disinfectants; chlorine dioxide; sars-cov; anti-microbial received: 9th october 2020 accepted: 19th october 2020 published online: 28th october 2020 citation: yee s, lim yc, goh cf, et al. efficacy of chlorine dioxide as a disinfectant. prog microbes mol biol 2020; 3(1): a0000128. https://doi.org/10.36877/pmmb.a0000128. introduction chlorine dioxide (clo2), a strong oxidant, has a long-standing significant role in the industrial settings as disinfectants, especially at water purification and food preparation areas, due to its wide spectrum antimicrobial property [1]. the significance of its antimicrobial role is further enhanced during the outbreak of bacillus anthracis in 2001, when fumigation of vaporous hydrogen peroxide and clo2 was used to destroy b. anthracis spores, along with a combination of hepa vacuuming, cleaning and bleach application. in this article, we revisit the efficacy and safety of clo2 as a suitable sanitizing agent during the current covid-19 pandemic crisis. recently, it is shown that clo2 is a potent disinfectant in preventing infectious disease outbreaks due to its oxidative properties in eliminating microbes [2]. in gaseous form, clo2 is able to eliminate culturable bacteria and detoxify bioterrorism agents such as bacillus spores. moreover, clo2, when used as a disinfectant on surfaces, has been reported to exhibit antimicrobial properties against various kinds of microbes efficiently even in wet environments. a concentration of 700-1100 ppm of clo2 is also a feasible alternative in replacing glutaraldehyde-based disinfectants [3, 4]. aside from its possibility as a potent disinfectant, clo2 has been reported to be a disinfectant without causing side effects due to its rapid action and safe antimicrobial properties [5]. furthermore, clo2 can be used a keratolytic compound with anti-inflammatory properties but is non-toxic to human tissue [6]. in addition, this chemical compound has a history of being used in water purification and disinfection process during food preparation due to its wide spectrum of antimicrobial effects. clo2 is also found to be more effective than hydrogen peroxide as bleaching agent of teeth [7]. a very recent review on dermatologic reactions to various types of disinfectants used to reduce the risk of coronavirus infection indicated that clo2 is safe, even with prolonged skin contact [8]. clo2 solution is also closely examined for its potential use to inactivate viruses, including severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [9] and human papillomavirus (hpv) [10]. interestingly, clo2 solution was also used to sterilize recycled kn95s or surgical face masks during critical shortage of such supplies [2]. mechanisms behind the efficacy of clo2 against microbes a study revealed that due to interactions between clo2 and biological thiol groups of amino acids, bacteria are unable to develop resistance against clo2 [5]. copyright @ 2020 by yee sm and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: choon fu goh, discipline of pharmaceutical technology, school of pharmaceutical sciences, universiti sains malaysia, penang, malaysia; choonfugoh@usm.my. long chiau ming, pap rashidah sa’adatul bolkiah institute of health sciences, universiti brunei darussalam, gadong, brunei darussalam; longchiauming@gmail.com. 2 clo2-based disinfectants have been shown to be effective in eliminating b. anthracis spores in solutions and less toxic than sodium hypochlorite in an in vitro study with human skin keratinocytes [11]. besides, another study has shown that virus inactivation by clo2 is achieved through denaturation of viral envelope proteins, thereby able to prevent aerosol-induced influenza virus infection at low concentrations [12]. the potential role of clo2 in completely inactivating porcine reproductive and respiratory syndrome virus (prrsv) was demonstrated through the action of degrading the genome and proteins of the virus [4]. this study also confirmed that the expression of inflammatory cytokines induced by this virus can be reduced by clo2. this is further supported by studies reporting protein-denaturing activities due to covalent oxidative modification of cysteine, tryptophan and tyrosine residues of model proteins (bovine serum albumin and g6pd of saccharomyces cerevisiae) as the mechanism behind the efficacy of clo2 against microbes [13,14]. furthermore, 0.03 ppm of clo2 has been indicated to prevent aerosol-induced influenza a virus by denaturing the envelope proteins of the virus [12]. the mechanism of norovirus inactivation by clo2 is attained through degradation of viral protein, including viral genomic rna and disruption of viral strucutre [15]. in addition, an observation on clo2reduced lysozyme activities showed the potential role of clo2 in denaturation and degradation of protein using raman spectroscopy and gel electrophoresis [16]. revisit the literature the wide spectrum of antimicrobial properties of clo2 is supported by its ability to inactivate various kinds of microbes including gram-positive and gramnegative bacteria, enveloped and non-enveloped viruses in low concentrations (as low as 0.05 ppm) and wet states [17]. in a quantitative bactericidal suspension test, it is demonstrated that vegetative forms of bacteria such as staphylococcus aureus and escherichia coli can be killed in the 100 mg/l of clo2 solution [18]. furthermore, clo2 concentration at as low as 0.03 ppm is effective against aerosol-induced influenza virus infection in a study with mice models [12]. however, concentrations of clo2 equal to or more than 0.6 mg/l are required for a complete inactivation of viruses such as hepatitis a viruses, norwalk and norwalk-like viruses [19]. a 3-log reduction in murine norovirus 1 (mnv-1) was found when stainless steel contact surfaces were treated with clo2 gas at 2 mg/l for 5 minutes and 2.5 mg/l for 2 minutes while a complete virus inactivation was shown in a 1minute treatment with 4 mg/l of clo2 gas [15]. although free residues of chlorine over a concentration of 2.19 mg/l of clo2 in wastewater do not entirely inactivating e. coli and f2 phage, it is able to completely inactivate sars-cov [20]. low-concentration clo2 gas (mean 0.05 ppmv, 0.14 mg/ m3) treatment in an area with a high humidity is useful in decreasing the risk of infection by norovirus without side effects [17]. furthermore, propidium monoazide (pmaxx)-viability rtqpcr assay revealed that clo2 is effective to a certain level in inactivating genogroup i and ii human norovirus (hunov) strains on contaminated food [21]. laboratory investigations also showed that the counts of natural or inoculated microbes including bacteria, yeast and mold can be reduced effectively in the range of 1-5 log by using 3-100 ppm of clo2 solution [22]. elimination of b. subtilis spores by clo2 was found due to damages to its membrane but no dna damage [23]. also, a study on the disinfection of wastewater revealed that clo2 is capable of inactivating bacteria such as coliforms although not as effective as chlorine of the same dosage [20]. concluding remarks the broad-range activity against microorganisms, effectiveness over a wide ph range (ph 4.0-10.0), rapid action together with the versatility of using it in either liquid or gaseous state have conferred clo2 as an important disinfectant for daily use in the past two decades. in comparison to other disinfectants, clo2 offers a higher antimicrobial performance at an extremely low concentration with no microbial resistance developed at present. funding the authors declare no funding support provided for this project. conflicts of interest the authors declare that there is no conflict of interest in this work. acknowledgment the authors declare no acknowledgement require for this project. authors’ contributions methodology, investigation, writing. sy, cfg, lcm; 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89: 60–67. 2007/12/20. doi: 10.1099/vir.0.83393-0. 13. ogata n. denaturation of protein by chlorine dioxide: oxidative modification of tryptophan and tyrosine residues. biochem, 2007; 46: 4898–4911. 2007/04/03. doi: 10.1021/bi061827u. 14. ison a, odeh in and margerum dw. kinetics and mechanisms of chlorine dioxide and chlorite oxidations of cysteine and glutathione. inorg chem, 2006; 45: 8768–8775. 2006/10/13. doi: 10.1021/ic0609554. 15. yeap jw, kaur s, lou f, et al. inactivation kinetics and mechanism of a human norovirus surrogate on stainless steel coupons via chlorine dioxide gas. appl environ microbiol, 2016; 82: 116–123. 2015/10/18. doi: 10.1128/aem.02489–15. © 2020 by the authors. submitted for possible open access publication under the terms and conditions of the creative commons attribution (cc by) license (http:// creativecommons.org/licenses/by/4.0/). pmmb 2023, 6, 1; a0000326. doi: 10.36877/pmmb.0000326 http://journals.hh-publisher.com/index.php/pmmb review article covid-19 pandemic in brunei darussalam wei shan ang1, jodi woan-fei law1,2, vengadesh letchumanan1,3, yong sze ong4, yatinesh kumari5, long chiau ming6,7, loh teng-hern tan1,8* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, selangor, malaysia; ang.weishan@monash.edu (wsa) 2next-generation precision medicine & therapeutics research group (nmet), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, selangor, malaysia; jodi.law1@monash.edu (jw-fl) 3pathogen resistome virulome and diagnostic research group (pathrid), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, selangor, malaysia; vengadesh.letchumanan1@monash.edu (vl) 4school of pharmacy, monash university malaysia, subang jaya 47500, selangor, malaysia; ong.yongsze@monash.edu (yso) 5neurological disorder and aging research group (nda), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, selangor, malaysia; yatinesh.kumari@monash.edu (yk) 6school of medical and life sciences, sunway university, 47500 sunway city, malaysia; chiaumingl@sunway.edu.my/longchiauming@gmail.com (lcm) 7paprsb institute of health sciences, universiti brunei darussalam, gadong, brunei darussalam 8innovative bioprospection development research group (inbiod), clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia *corresponding author: loh teng-hern tan, innovative bioprospection development research group (inbiod), clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) received: 16 january 2023; received in revised form: 30 january 2023; accepted: 02 february 2023; available online: 03 february 2023 abstract: within asean, brunei is one of the low population density nations successfully combating the first wave of the covid-19 pandemic in 2020. as of 4th january 2023, over 2.6 hundred thousand confirmed cases of covid-19, with 225 deaths, had been reported in brunei. this paper outlines the covid-19 trends in brunei and strategies taken by the health authorities to contain the pandemic. three waves of covid-19 have hit brunei, with the first case of covid-19 reported on 9th march 2020. the adoption of the “whole of nation approach” has proven to be effective in managing the outbreak. early and decisive interventions taken by authorities and the public’s cooperation have been a remarkable pmmb 2023, 6, 1; a0000326 2 of 16 success story. the key success factors are effective pandemic containment measures, public communication strategy, and enhanced surveillance mechanisms supported by the mass testing program and contact tracing. a national vaccination strategy ensuring adequate vaccine distribution and effective administration has been rolled out to render lasting protection against the infection. national covid-19 recovery plan framework and a gradual covid-19 de-escalation plan were implemented to ensure a smoother transition to the new normal. this review provides valuable insights into the development of a robust pandemic leadership model and highlights the lessons and strategies that other countries can adopt for any future uncertainties. keywords: covid-19; brunei; sars-cov-2; epidemiology; coronavirus; bruhealth 1. introduction three years have passed since the novel coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome-associated coronavirus 2 (sars-cov-2) was first reported in wuhan, china, in late december 2019 [1]. according to research studies, sars-cov-2 can be spread symptomatically or asymptomatically, spreading from person to person through respiratory droplets or touching contaminated surfaces [2]. this rapid spread of the highly contagious respiratory virus has caused severe illness and fatality [3], leading to the declaration of covid-19 as a global pandemic by world health organization (who) in march 2020 [4]. throughout the years, the whole world has been hit by three waves of covid-19 evolution as the viruses acquire many mutations generating multiple new variant strains, namely alpha, beta, gamma, delta (b.1.617.2) [5], omicron (b.1.1.529) [6] and their sub-variants [7-10]. this has taken a significant toll on social communities, the economy and healthcare systems globally [11-17]. taking a view at the asean countries, brunei was not spared from this novel coronavirus. brunei darussalam is a small and well-connected country located on the island of borneo in southeast asia, surrounded by the malaysian state of sarawak. it is a sultanate with a current population of 447,945 as of 3rd january 2023. it is also a high-income country with a gdp of usd 14.01 billion in 2021 [18, 19]. it has a well-developed healthcare system ensuring adequate access to healthcare services among its citizens and residents, contributing to infant mortality of 6.9/1000 live births and a life expectancy at birth of 76.4 years in 2022 [18, 20]. the government offers universal health coverage, including the provision of 15 comprehensive health centres, four hospitals, two air medical services, and two mobile health clinics. brunei has amassed 266,819 confirmed covid cases at the time of writing, with 225 deaths reported as of 4th january 2023 in the country [21, 22]. pmmb 2023, 6, 1; a0000326 3 of 16 2. covid-19 trend in brunei the first case of covid-19 in brunei was reported on 9th march 2020, involving a 53-year-old citizen returning from a tablighi jammat in kuala lumpur, malaysia, with three friends without any symptoms on 3rd march 2020 [23-25]. this 4-day tablighi jamaat mass gathering was attended by 16,000 members of the tablighi, an apolitical islamic group and has driven the global spread of corona internationally to other countries, including indonesia, singapore, thailand, brunei and many others in the region [26, 27]. the patient developed symptoms of fever, cough, runny nose, and body ache on the fourth day of arrival and was transferred to raja isteri pengiran anak saleha hospital for treatment. in the following weeks, 100 cases were reported, all acquired in malaysia, similar to the first case [28, 29]. in the late-march 2020, bruneian home affairs ministry imposed the outbound and inbound travel ban on all citizens and foreign residents with the exemptions of special circumstances to ensure the safety and well-being of the citizens and residents of brunei darussalam. the first covid-19 fatality of a 64-year-old man was reported on 28th march 2020. the number of new infections continued to increase at a slower rate, with the last local case on 6th may 2020 [30]. this was marked as the first wave of covid-19 in brunei. a deescalation plan through a gradual reduction of social distancing measures was put into action by the brunei government on 16th may 2020 to control the outbreak while maintaining its border control [31]. according to the guidelines, the four-stage de-escalation plan was implemented based on the capacity of the health system, the socio-economic, current situation of covid-19 in the country and at regional and global levels (table 1). the new guidelines stipulate four operational readiness levels with specific restrictions: level 0: very high restriction (closure); level 1: high restriction; level 2: medium restriction; level 3: low restriction; level 4: normal operation (pre-normal). progression from one stage to another and implementation of guidelines varies depending on the type of activities or premise along with the timelines [32]. pmmb 2023, 6, 1; a0000326 4 of 16 table 1. illustration of de-escalation plan implementation in brunei [31]. compared to other asean countries, brunei seems to have controlled the initial pandemic relatively rapidly, and the outbreak has been controlled for most of 2020 [33, 34]. an effective pandemic control could be shown when zero local transmission was reported over 15 months [35, 36]. subsequently, new cases reported were limited to imported infections, bringing the total number of confirmed cases to 338 as of 1st august 2021 [21]. the government was still at ease until the first local covid-19 transmission of the delta variant on 7th august 2021, marking the second wave of infection in brunei[37]. an exponential growth of covid-19 cases was triggered in the second wave due to the highly contagious delta variant, bringing the cumulative number of new cases surged to 1873 on 23rd august 2021 [38]. the devastating wave of infections due to the delta strain of rapid transmission, the severe acute respiratory syndrome and deaths led to overwhelming healthcare systems in brunei[39]. amid signs of a vaccine shortage and rising covid-19 cases, this situation set the whole nation on high alert and forced brunei into a two-month partial lockdown starting from 9th august 2021 in order to minimize the risk [40]. a strict curb was imposed, including curfews of 8.00 pm till 4.00 am called “operasi pulih”, closure of several public facilities or non-essential business, restrictions on gatherings and suspension of on-site activities at educational institutions [41]. food establishment was only limited to take away and delivery services [42-44]. the work-from-home policy was implemented in most companies, whereas pmmb 2023, 6, 1; a0000326 5 of 16 only workers in the essential fields were allowed to work in the office [38]. the number of cases decreased with the containment measures in place, and the curve flattened in november 2021. however, various challenges associated with economy lose, ineffective e-learning, psychological and emotional stress have been raised due to stricter control measures during the second wave [45, 46]. thereby, a national covid-19 recovery plan framework was announced on 25th october 2021 to ensure a safe transition and stable situation with minimal disruption to the daily activities of the community [41] (figure 1). despite the containment phase, this framework also includes the preparation phase, transition phase and endemic phase, whereby the commencement of each stage was based on vaccination coverage status and critical cases. government is ready to shift the zero-covid strategy to covid-19 protection, a new phase of safely living with this deadly virus [41]. figure 1. illustration of national covid-19 recovery framework in brunei [41]. meanwhile, the covid-19 steering committee has announced the early endemic phase started on december 2021. loose movement restrictions were imposed, including a shortened night curfew of 12 am to 4 am, but remained the restriction on leisure travel ban [47]. on late december 2021, brunei’s first imported case of omicron was detected when a traveller arrived from the united kingdom [48]. emerging of the new variant strains of omicron, with new cases increasing nearly six-fold than the peak of the delta wave in brunei, has introduced the third wave of infections as omicron replaced delta as the dominant strain[49]. the peak of the third wave was hit at the beginning of march 2022, with the reported number of cases around 4885 and a death tally of 135 [21]. amid high-vaccination rates, fatality rates had been dropping steadily since the beginning of the pandemic to 0.14 % by the end of june 2022 [30]. brunei commences its transition to the endemic phase starting on 1st june 2022. during the endemic phase, individuals had more freedom on inbound and outbound international travel, resuming of in-person learning, reopening of all public facilities, non-essential business, government premises, counter services, and private business offices, manufacturing and processing companies as well as no limitation on the pmmb 2023, 6, 1; a0000326 6 of 16 capacity of mass gatherings. [50]. starting from 22nd june 2022, the brunei government has halted the daily update of covid case numbers [51]. currently, brunei enters the fifth or “new normal” phase of its de-escalation plan (figure 2). figure 2. illustration of daily confirmed covid-19 cases, cumulative covid-19 cases, cumulative deaths due to covid-19, and a brief timeline of the events in brunei since the first case reported in the country. 3. measures taken by the government in addressing the impact of covid-19 in brunei the extreme quarantine implemented in wuhan and the entire hubei region of china mandated all residents to shelter in place without exception. singapore and vietnam’s strategy of comprehensive surveillance to detect and contain as many cases as possible has been highly successful in controlling or eliminating sars-cov-2 [52]. in brunei, the government has taken a ‘whole of nation’ approach in coping covid-19 pandemic effectively, whereby all the ministers and private sectors worked together as a team under the leadership of his majesty the sultan [53-55]. ministry of education (moe) implemented homebased learning and announced school closure [56, 57]. youth volunteer ad-hoc committee was established by the ministry of culture, youth and sports (mcys) to assist the ministry of health in handling the outbreak. ministry of home affairs (moha) implement strict monitoring measures such as proper sanitization, temperature screening and capacity limit permit to certain premises [58]. through the whole-of-government approach, brunei succeeded in suppressing the first wave of the covid-19 pandemic in 2020 by taking early and stringent containment measures without imposing lockdown rules [59]. since latejanuary 2020, prior to the confirmation of the first covid-19 cases in brunei, earlier travel bans on foreign visitors from italy, iran, china’s hubei, jiangsu and zhejiang provinces were imposed, and the returning citizens were required to undergo rt-pcr testing on arrival and a mandatory pmmb 2023, 6, 1; a0000326 7 of 16 quarantine at designated facilities for 14 days [58]. one of the factors that contributed to brunei’s success was adopting pt-pcr testing in the absence of clinical symptoms early in the outbreak and only trails singapore in the number of tests administered per 1,000 people within asean. under the infectious disease act, covid-19 testing is mandatory for anyone with influenza or pneumonia disease and travel or close-contact history, regardless of symptomatic or asymptomatic [60]. this enhanced surveillance mechanism, supported by a mass testing program and rigorous contact tracing, has been implemented across the country, resulting in a significant reduction in the effective reproduction number [61-63]. in responding specifically to the pandemic, the government swiftly dedicated a budget allocation of bnd15 million (us$10.5 million) for the viral outbreaks and emergencies. covid-19 relief fund has been set up to ease the financial burden of the public [60]. a bnd11 million (us$8.3 million) has been allocated to accelerate equitable access to new covid-19 tools. within weeks, the allocation contributed to a comprehensive health care system when key ministries collaborated effectively with sundry construction and engineering firms to pool resources supporting the conversion of the sports complex into a 24-hour testing facility and the construction of more monitoring centres for foreigners, citizens and residents of brunei darussalam from abroad to undergo self-isolation [64, 65]. new national isolation centre building in tutong district was also constructed, thereby increasing the hospital bed capacity by 7-folds. meanwhile, a new auxiliary virology laboratory has been constructed in bandar seri begawan, which is capable of increasing the covid-19 testing capacity in the country. starting in march 2020, the government of his majesty the sultan and yang di-pertuan of brunei darussalam has allocated a special monthly allowance of $400, specifically for medical staffs, volunteers, common workers in hospitals, isolation centre and quarantine centres as well as all staff under the ministry of health. this monthly allowance will be extended until the eradication of covid-19 in brunei darussalam [66]. on the other hand, brunei implemented a transparent public communication strategy through daily live press conferences on national television to provide the latest updates on covid-19 situation. the official launch of the gov.bn telegram channel also provides residents with official government information and announcements, including official press releases and latest covid-19 information. health advice line 148 has been set up and can be contacted for further information on covid-19 updates [67]. the ministry of health has collaborated with china’s yidu cloud technology, a medical technology company, to set up an artificial intelligence system to forecast infection trends and create medical resource mapping [66]. 4. contact tracking mobile apps in may 2020, following the steps of the neighbouring countries in developing contact tracing apps, namely mysejahtera by malaysia and pedulilindungi by indonesia on april 2020 and march 2020, respectively [68], a national mobile health application known as bruhealth developed by evyd technology team was launched by the ministry of health, pmmb 2023, 6, 1; a0000326 8 of 16 brunei darussalam (moh)[69]. it was launched to effectively trace infected individuals and potential risk exposure and identify high-risk areas using bluetooth and gps tracking features. it serves as a virtual platform to control the transmission of covid-19 in brunei via digital contact tracing and a qr code premise check-in [70]. the qr code comes with five colour codes, ranges of green, yellow, red, blue and purple, indicating individual health status, and each colour code denotes entry permissions to any premises [71]. to further ensure better accessibility of the bruhealth application to all residents of brunei darussalam, the premisescan application was launched, enabling the business owner to scan customers’ qr codes upon entry and exit of business premises [72]. under chapter 204 of the infectious diseases act, residents who failed to adhere to the regulation of entry premises based on colour health codes during the covid-19 pandemic will commit an offence. individuals will face a maximum compound fine of bnd5,000 if found guilty; if convicted in court can be fined up to bnd10,000 or sentenced to 6 months imprisonment or both [73]. on 25th september 2021, the bruhealth mandate was enforced to better regulate residents’ movement. the government imposed $100 on-the-spot fines on the individuals who failed to use the bruhealth app in public places [74]. the app has drawn a total of 436,047 registrations since its launch on 14th may 2020 [69]. bruhealth app is different from other contact tracing apps adopted in other countries; it initially provided pandemic updates and faqs on covid-19 and accessibility of medical resources. the new version of bruhealth was rolled out and turned into a healthcare management app which offers additional features such as personal health records, personal health plans, online appointments and online consultation. it also integrates with the brunei health information system (bru-hims), which adopts the concept of one patient one record, which links primary and secondary healthcare data across the entire government health network [75]. therefore, all residents are required to register with bru-hims before seeking medical services or treatment at any healthcare centre to ensure a smooth transition of medical records. a self-assessment tool was provided via healthinfo.gov.bn to self-assess risk factors for covid-19 and concurrently assist government’s initiative to provide real-time government dashboard. 5. vaccination program before vaccines for covid-19 were available, brunei was one of the many countries in the asia pacific region that had participated in the who-led solidarity trial, recognizing the critical importance of global collaborative efforts on the prevention and treatment of covid-19 [76]. the trial was a global effort to compare the safety and effectiveness of biotherapeutics using different combinations of remdesivir, iopinavir/ritonavir, interferon beta, chloroquine and hydroxychloroquine, as early as april 2020 [77, 78]. a wide range of vaccine types were authorized by the brunei darussalam medicines control authority (bdmca) under emergency use authorization to achieve herd immunity. the four vaccines approved for use are comirnaty from pfizer, spikevax from moderna, vaxzevria from astrazeneca and covilo from sinopharm [79-81]. covilo consists of inactivated sars-cov-2 virus in which the genetic materials have been destroyed, rendering it unable to be replicated pmmb 2023, 6, 1; a0000326 9 of 16 or causing disease but still able to stimulate the body’s immune response with the intact antigen protein on the virus surface. vaxzevria is a non-replicating viral vector that delivers modified genetic material to the host cell and triggers an immune response once viral spike proteins are produced in the host cell. comirnaty and spikevax utilise lipid-based nanoparticle encapsulated genetically engineered mrna encoding for the spike protein of sars-cov-2 that directs cells to produce spike protein. upon vaccination, the spike protein produced by the host cell induces the immune system to produce antibodies and other immune cells that protect host cells against the infectious virus. a covid-19 vaccine technical committee is established to study and evaluate the safety, efficacy and quality of covid-19 vaccine candidates for local authorization [82, 83]. vaccination strategy was developed and implemented starting from 3rd april 2021, in which all brunei residents, regardless of citizenship status, can receive the covid-19 vaccination for free. the distribution of the vaccine was divided into three phases. the first phase mainly targeted frontliners against the epidemic, senior citizens aged 60 and above, and students bound for overseas study. in the second phase, the jabs were administered to childcare staff, teachers and adults with comorbidities. in phase three of the vaccination program, the vaccine was available to all residents aged 18 and above [79]. the vaccination centre was allocated for each district in brunei, namely brunei-muara, belait, tutong and temburong. vaccination appointments assigned with a qr code were scheduled via bruhealth app[82]. astrazeneca and sinopharm vaccines were deployed in the beginning phase of the vaccination program. mass vaccination for all adult residents began on 5th july 2021, and the vaccination program was then set to roll out to the younger age group of 12 to 17 on 19th october 2021[84]. kids aged 5 to 11 were administered their first dose vaccine of the pfizer-biontech on 3rd april 2022 as the incidence of child hospitalisation rates quadrupled during the omicron-fuelled third wave, compared to the delta-driven second wave last year [85]. vaccines are not 100% effective at preventing covid infections, but immunization greatly reduces the risk of severe diseases, hospitalization and death in individuals who are fully vaccinated [86, 87]. around november 2021, frontline workers started receiving their booster dose and followed by the senior group later. following the emergence of the omicron variant in late 2021, adults who were fully vaccinated can get mrna vaccines as booster shots, and booster vaccination dosing interval varies depending on the type of vaccine in their second dose [88]. “mix and match” strategies were recommended as preliminary studies showed heterologous covid-19 prime-boost vaccination induces robust immune responses, characterized by high levels of both antibodies and t cells, which kill infected cells and support other antiviral responses [89]. the brunei government does not intend to make booster doses a mandatory condition; however, the administration of booster shots is encouraged to prolong protective immunity and provide maximum protection against covid-19 variants, including delta (b.1.617.2) and omicron (b.1.1.529) [90]. the administration of the third dose is on a walkin basis at all vaccination centres. booster shots for adolescents ages 12-17 were rolled out at the beginning of april 2022 [91]. on june 2022, a second booster shot was offered to elderly pmmb 2023, 6, 1; a0000326 10 of 16 aged 80 and above, adults aged 60 and over with chronic diseases, immunocompromised persons aged 18 and above, as well as healthcare workers and frontliners with high risk of exposure. the fourth jab will be voluntary and only administered to eligible individuals five months after their third dose. [92]. two bivalent vaccines from moderna and pfizer were authorised by the brunei darussalam medicines control authority and rolled out as a booster dose on late november 2022. these bivalent vaccines not only target the original covid19 virus strain and the omicron ba.1 subvariant concurrently it is expected to provide better protection against omicron subvariants, including against ba.4 and ba.5 than the original covid-19 vaccines [93]. as of 11th december 2022, 78.9% of the total population has received a third covid-19 vaccine dose under the national covid-19 vaccination programme. as of 24th november 2022, the vaccination program had delivered 1,283,392 vaccine doses with a vaccination rate of 285.83%, which contributing an immunisation coverage of 97.8% [21]. 6. conclusion as early as january 2020, brunei had implemented stringent precautionary measures early, including massive testing, implementation of travel bans and mandatory quarantine to prevent and suppress covid-19 before the first case was detected on 9th march 2020 [94]. brunei was hit by three covid-19 waves as follows: first wave occurred between march and may 2020; second was from the end of august 2021 to december 2021; third wave commenced in january 2022 until the present day. the key lessons of brunei’s success factors in managing the covid-19 pandemic are the adoption of “whole of government approach” and the “whole of nation approach”, which involves the engagement of all ministers, private sectors and residents in the country. successful containment of the covid19 pandemic in brunei was achieved through prompt actions from the government, speedy implementation of national policies and mutual adherence to government guidelines by the public. brunei government has taken a combination of pandemic containment measures such as travel restriction, social distancing, strict quarantine measures, and test-isolate-trace approach complemented with contact tracing applications, namely bruhealth and premisescan. this has resulted in the successful flattening of the curve of the first wave and maintaining zero local transmission for 15 months. at the same time, brunei deployed financial support to mitigate the economic fallout due to covid-19 pandemic [95]. adequate vaccine procurement, distribution, effective rollout and administration are also the key to securing a durable recovery. with the pandemic broadly under control, brunei implemented national covid-19 recovery plan framework and a gradual covid-19 de-escalation plan to bring the nation adapting to the new normal. the notable policies aforementioned can provide a framework for other nations to contain future pandemics. author contributions: wsa performed the literature search, critical data analysis as well as manuscript writing. jw-fl, vl, yso, yk and lcm performed editing and revision. jw-fl, vl, yso, yk, lcm and lt-ht provided technical support and proofreading. lt-th conceptualized this review writing project. conflicts of interest: the authors declare no conflict of interest. pmmb 2023, 6, 1; 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a0000339. doi: 10.36877/pmmb.0000339 http://journals.hh-publisher.com/index.php/pmmb review article the ameliorative role of probiotics in 5-fluorouracil induced intestinal mucositis alexandra arcilla xin hui sim1†, jia ying cheam1†, jodi woan-fei law1,2, vengadesh letchumanan1,3, yatinesh kumari4, satoshi ogawa5, sunny hei wong6, kok-gan chan1,7, loh teng-hern tan1,8* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia. asim0007@student.monash.edu (aaxhs); jche0331@student.monash.edu (jyc); jodi.law1@monash.edu (jw-fl) vengadesh.letchumanan1@monash.edu (vl); kokgan@um.edu.my (k-gc) 2next-generation precision medicine and therapeutics research group (nmet), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia 3pathogen resistome virulome and diagnostic research group (pathrid), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia 4neurological disorder and aging research group (nda), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor, malaysia. yatinesh.kumari@monash.edu (yk) 5jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia. satoshi.ogawa@monash.edu (so) 6lee kong chian school of medicine, nanyang technological university, singapore 308232, singapore; sunny.wong@ntu.edu.sg (shw) 7institute of biological sciences, faculty of science, university of malaya, kuala lumpur 50603, malaysia 8innovative bioprospection development research group (inbiod), clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia † these authors contributed equally to this work. *corresponding author: loh teng hern tan, innovative bioprospection development research group (inbiod), clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu received: 11 june 2023; received in revised form: 11 july 2023; accepted: 14 july 2022; available online: 16 july 2023 abstract: colorectal cancer (crc) remains one of the top cancers in the world. although early detection improves the survival rate to around 90%, late detection would mean the need to use chemotherapy or radiotherapy, especially if surgery is not feasible. 5-fluorouracil (5fu) is one of the common anti-cancer drugs used in treating crc. it is the drug that has the pmmb 2023, 6, 1; a0000339 2 of 34 greatest efficacy on crc. although it improves the survival rate, it has many detrimental side effects. one of these side effects is intestinal mucositis. it is described as having reduced villus height, villus atrophy, crypt fissure, abdominal discomfort, diarrhoea, and weight loss. clinically, there is no conclusive treatment therapy for mucositis. this is possibly due to the complex mechanism of the pathobiology of intestinal mucositis that includes the production of pro-inflammatory cytokines and increased epithelial cell apoptosis. 5-fu itself is known to cause gut dysbiosis. current studies revealed probiotics play a role in attenuating this inflammatory process of intestinal mucositis by reversing gut dysbiosis, reducing the expression of pro-inflammatory cytokines, and reducing intestinal damage. this review outlines the latest evidence supporting probiotic use in ameliorating 5-fu induced intestinal mucositis, thereby promoting good health and well-being in colorectal cancer patients receiving 5-fu chemotherapy. keywords: colorectal cancer; 5-fluorouracil; intestinal mucositis; gut dysbiosis; probiotic; sdg 3 good health and well-being 1. introduction colorectal cancer (crc) is the second leading cause of death due to cancer, with 935,000 deaths worldwide[1–3]. the survival rate of crc depends on the stages of cancer and when it was diagnosed[4]. generally, 63% of crc patients will live at least five years postdiagnosis. early stages can have survival rates up to 90%, while for later stages, especially those diagnosed with metastatic crc, the survival rates are only around or less than 20%[5,6]. when treating crc, the goal is to achieve as much total removal of tumours as possible while preserving patients' quality of life and survival. typically, like most cancers, conventional surgery or chemotherapy is the most common treatment option depending on the size, location, and extent of metastasis. typically, most early stages crc patients would undergo tumour resection, while those who cannot undergo surgery would undergo chemotherapy to hinder or kill cancer cells. 5-fluorouracil (5-fu) is the most common crc chemotherapy treatment. currently, 5-fu is either administered as a single drug or in combination with other drugs[7]. combination regimens available now are the folfox regimen (5-fu and oxaliplatin), folfiri regimen (5-fu, leucovorin, and irinotecan), folfoxiri (5-fu, oxaliplatin, and irinotecan) and others[7]. chemotherapy prolongs survival, reduces cancer-related symptoms, and preserves general well-being in advanced crc patients[8]. survival rates increase by almost 20% in high-risk crc patients that undergo chemotherapy[9,10]. being the backbone of crc chemotherapy, 5-fu improves overall survival and disease-free interval in stage 3 crc[10]. 5-fu does this by inhibiting the normal function of dna and rna. 5-fu interferes with thymidylate synthesis and inhibits dna synthesis during dna replication and repair[11]. eventually, 5-fu arrests the cell cycle and induces cell death[12]. although 5-fu is a key drug to treat crc, it comes with a cost. since chemotherapy targets tissues with a high cell division rate, other normally dividing cells with the same rate of division are also affected[13]. pmmb 2023, 6, 1; a0000339 3 of 34 repeated treatment with 5-fu, as frequently happens with chemotherapy, 5-fu causes changes to body weight, bowel movement and gut architecture. weight loss starts around day five post-5-fu, along with diarrhoea two days before. there is around a 10% risk of developing grade three or four diarrhoea (with severe diarrhoea until requiring hospital admission or severe diarrhoea with life-threatening results) in patients that undergo the folfox regimen, and it is doubled in patients receiving folfoxiri[14]. zooming into the gut's architecture, 5-fu causes villi shortening and reduced number of crypts and crypts cells. inflammation occurs, with neutrophil infiltration (measured by myeloperoxidase activity), an increase in pro-inflammatory cytokines (pic) and apoptosis. all these events can be categorised as intestinal mucositis (im). a common finding is the increased crypt apoptosis and villus atrophy leading to poor nutrition, malnutrition and bacteraemia[15,16]. having im is correlated with the application of total parenteral nutrition, analgesics and antibiotics, which are used to decrease the side effects of the inflammation and inflammation. however, in the long run, these are coupled with side effects that impact patients' quality of life, leading to a longer hospital stay, hospital fees and increases in the risk of nosocomial infections[17]. apart from that, im often results in dose reduction of chemotherapeutic agents or deferral of treatment, which further increases the mortality rate[18]. all these affect patients' ability to tolerate chemotherapy, affecting their chances of survival[17]. recognising these challenges, researchers are actively exploring natural resources to offer effective and less toxic alternatives to traditional chemotherapy[19–25], such as 5-fu in this case. the exploration of natural products, particularly those derived from microbes[26– 35], animals[36] and plants[37–40], holds promise in improving crc treatment. while these natural products offer the potential for reduced toxicity and enhanced therapeutic effects, 5fu remains the mainstay of chemotherapy used when crc is at the advanced stage or has a higher risk of recurrence[41]. among the many therapeutic alternatives, probiotics emerge as the most promising adjuvant in complementing and enhancing the effects of chemotherapy drugs. probiotics offer several mechanisms through the modulation of the gut microbiome, which plays a crucial role in overall health and immune function[42]. by promoting a healthy gut microbiome, probiotics may improve the body's response to chemotherapy and enhance treatment efficacy[43]. additionally, probiotics can support gastrointestinal health, alleviate chemotherapy-induced side effects such as diarrhoea, and enhance nutrient absorption[44–46]. by leveraging the symbiotic relationship between the gut microbiome and the body's response to cancer treatment, these studies aim to enhance the effectiveness of chemotherapy while reducing its associated toxicity, ultimately improving patient outcomes and quality of life[44,47]. gut microbiota influences the responses of a host to chemotherapeutic agents[48,49]. for instance, certain groups of gut microorganisms can enhance drug efficacy, while some promote chemoresistance and mediate chemotherapy-induced side effects[13]. one of the explanations is that chemotherapy causes ros-mediated dna and non-dna damage, which then leads to bacterial translocation through the gut epithelium, hence causing an pmmb 2023, 6, 1; a0000339 4 of 34 inflammatory reaction that can lead to systemic infections and other complications[13]. unsurprisingly, reports have shown the involvement of gut microbiota in the pathophysiology of im. specifically, the crucial role of gm in 5-fu induced im has recently been highlighted with the recent study on probiotics, showing that probiotics lessen side effects such as diarrhoea and weight loss, possibly because of restoration of altered gut microbiome composition caused by 5-fu[15]. therefore, this review discusses the pathophysiology of im induced by 5-fu and how gm can mediate the inflammatory process. although most of the studies are performed in animal models, overlapping features are shown in the studies and current understanding of the human body. key findings in these animal studies would serve as a good fundamental for future human studies. in short, gm can present as a target therapeutic approach in ameliorating chemotherapy-related side effects, especially in patients undergoing the 5-fu regimen. 2. the role of gut microbiome and their involvement in the pathophysiology of 5fu induced intestinal mucositis the microbiome is a huge and intricate microbial community comprising bacteria, viruses, archaea, fungi and parasites that inhabit the human body[50]. among them, actinobacteria, firmicutes and bacteroidetes are the most prevalent phyla of bacteria. there are more than 100 trillion microorganisms in the gastrointestinal tract, which serves as the site of primary communication between the microbiota, the host cells and the host immune system[51,52]. thus, the gut microbiome (gm) is associated with many functions in the human immune, metabolic, and structural functions. however, the occurrence of gut dysbiosis, specifically the disruption of the balance of beneficial bacteria in the gut microbiome, has been consistently linked to a range of human diseases[53–62]. recent evidence suggests that certain pathogenic gut microbiota play a role in carcinogenesis[63,64]. for example, sulfidogenic bacteria produce hydrogen sulphide which is capable of damaging dna. it also inhibits dna repair mechanisms, leading to extensive dna damage[65]. these sulfidogenic bacteria include fusobacterium, desulfovibrio and bilophila wadsworthia. besides that, gram-positive organisms such as streptococcus bovis or currently named s. gallolyticus are positively correlated with the development of adenomas and carcinomas. various studies show an estimated likelihood of a 6–71% chance of developing crc in patients that had s. gallolyticus bacteremia. infection with s. gallolyticus can occur via infectious endocarditis or the presence of s. gallolyticus in the blood[66,67]. im is a complex process that involves many different cells, tissues and signalling molecules. the pathophysiology of im is yet fully unravelled. sonis[68] proposed a 5step theory consisting of the i) initiation phase, ii) upregulation and message generating phase, iii) amplification and signalling phase, iv) ulcerative phase, and v) the healing phase. it is worth mentioning that the activators for every phase would be a good target for intervention. 5-fu induces dysbiosis in the gut, where it disturbs the delicate balance of homeostasis. gut dysbiosis is worsened when given antibiotics. antibiotics kill beneficial bacteria, such as those that secrete metabolites that protect the mucosa and those that influence the metabolism of 5-fu but spare pathogenic bacteria that contribute to decreasing the anti-tumour efficacy pmmb 2023, 6, 1; a0000339 5 of 34 of 5-fu[69]. with this being said, it could be thought that gm is important to humans and may play a role in preventing or treating im. we will discuss sonis[68] theory (summarised in figure 1) with a focus on how the gut microbiome plays a role in this inflammatory process. gm also influences intestinal permeability (ip) and mucus layer, which are the gut's defence barrier. 2.1. initiation phase based on clinical evidence, the onset of mucositis begins within 24 hours to 48 hours upon the initiation of the cancer treatment[18]. the first stage, which occurs seconds after exposure to chemotherapy, is marked by the production of reactive oxygen species (ros), direct dna and non-dna damage, and immune system activation[13]. it turns out that the human gut is constantly in a steady state of low-grade inflammation. this condition is due to the toll-like receptors (tlr) on epithelial cells and intestinal-associated immune cells. the conserved molecular motifs on bacteria are recognised by the tlrs outside the cell, activating several intracellular signalling cascades, such as the nf-κb signalling pathway[70]. tlr also maintains homeostasis by preserving the mucosal layer and preventing pathogenic bacteria from disrupting this homeostasis[70]. nf-κb is a transcription factor that upregulates pic, chemokines and adhesion molecules such as tnf-α, il-1β and il-6[68,70]. nf-κb is activated by chemotherapy or radiotherapy and leads to the generation of reactive oxygen species (ros). ros are capable of causing tissue damage in the gut epithelium, connective tissue and even blood vessels, eventually resulting in dna damage and cell death[68]. tlrs trigger a signalling cascade. the importance of tlr can be seen in tlr4 knocked-out mice exhibiting altered neutrophil recruitment and epithelial proliferation. it is thought that tlr4 is able to limit bacterial translocation and modulate intestinal response to injury[71]. this shows that intact tlr signalling might be crucial for the anti-inflammatory response. whether tlr agonism confers direct protection to either the gut itself or the intestinal microbiome is still unknown. it serves as a potential target site for treating im as tlrs are frequently exposed to bacteria parts, such as bacterial cell wall. due to the constant exposure between tlrs and bacteria, there is always a basal activation of pro-inflammatory transcription factor nf-κb. however, commensals do not trigger an inflammatory cascade[72]. it is postulated that the epithelium sequesters the commensals, preventing tlr activation, while pathogenic bacteria, equipped with virulence factors, are able to bypass the epithelial barrier to be detected by tlr ligands on immune cells[72]. it is also shown that commensals can suppress nf-κb, such as in the case of bacteroides thetaiotaomicron that targets active nf-κb subunit rela complex and decrease its activation. b. thetaiotaomicron also removes nf-κb complexes formed when epithelial cells were induced with virulent salmonella strains resulting in decreased pic production[73]. the full mechanism of how tlrs are able to differentiate pathogenic and commensal bacteria serves as a gap in present evidence, prompting further research. pmmb 2023, 6, 1; a0000339 6 of 34 figure 1. pathogenic bacteria bind to toll-like receptors (tlrs). tlrs activate ikk kinase. ikk phosphorylates nf-κb associated proteins, p50 and rela. the phosphorylated complex is targeted for polyubiquitination, allowing degradation by proteasome, leaving only p50 and rela. these free proteins translocate into dna and activate pro-inflammatory gene expression. commensal bacteria such as nonvirulent salmonella can prevent ikk complex from being ubiquitinated, preventing degradation and incorporation into dna. other commensals act at the nuclear level, acting on rela protein via transcription factor ppar-γ. these two bind together and dampen nf-κb pro-inflammatory cascade. besides b. thetaiotaomicron, nf-κb signalling can be suppressed directly by influencing other signalling pathways, as shown by bifidobacterium. il-10 is an antiinflammatory, having nf-κb inhibitory properties. il-10 can also block the transcription of il-1β, il-6, il-8, and tumour necrosis factor (tnf-α)[74]. il-10 maintains gut immune homeostasis by decreasing pic concentrations at the tissue damage site[75]. in il-10 deficient rats, bifidobacterium infantis managed to prevent proinflammatory-associated rearrangement of tight junction (tj) proteins and instead increase ter. the study also showed that long-term exposure to b. infantis reduces inflammation, maintains colon permeability to normal levels and reduces ifn-γ secretion[76]. ifn-γ is an important recruiter for macrophages, leading to immune cell infiltration[77]. moreover, faecalibacterium prausnitzii secretes an unknown molecule that promotes il-10 production[78]. short-chain fatty acids (scfa) such as butyrates by f. prausnitzii can also inhibit nf-κb and il-2[79,80]. butyrates are the main products excreted by microbes during the fermentation of dietary fibre (also known as prebiotics). it is found that butyrate suppresses inflammation by reducing tnf-α, ifn-γ, and il-12 and prevents carcinogenesis by strengthening the mucosal barrier and modulating oxidative stress[81,82]. probiotic supernatants have immune-modulating properties as well. supernatant of bacillus coagulans directly increases il-10 production, which leads to reduced ros production[11]. supernatant of escherichia coli nissle 1917 (ecn) was anti-inflammatory in rats with colitis, reducing tnf-α and increasing il-10[83]. in short, probiotic is capable of downregulating tissue-damaging ros by targeting upstream pmmb 2023, 6, 1; a0000339 7 of 34 factors, particularly nf-κb. the exact target to pinpoint in this pathway could be key in reducing im. 2.2. upregulation and message-generating phase after the initiation phase, begins the upregulation and message-generating phase. dna damage and ros production activate nf-κb similar to the initiation phase. tnf-α is also activated, leading to epithelial cell apoptosis. nf-κb can transcribe up to 200 genes, most of which are toxic to the gut mucosa. for example, pro-apoptotic proteins are produced by the upregulation of bcl2 genes and bax genes. the overall upregulation of genes such as bcl2 and bax genes hastens mucosal damage. nf-κb also can increase pic production in the mucosa. these cytokines damage the connective tissues and endothelium by initiating mesenchymal epithelial signaling and reducing oxygen concentrations in the epithelium resulting in epithelial cell death and injury[68]. this process causes thinning of the mucosa, making it erythematous. this condition can be ameliorated by supplementing with lactic acid bacteria (lab) such as bifidobacterium breve. lab protects against inflammation by releasing antimicrobial peptides, iga. its presence itself competes with pathogens to adhere to the gut epithelium. lab have a stronger adherence to epithelial cells by increasing the expression of binding proteins. besides that, labs are able to secrete antioxidant enzymes as well[84]. another study showed streptococcus thermophilus metabolites can cross the mucosa and suppress tnf-α[85]. 2.3. amplification and signalling phase the third phase is the amplification and signalling phase. there is positive feedback by the mediators released in previous phases that magnify other biological factors. chemotherapy such as 5-fu can activate nf-κb (a transcription factor), such as found in the gut's epithelial, endothelial, and mesenchymal cells. nf-κb in macrophages can be activated as well. activation of nf-κb leads to the production of pic such as tnf-α and interleukin1β (il-1β). they participate in positive feedback, further activating nf-κb. nf-κb upregulates transcription of mitogen-activated protein kinase (mapk), cyclooxygenase 2 (cox2) and tyrosine-kinase signalling molecules. these biological factors gather in the mucosa and target the tissue. for example, increased tnf-α expression has positive feedback on nf-κb activity. tnf-α can activate caspase 3 (pro-apoptotic protein), which causes cell apoptosis. upregulation of mapk also can lead to activation of pro-apoptotic protein caspase 3. additionally, upregulation of mapk, cox2 and tyrosine-kinase leads to the activation of matrix metalloproteinases (mmps) 1 and 3 present in epithelial cells and the lamina propria. mmps ultimately cause damage to the gut mucosa[86]. clinically, the gut mucosa may seem erythematous, and patients can manifest very few signs and symptoms while still in the early phases of inflammation[68]. pmmb 2023, 6, 1; a0000339 8 of 34 2.4. ulcerative phase the gut barrier consists of the mucus layer, tj and epithelial cells. disruption of any of these can impair the barrier. the mucus layer, secreted by goblet cells, provides a physical barrier around the mucosa lining. tj acts as a seal between epithelial cells comprising a bunch of proteins such as occludin and junctional adhesion molecules. tj interspersed between epithelial cells is a physical barrier to prevent unwanted substances such as pathogenic bacteria, toxins, or byproducts from penetrating the mucosa. zonula occludins (zo-1) tighten the tj seal at the cell junction[87]. 5-fu decreases both tj and zo-1 expression[88]. upstream inflammatory factors like tnf-α (through nf-κb activation) and il-1β exacerbate this cycle by down-regulating zo-1 proteins and altering the localisation of zo-1 proteins[89,90]. instead of the normal cohesive binding of zo-1, it becomes gaplike and zig-zag in appearance. disruption in both tj and zo-1 leads to a penetrable mucosal layer, leading to a more permeable intestine[89]. besides the connection between epithelial cells, epithelial cells themselves are damaged. cell apoptosis and villous atrophy occur, forming crypts. crypts serve as a harbour for pathogenic bacteria to grow on. crypts also leave nerve endings in the lamina propria exposed, making this phase painful for patients. besides losing the mucus layer, the epithelial barrier itself is damaged. the gut mucosa is very permeable in this phase, allowing bacteria to translocate across this barrier. the 'leaky' gut mucosa puts patients at risk of developing bacteraemia and sepsis. direct exposure of the gut to pathogenic bacteria causes the human body to recognise foreign bacteria and start another cascade of inflammatory processes. mononuclear infiltrates by chemotaxis lead to more inflammation, possibly promoting proapoptotic gene expression and pic, further potentiating further tissue injury (figure 2)[68]. figure 2. the gut mucosa is protected by one layer of epithelial cells with tight junction interspersed in between cells. this barrier is also reinforced with a mucus layer. bacteria bind to toll-like receptors (tlrs). the binding activates nf-κb. nf-κb causes an upregulation of pic. bacteria are also phagocytosed by dendritic cells. dendritic cells present bacteria parts to immunoglobins, subsequently setting off an inflammatory response as well. pmmb 2023, 6, 1; a0000339 9 of 34 regulating tj and zo-1 expression is vital to the progression of intestinal permeability. with the administration of bifidobacteria, there was increased expression tj forming proteins and decreased intestinal permeability[76]. lactobacilli also has been shown to elevate tj protein expression and restore intestinal permeability[91–94]. probiotic strains such as e. coli nissle 1917 and l. rhamnosus gg upregulates zo-1, protecting the gut from being more permeable[95,96]. upregulation of zo-1 is important as it protects the epithelial cells from damage such as those caused by pathogenic bacteria like escherichia coli o157:h7[96]. scfa produced by bacteria is able to reduce ip and increase cell viability[81]. it also helps modulate the intracellular environment, maintaining the intestinal structure, function and integrity and promoting gene expression and proliferation of the epithelial cells[97]. butyrate, propionate and acetate are usually produced due to the fermentation of undigested carbohydrates or prebiotics by b. breve and bacteroides ovatus[98]. 2.5. healing phase the last phase is the healing phase. gut mucosa self-heals itself once the causative factor is removed. in this phase, the extracellular matrix (ecm) releases molecules which promote the healing of the ulcer. first, the molecules migrate from ecm to the ulcer border. then, migration of the cells occurs to cover the ulcer. then these cells begin to differentiate into epithelial cells. normal architecture is then restored. other molecules that promote hyperplasia are downregulated. once the ulcer has healed, symptoms of im begin to resolve[68]. it is known that gut dysbiosis with increased numbers of pathogenic bacteria on an exposed intestinal surface can activate more tlrs. meanwhile, chemotherapy activates tlrs via nf-κb[70]. it turns out the steady-state inflammation by tlrs and commensal bacteria is proven to allow the repair of the epithelium. commensal bacteria help to induce the recovery of the gut mucosa. for instance, in a study on chicks, migration of the cells is faster by more than half the time of those in germ-free chicks[99,100]. this allows faster healing of the gut epithelium. in this case, tlr is also significant because it can sense injured or dead cells as tlr recognises and binds to heat shock protein (hsp) produced by dying epithelial cells[70,71]. cytoprotective effects of hsp are suppressed in tlr knockout mice. for example, a finding indicates that mice deficient in tlr (specifically tlr4) could not repair damaged cells. they experienced more severe damage in the epithelium, with decreased epithelial cell proliferation. tlr4 knockout mice show impaired tlr4 and hsp signalling, delaying response to dead or dying epithelial cells and leading to delayed epithelial healing[71]. as seen before, butyrate is a key player. butyrate promotes the migration of epithelial cells, thus enhancing mucosal healing[81,101]. other bacterial products, such as the peptides (p75 and p40) secreted by l. rhamnosus gg, prevent epithelial cell apoptosis and stimulate cell growth, hence stimulating the repair of the mucosal layer[102]. l. lactis nz900 producing pancreatitis-associated protein i (pap) also stimulates epithelial cell growth, including paneth cells. pap alters the expression of growth factors and proliferation-related molecules pmmb 2023, 6, 1; a0000339 10 of 34 along with antioxidant enzymes[103]. figure 3 summarises the 5 phases of mucositis induced by 5-fu. figure 3. the 5 phases of mucositis. the initiation phase begins with the exposure of gut mucosa to chemotherapy or radiotherapy. here, we show exposure of the gut to 5-fluorouracil. reactive oxygen species (ros) are then formed, promoting dna damage. ros activates nf-κb, a transcription factor that subsequently activates pic, such as interleukin 6 (il-6), interleukin 8 (il-8), il-1β, tnf-α, and cyclooxygenase 2 (cox-2). dna damage occurs and leads to apoptosis. in the third phase, there is a positive feedback loop in which pic further reinforce stimulation to produce more pic, causing further oxidative stress and worsening the injury. eventually, a vicious cycle is created, and ulcers form due to intestinal injury. after stopping stimuli, the mucosa begins to heal by itself in a few days until the mucosa is restored. 2.6. effects of 5-fu and its impact on gut microbiota composition 5-fu causes a shift in gut microbiota profile by decreasing the abundance of intestine firmicutes while increasing the abundance of bacteroidetes and verrucomicrobia[104]. specific strains were shown to decrease in abundance, such as clostridium spp., lactobacillus spp. and streptococcus spp., while some increased in abundance, like escherichia spp.[105]. the gut microbiome is in a dynamic state, allowing external factors to influence it, such as antibiotics[106]. studies have found antibiotics do not offer any clear protection against im. this was demonstrated by mice that were treated with both 5-fu and a cocktail of antibiotics; pathogenic bacteria such as escherichia, shigella, and enterobacter significantly increased in abundance while other commensals such as firmicutes decreased in abundance. the combination of antibiotics and 5-fu downregulates genes involved in amino acid metabolism, replication and repair translation and nucleotide metabolism, which contributed to overall reduced antitumor efficacy. the effect of gm on 5-fu antipmmb 2023, 6, 1; a0000339 11 of 34 tumour efficacy is evident with the administration of antibiotics. antibiotics use instead worsens patients' body weight in clinical trials. they also showed a higher tumour/body weight ratio in comparison to the 5-fu treatment alone[69]. therefore, since antibiotics are not working in ameliorating im, other strategies suitable for use along with 5-fu regimens to ameliorate im need to be investigated. 3. gut microbiota modulation-based strategies to ameliorate 5-fu-induced intestinal mucositis the commensals residing in the human gut are crucial for sustaining a healthy human body. healthy individuals usually contain bacterial species belonging to four phyla firmicutes, bacteroidetes, actinobacteria and proteobacteria[107,108]. gm helps hosts maintain immunity, protecting hosts from bacterial invasion and nutrient metabolism[47,109]. shifts in this dynamic structure are present in inflammatory diseases such as inflammatory bowel disease (ibd), psoriasis and multiple sclerosis[107,110,111]. neurological and psychiatric conditions, such as depression, obsessive compulsive disorder and myasthenia gravis, are also linked to gut microbiome changes[112,113]. although the full mechanism of all of these interactions has yet to be unravelled, associations between gm and many common illnesses have been evidenced in numerous studies. for instance, gut dysbiosis is also evident in crc patients. as mentioned earlier, 5-fu and antibiotics are also able to cause shifts in the gut microbial composition. various strategies such as probiotics, prebiotics, dietary modifications, postbiotics, and fecal microbiota transplantation are utilized for gut microbiota modulation to promote health and manage diseases[114–118]. among all of these strategies, probiotic use is one of the most common strategies, and it has been shown to prevent or ameliorate dysbiosis of the normal microbiota by maintaining a balanced gut microbiome by reducing potential pathogenic bacteria while increasing the number of beneficial bacteria[119–125]. with that, restoring the disturbed gut microbiota by supplementing probiotics can lessen the side effects of 5-fu, especially im[126]. 3.1. use of probiotics in intestinal mucositis probiotics are "live microorganisms when administered in adequate amounts that confer a health benefit on the host"[97]. they are non-invasive and safe when compared to pathogenic bacteria. only viable bacteria can be considered probiotics, not including bacteria components that are not viable[18]. some requirements that must be met to consider a bacterial strain as a probiotic include the ability to adhere to and colonise the gut mucosa to interact with the host to modulate the antagonism against pathogens and for immune system responses[44,127,128]. they colonise the gut temporarily and without releasing toxins or metabolites detrimental to the host. to ensure the viability of the bacteria for colonisation of the intestinal tract, they must be able to resist the environment of low ph and bile salt in the gastrointestinal tract[18]. they help to restore gut dysbiosis by cultivating good commensal bacteria. probiotics currently are used as adjuvants for many gastrointestinal disorders such as diarrhoea due to infections (rotavirus infection) or iatrogenic (antibiotic use) or due to other inflammatory diseases such as ibd[12,129]. it is thought that probiotics decrease pmmb 2023, 6, 1; a0000339 12 of 34 pathogenic bacteria that activate the immune system[107]. pic and ros have clearly been shown to be the main factors driving the inflammatory process of im. as such, probiotics can come into play by being an anti-inflammatory agent. since it has been shown that commensal organisms play a role in the inflammatory cascade, it would be a good target site to help ameliorate the manifestation of im. having said that, not only can probiotics suppress the inflammatory process in im, but they also promote healing. probiotics can improve host defence, such as mucus layer, tj proteins and immune defence, subsequently ameliorating the side effects of 5-fu by reducing apoptosis and improving mucin production. clinically, weight loss and diarrhoea were also attenuated with the administration of probiotics. together, probiotics are capable of ameliorating 5-fu induced side effects via different mechanisms[105,130,131]. 3.1.1. gut dysbiosis the composition of the gut microbiota is altered and modified through a process called dysbiosis due to undergoing chemotherapy, which is often related to the gastrointestinal tract's biochemistry and immunologic disorders[46]. gut dysbiosis is shown to be common among crc patients, indicating the possible relationship between the gut microbiome and the carcinogenesis of colorectal cancer[13,132]. a primary increase in gramnegative bacteria was found following 5-fu administration in rats, as shown in a preclinical study[13]. at the jejunal level, 5-fu decreases clostridium sp., lactobacillus sp. and streptococcus sp., but increases escherichia sp. in the colon, 5-fu caused decreases in enterococcus sp., lactobacillus sp. and streptococcus sp. faecal samples showed decreasing trends in lactobacillus sp. and bacteroides sp., and an increasing trend in e. coli and staphylococcus sp.[133]. this was also validated in another study of mice administered with 5-fu showing a shift away from good bacteria such as actinobacteria and towards bacteroidetes and verrucomicrobia phyla with pro-inflammatory traits[13]. hence, gut microbiota modulation-based strategies which can shift dysbiosis towards eubiosis or achieve gut microbiota homeostasis should be developed[46]. probiotics have evidently been able to change this shift in organisms. probiotic bacteria that can colonise the intestinal tract have been shown to restore eubiosis by releasing antimicrobial compounds such as bacteriocins and decreasing the ph to limit the development of other harmful bacteria[36]. gut microbiota is brought back into balance when the amount of pathogenic bacteria reduces due to their inability to survive in the acidic environment while the amount of good bacteria which flourish in the acidic environment increases[18]. this was apparent when researchers used a probiotic mixture in mice and showed a decrease in opportunistic pathogens[69]. in male mice that received both 5-fu and lactobacillus reuteri dsm 17938 (bg), it was found bacteroides and bacteroidaceae were most bountiful in 5-fu treated rats supplemented with the mixture, while those that received only saline solution showed that muribaculaceae (s24_7) was most abundant[134]. similarly, lactobacillus plantarum 299v reduced 5-fu stimulated the growth of facultative anaerobes in the intestine[135]. both probiotic strains, lactobacillus casei pmmb 2023, 6, 1; a0000339 13 of 34 variety rhamnosus (lcr35) and bifidobacterium bifidum g9-1 (bbg9-1), also can significantly reverse the perturbed microbiota composition of firmicutes and bacteroidetes induced by folfox. however, supplementation of lcr35 itself did not affect faecal gm composition[12,131]. this shows that probiotics are straindependent. hence, further research must be carried out in the future to find out more functional probiotic strains. another strain, rhodotorula mucilaginosa ufmgcb 18377, was also found to be able to reduce intestinal enterobacteria levels in 5-fu treated mice with induced mucositis[136]. in vitro studies, non-recombinant l. lactis nz9000 showed antagonistic activity against the invasive gut pathogen listeria monocytogenes and its secreted compound called pap had an inhibitory effect against opportunistic enterococcus faecalis[137,138]. pap is not the only property possessed by l. lactis nz9000; it is thought that its antagonistic actions against pathogens could also be due to the secretion of lactic acid, bacteriocins or metabolites[137]. 3.1.2. pro-inflammatory cytokines in the pathobiology of im, tnf-α is a key pic that causes im. other cytokines involved include interleukin (il)-4, il-6, il-10 and il-17. il-17 is an inflammatory mediator. it acts together with ifn-y to synergistically enhance il-8 secretion by human fetal intestinal epithelial cells[139]. when supplemented with probiotics lcr35 and bifidobacterium bifidum (labi, infloran®), levels of pic decreased[12,140,141]. the study shows lcr35 has the ability to suppress nf-κb activity, also leading to suppression of inflammation and subsequently reduced im in the intestine[12]. other lactobacillus strains, such as lactobacillus delbrueckii cidca 133 (pexu:hsp65), a recombinant strain of l. delbrueckii is able to increase serum il-10 in ileum tissue while reducing inflammatory infiltrate and cytokines such as tnf-α, il-6, il-12, and il-1β[142]. other strains s. thermophilus crl 808 decreases il-6 but increases il-10. this vitamin-overproducing strain can reverse the rise of the anti-/proinflammatory cytokines ratio. it increases concentrations of il-10 in the serum along with decreased ifn-γ levels[143]. in another experiment on a mouse model, it was found that the levels of tnf-α, il-1β and il-6 were higher in the 5-fu induced mouse group than the control group. however, those concentrations markedly decrease in the use of s. thermophilus st4 along with 5-fu treatment[97]. b. infantis also successfully reduced high levels of pic caused by 5-fu in crc rats. as mentioned, ifn-γ recruits macrophages[77]. il-2 is important for ifn-γ stimulation while il-12 stimulates production of ifn-γ by naïve t cells[144] . in this case, it is found that th1 cells and their cytokines, il-2, il-12 and ifn-γ, which are upregulated during chemotherapy, can be reversed by b. infantis[145]. pmmb 2023, 6, 1; a0000339 14 of 34 3.1.3. intestinal damage as mentioned before, changes in the architecture of the intestinal mucosa are side effects of 5-fu, including a decrease in villus high, an increase in crypt depth and intestinal permeability and a shortening of the intestinal length. villus blunting and crypts fissure in the small intestine are mainly caused by an increase in apoptosis and a decrease in cell proliferation as a result of chemotherapy[97]. restoration of the intestinal structure is very important as it influences the ability of nutrients to be absorbed by the body. a taller villus has more surface area for the absorption of nutrients, and it also lessens water loss and electrolyte loss[146]. inefficient nutrient absorption can lead to loss of weight (low) in cancer patients[142]. hence, probiotics have been proven to play a role in restoring intestinal damage. in an in vivo experiment, s. thermophilus st4 was proven to be able to ameliorate the shortening of colon length of mice treated with 5-fu. this is shown by a shorter average colon length in the mice treated only with 5-fu when compared to the 5-fu + s. thermophilus st4 group. it was also found that the administration of s. thermophilus st4 had also mitigated the decrease in the villus heights and crypt depths[97]. another probiotic strain, r. mucilaginosa ufmgcb 18377, was also demonstrated to be able to decrease intestinal shortening of mice induced with mucositis by 5-fu from around 17% shortening compared to the control group to 7.5% only. it is also found that this probiotic strain is also efficient in ameliorating the structure of the damaged intestinal mucosa, restoring its architecture and villus to crypt ratio, and reducing the increasing permeability of the intestine as a result of 5fu treatment[136]. mucosal damage, such as crypt depths, has been shown to be significantly restored, even to levels similar to those only injected with saline, with lcr35 and labi supplementation. villus height to crypt depth ratio managed to return to normal[140], although another study by huang et al.[141] has shown similar results without it being statistically significant. other strains also showed similar results, with saccharomyces boulardii and lactobacillus fermentum br11[130,147]. s. boulardii also induced the recovery of intestinal permeability by normalising the lactulose: mannitol ratio[130]. effects of supernatant and live s. thermophilus th-4 reduced ileal crypt fissure by at least half of the controls, although it unsuccessfully ameliorated 5-fu induced mucositis[148]. lactobacillus plantarum atcc 8014 showed a potential beneficial effect on ameliorating 5fu induced microscopic changes in the intestinal mucosa[149]. lactobacillus delbrueckii (pexu:hsp65), l. delbrueckii subsp. lactis cidca 133 strain, l. acidophilus or milk fermented with l. rhamnosus flrh93 all were able to stop the shortening of the gut due to 5-fu. these probiotic strains also reduced 5-fu's adverse effects on ip, crypt depth, villus height, as well as villus/crypt ratio and goblet cells[142,146,150–152]. l. rhamnosus flrh93 has also managed to prevent small intestine shortening, with an almost 5% difference compared to mice with 5-fu alone. this is consistent with the finding from the same study that the mice had less low[150]. pmmb 2023, 6, 1; a0000339 15 of 34 two tested probiotic mixtures, such as lactobacillus acidophilus and b. lactis, or a mix of four strains (lactobacillus acidophilus, lactobacillus paracasei, l. rhamnosus, and b. lactis) partially reversed histopathological changes induced by 5-fu[153]. bbg9-1 as mentioned before, prevented the increase in crypts quantity and reduction of small intestine length (although not statistically significant) but could not stop the induction of crypt cell apoptosis, as shown with b. infantis supplementation[145,154]. supernatants from e. coli nissle 1917 and f. prausnitzii enhanced barrier integrity by normalising crypt depth and prevented the change in barrier by 5-fu. s. thermophilus crl 808 and s. thermophilus crl 415 are strains that can produce folate, which was shown to induce less inflammation in the jejunum and preserved villus height/crypt depth ratio[143]. improvement in the histopathological score was also apparent with p. freudenreichii wt[155]. treatment with saccharomyces cerevisiae ufmg a-905 successfully re-established a normal state in the intestine. intestinal permeability returned to normal. s. cerevisiae ufmg a-905 has antioxidant and anti-inflammatory properties. inflammatory markers were further reduced after giving selenium-enriched yeast[156]. 3.1.4. immune cells infiltration the infiltration of inflammatory cells is broadly related to mucosal damage[136]. neutrophil infiltration, associated with intestinal damage and its recruitment, can be measured quantitatively by myeloperoxidase activity (mpo). in a recent study, 5-fu can increase this by almost 500 times with an 83% decrease in sucrase[157]. on the other hand, eosinophil peroxidase (epo) activity can be used to quantify the accumulation of another inflammatory cell called eosinophils. eosinophils play an important role in inducing inflammation in the intestine by acting as antibacterials through the production of pic and epo activity. 5-fu induced mucositis often results in the recruitment of neutrophil and eosinophil infiltrates or causing an increase in mpo and epo levels, hence triggering inflammatory reactions in the tissues[136]. in a mucositis mouse model, mpo level has been shown to decrease with l. delbrueckii subsp. lactis cidca 133 strain, s. boulardii, lactococcus lactis nz9000, lactobacillus acidophilus a4 (a4), bbg9-1, r. mucilaginosa ufmgcb 18377 and probiotic mixtures[130,131,136,137,151,153,158,159]. r. mucilaginosa ufmgcb 18377 can also reduce epo levels in the ileum and jejunum of mice with induced mucositis due to 5-fu[136]. lower mpo and epo levels also indicate that there is lesser immune cell infiltration, which will reduce inflammatory responses. besides, fewer polymorphonuclear cells in the lamina propria lead to less inflammatory activity by these cells, following fewer lesions occurring[137]. l. delbrueckii subsp. lactis cidca 133 has also been shown to minimise 5-fu induced leukopenia[151]. intestinal microbiota plays a part in the adaptive immune system by regulating iga secretion. iga is secreted by immune cells scattered across the mucosa[142]. iga prevents bacteria colonisation and invasion into the mucosa. toxic materials are blocked by iga from penetrating the mucosa. iga modulates the inflammatory response by inhibiting inflammatory response by activating regulatory t-cells[160]. clearly, serum iga has a dual pmmb 2023, 6, 1; a0000339 16 of 34 function in modulating microorganisms present in the gut. it protects the gut from pathogens but also regulates good bacteria. it seems that iga has a different affinity for different microorganisms. there is a high affinity for pathogenic microorganisms but a low affinity for commensal bacteria[161]. iga and commensals also regulate each other. bifidobacteria is capable of modulating the expression of secretory iga. serum iga was increased after 3 days of bifidobacteria supplementation in rats that suffered burn injuries in the gut, but these levels were returned to normal by day 5. this self-modulating expression reduced incidences of bacteria translocated and reduced counts of e. coli and fungi[162]. clearly, strains that increase iga is a good probiotic to consider as well. l. delbrueckii cidca 133 (pexu:hsp65), a recombinant strain can also increase serum iga[142]. 3.1.5. apoptosis 5-fu is capable of inducing cell apoptosis by inducing the expression of il-1β and tnf-α[15]. this is shown by its antagonist, interleukin-1 receptor (il-1r) antagonist, in successfully reducing apoptosis after chemotherapy. up-regulation of bax and caspase 3 (pro-apoptotic proteins) along with downregulation of bcl-2 and bcl-xl (antiapoptotic proteins) occur during 5-fu induced intestinal apoptosis[163]. caspases and bcl-2 proteins are important in early apoptosis[164]. bcl-2 can prevent cells from harsh environments such as radiation, heat and chemotherapy. bcl-2 prevent these cells from undergoing apoptosis[163]. in vitro study shows that il-1r antagonist inhibits apoptosis without affecting its anti-tumour efficacy. this shows that antagonising il-1β activation can protect the gut from chemotherapy-induced im[163]. lcr35 has been shown to suppress apoptosis caused by folfox administration. the reduction of the increased bax/bcl-2 ratio induced by folfox and a shift toward antiapoptosis when lcr35 is given[12]. besides that, s. boulardii, a probiotic yeast, has been shown to reduce 5-fu induced intestinal cell apoptosis. 5-fu modifies the tlr response to activate pic. when this cascade is activated, there is phosphorylation of erk1/2, p38 and jnk. in particular, tnf-α and il-1β activate these protein kinases in response to oxidative stress. the il-1β induced myd88 pathway and phosphorylation of protein kinases are inhibited by s. boulardii[165]. in another case, the probiotic propionibacterium freudenreichii requires slpb protein to alleviate mucositis induced by chemotherapy[130]. 3.1.6. mucin production the human gut mucosa is protected by a mucus layer. this mucus layer comprises water, glycoproteins, trefoil factors, defensins and mucins. mucin is an important component, it being the first-line defence in the gut. mucins are produced by goblet cells that reside all over the small and large intestines and produce mucin[166]. bacteria are able to adhere to mucin oligosaccharides. commensals can compete with pathogens to bind and colonise the mucin, preventing pathogens from attaching to the mucosal surface. for example, the parasite entamoeba histolytica requires contact with epithelial cells in order to invade it. having a thick mucus layer prevents it from having contact with the epithelium. once this mucus layer pmmb 2023, 6, 1; a0000339 17 of 34 is disrupted, e. histolytica can easily penetrate through the gut barrier leading to phagocytosis of epithelial cells[167]. our gut epithelium is endlessly exposed to various noxious chemicals and physical insults such as digestive enzymes, faecal material, resident bacteria, and intestinal pathogens and their products. mucin blocks the mucosa from bacterial enzymes by acting as a substrate for enzymes such as α-galactosidase, β-n-acetylgalactosaminidase, sialidase, β-glucuronidase, blood group degrading enzymes, and proteases[166]. having a mucosal shield as a barrier between underlying epithelium and these substances is essential in preventing im. the mucus layer is also thickened in response to the presence of pathogens. this quick response is key to eliminating pathogens. in other words, pathogens and their toxins have a positive stimulation on mucin production. thick mucus allows easier excretion of pathogens but also carries a risk of diminished mucin[167,168]. vibrio cholerae enterotoxin stimulated mucin production[169]. however, helicobacter pylori downregulate mucin-producing genes muc1 and muc5ac resulting in reduced mucin synthesis[170]. another important gene is muc2. muc2 is a key player in the formation of the mucus layer. it is stored in goblet cells and is key in determining goblet cell shape[171]. in muc2 knockout mice, defective or depleted mucus defence layer showed bacteria colonisation and inflammation (with multiple crypts), subsequently leading to carcinogenesis[168,171]. for example, lactobacillus plantarum 299v, l. rhamnosus strain gg and l acidophilus strain dds-1 are shown to increase gene expression of muc2 and muc3 at the mrna level[172,173]. this managed to prevent adherence of pathogenic e. coli onto gut epithelium[172]. besides, non-pathogenic commensal e. coli nissle 1917 regulates multiple genes. besides muc2 and muc3, it also upregulates muc5ac and muc5a[174]. butyrate-producing organisms also have shown abilities to increase muc2 gene expression in in vitro studies. human colon biopsies in ex vivo studies showed increased mucous secretion in the presence of butyrate-producing organisms. trefoil factors, a contributor to the viscosity and elasticity of mucus, can reduce the chemotaxis of inflammatory cells and are thought to repair damaged mucosa. like mucin, it is only produced by gc. trefoil factors are reduced in rats with colitis, but with butyrate administration, these levels increased[175]. 5-fu treatment has been shown to influence the mucus layer and decrease goblet cells while increasing the number of crypts in the jejunum[176]. this is possibly due to the proinflammatory state 5-fu induces. it is found that rhodotorula mucilaginosa ufmgcb 18377 decreases the loss of goblet cells in mice induced with mucositis by 5-fu[136]. other than that, treatment with l. delbrueckii rcidca 133:hsp65 also increases goblet cells and expression of the muc2 gene. a higher quantity of goblet cells and increased muc2 gene expression can reinforce each other's positive effect in protecting the mucus layer[142]. 3.1.7. weight loss regarding weight loss, probiotic strains such as lcr35 and labi have been shown to reduce body weight loss by 20%. mice in the lcr35 probiotic group showed a lesser degree of low compared to 5-fu and saline group[177]. other lactobacillus strains, such as l. pmmb 2023, 6, 1; a0000339 18 of 34 plantarum 299v, have been shown to improve food intake, resulting in less low, while others, such as l. delbrueckii subsp. lactis cidca 133 strain and l. acidophilus could increase food and milk intake but still reduce low[135,142,151]. riboflavin overproducing l. plantarum crl2130 also showed similar results[152,178]. besides these, l. fermentum br11 and l. rhamnosus gg increase colon weight in conjunction with 5-fu treatment[157]. lactobacillus plantarum 299v and bbg9‐1, at a concentration of 1 × 109 cfu/ml, improved both food consumption and body weight post 5-fu administration[131,147]. interestingly, high doses resulted in low, while low doses resulted in body weight gain after one week of 5-fu treatment. on day 8 after the treatment, low doses increased body weight compared to high doses of the probiotic, which decreased food intake[147]. b. infantis also has shown weight-gaining properties[145,154]. in another in vivo experiment in mice, it was found that mice with 5-fu induced im showed a significant decrease of about 50% in food intake and about a 20% decrease in their body weight in relation to the initial weight on the 18th day. however, in the 5-fu + s. thermophilus st4 mice group, there was no big difference in body weight loss (only 1.85%) and food intake compared to the control group[97]. another strain of probiotic, rhodotorula mucilaginosa ufmgcb 18377 ameliorated the decrease in food consumption of mice induced with mucositis by 5-fu hence reducing the weight loss in comparison to their initial weight from 20.3% to 12.4% in the probiotic-treated group as compared to the mucositis group[136]. a probiotic mixture (l. acidophilus, l. paracasei, l. rhamnosus, and b. lactis) reduced body weight loss[153]. however, the addition of streptococcus genus, resulting in a mixture of b. breve dm8310 + l. acidophilus dm8302, l. casei dm8121 + s. thermophilus dm8309 showed no differences in faecal output, and food intake[158]. e. coli nissle 1917 supernatant and f. prausnitzii supernatant prevented further weight loss from happening post 5-fu induction resulting in normal urine and faecal output[179]. these mixed results show that, clinically, some probiotic strains are better than others at preventing weight loss. none of the probiotics mentioned exacerbated weight loss, suggesting its importance in maintaining patients' weight. further work should be done to understand the underlying mechanisms exhibited by some strains to preserve the body weight, potentially via improved feeding behaviour and the host's metabolism[180]. 3.1.8. diarrhoea severe diarrhoea is presumed to be closely related to the shortened colon, one of the side effects of the administration of 5-fu[97]. diarrhoea occurs when the colon's absorption capacity is surpassed by the rising fluid volume out of the small intestine[44]. rat studies show that probiotics can ameliorate diarrhoea. the research was carried out on a mice model by assessing the severity of diarrhoea using bowen's score system, in which diarrhoea is categorised into 4 grades based on the consistency of stool (0 — normal stool; 1 — mild diarrhoea, the stool is slightly soft and wet; 2 — moderate diarrhoea, stool are wet and unformed; 3 — severe diarrhoea, stool are watery). it is found that the 5-fu + s. thermophilus pmmb 2023, 6, 1; a0000339 19 of 34 st4 group shows a lower mean diarrhoea score that changed from 1.0 to 0.1 when compared to the group of mice induced with 5-fu only that scored 2.5 and 2.5 respectively, on the most severe day 14th to 15th[97]. for example, lcr35 group and labi group, l. delbrueckii cidca 133 (pexu:hsp65), l. plantarum crl2130, bbg9-1 and also folate-producing s. thermophilus crl 808 were shown to successfully reduce the incidence of diarrhoea[140,143,178]. probiotics reduced the incidence of diarrhoea in human patients. in a systematic review, researchers found probiotics and fibre reduced the incidence of grade 3 or 4 abdominal discomfort (flatulence, borborygmi and abdominal distension) and the need for chemotherapy dose reduction, compared with placebo[14,181]. l. rhamnosus gg decreased the incidence of diarrhoea in grades 3 or 4 by around 15%. in patients with severe diarrhoea, metabolic and nutritional imbalances may occur, which may further worsen their condition. it is evident that probiotics show improvement in diarrhoea in animal models and humans[45]. in another clinical trial on patients treated with 5-fu based chemotherapy and administered with probiotic lactobacillus kefiri lkf01, it was found that the incidence of diarrhoea was reduced. only 4.7% and 8.7% of patients treated with 5-fu and folfoxiri respectively developed g3-4 severe diarrhoea [ctcae grade [g] 3-4]. in contrast, no incidence of highgrade diarrhoea was reported by patients treated with folfox and folfiri, respectively. since their results also showed that the onset of diarrhoea mostly started at the early stages of chemotherapy, hence it is hypothesised that the intake of probiotics earlier before the treatment begins and not concomitantly as they did in their studies could possibly work as a preventive measure to reduce the rates of early-onset diarrhoea, but further studies are required to validate it[44]. table 1. probiotic genus, strains and mixtures that show beneficial effects toward im. different probiotics ameliorate im in various aspects are summarised in this table. probiotic effects of probiotic on ameliorating intestinal mucositis reference lactobacillus acidophilus • preserved villus and crypt length ratio • reduced gsh levels • reduced myeloperoxidase activity • reduced nitrite levels • reduced level of pic, tnf-α, il-1β • reduced levels of chemokines cxcl-1 • increased levels of anti-inflammatory cytokines il-10 [152] lactobacillus acidophilus a4 • prevented attachment of escherichia coli o157:h7 • reduced level of pic, il-8, tnf-α, il-1β • induced the expression of muc2 [182] lactobacillus brevis 47 • partially restored expression of ki-67 epithelial proliferation cell marker [183] lactobacillus plantarum crl2130 • reduced weight loss • reduced diarrhoea scores • maintained mucosal architecture • reduced mucosal inflammation [178] pmmb 2023, 6, 1; a0000339 20 of 34 probiotic effects of probiotic on ameliorating intestinal mucositis reference • preserved villus and crypt length • reduced level of pic, il-10 lactobacillus plantarum supernatant • increased sensitivity of colorectal cancer cells to 5fluorouracil • inhibited cd44 gene expression • inhibited cd133 gene expression • inhibited cd166 gene expression • inhibited aldh1 gene expression • increased caspase 3 activity • inhibited signalling pathway wnt/β-catenin [43] lactobacillus casei variety rhamnosus (l cr35) • reduced diarrhoea scores • reduced loss of weight • normalised crypt depth • preserved villus and crypt length ratio • reduced villous inflammation • diminished apoptosis • reduced level of pic, tnf-α and il-6 • reduced levels of nf-κb-, and bax-activated cells • restored composition of fecal gut microbiota • decreased firmicutes and bacteroidetes ratio • reduced cd3+/cd8+ count • increased cd3+cd4+/cd3+cd8+ • reduced cd44 the number of ki67 proliferative cells [177] lactobacillus delbrueckii cidca 133 (pexu:hsp65) • reduced inflammatory infiltrate • reduced intestinal permeability • increased serum iga in the intestinal fluid • improved histological score • prevented shortening of the intestine • preserved villus and crypt length ratio • reduced myeloperoxidase activity • reduced level of pic, tnf-α, il-6, il-12, il-1β • reduced level of toll-like receptors • increased level of anti-inflammatory cytokines il-10 • increased gene expression of muc2 • increased gene expression of claudin 1 • increased the number of goblet cells [142] lactococcus lactis nz9000 • prevented growth of listeria monocytogenes • pancreas-associated peptide secretion prevented growth of e. faecalis • improved histological score • reduced infiltration of neutrophils • reduced infiltration of eosinophils • reduced secretion of immunoglobulin-a in the gut • reduced gut inflammation [137] lactobacillus sp. • induced expression of muc3 [173] lactobacillus rhamnosus flrh93 • preserved villus and crypt length ratio • lessen the decrease of goblet cells [150] pmmb 2023, 6, 1; a0000339 21 of 34 probiotic effects of probiotic on ameliorating intestinal mucositis reference • increased the expression bcl-2 in the intestinal tract • decreased expression of nlrp3 • reduced level of pic, tnf-α, il-1β • increased survival rate of mice lactobacillus rhamnosus gg • reduced diarrhea scores • reduced incidence of abdominal discomfort [45] streptococcus thermophilus crl 808 • enhanced chemotherapeutic effect of 5-fu • reduced diarrhoea scores • reduced jejunal inflammation • increased histological score • reduced level of pic, il-6 • increased level of anti-inflammatory cytokines il-10 [143] streptococcus thermophilus st4 • increased food intake of mice • decreased body weight loss • reduced mean diarrhoea score • reduced shortening of colon length • restored villus heights and crypts depths • reduced level of pic, tnf-α, il-1β and il-6 [97] bifidobacterium infantis • mice showed higher body weight • mice had taller villus • reduced level of pic, il-6, il-1β and tnf-α • down-regulated levels of t-bet (th transcription factor) • increased cd4+ levels • increased cd25+ levels • increased foxp3+ levels • increased tregs levels • reduced cd4+ il17a+ cells [145] saccharomyces boulardii • changed expression of toll-like receptors, tlr2, tlr4 • changed expression of myd88 • changed expression of nf-κb • changed expression of erk1 • changed expression of phospho-p38 • changed expression of phospho-jnk • reduced level of pic, tnf-α, il-1β • reduced level of chemokine cxcl-1 [165] saccharomyces cerevisiae ufmg a-905 • partially reduced intestinal permeability • reduced myeloperoxidase activity • reduced level of chemokine cxcl-1 • reduced mucosal inflammation • reduced oxidative stress [156] propionibacterium freudenreichii • reduced level of pic, il-12, il-17a, il-8, tnfα • changed expression of cld1 [155] pmmb 2023, 6, 1; a0000339 22 of 34 probiotic effects of probiotic on ameliorating intestinal mucositis reference wild strain of propionibacterium freudenreichii • reduced weight loss • reduced gut inflammation • increased histopathological scores • induced production of th17 cells • reduced level of iga [155] rhodotorula mucilaginosa ufmgcb 18377 • ameliorated the decrease in food consumption • reduced weight loss • reduced shortening of intestinal length • restored villus to crypt ratio • reduced intestinal permeability • reduced goblet cells loss • reduced enterobacteria in intestine • reduced mpo and epo levels / reduced neutrophils and eosinophils infiltration [136] probiotic mixture : • bifidobacterium breve dm8310 • lactobacillus acidophilus dm8 302 • lactobacillus casei dm8121 • streptococcus thermophilus dm 8309 • reduced level of pic • reduced infiltration of neutrophils • reduced intestinal permeability • changes of toll-like receptors, tlr2, tlr4 signalling pathway [158] 4. discussion and future direction despite extensive evidence shown in this review, there is no solid guideline for treating intestinal mucositis due to chemotherapy. current medications or other ways to combat chemotherapy-induced side effects are generally not completely effective, mostly fail to address possible long-term impacts, and may even cause additional side effects that merely exacerbate patients' sufferings[13]. some of the interventions being used currently are limited to ice, analgesics, barrier protectors and topical antimicrobials. hence, it is necessary to look for other possible alternatives to treat im[136]. probiotics have clearly shown to be a valuable adjuvant therapy in treating intestinal mucositis. many strains successfully reduce levels of pic, inhibit inflammatory pathways, and strengthen the gut's physical defence system. probiotics also manage to prevent adherence of pathogenic bacteria and prevent gut cell apoptosis. when patients undergo cancer treatment, they are at risk of gut dysbiosis. they will be less protected from harmful external factors due to lack of commensal gm and also due to a weak immune system from cancer itself as well as the chemotherapy drug. pmmb 2023, 6, 1; a0000339 23 of 34 supplementing with probiotics before, during and after chemotherapy can increase one more level of protection for cancer patients. further research is required to investigate the integration of probiotic interventions into the clinical management of im, effectively halting mucositis in its track. with the current understanding that the gut microbiome plays a crucial role in all stages of intestinal mucositis, including initiation, upregulation, message generation, amplification, signaling, ulceration, and healing, it is anticipated that this integration will be realised in the near future. there is also a risk of bacterial translocation, causing invasive infections. immunocompromised cancer patients would be particularly vulnerable, especially the risk of bacteraemia or sepsis, worsening patients' survival and mortality rate. however, with all of these studies mentioned, there were no events of bacteraemia or sepsis. probiotics are beneficial. they inhibit the invasion of pathogens and strengthen the immune system. it is also important to note that probiotics do not compromise 5-fu efficacy, supporting their further use[17]. as shown in this review, there is a large shortage of studies conducted on humans. although some of the preclinical studies show promising results, they might not translate similarly to humans. most of the studies are also done in mice induced for mucositis or induced with carcinoma, which could be different from humans with chemotherapy-induced mucositis with underlying crc. perhaps this is why there is currently no definitive proof supporting the use of probiotics in treating im. therefore, this review calls for future study of ethical, large, randomised, double-blind human trials. this is to further assess the capability of probiotics in real-life clinical settings. studies should include isolated strains that can ameliorate all side effects of im rather than just ameliorating one specific side effect. other than that, the most suitable doses along with the most effective probiotic strains or combination of different probiotic strains, should also be further evaluated to maximise the beneficial effects of probiotics on the host. also, human factors such as genetics[184,185], race, and environmental factors could affect patients' response to probiotics supplementation. thus, knowing the correlation between these external factors and response to probiotics should be studied as well. by doing this, we might be able to predict which patients are specifically at risk of developing certain side effects. perhaps we can tailor the probiotic supplementation to patients' needs, thus developing a personalised probiotic regimen for each patient to reach optimal treatment outcomes. individual variability is impossible to predict; however, more studies on the association of certain external factors or internal factors, such as gene polymorphism, can be a starting point for having proper guidelines for im therapy. pmmb 2023, 6, 1; a0000339 24 of 34 a challenge in using probiotics as a therapeutic method would be modulating its effects to target inflamed tissues while leaving normal tissues undisturbed and, most importantly, without disturbing the efficacy of 5-fu. evidence showed probiotics do not affect the efficacy of 5-fu and its actions on tumour cells. however, similar to other studies, probiotics' influence on 5-fu's mode of action is only primarily known in rat studies. there has yet to be a large human trial to allow full justification. the challenge of maximising probiotic protection of mucosa while minimising the protection of tumour will need to be overcome. perhaps, new techniques or technology should be used to overcome this challenge. also, probiotics are required to remain in the body for a considerable amount of time. so far, it is uncertain how long the effects of probiotics will last and whether the effects will continue to last even after probiotics are no longer in the body[186]. this review also does not discuss the viability of probiotics after going through the digestive tract, such as stomach acid and digestive enzymes[146]. 5. conclusion in conclusion, this review shows promising evidence that would encourage future studies on the use of probiotics in ameliorating the side effects of 5-fu or chemotherapy in general. more studies are important, as we have seen that im is a complex process, and its association with gut microbes is even more complex. 5-fu continues to be used today, and yet there is only a superficial understanding of the relationship between gm and 5-fu. this review highlights that gm plays an important role in the host's response to 5-fu. also, this review gives a framework on how probiotics may increase the efficacy and lessen the side effects of 5-fu; thus, future studies on this will be beneficial to many patients that use 5-fu. there has yet to be fully validated that probiotics could be the magic bullet, and for now, it serves better as an adjuvant targeted therapy. nevertheless, this emerging field of research provides hope for finding safer and more effective treatment options for crc and mitigating the adverse effects associated with traditional chemotherapy. these approaches not only combat cancer but also preserve patients' quality of life during treatment. continued efforts in this direction may open new avenues for personalized and targeted approaches to crc treatment, revolutionizing the way we combat this devastating disease. author contributions: aax-hs and j-yc performed the literature search, critical data analysis as well as manuscript writing. j-yc, jw-fl, vl, yk, so, shw, and k-gc performed editing, revision and proofreading. jw-fl, vl, yk, so, shw, k-gc and lt-ht provided technical support and expertise. lt-th conceptualised this review writing project. acknowledgments: this work was inspired by the jeffrey cheah school of medicine and health sciences (jcsmhs) "med5101 scholarly intensive placement (sip)". the author also would like to acknowledge and sincerely thank the young scholar’s program (ysp) conducted at jcsmhs, monash university malaysia. pmmb 2023, 6, 1; 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78(4): 675–683. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2023, 6, 1; a0000331. doi: 10.36877/pmmb.0000331 http://journals.hh-publisher.com/index.php/pmmb original research article hplc-uv-ms/ms profiling of phenolics from euphorbia nicaeensis (all.) leaf and stem and its antioxidant and anti-protein denaturation activities khawla bouaouda1, chaimae elagdi2, naoufal el hachlafi3, karima mohtadi1, mohammed hsaine2, anass kettani1, rachid flouchi3, khang wen goh4, abdelhakim bouyahya5, hanae naceiri mrabti6, rachid saile1, hassan taki1* article history 1laboratory of biology and health, university hassan ii of casablanca, faculty of sciences ben m’sik, casablanca, morocco, p.o.box 7955; khawla.bouaouda-etu@etu.univh2c.ma (kb); karima.mohtadi@univh2c.ma (km); anass.kettani@univh2c.ma (ak); rachid.saile@univh2c.ma (rs) 2 laboratory of ecology and environment, university hassan ii of casablanca, faculty of sciences ben m’sik, casablanca, morocco, p.o.box 7955; chaimaa.elagdi-etu@etu.univh2c.ma (ce); mohammed.hsaine@univh2c.ma (mh) 3laboratory of microbial biotechnology and bioactive molecules, sciences and technologies faculty, sidi mohamed ben abdellah university, p.o. box 2202, imouzzer road, fez, morocco; naoufal.elhachlafi@usmba.ac.ma (neh), racinf@gmail.com (rf) 4faculty of data science and information technology, inti international university, 71800 nilai, malaysia; khangwen.goh@newinti.edu.my (kwg) 5laboratory of human pathologies biology, department of biology, faculty of sciences, mohammed v university in rabat, rabat 10106, morocco; a.bouyahya@um5r.ac.ma (ab) 6high institute of nursing professions and health techniques casablanca, morocco; naceiri.mrabti.hanae@gmail.com (hnm) *corresponding author: hassan taki, university hassan ii of casablanca, faculty of sciences ben m’sik, casablanca, morocco; hassan.taki@univh2c.ma (ht) received: 19 march 2023; received in revised form: 21 april 2023; accepted: 10 may 2023; available online: 05 june 2023 abstract: this work focuses on the leaves and stems of euphorbia nicaeensis all. to confirm its historical use by the moroccan population. the phytochemical profile of the plant by hplc-uv-ms/ms was identified for the first time, and the biological activities of each plant organ was evaluated separately by maceration and ultrasonic-assisted extraction. the evaluation of antioxidant activity based on dpph assay and hydrogen peroxide scavenging assay showed that leaves could be used as a natural source of antioxidants as they provide a potent antioxidant effect (dpph ic50 = 23.47±0.62 µg/ml), (h2o2 ic50 = 110.27 ± 3.59 µg/ml) compared to stems. these results were proven by hplc-uv-ms/ms analysis and revealed that e. nicaeensis leaves are richer in phenolic compounds, especially quercetin and derivatives, known for their antioxidant properties. in contrast, the stems could be considered a potential anti-inflammatory agent considering their solid anti-inflammatory activity. the pmmb 2023, 6, 1; a0000331 2 of 20 most potent effects were obtained at a concentration of 2 mg/ml, which induced 83.98% and 82.04% inhibition against bovine albumin and egg albumin denaturation, respectively, compared to the control. in addition, the stem phytochemical profile indicated the presence of some compounds with anti-inflammatory effects, such as fargesin and nuciferine. likewise, the findings showed that ultrasound-assisted extraction was more effective than maceration. keywords: euphorbia nicaeensis all., maceration, ultrasound extraction, antioxidant activity, anti-inflammatory activity, hplc-uv-ms/ms 1. introduction aromatic and medicinal plants have long been regarded as a vital source of therapeutics and curative remedies due to the presence of bioactive compounds and phytochemical constituents. medicinal herbs have proven to be the basis of traditional medicine worldwide. curing and healing human diseases has always been related to using entire plants or parts of them in remedy preparation [1]. moroccan population has traditionally employed aromatic and medicinal plants; over time, people have gained knowledge of old cosmetics and pharmacopoeia. oral communication is still used to pass on essential information about these activities from generation to generation [2]. however, several medicinal plants are currently underexploited in morocco despite their ancestral use in treating human diseases. euphorbia nicaeensis all. is one of these plants on which this investigation focuses. it is a species of euphorbiaceae family, a perennial spurge herbaceous plant that prefers calcareous soils. it grows in sunny and dry places and is distributed in the mediterranean and central europe with a strong morphological variability in leaves and bracts [3]. e. nicaeensis is an abundant species in the rocky lawns of morocco [4], latex is the most traditionally used part of the plant, and it has been used in the past to attack warts and erase dead flesh. the population was aware of the vesicant properties of latex on the skin, eyes, and mucous membranes [5]. there are also other uses of leaves and stems against bacterial infections, hepatitis, tuberculosis, typhoid, and other diseases [6–8]. numerous studies have proven the traditional use of e. nicaeensis and its anthelmintic, antifungal, and anticancer activities due to the jatrophane diterpenoid isolated from the root extracts and latex [9–11]. previous studies have also evaluated some biological activities of e. nicaeensis aerial parts and detected its anti-inflammatory properties [12,13]. this study aims to assess the phytochemical profile of euphorbia nicaeensis by hplc-uvms/ms analysis for the first time and to evaluate the anti-inflammatory properties and antioxidant activity of leaves and stems. therefore, two extraction methods were compared, probe ultrasonic-assisted extraction and maceration. pmmb 2023, 6, 1; a0000331 3 of 20 2. materials and methods 2.1. plant material the plant was harvested from the ifrane region (figure 1). the lambert coordinates: 33° 32′ 44.4″ n, 5° 19′ 17.89″ w. and the plant have undergone drying after separation of the leaves. figure 1. photo of the species euphorbia nicaeensis all. 2.2. extraction procedure 2.2.1. maceration an amount of 2.5 g of ground plant powder (leaves and stems) was extracted by 50 ml (methanol water) (80:20) v/v. the maceration extracts were kept under stirring for 24 hours, then filtered under a buchner funnel and concentrated by rotary evaporation. the residues were collected and stored until further analysis. 2.2.2. probe ultrasonic-assisted extraction (puae) probe ultrasonic-assisted extraction (puae) is one of the main extraction methods used for plant materials. sonication causes cavitation and implosion, which causes cell-wall rupture and increases the number of disturbed cells. when disturbed, the solvent penetrates the cell, and the intracellular plant material is absorbed into the solvent [14]. the extraction was done using an ultrasonic probe system (bioblock scientific, vibra cell 75042). the probe was submerged 1.5 cm under the surface of the mixture (methanolwater) (80:20) v/v and 2.5 g of plant powder. the extraction was performed at the maximum power settings of the transducer (100%, 400 w), at 24 khz, for 15 min, with a 20 s pulse. following extraction, the extract was filtered and concentrated by rotary evaporation until dryness and was stored for further use. 2.3. phytochemical screening phytochemical screening of euphorbia nicaeensis all. leaves and stems were performed using standardized laboratory protocols. the presence or absence of phenols, tannins [15], flavonoids [16], saponins [17], flavonol, carbohydrates, alkaloids, gums, amino acids [18], sterols and polyterpenes [19], was analyzed accordingly. pmmb 2023, 6, 1; a0000331 4 of 20 2.4. total phenolic content (tpc) the phenolic compounds were measured using folin-ciocalteu method [20]. in brief, 50 µl of folin-ciocalteu reagent was added to the sample, followed by 150 µl of na2co3 after 10 min, and the volume was made up to 1 ml with water. the absorbance was measured in a spectrophotometer reader at 760 nm after 2 hours of incubation and compared to the gallic acid calibration curve (5 – 100 µg/ml), r2= 0.998. the results were expressed as mg gallic acid equivalents /g dry weight (mg gae/g dw). 2.5. total flavonoid content (tfc) according to [21], 250 µl of the extract solution was mixed with 1 ml of distilled water and 75 µl of nano2 solution (5 %). after 6 min, 75 µl of alcl3 solution (10 %) was added. the mixture was allowed to stand for 6 min before adding 1 ml of naoh solution (4 %) and bringing the final volume to 2.5 ml with distilled water. the mixture was properly vortexed and allowed to stand for 15 min in darkness. the absorbance was measured at 510 nm and compared to the catechin standard curve (5 – 400 µg/ml) r2= 0.997. the results were expressed as mg of catechin equivalent per g dry weight (mg ce/g dw). 2.6. flavonol content (fc) the content of flavonols was determined according to the method described by [22]. an aliquot of 500 µl of extract was added to 500 µl of aluminium chloride (20 mg/ml) and 1.5 ml of sodium acetate (50 mg/ml). the mixture was properly vortexed and left to stand in darkness for 2.5 h. the absorbance was read at 440 nm, and the result was expressed referring to the absorbance of standard quercetin solution prepared in the same conditions (5 – 200 µg/ml), r2= 0,995. the flavonol content is expressed in mg quercetin equivalents per g dry weight (mg qe/g dw). 2.7. condensed tannin content (ctc) the assay was executed as reported previously [23]. an aliquot of 100 µl of each extract was added to 1.5 ml of vanillin (4 %) and 750 µl of concentrated hydrochloric acid (hcl). the mixture was vortexed, left to stand in the dark for 20 min, and the absorbance was recorded at 500 nm. the condensed tannin content was counted related to the catechin calibration curve (5 – 400 µg/ml) r2= 0.985, elaborated in the same manner. the results are expressed as mg of catechin equivalent per 100 g dry weight (mg ce/100g dw). 2.8. lipid-soluble pigment content according to [21], 150 mg of vegetal powder (leaves and stems) was mixed for 1 min with 10 ml of acetone/hexane (4:6). the mixture was filtered through whatman filter paper grade 4. the absorbance was recorded at 453,505,645, and 663 nm. the content of β-carotene, lycopene, chlorophyll a, and chlorophyll b was calculated according to the following equations, expressed in µg per g dry weight (dw): pmmb 2023, 6, 1; a0000331 5 of 20 β-carotene = 0.216 × 𝐴663 − 1.220 × 𝐴645 − 0.304 × 𝐴505 + 0.452 × 𝐴453 lycopene = −0.0458 × 𝐴663 + 0.204 × 𝐴645 − 0.304 × 𝐴505 + 0.452 × 𝐴453 chlorophyll a = 0.999 × 𝐴663 − 0.0989 × 𝐴645 chlorophyll b = 0.328 × 𝐴663 + 1.77 × 𝐴645 a453, a505, a645, and a663 are the absorbance measured at 453,505,645, and 663 nm, respectively. 2.9. in vitro antioxidant activity 2.9.1. 2,2-diphenyl-1-picrylhydrazyl (dpph) radical-scavenging assay the radical scavenging ability of the extract was carried out as described by [24–26]. 500 µl of extract solution was mixed with 1 ml of dpph solution (0.1 mm in methanol) freshly prepared. the mixture was vortexed properly and left to stand in the dark for 60 min. the reduction of dpph was measured at 515 nm, and the scavenging effect was estimated based on the percentage of scavenging dpph radicals using the following equation: % 𝐃𝐏𝐏𝐇 𝐬𝐜𝐚𝐯𝐞𝐧𝐠𝐢𝐧𝐠 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 = 𝑨𝐃𝐏𝐏𝐇 − 𝑨𝐬 𝑨𝐃𝐏𝐏𝐇 × 𝟏𝟎𝟎 adpph is the absorbance of dpph and as is the absorbance of dpph when the sample has been added at different concentrations. the ic50 value is the concentration that scavenges 50 % of dpph radicals, and it is calculated from the scavenging effect percentage graph. ascorbic acid was used as a standard. 2.9.2. scavenging of hydrogen peroxide assay the performance of hydrogen peroxide (h2o2) scavenging was investigated as follows [27]. the phosphate buffer (50 mm, 7.4 ph) received a 40 mm h2o2 solution. in order to quantify the mixture's absorbance spectrophotometrically at 230 nm, all experimental samples were combined with 0.6 ml of h2o2 solution. the mixture was then incubated for 10 minutes. ascorbic acid served as the standard, while phosphate buffer functioned as the control. hydrogen peroxide scavenging (%) was calculated using the formula below. % 𝐇𝐲𝐝𝐫𝐨𝐠𝐞𝐧 𝐩𝐞𝐫𝐨𝐱𝐢𝐝𝐞 𝐬𝐜𝐚𝐯𝐞𝐧𝐠𝐢𝐧𝐠 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 = 𝑨𝟎 − 𝑨𝐬 𝑨𝟎 × 𝟏𝟎𝟎 where as is the absorbance in the presence of the ascorbic acid standard or samples, and ao is the absorbance of the blank. ascorbic acid was used as a standard. pmmb 2023, 6, 1; a0000331 6 of 20 2.10. in vitro anti-inflammatory activity 2.10.1. bovine serum albumin assay (bsa) protein denaturation is one of the causes of inflammatory and rheumatic diseases, as reported by a previous study [28]. any compound that provides greater than 20% inhibition of protein denaturation is considered a potential anti-inflammatory agent and could be useful for treating several diseases [29]. bsa assay of leaf and stem extracts was determined using the method reported by [27]. 0.45 ml of bovine serum albumin was mixed with 0.05 ml of samples at various concentrations (200 2000 μg/ml). the mixture was incubated for 25 min at 40°c, and phosphate buffer saline (2.5 ml; ph 6.3) was added to tubes. the control received phosphate buffer solution (0.05 ml) instead of extract. the absorbance was measured using a spectrophotometer at 660 nm, and the percentage inhibition of bsa denaturation was calculated using the following equation: % 𝐢𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧 𝐨𝐟 𝐁𝐒𝐀 𝐝𝐞𝐧𝐚𝐭𝐮𝐫𝐚𝐭𝐢𝐨𝐧 = 𝑨𝟏 − 𝑨𝟐 𝑨𝟏 × 𝟏𝟎𝟎 where a1 = absorbance of the control and a2 = absorbance of the test sample. 2.10.2. chicken egg albumin assay (cea) using the technique described by [30], the ability of our extracts to inhibit protein denaturation was examined. to 2.8 ml of pbs solution, 2 ml of extract and 0.2 ml of chicken egg albumin were added. all samples were kept at room temperature for 15 minutes before being heated to 70 °c for 10 minutes. diclofenac was employed as standard, and the absorbance was measured at 660 nm. the equation below was used to determine the inhibition of egg albumin denaturation: % 𝐢𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧 𝐨𝐟 𝐞𝐠𝐠 𝐚𝐥𝐛𝐮𝐦𝐢𝐧 𝐝𝐞𝐧𝐚𝐭𝐮𝐫𝐚𝐭𝐢𝐨𝐧 = 𝑨𝟏 − 𝑨𝟐 𝑨𝟏 × 𝟏𝟎𝟎 a1 is the control's absorbance and a2 is the test sample's absorbance. 2.11. phenolic profile analysis by hplc-uv-ms/ms phenolic compounds were qualitatively analyzed using a liquid chromatography system coupled with a triple quadrupole mass spectrometer (thermo fisher scientific, san jose, ca, usa). the suggested technique was carried out using a kinetex c18 reversedphase column (100 x 4.6 mm, 2.6 m particles). from solvent a (0.1% formic acid aqueous solution) and solvent b (methanol), gradient separation was created following the method described by [31]. retention time and spectrum matching with nine standards were used to identify the phenolic compounds, along with the nist-ms/ms library. pmmb 2023, 6, 1; a0000331 7 of 20 2.12. statistical analysis t-test was used to evaluate the statistical differences between extraction methods (maceration and probe-assisted ultrasound extraction) and organs (leaves and stems). experiments were run in triplicate, and the results are expressed as mean values of three analyses. data are statistically significant at p<0.05 and highly significant at p<0.001. pearson correlation coefficient (r), dpph (ic50), and h2o2 (ic50) were determined using graphpad prism 8.0.2. 3. results 3.1. phytochemical screening the results of phytochemical screening of euphorbia nicaeensis all. leaves and stems are shown in table 1. phenols, tannins, saponins, and carbohydrates are strongly present in all plant organs. flavonoids, flavonol glycosides, sterols, and polyterpenes are highly present in leaves and small amounts in stems. alkaloids and amino acids are present in tiny quantities, quinones and mucilage are absent in all plant organs. table 1. phytochemical screening results of leaves and stems of euphorbia nicaeensis all. phytochemical compounds leaves stems phenols / tanins (++) (++) flavonoids (++) (+) flavonol glycosides (+) (+/-) alkaloids (+/-) (+/-) saponins (++) (++) carbohydrates (++) (++) quinones (-) (-) gums and mucilages (-) (-) amino acids (+) (+) sterols and polyterpenes (++) (+) (+): presence of phytochemicals, (-): absence of phytochemicals, (++): presence in significant amounts, (+/-): presence in small amounts. pmmb 2023, 6, 1; a0000331 8 of 20 3.2. total phenol (tpc), total flavonoid (tfc), flavonol content (fc), and condensed tannin content (ctc) the carried-out tests indicated that the leaves and stems of euphorbia nicaeensis are rich in phenolic compounds. the percentage yield of leaf and stem extracts by maceration (3.32%, 1.68%), and ultrasonic-assisted extraction (12.69 ± 0.17%, 4.62 ± 1.55%), respectively, showed that the highest extraction yield of both leaves and stems was obtained with probe ultrasonic-assisted extraction (puae). the (tpc) values of puae extract of leaves and stems (80.13 ± 1.61 mg gae/g dw, 77.79 ± 2.29mg gae/g dw), respectively, were statically higher (t-test, p<0.05) than that of maceration extracts (68.69 ± 0.91mg gae/g dw, 68.31 ± 1.83 mg gae/g dw). this data also reveals that the leaves are richer in flavonoids, condensed tannins, and flavonols than the stems as presented in table 2. table 2. total phenol, flavonoids, condensed tannins, flavonols contents and extraction yield of maceration extracts and probe ultrasonic-assisted extract of euphorbia nicaeensis all. leaves and stems. elm* esm* elu* esu* p1 p2 p3 p4 tpc (mg gae/g dw) 68.69±0.91 68.31± 1.83 80.13± 1.61 77.79±2.29 0.574 0.042 <0.001 <0.001 tfc (mg ce/g dw) 32.83 ±3.84 21.83 ±9.93 49.22 ±0.192 45.27 ±2.5 0.047 0.031 0.004 0.010 ctc (mg ce/100g dw) 1.33±1.1 1.88±1.92 7.61±1.92 1.22±0.92 0.450 0.002 0.001 0.361 fc (mg qe/g dw) 4.80±0.08 1.23 ±0.27 36.14±0.08 5.88±0.33 <0.001 <0.001 <0.001 <0.001 extraction yield (%) 3.32±0.59 1.68±0.28 12.69±0.17 4.62±1.55 0.024 0.011 <0.001 0.077 elm: euphorbia leaf maceration, esm: euphorbia stem maceration, elu: euphorbia leaves ultrasonicassisted extraction, esu: euphorbia stems ultrasonic-assisted extraction, tpc: total flavonoid content, tfc: total flavonoid content, ctc: condensed tannin contents, fc: flavonols content, p1: p-value (elm – esm), p2: p-value (elu – esu), p3: p-value (elm – elu), p4: p-value (esm – esu). *means ± sd from triplicate determinations. p-value is considered significant at p<0.05 and highly significant at p<0.001. pmmb 2023, 6, 1; a0000331 9 of 20 3.3. lipid-soluble pigment content as shown in figure 2, this study revealed that leaves and stems differed significantly (p<0.05) in the content of chlorophyll pigment. the leaves are richer in chlorophyll a and b (0.167±0.001 µg/g dw, 0.108± 0.002 µg/g dw) than the stems (0.016 µg/g dw, 0.049± 0.001 µg/g dw). these findings also showed that leaves have significantly higher (p<0.05) β-carotene content (0.035 ± 0.001 µg/g dw) compared to stems (0.006 ± 0.001 µg/g dw). in addition, low lycopene concentration was detected in both leaves and stems (0.033 ±0.001 µg/g dw, 0.036 µg/g dw), respectively, with no significant difference (p>0.05). figure 2. lipid-soluble pigment content of leaves and stems of euphorbia nicaeensis. values are means ± s.d of three independent measurements. el: euphorbia leaves, es: euphorbia stems. the values with the same superscript letters are not significantly different (p>0.05). 3.4. in vitro antioxidant activity the evaluation of the antioxidant activity of e. nicaeensis leaf and stem extracts was screened by 2,2-diphenyl-1-picrylhydrazyl (dpph) assay and scavenging of hydrogen peroxide (h2o2) assay. all extracts showed antioxidant activity in figure 3. the results were positively correlated to the concentration, and the maximum scavenging activity was recorded at the highest concentration for all extracts. furthermore, it was observed that puae provided a strong significant dpph scavenging effect (p<0.01), translated by a low ic50 value of leaf extracts (ic50 = 23.47±0.62 µg/ml) closely compared to ascorbic acid (ic50 el es β-carotene 0.035 0.006 lycopene 0.033 0.036 chlorophyll a 0.167 0.016 chlorophyll b 0.108 0.049 0.000 0.020 0.040 0.060 0.080 0.100 0.120 0.140 0.160 0.180 li p id -s o lu b le p ig m e n ts c o n te n t µ g /g ± 0.001 ±0.000a ±0.000 ±0.002 ± 0.001 ±0.001a ±0.001 ±0.002 pmmb 2023, 6, 1; a0000331 10 of 20 =19.89±0.14 µg/ml). while the maceration leaf extract showed a low antioxidant effect (ic50 = 79.76±1.93 µg/ml). figure 3. dpph radical scavenging activity (a) and h2o2 scavenging activity (b) versus concentration (μg/ml). elm: euphorbia leaf maceration, esm: euphorbia stem maceration, elu: euphorbia leaf ultrasonicassisted extraction, esu: euphorbia stem ultrasonic-assisted extraction. the results are expressed as means ± sd of three independent measurements. the stem extract of puae also exhibited a moderate antioxidant effect (ic50 = 42.45 ± 2.30µg/ml) compared to the maceration extract (ic50 = 61.16 ± 1.53µg/ml). however, it was observed that stem maceration extract has a significantly (p<0.01) lower ic50 value than leaf maceration extract, which implies a higher antioxidant capacity, as shown in table 3, similarly, for h2o2 scavenging results. the lowest ic50 was observed in puae leaf extract (ic50=110.27 ± 3.59 µg/ml) followed by stem extract (ic50 = 131.51 ± 1.80 µg/ml) in comparison to maceration (ic50 = 678.32 ± 7.22 µg/ml, ic50 = 1073.63 ± 1.08 µg/ml, respectively). table 3. scavenging activity of leaf and stem extracts of euphorbia nicaeensis all. scavenging activity (ic50 µg/ml) esm* dpph assay h2o2 assay 61.16 ± 1.53 1073.63 ± 1.08 elm* 79.76 ± 1.93 678.32 ± 7.22 esu* 42.46 ± 2.30 131.51 ± 1.80 elu* 23.48 ± 0.62 110.27 ± 3.59 aa* 19.90 ± 0.14 35.41 ± 0.23 *means ± sd from triplicate determinations, p < 0.001. (a) (b) pmmb 2023, 6, 1; a0000331 11 of 20 the correlation analysis between antioxidant activity and phenolic content of the extracts is presented in table 4. pearson's coefficient revealed a positive relationship between antioxidant activity and total phenolics and flavonoids' plant content. the dpph assay of antioxidant activity and tpc showed a pearson coefficient of r=0.8827 and r=0.7768 for total flavonoids content with no significant correlation (p>0.05). moreover, a highly significant correlation was observed between the h2o2 assay and the composition of the plant in both tpc (p < 0.001) and tfc (p < 0.05). table 4. pearson correlation test between total phenolic content, total flavonoids content, and antioxidant activity. *significant at p <0.05; **significant at p<0.001; dpph: dpph scavenging activity; h2o2: hydrogen peroxide scavenging activity; tpc: total flavonoid content; tfc: total flavonoid content. 3.5. in vitro anti-inflammatory activity according to the results presented in table 5, all extracts showed an inhibition of bovine serum albumin (bsa) and chicken egg albumin denaturation (cea), which increased with the sample concentration. the highest inhibitions were observed at the 2000 µg/ml dose for all extracts. the findings revealed a notable inhibition of serum albumin denaturation (83.98% and 75.39 %, p<0.001) and egg albumin denaturation (82.04% and 78.87%, p<0.001) induced by puae extracts of stems and leaves, respectively, at the dose of (2000 µg/ml). the maceration extracts of leaves and stems showed a lower inhibition of bovine serum albumin (38.28% and 43.36%, p<0.001) at the same concentration. the inhibition of egg albumin revealed similar results. maceration extracts from stems and leaves showed a weak inhibition (49.85%, 62.52%, respectively). while the standard drug diclofenac sodium exhibited potent inhibition (97.73%, p<0.001) at the same concentration when compared to the control. pearson's correlation (r) dpph (1/ic50) h2o2 (1/ic50) tpc 0.8827 0.9995** tfc 0.7768 0.9505* pmmb 2023, 6, 1; a0000331 12 of 20 stem extracts from maceration and ultrasound-assisted extraction showed a significantly higher level of protein denaturation inhibition (p<0.05) than leaf extracts at the concentration range of (2000500 µg/ml). table 5. in vitro anti-inflammatory activity of euphorbia nicaeensis all. treatment dosage (µg.ml-1) bovine serum albumin denaturation chicken egg albumin denaturation absorbance % inhibition absorbance % inhibition control 0.917 ± 0.015 0.913 ± 0.015 _ elm 200 0.652 ± 0.0006 28.90 0.286 ± 0.003 72.48 300 0.591 ± 0.002 a 35.54 0.221 ± 0.001 72.08 500 0.571 ± 0.0026 37.77 0.218 ± 0.003 70.95 1000 0.570 ± 0.0021 37.89 0.197 ± 0.001 68.80 2000 0.566 ± 0.001 38.28 0.193 ± 0.001 62.52 esm 200 0.677 ± 0.0072 26.17 0.279 ± 0.001 73.32 300 0.570 ± 0.006 37.89 0.223 ± 0.001 63.94 500 0.541 ± 0.0066 41.02 0.206 ± 0.004 59.82 1000 0.536 ± 0.0061 42.19 0.192 ± 0.001 52.04 2000 0.519 ± 0.009 43.36 0.164 ± 0.004 49.85 elu 200 0.638 ± 0.003 30.47 0.342 ± 0.003 68.72 300 0.605 ± 0.0096 a 33.98 0.285 ± 0.004 b 75.77 500 0.390 ± 0.0046 57.42 0.265 ± 0.003 76.13 1000 0.276 ± 0.0053 69.92 0.255 ± 0.004 78.43 2000 0.226 ± 0.001 75.39 0.251 ± 0.001 78.87 esu 200 0.613 ± 0.0044 33.20 0.458 ± 0.006 69.49 300 0.537 ± 0.001 41.41 0.438 ± 0.012 b 75.62 500 0.308 ± 0.0053 66.41 0.367 ± 0.002 77.48 1000 0.208 ± 0.000 77.34 0.329 ± 0.003 78.98 2000 0.147 ± 0.0035 83.98 0.244 ± 0.002 82.04 diclofenac sodium 200 0.079 ± 0.0026 91.41 0.054 ± 0.004 94.12 300 0.062 ± 0.001 93.23 0.047 ± 0.002 94.89 500 0.057 ± 0.0036 93.75 0.041 ± 0.002 95.47 1000 0.050 ± 0.0017 94.53 0.035 ± 0.004 96.17 2000 0.021 ± 0.0044 97.73 0.022 ± 0.004 97.59 the results are expressed as means ± sd of three independent measurements. elm: euphorbia leaf maceration, esm: euphorbia stem maceration. elu: euphorbia leaf ultrasonic-assisted extraction, esu: euphorbia stem ultrasonic-assisted extraction. the values with the same superscript letters are not significantly different (p>0.05). pmmb 2023, 6, 1; a0000331 13 of 20 3.6. phenolic profile analysis by hplc-ms figure 4. hplc-ms/ms chromatogram of uae stem extract. figure 5. hplc-ms/ms chromatogram of uae leave extract. based on the previous results of the phenolic compounds assay, uae leaf, and stem extracts were retained for hplc-uv-ms/ms analysis. the presence of the compounds identified by hplc-uv analysis was confirmed using hplc-ms/ms. comparisons with reference standards, nist library, and earlier literature reports helped identify. the retention times of the standards tested did not match the peaks detected in the chromatograms of the leaf and stem extracts (figures 4 and 5). this is because many are in the form of sugar derivatives that are rarely available. therefore, mass spectrometry was utilized to characterize the peaks and allowed further identification of the compounds. comparing the ms/ms data with those in the nist library and other references revealed several phenolic compounds. pmmb 2023, 6, 1; a0000331 14 of 20 table 6 demonstrates the abundance of different phytochemicals present in both extracts, with a slight difference in composition, including flavonols, phenolic acids, lignan, calchone, carotenoids, and alkaloids. fargesin, 2’-hydroxy-3,4,4’,6’tetramethoxychalcone, nuciferin, quercetin 3'-methyl ether, and isoquercetin are detected in both extracts with different peak airs. isoquercetin was detected as the predominant compound in the leaf extract, followed by quercitrin hydrate and guaiaverin. the stem extract is rich in 2’-hydroxy3,4,4’,6’tetramethoxychalcone, representing the highest peak (22.04%), followed by quercetin 3'-methyl ether. table 6. hplc-ms/ms tentative identification of phenolics and derivatives in e. nicaeensis leaf and stem extracts. leave stem n° rt tentative identification air % ref n° rt tentative identification air% ref 1 1.70 fargesin 5.72 n 1 1.70 fargesin 3.34 n 2 1.92 2’-hydroxy-3,4,4’,6’ tetramethoxychalcone 4.51 n 2 1.96 2’-hydroxy-3,4,4’,6’ tetramethoxychalcone 22.04 n 3 3.18 nuciferine 2.08 n 3 3.18 nuciferine 8.14 n 4 4.86 quercetin 3’-methyl ether 3.90 n 4 4.78 quercetin 3’-methyl ether 10.02 n 5 10.08 5,7,3’,4’ tetramethoxyisoflavone 2.11 n 5 10.38 lutein 3.65 n 6 10.97 galloylquinic acid 1.79 [32] 6 17.77 dilinolenin 3.11 n 7 19.90 quercetin-galactosidegallate 8.70 [33] 7 21.11 isoquercetine 0.94 n 8 20.47 myricitrin 6.59 n 8 21.52 2'-hydroxy-2,4,4'trimethoxychalcone 9.59 n 9 21.11 isoquercetin 27.49 n 10 22.57 guaiaverin 11.78 n 11 22.99 quercitrin hydrate 14.49 [31] rt: retention time, n: nist-ms/ms library, air %: relative peak area pmmb 2023, 6, 1; a0000331 15 of 20 4. discussion most of the phytochemical screening findings align with a previous investigation conducted in serbia for euphorbia nicaeensis ssp. glareosa [34]. however, some differences were observed, namely, the presence of alkaloids and the absence of quinones in e. nicaeensis all. leaves and stems, compared to this subspecies. this could be due to several parameters, including genetics, differences in harvesting site, rainfall, light, season, harvesting period, topography, soil type, and extraction method [35]. the phytochemical assay showed that the leaves and stems of e. nicaeensis are rich in total phenolic, flavonoids, and flavonols and contain a moderate amount of condensed tannins. flavonoids, particularly flavonols, are effective antifungal agents against various pathogens, including candida albicans, candida glabrata, candida krusei, candida parapsilosis, candida tropicalis, trichophyton rubrum and trichophyton beigelii.[36–40]. condensed tannins have also been demonstrated to have potent anthelmintic properties against worm infections. these findings support the traditional usage of euphorbia nicaeensis to treat fungal infections and parasite disorders [41]. by comparing the results of total phenolic content (tpc), total flavonoid content (tfc), condensed tannin content (ctc), flavonol content (fc), and the yield of the two extraction methods, it can be concluded that probe ultrasound-assisted extraction (puae) is more efficient than maceration. these might be linked to acoustic cavitation processes, which could cause a forceful impact on the solid surface, resulting in an enhanced extraction rate, as already reported [42]. it is therefore inferred that puae could be a convenient technique for phenolic compound extraction. however, the maceration extract of stems proved to have a higher antioxidant effect than leaf maceration extract. these results can be interpreted by the variation of the nature and chemical structure of the phenolic compounds present in each sample. in some cases, the highest antioxidant activity may be observed in extracts with a low phenolic content. synergy may occur between the major antioxidants (phenolic compounds) and other minor constituents of the plant, so this could significantly impact the differences in their antioxidant activity [43]. this study revealed that e. nicaeensis all. leaves possess a potent antioxidant effect close to ascorbic acid activity. a higher free radical scavenging and hydrogen peroxide scavenging effect were observed in probe ultrasound-assisted extraction leaf extracts, compared to stems. the difference between leaves and stems in extraction yield and total phenolic contents could partially explain this. studies have reported that leaves contain a high amount of phenolic compounds than stems [44]. a potent correlation was observed between phenolic compounds and h2o2 scavenging activity, as reported by reports [45,46]. however, the inclusion of other substances, particularly sugars, which, according to [47], can interfere with quantification and antioxidant activity assays, may cause a non-significant correlation between tpc and dpph. hplc-uv-ms/ms analysis showed that the tested extracts are rich in phenolic compounds, especially flavonoids (flavonol glycosides). quercetin and its derivatives are the pmmb 2023, 6, 1; a0000331 16 of 20 most abandoned compounds in the tested extracts, especially in the leaves, and studies have reported their different biological virtues [48,49]. the presence of isoquercetin and quercitrin hydrate represented by the highest peaks, may be among the leading causes of the significant antioxidant properties of the leaves. fargesin, myricitrin, and guaiaverin also play a crucial role in plant health and display several therapeutic values due to their antioxidant or/and antiinflammatory properties [50,51]. from this, we can infer that the phenolic compounds of the plant are the major cause of the scavenging effect of the extracts, supporting the results obtained and explaining why the leaves have a higher antioxidant power than the stems. therefore, leaves are a potential source of natural antioxidants and bioactive compounds. the results of previous research on different species and subspecies of the euphorbiacea family have demonstrated that e. nicaeensis all. has a higher dpph radical inhibition potential than e. retusa forssk. (ic50 leaf = 287.52±2.92 µg/ml, ic50 stem=225.87±3.88 µg/ml [52] and e. hirta l. (ic50 leaf = 803 µg/ml, ic50 stem= 1358 µg/ml) [53], as well as e. heterophylla (ic50 = 194.28±0.22 µg/ml) [54]. the subspecies euphorbia nicaeensis ssp. glareosa showed more than 50% inhibition of dpph (56.5%), which qualifies the plant as moderately active [34]. yet no previous study on the in-vitro antioxidant activity of e. nicaeensis all. has been published. the aerial part of e. nicaeensis all. is recognized as having anti-inflammatory virtue as it contains glyceroglycolipids tested for their anti-inflammatory activity [55]. this study's results agree with what has been previously published. in vitro tests showed that the plant has a strong anti-inflammatory effect, inhibiting the degradation of bovine serum albumin (bsa) and chicken egg albumin (cea), observed mainly in the stem extracts. nuciferine (alkaloid) and fargesin (lignan) are among the main compounds identified in the stem extract according to chromatograms and ms/ms identification; they are phytochemical compounds known for their anti-inflammatory virtues [50,56] which proves the results of the antiinflammatory activities performed. the inhibition of thermal degradation of proteins by extract can be explained by several mechanisms, depending on the type of plant extract and the protein studied [57]: proteins can be protected from thermal degradation by antioxidants because they can scavenge reactive oxygen species (ros) and reduce oxidative degradation [58]. when phenolic chemicals interact with proteins, their properties, such as solubility, digestibility, and thermal stability, may alter. however, extracts rich in phenolic compounds have been employed to inhibit protein denaturation and improve thermal stability [59]. the antioxidant effect of e. nicaeensis extracts resulting from the presence of phenolic compounds, specifically quercetin, and derivatives, play an essential role in the stability of proteins and inhibit denaturation caused by heat treatment, as already reported. based on the results of anti-inflammatory activity and those reported by a previous study, we can deduce that the aerial part of e. nicaeensis, especially the stems, provides a pmmb 2023, 6, 1; a0000331 17 of 20 potent anti-inflammatory activity and could be considered a potential antiinflammatory agent. 5. conclusion this study aimed to investigate the phytochemical profile and determine the antioxidant and anti-inflammatory activities of leaf and stem extracts of euphorbia nicaeensis all. it has been observed that ultrasound-assisted extraction is an efficient and recommended method for phenolic compound extraction. this work also showed that all extracts are rich in phenolic compounds and exhibited efficient antioxidant and antiinflammatory properties with significant differences between organs. hplc-uv-ms/ms confirmed the presence of phenolic compounds, particularly quercetin and its derivatives, present mainly in the leaf extracts. the leaves showed the highest antioxidant activity, while the most potent anti-inflammatory effect was observed in the stem extracts. the phenolic composition indicated the presence of several compounds with anti-inflammatory properties. these findings confirmed e. nicaeensis historical medicinal uses and opened up new ways and possibilities of developing this plant in the pharmaceutical and food fields as a new source of bioactive compounds unaffected by commercial breeding. however, it is evident that additional research, such as the molecular identification of the bioactive substances responsible for these biological activities, is required to offer a more satisfactory and clearer perspective to this study. author contributions: conceptualization, kb and ht.; methodology, kb, ht, ce, rs; software, neh, ce.; validation, x.x., y.y. and z.z.; formal analysis, neh, hnm, kb, km, mh, rf; investigation kb, ht, ce; resources kwg, ab, hnm; data curation, ak and kb.; writing—original draft preparation, kb and ce; writing—review and editing ab, neh, ht, kwg. funding: no external funding was provided for this research. acknowledgments: authors of this work express their sincere thanks to everyone who contributed to the realization of this study, especially abderrahmane belhouari, professor of statistics at the faculty of science ben msik and said zaidoune, professor of english at the faculty of letters ben msik. conflicts of interest: the authors declare no conflict of interest. references 1. garg ak, faheem m, and singh s. role of medicinal plant in human health disease. asian j plant sci 2021; 11(1). doi:10.36648/2249-7412.11.1.19-21 2. elachouri m, kharchoufa l, fakchich j, et al. ancestral phytotherapeutic practices in morocco: regards on history, current state, regulatory and safety of commonly used herbal medicine. arab j chem 2021; 8(1): 133-149. 3. samman na, martin a, and puech s. inflorescence architecture variability and its possible relationship to environment or age in a mediterranean species, euphorbia nicaeensis all. 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2: 53. 59. cirkovic velickovic td, and stanic‐vucinic dj. the role of dietary phenolic compounds in protein digestion and processing technologies to improve their antinutritive properties. compr rev food sci 2018; 17(1): 82-103. pmmb 2022, 5, 1; a0000281. doi: a10.36877/pmmb.0000281 http://journals.hh-publisher.com/index.php/pmmb systematic review article the antibacterial activities of secondary metabolites derived from streptomyces sp. rabia mrehil elsalami1, khang wen goh2, mahani mahadi1, najwa mohammad1*, yaman walid kassab3, noraziah mohamad zin4*, kok-yong chin5 article history 1department of pharmaceutical sciences, faculty of pharmacy, university of cyberjaya, 63000 cyberjaya, malaysia; elzaidi79@yahoo.com (rme); mahani@cyberjaya.edu.my (mm) 2faculty of data science and information technology, inti international university, 71800 nilai, malaysia; khangwen.goh@newinti.edu.my (kwg) 3faculty of pharmacy, syrian private university, syria; dryamankassab@yahoo.com (ywk) 4center for diagnostic, therapeutics & investigative studies, faculty of health sciences, universiti kebangsaan malaysia, kuala lumpur 50300, malaysia 5department of pharmacology, faculty of medicine, university kebangsaan malaysia, cheras 56000, kuala lumpur, malaysia; chinkokyong@ppukm.ukm.edu.my (kyc) *corresponding author: najwa mohammad, department of pharmaceutical sciences, faculty of pharmacy, university of cyberjaya, 63000 cyberjaya, malaysia; najwa@cyberjaya.edu.my (nm); noraziah mohamad zin, center for diagnostic, therapeutics & investigative studies, faculty of health sciences, universiti kebangsaan malaysia, kuala lumpur 50300, malaysia; noraziah.zin@ukm.edu.my (nmz) received: 25 september 2022; received in revised form: 01 december 2022; accepted: 02 december 2022; available online: 04 december 2022 abstract: the spreading of infectious diseases caused by the emergence of multidrugresistant (mdr) pathogens is a global threat that has led to numerous deaths annually. in view of this, there is an overwhelming need to discover new bioactive compounds with effective antimicrobial properties. concurrently, the genus streptomyces has a growing reputation as a potential biological source of various antibiotics and other bioactive metabolites. streptomyces sp. has been isolated from different sources, including terrestrial and marine habitats with a myriad of promising compounds that could be used to treat mdr pathogens. therefore, this study presents a systematic review of the antibacterial activities of streptomyces-derived secondary metabolites. the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines and checklist were employed in this study to collect relevant articles from two research databases, namely pubmed and science direct. the selection process includes identification, screening, eligibility, and inclusion of articles. several keywords and criteria were established for the screening and selection process. based on the results, a total of 26 articles were selected from 70 potential articles. the articles were published between 2015 and 2020 with most studies being published in 2020, indicating an increased interest in streptomyces and its derived compounds. approximately 51 different mailto:elzaidi79@yahoo.com mailto:mahani@cyberjaya.edu.my mailto:khangwen.goh@newinti.edu.my mailto:dryamankassab@yahoo.com mailto:chinkokyong@ppukm.ukm.edu.my mailto:najwa@cyberjaya.edu.my pmmb 2022, 5, 1; a0000281 2 of 25 streptomyces-derived compounds have been identified, ranging from crude extracts, pure compounds, and partially purified compounds. various parameters were also used to assess their antibacterial activities, particularly the minimum inhibitory concentration (mic) (69%) and the zone of inhibition (11%). moreover, the antibacterial activities of these compounds were effective on numerous gram-positive and gram-negative bacteria. furthermore, 46% and 54% of the selected studies were focused on inhibiting mdr and nonmdr pathogens, respectively. in conclusion, both crude and purified compounds from streptomyces sp. exhibited strong antibacterial effects. it is expected that extensive future research would develop a standard method to compare the antibacterial strength of each extracted compound from streptomyces sp. and determine the most effective bioactive compounds to treat diseases caused by mdr pathogens. keywords: streptomyces; secondary metabolites; bioactive compounds; multidrugresistant pathogens; antibacterial activity; in vitro 1. introduction the re-emergence and transmission of multidrug-resistant (mdr) pathogens have become an alarming global health crisis, particularly in developing countries with a high prevalence in hospitals, convalescent homes, and community settings [1]. these human pathogens lead to severe infections of the skin and soft tissues as well as systemic chronic diseases, which are responsible for significant mortalities [2]. the increased bacterial resistance is associated with several factors, such as misuse of antibiotics to treat infections and accelerated transmission of vertical and horizontal genes between bacterial species [3]. nevertheless, the growing concern over the spreading of mdr pathogens is related to the limited novel antimicrobial agents to substitute those made ineffective by mdr strains [4]. hence, the discovery, design, and development of new antibacterial drugs have become a more relevant topic in the scientific community to combat emerging mdr pathogens [5]. streptomyces, which belongs to the phylum actinobacteria and is a member of the order of actinomycetales, is a prolific source of antibiotics and other bioactive secondary metabolites [6] . the aerobic gram-positive streptomyces is rich in guanine-cytosine content (gc-content) and possesses both aerial and substrate mycelium [7]. streptomyces have been isolated from numerous sources, including terrestrial (soil, insects, animals, and plants) as well as marine (sediment, fish, corals, sponge) habitats [8]. to date, over 800 species have been discovered with valid names [9,10]. it is understood that members of the streptomyces generate a plethora of secondary metabolites with unique structures and exhibit antimicrobial activities [11]. almost 75% of the commercially available antibacterial drugs in the market have been developed by this genus alone [12]. these bioactive compounds are primarily extracellular, produced during the growth phase, and are not necessary for growth and reproduction, providing them with a competitive advantage over other microorganisms [13,14]. they also produce numerous secondary with pmmb 2022, 5, 1; a0000281 3 of 25 unique structures belonging to alkaloids, flavonoids, terpenoids, amino acids, and steroids, which have potentially useful medical benefits to humans [15]. in view of the remarkable properties of streptomyces sp. and the essential need to seek alternative bioactive compounds to suppress the emergence of mdr pathogens, this review was carried out to provide the latest insight on the available secondary metabolites derived from streptomyces sp. and their antibacterial activities. the preferred reporting items for systematic reviews and meta-analyses (prisma) method was implemented to gather relevant literature studies from several research databases. following the selection of articles, a thorough review of each article was conducted to obtain important information regarding the outlined topic. 2. materials and methods 2.1. literature search strategies a systematic review was performed according to the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines and checklist [16]. an electronic literature search was conducted using two databases, namely pubmed and science direct. the search was performed using the following search string: ‘streptomyces’ and ‘secondary metabolites’ or ‘bioactive compounds’ and ‘pathogens’ and ‘antibacterial activity’ and ‘in vitro’. 2.2. eligibility criteria and study selection the criteria for the review process included in vitro studies on the antibacterial activities of streptomyces-derived secondary metabolites against pathogens. in addition, articles that were (1) only available in abstract form; (2) not written in english; and (3) books and books chapters, reviews, meta-analyses, conference/proceeding papers, letters to the editor, commentaries, and thesis, were excluded from the search process. the bibliographies of relevant articles were also examined to identify potential articles that were overlooked during the database search. figure 1 depicts the selection process, from identification to screening, eligibility, and inclusion of articles. 2.3. study exclusion three independent reviewers (rme, ywk, and mm) screened and extracted the search results by referring to the titles and abstracts, followed by a full-text screening process. articles that failed to meet the selection criteria were excluded. in case of a disagreement regarding the selection of an article, a group discussion was held with three more reviewers (kyc, nmz, and nm). pmmb 2022, 5, 1; a0000281 4 of 25 figure 1. prisma flowchart of the systematic literature search. 3. results 3.1. extraction of articles the preliminary literature search found 70 possible articles, which only include in vitro studies and excluded those related to animal or human studies. three redundant articles were found and immediately removed. the remaining 67 articles were then screened by reviewing the titles and abstracts. eventually, 41 articles were removed since they did not meet the review criteria. therefore, 26 articles were selected for the final full-text review process. figure 2a depicts the number of articles that were published between 2015 and 2020, while figure 2b shows the number of articles based on the country of origin with the highest number of published articles in india, followed by china. figure 2. number of published articles based on (a) year throughout 2015–2020 and (b) publication distribution according to the country of origin. 2 3 5 3 2 11 0 2 4 6 8 10 12 2015 2016 2017 2018 2019 2020 n u m b e r o f p a p e r s years articles after duplicates removed (n = 67) full-text articles assessed for eligibility (n = 67) full-text articles excluded with reasons articles included in the final review (n = 26) id e n ti fi c a ti o n e li g ib il it y in c lu d e d articles identified through databases searching (n = 70) pmmb 2022, 5, 1; a0000281 5 of 25 3.2. general findings the systematic review focused only on the antibacterial properties of streptomyces species. the extracted data include the author’s name, year of publication, streptomyces species, source/ location of isolation, extracted secondary metabolites, pathogens, type of substance, and strength of the antibacterial activity, as presented in table 1. approximately 51 extracted compounds were utilised in the 26 reviewed articles. the chemical structures of each extracted secondary metabolite in the selected articles are shown in figure 3. the compound of interest was used in the form of crude extracts (8 studies), both crude and pure compounds (3 studies), and partially purified compounds (2 studies). the remaining 13 studies used different pure compounds, as depicted in figure 4. moreover, 50% of the streptomyces studies were related to terrestrial sources, while the rest were marine-based sources. of the terrestrial species, 61% of them were found in soil, followed by 31% in plants and 8% in insects (figure 5). pmmb 2022, 5, 1; a0000281 6 of 25 table 1. extracted secondary metabolites from streptomyces sp. and their strength of antibacterial activity. streptomyces species source/ location extracted secondary metabolites type of bacteria type of substance strength of antibacterial activity ref. streptomyces sp. strain sbt343 marine sponge, greece 1. azalomycin 2. streptocytosine 3. streptocytosine c 4. daryamide a 5. azmerone 6. antimycin b1 7. usabamycin a 8. actinoramide d gram-positive bacteria: staphylococcus aureus and staphylococcus epidermidis crude extract • bic: bic50 (62.5 µg/ml), bic71.35 (250 µg/ml) • mic: nm • inhibition zone: nm [17] streptomyces sp. strain sbt348 marine sponge, greece 9. compound skc3 gram-positive bacteria: s. aureus and s. epidermidis. skc3 is more effective against msra, mssa, and vrsa but not against gram-negative bacteria: pseudomonas aeruginosa crude extract and pure compound • mic of pure compound (31.25 µg/ml) • bic90 (3.95 µg/ml) • bic90 of crude extract (62.5 µg/ml) on s. epidermidis • inhibition zone: nm [18] streptomyces griseorubens strain dsd069 marine sediment, the philippines anthracycline shunt metabolites: 10. bisanhydroaklavinone 11. hydroxy bisanhydroaklavinone gram-positive bacteria: mrsa crude extract and pure compounds • mic of crude extract (2.44 µg/ml) • mic of both compounds (6.25 µg/ml and 50.00 µg/ml, respectively) • inhibition zone of crude extract (15 mm) [13] streptomyces sp. strain al-dhabi100 (novel strain) marine soil sediment, saudi arabia 12. benzenebutanoic acid 13. benzestrol 14. 1-(2,6-dimethyl-4propoxyphenyl) propan1-one 15. phenol, 4-(1,1dimethylpropyl) 16. 1-(2,6-dimethyl-4-pro poxyphenyl) propan-1one 17. ethyl 2-propylphenyl ester gram-positive bacteria: enterococcus faecalis, bacillus subtilis, s. aureus, and s. epidermidis gram-negative bacteria: klebsiella pneumoniae acid-fast bacteria: mycobacterium tuberculosis partially purified compounds • mic of the fractions (62.5, 31.25, 125, and 250, 125 µg/ml, respectively) • mbc of the fractions (62.5 to 500 µg/ml) • mic against m. tuberculosis was not stated • inhibition zone: nm [19] pmmb 2022, 5, 1; a0000281 7 of 25 18. phenol, 2-methyl-4(1,1,3,3-tetramethyl butyl) 19. androst-5,16-diene3.beta.-ol 20. beta.-carotene-3,30-diol, (3r, 30 r)-all-trans streptomyces zhazhouensis strain ca-185989 marine sediment, equatorial guinea 21. isoikarugamycin 22. 28-nmethylikarugamycin 23. 30-oxo-28-nmethylikarugamycin gram-positive bacteria mrsa pure compounds • mic of the pure compounds (2–4, 1–2, and 2–4 µg/ml, respectively) • mbc: nm • inhibition zone: nm [20] streptomyces sp. strain eg1 marine wet sediment, egypt new tetracene derivatives: 24. mersaquinone 25. tetracenomycin d 26. resistoflavin 27. resistomycin gram-positive bacteria mrsa pure compounds • mic of the pure mersaquinone (3.36 µg/ ml) • bic: nm • inhibition zone: nm [21] streptomyces xinghaiensis strain oy62 and streptomyces rimosus strain og95 marine sediment, nigeria 28. phenol,2,4-bis (1,1dimethyl ethyl) 29. 1,2-benzene dicarboxylic acid, bis(2-methyl propyl ester 30. phthalic acid, isobutyl 2pentyl ester 31. 1,2-benzene dicarboxylic acid, butyl octyl ester 32. 9-octadecenoic acid, methyl ester 33. 9-octadecenamide 34. dibutyl phthalate 35. bis(2-ethylhexyl) phthalate gram-positive bacteria: e. faecalis and b. subtilis gram-negative bacteria: campylobacter jejuni, p. aeruginosa, and salmonella typhimurium partially purified compounds • mic of partially purified extract of co-cultured (3.12–6.25) • mbc: (12.5–25.0 µg/ ml) [22] streptomyces sp. strain al-dhabi97 marine sediment, saudi arabia 36. 1-phenanthrenemethanol 37. phthalic acid, di(2propylpentyl) ester 38. benzenebutanoic acid 39. podocarp-7-en-3-one 40. indole-3-carboxaldehyde gram-positive bacteria: b. subtilis, e. faecalis, s. epidermidis, and s. aureus gram-negative bacteria: crude extract • mic value of the crude extract against grampositive bacteria (500, 250, 125, and 62.5 µg/ml, respectively) and gram-negative bacteria [23] pmmb 2022, 5, 1; a0000281 8 of 25 p. aeruginosa, k. pneumoniae, escherichia coli, and salmonella paratyphi (500, 500, 250, and > 250 µg/ml, respectively) • bic: nm • inhibition zone: nm streptomyces sp. strain al-dhabi90 marine sediment, saudi arabia 41. 3-methylpyridazine 42. n-hexadecenoic acid 43. indazol-4-one 44. octadecanoic acid 45. 3a-methyl-6-(4-methyl phenyl) sul gram-positive bacteria: s. aureus gram-negative bacteria: k. pneumoniae and esbl (e. coli, p. aeruginosa, and proteus mirabilis) gram-positive bacteria: vancomycin-resistant enterococcus faecium crude extract • mic of the crude extract (12.5, 50, 12.5, 25, and 50 µg/ml, respectively) • bic: nm • inhibition zone: nm [24] streptomyces sp. strain kcb132 marine sediment, china five angucyclinones: 46. (±)-pratensilin d 47. pratensilin a 48. kiamycin e 49. two angucyclinones tetrangulol 50. 8-o-methylteterangulol gram-positive bacteria: s. aureus and bacillus cereus pure compounds • mic of (-) pratensilin d (4 µg/ml). in contrast, its (+) enantiomer showed no mic value • bic: nm • inhibition zone: nm [25] streptomyces sp. strain adi95-16 marine sponge, tautra 51. echinomycin 52. linearmycin gram-positive bacteria: b. cereus pure compounds • mic: nm • bic: nm • inhibition zone: nm [9] streptomyces sp. strain ask2 rhizospher e soil, india 53. ask2 gram-negative bacteria: mdr k. pneumoniae pure compound • mbec of the pure compound (240 µg/ml) • mic: nm • inhibition zone: nm [26] streptomyces sp. strain hnm0039 (novel strain) marine sponge, china 54. tirandamycins a 55. tirandamycins b gram-positive bacteria: streptococcus agalactiae pure compounds • mic of purified compounds a and b (2.52 and 2.55 µg/ml, respectively) • bic: nm • inhibition zone: nm [27] streptomyces sp. strain scsio11594 deep sea sediment, south china 56. marangucycline a 57. marangucycline b 58. dehydroxyaquayamycin 59. undecyloprodigiosin 60. metacycloprodigiosin gram-positive bacteria: e. faecalis and methicillinresistant s. epidermidis strain shhs-e1 pure compounds • mic of compounds 1–3 (64.0 µg/ml) and e. faecalis (16.0 µg/ml) against s. epidermidis • bic: nm • inhibition zone: nm [28] pmmb 2022, 5, 1; a0000281 9 of 25 streptomyces levis agricultura l soil, north india 61. 2,6-disubstituted chromone derivative gram-positive bacteria: s. aureus gram-negative bacteria: p. aeruginosa and k. pneumoniae pure compounds • mic of active compounds (6.25, 12.5, and 6.25 µg/ml, respectively) • bic: nm • zone of inhibition of the pure compounds (24, 20, and 23 mm, respectively) [29] streptomyces sp. strain eri-15 soil, india 62. dibutyl phthalate 63. 8-hydroxyquinoline 64. 2-amino-3-chlorobenzoic acid gram-positive bacteria: s. aureus, b. subtilis, and mrsa gram-negative bacteria: e. coli crude extract • mic: nm • bic: nm • zone inhibition of fractions (12–16 mm) [30] streptomyces misionensis strain v16r3y1 soil, tunisian oasis 65. cyclo-(l-prolyl-lleucine), cyclo-(l-leu-lpro) 66. phenylacetamide gram-positive bacteria: s. aureus, e. faecalis, and b. cereus gram-negative bacteria: p. aeruginosa, escherichia ferusonii, and salmonella enterica pure compounds • mic of pure compounds (30, 12, 16, 34, 230, and 11 µg/ml, respectively) • bic: nm • inhibition zone: nm [31] streptomyces sp. strain s17 soil, egypt 67. behenic acid (docosanoic acid) 68. borrelidin 69. 1h-pyrrole-2-carboxylic acid gram-negative bacteria: p. aeruginosa pure compounds • mic: nm • bic: nm • inhibition zone: nm • quorum sensing inhibitory concentration (1 mg/ml) [32] streptomyces sp. strain at37-1 (novel strain) soil, algeria 70. furanone derivative gram-positive bacteria: mrsa pure compound • mic of pure compound (15–30 µg/ml) • mbic: 10–15 µg/ml • inhibition zone: nm [3] streptomyces sp. strain musc125 mangrove soil, malaysia 71. thiophene,2-butyl-5ethyl 72. 8-inaziridylethylaminol-2-6, dimethyloctene-2 73. pyrroli1,2-alpyrazine1,4-dion, hexahydro gram-positive bacteria: mrsa and biofilm crude extract • mic of crude extract (12.5–25 mg/ml) • mibc: 1.5625 mg/ml • inhibition zone: nm [33] pmmb 2022, 5, 1; a0000281 10 of 25 74. 9,9-dimethyl-3,7diazabicyclo [3.3.1] nonane streptomyces cuspidosporus strain sa4 agriculture soil, egypt 75. 1,2-benzene dicarboxylic acid 76. bis(2-methylpropyl) ester gram-positive bacteria: s. aureus and b. subtilis gram-negative bacteria: e. coli, k. pneumoniae, salmonella typhi, proteus vulgaris, shigella flexneri, and p. aeroginosa crude extract and pure compound • mic: nm • bic: nm • zone of inhibition of partially purified and crude extract at 75 µg (16, 20, 22, 19, 20,17, 18, and 7 mm, respectively) [34] endophytic streptomyces coelicolor strain azra37 medicinal plant, azadiracht a indica, india 77. cryptic metabolites gram-negative bacteria: aeromonas hydrophilia, s. typhi, and s. flexneri gram-positive bacteria: e. faecalis and s. aureus crude extract • mic of crude compounds (40 µg/ml) against gramnegative bacteria and (60 µg/ml) against grampositive bacteria • bic: nm • inhibition zone: nm [35] streptomyces sp. strain suk25 beehive ginger plant (zingiber spectabile), malaysia 78. cyclo-(tryptophanylprolyl) 79. chloramphenicol gram-positive bacteria: mrsa pure compounds • mic of pure compounds (8 and 16 µg/ml, respectively) • bic: nm • inhibition zone: nm [36] s. ceolicolor strain aobkf977550 sawdust, lagos lagoon, nigeria 16 secondary metabolites: 80. mutamicin 81. hyberimycin 82. kanamycin 83. daunorubicin 84. indolyl-3-carboxylic acid 85. mitomycin 86. 2-phenylacetamide 87. streptomycin 88. mithramycin 89. pilacamycin 90. gentamicin 91. etamycin 92. chloromycetin 93. hydroxygentamycin 94. tetracycline 95. pimprinine gram-positive bacteria: mrsa and bacillus coagulans gram-negative bacteria: e. coli and k. pneumoniae crude extract • mic: nm • bic: nm • inhibition zone range from 16 to 21 mm [37] pmmb 2022, 5, 1; a0000281 11 of 25 streptomyces olivaceus strain lep7 lichen, tree bark, nilgiris, tamilnadu 96. cyclopentene gram-negative bacteria: e. coli, p. aeruginosa, klebsiella sp, and acinetobacter sp gram-positive bacteria: s. aureus crude extract • mic of partially purified compound (7.81 µg/ml against e. coli and p. aeruginosa) • bic: nm • inhibition zone range between 6 and 12 mm, while no zone of inhibition against acinetobacter sp. [38] streptomyces globisporus strain wa5-2-37 intestinal tract of american cockroach (periplanet a americana) , china 97. actinomycin x2 98. collismycin a gram-positive bacteria: mrsa pure compounds • mic of both compounds (0.25 and 8 µg/ml, respectively) • bic: nm • inhibition zone: nm [39] note: mic = minimum inhibitory concentration; bic = biofilm inhibitory concentration; mbic50 = minimal biofilm inhibition concentration at 50%; nm = not measured, mrsa = methicillin-resistant staphylococcus aureus pmmb 2022, 5, 1; a0000281 12 of 25 1. azalomycin o oh o oh o oh o oh oh oh oh oh oho o oh oh n h nh2 n o n n n h s o o oh 2. streptocytosine b o n n n h o o oh 3. streptocytosine c n h o o oh oh nh2 o 4. daryamide a n n o o o oh oh cl 5. azamerone n h o n h o o oh o o o oh oh oh 6. antimycin b1 7. usabamycin a n h n o h o n n oh o o nh o o nh oh nh o nh2 o oh 8. actinoramide d o o oh o o oh 9. bisanhydroaklavinone o o oh o o oh oh 10. 1-hydroxybisanhydroaklavinone 19 14 16 o 8 n h 1 3 26 nh 28 oh 23 30 31 o o 11. isoikarugamycin o n h n 28 oh o o 12. 28-n-methylikarugamycin o n h n 28 oh 30 o o o 13. 30-oxo-28-n-methylikarugamycin figure 3. chemical structures of extracted secondary metabolites that included in the selected articles. pmmb 2022, 5, 1; a0000281 13 of 25 o o oh oh oh oh 14. mersaquinone oh oh o oh oh o 15. tetracenomycin o oh ohooh ch3 o ch3ch3 oh 16. resistoflavin o oh ohooh ch3 oh ch3ch3 17. resistomycin oh 18. phenol, 2,4-bis (1,1-dimethyethyl) o oo o 19. 1,2-benzene dicarboxylic acid o oo o 20. phthalic acid isobutyl o o o o 21. 1,2-benzene dicarboxylic acid, 22. 9-octadecenoic acid, methyl ester o o o nh2 23. 9-octadecenamide o oo o 24. dibutyl phthalate o oo o 25. bis (2-ethylhexyl) phthalate bis (2-methyl propyl ester) 2-pentyl ester butyl octyl ester n o o o o o oh 26. (r,s)-pratensilin d oh o oh oh o 27. pratensilin a o o o o 28. kiamycin e oh o oh o 29. tetrangulol figure 3. cont. pmmb 2022, 5, 1; a0000281 14 of 25 figure 3. cont. pmmb 2022, 5, 1; a0000281 15 of 25 49. dehydroxyaquayamycin o oh ooh o oh oh nh nh o n c 11 h 23 50. undecycloprodigiosin 51. metacycloprodigiosin nh nh o n figure 3. cont. figure 4. percentage of reviewed articles that utilised crude extracts, pure compounds, both (crude extract and pure compounds), and partially purified compounds. figure 5. percentage of terrestrial sources of different streptomyces species, including soil, plant, and insect. 31% 50% 11% 8% type of substance crude extract pure compound crude extract and pure compound partially purified compound pmmb 2022, 5, 1; a0000281 16 of 25 3.3. streptomyces as a biological source of secondary metabolites the comprehensive analysis of the selected articles revealed the implementation of various analytical methods to assess the antibacterial properties of streptomyces-derived compounds. the minimum inhibitory concentration (mic) is the most commonly employed analysis (69%), followed by the zone of inhibition (11%). interestingly, several studies applied multiple analyses to determine the bacteriostatic or bactericidal properties of the tested compounds. furthermore, both gram-positive and gram-negative bacteria were involved in these studies, where 46% and 54% of the selected studies emphasised the inhibition of mdr and non-mdr pathogens, respectively. figure 6 portrays the numerous analytical methods used to measure the antibacterial properties of streptomyces-derived compounds based on the 26 selected articles. figure 6. numerous analytical methods used to measure the antibacterial properties of streptomyces-derived compounds. 4. discussion despite the breakthrough in the development of novel antibiotics and their successful commercialisation, infectious diseases are still considered the leading cause of death worldwide [18]. one of the main factors is the emergence of mdr pathogens among pathogenic microorganisms. consequently, this has triggered the urgent need to continuously seek new potential bioactive compounds. ironically, various microbial strains were found to pmmb 2022, 5, 1; a0000281 17 of 25 produce bioactive compounds with significant antimicrobial properties. in fact, about 75% of the available antibiotics were isolated from the genus streptomyces [40]. 4.1. isolation and source of streptomyces species a plethora of streptomyces sp. has been isolated from different habitats, including marine, soil, plant debris, dung, and house dust. usually, streptomyces sp. can adapt to various environmental conditions, such as different temperature ranges, varying nutrient availability, and diverse dissolved oxygen levels and pressure, which allows them to produce a wide range of metabolites that are beneficial to human health [41,42]. for example, balasubramanian et al. [17] isolated demonstrated the anti-biofilm efficacy of an organic extract from streptomyces sp. strain sbt343, which was isolated from marine sponge petrosia ficiformis. in a separate study, balasubramanian et al. [18] described the antistaphylococcal activity of an organic extract from streptomyces sp. strain sbt348, which was isolated from the same sponge. furthermore, rahman et al. [43] revealed the antibacterial activity of an organic extract from streptomyces sp. strain mars-17 isolated from streptomyces parvulus. it was noted that environmental factors had somehow minor effects that lead to some differences in the antibacterial activities observed between different streptomyces species. for example, streptomyces isolated from marine sources showed better antibacterial activity as compared to streptomyces derived from other sources such as plants. concurrently, research efforts have focused on the exploration of bioactive substances from other sources of streptomyces, such as insects, soils, and plants. for instance, chen et al. [39] investigated the anti-mrsa efficacy of purified extracts from s. globisporus strain wa5-2-37 from the intestinal tract of american cockroaches (p. americana). similarly, alshaibani et al. [36] examined the anti-mrsa efficacy of endophytic streptomyces sp. strain suk25 isolated from the root of the beehive ginger plant (z. spectabile). meanwhile, streptomyces sp. strain musc135t was isolated from a soil sample collected from a mangrove forest on the east coast of peninsular malaysia and exhibited a broad spectrum of bacitracin against mrsa atcc baa-44.40. 4.2. antibacterial activity of streptomyces-derived crude extracts in general, the present review was unable to suggest a solid finding regarding the antibacterial activities of streptomyces-derived compounds due to various factors, such as follows: (1) each literature study utilised different types and strains of pathogens; (2) various pmmb 2022, 5, 1; a0000281 18 of 25 positive controls (antibiotics) were used for comparison in each article; (3) the inconsistent use of analytical method to assess the antibacterial activity. in fact, even similar parameters were measured differently according to each article; and (4) the addition of external substances to the growth medium that may induce or inhibit the antibacterial activities of streptomyces-derived extracts. for example, abdullah al-dhabi et al. [23] used a micro-broth dilution assay for the mic analysis, while adeyemo et al. [22] used a macro-broth dilution method for the mic testing to assess the antibacterial activity of streptomyces-derived compounds. furthermore, 5 out of 8 studies (62.5%) that used crude extracts showed significant antibacterial activities compared to the positive controls used in each study. as reported by paderog et al. [12], the crude extract of s. griseorubens strain dsd069 recorded the highest antibacterial activity against mrsa with an mic value of 2.44 µg/ml compared to tetracycline (positive control). however, the result could be misleading as the considerably high antibacterial activity could be attributed to the weak effect of tetracycline against mrsa. another article used a different strain of mrsa to examine the antibacterial activity of a crude extract of streptomyces sp. strain musc125. the result showed a lower antibacterial activity with an mic range of 12.5–25 mg/ml, which was weaker compared to vancomycin [33]. thus, it would be more practical to use mic with a standard positive control to measure and compare the antibacterial strength of streptomyces-derived extracted secondary metabolites. apart from that, al-dhabi et al. [24] studied the antibacterial activity of crude extract from streptomyces sp. strain al-dhabi-90 towards different drug-resistant extendedspectrum beta-lactamase (esbl) pathogens. the recorded antibacterial activity with mic values varying from 12.5 to 50 μg/ml could be attributed to the effect of crude extracts that altered the membrane integrity and blocked the cellular constituents of the pathogens. as a result, the growth of pathogens was effectively inhibited. a more recent study by al-dhabi et al. [23] demonstrated a greater antibacterial activity of a crude extract from streptomyces sp. strain al-dhabi-97 against gram-positive pathogens compared to gram-negative pathogens with mic values of 62.5–500 µg/ml but weaker than streptomycin (positive control). despite the thicker cell wall of gram-positive bacteria compared to that of gramnegative bacteria, the extremely complex structure of the cell wall in gram-negative bacteria that contains various viscous components, such as lipids, lipoproteins, and lipopolysaccharides, makes it more difficult for the active compounds to penetrate the cells, thus, reducing the antibacterial activity against gram-negative bacteria. pmmb 2022, 5, 1; a0000281 19 of 25 regarding the addition of inducers to the growth media, kumar et al. [35] supplemented 25 µm 5-azacytidine in the crude extract of s. coelicolor strain azra37. the extract showed high antibacterial activity against both gram-positive and gram-negative bacteria with mic values of 40 µg/ml compared to that of untreated control with an mic value of 60 µg/ml. nevertheless, the enhanced activity might be due to the presence of 5azacytidine in the growth medium that stimulated the production of additional compounds, which in turn contributed to the favourable outcome [35]. in one study, balasubramanian et al. [17] employed the biofilm inhibitory concentration (bic) as an alternative method to evaluate the antibacterial activity of marine streptomyces sp. strain sbt343 extract. the bacterial extract showed a significant reduction in staphylococcal biofilm formation after 24 hours of growth. although the anti-biofilm effect exhibited by streptomyces sp. strain sbt343 was at a much lower concentration (62.5– 250 µg/ml) compared to sodium metaperiodate at a concentration of 40 mm (positive control), the anti-biofilm activity of the extract was dose-dependent and not due to growth effect [17]. balasubramanian et al. [18] also proved the anti-biofilm activity of streptomyces sp. strain sbt348 isolated from the same marine sponge against staphylococcal epidermidis at a concentration of 62.5 µg/ml. the biofilm formation was reduced by ∼90% and was designated as the bic90 [18]. 4.3. antibacterial activity of purified streptomyces-derived secondary metabolites past studies have also screened the antibacterial activities of pure compounds isolated from streptomyces sp. through various methods, particularly the microor macro-dilution method. out of the 26 selected articles, 12 of them (46.15%) showed the antibacterial activity of pure compounds at different concentrations, incubation times, and types of bacterial pathogens. as shown in figure 2, approximately 51 extracted secondary metabolites have been identified and reported in past studies. in one study, chen and colleagues reported that actinomycin x2 was more potent against mrsa after 12 hours of incubation at a lower mic value of 0.25 μg/ml compared to collismycin a (mic value of 8 μg/ml) with vancomycin as the positive control. the result could be due to the destruction of the mrsa cell membrane after treatment with these two compounds [35]. this study was also the first to derive actinomycin x2 and collismycin a produced by s. globisporus strain wa5-2-37, which was isolated from the intestinal tract of american cockroaches (p. americana). pmmb 2022, 5, 1; a0000281 20 of 25 in another study, wang et al. [44] evaluated the antibacterial activity of actinomycins d, x2, and two new natural neoactinomycins a and b against various strains of e. coli, k. pneumoniae, mrsa, and vancomycin-resistant enterococcus (vre). comparatively, actinomycins d and x2 were very effective against mrsa and vre with mic values of 0.125–0.25 μg/ml, while neoactinomycins a and b showed moderate to weak antibacterial activities against mrsa and vre with mic values of 16–64 μg/ml and 128 μg/ml, respectively. nevertheless, all actinomycins recorded weak activity against the different strains of e. coli and k. pneumoniae with mic values of greater than 128 μg/ml [44]. a study by lacret et al. [20] reported the first findings on the chemical composition of marine strain extracts closely related to the recently published terrestrial species s. zhaozhouensis [20]. it was found that the isoikarugamycin, 28-n-methyllikaguramycin, and ikarugamycin extracts exhibited high growth inhibition of mrsa after 24 hours of incubation with mic values of 2–4, 1–2, and 2–4 μg/ml, respectively. the strong antibacterial activity was attributed to the presence of ethyl group in the molecules of these compounds. furthermore, paderog et al. [12] reported that the strong activity of s. griseorubens strain dsd069 crude extract against mrsa was associated with one of the identified compounds, namely bisanhydroaklavinone (9), which strongly inhibited mrsa with an mic value of 6.25 μg/ml compared to 1-hydroxybisanhydroaklavinone (10) and tetracycline (positive control) that displayed a relatively weak antibacterial activity of 50 μg/ml each. the occurrence may be due to the degradation effects of streptomyces compounds on the cell membrane of mrsa, as demonstrated by the protein and dna leakage and loss of essential cell components, abnormal cell shrinkage, and increased membrane permeability [12]. recently, kim et al. [21] isolated mersaquinone (14) from marine-derived streptomyces sp. strain eg1. the extracted compound showed moderate antibacterial activity against mrsa compared to ciprofloxacin hydrochloride hydrate with mic values of 3.36 µg/ml and 0.93 µm, respectively. this phenomenon was probably due to the carbon skeleton of tetracenomycin derivatives, which have been reported to exhibit considerable anti-mrsa activities [21]. meanwhile, alshaibani and his colleagues extracted two compounds from endophytic streptomyces suk25 comprising cyclo-(tryptophanyl-prolyl) (46) and chloramphenicol. both extracts showed good antibacterial activity against mrsa with mic values of 16 µg/ml and 8 µg/ml, respectively. the strong antibacterial activity could destroy the cell membrane of mrsa, subsequently inhibiting its growth [36]. pmmb 2022, 5, 1; a0000281 21 of 25 besides that, driche et al. [3] reported the isolation of a furanone derivative from a saharan soil-derived streptomyces sp. strain at37. the compound was considered to belong to the furanone heterocyclic family group of antibiotics e-975, which comprised numerous natural products and medicines with various biological activities [3]. based on the results, the compound exhibited strong antibacterial activity against several mrsa and vancomycinresistant s. aureus (vrsa) strains. the extracted compound also recorded a significant decrease in the formation of biofilm by mrsa and methicillin-sensitive staphylococcus aureus (mssa) with an mbic50 of 15 µg/ml and 10 µg/ml, respectively. interestingly, song and colleagues discovered that dehydroxyaquayamycin (49) extracted from deep-sea streptomyces sp. strain scsio 11594 exhibited a selective antibacterial activity against methicillin-resistant s. epidermidis-strain shhs-e1 with an mic of 16 µg/ml. the result could be due to the presence of cyclic peptides [28]. guo and colleagues reported the characteristics of two new cleavage angucyclinones that were isolated from a chinese marine streptomyces sp. the (-) pratensilin d (26) was effective against b. cereus with an mic value of 4 µg/ml [25]. in contrast, its enantiomer (+) structure showed no effect against all tested bacteria with an mic of up to 64 µg/ml. previously, angucyclinones have been proven to be a prolific source of antibiotics for the production of 45% of all reported active metabolites [25]. huang and colleagues also identified two active compounds derived by streptomyces sp., which consist of tirandamycins a and b (31,31), isolated from a novel marine sponge. both bioactive compounds displayed potent inhibitory activities against s. agalactiae with mic values of 2.52 and 2.55 µg/ml, respectively, compared to tobramycin (positive control). the antibacterial activity of tirandamycins a and b against gram-positive bacteria has been highlighted in past studies, which showed their role in inhibiting bacterial rna polymerase [27]. in addition, saadouli et al. reported the isolation and antibacterial evaluation of cyclo-(l-leu-l-pro) (37) derived from s. misionensis v16r3y1, which was isolated from soil in the tunisian oasis. the cyclo dipeptide compound was most susceptible to s. enterica, followed by e. faecalis, b. cereus, and s. aureus with mic values of 11,12, 16, and 30 µg/ml, respectively. the pyrrolo pyrazine was recognised as the main contributing component to the strong antibacterial activity of the cyclo dipeptide compound [31]. 5. conclusion and future perspectives based on the 26 selected articles, this systematic review highlighted the significant antibacterial activities of approximately 51 streptomyces-derived crude extract and pure compounds against gram-positive and gram-negative pathogens. various streptomyces sp. pmmb 2022, 5, 1; a0000281 22 of 25 have been isolated from a diverse range of habitats and showed promising organic extracts with strong antibacterial activities. nevertheless, the efforts of most antibiotic screening programs were not fully utilised as most research persistently focused only on the already known secondary metabolites. therefore, further research is required to compare the antibacterial activity of both crude extract and pure compounds. with the rapid emergence of mdr pathogens, it is essentially vital to put more effort into isolating novel classes of antimicrobial compounds. for this reason, investigative studies should be intensified to induce novel secondary metabolite production from streptomyces biosynthetic silent gene cluster. in addition, the nature and chemical structure of the purified compounds should be thoroughly studied to determine the compound with the highest antibacterial activities. their ability to react with extracellular and soluble proteins as well as the complex bacterial cell wall should also be explored so that the mechanism of action can be improved to achieve effective medical treatment against mdr-related infectious diseases. author contributions: conceptualization, rme, nm, nmz, k-yc; 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a0000330. doi: 10.36877/pmmb.0000330 http://journals.hh-publisher.com/index.php/pmmb genome report whole-genome sequence of streptomyces pluripotens strain mum 16j, a potential resource of glycopeptide antibiotic and biocontrol agent against biofilm-forming bacteria priyia pusparajah1, jodi woan-fei law2,3, kok-gan chan3,4,5,, bey-hing goh6,7, karwai hong3*, learn-han lee2,3,8*, loh teng-hern tan3,8* article history 1medical health and translational research group (mhtr), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; priyia.pusparajah@monash.edu (pp) 2next-generation precision medicine and therapeutics research group (nmet), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia; jodi.law1@monash.edu (jw-fl) 3novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia 4international genome centre, jiangsu university, zhenjiang, china. 5institute of biological sciences, faculty of science, university of malaya, 50603, kuala lumpur, malaysia; kokgan@um.edu.my (kgc) 6biofunctional molecule exploratory research group (bmex), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; goh.bey.hing@monash.edu (b-hg) 7college of pharmaceutical sciences, zhejiang university, hangzhou, zhejiang, people’s republic of china 8innovative bioprospection development research group (inbiod), clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia *corresponding author: kar-wai hong, learn-han lee; novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor, malaysia; hong.karwai@monash.edu (kwh), lee.learn.han@monash.edu (l-hl); loh teng-hern tan; innovative bioprospection development research group (inbiod), clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) received: 28 march 2023; received in revised form: 20 april 2023; accepted: 29 april 2023; available online: 02 may 2023 pmmb 2023, 6, 1; a0000330 2 of 8 abstract: streptomyces pluripotens strain mum 16j is a gram-positive filamentous bacteria isolated from mangrove soil in kampung trombol area of kuching, sarawak. the draft genome of streptomyces pluripotens strain mum 16j consists of 7,671,699 bp assembled into 164 contigs. the gc content of the genome is 70.06 %, and the sequencing coverage of 108×. its genome consists of 6,781 predicted genes, 6,459 protein-coding genes, and 79 rna-coding genes (trna: 71, rrna: 8). here we report the draft genome of this strain to highlight its potential in producing glycopeptide antibiotic as well as catechol, a biocontrol agent of biofilm-producing bacteria. keywords: streptomyces pluripotens; genome; antibiofilm; glycopeptide; catechol; malaysia 1. introduction streptomyces are traditionally considered as gram-positive spores-producing, soildwelling and mycelium-forming filamentous bacteria with enormous potential for biotechnological and pharmaceutical applications [1-4]. streptomyces has been a rich source of valuable medicinal chemicals, ranging from antibiotic to antifungal, antioxidant, antiparasitic to even anticancer compounds [5-12]. other than medical and pharmaceutical industry, streptomyces also play an important role in various industrial bioprocesses such as industrial fermentative production [13-18]. the study of microbes in pristine environments such as the unexplored regions of rainforest, peat swamp forest and mangrove forest represent a promising avenue of research for the discovery of new biologically active compounds [19-23]. in this study, we present the genomic features of s. pluripotens strain mum 16j derived from an unexplored mangrove environment and highlight its capability to produce glycopeptide antibiotic and catechol (biocontrol agent of biofilm-forming bacteria). 2. data description streptomyces sp. strain mum 16j was isolated from the mangrove soil in kampung trombol area of kuching, sarawak. bacteria were routinely grown in international streptomyces project (isp) 2 medium at 28°c [24]. the genomic dna was extracted using the masterpure gram-positive dna purification kit (lucigen, middleton, wi, usa), according to the recommended procedures of the manufacturer [25]. subsequently, the extracted dna was subjected to rnase treatment. prior to sequencing library construction, the quantity and quality of the extracted genomic dna were measured using a qubit 2.0 fluorometer and a nanodrop 2000 spectrophotometer (both thermo fisher scientific, waltham, ma, usa), respectively [26]. the sequencing library was constructed using nextera™ dna sample preparation kit (nextera, usa) [27]. the library profile, size distribution and concentration of the sequencing library were evaluated using bioanalyser 2100 high sensitivity dna kit (agilent technologies, palo alto, ca) prior to performing pmmb 2023, 6, 1; a0000330 3 of 8 paired-end sequencing on miseq platform with miseq reagent kit 2 (2×250bp; illumina inc., madison, wi, usa) [28, 29]. upon completion of the whole genome sequencing, the quality of the sequencing raw reads was evaluated using fastqc (version 0.11.9) [30]. subsequently, sequencing reads with a mapping quality below q20 were removed from subsequent analysis using trimmomatic (version 0.39) [31]. the quality-trimmed reads were assembled de novo using spades (version 3.14.1) [32]. the assembled genome was evaluated based on contiguity and completeness with single-copy orthologs using the quast (version 5.2.0) [33] and benchmarking universal single-copy orthologs (busco) (version 5.4.4) [34], respectively. the lineage datasets in the busco analysis were streptomycetales_odb10. the assembled sequences were annotated using prokka (version 1.14.6) and ncbi prokaryotic genome annotation pipeline (version 4.13). the genomic features of s. pluripotens strain mum 16j are shown in table 1. from the busco analysis, a total of 1,579 busco markers were searched, of which there are 1,571 complete and single-copy busco markers identified, and 6 complete and duplicated busco markers found. no fragmented busco markers were found and 2 busco markers were missing. table 1. genomic features of streptomyces pluripotens strain mum 16j. streptomyces pluripotens mum 16j strain mum 16j total number of contigs 164 genome size (bp) 7,671,699 sequencing coverage 108× gc (%) 70.06 n50 (bp) 164,408 n90 (bp) 40,084 l50 (bp) 15 l90 (bp) 47 total number of predicted genes 6,781 total number of protein-coding genes 6,459 total number of rna-coding genes 79 (trna-coding genes: 71, rrna-coding genes: 8) total number of ncrna-coding genes 3 total number of pseudogenes 243 biosample samn16860772 bioproject prjna679886 genbank assembly accession number gca_022414615.1 genbank nucleotide accession number jadwyn000000000.1 pmmb 2023, 6, 1; a0000330 4 of 8 from the assembled genome, the 16s ribosomal rna (rrna) gene of streptomyces pluripotens strain mum 16j was predicted using barrnap (version 0.9) [35], and the predicted 16s rrna gene sequence was searched against ezbiocloud database [36, 37], a well-curated database of 16s rrna sequences and bacterial genomes. from the 16s gene analysis using the ezbiocloud database, the 16s rrna gene of strain mum 16j has a similarity and completeness of 100%, respectively, with s. pluripotens strain musc 135, suggesting strain mum 16j is very likely to be s. pluripotens. figure 1. position of streptomyces sp. strain mum 16j (arrow) in phylogenetic tree comprising selecting species of streptomyces, in which the phylogenetic tree was generated using the ggdc distance matrix. the identities of the strain mum 16j were also determined by a whole genome-based taxonomic analysis via type (strain) genome server (tygs) [38], and digital dna-dna hybridization (dddh) calculations via genome-to-genome distance calculator (ggdc) (version 3.0) [39-41]. the digital ddh values were calculated using ggdc distance formula d4, which is the sum of all identities found in the high-score segment pairs (hsps) divided by the total length of all hsps. supplementary table s1 shows the analysis findings of ggdc. figure 1 shows the position of strain mum 16j in the phylogenetic tree comprising selecting species of streptomyces, in which the phylogenetic tree was generated using the ggdc distance matrix (kendall-colijn test, p < 0.05). the genome of streptomyces pluripotens strain mum 16j was used as a query to compare against the reference strains, namely s. pluripotens strains musc 135 (ncbi accession number: cp021080.1) and strain musc 137 (ncbi accession number: cp022433.1), using blast [42]. this is to identify the accessory genomes or non-core genome of strain mum16j [43]. the accessory genome of strain mum16j was analyzed using pmmb 2023, 6, 1; a0000330 5 of 8 antismash (version 7.0) [44-46] and sempi (version 2.0) [47, 48] in order to identify its biosynthetic gene clusters. from the analysis output of antismash, a glycopeptide antibiotic-producing bgs was identified on nz_jadwyn010000070.1. from the analysis output of sempi, two contigs, namely nz_jadwyn010000151.1 and nz_jadwyn010000152.1, were predicted to carry the gene which is responsible for the biosynthesis of catechol, in which catechol is a biofilm-inhibiting compound [49, 50]. the draft genome sequence of s. pluripotens strain mum 16j has been deposited at ddbj/embl/genbank under the accession number jadwyn010000000.1. the version described in this paper is the first version. the data are publicly available at ncbi genbank under the bioproject accession number prjna679886, and the biosample accession number samn16860772. author contributions: writing—original draft preparation, pp; conceptualization, k-wh and b-hg, l-hl, lt-ht; methodology and data analysis, pp, lt-th, jw-fl and k-wh; 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26(3): 559. 50. kemung hm, tan lth, khaw ky, et al. an optimized anti-adherence and anti-biofilm assay: case study of zinc oxide nanoparticles versus mrsa biofilm. prog microbes mol biol 2020; 3(1). pmmb 2023, 6, 1; a0000330 8 of 8 author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology genome report 1 complete genome of mangrove-derived anti-mrsa streptomycete, streptomyces pluripotens musc 135 t hooi-leng ser 1,2,3,4, kok-gan chan 5,6, wen-si tan 5, wai-fong yin 5, bey-hing goh 2,3,4,8*, nurul-syakima ab mutalib 7, learn-han lee 2,3,4,8* 1institute of biomedical and pharmaceutical sciences, guangdong university of technology, guangzhou 510006, pr china. 2novel bacteria and drug discovery (nbdd) research group, biomedicine research advancement centre (brac), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 3biofunctional molecule exploratory (bmex) research group, biomedicine research advancement centre (brac), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 4biomedical research laboratory, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 5division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia 6international genome centre, jiangsu university, zhenjiang 212013, pr china 7ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia 8center of health outcomes research and therapeutic safety (cohorts), school of pharmaceutical sciences, university of phayao, phayao, thailand abstract : microorganisms serve as attractive resources, owing to their ability to synthesize structurally-diverse substances with various bioactivities. within the bacteria domain, members of the genus streptomyces have demonstrated remarkable ability to produce clinically useful, secondary metabolites such as anticancer, antioxidants, antivirals and antibacterials. streptomyces pluripotens musc 135t was isolated as novel strain from mangrove forest in malaysia. this strain exhibited broad spectrum bacteriocin against several pathogens including methicillin-resistant staphylococcus aureus (mrsa) strain atcc baa-44, salmonella typhi atcc 19430t and aeromonas hydrophila atcc 7966t. thus, the strain was selected for whole genome sequencing as an attempt to explore its bioactive potential. here we report the first complete genome of s. pluripotens musc 135t genome which comprise of 7.35 mbp with g+c content of 69.9 %. a total of 6,404 open reading frames (orfs) were predicted, along with 18 rrna and 69 trna genes. using bacteriocin mining tool, bagel detected eights gene clusters associated with bacteriocin production including lanthipeptides and linear azol(in)e-containing peptides (laps). members of streptomyces have contributed greatly towards improving lives, particularly against deadly infections and chronic diseases. the availability of s. pluripotens musc 135t genome sequence has opened new window for drug discovery, particularly for effective drugs against harmful pathogens such as mrsa and certainly deserves further detailed study. keywords: streptomyces; mrsa; mangrove; bioinformatics; genome received: 5th september 2018 accepted: 20th september 2018 published online: 10th october 2018 *correspondence to: learn-han lee, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; lee.learn.han@monash.edu; leelearnhan@ yahoo.com; bey-hing goh, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; goh.bey.hing@monash.edu citation: ser hl, chan kg, tan ws, et al. complete genome of mangrove-derived anti-mrsa streptomycete, streptomyces pluripotens musc 135t. prog micobes mol biol 2018; 1(1): a0000004. short introduction members of genus streptomyces are known to produce various types of compounds with bioactivities including anticancer, antioxidant, antifungal and antibacterial properties[1-7]. the unique life cycle of streptomycetes have enhanced their ability to persist in nature and to survive in harsh environmental conditions[8-13]. streptomyces pluripotens musc 135t was firstly isolated as copyright 2018 by ser hl et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) 2 novel strain from mangrove forest in peninsular malaysia during a screening program for antibiotic producers[14,15]. this strain exhibited broad spectrum bacteriocin against several pathogens including methicillin-resistant staphylococcus aureus (mrsa) strain atcc baa-44, salmonella typhi atcc 19430t and aeromonas hydrophila atcc 7966t. in order to gain insights on its bioactive potential, strain musc 135t was subjected to complete genome sequencing and annotation to identify presence of biosynthetic gene clusters. data description streptomyces pluripotens musc 135t was isolated from tanjung lumpur mangrove forest located in the city of kuantan, state of pahang, in december of the year 2012[14,15]. purified cultures were maintained on isp medium 2 slants at room temperature for short-term storage and as glycerol suspensions (20%, v/v) at −80°c for long-term storage[16]. the general features of the strain are summarized in table 1[17]. the strain is available from culture collection centers under accession number of mccc 1k00252t = dsm 42140t. table 1: general features of streptomyces pluripotens musc 135t and migs mandatory information. property description classification domain bacteria phylum actinobacteria class actinobacteria order actinomycetales family streptomycetaceae genus streptomyces species pluripotens strain: musc 135t gram stain positive cell shape branched mycelia motility dispersion of spores sporulation yes temperature range 20–40°c optimum temperature 28–32°c ph range; optimum 5.0–9.0; 5.0–8.0 carbon source varied oxygen requirement aerobic pathogenicity non-pathogenic geographic location tanjung lumpur, pahang, malaysia latitude 3° 48’ 3.2”n longitude 103° 20’ 22.7”e altitude 0 above sea level the genomic dna of musc 135t was extracted with wizard® genomic dna purification kit (promega) prior to cleanup process using ampure® pb beads. dna quality was examined using nanodrop spectrophotometer (thermo scientific, waltham, ma, usa) and a qubit version 2.0 fluorometer (life technologies, carlsbad, ca, usa)[18]. smrtbell dna libraries (pacific biosciences) were constructed according to the pacbio standard protocol with the bluepippin size-selection system (sage science). whole genome sequencing of musc 135t was performed with pacbio rsii sequencing technology using p6-c4 chemistry, yielding output data with an average genome coverage of 217.22-fold (table 2). table 2: genome statistics of streptomyces pluripotens musc 135t sequencing platform pacbio rsii assembly hgap3 number of replicon 1 accession number cp021080 genome size (bp) 7,346,075 g + c content % 69.9 protein coding genes 6,404 trna 69 rrna 6, 6, 6 (5s, 16s, 23s) upon sequencing, the raw reads were assembled using hgap3. de novo assembly of the insert reads was performed with the hierarchical genome assembly process (hgap) algorithm. the genome sequence of musc 135t was assembled into a single gc-rich (69.9 %) contig with genome size of 7,346,075 bp. rrna and trna predictions were performed using aragorn and rnammer, respectively[19,20]. using these tools, a total of 77 trna genes and 18 rrna operons was predicted in musc 135t genome. gene prediction was conducted using prodigal (version 1.20), which 6,404 open reading frames (orfs) were predicted in musc 135t genome[21]. the whole genome project of musc 135t was deposited at ddbj/ embl/genbank under accession number cp021080. the assembly was uploaded for annotation to rapid annotation using subsystem technology (rast)[22]. from rast annotation system, most of the genes in musc 135t were involved in amino acids and derivatives metabolism, followed by carbohydrates metabolism and protein metabolism subsystems (figure 1). interestingly, one of the genes belonging to virulence, disease and defense subsystem was predicted to encode for colicin e2 tolerance protein cbrc-like protein. the detection of this gene suggests ability of the strain to survive against action of dna endonuclease, colicin e2 and persist in the environment[23,24]. in addition to that, a webbased bacteriocin mining tool, bagel identified eights gene clusters associated with bacteriocin production[25]. among these gene clusters, one of them was found to be responsible for production of linear azol(in)e-containing peptides (laps). the members of laps family, such as goadsporin and plantazolicin have previously been detected from microbes have shown potent antibacterial activities against pathogens including bacillus anthracis and mrsa[26-28]. in conclusion, we report the complete whole genome of streptomyces pluripotens musc 135t... a sequence of s. pluripotens musc 135t. the availability of its genome sequence has suggested potential exploitation of the strain for potentially useful compounds, particularly against pathogens such as mrsa and certainly deserves further detailed study. acknowledgement this work was supported by industry grant (biotek abadi vote no. gba-808813), mosti escience funds (project no. 06-02-10-sf0300) awarded to l.-h.l. and a university of malaya for high impact research grant (um-mohe hir nature microbiome grant no. h50001-a000027 and no. a000001-50001) and ppp grant (pg090-2015b) awarded to k.-g.c. conflict of interest the authours declared that there is no conflict of interest. reference 1. berdy j. bioactive microbial metabolites. j antibiotics 2005; 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2(3): 207–220. doi: 10.1021/ acsinfecdis.5b00115. whole genome of streptomyces pluripotens musc 135t... pmmb 2023, 6, 1; a0000334. doi: 10.36877/pmmb.0000334 http://journals.hh-publisher.com/index.php/pmmb review article vibrio parahaemolyticus: exploring its incidence in malaysia and the potential of streptomyces sp. as an antivibrio agent ke-yan loo1, loh teng-hern tan1,2, jodi woan-fei law1,3, priyia pusparajah4, sunny hei wong5,6, kok-gan chan1,7,8, learn-han lee1,9,10*, vengadesh letchumanan1,10* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia; ke.loo@monash.edu (k-yl) 2innovative bioprospection development research group (inbiod), clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) 3next-generation precision medicine and therapeutics research group (nmet), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia; jodi.law1@monash.edu (jw-fl) 4medical health and translational research group (mhtr), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, 47500, selangor darul ehsan, malaysia; priyia.pusparajah@monash.edu (pp) 5lee kong chian school of medicine, nanyang technological university, singapore 308232, singapore; sunny.wong@ntu.edu.sg (shw) 6state key laboratory of digestive disease, department of medicine and therapeutics, the chinese university of hong kong, hong kong sar, china 7institute of biological sciences, faculty of science, university of malaya, 50603, kuala lumpur, malaysia; kokgan@um.edu.my (k-gc) 8international genome centre, jiangsu university, zhenjiang, china 9sunway microbiomics centre, school of medical and life sciences, sunway university, sunway city 47500, malaysia 10pathogen resistome virulome and diagnostic research group (pathrid), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia *corresponding author: vengadesh letchumanan and learn-han lee, pathogen resistome virulome and diagnostic research group (pathrid), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia; vengadesh.letchumanan1@monash.edu (vl); lee.learn.han@monash.edu (lhl) received: 13 april 2023; received in revised form: 28 may 2023; accepted: 6 june 2023; available online: 10 june 2023 pmmb 2023, 6, 1; a0000334 2 of 17 abstract: as the world recovers from the covid-19 pandemic, concern remains for future potential outbreaks because of the persisting effects of climate change, including the proliferation of infectious diseases. the frequent isolation of vibrio parahaemolyticus in the surrounding environment is of concern as it can cause infections in marine animals and transmitted to humans. v. parahaemolyticus is the leading cause of foodborne gastroenteritis worldwide. malaysia is one of the top seafood consumers and this places us at a higher risk of exposure to v. parahaemolyticus infections. over the years, this foodborne pathogen has been isolated from various sources in malaysia, mainly from seafood such as shellfish, shrimps, and fish. to make matters worse, there has been an emergence of antibiotic-resistant v. parahaemolyticus worldwide, which is attributed to the uncontrolled use of antibiotics in aquaculture to prevent and treat vibriosis. therefore, it is vital to utilize alternatives such as probiotics to control v. parahaemolyticus to prevent further propagation of antibioticresistant strains of the bacteria. a potential candidate for probiotics is streptomyces sp., a class of filamentous, gram-positive bacteria that produce a variety of bioactive compounds during their life cycle, which can be useful in drug discovery. the bioactive compounds produced by streptomyces sp. have been proven to have microbiota-modulating and stimulatory effects on the host, enhancing immunity and providing protective effects against v. parahaemolyticus infections. with the application of streptomyces sp. as probiotics in aquaculture, the efficacy of the available antibiotics can be preserved, and the further spread of antibiotic resistance in the environment can be reduced. keywords: vibrio parahaemolyticus; anti-vibrio; streptomyces; prevalence; probiotics 1. introduction the fear of an endemic or pandemic will always linger since we are slowly recovering from the covid-19 pandemic. this zoonotic-transmitted disease was discovered in china and moved rapidly from person to person [1, 2]. as we scrambled to find the treatment for this pneumonia-like infection, the virus continued to travel to other countries worldwide [3, 4]. covid-19 infected millions, causing a rise in death cases and paralysis of the healthcare system [3, 5-7]. although vaccination programs were initiated, infection cases are still being reported due to emerging new variants of concern (voc) [8-14]. we have yet to be entirely freed from the covid-19 pandemic and have already started addressing another primary public concern; climate change. climate change is a pressing global challenge impacting the environment, ecosystems, and human health [15, 16]. among the many consequences of climate change, the proliferation of infectious diseases is a growing concern [17]. recently, the rise of vibrio parahaemolyticus infections in animals and humans has gained attention [18-20]. the situation is further worsened by the spillover of antibiotic-resistant v. parahaemolyticus to animals and humans. pmmb 2023, 6, 1; a0000334 3 of 17 vibrio parahaemolyticus is a halophilic, gram-negative bacteria that naturally reside in bodies of water such as estuaries, rivers, and oceans [21-23]. as they share a natural habitat with aquatic animals, v. parahaemolyticus can infect these animals and cause a disease known as vibriosis. symptoms of vibriosis in marine animals can manifest as skin ulcerations, exophthalmia, necrosis of the appendages, and death in severe cases [24]. vibriosis has significantly impacted the aquaculture industry as it causes high mortality rates in farmed marine life, thus reducing the production of farmed marine fish and shrimp, leading to severe economic losses [24-26]. most notably, v. parahaemolyticus is responsible for acute hepatopancreatic necrosis disease (ahpnd) in shrimps, which causes death in the early stages of the life of shrimps [27, 28]. outbreaks of ahpnd have significantly reduced the production of shrimps worldwide; for example, in thailand, there was a decrease in approximately 30% of shrimp production in 2013 compared to 2012 [29]. it is estimated that ahpnd costs the shrimp aquaculture industry about usd 1 billion annually [30]. moreover, these pathogenic bacteria can be transmitted to humans via consumption or exposure to contaminated seafood, resulting in gastroenteritis in humans which manifests as fevers, nausea, stomach cramps, diarrhea, and vomiting [31-34]. mild cases of gastroenteritis caused by v. parahaemolyticus is generally self-limiting, with rehydration being the focus for recovery. with severe cases of gastroenteritis due to the foodborne pathogen, antimicrobial therapy is guided by comparable clinical syndromes caused by other vibrio species, such as vibrio cholerae. thus, the antibiotic of choice is doxycycline, fluoroquinolones, and macrolides [35]. however, due to the uncontrolled use of antimicrobials for prophylaxis and management of diseases in aquaculture farms [36, 37], the environmental pressure from antimicrobial residues enables the emergence of antibiotic-resistant strains of v. parahaemolyticus. the pathogen has since developed antimicrobial resistance against a myriad of antibiotics such as penicillin, cephalosporins, aminoglycosides, tetracyclines, macrolides, quinolones, fluoroquinolones, sulfonamides, and carbapenems [38-45]. the rapid emergence of antimicrobial resistance in v. parahaemolyticus decreases the efficacy of antibiotics in treating its infections. thus, to better manage v. parahaemolyticus infections, alternatives to antibiotics need to be explored to prevent the further spread of antibiotic resistance genes within the environment. recently, probiotics have gained popularity in their application in managing v. parahaemolyticus infections [46-49]. probiotics are live microorganisms that can benefit the host when administered adequately [50, 51]. they modulate the host microbiota to promote growth and increase disease resistance [48, 52]. studies have also reported that probiotics can have many functionalities in aquaculture. for instance, they can act as growth promoters, stimulate the production of inhibitory compounds, improve nutrient digestion in the host, strengthen immune response, and improve water quality [53, 54]. the microorganisms which are commonly known to be probiotics are lactobacillus acidophilus, lactobacillus casei, bacillus sp., bifidobacterium bifidum, lactococcus lactis, and saccharomyces cerevisiae [47, 49]. however, recent studies point to streptomyces sp. as a potential probiotic to be used in aquaculture due to its ability to produce many secondary metabolites, which can elicit pmmb 2023, 6, 1; a0000334 4 of 17 beneficial effects in the host [55-57]. they are potential probiotics for the prophylaxis and treatment of vibriosis in aquatic animals. continuous monitoring and surveillance on the prevalence of v. parahaemolyticus remain crucial to ensure the microbial load in the seafood supplied to consumers is safe for consumption. in addition, consistent efforts to monitor the prevalence of v. parahaemolyticus in our surrounding environment can prevent gastroenteritis outbreaks and maintain public health. the exploration of alternatives to antibiotics is also essential to preserve the efficacy of the currently available antibiotics and prevent the spread of antibiotic-resistance genes. probiotics, such as streptomyces sp., help to prevent and manage v. parahaemolyticus infections in farmed marine animals, reducing the probability of disease transmission from seafood to humans. in addition, probiotics can promote the growth of farmed marine animals and produce safe-to-consume seafood products for the general public. this review aims to provide insight into the prevalence of v. parahaemolyticus in malaysia and the anti-vibrio properties of streptomyces sp., which make them potential agents in managing v. parahaemolyticus infections. 2. incidence of vibrio parahaemolyticus in malaysia seafood is a nutrient-rich source of food that is vital in maintaining a healthy, balanced diet as they provide protein, unsaturated fatty acids, minerals, and vitamins [58]. the consumption of seafood has also been associated with reduced cholesterol levels and the prevention of cardiovascular diseases due to omega-3 fatty acids in seafood [59]. in malaysia, seafood consumption has been consistently on an upwards trend. for instance, seafood consumption increased from 42.7kg per capita in 2020 to 43.38kg per capita in 2021 [60]. the high seafood consumption in malaysia puts malaysians at risk of foodborne diseases from seafood, given there is an increased risk of transmission and/or exposure to v. parahaemolyticus. moreover, the foodborne pathogen has been frequently isolated from various sources such as seafood and the water environments in malaysia [39, 43, 44, 61-63] (table 1). v. parahaemolyticus are halophiles gram-negative bacterium that favor warm, brackish waters as their natural habitat [64, 65]. it has been reported that v. parahaemolyticus thrive in warmer seawater temperatures where the expression of virulence factors is upregulated, thus promoting the propagation of pathogenic bacteria [66, 67]. the tropical climate in malaysia makes it hot and humid year-round, thus resulting in higher temperatures in bodies of water around the country. this creates a favorable environment for v. parahaemolyticus to grow and increases the risk of transmission to marine animals and humans. therefore, studies have been done to determine the prevalence of these foodborne pathogens in malaysia. a quick search was performed across three databases, embase, medline, scopus, and google scholar, using the keywords: “prevalence or incidence or occurrence and vibrio parahaemolyticus and malaysia” to determine relevant original articles published between 2005 and 2023. results from the search found that v. parahaemolyticus are most commonly isolated from seafood such as shellfish [39, 41, 43, 62, 68pmmb 2023, 6, 1; a0000334 5 of 17 76], shrimps [42, 63, 68-70, 77-81], squids [68, 69, 72], fishes [40, 44, 61, 69, 72, 75, 82-90], and sea cucumber [91]. the pathogen has also been isolated from vegetables [92, 93] and environmental sources such as seawater [44, 94], coastal waters [73, 74, 95], and water from shrimp farms [63, 77]. in a study done to determine the prevalence of v. parahaemolyticus in shellfish in selangor, malaysia, 450 shellfish samples were screened, and based on colony morphology, all these samples were positive for vibrio sp. upon further confirmation via the toxr gene polymerase chain reaction assay [96], 44.4% (200/450) of the samples were confirmed to have v. parahaemolyticus [39]. tan et al. also reported 61/75 shellfish samples were positive for v. parahaemolyticus [68], indicating a high prevalence of the foodborne pathogen in shellfish. moreover, a study done in kuala terengganu, malaysia, where 80 shellfish samples, including mussels, carpet clams, cockles, and scallops, were obtained, found that more than half (57.5%, 46/80) of the samples were contaminated with v. parahaemolyticus [71]. the high prevalence of v. parahaemolyticus in shellfish can be attributed to their natural habitat and filter feeding. shellfish typically reside on the sea floors, and due to their filter feeding, they can garner a higher concentration of v. parahaemolyticus up to 100-fold higher than their surrounding waters [97]. in shrimps, letchumanan et al. reported a prevalence of 57.8% (185/320) for v. parahaemolyticus isolates found in samples obtained from retail supermarkets [42]. in comparison, tan et al. reported 88.57% (31/35) of shrimp samples were positive for the marine pathogen [68]. a prevalence study on cultured shrimps reported that over half (55%) of the 225 samples tested positive for v. parahaemolyticus, with the rest testing positive for other species in the vibrio family [41]. the high prevalence of v. parahaemolyticus is of concern as these bacteria can cause ahpnd in the shrimps, which primarily targets the hepatopancreas of the shrimp in the early stages of life, resulting in early mortality [98]. outbreaks of ahpnd in malaysia have caused a reduced production of shrimp and cost the industry a loss of approximately usd 0.49 billion from 2011 to 2014 [99]. v. parahaemolyticus has also been isolated from fish; for example, the pathogen has been isolated from 116 of 130 short mackerel samples, indicating that 89.2% of the fish samples were contaminated with bacteria [61]. meanwhile, noorlis et al. reported that 49 isolates of v. parahaemolyticus were found in 300 freshwater fish purchased from retail levels [82]. in a separate study, 120 finfish samples were tested, and the results show that v. parahaemolyticus was present in 48.33% of the samples [84]. researchers also studied the occurrence of v. parahaemolyticus in cultured groupers in peninsular malaysia, whereby they found that 25% of the 270 grouper samples were contaminated with v. parahaemolyticus. the foodborne pathogen can also be isolated from environmental sources such as seawater; for instance, 50 water samples, including river water, seawater, and water from a waterfall in kelantan were tested and 25 of the samples were positive for v. parahaemolyticus [100]. besides, 21 v. parahaemolyticus isolates were also detected from 21 coastal seawater samples from three beaches in peninsular malaysia [94]. the incidence of v. parahaemolyticus is unavoidable in aquatic environments, hence marine life such as fishes and shrimps can accumulate v. parahaemolyticus before being harvested. the high pmmb 2023, 6, 1; a0000334 6 of 17 prevalence of v. parahaemolyticus detected in malaysia shows the need for constant surveillance of the microbial load of this foodborne pathogen in seafood and the environment. this is to ensure the seafood supplied to consumers is safe for consumption, and gastroenteritis outbreaks can be avoided. moreover, by monitoring the microbial load of v. parahaemolyticus in the aquaculture system, vibriosis outbreaks can be detected earlier to prevent high mortality, reduce economic losses and increase seafood production to meet supply demands. table 1. sources of v. parahaemolyticus in malaysia. source references aquatic animals blood clams [68, 69] fish [40, 44, 69, 72, 75, 82-90] oyster [69] prawn [69] sea cucumber [91] shellfish [39, 41, 43, 62, 70-76] short mackerels [61] shrimps [42, 63, 68-70, 77-81] squids [68, 69, 72] surf clams [68] vegetables cabbage [92, 93] carrot [92, 93] cucumber [92, 93] four-winged bean [92] indian pennywort [92] japanese parsley [92] lettuce [92, 93] long bean [92] sweet potato [92] tomato [92, 93] wild cosmos [92] environmental river water [100] sea water [44, 73, 74, 94, 95, 100] shrimp farm water [63, 77] pmmb 2023, 6, 1; a0000334 7 of 17 3. harnessing the anti-vibrio properties of streptomyces sp. there is a dire need for antibiotic alternatives because of the rapid emergence of antibiotic-resistant genes and antibiotic-resistant v. parahaemolyticus in the environment [38, 101, 102]. in aquaculture, the uncontrolled use of antibiotics has further accelerated the development of antibiotic resistance within these bacteria, thus rendering the effects of antimicrobial agents in these systems useless [36]. henceforth, researchers are looking for alternatives to antibiotics to be used in the aquaculture industry to better combat diseases caused by v. parahaemolyticus. by finding alternatives to antibiotics, the efficacy of the currently available antibiotics can be preserved, and they can remain effective in treating infections caused by the pathogen. in recent years, streptomyces sp. has been the microorganism of interest for aquaculture use as a probiotic [48, 49, 54, 103]. in 1943, streptomyces sp. was first discovered as the source of the antibiotic streptomycin by waksman et al. [104], and to date, over 700 known species have been isolated from the environment [105, 106]. streptomyces, belonging to the actinobacteria class [107, 108], is a group of complex filamentous, sporulating, gram-positive bacteria which resemble fungi in their morphology [109-112]. they are highly abundant in soils and are also found in the sediments of marine environments, as they play a vital role in the ecosystem due to their broad range of metabolic processes [113-115]. their ability to produce a variety of secondary metabolites and bioactive compounds makes them essential microorganisms in drug discovery [116-123]. the metabolites are typically produced during the life cycle of streptomyces sp. driven by environmental factors [124], and over 10,000 bioactive compounds have been retrieved from these species [125]. the bioactive compounds produce possess a multitude of functionalities, including antibacterial [126-128], anticancer [129-131], antioxidant [132-134], and antifungal properties [135-137]. moreover, the potential of cultured streptomyces in producing bioactive secondary metabolites is yet to be fully realized, as environmental factors such as ph, temperature, and incubation times can affect metabolite production [138, 139]. studies have shown that cocultivation with other bacterium produces secondary metabolites that would otherwise not be found when culturing streptomyces under standard lab conditions [140-142]. furthermore, genome mining studies on streptomyces have shown that they possess biosynthetic gene clusters that are not known to produce any secondary metabolites, and they are also commonly called cryptic clusters. nonetheless, these cryptic biosynthetic clusters may not be cryptic in a second species of streptomyces [143]. therefore, it is suggested that a good fraction of undiscovered secondary metabolites may potentially become therapeutic agents [144]. this makes streptomyces sp. and its bioactive compounds good candidates in drug discovery, as its potential has yet to be fully exploited. from the plethora of bioactive compounds produced by streptomyces, several have been identified as a source of antibacterial agents, specifically against vibrio species [145]. this group of bacteria has been identified as potential candidates for probiotics to be used in aquaculture [48]. marine streptomyces sp. isolated by yang et al. demonstrated strong pmmb 2023, 6, 1; a0000334 8 of 17 antagonistic activity towards pathogenic v. parahaemolyticus. by using the agar diffusion method, they reported clear inhibition zones of 33 mm and 15 mm in diameter after 96 hours of incubation in both solid and liquid cultures of the selected streptomyces sp. strain, s073, against pathogenic v. parahaemolyticus [56]. the antagonistic activity of the chosen streptomyces sp. could be attributed to its production of carboxylate-type siderophore to create a lethal iron-limiting condition for v. parahaemolyticus isolates [56]. in a follow-up study with the same strain of streptomyces sp., the researchers identified dibutyl phthalate (dbp) as another compound that elicits inhibitory effects against v. parahaemolyticus. it was concluded that the antagonistic effects of s073 were dependent on the synergistic effects of dbp-mediated antagonism and siderophore-governed iron competition with v. parahaemolyticus [146]. the vibriocidal activity of streptomyces sp. was also discovered when an inhibition zone of 20mm was observed during the agar well diffusion method when tested against pathogenic v. parahaemolyticus isolated from fish [147]. in india, streptomyces rubrolavendulae m56 isolated from the sediments of the bay of bengal also showed antagonistic effects towards v. parahaemolyticus. co-culture experiments with medium-sized biogranules produced by m56 and v. harveyi, v. parahaemolyticus, v. alginolyticus, and v. fluvialis showed a gradual decline in viable vibrio count. upon day three of the co-culture experiment, no viable vibrios were detected in the water tanks. in addition, m56 was reported to be non-pathogenic to post larvae shrimp under experimental setup, making them a safe option for use in aquaculture [55]. it is suggested that the enzymes produced by m56, including protease, amylase, lipase, dnase, and phosphatase could inhibit the growth of vibrio sp. in addition, competition for colonization between m56 and vibrio sp. in the natural system also reduces the vibrios available to invade the postlarvae [55]. a separate study reported that supplementation of streptomyces sp., rl8, as a probiotic stimulated the growth of bacteriovorax, a group of predator bacteria [57]. they can invade the periplasmic space of gram-negative bacteria such as v. parahaemolyticus, and alter the vibrio cell wall to consume their cytoplasmic content, resulting in cell lysis [148]. rl8 also stimulated the antimicrobial producers, which protected the white shrimps by preventing them from v. parahaemolyticus infections [57]. when rl8 was used with other probiotics, such as bacillus sp., higher bacterial diversity and significant stimulation of bacterivorax population were also reported [57]. this indicates that rl8 is a potential candidate to be used in synergy with other commonly known probiotics to produce beneficial effects on the host. given the current evidence of the anti-vibrio properties of streptomyces sp. in animal model studies, streptomyces sp. possesses the excellent potential to become probiotics that can be utilized in aquaculture to prevent and control vibrio infections. in addition, streptomyces sp. can stimulate diversity in the gut microbiome of aquatic animals such as shrimp, leading to increased antimicrobial producers within the gut, thereby providing a protective effect against infections [57]. furthermore, streptoymces sp. has also been shown to enhance the growth of red swordtail fish via the production of indoleacetic acid, a growthpromoting hormone [149]. supplementation of streptomyces sp. in oral shrimp feed also promoted the growth rates of post-larval shrimp [150]. moreover, in aquaculture systems, the pmmb 2023, 6, 1; a0000334 9 of 17 accumulation of organic waste, such as ammonia and nitrite can cause water quality problems, making the farmed animals susceptible to diseases and infections [151]. studies have shown that the administration of streptomyces sp. within these culture systems increased the population of heterotrophic bacteria, thereby increasing the decomposition rate of organic waste, and the ammonia levels were reduced, ultimately improving the water quality [150, 152, 153]. the multi-faceted beneficial qualities of streptomyces sp., including its capabilities in vibrio inhibition, gut modulation, water quality improvement, and growth promotion, can be harnessed to develop probiotics that can be used in aquaculture systems. the synergistic effects of streptomyces sp. can protect populations of farmed animals within culture systems from infections, thereby preserving the stability and sustainability of the aquaculture industry. 4. conclusion in conclusion, the prevalence of v. parahaemolyticus in malaysia's seafood and the environment remains of concern as the pathogen is frequently isolated from these sources. however, to cause disease in humans, the v. parahaemolyticus isolates would have to express the virulence genes such as the thermostable direct hemolysin (tdh) gene and the tdh-related hemolysin (trh) gene to cause symptoms of gastroenteritis [154]. the isolates must express the photorhabdus insect-related (pir) toxin genes, pira and pirb genes to cause ahpnd in shrimps [63, 155]. nevertheless, these foodborne pathogens are still frequently detected in our surroundings, thus increasing the probability of disease transmission, which could have detrimental effects on the aquaculture industry and the country’s public health. in addition, the rapid emergence of antibiotic resistance among v. parahaemolyticus isolates has reduced the efficacy of the available antibiotics. the process of developing a new antimicrobial agent is lengthy and time-consuming. thus, alternatives such as probiotics are being explored to replace antibiotics to prevent vibriosis in aquatic animals. streptomyces sp. has been a focal point of drug discovery in aquaculture, as it produces many bioactive compounds during its life cycle that could be useful in modulating the microbiota of farmed marine life. the various bioactive compounds produced can have antagonistic or vibriocidal effects on v. parahaemolyticus, which can be useful in deploying them in aquaculture. streptomyces sp. has been found to stimulate growth and provide protective effects in shrimps. however, further studies and trials need to be done to determine the most effective dosage for administration to other species of aquatic animals. currently, animal models of the effects of streptomyces sp. as a probiotic have been producing promising results. thus researchers are optimistic that these beneficial effects can be proven in clinical trials so that v. parahaemolyticus infections in humans can also be prevented with the application of streptomyces sp. author contributions: k-yl conducted the literature search, critical data analysis, and manuscript wiring. lt-ht, jw-fl, pp, shw, kcg, l-hl and vl provided proofreading, comprehensive editing and technical support. vl and l-hl conceptualized and founded this writing project. all authors have read and agreed to the published version of the manuscript. funding: this study was supported by the jeffrey cheah school of medicine and health sciences strategic grant 2021 (vote number: stg-000051). pmmb 2023, 6, 1; 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305(1): 32-41. 154. wang h, tang x, su y-c, et al. characterization of clinical vibrio parahaemolyticus strains in zhoushan, china, from 2013 to 2014. plos one 2017; 12(7): e0180335. 155. lin sj, huang jy, le pt, et al. expression of the ahpnd toxins pira(vp) and pirb(vp) is regulated by components of the vibrio parahaemolyticus quorum sensing (qs) system. int j mol sci 2022; 23(5). author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2022, 5, 1; a0000323. doi: a10.36877/pmmb.0000323 http://journals.hh-publisher.com/index.php/pmmb review article anti-sars-cov-2 biotherapeutics and chemotherapeutics: an insight into product specifications and marketing dynamics vijay kotra1*, dinakar mallem2, anil kumar kanuri3, mahesh reddy burra3, shaik nyamathullah3*, looi shu ying1, khairul anwar husain1, ravi varala4, madhavedi sudhakar5, khang wen goh6, toong hai sam7*, long chiau ming8 article history 1faculty of pharmacy, quest international university, ipoh, perak, malaysia; shuying.looi@qiu.edu.my (lsy); khairulanwar.husain@qiu.edu.my (kah) 2department of forensic medicine, apollo institute of medical sciences & research, hyderabad; mallemdinakar@gmail.com (dm) 3department of pharmaceutical technology, faculty of pharmacy, universiti malaya, kuala lumpur, malaysia; anilkumarkanuri88@gmail.com (akk), burra.maheshreddy1995@gmail.com (mrb), 4scrips pharma, hyderabad, telangana, india; ravivarala@gmail.com (rv) 5department of management, kshatriya college of engineering, nizamabad, telangana, india; reachfirst@gmail.com (ms) 6faculty of data science and information technology, inti international university, nilai, malaysia; khangwen.goh@newinti.edu.my (kwg) 7faculty of business and communications, inti international university, nilai, malaysia 8school of medical and life sciences, sunway university, sunway city, malaysia; chiaumingl@sunway.edu.my (lcm) *corresponding authors: vijay kotra; faculty of pharmacy, quest international university, ipoh, perak, malaysia; vijay.kotra@qiu.edu.my (vk); shaik nyamathullah; faculty of pharmacy, universiti malaya, kuala lumpur, malaysia; nyamathullask@um.edu.my (sn); toong hai sam; faculty of business and communications, inti international university, nilai, negeri sembilan, malaysia; toonghai.sam@newinti.edu.my (ths) received: 30 november 2022; received in revised form: 23 december 2022; accepted: 27 december 2022; available online: 30 december 2022 abstract: chemotherapeutics and biotherapeutics agents have been explored as a potential treatment for covid-19. this study was aimed to elucidate latest update related to biotherapeutics and chemotherapeutics against covid-19 and its variants as well as the product specifications and marketing dynamics of its pharmacotherapies. there are several chemotherapeutics that have been studied for the treatment of covid-19, including paxlovid (nirmatrelvir & ritonavir) fixed dose combination, nirmatrelvir and ritonavir fixed dose combination, remdesivir (vekulary/covifor) fixed dose combination, chloroquine and hydroxychloroquine, molnupiravir, favipiravir, infliximab (remsima®) fixed dose mailto:shuying.looi@qiu.edu.my mailto:mallemdinakar@gmail.com mailto:ravivarala@gmail.com mailto:reachfirst@gmail.com mailto:vijay.kotra@qiu.edu.my mailto:nyamathullask@um.edu.my pmmb 2022, 5, 1; a0000323 2 of 25 combination tocilizumab (actimera), casirivimab+imdevimab (ronapreve) fixed dose combination, ivermectin, methylprednisole. these drugs have been repurposed for use in covid-19 due to their antiviral properties and ability to reduce inflammation. the covid19 death cases have significantly reduced because of tested efficacy of advanced biotherapeutics and chemotherapeutics. as for marketing dynamics, the demand for chemotherapeutics for the treatment of covid-19 has increased significantly since the outbreak of the pandemic. hence, the prices of anti-viral and monoclonal antibodies in top covid-19 affected countries is also presented here. considering the molecular interactions, therapeutic activity, efficacy, and adverse effects, the usfda and the who recommended the aforementioned drugs. the chances of approval seems favouring biotherapeutics as these have the best characteristics among the chemotherapeutics. overall, this review contributes to the scientific understanding of the covid-19. this can help to inform future research and development efforts and contribute to the overall advancement of knowledge in the field of medicine. keywords: paxlovid ; nirmatrelvir and ritonavir; remdesivir; chloroquine and hydroxychloroquine; molnupiravir; favipiravir; infliximab; ivermectin 1. introduction severe acute respiratory syndrome coronavirus 2 (sars‑cov‑2) or commonly known as covid-19 has claimed millions lives worldwide [1, 2]. the coronavirus causing covid-19 mutated over time, resulting in genetic variation in the population of revolving viral strains across the host. the continuously changing mutations will adversely affect human health and disease progression. due to this, the drug development for covid-19 is challenging for scientists and industries worldwide; therefore, medical professionals serve patients with prequalified treatment protocols by using existing therapeutics, including convalescent plasma treatment rather than developing new drugs [3, 4]. with the support of a professional team of methodologists, the who ‘guideline development group’ (gdg) meets regularly to prepare for and review evidence summaries from international clinical trials. the gdg then develops clinical practice recommendations for the use of therapeutics to treat patients with covid-19 of any disease severity. to improve patient outcomes, it is crucial that who-recommended medications are promptly adapted and integrated into the covid-19 clinical care pathways at both the national and local levels [5-8]. in december 2021, the united states food and drug administration (us fda) approved the ‘emergency use authorization’ (eua) of the antiviral chemotherapeutic drug paxlovid, along with a few other chemotherapeutics to incorporate in standardized treatment protocols for covid-19 [9]. the significant concerns raised with chemotherapeutics are due to the potential reactivity to synthetic chemicals. on the other hand, biotherapeutics are made pmmb 2022, 5, 1; a0000323 3 of 25 up of natural agents, which are more effective and exhibit almost no or lesser side effects than chemotherapeutics [9]. tocilizumab, a monoclonal antibody that inhibits the interleukin6 (il-6) receptor activity, was the first biologic agent to be largely evaluated in covid-19 patients; and was also approved by the who for prequalified treatments for n-covid-19 [10]. two biosimilars are under clinical trials; one is celltrion healthcare's infliximab biosimilar, remsima®, and cinnagen’s biosimilar interferon (ifn) β-1a (recigen®). recigen is an anti-inflammatory drug prescribed for patients with multiple sclerosis. recigen® was part of the first published study to evaluate the therapeutic efficacy of interferon (ifn) molecules in patients with covid-19 [11, 12] . before analyzing the findings of current research work, it is essential to understand the history, structure and morphology of the novel coronavirus (severe acute respiratory syndrome coronavirus 2 (sars-cov-2), in brief. investigations associated with the structure and morphology of the novel coronavirus revealed that the viral strain belongs to the oldest group of viruses dates back to the late 1920s rather than a new class of virus. however, there was no evidence of coronaviruses that infects humans until the discovery of human coronaviruses in the late 1960s. coronaviruses belong to a group of rna viruses that infects mammals; they cause respiratory tract infections that may last from mild to lethal. mild illnesses in humans include the common cold and fever [13, 14]. in contrast, lethal varieties are sars, middle east respiratory syndrome (mers), and an ongoing pandemic covid-19, which can infect the lower respiratory tract and lungs and eventually creates respiratory distress and kill human beings. this virus can commonly infect diarrhea in pigs and cows; hepatitis and encephalomyelitis in mice and rats [15].meanwhile, the emergence of monkeypox also caused a menace to the public health which casualty is on the rise [16]. various drugs have been repurposed for use in covid-19 due to their antiviral properties and ability to reduce inflammation. the analysis revealed that the demand for covid-19 therapeutics is growing as the covid-19 pandemic has not subsided yet [2]. pandemic is rising continuously in some or other corners of the world despite the availability of multiple varieties of vaccines [17-20]. however, the death cases are significantly reduced because of release of advanced biotherapeutics and chemotherapeutics into the market to treat the disease effectively this study was aimed to elucidate latest update related to biotherapeutics and chemotherapeutics against covid-19 and its variants as well as the product specifications and marketing dynamics of its pharmacotherapies. 2. literature search we performed a comprehensive review of literature from all peer-reviewed journals published since january 2020 when covid-19 first made the headline. we reviewed the articles from different journal databases like nature, lancet, pubmed, scopus, science direct, bentham science, doaj, clarivate web of science, and many more, used google translator to translate the foreign language into english. the keywords used for the search include, "covid-19", "biosimilars", "biologics", "covid-19 treatment", "adverse effects of covid19 drugs" etc. pmmb 2022, 5, 1; a0000323 4 of 25 3. classification coronaviruses constitute the subfamily orthocoronavirinae, in the family corona viridae with order nidovirales and realm riboviria. they are enveloped viruses with a positive-sense single-stranded rna genome and a nucleocapsid of helical symmetry. the genome size of coronaviruses ranges from nearly 26 to 32 kb, one of the largest among rna viruses. they have characteristic club-shaped spikes that project from their surface, which in electron micrographs create an image reminiscent of the solar corona, from which their name derives. they are further classified into four genera: α-coronavirus, β-coronavirus, γ-coronavirus and δ-coronavirus. αand β-coronaviruses infect mammals, while γ and δ coronaviruses primarily infect birds [21]. 3.1. structure & morphology coronavirus is structurally large and morphologically spherical particles with unique surface projections. the size of each virus particle varies from 50 to 120 nm in diameter. the virus is mainly composed of different proteins which make up the structure and function for the maximum survival rates and viral replication, which are the lipid bilayer viral envelope, membrane proteins, and nucleocapsid proteins [22]. 3.2. structure of a covid-19 virus the external envelope of the virus is purely made up of lipid bilayer, which is mainly composed of structural proteins, namely membrane protein (m), envelope protein (e), and functional proteins, namely spike glycoprotein (s). the structural proteins membrane protein (m) and envelope protein (e) form the shape of the viral envelope and maintain a specific size and shape for the identity of the virion. on the other hand, the spike glycoprotein (s) is functionally required for host cell interaction. the nucleocapsid resides inside the envelope, formed by multiple copies of nucleoproteins (n) bound to the genetic material that is the single-stranded genomic rna. most coronavirus strains have spike-like proteins called hemagglutinin esterase (he); these are functional proteins consisting of about 400 amino acids per single peptide chain that helps to interact with host cells and spike glycoproteins [22] . 3.3. viral transmission when covid-19 infected person coughs, sneezes, or communicate without personnel protective equipments such as nose masks, face shield, hand gloves, etc., the tiny particles known as aerosols carry the viral strains into the environment from their nose and mouth. the healthy person who is in contact with the infected person without social & physical distancing and personnel protective equipments is susceptible to becoming infected by covid-19. researchers demonstrated that the viral particles could live in the air for nearly three hours [23]. pmmb 2022, 5, 1; a0000323 5 of 25 3.4. lifecycle the lifecycle of covid-19 is a three-phase process. the first phase of the process initiates with the entry of a viral particle into the host cell through cellular receptors on the host cell, followed by several biosynthetic pathways of the virus in the cytoplasm of the host cell with the help of some cell organelles and enzymes in the host cell favoring the biosynthesis of the foreign viral particle. the final stage of viral replication in the host cell ends with the assembly and exit of the new virions through budding [6, 24]. 3.5. viral entry the infection in the body begins when the virus enters and starts to bind with the host cellular receptors on the cell. the spike glycoprotein (s) located on the virus's surface helps the virus attach the receptors, namely angiotensin-converting enzyme 2 (ace2), to the complementary host cell. after successfully attaching the virus to the ace2 receptor, an enzyme called protease from the host cell cleaves and activates spike glycoprotein (s) attached to the ace2. when the attachment and activation of the spike glycoprotein (s) are completed, the virus crosses the cell wall and enters the host cell through direct fusion or endocytosis [25, 26]. 3.6. biosynthesis upon entry into the host cell's cytoplasm, certain enzymes cleave the virus's membrane and help to release genetic material into it. covid-19 belongs to rna viruses, and the rna of its genome allows it to act like a messenger rna or mrna and helps in the translation into polyproteins, namely pp1a and pp1ab, by the host cell ribosomes. these polyproteins undergo proteolysis, which is a hydrolysis reaction of peptide bonds in which complex protein molecules break down into smaller peptides or individual proteins to yield small proteins [27]. 3.7. replication & transcription the replicase transcriptase complex is an enzyme responsible for the replication and transcription of the viral genome into genomic rna and mrna, respectively, which will enhance the translation process further. the enzyme transcriptase converts the genomic rna into four mrnas, which will be translated into respective proteins for packing and assembly. the subgenomic rna is replicated over time to produce the genomic rna [27]. 3.8. translation the four transcribed spike glycoprotein (s), envelope protein (e), membrane protein (m) and nucleocapsid protein (n) mrnas then translated into proteins by two different pathways. nucleocapsid protein (n) mrna is directly translated into nucleocapsid proteins (n) by attaching with the genomic rna replicated from the virus's genome. the remaining three mrna's translated through endoplasmic reticulum and golgi complex mediated pmmb 2022, 5, 1; a0000323 6 of 25 biosynthesis. during this process, three mrna's go inside er and subsequently with the golgi resulting in the formation of endoplasmic reticulum golgi intermediate complex (ergic). 3.9. assembly and exit assembly is a featured step in the viral life cycle, which is a dynamic process through morphogenetic reactions involving peptide associations between viral genome (n) and core structural proteins. cellular vesicles help to deliver the viral genome (n) into the core structure. this ergic packed with the nucleocapsid proteins (n) is assembled in a vesicle, ready to exit the host cell. the exit of progeny viruses is by means of exocytosis through budding to cross the cell wall once the new virus exits the host cell, which enables viruses to infect other cells in the body [25]. 4. drugs for treatment of covid-19 unfortunately, there is no specific drug available on the market till now to treat covid-19, except prequalified and emergency authorized medications; due to this, scientists are unable to find the efficacy and adverse effects of these drugs used for covid-19 treatment. we can estimate the quality and quantity of use for these drugs as these are within our hands. developing an efficient drug for the treatment of covid-19 is now a global challenge, but unlike many other diseases, it is a severe pandemic and spreads quickly; thus, there is not enough time to spend researching a new drug instead using the existing drugs. the us fda approved a few drugs for emergency use authorization, but one does not know the efficacy of these drugs [28]. developing a new drug for a specific disease takes several years to study the physicochemical properties, efficacy, and adverse reactions. in the covid-19 case, there is no choice for who and usfda except for the approval of prequalified and emergency use medicines. here, in this case, those drugs are tested preliminary on the covid-19 virus and related proteins to determine the activity; once they ascertain the beneficial results, they can be approved for emergency use. of all the drugs, chemotherapeutics or chemo drugs have more adverse effects than biotherapeutics or biobased drugs. thus, here in this article, we present the drugs available for covid-19 treatment, which are already approved or under research, and their adverse and positive effects are identified, as summarized in table 1. pmmb 2022, 5, 1; a0000323 7 of 25 table 1. characteristics and summaries of chemotherapeutics and biotherapeutics agents. no. name category activity side effects status 1 paxlovid (nirmatrelvir + ritonavir) chemotherapeuti cs (antiviral) protease inhibitor alteration in sense of taste, diarrhea increased blood pressure. muscle aches approved 2 remdesivir chemotherapeuti cs (antiviral) nucleotide analog rna polymerase inhibitor respiratory failure, electro cardiogram abnormalities, juandice approved 3 favipiravir chemotherapeuti cs (antiviral) rna dependent rna polymerase inhibitor diarrhea, hyperuricemia (elevated uric acid), reduced neutrophil count, nausea, vomiting, abdominal pain not approved 4 molnupiravir chemotherapeuti cs (antiviral) protease inhibitor diarrhea, nausea, dizziness not approved 5 chloroquinine and hydroxy chloroquine chemotherapeuti cs (antimalarial) lysozomal enzyme inhibitor uncontrolled movements, deafness, nausea, diarrhea, abdominal cramps, head ache, mentalillness, serious infecton, skin rashes, itchiness, hair loss, taste loss recalled 6 ivermectin chemotherapeuti cs (anti parasitic) protease inhibitor nausea, vomiting, diarrhea, hypotension( low blood pressure), allergic reactions (itching and hives), dizziness, afaxia (problems with balance), seizures, coma not approved 7 tocilizunab (biologic) biotherapeutics (immunosuppressive) interleukin-6 inhibitor a cough or sore throat, blocked or runny nose. headaches or dizziness. mouth ulcers. high blood pressure. hypercholesterolaemia (increased cholesterol in the blood) approved 8 infliximab (biosimilar) biotherapeutics (immunosuppressive) tumor necrosis is a factor – alpha (tnf-alpha) inhibitor sinus pain, fever, cough, shortness of breath; high or low blood pressure, rash, itching. stomach pain approved 9 dexamethasone (carticosteroid) biotherapeutics (immunosuppressive) human steroidogenic enzyme inhibitor upset stomach. stomach irritation. vomiting, headache, dizziness insomnia, restlessness, depression. approved 10 biontech (mrna vaccine) biotherapeutics (immunomodulatory) proteasome inhibitor head ache, fatigue, muscle and joint pain, fever and chills, pain at the site of injection approved 11 plasma therapy (immunoglobulin s) biotherapeutics (immunomodulatory) cell proliferation inhibitor transfusion related dyspnea and severe allergic reactions with association bronchospasm approved pmmb 2022, 5, 1; a0000323 8 of 25 4.1. classification of covid-19 therapeutics drug discovery and development is a complex process that takes decades to develop a branded drug, the reason why most pharmaceutical companies and research and development centers develop generic drugs, which are proven to show the efficacy of branded drugs. based on the structure and nature of the drug classification, the covid-19 drugs fall into two classes, i.e., chemotherapeutics and biotherapeutics (figure 1). as chemotherapeutic drugs are synthetic chemicals in nature due to their potential reactivity, adverse reactions are mostly expected while using these drugs [29, 30]. on the other hand, biotherapeutics/biosimilars, biologics, and vaccines, derived from natural resources like microorganisms or animals and are generally considered as safe due to their mimicking nature against proteins synthesized in the human body [29, 30]. these products provide cost-effective solutions with minimum adverse effects. figure 1. classification of covid-19 drugs. multiple symptoms are recorded globally for covid-19 infected patients, so there is a need to use different therapeutics to meet the covid-19 patient's requirement for better treatment. for example, patients with some strains of covid-19, it is required to use specific antibiotics such as anti-malarial and anti-inflammatory to get relief from the cold, fever, and pain. so it is a global demand to approve those classes of therapeutics for use in covid-19 treatment. on 22nd october 2020, us fda approved the first antiviral drug remdesivir for the treatment of covid-19, and it was initially developed to treat hepatitis-c [31] . however, due to the exigencies of the covid-19 pandemic, the drug authorities authorized emergency use for treating patients in critical stages. after the approval of remdesivir®, many therapeutics like antibiotics, hormonal, anti-inflammatory, nutraceutical, etc., were developed and approved. some are still under the consideration of approval. considering the extensive use of chemo drugs during api (active pharmaceutical ingredient) manufacturing process, potential impurities, also called related substances, arise from the materials used like raw materials, isomers, intermediates, reagents, solvents, catalysts, etc. few chemo drugs are pmmb 2022, 5, 1; a0000323 9 of 25 genotoxic and mutagenic, which may alter the dna sequence and lead to mutations resulting in many life-threatening disorders, including cancer and ulcers [31]. us fda focused on controlling these gtis (genotoxic impurities) at the api level. many methods and techniques were developed from pharmacopeias based on ich (international conference on harmonization) guidelines. in contrast, biotherapeutics were found to be effective with high-quality standards with minimized adverse effects by reduced impurity percentage to meet the global pharmaceutical market demands for better patient care. this review proposes evaluating the effectiveness and drawbacks of chemo and biobased drug treatments available for covid-19 [29, 30]. covid-19 created plenty of opportunities for the pharmaceutical sector in terms of diagnostic, ppe kits, and therapeutics. the pharmaceutical corporations have scaled up their r&d capabilities, production capacities, technology upgradation, and marketing collaboration during the last two years. the rapid recovery of companies to pre-covid levels, adopting new normals, and adopting preventive measures can slower and minimize the covid-19 transmission; thereby, covid-19 therapeutic markets continue to decline further at -44% compound annual growth rate (cagr). the covid-19 therapeutics are broadly classified into anti-viral therapies (lopinavir, ritonavir, remdesivir, molnupiravir and paxlovid), monoclonal antibodies (lerolimilab, bamlanivimab+cetesevimab, casirivimab+imdevimab cocktail combinations), cell-based therapies (mesenchymal stem cells therapy), immunomodulators (prednisone, tocilizumab, methylprednisolone and hydrocortisone) and supplemental therapies (chloroquine and hydroxychloroquine). the near-term projection for covid therapeutics is highly contradictory from company to company. the current raising of covid-19 waves coupled with research in therapeutics, disease awareness, and rising healthcare expenditure will drive the covid-19 market across the globe (bloomberg). a group, namely, the peoples vaccine alliance, comprises of 80 low and middle-income (lmi) countries demanding the suspension of intellectual property rights (ipr) for covid vaccines, tests, and treatment. another group led by india and south africa, with the active support of other 100 nations, including the usa and nobel laureates bidding for ipr weaver, could not make progress due to the blockade of the uk and germany. as per the plead from international federation of pharmaceutical manufacturers & associations (ifpma), the who approved seven therapeutic formulations for covid-19, and the same are produced in 150 manufacturing facilities around the globe [32]. the industry had brought vaccines within 12 months of the declaration of covid-19 as a pandemic. biovaxys technology corp, merck, moderna, sorrento therapeutics, pfizer, astrazeneca, cipla, hetero, dr. reddy’s, cadila healthcare, johnson &johnson, cansino, eli lilly, novartis and glenmark are the leading biotechnology companies offer wide varieties of covid-19 therapeutics [33]. the merck and the pfizer corporations have initiated voluntary licensing (vl)/ sub-licensing model to offer generic therapeutics to the lmic nations [34]. however, the critics are opposing this model, as it will narrow down the possibility of trips exemption such as compulsory licensing, patent opposition and bolar provisions. the countries such as argentina, brazil, thailand, russia, colombia, ukraine, peru, turkey, pmmb 2022, 5, 1; a0000323 10 of 25 mexico, and many other countries are excluded from this agreement due to the nonpermissibility of generic drugs in their countries due to the statutory provisions. [34] the price data relating to covid-19 therapeutics were collected from the top covid-19 hit countries ever since their introduction into market. the price information published in their national newspapers and official reports are considered. this study has attempted to know the price variations in different countries and their impact on the patients (table 2). several drugs of chemo and biological origin are approved, and few are under approval with different activities and different side effects. the above describes all the details about the different drugs for covid-19 treatment. table 2. the prices of anti-viral and monoclonal antibodies in top covid-19 affected countries. nirmatrelvir with ritonavir remdesivir per vial molnupiravir favipiravir/ course of 122 tabs hydroxy chloroquin 200mg (100 tab) ivermectin (10 tabs) tocilizumab casirivimab& imdevimab methyl prednisolone 4mg (10 tabs) $529.00 $320.00 $700.00 nac $37 $35 (17.5) $527.00 $2,100.00 $9.00 $18.00 $69.23 $38.46 $20 $9 $5.0 $416.00 $765.38 $0.50 unspecified na $600.00 nac $17 $30.0 $500.00 na $1.25 $529.00 $390.00 na na $33 na na $2,000.00 na $530.00 na $700.00 nca $31 $17.5 $500.00 $2,000.00 na $502.50 $390.00 $700.00 nca $12 $42.0 na $1,500.00 $5.00 $529.00 na na na $37 na na $2,100.00 na na $390.00 $700.00 nac na na $500.00 na na na na $750.00 $155 $15 $30 na $2,730.00 $5.00 na $390.00 $20.00 $15 $4 na $359.00 na na $1,000.00 na $1,000.00 nac $20 $54 $410.00 na $5.00 na indicates, the prices are not revealed by the public health authorities openly. 4.2. paxlovid (nirmatrelvir & ritonavir) the paxlovid (nirmatrelvir and ritonavir) is a leading player which can reduce the probability of death rate by 66% in severe covid patients; hence this drug is an obvious choice for the medical professional in case of severe and high-risk patients (bloomberg, 2021). us fda granted eua for paxlovid in december 2021 for the covid-19 treatment. hence, the us fda accorded permission to specific pharmaceutical companies to manufacture and market with prior approval from the local regulatory agencies for the quality and efficacy testing to meet the patient’s demands. in order to make the drug available in the market, it requires rapid research & development followed by commercial manufacturing until the release of the finished product into the market [35, 36]. the drug maker pfizer offers paxlovid by adopting a differential pricing policy based on the nation's economic status, i.e., high-income countries; and low and middle-income pmmb 2022, 5, 1; a0000323 11 of 25 (lmic) countries. the firm had discussions with 100 low and middle-income (lmic) countries to sub-license its hallmark drug, paxlovid, with the support of un-backed mpp (medicines patent pool) for affordable prices of just below $60 for the entire course (figure 2) [35, 36]. to bring this humanitarian arrangement into effect, 35 generic manufacturers in 12 countries signed a contract with pfizer, but this initiative can come into reality from 2023 onwards. paxlovid is offered to all the top 15 covid-19 affected countries in the range of $529 to $1000 for developed nations, where the same generic version is manufactured in india and sold for $18, which gives more access to the population. within 2021-22, pfizer generated $37 billion and is expected to accrue $22 billion in the current financial year 202223 [35, 36]. figure 2. nirmatrelvir with ritonavir (paxlovid) prices in top covid-19 affected countries. 4.2.1 chemistry & pharmacology chemically paxlovid is a combination of two antiviral drugs, nirmatrelvir and ritonavir, a co-packaged solid oral investigational medication developed for covid-19 [37]. 4.2.2. biological activity nirmatrelvir is a covalent protease inhibitor that binds directly to the catalytic cysteine residue of the cysteine protease enzyme during translation. in the combination medication nirmatrelvir+ritonavir (paxlovid®), ritonavir acts to slow down the metabolism of nirmatrelvir through cytochrome enzyme inhibition, resulting increase of the nirmatrelvir concentration, thereby enhancing the pharmacokinetics [37]. 4.2.3. adverse effects as a chemically synthesized drug, paxlovid® does not have serious side effects. the most common side effects include • alteration in the sense of taste $529 $18 $529 $530 $503 $529 $1,000 $0 $200 $400 $600 $800 $1,000 $1,200 usa india brazil germany uk italy australia pmmb 2022, 5, 1; a0000323 12 of 25 • diarrhea • increased blood pressure • muscle aches 4.3 remdesivir (veklury) remdesivir is a parenteral prodrug medication initially developed to treat hepatitisc infection, and later the drug was tested for ebola and other viral diseases. due to its broad spectrum of antiviral properties, us fda & who approved under eua for covid-19 treatment . however, this drug is mainly used for hospitalized severe covid-19 infected patients. remdesivir is widely used in the covid-19 treatment all over the world [38, 39]. 4.3.1. chemistry & pharmacology chemically remdesivir is a protide (prodrug of nucleotide) broad-spectrum antiviral therapeutic which can diffuse into the cells. the drug mainly interferes with the action of rna-dependent rna polymerase and inhibits the rna polymerase activity [38, 39]. 4.3.2. adverse effects the following are the side effects reported for the remdesivir [38, 39]. • back pain. • chest tightness. • dark-colored urine. • flushing. • headache. • hives and itching. • light-colored stools. • nausea and vomiting. 4.3.3. market status this drug’s patent was awarded to gilead corporation initially for treatment of ebola virus in 2017, but this drug is also used in covid-19 treatment due to the extended benefits. remdesivir has not received any recalls from the regulatory agencies except the voluntary recall for a customer complaint of two lots (remdesivir 100 mg for injection) by the gilead sciences inc. due to glass particulates observed in the product by the customer. the firm investigation confirmed it. the risk involved in such issues results in local irritation and swelling due to the foreign nature of glass particulates. the route of administration of pmmb 2022, 5, 1; a0000323 13 of 25 remdesivir is parenteral, so the drug directly enters into the bloodstream. if the glass particulates enter the blood vessel, they can travel through different organs and block the blood flow, resulting in stroke and even death. the recall has been completed, and us fda terminated [38, 39]. the price was offered at $390 per single vial to the usa, germany, uk, south korea, and vietnam. in india, many companies offer the generic version of this medicine at a cheaper price ranging from $40 to $69 (figure 3). in similar lines to pfizer's paxlovid, the generic version of remdesivir was launched within the price range of $390 to 780$ per entire ten days course. the manufacturer gilead had a tie-up with nine major generic drug makers in asia. since the remdesivir is to be administered under medical supervision, the additional hospitalization cost will become an overhead in treatment expenditure [38, 39]. figure 3. remdesivir prices/ vial in top covid-19 affected countries. 4.4. chloroquine and hydroxychloroquine chloroquine and hydroxychloroquine are anti-malarial medications used against plasmodium vivax, a parasite that causes malaria. these therapeutics are also used in autoimmune diseases. a combination of these two drugs is used as a treatment for covid-19 infection, and in fact, neither drug prevents covid-19 infection. hydroxychloroquine is a derivative of chloroquine phosphate, which is less toxic than chloroquine. it is used for the treatment of malaria parasites and rheumatoid arthritis. hydroxychloroquine was approved for medical uses more than 60 years ago. it was used to treat covid-19 during the initial days of pandemic. it is used as a prophylaxis dosage for health workers and in direct contact with patients suffering from covid-19. however, the studies revealed that it is effective against covid-19, and is low effective in preventing covid-19 transmission, illness, and hospitalization and death events. therefore, the who has not approved hydroxychloroquine for covid19 treatment [40, 41]. pmmb 2022, 5, 1; a0000323 14 of 25 4.4.1. chemistry & pharmacology both molecules are amino quinoline derivatives that act against malaria, rheumatoid arthritis, and other diseases. cathepsins are a type of proteases in all animals on the cell membrane. the viral entry into the cell requires the cleavage of spike protein can be accomplished by the cathepsins which are present in the lysosomes. the action of these drugs against cathepsins gives more benefits to the patients of covid-19 treatment [40, 41]. 4.4.2. adverse effects although it is under eua approval, unlike other medications used in the treatment protocols for covid-19, hydroxychloroquine has a possible risk of side effects. the clinical research showed the ineffectiveness of these drugs in the treatment of covid-19. in april 2020, us fda announced severe heart arrhythmia (rhythm problems) observed in covid19 patients treated with hydroxychloroquine. hydroxychloroquine can exhibit contraindications such as sleep difficulty, hallucinations, paranoia, depression and other problems like kidney injuries and hot rhythm problems [40, 41]. 4.4.3. market status as these drugs showed serious side effects in covid-19 patients, usfda revoked eua on 15th june 2020 based on the scientific and clinical data reports. the primary purpose of the revoke is to stop the use of these drugs to minimize the risk of adverse effects specific to the covid-19 patients [40, 41]. figure 4. hydroxychloroquine prices (100 tablets) in top covid-19 affected countries. since hydroxychloroquine failed to show efficacy in trials, the world health organization stopped its clinical trials. since the hydroxychloroquine is an off-patent drug it is available in innovator and generic versions. the innovator version is used in usa, brazil, germany and italy in these countries. hydroxychloroquine 100 tablets cost $37, $33, $31, $37, whereas the generic version is available in india at $17, uk $12, japan $15, and pmmb 2022, 5, 1; a0000323 15 of 25 australia $20, and it is lowest in vietnam for a meager price of $4 for 100 tablets. during covid-19 pandemic, non-malaria countries suffered with acute shortage of hydroxychloroquine as it is a prime medication in the treatment of malaria and rheumatic diseases. the hydroxychloroquine is available for sale in almost all countries (figure 4). due to the elapsed patent period for this drug, during the first wave of covid-19, india has played a significant role in exporting hydroxychloroquine to the countries affected severely by covid-19. many corporate companies came forward to offer hydroxychloroquine as part of their charity objectives after discontinuing hydroxychloroquine from treatment protocols [42]. 4.5. molnupiravir the antiviral medication molnupiravir inhibits the rna viral replication of the infectious viruses in the host cell. the primary purpose of this drug is to treat the influenza virus. based on the positive results against covid-19, usfda granted eua for molnupiravir to treat covid-19 patients. this anti-viral drug was developed by merck corporation to treat covid-19. it is the first medication that can be administered orally and taken in isolation treatment. as per the manufacturer’s clinical data, molnupiravir can reduce the probability of hospitalization and death in moderate to severe cases by approximately 50% (merck, 2021). the patent protection was sought for 20 years; however, the company arranged to reach out the medicines to a larger population at an affordable price [43, 44]. 4.5.1. chemistry & pharmacology like remdesivir, molnupiravir is also a prodrug of synthetic nucleoside derivative of n-hydroxy cytidine. the drug mainly acts by interfering with the rna of viruses during viral rna replication. molnupiravir can exist in two tautomeric forms one mimics cytidine (nucleoside analogue of cytosine), and another mimics uridine (nucleoside analogue of uracil). molnupiravir acts by directly attaching to the rna when the rna polymerase of the virus tries to copy the host rna containing molnupiravir, which exists in two tautomeric forms; this causes over mutation of the genetic material, and this effect is known as error catastrophe or lethal mutagenesis [43, 44]. 4.5.2. adverse effects the common side effects like diarrhea, nausea, and dizziness are reported in the covid-19 patients. however, if any symptoms of serious allergic reactions are observed, there is a need to seek medical attention [43, 44]. 4.5.3. market status the antiviral agent molnupiravir is not approved but has been authorized as eua by us fda. the approval is purely based on the phase-iii clinical trial, and the significant activity of molnupiravir against covid-19 patients rendered the best marketing strategies. like pfizer's paxlovid, the molnupiravir drug maker merck had entered into an agreement pmmb 2022, 5, 1; a0000323 16 of 25 with 105 lmic nations to supply its molnupiravir at generic rates (figure 5). the molnupiravir prices in all developed nations were in the range of $600 to $1000 per course, which is high in australia, whereas the same medicine in the generic version is much cheaper in india at $38 and in vietnam at $20, which is lower than the recommended price for developing countries. france cancelled the order of 50,000 molnupiravir doses due to the non-conformance of molnupiravir in reducing hospitalization in high-risk patients by 30% is not demonstrated. the perception of medical and healthcare experts is that the price of molnupiravir is double that of remdesivir and 30% higher than the paxlovid [45]. figure 5. molnupiravir prices in top covid-19 affected countries. 4.6. favipiravir favipiravir is an antiviral medication used primarily in the treatment of influenza. us fda granted permission to favipiravir for covid-19 treatment under the eua category [46, 47]. 4.6.1. chemistry & pharmacology favipiravir, a nucleoside analogue, mimics both guanosine (nucleoside of guanine) and adenosine (nucleoside of adenine). it is a selective inhibitor of viral rna polymerase [46, 47]. 4.6.2 adverse effects the most common side effects of favipiravir are as follows [46, 47]. • increased blood uric acid levels • diarrhea • dizziness • elevated liver enzymes • neutropenia pmmb 2022, 5, 1; a0000323 17 of 25 4.6.3 market status figure 6. favipiravir prices (per course) in top covid-19 affected countries. this molecule is invented by fujifilm toyama chemical and sold under the brand name avigan. this medicine is efficacy against ebola virus but unable to demonstrate outcomes in the covid-19 clinical trials. except in india, japan and vietnam, this drug is basically used for clinical trials. all developed nations have not authorized to use this medicine for commercial use. the prices of favipiravir (avigan) in japan is $155, and the same is available in india under the brand name of fabiflu is below $20 and in vietnam is $15 (figure 6) [46, 47]. 4.7. infliximab (remsima®) infliximab is a high molecular weight macro molecular chimeric monoclonal antibody biologic. it is mainly used to treat many auto-immune diseases. the biologic was originally developed in mice as mouse antibodies. however, humans also have some immune reactions to mouse proteins. the mouse monoclonal antibodies were replaced with human antibodies. the us fda also approves the biosimilars of the biologic. in fact, it is listed in the who's list of essential medicines [48]. 4.7.1. structure and activation of infliximab the biologic is neither approved nor granted for eua in treating covid-19, but the drug is authorized to test on covid-19 patients in the form of clinical trials. the molecule is in the phase-iii clinical trial, and if it shows efficacy, it would be the future therapeutic option for covid-19 treatment. not many side effects are reported for this biologic while used in other treatments [48-50]. 4.8. tocilizumab (actimera) when responding to covid-19, the human body immunologically secretes lifethreatening cytokines in the lower respiratory tract. the abnormal release of cytokines (cytokine storming) blocks alluvia, eventually creating acute respiratory distress, leading to pmmb 2022, 5, 1; a0000323 18 of 25 fatality. in light of this biological response to the pathogen, the concept of monoclonal antibody treatment has emerged. tocilizumab is an immunoglobulin antibody that blocks interleukin-6, used in rheumatoid arthritis to treat the life-threatening cytokine release; thus acute respiratory distress subsided [10, 51]. the drug is patented by roche corporation [52]. 4.8.1. chemistry & pharmacology tocilizumab is a humanized monoclonal antibody immunosuppressive drug which targets to interleukin-6 receptor. the drug is produced by recombinant dna technology, chemically it is an antibody consisting of 2 heavy chains and 2 light chains with 12 intrachain, 4 inter-chain disulphide bridges [53, 54]. 4.8.2. adverse effects although tocilizumab has some common side effects reported in other infections, there are no adverse effects revealed by the initial research on covid-19 patients by the researchers [53, 54]. 4.8.3. market status usfda granted eua for tocilizumab to use on covid-19 hospitalized adults and pediatric patients (2 years and above). this shows that the efficacy of the drug might gain good market and production facilities [55, 56]. figure 7. tocilizumab prices in top covid-19 affected countries. the monoclonal drug market continues to dominate in north america, europe, asia pacific, africa, and the middle east and latin american countries. the price of tocilizumab is close to paxlovid (nirmatrelvir and ritonavir), in the range of $359 to $527 (figure 7). so far, tocilizumab is not available in generic form for lmi countries; the lower price version of tocilizumab is not available in the market; nevertheless, this drug price used to be two to pmmb 2022, 5, 1; a0000323 19 of 25 three times higher than the current price before the covid-19 pandemic. due to the higher demand and increased production, bulk procurement of apis, the manufacturing cost was decreased to $40 but the manufacturer capitalized with 12 times margins due to patent protection. the treatment for tocilizumab is expensive for lmi countries and patients, but buying this is due to promising results in severe illness patients. since the drug is to be given along with other immune-suppressant medicines under medical supervision, the cost of hospitalization will have added effect on the patient economic conditions. hence, the drug maker roche needs to consider making the drug in a generic version immediately to offer lmi countries [55, 56]. 4.9. casirivimab+imdevimab (ronapreve) 4.9.1. chemistry & pharmacology regn-cov2 is the brand name for ronapreve, which is a combination drug consisting of two human monoclonal antibodies, namely casirivimab and imdevimab. the combination mainly works against the spike protein of the covid-19 virus. as a parenteral drug, this combination gives immediate action in the covid-19 patients who are hospitalized [57, 58]. 4.9.2. adverse effects from phase one to three, there is not even a single death or serious adverse reactions observed with the regn-cov2, due to the reason the clinical research is still going on for the same [57, 58]. 4.9.3. market status usfda limited this combination to the specific variant like omicron, which is susceptible to this drug. in fact, this combination is approved by the ema in the european countries to treat the covid-19 patients and has been issued market authorization for the same. by this date, it is evident that in the future, the use of this combination will bring a huge market demand [57, 58]. another monoclonal antibodies cocktail demonstrating the promising result is casirivimab and imdevimab. this drug is patented by roche corporation. out of all the existing therapies, this mab is expensive; however, quite effective for severe illness patients. the price of ronapreve ranges from $1500 to $2730 in developed countries, but the same medicine is offered in india for a generic version at $765 (figure 8). even though the medicine is lower compared to developed countries, still not accessible to the large populations in lmi countries. therefore, the generic version must be within the limits of accessibility to reach out to the mass population [57, 58]. pmmb 2022, 5, 1; a0000323 20 of 25 figure 8. casirivimab and imdevimab cocktail prices in top covid-19 affected countries. 4.10. ivermectin ivermectin was patented under merck and sold under the brand name stromectol. it is an anti-parasite medicine and used for veterinary deworming. due to the misusage, this medicine is highly used in covid-19 protocols, but it has low and limited outcomes in the pandemic control. the price of branded ivermectin is in the range of $30 to $54 in patent protection countries. however, the generic version of this medicine is in the range of $5 to $ 75 (figure 9). the who removed this medicine from its treatment protocols upon the detailed studies of its outcomes. now the price of this medicine is under control due to the decline in the covid-19 therapeutic consumption, and it is exclusively used for worm related infections [59, 60]. figure 9. ivermectin prices (10 tablets) in top covid-19 affected countries. 4.11. methylprednisolone methylprednisolone is a synthetic glucocorticosteroid which was patented under pfizer and sold under the brand name medrol. it is widely used in the health conditions like arthritis, allergies and blood related disorders. it is a proven and effective drug for the pmmb 2022, 5, 1; a0000323 21 of 25 immune related diseases. this is considered for the treatment due to its effectiveness in the treatment of covid-19. since the price of medrol is fairly less, 10 tablets of 4 mg pack is in the range of $5 to $9 in uk, japan, australia, and usa. however, the generic version of this drug is available at just below $1 in india and vietnam, and $1.3 in france (figure 10). as the medicine is widely available and used around the world, many generic brands exist for this drug. the covid-19 has given a sudden boost in sales for this product due to its efficacy in treatment [61, 62]. figure 10. methylprednisolone (10 tablets) prices in top covid-19 affected countries. 5. conclusion the analysis revealed that the demand for covid-19 therapeutics is growing as the covid-19 pandemic has not subsided yet. pandemic is rising continuously in some or other corners of the world despite the availability of multiple varieties of vaccines. however, the death cases are significantly reduced because of the release of advanced bio-therapeutics and chemo-therapeutics into the market to treat the disease effectively. these therapeutics are highly expensive and far from ordinary people in lmi countries. the medicines that are to be administered under medical supervision will worsen the financial condition of the patients due to increased hospital expenditure. however, there are positive signs of bringing down these medicine prices to lmi countries by sub-licensing patented medicines to offer them in a generic version under medicines patent pool. paxlovid, molnupiravir, and remdesivir manufacturers had collaborated with generic manufacturers in south asia, south america, and latin american countries around the globe. therefore, there is a massive demand for therapeutics which can be used in home isolation. the most effective bio-therapeutics, such as monoclonal antibodies, are popular these days, but the costs of these therapeutics are yet to be reduced for lmi countries at the earliest possible in the interest of humanity. the concept of sub-licensing somehow dilutes the compulsory licensing/bolar provisions, still helping the lmi countries to get medicines at affordable prices. the review outlined important information about the effectiveness and safety of the newly approved covid-19 medicine, which can help healthcare providers make informed decisions about treatment pmmb 2022, 5, 1; 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28(3): e299. 60. popp m, stegemann m, metzendorf m-i, et al. ivermectin for preventing and treating covid‐19. cochrane database syst rev 2021(7). 61. chaudhuri d, sasaki k, karkar a, et al. corticosteroids in covid-19 and non-covid-19 ards: a systematic review and meta-analysis. intensive care med 2021; 47(5): 521-537. 62. corral-gudino l, bahamonde a, arnaiz-revillas f, et al. methylprednisolone in adults hospitalized with covid19 pneumonia. wien klin wochenschr 2021; 33(7): 303-311. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2023, 6, 1; a0000270. doi: 10.36877/pmmb.0000270 http://journals.hh-publisher.com/index.php/pmmb original research article streptomyces griseiviridis sp. nov., a novel “modern actinobacteria” isolated from malaysia mangrove soil jodi woan-fei law1*, loh teng-hern tan1,2, vengadesh letchumanan1, kar-wai hong1, hooi-leng ser3, bey-hing goh4,5, nurul syakima ab mutalib1,6, kok-gan chan7,8*, learn-han lee1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, selangor, malaysia; vengadesh.letchumanan1@monash.edu (vl); hong.karwai@monash.edu (k-wh) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) 3department of biological sciences, school of medical and life sciences, sunway university, kuala lumpur, malaysia; hooilengs@sunway.edu.my (hls) 4biofunctional molecule exploratory research (bmex) group, school of pharmacy, monash university malaysia, subang jaya 47500, selangor, malaysia; goh.bey.hing@monash.edu (b-hg) 5college of pharmaceutical sciences, zhejiang university, 866 yuhangtang road, hangzhou 310058, china 6ukm medical molecular biology institute (umbi), universiti kebangsaan malaysia, 56000 kuala lumpur, malaysia; syakima@ppukm.ukm.edu.my (nsam) 7division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia 8international genome centre, jiangsu university, zhenjiang, china *corresponding author: jodi woan-fei law and learn-han lee; novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, selangor, malaysia; jodi.law1@monash.edu (jw-fl); lee.learn.han@monash.edu (l-hl); kokgan chan; division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia; kokgan@um.edu.my (kgc) received: 18 november 2022; received in revised form: 03 january 2023; accepted: 10 january 2023; available online: 11 january 2023 abstract: a novel strain, streptomyces griseiviridis mum 136jt was recovered from a mangrove forest soil in malaysia. the gram-positive bacterium forms strong yellow aerial mycelium and moderate yellow substrate mycelium on isp 2 agar. a polyphasic approach was used to determine the taxonomy status of strain mum 136jt. the strain showed a spectrum of phylogenetic and chemotaxonomic properties consistent with those of the members of the genus streptomyces. the cell wall peptidoglycan was determined to contain pmmb 2023, 6, 1; a0000270 2 of 20 ll-diaminopimelic acid. the predominant menaquinones were identified as mk-9(h8) and mk-9(h6), while the identified polar lipids consisted of lipid, aminolipid, phospholipid, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylinositolmannoside. the cell wall sugars consist of ribose, mannose, and galactose. the predominant cellular fatty acids (>10.0 %) were identified as iso-c16:0 (31.6 %), anteiso-c15:0 (14.8 %), iso-c15:0 (12.0 %), and anteiso-c17:0 (11.1 %). phylogenetic analysis identified that closely related strains for mum 136jt are streptomyces leeuwenhoekii dsm 42122t (98.9 %), streptomyces erythrogriseus jcm 9650t (98.4 %), streptomyces griseoincarnatus jcm 4381t (98.5 %). the dna-dna relatedness values between mum 136 jt and closely related type strains ranged from 13.3 ± 1.5 % to 17.4 ± 2.0 %. the name streptomyces griseiviridis sp. nov. is proposed, and the type strain is mum 136jt (= nbrc 114249t = mccc 1k04199t). keywords: streptomyces griseiviridis; actinobacterial; mangrove; antioxidative; polyphasic taxonomy; mod-actino 1. introduction actinobacteria have never ceased to gain the attention of researchers worldwide due to their astonishing abilities to produce valuable biologically active compounds. the “modern actinobacteria” (mod-actino) has been the focus lately to uncover actinobacteria from unique sources with bioactive potentials [1, 2]. actinobacteria are present in diverse habitats such as terrestrial soil [3, 4], marine [5, 6], pond [7, 8], desert [9-11], cave [12-14], glacier [15, 16], hot spring [17, 18], artic and antarctic zones [19-22], and mangrove [23-26]. this phylum of bacteria can survive in a wide range of environmental conditions through their complex multicellular life cycle and the development of unique defence mechanisms, notably observed in the genus streptomyces [27-29]. the largest genus in the phylum actinobacteria is streptomyces, which has largely contributed to improving our health and well-being. this genus consists of bacteria that are producers of important antibiotics currently being used for treating infections in animals and humans [30-32]. streptomyces is well-known for their antimicrobial activity, and many studies have investigated their antimicrobial effect against various pathogens in hopes of searching for new effective antibiotics [33-36]. for instance, the increasing morbidity and mortality burden of life-threatening infection caused by methicillin-resistant staphylococcus aureus (mrsa) has urged the need for more effective antibiotics. studies have reported that streptomyces spp. could produce compounds with promising anti-mrsa activity [37-41]. another example of a recently targeted pathogen is the sars-cov-2 virus ⸺ a causal pathogen of coronavirus disease (covid-19) [42-45]. the covid-19 pandemic has caused a tremendous loss of human life and reduced the population’s quality of life globally [46-51]. ivermectin, refined from avermectin (a streptomyces-derived antiparasitic drug), is proposed as a potential drug candidate for treating (covid-19) [52, 53]. efforts taken into repurposing existing drug agents (e.g., ivermectin, hydroxychloroquine, etc.) aim to offer treatment pmmb 2023, 6, 1; a0000270 3 of 20 options for covid-19 [54-56]. additionally, a genomic and metabolomic study conducted by melinda et al. [57] reported the production of antiviral agents, echoside a and echoside b, against sars-cov-2 by streptomyces sp. gmr22. furthermore, the emerging roles of streptomyces as probiotics for aquaculture applications have been discussed [58-60]. marine fishes and shrimps are prone to infection caused by vibrio spp. pathogens (e.g. vibrio parahaemolyticus, vibrio vulnificus, vibrio alginolyticus), resulting in an illness known as vibriosis with a high mortality rate [61]. consequently, the occurrence of these pathogens in seafood has led to foodborne diseases [6265]. several studies have provided evidence that streptomyces spp. exerted significant antivibrio activity [58, 66-68]. the antibacterial property of streptomyces spp. is closely associated with their role as probiotics that protect fishes and shrimps against infectious diseases such as vibriosis [69-71]. it is crucial to continue finding new sources of these beneficial actinobacteria. mangrove forests have unique and dynamic environmental characteristics as they are located in intertidal zones. the mangroves in malaysia remain largely unexplored, and they are excellent sources for discovering novel and bioactive streptomyces species. numerous novel streptomyces species have been found in mangroves, and they are capable of producing bioactive compounds associated with antimicrobial, anticancer, antioxidant, and neuroprotection activities [1, 27, 72, 73]. some examples of these strains are streptomyces colonosanans (anticancer and antioxidant) [74], streptomyces pluripotens (antibacterial) [75], streptomyces antioxidans (antioxidant and neuroprotection) [76], and streptomyces nigra (antitumor) [77]. the streptomyces bacteria possess large genome sizes, typically ranging from 7 to 10 mbp, which account for their secondary metabolites production capabilities [7880]. therefore, research exploring novel species of streptomyces remains worthwhile, considering the great benefits these bacteria can offer and the chances of finding new or valuable compounds. this study aims to identify and characterize a novel streptomyces strain, mum 136jt, isolated from the soil of a malaysian mangrove forest. a polyphasic approach was carried out to investigate the genotypic, genomic, phenotypic, and chemotaxonomic features of the strain. next-generation sequencing technique was applied to determine the whole genome sequence of strain mum 136jt for further bioinformatic analyses. streptomyces griseiviridis sp. nov. mum 136jt is a malaysian mod-actino that can serve as a new microbial source of bioactive agents. 2. materials and methods 2.1. soil sampling, isolation, and maintenance of the strain soil samples were collected from a mangrove forest located on the east coast of malaysia, in june 2015. strain mum 136jt was isolated from a soil sample collected at the mangrove site labelled as kttas 4 (1º41’48.48”n 110º11’13.40”e). the soil samples were pmmb 2023, 6, 1; a0000270 4 of 20 processed by air-drying, followed by mixing and selective pretreatment through wet heat for 15 min at 50 ºc [81]. strain mum 136jt was isolated from a nutrient agar (na) plate supplemented with cycloheximide (50 mg/l) and nalidixic acid (20 mg/l). the strain was purified on isp 2 plate. pure cultures of strain mum 136jt were maintained on isp 2 agar slants at 28 °c and kept in glycerol suspensions (20 %, v/v) at -20 °c for long-term storage. 2.2. genotypic identification and phylogenetic analysis genomic dna extraction and 16s rrna gene pcr amplification were conducted according to established protocols [74, 75, 82]. the 16s rrna gene sequence of strain mum 136jt was obtained and manually aligned with representative sequences of related type strains of streptomyces genus retrieved from genbank/embl/ddbj databases using clustal-x software. phylogenetic trees were reconstructed with neighbour-joining [83] and maximum likelihood [84] algorithms using mega version 7.0. kimura’s two-parameter model [85] was applied for the computation of evolutionary distances in the neighbour-joining phylogenetic tree, and felsenstein’s method [86] of bootstrap analysis based on 1000 resamplings was applied to evaluate the tree topologies. the sequence similarities of strain mum 136jt and related type strains were analyzed by ezbiocloud server (http://www.ezbiocloud.net/). dna-dna hybridization (ddh) was conducted by the identification service of the dsmz, braunschweig, germany [87, 88], on strain mum 136jt and its closely related type strains streptomyces leeuwenhoekii dsm 42122t, streptomyces erythrogriseus jcm 9650t, and streptomyces griseoincarnatus jcm 4381t. 2.3. next generation sequencing and bioinformatic analysis genomic dna extraction was done using masterpure™ gram positive dna purification kit (epicentre, illumina inc., madison, wi, usa). the quality of the bacterial dna was verified using nanodrop 2000 spectrophotometer (thermo scientific, waltham, ma, usa ) and qubit 2.0 fluorometer (life technologies, carlsbad, ca, usa). dna library construction was carried out using nextera dna flex library prep kit (nextera, usa), and the constructed sequencing libraries were loaded into illumina miseq platform with miseq reagent kit v3 (illumina inc., madison, wi, usa). the sequencing quality was evaluated using fastqc (version 0.11.9) [89]. subsequently, along with the adapter sequences, the raw reads were trimmed using bbduk of bbtools (v36). the trimmed raw reads were then assembled using st. petersburg genome assembler (spades) (v3.14.1) [90]. the assembled genomic sequence was submitted to rapid annotation using subsystem technology (rast) database (https://rast.nmpdr.org/) for annotation, with the following settings: default pipeline for rasttk, domain bacteria, and automatically fixed error options turned on [91, 92]. the genome assembly was compared with genomes of closely related streptomyces species (retrieved from ncbi database) using fastani (version 1.33) [93]. the genome assembly was uploaded to type strain genome server (https://tygs.dsmz.de) for http://www.ezbiocloud.net/ https://rast.nmpdr.org/ https://tygs.dsmz.de/ pmmb 2023, 6, 1; a0000270 5 of 20 phylogenomic analysis [94]. the detection of biosynthetic gene clusters related to secondary metabolite production was analyzed using antismash (version 6.1.1) [95]. 2.4. phenotypic analysis the cultural morphology of strain mum 136jt was observed on yeast malt agar (isp 2), oat meal agar (isp 3), inorganic salt starch agar (isp 4), glycerol asparagine agar base (isp 5), peptone yeast extract iron agar (isp 6), tyrosine agar base (isp 7), actinomycetes isolation agar (aia), streptomyces agar (sa), starch casein agar (sca), nutrient agar (na), luria-bertani agar (lba), and mueller hinton agar (mha), at 28 c for 14 days [74, 82]. the colony colors were determined according to iscc-nbs color charts. the cellular morphology of strain mum 136jt was observed under light microscopy (80i, nikon) and scanning electron microscopy (jeol-jsm 6400) after growing on isp 2 plate at 28 c for 7–14 days. the growth of strain mum 136jt at different ph ranges (ph 2–10) and nacl concentration (0–10 %) were examined using tryptic soy broth (tsb) incubated at 28 c for 14 days. the effect of different temperatures (4–50 c) on the growth of strain mum 136jt was tested using isp 2 agar plates, and the responses were recorded for 14 days. melanoid pigments were produced using isp 7 medium after incubation at 28 c for 7–14 days, and hemolytic activity was observed using horse blood agar medium after incubation at 32 c for 7–14 days [74, 75]. enzymatic activities of strain mum 136jt including amylolytic, cellulase, chitinase, catalase, protease, and xylanase were investigated according to the previously described protocol [75]. all phenotypic tests were conducted simultaneously on strain mum 136jt, s. leeuwenhoekii dsm 42122t, s. erythrogriseus jcm 9650t, and s. griseoincarnatus jcm 4381t. 2.5. chemotaxonomic evaluation the evaluation of cell wall peptidoglycan, whole cell sugars, respiratory quinones, fatty acids, and polar lipids were done by the identification service of the dsmz, braunschweig based on established protocols [74, 75, 82, 96]. 3. results 3.1. genotypic identification and phylogenetic analysis of strain mum 136jt the nearly full-length 16s rrna gene sequence was attained for strain mum 136jt (1488 bp; genbank/embl/ddbj accession number mk368433). the alignment of the sequence with the corresponding partial 16s rrna gene sequences of the type strains of representative members of the genus streptomyces retrieved from genbank/embl/ddbj databases was conducted manually. based on the 16s rrna gene sequences, phylogenetic trees were reconstructed to determine the phylogenetic position of this strain (figure 1 and supplementary figure s1). the phylogenetic analysis demonstrated that the most closely pmmb 2023, 6, 1; a0000270 6 of 20 related strain is streptomyces leeuwenhoekii dsm 42122t (98.9 % sequence similarity) with shortest evolutionary distance (fig. 1). the 16s rrna gene sequence analysis for strain mum 136jt revealed that this strain exhibited the highest similarity to strain s. leeuwenhoekii dsm 42122t (98.9 %), s. erythrogriseus jcm 9650t (98.4 % similarity), and s. griseoincarnatus jcm 4381t (98.5 %). furthermore, the results of ddh revealed that the dna–dna relatedness levels between strain mum 136jt, s. leeuwenhoekii dsm 42122t (17.4 ± 2.0 %), s. erythrogriseus jcm 9650t (15.7 ± 1.8 %), and s. griseoincarnatus jcm 4381t (13.3 ± 14.8 %) were significantly below 70 % which has been designated as the threshold value for the delineation of bacterial species [97]. figure 1. neighbour-joining phylogenetic tree based on 1488 nucleotides of 16s rrna gene sequence showing the relationship between strain mum 136jt and representatives of related taxa. numbers and nodes indicate percentages (> 50 %) of 1000 bootstrap re-sampling. bar, 0.002 substitutions per site. pmmb 2023, 6, 1; a0000270 7 of 20 3.2. whole genome sequence and bioinformatic analysis of strain mum 136jt a total of 6,798,218 reads have been generated from the sequencing experiment. after adapter trimming and trimming the sequencing raw reads, the whole genome sequence was assembled using spades, resulting in a total of 169 contigs and n50 of 100,013 bp. the total genome size of strain mum 136jt is 7,180,176 bp, with a g+c content of 72.32 % and the calculated sequencing coverage of 144.75-times (table 1). the genome sequence of streptomyces griseiviridis mum 136jt has been deposited at ddbj/embl/genbank under accession number jadwyp000000000. table 1. general features of streptomyces griseiviridis mum 136jt genome. streptomyces griseiviridis mum 136jt genome size (bp) 7,180,176 contigs 169 contigs n50 (bp) 100,013 g + c content 72.32 % genome coverage 144.75x cds 6637 trna 66 rrna 2(5s), 1(16s), 1(23s) based on the rast system, a total of 6637 coding sequences assigned to the 1252 subsystem, 66 trna, and 4 rrna, were detected in the genome of strain mum 136jt (table 1). the majority of the genes are involved in amino acids and derivatives metabolism (6.03 %), carbohydrate metabolism (4.81 %), and protein metabolism (3.30 %) (figure 2). besides, antismash analysis had identified the presence of gene clusters account for the biosynthesis of compounds such as ectoine (100 % known gene cluster similarity), albaflavenone (100 % known gene cluster similarity), γ-butyrolactone (100 % known gene cluster similarity) and paenibactin (83 % known gene cluster similarity). pmmb 2023, 6, 1; a0000270 8 of 20 figure 2. rast output on subsystem category distribution of streptomyces griseiviridis mum 136jt. fastani demonstrated that comparing whole genome sequences between strain mum 136jt and its closely related type strain streptomyces griseosporeus jcm 4766t resulted in ani value of 87.36 % (table s1). the ani value between strain mum 136jt and s. leeuwenhoekii dsm 42122t was also calculated and resulted in 84.54 %. the phylogenomic analysis of the whole genome sequences with tygs showed that strain mum 136jt is closely related to s. griseosporeus jcm 4766t and s. leeuwenhoekii dsm 42122t, with digital ddh (dddh) values (formula d4) of 31.6 % and 27.2 % respectively (figure 3). figure 3. the whole genome sequence tree constructed using tygs web server for streptomyces griseiviridis mum 136jt and closely related type strains. tree inferred with fastme 2.1.6.1 [98] from genome blast distance phylogeny (gbdp) distances calculated from genome sequences. the branch lengths are scaled in terms of the gbdp distance formula d5. the numbers above branches are gbdp pseudo-bootstrap support values > 60 % from 100 replications, with an average branch support of 96.5 %. the tree was rooted at the midpoint. pmmb 2023, 6, 1; a0000270 9 of 20 3.3. phenotypic characteristics of strain mum 136jt based on the growth observation of strain mum 136jt on different media after incubation at 28 c, for 7–14 days, strain mum 136jt was able to grow well on isp 2, isp 6, sa, na, lba, and mha; grow moderately on isp 5, isp 7, aia, and sca; but unable to grow on isp 3 and isp 4. the aerial and substrate mycelium colors were media dependent, as shown in table 2. the cellular morphology of strain mum 136jt matched the typical features observed in genus streptomyces (figure 4). for the effects of temperature, ph, and nacl tolerance on the growth of strain mum 136jt, the results demonstrated that growth was found to occur at 26–32 °c (optimum 26–28 °c), at ph 6.0-8.0 (optimum ph 8.0), and with 0-6 % nacl tolerance (optimum 0-2 %). cells were positive for catalase activity, but no hemolytic activity was observed. moreover, the cells were capable of hydrolyzing casein. table 2. the colony appearance of streptomyces griseiviridis mum 136jt after growing on different culture media. medium growth colony colour aerial mycelium substrate mycelium yeast malt agar (isp 2) good strong yellow moderate yellow oat meal agar (isp 3) no growth inorganic salt starch agar (isp 4) no growth glycerol asparagine agar base (isp 5) moderate yellowish white pale yellow peptone yeast extract iron agar (isp 6) good yellowish grey moderate olive brown tyrosine agar base (isp 7) moderate yellowish white yellowish white actinomycete isolation agar (aia) moderate pale yellow pale yellow streptomyces agar (sa) good strong yellow dark yellow starch casein agar (sca) moderate pale yellow greyish yellow nutrient agar (na) good pale yellow light yellow luria bertani agar (lba) good pale yellow moderate yellow mueller hinton agar (mha) good greyish yellow deep greenish yellow -, not detected figure 4. morphology of streptomyces griseiviridis mum 136jt as observed by scanning electron microscopy. pmmb 2023, 6, 1; a0000270 10 of 20 3.4. chemotaxonomic characteristics of strain mum 136jt the cell wall peptidoglycan analysis of strain mum 136jt showed that it presented a type i cell-wall as it contained ll-diaminopimelic acid. the predominant menaquinones of strain mum 136jt were identified as mk-9(h8) (68 %) and mk-9(h6) (10 %), with other minor menaquinones identified as mk9(h10) (5%), mk10 (4%), mk10(h2) (3%), and mk9(h4) (2%). the whole-cell sugars detected were ribose, mannose, and galactose. fatty acid profiles of strain mum 136jt and its closely related type strains are compiled in table 3. the major cellular fatty acids in strain mum 136jt were identified as iso-c16:0 (31.6 %), anteiso-c15:0 (14.8 %), iso-c15:0 (12.0 %), and anteiso-c17:0 (11.1 %). the fatty acid profile of strain mum 136jt displayed high levels of similarities with those of closely related phylogenetic neighbors, s. leeuwenhoekii dsm 42122t, s. erythrogriseus jcm 9650t, and s. griseoincarnatus jcm 4381t, as they also contain iso-c16:0 (21.9–35.3 %) as their major fatty acid (table 3). however, quantitative differences can be observed in the fatty acid profiles of strain mum 136jt and its closely related type strains. for instance, isoc16:0 (31.6 %) was found to be predominant in strain mum 136j t, but the quantity of the same fatty acid was higher in streptomyces leeuwenhoekii dsm 42122t (35.3 %). as for polar lipids analysis, there were lipid, aminolipid, phospholipid, phophatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylinositolmannoside present in strain mum 136jt (figure 5). figure 5. total lipid profile of streptomyces griseiviridis mum 136jt. l, lipid; al, aminolipid; pl, phospholipid; pg, phophatidylglycerol; pi, phosphatidylinositol; pe, phosphatidylethanolamine; dpg, diphosphatidylglycerol; pim, phosphatidylinositolmannoside. pmmb 2023, 6, 1; a0000270 11 of 20 table 3. fatty acid profiles of streptomyces griseiviridis mum 136jt and closely related type strains streptomyces leeuwenhoekii dsm 42122t, streptomyces erythrogriseus jcm 9650t, and streptomyces griseoincarnatus jcm 4381t. fatty acid streptomyces griseiviridis mum 136jt streptomyces leeuwenhoekii dsm 42122t streptomyces erythrogriseus jcm 9650t streptomyces griseoincarnatus jcm 4381t iso-c12:0 0.1 iso-c13:0 0.1 0.2 anteiso-c13:0 0.1 0.1 iso-c14:0 3.5 6.5 8.2 3.1 c14:0 0.2 0.2 0.2 iso-c15:0 12.0 3.7 7.8 8.1 anteiso-c15:0 14.8 19.6 20.4 17.2 c15:1 b 0.1 0.1 c15:0 1.7 0.3 0.7 0.9 anteiso-c15:0 2oh 0.1 0.2 iso-c16:1 h 4.4 1.4 2.3 2.0 iso-c16:0 31.6 35.3 26.2 21.9 c16:1 cis 9 0.5 0.1 2.4 2.2 c16:0 4.7 2.8 7.1 5.9 c16:0 9methyl 2.3 0.7 1.7 4.2 anteiso-c17:1 c 4.2 2.8 1.5 4.1 iso-c17:0 5.2 5.2 6.8 8.1 anteiso-c17:0 11.1 15.7 12.4 15.8 c17:1 cis 9 0.2 0.3 0.4 c17:0 cyclo 0.7 1.7 1.8 c17:0 0.4 0.3 0.7 0.7 c17:0 10methyl 0.1 0.2 0.2 0.2 iso-c18:1 h 0.2 0.6 iso-c18:0 0.5 0.4 c18:1 cis 9 0.1 0.1 1.2 iso-c17:0 2oh 0.2 0.4 c18:0 0.2 0.2 -, <0.1% or not detected. all data are obtained concurrently from this study. 4. discussion due to the tremendous number of existing streptomyces species (approximately 1140 validly identified species), determining a novel strain in this genus is more challenging and complicated. nonetheless, the standard process for identifying and characterizing a novel strain required a polyphasic approach involving a combination of genotypic, phylogenetic, pmmb 2023, 6, 1; a0000270 12 of 20 and phenotypic tests [99, 100]. the advances in molecular techniques, such as next-generation sequencing, have allowed higher efficiency and better characterization of bacterial species. in this study, the novelty of strain mum 136jt is firmly proven and supported by a series of phylogenetic, phylogenomic, genomic, phenotypic, and chemotaxonomic analyses. based on 16s rrna gene similarity according to the ezbiocloud server, strain mum 136jt and s. leeuwenhoekii dsm 42122t exhibited the highest similarity of 98.9 %. the outcomes of phylogenetic analysis based on neighbour-joining and maximum likelihood algorithms were consistent, illustrating that the strain mum 136jt formed a clade with s. leeuwenhoekii dsm 42122t. nevertheless, the ddh value of laboratory-based genome-wide comparison between strain mum 136jt and its closest related type strain s. leeuwenhoekii dsm 42122t was 17.4 ± 2.0 %, significantly below the 70 % threshold of dna-dna relatedness. ddh has been the gold standard for the taxonomic evaluation of strain, and if the value is less than 70 %, the two strains can be categorized as two different species [97, 100, 101]. furthermore, phylogenomic analysis by tygs illustrated that the closely related type strain for strain mum 136jt is s. griseosporeus jcm 4766t with a dddh value of 31.6 %. therefore, strain mum 136jt is a distinct species from s. leeuwenhoekii dsm 42122t and s. griseosporeus jcm 4766t. average nucleotide identity (ani) analysis was performed in this study by comparing the genome sequences of the strain mum 136jt and closely related type strains to determine their genetic relatedness. ani technique has become increasingly popular as whole genome sequencing is now more accessible, and it is a promising substitute for the labour-intensive ddh technique [102]. fastani is a new method that can be applied to both complete and draft genomes to calculate ani using alignment-free approximate sequence mapping [93]. the estimated ani values between mum 136jt and its closely related type strains s. griseosporeus jcm 4766t, and s. leeuwenhoekii dsm 42122t were significantly below 95 %. goris et al. [103] reported that ani values of 95 % and 69 % conserved dna are equivalent to 70 % ddh cut-off for species delineation. thus, strain mum 136jt is a novel species of the genus streptomyces, as supported by the data obtained from laboratory-based ddh, tygs (dddh), and fastani. the phenotypic and chemotaxonomic information serve as important supplementary data to confirm that mum 136jt belongs to the genus streptomyces. strain mum 136jt exhibits typical colony and cellular morphology of streptomyces, for example, the formation of aerial and substrate mycelia. moreover, the detection of ll-diaminopimelic acid in cell wall peptidoglycan, accompanied by mk-9(h8) and mk-9(h6) predominant menaquinones of strain mum 136jt further corroborates the strain as streptomyces species. lldiaminopimelic acid, mk-9(h8) and mk-9(h6) menaquinones are typically found in the genus streptomyces, and similar findings have been reported by many relevant studies [76, 104109]. meanwhile, antismash detected the presence of biosynthetic gene clusters accounting for compounds such as ectoine and albaflavenon. ectoine is an extremolyte pmmb 2023, 6, 1; a0000270 13 of 20 naturally found in bacteria that thrive in extreme environmental conditions such as high salinity, irradiation, drought, extreme ph, and temperature [110, 111]. ectoine confers protection to the bacteria by regulating osmotic stress, stabilizing lipid bilayers, preventing dna and protein damage, and offering hydroxyl radical scavenging activity [112]. the presence of ectoine biosynthetic gene cluster in mum 136jt could be explained by the need for this bacterium to survive in a dynamic mangrove environment consisting of constant changes in salinity and tidal gradient. ectoine is a compound commonly used in cosmetic products to promote anti-aging and whitening effects and prevent skin dehydration [113, 114]. studies have reported anti-inflammation and cell protection properties exhibited by ectoine [113, 115-117]. another compound, albaflavenon, is a tricycle sesquiterpene antibiotic initially discovered from streptomyces albidoflavus [118]. the biosynthetic gene clusters for albaflavenon was also detected in strain mumm 136jt. given the availability of the whole genome sequence of mum 136jt, the genomic information obtained revealed the potential of this mangrovederived novel strain and its role as mod-actino. further studies on the production of compounds such as ectoine and albaflavenone could provide better insights into strain mum 136jt as a valuable source of cosmeceutical or pharmaceutical agents. 5. conclusion and description of streptomyces griseiviridis sp. nov. mum 136jt streptomyces griseiviridis sp. nov. (gri.se. i.vi’ri.dis. l. adj. griseus, grey; l. adj. viridis, green; n.l. masc. adj. griseiviridis, grey-green, referring to the color of the mycelia). the type strain is mum 136jt (=nbrc 114249t = mccc 1k04199t) isolated from soil sample collected from the malaysia mangrove forest. the 16s rrna gene sequence of strain mum 136jt has been deposited in genbank/embl/ddbj under the accession number mk368433. the cells of strain mum 136jt appear with strong yellow aerial mycelium and moderate yellow substrate mycelium on isp 2 agar plate. the cells grow well on isp 2, isp 6, sa, na, lba, and mha media. the strain is capable of growing at 26–32 °c (optimum 26–28 °c), ph 6.0-8.0 (optimum ph 8.0), and 0-6 % nacl (optimum 0-2 %), with positive casein hydrolysis. the cell wall peptidoglycan consists of ll-diaminopimelic acid. major menaquinones of strain mum 136jt includes mk-9(h8) and mk-9(h6), and the major cellular fatty acids (>10 %) are iso-c16:0, anteiso-c15:0 , iso-c15:0, and anteiso-c17:0. strain mum 136jt has ribose, mannose and galactose as its whole cell sugars. the polar lipids comprise lipid, aminolipid, phospholipid, phophatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylinositolmannoside. the genome size of strain mum 136jt is 7,180,176 bp, with g+c content of 72.32 % and coverage of 144.75-times. the genome has been deposited at ddbj/embl/genbank under accession number jadwyp000000000. rast system predicted a total of 6637 coding sequences assigned to 1252 subsystem, 66 trna, and 4 rrna in the genome of strain mum 136jt. most of the genes are involved in amino acids and derivatives metabolism, carbohydrates metabolism, and protein metabolism. pmmb 2023, 6, 1; a0000270 14 of 20 author contributions: writing—original draft preparation, jw-fl; conceptualization, jw-fl, and l-hl; methodology and data analysis, jw-fl, h-ls, and k-wh; validation, l-hl, b-hg, and nsam.; review and editing, lt-ht, vl, 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allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology original article 1 risk factors affecting covid-19 case fatality rate: a quantitative analysis of top 50 affected countries hui poh goh1*, wafiah ilyani mahari1, norhadyrah izazie ahad1, li ling chaw1*, nurolaini kifli1, bey hing goh2,3, siang fei yeoh4, khang wen goh5; long chiau ming1 1pap rashidah sa’adatul bolkiah institute of health sciences, universiti brunei darussalam, gadong, brunei darussalam 2college of pharmaceutical sciences, zhejiang university, hangzhou, china 3biofunctional molecule exploratory (bmex) research group, school of pharmacy, monash university malaysia, selangor, malaysia 4department of pharmacy, national university health system, singapore 5faculty of science and technology, quest international university, ipoh, perak, malaysia abstract: background: latest clinical data on treatment on coronavirus disease 2019 (covid-19) indicated that older patients and those with underlying history of smoking, hypertension or diabetes mellitus might have poorer prognosis of recovery from covid-19. we aimed to examine the relationship of various prevailing population-based risk factors in comparison with mortality rate and case fatality rate (cfr) of covid-19. methods: demography and epidemiology data were used, which have been identified as verified or postulated risk factors for mortality of adult inpatients with covid-19. the number of confirmed cases and the number of deaths until april 16, 2020 for all affected countries were extracted from johns hopkins university covid-19 websites. datasets for indicators that are prevailing or postulated factors of covid-19 mortality were extracted from the world bank database. out of 185 affected countries, the top 50 countries were selected for analysis in this study. the following seven variables were included in the analysis, based on data availability and completeness: 1) proportion of people aged 65 above, 2) proportion of male in the population, 3) smoking prevalence, and 4) number of hospital beds. linear regression analysis was carried out to determine the relationship between cfr and the aforementioned risk factors. results: united states shows approximately 0.20% of confirmed cases and it has about 4.85% of cfr. luxembourg shows the highest percentage of confirmed cases of 0.55% but a low 2.05% of cfr, showing that a high percentage of confirmed cases does not necessarily lead to high cfr. there is a significant association between cfr, people aged 65 and above (β=4.70; p = 0.035). conclusion: countries with high proportion of older people above 65 years old have a significant risk of having high cfr from covid-19. nevertheless, gender differences and smoking prevalence failed to prove a significant relationship with covid-19 mortality rate and cfr. keywords: covid-19, risk, epidemiology, demography, fatality, age, diabetes received: 19th october 2020 accepted: 19th november 2020 published online: 28th november 2020 introduction on february 11, 2020, the world health organisation (who) renamed the highly contagious respiratory disease, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), as coronavirus disease-19 (covid-19) [1]. the unprecedented increase in covid-19 cases has led who to call it as a pandemic on march 11, 2020 [2,3]. as this pandemic continues to evolve, researchers are learning more about sars-cov-2 every day, including the fact that it can be transmitted from symptomatic, presymptomatic and asymptomatic people infected with covid-19 [4]. studies have shown that covid-19 is primarily transmitted from symptomatic people to people who are in close contact through respiratory droplets, by copyright @ 2020 by si tw and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: hui poh goh / li ling chaw, pengiran anak puteri rashidah sa’adatul bolkiah institute of health sciences universiti brunei darussalam, tungku link road, be1410 gadong, brunei darussalam. telephone: +6737213663 email: pohhui.goh@ubd.edu.bn/ liling.chaw@ubd.edu.bn citation: goh hp, mahari wi, ahad ni et al. risk factors affecting covid-19 case fatality rate: a quantitative analysis of top 50 affected countries. prog microbes mol biol 2020; 3(1): a0000171. https://doi.org/10.36877/ pmmb.a0000171. 2 risk factors affecting covid-19... direct contact with infected persons or by contact with contaminated objects and surfaces [5]. fever, tiredness and dry cough are the most common symptoms in symptomatic covid-19 patients [6,7]. current evidence indicated that certain group of people are at a higher risk of suffering from severe illness from covid-19 [8,9]. such risk factors include gender, bacilllus calmette-guerin (bcg) vaccination, smoking and malaria prevalence [10,11]. older people have a higher risk due to the decreasing function of the agedependent lymphocytes, which resulting in their increased susceptibility to covid-19 disease [7]. in addition, a study shows that there is a higher percentage of death among patients aged over 65 years (62%) than patients aged below 65 years old (37%) [12]. furthermore, male gender is commonly observed in covid-19 patients (73%) according to a retrospective study done on 113 deceased patients [13]. another risk factor for covid-19 mortality is existing comorbidities. a study by guan et al. shows that covid-19 are more commonly seen in patients with hypertension, diabetes, cardiovascular disease and a history of smoking [14]. not only were these patients susceptible to the disease, they also had a higher chance of obtaining poor health outcomes after immediate care unit (icu) admission and may lead to death [12]. moreover, a study on the correlation between covid-19 mortality and bcg vaccination suggested that early bcg vaccination could help to decrease the mortality rate [10]. other than that, malaria prevalence is also another risk factor of covid-19 mortality. a higher number of covid-19 cases were reported in countries with low malaria prevalence than countries that had higher malaria prevalence [15,16]. apart from addressing risk factors, there are also parameters that may affect the covid-19 mortality rate such as shortage of staff, lack of medical supply or equipment, insufficient hospital beds and the country’s health expenditure. as of late april 2020, sars-cov-2 virus has resulted in more than 3.1 million infections and over 217,000 deaths globally [1]. as covid-19 has become a global pandemic issue, implementation of suitable interventions will be needed for the public, healthcare professionals and patients and also to ensure all sectors to work together cohesively and efficiently [17]. even though covid-19 originate from the same family as other known coronaviruses, sars-cov-2 has very different severity and contagion characteristics and much still needs to be learned about it. thus, it is imperative to evaluate the relationship of postulated or verified risk factors with covid-19 mortality, as presented in a recent analysis based on united kingdom [18] and spain [19]. it is absolute crucial to evaluate the risk factors of mortality among patients infected with covid-19 at population level. by validating the relationship, patients with covid-19 can be treated more aggressively than those without the risk factor [20]. the findings of the current study provide a picture of covid-19 case fatality rate (cfr) in top 50 affected countries. we aimed to determine the association between specific risk factors and covid-19 cfr. these findings consolidate the evidence of crucial risk factors that front liners need to prioritise to decrease the covid-19 mortality globally. this is an ecological study that examines indicators or variables that could be associated with covid-19 mortality. the unit of observation in an ecological study is the population of the particular area or specific country in which the disease rates were measured. one of the advantages of an ecological study is it provides a snapshot of a transforming event, in additional to the fact that the disease rate and indicator statistics could be mined from existing databases thus saving time [21]. the compared populations and disease of interest are normally defined based on temporal and spatial variation. it is apparent that the reported cases of covid-19 tend to fluctuate and the sudden spike could be linked to so local transmission cluster. in term of geographical comparisons, epidemiologists are also interested to determine the geographical associations between disease incidence or mortality and the prevalence of risk factors, as exemplified by recent covid-19 study by whittle and diaz-artiles [22]. methods data extraction demography and epidemiology data which have been identified as verified or postulated risk factors for mortality among adult inpatients with covid-19 were used. the data were collected from world bank (https:// data.worldbank.org/) and johns hopkins university covid-19 (https://coronavirus.jhu.edu/map.html) websites. the number of confirmed cases and the number of deaths for all affected countries were extracted from the latter [23], while datasets for indicators that are prevailing or postulated as the risk factors of covid-19 mortality were extracted from the world bank database [24]. data extracted for this study was up until april 16, 2020. all data acquired were exported in excel format and arranged according to country rankings with the top having the highest number of confirmed cases as of april 16, 2020 and the bottom having the least. to facilitate comparison, out of about 185 affected countries, only top 50 countries were selected to be analyzed in this study. the following seven variables were included in the analysis, based on data availability and completeness: 1) proportion of people aged 65 above, 2) proportion of male in the population, 3) smoking prevalence, and 4) number of hospital beds. data analysis for each country, the percentage of confirmed covid-19 case per country was calculated by dividing the number of confirmed covid-19 cases by the total population for each country. also, cfr was calculated by dividing the number of deaths related to covid-19 by the confirmed covid-19 cases. linear regression analysis was conducted to determine the risk factors of cfr for covid-19. this method was 3 goh hp et al. chosen so that the degree of association can be quantified and that this estimate can be adjusted with other potential risk factors. for this analysis, two variables (cfr and number of hospital beds) were standardized due to differences in scale and very large range. standardization was done by subtracting each value by the mean and then dividing it with the standard deviation. however, data for four variables (diabetes prevalence, current health expenditure, and number of nurses and midwives) were not normally distributed. transforming these variables to normality were performed but model over-fitting occurred, therefore further analysis was not pursued. all analyses were conducted using microsoft excel and r (ver. 3.6.0). a p-value < 0.05 was considered as statistically significant. results information of 2,017,444 confirmed covid-19 cases and 137,166 deaths from each of the 50 top countries (supplementary information) were extracted. this constitutes about 93.3% and 92.8% of the global confirmed cases and and deaths on the data collection date (april 16, 2020). case fatality rate (cfr) and mortality rate of covid-19 in the top 50 countries from data extracted up until april 16, 2020, the united states (us) reported to have the highest number of total confirmed cases and the highest total number of deaths of 639,733 cases and 31,002 cases respectively. despite that, the us accounted about 0.20% (table 1) of confirmed cases and it had about 4.85% of cfr (table 1), indicating a moderate mortality rate of covid-19 in comparison to other countries. luxembourg, ranked 47th in the list of top 50 covid-19 countries, had the highest percentage of confirmed cases of 0.55% but a low 2.05% of cfr (table 1), indicating that a high percentage of confirmed cases does not necessarily lead to a high cfr. this is due to variations in number, transmission rate and severity of the disease regardless of the rankings [25]. hence, it is important to evaluate the possible factors that can affect the increase of covid-19 mortality rate globally. no. countries confirmed covid-19 cases (%) case fatality rate (%) 1 belgium 0.3 13.95 2 united kingdom 0.15 13.82 3 italy 0.27 13.11 4 france 0.2 12.77 5 netherlands 0.17 11.32 6 sweden 0.12 10.63 7 spain 0.39 10.46 8 indonesia 0.002 8.99 9 mexico 0.005 7.68 table 1: percentage of confirmed cfr and covid-19 cases (in sequence of the highest to lowest cfr%) 10 philippines 0.01 6.4 11 iran 0.1 6.24 12 brazil 0.01 6.07 13 dominican republic 0.03 5.23 14 romania 0.04 5.09 15 ecuador 0.05 4.94 16 united states 0.2 4.85 17 switzerland 0.31 4.8 18 denmark 0.12 4.54 19 colombia 0.01 4.22 20 china 0.01 4.01 21 poland 0.02 3.76 22 canada 0.08 3.56 23 ireland 0.26 3.54 24 india 0.001 3.4 25 portugal 0.18 3.33 26 germany 0.1 2.86 27 ukraine 0.01 2.79 4 28 panama 0.09 2.75 29 austria 0.16 2.73 30 czech republic 0.06 2.63 31 finland 0.06 2.23 32 norway 0.13 2.21 33 peru 0.04 2.21 34 turkey 0.08 2.19 35 korea, rep 0.02 2.16 36 japan 0.01 2.06 37 luxembourg 0.56 2.05 38 serbia 0.07 2.03 39 pakistan 0.003 1.85 40 thailand 0.004 1.72 41 malaysia 0.02 1.62 42 saudi arabia 0.02 1.3 43 chile 0.04 1.14 44 israel 0.14 1.11 45 australia 0.03 0.97 46 belarus 0.04 0.95 47 russian federation 0.02 0.83 48 united arab emirates 0.06 0.62 49 singapore 0.07 0.27 50 qatar 0.15 0.17 relationship between the different risk factors and covid-19 cfr the proportion of people aged 65 and above had a significant association with cfr (4.70 [95% ci: 0.34, 9.06], p = 0.04, table 2). for every 1-unit increase in the proportion of people aged 65 years, the cfr increased by 4.7 units. this relationship is illustrated in figure 1, where cfr sharply increases when the proportion of people aged 65 years is 0.18 and above. risk factors affecting covid-19... table 1: percentage of confirmed cfr and covid-19 cases (in sequence of the highest to lowest cfr%) (continued) independent variable β estimate (95% cl) standard errors p-value age (above 65 years) 4.70 (0.34, 9.06) 2.17 0.035 gender male -0.33 (-0.92, 0.26) 0.29 0.26 smoking prevalence (total % of people aged 15 and above) 0.009( -0.03, 0.04) 0.02 0.60 hospital beds (per 1,000 people) -0.097 (-0.39, 0.19) 0.14 0.50 table 2: linear regression analysis for different risk factors (independent variable) and covid-19 cfr (dependent variable) figure 1: association between case fatality rate and proportion of people aged 65 and above. discussion there are still a lot of unknown regarding covid-19 disease. however, good clinical findings are made available now to understand the risk factors that could affect its treatment outcomes. studies have shown that age is a clear risk factor for severe covid-19 disease. this has been confirmed by our study where the proportion of people aged 65 and above has shown a significant association with cfr. this indicates that countries with a higher proportion of people aged 65 and above may result in higher covid-19 mortality 5 goh hp et al. figure 1: association between case fatality rate and proportion of people aged 65 and above. rate (figure 1). bhatraju et al. (2020) has shown that in seattle, the us reported more than 60% of covid-19 deaths in patients aged 65 years and above than those who are younger than 65 years old [12]. verity et al. (2020) has shown that the a stark difference in the cfr between those aged below and above 60 years (1.4% versus 4.5%, respectively) [9]. this suggests that the older the country population, the higher the cfr. for those 80 years old and above, covid-19 appears to have a 13.4% fatality rate [9]. notably, the average age of deceased and recovered patients were found to be 68 and 51 years, respectively [13]. these studies show that covid-19 disproportionately impacts certain groups, and that older people [26] and pregnant women [27] are among the vulnerable groups. this could be due to the weakening effects of ageing on the immune system. as age increases, there is an increase of deficiency in t-cell and b-cell function and overproduction of type 2 cytokines [7]. this may promote viral replication and extend the duration of pro-inflammatory responses, leading to poor prognosis [7]. furthermore, older people tend to have more underlying conditions that may also be risk factors for severe covid-19 [9,14]. studies have shown that many of the severe covid-19 patients also have underlying medical conditions, such as diabetes and cardiovascular diseases [13,28]. patients with existing comorbidities, including hypertension, diabetes, cardiovascular disease and history of smoking, seems to be associated with covid-19 more severely [14]. with reference to a retrospective study of 113 deceased patients from covid-19, 48% of the patients had chronic hypertension and 14% of them had cardiovascular diseases [13]. in addition to that, covid-19 patients who have hypertension were closely associated with poor health outcomes after hospital admission. this may be due to factors such as vascular aging, reduced renal function and medication interactions [29]. although smoking prevalence has shown no significant association with covid-19 (p=0.60), it cannot be assumed that there is no association between other comorbidities and covid-19 cfr since not all factors were considered in this study, such as hypertension and cardiovascular diseases [6]. covid-19 is a rapid spreading communicable disease and general public are responsible in controlling this covid-19 pandemic. various levels of wellpreparedness plans are needed to tackle this pandemic situation [30]. these include availability of medicines or medical supplies to treat covid-19 patients, availability of suitable places to quarantine or self-isolate these patients, number of healthcare professionals and a strategized interventions, such as social distancing, quarantine, isolation actions and proper management, for the patients and public to flatten the curve and to reduce healthcare burden. it is necessary for some countries which may need more supplies than others to cater all sick patients, and thus increasing the health expenditure of the country [2]. some of the medical supplies include personal protective equipment, mechanical ventilators, covid-19 testing kits and extracorporeal membrane oxygenation. due to surge of demand on healthcare system, countries with low income and poor healthcare infrastructure suffer the most [14]. however, in our study, current health expenditure of the top 50 countries was not tested due to aforementioned reason. sufficient hospital capacity in term of hospital bed is necessary to accommodate unforeseen pandemic situations [31]. despite the insignificant association to covid-19 cfr (p=0.50), accessibility to adequate hospital beds for covid-19 patients can potentially affect the cfr as the number of beds required depends on the number of confirmed cases in each country. apart from hospital beds, other medical supplies such as medical grade face mask and mechanical ventilators must be sufficient as they are the key equipment for frontline healthcare workers [32–34]. evidence of high numbers of infections and deaths among healthcare workers due to lack of face mask and medical gowns were reported in italy [32]. in the us, recent estimates have suggested that the estimated ventilators needed is ranging from several hundred thousand to a million, far more than what are currently available [32]. it is difficult to estimate the exact number of ventilators needed as it depends on the number, transmission rate and severity of the disease in each country. 6 there are a number of limitations in this study. the nature of the study design (ecological study) and type of analysis used (linear regression analysis) allowed us to use countrylevel aggregated data to determine the relationship between cfr and specific risk factors. since aggregated data were used, this means that the results are only applicable at a country-level, instead of individual-level. some factors had to be excluded due to incomplete data such as malaria prevalence and bcg vaccination. even if the data were available, the data may come from different years. the years from which the data were retrieved were not consistent for all indicators. the data collected were also limited by unavailability of certain data to sufficiently make an overall conclusion for several factors, including comorbidities. there were four other proposed comorbidities to be analyzed but only two indicators’ datasets were available in world bank data, which are diabetes and smoking prevalence. of note, our study did not able to test the association between diabetes prevalence and cfr. therefore, more research should be conducted to further understand the relationship between comorbidities and cfr. this would help to identify and to better understand other possible factors that may also affect cfr. however, even with these limitations, it is important to note that the aim of our report is to determine the association between specific risk factors and covid-19 case fatality rate globally. also, our analysis is limited by data derived from confirmed covid-19 cases as of april 16, 2020, yet the number of global cases continues to increase. it is also important to note that transmissibility rate and various periods of the pandemic were not taken into account, as it may differ at different countries or even area within the same country [6,35,36]. conclusion as covid-19 is such a new disease, much still needs to be learned about it. age is a clear risk factor for severe covid-19 and death. covid-19 is an illness that disproportionately impacts older people. however, other risk factor such as smoking should not be neglected. prediction alone is not efficient, but well-planned and suitable interventions should also be carried out. in addition to that, potential risk factors need a lot more research in order to understand the risks for the worst forms of covid-19 and what we ought to learn to best protect the people with higher risk. participation and involvement of every individual, including patients, public and healthcare professionals, is necessary and everyone should work together towards combating covid-19 disease. conflict of interest none declared. funding none. author 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the creative commons attribution (cc by) license (http:// creativecommons.org/licenses/by/4.0/). 31. hick jl and biddinger pd. novel coronavirus and old lessons preparing the health system for the pandemic. n engl j med 2020; 382: e55. doi:10.1056/nejmp2005118. 32. ranney ml, griffeth v, jha ak. critical supply shortages the need for ventilators and personal protective equipment during the covid-19 pandemic. n engl j med 2020; 382: e41. doi: 10.1056/ nejmp2006141. 33. adams jg and walls rm. supporting the health care workforce during the covid-19 global epidemic. jama j am med assoc 2020; 323: 1439–1440. progress in microbes and molecular biology review article 1 an overview of the human immune system and the role of interferon regulatory factors (irfs) ashwinder kaur1, chee-mun fang2* 1school of pharmacy, the university of nottingham malaysia, selangor darul ehsan, malaysia 2division of biomedical sciences, school of pharmacy, the university of nottingham malaysia, selangor darul ehsan, malaysia abstract: the immune system consists of a dynamic network of cells, proteins, tissues, and organs that communicate to provide adequate defense responses against pathogenic agents. the immune system divide into the non-specific (innate) and the specific (adaptive) components, where the interactions between these two arms are intricately regulated. to deploy effective immune responses, immune systems comprise various cells and molecules that communicate with each other via signaling pathways coordinated by gene regulatory networks. the interferon regulatory factors (irfs) are critical regulators of both the immune system’s development and activation of different cells. to better understand the essential components of the normal immune system, this review essentially aims to cover the current knowledge of individual components of the immune system and the important role of irfs in regulating the immune system. keywords: interferon regulatory factor; innate; adpative; transcription factor; t cells received: 9th october 2020 accepted: 19th october 2020 published online: 28th october 2020 citation: kaur a and fang c-m. an overview of the human immune system and the role of interferon regulatory factors (irfs). prog microbes mol biol 2020; 3(1): a0000129. https://doi.org/10.36877/pmmb.a0000129. introduction an effective host defense system depends on prompt recognition and response against invaders. thus, the immune system produces various cells and molecules that communicate with each other via signaling pathways coordinated by gene regulatory networks. in this regard, the immune system, which is the host defense mechanism, is controlled by transcription factors and other elements that activate or repress their target genes in determining cell fate or effector state to ensure effective immune responses[1]. this gene regulatory network is controlled by transcription factors such as interferon regulatory factors (irfs), comprised of 9 family members (irf1-9) in mammals. although this family was initially identified in the type i interferon system, subsequent studies have revealed much broader functions performed by irf members in host defense. the irfs play crucial roles in the regulation of immune responses[2]. irfs function as central molecules that mediate different signaling pathways to induce the expression of interferons and inflammatory cytokines. furthermore, irfs also regulate immune cells’ development and activation and act as a bridge between innate and adaptive responses. the irfs family possesses a turn-helix turn motif that recognizes dna consensus, known as the ifn sensitive response element (isre), which can be found in the promoters of many genes involved in immune responses[3]. the immune responses and pathogenesis of certain diseases correlate with the balance of th1 and th2 responses[4,5]. for instance, an imbalance of th1/th2 responses, with th1 bias linked to autoimmune diseases such as systemic lupus erythematosus (sle) and rheumatoid arthritis (ra). on the other hand, th2-dominated responses are associated with allergic conditions such as asthma. interestingly, many irfs are involved in t helper (th) differentiation. for instance, irf1, irf2, and irf8 are mainly engaged in th1 differentiation[6,7]. meanwhile, irf4 that shares several similar biological activities with irf5 is critical for th2 cell development [6, 7]. irf4 and irf5, which are both involved in myd88-dependent toll-like receptor (tlr) signaling, found to interact with each other in the induction of proinflammatory cytokines and type i interferons[8]. furthermore, both of these transcription factors are shown to directly regulate blimp1, a master regulator of plasma differentiation[9,10]. therefore, we aim to provide an overview of the current knowledge of their roles in immune responses and immune cell development. the human immune system immune responses involve recognition of any “nonself” substances, including pathogens. these foreign pathogens or modified particles present in the host body, resulting in activation of cascade complex events copyright @ 2020 by kaur a and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) * correspondence: chee-mun fang, division of biomedical sciences, school of pharmacy, the university of nottingham malaysia, selangor darul ehsan, malaysia; cheemun.fang@ nottingham.edu.my 2 known as the inflammatory process to eliminate the nonself-substances[11]. following the inflammatory process, the immune system initiates restoration mechanisms involving a series of cellular and molecular events that mitigate the restoration of tissue homeostasis and resolve the inflammatory process[12]. on that note, immune responses are tightly regulated, involving various mechanisms to ensure normal homeostasis. when there is an imbalance of the immune system activity, it can lead to diseases such as autoimmune, chronic inflammatory, and cancer that can potentially be life-threatening[13]. in general, the immune system can be subdivided into two forms of protection known as innate and adaptive immune responses that work closely together to provide effective host defenses (figure 1). an overview of the human... figure 1. the innate and adaptive immunity in human. innate immunity innate immune response acts as the first line of defense. it is also known as non-specific defense mechanism that responds immediately upon recognizing a diverse array of microbial or “danger” signals due to changes in the homeostasis via the pattern recognition receptors (prrs) such as toll-like receptors (tlrs), nucleotide oligodimerization domain (nod) -like receptors (nlrs) and retinoic acid-inducible gene 1 (rig-1) like receptors (rlrs), which recognize pathogenassociated molecular patterns (pamps) and dangerassociated molecular pattern (damps)[14]. these recognition systems elicit distinct cellular responses depending on the nature of stimuli and responding cells. the significant roles of innate responses involve; a) prevent the entry of any foreign substances, b) initiate complement pathway for getting rid of pathogens, c) generation of local inflammatory responses, d) induce phagocytosis and cytotoxicity activities, e) facilitate wound healing and tissue repair and, f) activation of adaptive immune responses through antigen-presenting cells[11,15–17]. the innate immune system components consist of physical and chemical barriers, immune cells, and soluble factors that orchestrate rapid immune responses[18]. the physical barriers include epithelial layers of skin and the mucosal membrane that protect the external environment and exposure to foreign substances, including pathogens[18]. any breakdown or defect in the physical barrier increases the susceptibility of infection and leads to immune responses. chemical barriers consist of anti-microbial proteins and peptides, such as defensins permeable to microbes and induce cell death [18]. thus, physical and chemical barriers both play a crucial role in preventing the entry of foreign substances and infection. when pathogens breach protective physical and chemical barriers, they are combated through innate immune cells and soluble proteins. upon recognition of foreign molecules via the pprs, innate cells are activated following a cascade of signaling pathways that trigger transcription factors and other proteins to regulate various soluble factors’ gene expression to mount defense responses against the pathogens[18,19]. one of the key soluble factors of immune responses is cytokines. cytokines are peptides that act as mediators in cell communication and signaling. the role of cytokines is not isolated for innate immune responses, as they also play vital roles in adaptive immune responses. cytokines are broadly classified based on their immune responses and often have overlapping functions[17]. most cytokines are primarily but not exclusively produced by leukocytes called interleukin (il)[20–22]. interleukins can amplify their production in an autocrine or paracrine manner and induce or inhibit other cytokines’ production. they bind to their receptors on the cells that produce them (autocrine) or other cells (paracrine) and modulate the transcriptional program in determining the cells’ fate[20]. a distinct subset of cytokines named chemotactic cytokines or chemokines is predominantly involved in trafficking activation of 3 kaur a et al. cytokines for modulating other immune cells’ function to mount against the antigens. the principal cells involved in the adaptive immune response are apcs, b, and t-lymphocytes[23]. apcs refers to the specialized cells that internalize and process antigen, concomitantly presenting the antigen as peptide within mhc (also known as human leukocyte antigen, hla, the term designated for humans) on their cell surface[16,23]. there are two types of mhc complexes. mhc class i is expressed on all nucleated cells and present peptide antigens derived from intracellular antigens (e.g., viral proteins, autologous proteins, and tumor antigens)[21,23,27]. on the contrary, mhc class ii expression is predominantly restricted to apcs and presents peptide antigens synthesized from extracellular antigens (e.g., extracellular microbes, toxins, and allergens). examples of prominent apcs are dcs, macrophages, b cells, and thymic epithelium[27]. antigens processed by dendritic cells are displayed on their mhc class i and class ii are capable of activating naïve t cells into helper t cells (cd4+) or cytotoxic t cells (cd8+) subsets, respectively[15,26,27]. macrophages and b cells can also serve as apcs, presenting antigens to t cells during different type of immune responses[26]. a subset of dcs known as follicular dendritic cells can present antigens to b cells to establish the humoral immune response[20]. as mentioned previously, b cells and t cells are the lymphocytes which developed from lymphoid lineage originated from the hematopoietic stem cells (hscs) that share the same common lymphoid progenitors with innate lymphoid cells (e.g., nk cells). these lymphocytes undergo complex maturation by which they express surface receptors that dictate their functions and phenotypes. upon recognizing and binding the antigen-specific to their surface receptors, b and t cells undergo activation, proliferation (clonal expansion), and differentiation to effector cells and memory cells[11]. unlike b and t cells, innate lymphoid cells are not clonally expressed for specific antigens, serving the innate defense system[23]. b cells comprise several subsets classified based on their ontogeny and anatomical location in[28]. for example, b1 and b2 b cells are associated with antibody productions and regulatory b cells (bregs), which are essential for suppressing autoimmune and inflammatory responses. b cells’ developmental and maturation process involves structural and functional rearrangement of their receptors within the bone marrow[29]. b cells express membranebound immunoglobulin (ig) receptors on their surface and produce soluble antibodies of the receptor’s same antigenic specificity. mature b cells (express membrane-bound igm and igd) migrate to peripheral lymphoid organs or lymph nodes via the circulatory, where they encounter antigens to establish humoral immunity[28,29]. depending on the nature of antigens encountered and the subset of b cells involved. these b cells can be activated either with the involvement of activated helper t cells that express cd40l and the cytokines produced by them (t-dependent b cell activation) or without the involvement of helper t cells (t-independent b cell activation), which is usually facilitated by tlr stimulation[29,30]. following the activation of b cells, the b cells undergo leukocytes to inflammatory sites[17,22,23]. concerning immune cells, the cells are developed from hematopoietic stem cells (hscs) within the bone marrow[24]. as these cells mature, they can be differentiated into two main lineages, myeloid progenitor cells (neutrophils, basophils, eosinophils, monocytes, macrophages, mast cells, and dendritic cells) and lymphoid progenitor cells (b cells, t cells, and innate lymphoid cells)[24]. some of the primary innate immune cells include the granulocytes that contain granules sacs containing enzymes and inflammatory proteins (neutrophils, basophils, eosinophils, and mast cells)[24]. among these cells, neutrophils are the most abundant leukocytes whose primary function in host defense is to patrol and guard the immune system against invaders[17,24]. while basophils and eosinophils are responsible for defense against helminth and allergic-related diseases through the degranulation process[20,24]. mast cells primarily reside in the tissues rather than in the circulatory, unlike the other granulocytes[20]. mast cells play an important role in triggering inflammatory response as well as participate in wound healing [24]. meanwhile, another group of innate cells; monocytes, macrophages, and dendritic cells, belong to the mononuclear phagocyte system that plays multiple roles during inflammation[25]. monocytes circulate in the circulatory with a rather short life span and may differentiate into tissue macrophages or dendritic cells depending on the surrounding stimuli[17,25]. both macrophages and dendritic cells are also known as the antigen presenting cells (apcs), as they capable of processing and presenting foreign protein-based molecules (antigens) to lymphocytes[20,26]. in terms of protection against virally infected cells and tumor cells, other innate cells known as natural killer (nk) cells are well known for its function in killing infected and transformed cells that managed to escape t cell recognition[24]. adaptive immunity the adaptive immune response is highly specific. it involves recognizing antigens (foreign agents and particles) via the receptors bound to the surface of b lymphocytes and t lymphocytes, which are unique to different antigens[23]. this second defense mechanism is a contingent of the innate immune system and initiated in the later onset of infection. it provides excellent defense responses against persistent infection and, importantly, possesses immunological memory[15,23]. when the same or closely related antigens are encountered, the immunological memory program is activated, and the adaptive immune system provides rapid and enhanced protection. the adaptive immunity is broadly divided into humoral-mediated immune responses and cell-mediated immune responses coordinated by b cells and t cells, respectively[21,23]. humoral mediated immune responses involve the production of antibodies by b cells against soluble antigens such as extracellular microbes and toxins. on the contrary, cell-mediated reactions involve activation of effector t cells such as cytotoxic t cells that kill intracellular microbes and tumor cells. these are inaccessible to antibodies and t helper cells that produce 4 clonal expansion and differentiate into plasma cells that produce igm and igd type antibodies designed to mount against the particular antigens[28]. besides, most of the b cells become effector cells, plasma cells that further interact with other stimuli such as cytokines in the local microenvironment. while the plasma cells capable of producing different classes of antibodies other than igm and igd (iga, igg, and ige) through the process called immunoglobulin class switching[28,29]. some of the b cells become memory cells that preserve the “information” for those successful antibodies generated against the antigen and provide robust protection if the same antigens are encountered[29]. the functions of antibodies are to neutralize virulence factors of antigens and enhance the complement pathway and phagocytes’ activation to eliminate the antigens[11,18,23]. however, antibodies can also contribute to autoimmune diseases’ pathogenesis due to disrupted self-tolerance mechanisms leading to the generation of autoantibodies as a consequence of b cells reacting against self-antigens (particles of host body)[28]. unlike b cells that can recognize antigens in the extracellular spaces, t lymphocytes have restricted specificity for antigens as they identify and respond to surface-bound antigens displayed on the mhc of apcs. interferon regulatory factors (irfs) family transcription factors are key players during gene expression as they facilitate when and what gene is “turn on or off” by binding to dna sequences, acting as coactivator or co-repressor of the gene response to various signals[31]. structurally they comprise of two domains: a) dna binding domain that recognizes and bind to dna sequences, b) activation (effector) domain which interacts with cofactors or other transcription factors[32–34]. some of them also have an additional domain (signalsensing domain) that binds to ligands to modulate their activity response to environmental cues[32,33]. transcription factors are categorized into several groups based on their dna binding domain structures and their interaction with dna sequences[33–35]. some of these transcription factors are known as general transcription factors (gtfs), which are ubiquitous and essential for initiating transcription in the protein-coding gene[31,34]. other transcription factors are either constitutive or inducible and are specific to certain cell types and stages of organism development[31,34]. several of these transcription factors function as master transcriptional regulators in controlling signal responses and specifying cells’ lineage[31,36]. also, transcription factors play a crucial role in interacting with histone proteins, which influence chromatin state and establish the environment that allows interactions for activation or repression of gene transcription[33,37]. one group of transcription factors that have been extensively studied for their crucial role in regulating gene networks in the immune system is the interferon regulatory factors (irfs), which possess the turn-helix turn motif[38]. the first irfs member, irf1, was initially identified as a factor that binds to the human interferon’s upstream regulatory region (ifn) β gene and induced its expression[39]. over decades, the family of irfs has been expanded to ten members, to which irf1 to 9 are found in mice and humans, whereas irf10 is only found in chickens[2]. also, several viral irfs that interact with cellular irfs have been identified[40]. all irfs consist of two domains, the n-terminal (dna binding domain) and c-terminal (regulatory domain). the n-terminal binding domain is enriched with five tryptophan repeats that are well conserved among all irf. this region recognizes a dna consensus, known as ifn sensitive response element (isre) found in the promoters of type 1 ifn, ifninducible genes, and many other genes involved in immune responses[3]. on the other hand, the c-terminal consists of the irf association domain (iad) responsible for homo and heteromeric interaction with other family members or transcription factors[38,41]. irfs functions are not limited to, regulating innate and adaptive immune responses. these transcription factors also play a crucial role in controlling type 1 ifn induced by viruses and pathogens involved in the cell cycle, apoptosis, and oncogenesis[2,3,38,41]. some of these irfs regulate immune cell development and functions[2,6,7,42]. accumulating data from several studies has shown that irf5 is a critical mediator in developing th1 responses associated with the pathogenesis of various diseases, including autoimmune, metabolic, and infectious diseases[43]. conversely, studies have also demonstrated that irf5 plays a role in inducing th2 responses[44,45]. strikingly, most studies have attributed irf5 in regulating th1/th2 reactions by altering antigen-presenting cells (e.g., macrophages and dendritic cells) rather than irf5 intrinsic properties in t cells. perhaps because early studies reported that expression of irf5 is barely detected in t cell[46]. nonetheless, recent studies have detected elevated irf5 expression in parasitic and viral infected t cells[47,48] and have highlighted its direct role in a t cell subset during chronic parasitic infection[49]. fabié et al. (2018) [49] demonstrated that tlr-7 mediated activation of irf5 promoted ifnγ+ cd4 t cell death because of its ability to enhance death receptor 5 (dr5)-induction cell apoptosis in cd4 t, thus promoting persistent l. donovani infection. however, the role of t cell-intrinsic dependent on irf5 in modulating th cytokine production remains uncertain. roles of irfs in t helper differentiation over the years, many irfs are involved in t helper differentiation either by modulating the functions of antigen presenting cells or directly altering the transcription of cytokine genes of th cells[7]. irf1 is a crucial factor in promoting th1 differentiation and its absence results in predominant th2 responses as best characterized using experimental mice disease model. for example, irf1 deficient mice (irf1-/-) were vulnerable to leishmania major (l. major) and listeria monocytogenes (l. monocytogenes) because the host could not control the parasitic infection due to a lack of th1 responses, which is needed for protection against the intracellular parasitic infections[50,51]. collectively, irf1, irf2, and irf8 directly participated in th1 differentiation predominantly by regulating the production of il-12, the th1 signature cytokine. an overview of the human... 5 on the other hand, irf4 is critical for th2 cell development. mice lacking irf4 showed to be able to mount th1 immune response against l. major, but the th2 development was impaired[59]. also, cd4+ t cells of irf4-/mice were unable to differentiate into th2 cells and displayed impaired production of th2 cytokines; il-4, il-5, and il-13[60]. moreover, overexpression of irf4 in human jurkat t cells activated th2 cytokines’ expression in response to mitogenic stimulation[61]. consistently, in the absence of irf4, il-4 failed to induce th2 cytokine, instead induced th1 response by upregulating ifnγ and tnf expression, thereby implying the importance of irf4 in th2 differentiation[62]. in accordance with the role of irf4 in th2 differentiation, honma et al. (2008)[63] reported that irf4 plays a dual role in different stages of cd4+ t cells. irf4 inhibited th2 cytokine production in naïve cd4+t cells but induced th2 cytokine production in effector/ memory cd4+ cells. however, irf4 function is restricted to th2 cell differentiation since several studies have shown that irf4 is vital in generating th9, th17 and tregs cells[6]. taken together, irf4 is able to differentially control the th subsets and act as th modulator. on the other hand, irf5 plays a pivotal role in modulating t helper responses by altering the functions of antigen-presenting cells such as macrophages and dendritic cells and influencing the cytokine production drives the th differentiation as well as participates in activation downstream of tlr-mediated signaling[3,64]. conclusion the immune system protects against invaders such as microbial pathogens, toxic agents, and transformed malignant cells. the crosstalk between innate and adaptive immunity ensures an effective host defense system, mediated by various cells and molecules that work dynamically. in terms of t cells, upon activation, naïve cd4+ t cells acquire the ability to differentiate into several subsets, including t helper 1 and t helper 2 cells mediated by specific cytokine signaling and transcription factor. the positive vital regulators involve in th1 differentiation are t-bet, ifnγ/stat1, and il-12/stat4; meanwhile, for th2 differentiation are gata3 and il-4/stat6. besides the master transcription factor, several other transcription factors such as the runx3, hlx, irf1, dec2, irf4, nfat, gif-1, c-maf, and jun-b are known to cooperate with the subset specific master transcription factor that collectively involved in establishing th1/th2 differentiation. although specific cytokines expressions and transcription factors of t helper subsets are exclusive to each subset, they can still present in both states and fine-tune the t helper cells capabilities for polarization when the situation requires. this can be exemplified clearly by il-2, which is essential for both th1 and th2 differentiation. collectively, the immune cells orchestrate a complex network made of transcription factors, in cooperation with other proteins involved in cytokine signaling. in which subsequently influences the chromatin state of the targeted genes, resulting in activation or repression of the genes, thereby eliciting the required immune responses in a given state. authors contributions ak and c-mf performed the literature review and manuscript writing. c-mf conceptualizes the research project. conflict of interest the authors declare that there is no conflict of interest in this work. acknowledgement this project is funded by the ministry of higher education, malaysia, fundamental research grant scheme (frgs/1/2013/skk01/musm/03/03). references 1. singh h, khan aa, dinner ar, et al., gene regulatory networks in the immune system. trends immunol, 2014; 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nsyaza7@graduate.utm.my (nsa); jitonglin1998@gmail.com (jtl); hairudin@utm.my (ham); aszuraini@utm.my (zas); huiwennies@utm.my (nhw); cwenghowe@utm.my (cwh) *corresponding author: azurah a samah; faculty of computing, universiti teknologi malaysia, 81310 johor bahru, johor, malaysia; azurah@utm.my (aas) abstract: biological data obtained from sequencing technologies is growing exponentially. multi-omics data is one of the biological data that exhibits high dimensionality, or more commonly known as the curse of dimensionality. the curse of dimensionality occurs when the dataset contains many features or attributes but with significantly fewer samples or observations. the study focuses on mitigating the curse of dimensionality by implementing support vector machine – recursive feature elimination (svm-rfe) as the selected feature selection method in the lung cancer (lusc) multi-omics dataset integrated from three single omics dataset comprising genomics, transcriptomics and epigenomics, and assess the quality of the selected feature subsets using sdae and vae deep learning classifiers. in this study, the lusc datasets first undergo data pre-processing, including checking for missing values, normalization, and removing zero variance features. the cleaned lusc datasets are then integrated to form a multi-omics dataset. feature selection was performed on the lusc multi-omics data using svm-rfe to select several optimal feature subsets. the five smallest feature subsets (fs) are used in classification using sdae and vae neural networks to assess the quality of the feature subsets. the results show that all 5 vae models can obtain an accuracy and auc score of 1.000, while only 2 out of 5 sdae models (fs 1000 & 4000) can do so. 3 out of 5 sdae models have an auc score of 0.500, indicating zero capability in separating the binary class labels. the study concludes that a fine-tuned supervised learning vae model has better capability in classification tasks compared to sdae models for this specific study. additionally, 1000 and 4000 are the two most optimal feature subsets selected by the svm-rfe algorithm. the sdae and vae models built with these feature subsets achieve the best classification results. pmmb 2023, 6, 1; a0000327 2 of 23 keywords: multi-omics analysis, support vector machine – recursive feature elimination (svm-rfe), stacked denoising autoencoder (sdae), variational autoencoder (vae) 1. introduction despite the availability of various cancer treatments, cancer continues to be a leading cause of death worldwide. among the fatal types of cancer is lung cancer[1]. in 2020, it accounted for 11.4% of total cancer cases and took the lives of around 1.8 million people[2]. omics refers to several fields of study in life sciences that focus on much information to understand life[3]. multi-omics, on the other hand, is formed when two or more omics types are combined to allow the study of the biological phenomenon in a more holistic way, which in turn improves the prognosis and predictive accuracy of disease phenotype, allowing a better treatment and prevention of cancers to be facilitated[4]. the study primarily focuses on the curse of dimensionality of multi-omics data, also known as the large p small n problem, whereby the multi-omics dataset has a small number of samples (n) and a large number of features (p)[5]. the nature of multi-omics data analysis that requires the researchers to merge multiple omics data into one usually limits the number of observations for the multi-omics data[6], as the integration process requires the data from the same individual or patient to exist in every omics type involved in the study[7]. the study employs support vector machine – recursive feature elimination (svmrfe) as the feature selection algorithm to address this problem. the study aims to use svmrfe to select only the relevant features from the lung cancer multi-omics data to develop better deep-learning classifiers. next, stacked denoising autoencoder (sdae) and variational autoencoder (vae) are used in the binary classification of the selected feature subsets. the objectives of the study include: 1) to study and understand the algorithm of svm-rfe, sdae, and vae, 2) to determine suitable parameters for the selected algorithms and apply appropriate fine-tuning methods to them, and 3) to validate and verify the performance of svm-rfe using sdae and vae. the remaining sections of this paper are structured as follows: section ii provides a comprehensive review of relevant literature for this study, section iii outlines the methods and procedures employed to conduct the analysis, section iv presents the results and subsequent discussion of the model performance, and finally, section v provides the conclusion. 2. materials and methods the experimental workflow of the research is summarized in figure 1. in general, the procedure of the study starts with data acquisition, followed by data cleaning, multi-omics integration, feature selection, and classification. the results and findings of the study are then discussed. pmmb 2023, 6, 1; a0000327 3 of 23 kl figure 1. the experimental workflow of the research. 2.1. data acquisition this study's lung cancer omics dataset is retrieved from an open-source website http://acgt.cs.tau.ac.il/multi_omic_benchmark/download.html. the dataset comes in a package of 4 files: the 3 omics datasets (i.e., gene expression, dna methylation expression & mirna expression) and 1 clinical dataset. all 4 datasets contain the patient id, vital for http://acgt.cs.tau.ac.il/multi_omic_benchmark/download.html pmmb 2023, 6, 1; a0000327 4 of 23 column concatenation in multi-omics integration. a quick summary of the dimensions of each dataset is tabulated in table 1. table 1. summary of the dimensions of the raw lung cancer omics datasets. data omics field num. of features num. of patients gene expression genomic 20531 553 dna methylation epigenomics 5000 413 mirna transcriptomics 1046 388 clinical data 626 127 the class labels in the clinical dataset are binary, containing one positive outcome (has lung cancer) and one negative outcome (no lung cancer). the positive outcome is denoted as "primary tumour" while the negative outcome is denoted as "solid tissue normal". 2.2. data preprocessing the acquired datasets undergo a data-cleaning process to prepare the data for further analysis. the 2 types (i.e., omics dataset & clinical dataset) are cleaned differently. the omics datasets are cleaned by performing data transposition, imputation, normalization, and variance threshold analysis. the patient id and the class label are extracted from the clinical data. 2.2.1. data transposition despite being labeled as pre-processed, the omics datasets still contain certain caveats which require further processing. first, the rows and columns of the data of the raw omics dataset are inverted and misleading. data transposition is performed on all 3 omics datasets to correct the orientation of the data to be represented. after data transposition, the rows now represent the samples/instances corresponding to the patient id, while the columns now represent the omics expression values. the dimensions of the omics datasets before and after data transposition is summarized in table 2. table 2. the dimensions of the omics datasets before and after data transposition. dataset dimension (row, column) raw dataset after data transposition gene expression (20531, 552) (552, 20531) dna methylation (5000, 412) (412, 5000) mirna (1046, 387) (387, 1046) pmmb 2023, 6, 1; a0000327 5 of 23 2.2.2. data imputation the next step was data imputation. these steps involved checking for duplicated rows and missing values (or nan) and imputing them with a value zero, as missing values tend to reduce the study's statistical power and produce biased estimations[8]. the result of the checking shows that all 3 omics datasets contain neither duplicated rows nor missing values. 2.2.3. data normalization and variance threshold analysis the omics datasets then undergo data normalization. the values of each feature in each omics dataset are adjusted and scaled between 0 and 1 to improve the data quality and the machine learning model[9]. the data cleaning phase ends with variance threshold (vt) analysis. zero variance features (i.e., features with only one unique value or the value for each sample in a particular feature are the same) are dropped as they do not provide any predictability to the output class[10]. a total of 287 and 160 zero variance features are removed from the gene expression and mirna expression omics data, respectively. the dna methylation expression dataset contains no zero-variance feature. the summary of the vt analysis is shown in table 3. table 3. the dimensions of the omics datasets before and after variance threshold analysis. dataset dimension (row, column) before vt after vt gene expression (552, 20531) (552, 20244) dna methylation (412, 5000) (412, 5000) mirna (387, 1046) (387, 886) clinical data (626, 127) (626, 127) 2.3. multi-omics integration in multi-omics integration, the columns from each cleaned single omics dataset are concatenated by using the patient id as an index. meaning the integrated multi-omics dataset will only contain the information of the patients whose information is present in all 4 datasets. the summary of the datasets before and after data preparation and multi-omics integration is shown in table 4. pmmb 2023, 6, 1; a0000327 6 of 23 table 4. summary of the datasets before and after data preparation and multi-omics integration. dataset dimension of the dataset (row, column) raw dataset data transposition variance threshold multi-omics integration gene expression (20531, 552) (552, 20531) (552, 20245) (344, 26131) dna methylation (5000, 412) (412, 5000) (412, 5000) mirna (1046, 387) (387, 1046) (387, 886) clinical data (626, 127) (626, 127) (626, 1) it is worth noting that the class label distribution before and after multi-omics integration has changed drastically. table 5 summarizes the class label distribution for each omics dataset, including the integrated multi-omics data. before integration, each single omics dataset has a class label distribution of around 90:10 for primary tumour and solid tissue normal. the distribution changed to 99:1 when the multi-omics data was integrated. the multi-omics data is now severely imbalanced. table 5. class label distribution with percentage for each omics dataset. omics primary tumour solid tissue normal total sample gene expression 501 (90.8%) 51 (9.2%) 552 dna methylation 370 (89.8%) 42 (10.2%) 412 mirna expression 342 (88.4%) 45 (11.6%) 387 multi-omics data 341 (99.1%) 3 (0.9%) 344 the integrated multi-omics dataset undergoes data splitting, as shown in figures 2a and 2b. first, the multi-omics data is split into the train-test set with a ratio of 70:30, which empirically produces the best result[11]. for feature selection with svm-rfe (figure 2(a)), the train set is used in stratified 2-fold cross-validation (cv). for classification with sdae and vae (figure 2(b)), the train set is further split into train and validation sets with a 70:30 ratio. the class distribution of the train and test set after data splitting are shown in figure 3. pmmb 2023, 6, 1; a0000327 7 of 23 figure 2a. the data splitting of the multi-omics data for feature selection. figure 2b. data splitting for deep learning unsupervised training. figure 3. the class distribution of the multi-omics data for the train and test set. pmmb 2023, 6, 1; a0000327 8 of 23 2.4. feature selection with the integrated multi-omics data, the study proceeds with a wrapper feature selection svm-rfe. wrapper methods use a classification algorithm to assess the significance of data features. the classifier is encapsulated in a search algorithm to identify the optimal subset of features[12]. svm-rfe is responsible for selecting the 𝑛 most relevant features, whereby 𝑛 is the number of features to be selected. the study aims to select several subsets of features to assess the most optimal number of features to be explicitly selected for this study. a total of 20 feature subsets (abbreviated as fs from now on) are selected, which range from 20000, 19000, 18000, …, 1000. the most optimal set of hyperparameters for svm-rfe is determined using grid search. the hyperparameter grid used in the search is summarized in table 6[13]. the "c" parameter controls the tradeoff between the correctly classified instances and the capability of the hyperplane to separate instances. "linear" kernel is the only kernel that produces feature importance as one of its outputs for the rfe algorithm to rank the feature. "step" is the hyperparameter for rfe, whereby it decides the number of features to remove in each iteration. table 6. hyperparameter grids used for svm-rfe. hyperparameter values c 0.1, 1, 10, 100 kernel linear step 1, 2, 3 to obtain early insight regarding the 20 selected feature subsets, an svm model with a similar set of hyperparameters shown in table 6 is used to classify each feature subset. a 2-fold cv is employed to obtain a more generalized result. the omics composition is also observed for each feature subset. ultimately, the output of the svm-rfe algorithm is the 20 selected feature subsets, which are used as inputs for the deep learning models for classification. 2.5. smote the issue with data imbalance for the integrated multi-omics data, addressed in 2.3 is handled here. the study employs a data oversampling, namely smote, on the training set. smote creates synthetic examples to oversample the minority class instead of replacing them with unfamiliar samples[14]. the hyperparameter chosen for smote is listed in table 7. "sampling_strategy" is set to 1 so that the newly synthesized instances with minority class label (solid tissue normal) will match the number of the instances with "primary tumour". "k_neighbors" decides the number of nearest data points to use as references to synthesize new data points. it is forced to set to 1 since "k_neighbour" has to be smaller than the number of minority classes. "random_state" is set to 42 to allow reproducible results. pmmb 2023, 6, 1; a0000327 9 of 23 table 7. hyperparameters used for smote on train data. hyperparameters settings sampling_strategy 1 k_neighbors 1 random_state 42 the result of smote on the training set is depicted in figure 4. now, the training set for the multi-omics data is balanced with the equal number of samples on either class label. however, the testing set is still severely imbalanced. figure 5 compares the class distribution between the training and testing set. figure 4. the class distribution for the multi-omics train set before and after smote. figure 5. the comparison of class distribution for the training and testing set of the multi-omics data. 2.6. deep learning models deep learning, which involves using artificial neural networks structured in layers to enable learning, has found application in cancer research[15–17] and other medical fields such as dental research and molecular biology[18–24]. the study includes two deep learning models: sdae and vae, to validate and assess the feature subsets selected by svm-rfe in 2.4. at pmmb 2023, 6, 1; a0000327 10 of 23 the same time, due to hardware constraints, whereby the memory for the gpu used is insufficient for large neural networks, the study only incorporates fs 5000, 4000, 3000, 2000, and 1000 in classification. the setup for the experiment using the two models is specified in their respective subchapters. 2.6.1. sdae the sdae model building starts with unsupervised learning. the main function for unsupervised learning is to train the sdae model to learn the important features from each feature subset by encoding them into a smaller dimension (latent layer). the model loss during the unsupervised learning phase is recorded to assess the capability of the model to reconstruct the given inputs. figure 6(a) shows the neural network of the sdae model during unsupervised learning. figure 6a. the neural network structure of the sdae model for unsupervised training. pmmb 2023, 6, 1; a0000327 11 of 23 figure 6b. the neural network structure of the sdae model for supervised training. the hyperparameters used for the sdae model during unsupervised training is tabulated in table 8. there are 8 layers in the neural network, including the gaussian noise layer. each layer in the encoder part of the model reduces the dimension of the input by 30%. the original input is encoded into 10% of its original dimension at the latent layer. for the decoder part, each layer increases the dimension by 30%. the dimension is restored to 100% at the output layer, in which the original input is attempted to be reconstructed. the epoch and batch size are set to 50 and 16, respectively, which is observed to allow the model to minimize the model loss to a converging point[25]. with reference to[26], the activation functions used the hidden layers, and the output layer is set to "relu" and "sigmoid" respectively. "adam" optimizer is used as it is the most recommended optimizer and is being adapted as the benchmark for deep learning models[27]. binary cross entropy is used as the loss function as the objective of the sdae model is classification[28]. the gaussian noise layer introduced at the input layer uses 10% of the dropout rate to aid the model learning[29]. the unsupervised learning sdae model is then fine-tuned into a supervised learning model. this is done by replacing the decoder part of the model with a new layer that contains only 1 node with a sigmoid activation function, as shown in figure 6(b). this layer acts as the new output layer. it allows the sdae model to output values between 0 and 1 to represent the binary classes (solid tissue normal & primary tumour), which turns the model into a supervised learning model capable of performing classification. the hyperparameters used during the unsupervised training phase are kept unchanged except for the layers. pmmb 2023, 6, 1; a0000327 12 of 23 table 8. hyperparameters used for sdae during unsupervised and supervised training. hyperparameters unsupervised learning supervised learning layers 5000, 5000 (noisy), 3500, 2000, 500, 2000, 3500, 5000 100%, 100% (noisy), 70%, 40%, 10%, 40%, 70%, 100% 5000, 5000 (noisy), 3500, 2000, 500, 1 100%, 100% (noisy), 70%, 40%, 10%, 1 epoch 50 50 batch size 16 16 optimizer adam adam activation functions relu – hidden layers sigmoid – last layer (output layer) relu – hidden layers sigmoid – last layer (output layer) loss function binary cross entropy binary cross entropy gaussian noise dropout rate 10% 10% 2.6.2. vae the vae model building follows a similar fashion. it also starts with unsupervised learning to learn the features from each feature subset and encode them into a smaller dimension. a sampler is incorporated to generate new data points according to the mean and variance learned from the previous layers. the model then reconstructs the original input according to the sampled data[30]. similarly, the vae model is assessed based on the model loss. figure 7a shows the neural network of the vae model during unsupervised learning. pmmb 2023, 6, 1; a0000327 13 of 23 figure 7a. the neural network structure of the vae model for unsupervised training. figure 7b. the neural network structure of the vae model for supervised training. pmmb 2023, 6, 1; a0000327 14 of 23 table 9 shows the hyperparameters used for the unsupervised training of the vae model. the hyperparameters used are very similar to those used in the unsupervised learning of the sdae model. however, the difference between the two models lies in the activation and loss functions. with reference to[29] in their work, the activation function for the sampler is set to "linear". two different loss functions are used for the vae model. the generative loss measures the overall reconstruction loss of the model, while the kullback-leibler (kl) loss measures the difference between the two probability distributions. the total loss of the vae model is the combination of both loss functions[29]. table 9. hyperparameters used for vae during unsupervised training. hyperparameters unsupervised learning layers 5000, 3500, 2000, 500, 2000, 3500, 5000 100%, 70%, 40%, 10%, 40%, 70%, 100% epoch 50 batch size 16 optimizer adam activation functions relu (rectified linear unit) – hidden layers linear – bottleneck layer (latent layer) sigmoid – last layer (output layer) loss function generative loss kullback-leibler (kl) loss classification cannot be done directly by the vae model itself by using a similar fine-tuning method in the supervised learning of the sdae model. this is because the accuracy produced fluctuates around 50%, which is not in line with the baseline accuracy of the data. an external classifier is used to aid the vae model in classification. this is done by extracting the data points sampled by the sampler in the latent layer and feeding them to the external classifier. in this study, svm is the chosen external classifier. to keep it simple, the hyperparameters of the svm model are kept as default as shown in table 10. table 10. hyperparameters used for svm as external classifier. hyperparameters description c 1.0 kernel linear pmmb 2023, 6, 1; a0000327 15 of 23 3. results the output of the svm-rfe algorithm is shown here. besides that, the sdae and vae models are built with the selected feature subsets by the svm-rfe, and the classification results are shown. 3.1. svm-rfe with the grid search implemented to determine the best set of hyperparameters for the svm-rfe, it has been determined that the most optimal set of hyperparameter are 𝐶 = 0.1, linear kernel and 𝑠𝑡𝑒𝑝 = 1 as shown in table 11. the total computation time for the svm-rfe to finish selecting 20 feature subsets is recorded at 3 hours and 9 minutes. table 11. the selected set of hyperparameters for the feature selection using svm-rfe for each feature subset. feature subset computation time c 0.1 kernel linear step 1 the initial classification result on the 20 feature subsets of multi-omics data is shown in figure 8. it is observed that from fs 20000 to 14000, the mean accuracy is recorded at 0.996. while the mean accuracy for fs 13000 to 1000 is recorded at 1.000. figure 8. boxplot for the cv result (accuracy) for each feature subset. the omics composition for each feature subset is depicted in figure 9. the general trend observed is that the composition of gene expression omics goes down (75.08% to 70.50%) as the size of the feature subset reduces, while the composition of dna methylation expression rises (22.77% to 27.94%). this trend is observed until fs 5000, in which the trend is observed to go the opposite way, whereby the composition of gene expression goes up while the composition of dna methylation goes down. the initial composition of mirna pmmb 2023, 6, 1; a0000327 16 of 23 expression at 2.15% fluctuates as the size of the feature subset goes down until it settles at 1.8%. figure 9. omics composition after svm-rfe represented in bar chart. 3.2. sdae the model loss of the developed sdae models using fs 1000 to 5000 during the unsupervised learning phase is recorded in figure 10. generally, the model loss for each sdae model is low at below 0.5. it is noted that only the model loss for the validation in fs 5000 resembles closer to the training set compared to the sdae models built with other feature subsets. figure 10. model loss for the unsupervised learning for the sdae model. pmmb 2023, 6, 1; a0000327 17 of 23 the classification results of the fine-tuning supervised learning sdae models are recorded. figure 11 shows the accuracy of the sdae models with respect to epoch, while figure 12 shows the confusion matrix obtained from the classification result of the last epoch. it is observed that fs 1000 and 4000 can correctly classify all the instances, while fs 2000, 3000, and 5000 cannot classify the negative class label correctly, resulting in one false positive. the confusion matrix tabulates the accuracy, auc score, precision, recall, and f1 score in table 12. figure 11. the classification result for the fine-tuned supervised learning sdae model. the accuracy of supervised learning for the sdae model. figure 12. the confusion matrix from the classification using the fine-tuned sdae model. pmmb 2023, 6, 1; a0000327 18 of 23 table 12. metrics obtained from the classification result of the sdae model. feature subset accuracy auc score precision recall f1 score 5000 0.9904 0.5000 0.9904 1.0000 0.9951 4000 1.0000 1.0000 1.0000 1.0000 1.0000 3000 0.9904 0.5000 0.9904 1.0000 0.9951 2000 0.9904 0.5000 0.9904 1.0000 0.9951 1000 1.0000 1.0000 1.0000 1.0000 1.0000 3.3. vae the unsupervised learning vae models are developed using fs 1000 to 5000. the total model loss of the model is obtained using the combination of the generative loss and the (kullback-leibler) kl loss, as shown in figure 13. generally, the total model loss for the vae models decreases as the size of the feature subset used to develop the vae models decreases. it is observed that both the total model loss for the training and validation sets can converge, but fluctuation is also observed for the validation sets. figure 13. the overall loss of the vae model during the unsupervised learning phase. the classification of the vae model involves using an external classifier, an svm model. the study first built a supervised learning vae model using the same method applied for fine-tuning the sdae model. however, it is observed that the accuracy produced by the fine-tuned vae models is around 50%, which is far below the baseline accuracy for this testing set at 99.04% (refer to table 13). this leads to the use of an external classifier for the classification of the vae model. pmmb 2023, 6, 1; a0000327 19 of 23 to achieve this, the sampled data by the sampler in the latent space for each vae model are extracted and fed to the svm classification model. the hyperparameters used for the svm model are kept as default at c = 1 with linear kernel. the classification of the sampled data by the vae model using the svm classifier is shown as a confusion matrix in figure 14. figure 14. confusion matrices were produced using svm's classification results as the external classifier on the encoded inputs in the vae model. the accuracy, auc score, precision, recall, and f1 score are calculated from the confusion matrix. the results are tabulated in table 13. in table 13, the metrics from the fine-tuned vae model and the svm model are displayed side-by-side to demonstrate the difference in performance between the two classification methods. table 13. comparison between the metrics obtained from the fine-tuned supervised learning vae model's classification result and the svm classification. feature subset accuracy auc (roc) precision recall f1 score ft svm ft svm ft svm ft svm ft svm 5000 0.500 1.000 0.252 1.000 0.981 1.000 0.505 1.000 0.667 1.000 4000 0.481 1.000 0.738 1.000 1.000 1.000 0.476 1.000 0.645 1.000 3000 0.462 1.000 0.728 1.000 1.000 1.000 0.456 1.000 0.627 1.000 2000 0.529 1.000 0.267 1.000 0.982 1.000 0.534 1.000 0.692 1.000 1000 0.471 1.000 0.238 1.000 0.980 1.000 0.476 1.000 0.605 1.000 pmmb 2023, 6, 1; a0000327 20 of 23 4. discussion according to the boxplot in figure 8, it could be understood that the svm-rfe has removed many irrelevant features as the size of the feature subsets decreases. therefore, the smaller feature subsets contain a higher density of relevant features, which allows the svm classifier to obtain 100% accuracy. as for the omics composition after feature selection, the decline of the composition of gene expression and the rise of the composition of dna methylation expression from fs 20000 to 5000 could indicate that, as the feature subset becomes smaller, the svm-rfe algorithms has removed a lot of irrelevant features from gene expression omics while keeping the more relevant features from dna methylation expression omics. as the size of the feature subsets continues to decline, the opposite is observed, whereby the gene expression features become more relevant than that of the dna methylation expression, causing the svm-rfe to remove more features from dna methylation expression, which results in the decline in composition. the baseline accuracy for the testing set used is 0.9904 or 99.04%. the testing set with 104 instances only has 1 example with the negative class label (solid tissue normal). therefore, by simply predicting only the positive class (primary tumour), an accuracy of 0.9904 can be achieved. from the classification results of the sdae models built with fs 1000 to 5000, only fs 1000 and 4000 can achieve 100% accuracy with all correctly classified instances. on the other hand, fs 2000, 3000, and 5000 failed to predict the negative class label accurately, resulting in 1 false positive prediction. despite that, these models still achieved an accuracy of 0.9904. in this case, accuracy might not be a useful metric as the concern is to predict the minority negative class correctly. the auc score is a more useful metric since it scores according to the predicted outcome for both class labels. the auc score for fs 1000 and 4000 are recorded at 1.000 since all the instances are correctly classified. meanwhile, fs 2000, 3000, and 5000 achieved 0.5000 for their auc score, indicating zero capability in separating the class labels. according to the classification result on the extracted sampled data from the vae models for each feature subset using the svm model as an external classifier, each feature subset can achieve 1.000 accuracies. with all correctly classified instances, the rest of the metrics are also measured at 1.000. when comparing the classification results of the sdae and vae models, it could be deduced that the vae models are more capable of learning the valuable feature of each feature subset during the encoding process. fs 1000 and 4000 are the two feature subsets that allowed both the sdae and vae models to achieve a score of 1.000 for each metric. this could indicate that fs 1000 and 4000 are the two most optimal feature subsets selected by the svm-rfe algorithm. pmmb 2023, 6, 1; a0000327 21 of 23 5. conclusions the study has employed svm-rfe for feature selection to extract the most relevant features from the multi-omics data with large dimensions. the output obtained from the svm-rfe are the 20 most optimal feature subsets the algorithm selects. the 5 feature subsets with the smallest size are then used in cancer classification using the fine-tuned supervised learning sdae and vae deep learning models. the result suggests that fs 1000 and 4000 are the two most optimal feature subsets selected by the svm-rfe algorithm. the sdae and vae classifiers can correctly classify all the instances using the testing set. the suggestions for future work for this study include 1) the use of new and updated multi-omics data to cater to the severe data imbalance problem and 2) the use of better hardware, including a gpu with larger memory capacity, to allow the development of the neural network of larger feature subsets (e.g., fs 20000) in deep learning classification. author contributions: conceptualization, methodology, aas, nsa, jtl, ham; literature search aas, nsa, jtl, ham; zas experiment, result analysis and validation, aas, nsa, jtl, ham; nhw writing—original draft preparation, aas, nsa, jtl, cwh writing—review and editing, aas, nsa; proofread, aas, ham, zas, cwh. funding: this work was funded by the malaysian ministry of higher education through the utm fundamental research (grant number: q.j130000.2551.21h71). acknowledgments: the authors would like to express gratitude to the malaysian ministry of higher education for the financial sponsorship of this study through the utm fundamental research (grant number: q.j130000.2551.21h71). the faculty of computing, universiti teknologi malaysia also 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variational autoencoders for cancer data integration: design principles and computational practice. front genet 2019; 10. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology methods article 1 generation of stably expressing irf5 spliced isoform in jurkat cells ashwinder kaur1, chee-mun fang2* 1school of pharmacy, the university of nottingham malaysia, selangor darul ehsan, malaysia 2division of biomedical sciences, school of pharmacy, the university of nottingham malaysia, selangor darul ehsan, malaysia abstract: lentiviral transduction enables the generation of gain-of-function of a targeted gene in mammalian cells. single cell cloning through limiting dilution can establish a population of cells with homogenous transgene expression for exploring protein function. here, we describe step by step optimized protocols for generating clonal stably expressing using crude lentiviral supernatant in jurkat cells. although the protocol is for general use, we will detail how to create stable cell lines based on jurkat cells expressing irf5 spliced isoform. these protocols will be broadly useful for researchers seeking to apply overexpression by viral transduction and generation of stable clone to study gene function in mammalian cells. keywords: lentiviral; irf5; viral transduction; transgene; jurkat cells received: 9th october 2020 accepted: 29th october 2020 published online: 5th november 2020 citation: kaur a and fang c-m. generation of stably expressing irf5 spliced isoform in jurkat cells. prog microbes mol biol 2020; 3(1): a0000131. https://doi.org/10.36877/pmmb.a0000131. introduction irf5 has a diverse role in the induction of pro-inflammatory cytokines and chemokines downstream to various signalling pathways contributing to the pathogenesis of various autoimmune and inflammatory diseases [1-3]. in humans, irf5 gene exists as multiple spliced variants that give rise to at least nine isoforms [2-4]. out of these nine isoforms, four are known as the functional isoforms (v1, v3, v4, v5 and v6), the rest are either transcriptionally inactive or lack certain functional elements, resulting in mutant irf5 [4-6]. we utilised one of the predominant functional irf5 spliced isoforms; irf5 variant 4 (irf5v4) in our study. the irf5v4 is among the first to be cloned variant, and it exhibits similar characteristics to another spliced variant, irf5v3 in terms of identical deletion pattern in exon6, encodes identical polypeptide sequences as well as their functions [4-7]. the irf5v4 are widely characterised in early studies in defining irf5 roles in regulating type 1 interferons in response to viral infection [8, 9]. besides that, irf5v4 was found to be involved in ubiquitination, a post-transcriptional process critical for the nuclear translocation and target gene regulation of irf5 [10]. gene overexpression is a method to produce many targeted gene products for several applications. these include studying gene function, recombinant protein production, screening tools for drug targets, connecting genes in biological pathways, and finding genetic modifiers overexpression phenotypes [11]. the use of mammalian cells as expression systems has been widely established to study the function of gene and production of recombinant protein [12]. delivery of exogenous genetic materials (e.g., dna, sirna) can be accomplished using viral or non-viral vectors that act as vehicles to carry genes of interest into host cells. viral vectors consist of viruses such as adenoviruses, retroviruses, and lentiviruses, which deliver nucleic acids efficiently into target cells through infection [13-16]. lentiviral vector derived from hiv has been widely used as a gene delivery tool. it provides stable and long-term gene expression in various mammalian cells, including non-dividing primary cells like neurons [17]. it also serves several benefits, such as carrying large inserts and exhibiting low immunogenicity after some modification in the vector design. gene transfer mediated by viruses is highly efficient due to their natural ability to infect cells. however, concern over safety and toxicity issues due to the possibility of producing wild-type infectious viruses has led researchers to explore various strategies to improvise the use of viruses as a vector by generating a recombinant viral vector. generation of viral vector particles depends on multiple plasmid proteins of which the genes are genetically separated, for instance, a) packaging plasmids that consist of gag, pol, tat, and rev genes that are required viral particle formation, b) transfer vector bearing the expression cassette for copyright @ 2020 by kaur a and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: chee-mun fang, division of biomedical sciences, school of pharmacy, the university of nottingham malaysia, selangor darul ehsan, malaysia; cheemun.fang@ nottingham.edu.my 2 generation of stably... transgene insert, c) envelope glycoprotein for infectivity [18-20]. to date, three-generation systems of hiv type 1 based lentiviral vector have been generated. the first generation system closely resembles the wild-type hiv genome except that the packaging systems are modified whereby the helper plasmid that encodes gal-pol and the envelope plasmid are driven by heterologous promoter rather than the viral ltr. the hiv glycoprotein (gp120) envelope is usually replaced with vesicular stomatis virus glycoprotein (vsv-g) to target broader host cells [18, 21]. the second and third-generation vectors are designed so that the necessary components for virus production are split for increased biosafety of the use of lentiviral in the laboratory. in the second generation systems, the accessory genes are removed from the packaging systems, whereas in the third generation, the packaging systems are separated into two plasmids, and tat gene is removed; (gal and pol) and (rev) [18, 19, 21, 22]. besides, the transfer vector in the third generation is modified such that a chimeric 5’ltr is fused to a heterologous promoter such as cytomegalovirus (cmv) or rovs sarcoma virus (rsv), and the u3 3’ltr is deleted from the viral genome to create a self-inactivating vector which is replication-incompetent [15, 20-22]. of note, the replacement of a strong viral promoter or enhancer with a hybrid heterologous constitutive promoter reduces insertional mutagenesis and immune genotoxicity because of the absence of virulence factors. the third generation lentiviral systems are considered safe, even to be used in clinical studies [22]. the lentivirus production is accomplished through transcomplementation, whereby by change, all three plasmids get contained into a single cell and together express all the viral proteins that assemble into infectious lentiviral particles [23]. the 293 cells are derived from human embryonic kidney cells, which were transformed by adenovirus type 5 fragments [24] followed by the insertion of a temperature-sensitive version of the simian virus 40 (sv-40) large t tumor antigen [25, 26]. the permissibility of transfection of 293t cells and sv40t, which aids in the extra-chromosomal amplification of hiv-1 plasmid containing sv40 origin of replication, makes it suitable for the production of hiv-1 based lentiviral vector particles [26]. crude viral supernatant is usually sufficient for in vitro transduction [27-30]. the titer of crude viral supernatants usually ranged from 1 to 5x107 infectious particles [31]. although several studies have employed the technique of concentrating lentivirus and titer, this is particularly important for some difficult to transfect cells such as primary cells and the use in vivo experiment that requires control of transduction rate [30, 32-35]. nonetheless, many studies have reported the use of fresh viral supernatant to transduced cells [30, 32, 33, 35]. moreover, the use of virus supernatant is a faster and simpler approach to transduce cells. most of the existing protocols used polybrene to enhanced transduction efficiency [32, 36-38]. this is because both cells and virus lipid confers net negative charges, and polybrene a cationic polymer function as counteracting repulsive electrostatic effect, which mediates virus adsorption to the cells, thereby increasing the transduction rate [39]. in this study, we used plenti cmv gfp puro (addgene 17448), a third-generation lentiviral vector to carry the irf5 cdna construct into jurkat cells to achieve stably expressing jurkat cells. this lentiviral vector is a tat independent and self-inactivating lentiviral vector with enhanced green fluorescence protein, (gfp) under cmv promoter and confers puro gene for puromycin antibiotic selection. to do so, dna fragment encoding gfp was removed and replaced with irf5v4. the production of recombinant lentivirus was carried out by transient cotransfection of three plasmids; plenti expressing gfp or plenti expressing irf5v4 was co-transfected with packaging plasmid, pspax2 (addgene 12260) and envelope plasmid, pmd2g (addgene 12259) into hek 293t cells, mediated by trans-it x2 transfection reagent (mirus bio llc). production of experimental lentiviral constructs of irf5v4 was done in parallel with lentiviral containing gfp in separate flasks. unlike the transfer vector that carries gfp in its backbone, no reporter gene was conjugated in these experimental lentiviral constructs of irf5v4. to ensure that the experimental conditions during this transient transfection experiment were conducive, the expression of gfp was evaluated in the cells transfected with plenti-gfp. prior to generating stably expressing irf5v4 in jurkat cells, the transduction protocol was optimized using plenti-gfp. this methodology article aims to present the optimized protocol for evaluating the use of crude viral supernatant for generating stably expressing irf5v4 in jurkat cells. moreover, this experiment was conducted to investigate whether the virus packaging was an indeed successful and infectious virus was released into the culture medium. for this experiment, the easy-to transduce cells, hek 293t cells, were used for transduction with lentivirus expressing gfp. aside from checking the infectious virus produced, we also titer different volumes of the viral supernatant to test an adequate amount for transduction without causing any toxicity. following the optimized protocol, the transduction of lentiviral irf5v4 construct was done parallel to lentiviral encoding gfp that served as a positive control. the success of the generation of stable clones of irf5v4 was validated through western blot analysis. the flow of the experiment is illustrated in figure 1. method details construction of lentiviral vector carrying irf5v4 primers for human irf5 coding sequences [8] were designed with restriction enzymes bamhi and sali, incorporated at the 5’ and 3’ ends, respectively. amplified fragments of irf5v4 (1467 bp) were cloned into pcr blunt ii topo vector (invitrogen) and subsequently cloned into plenti gfp puro (addgene 17448), replacing the gfp fragment. 3 kaur a et al. a) procedure for recombinant lentivirus production 1. one day before transfection, seed 5x106 hek293t cells in t-75 cell culture flask containing 15 ml of growth media, dmem +10% fbs media (*no antibiotic) to achieve 70-80% of confluency on day of transfection. *notes: antibiotic may interfere with a transfection complex 2. add 1.85 ml of serum-free media optimem into falcon tube followed by all three plasmid dna as below: 3. add 45 ml of mirus transit-x2 by dropwise into the falcon tube and gently mix with the diluted plasmids and incubate for 30 minutes undisturbed for complex formation. 4. aspirate the mixture gently* and add dropwise into the flask using 1000 µl pipette. rock flask frontback and sideways to distribute the complex evenly over the cells. incubate the cells at 37°c, 5% co2 incubator for 16 hours. *note: do not pipette up and down this time as it will dislodge the formed complex between the plasmids and transfection reagent. cell line and conditions human embryonic kidney (hek) 293t cells (generously provided by dr. leong chee onn, international medical university) were maintained in dmem (cellgro mediatech, us) supplemented with 4.5g/l glucose, l-glutamine, sodium pyruvate, 10% heat-inactivated fetal bovine serum, fbs (sigma aldrich), and 1% penicillin/ streptomycin (gibco, thermo fisher scientific, us). jurkat cells (obtained from cell culture bank of university of nottingham malaysia campus) were cultured in rpmi medium (cellgro mediatech, us) supplemented as above, with an additional component of 25mm hepes. determination of infectivity and optimal inoculum of crude supernatant for transduction using lentiviral expressing gfp materials • serological pipettes, pipettor • pipettes, tips • microcentrifuge tubes and rack • falcon tubes and holder • dmem (cellgro mediatech, us) • optimem (gibco, thermo fisher scientific, us) • heat inactivated fbs (sigma aldrich) • 0.25 % trypsin-edta (gibco, thermo fisher scientific, us) • 1xpbs without ca2+ and mg2+ (cellgro mediatech, us) • mirus transit-x2 dynamic delivery system • plasmids (plenti-gfp, pspax2, pmd2g) • petri dish • 30 ml syringes • 0.45µm pes filter • t-75 flask • 6 well plate figure 1. the schematic diagram for the flow of the experiment plasmids concentration ratio transfer vector (plenti-gfp) 3750 ng 4 packaging vector (pspax2) 2812.5 ng 3 envelope vector (pmd2g) 937.5 ng 1 4 5. after 16 hours of post-transfection, remove media and replace* with fresh 15 ml of complete media. return the flask into the incubator for 24 hour incubation. *note: it is essential to do all media changes with extreme gentleness, as the cells have been sensitized and can easily detach from the plate. if needed, use two serological pipettes, to add media (first 10 ml and then 5 ml, to prevent cells detaching, insert the serological pipette fully inside the flask and add media to edge of flask, dropwise). 6. at day two post-transfection (48 hours after transfection). observe cells for gfp expression using a fluorescence microscope and capture images to evaluate transfection efficiency. 7. harvest the supernatant containing viral particles by gently aspirating the medium from the flask with a serological pipette into a 50 ml centrifuge tube. replace 15 ml of complete media into the flask and put back in the incubator. 8. get rid of debris* from the supernatant containing viral particles by filter syringe. this can be done by gentle aspiration of supernatant from the petri dish with a syringe, invert syringe several times to mix the solution well. pass supernatant through 0.45 µm filter and drain the filtered supernatant into a falcon tube. *notes: ideally, there should be no debris of cells in the virus supernatant. if there are some small cells, gentle centrifugation of 1500 rpm for 5 minutes at 40c before filtering the supernatant can be done to prevent the filter from getting blocked by the cells’ debris. 9. on day three post-transfection (72 hours after transfection). repeat step 6-7. 10. pool the 48 and 72 hours of viral supernatant collection. use immediately or store in 40c for not more than 24 hours and proceed with transduction. avoid freeze-thaw as it can cause a lower transduction rate. *notes: the ratio of transfer plasmids to packaging plasmid and envelope plasmid used for transfection is 4:3:1 was adapted from previous studies[29,33]. the ratio of plasmids dna to transfection reagent used is 1:2:4 and the seeding number of cells was determined in preliminary expreiment done in out lab. the steps shown here is for t-75 flask, to scale down to 6 wells, divide by 2.5. number of cells for 6 wells to use: 0.5x106 cells/well. b) procedure of lentiviral titer using crude viral supernatant 1. prepare complete media containing polybrene to a final concentration 8 µg/ml. 2. seed 2x105 hek 293t cells into 6 wells of the 6 well culture plate containing 2 ml of growth media and polybrene. 3. bring virus supernatant to room temperature* and mix gently. add 0.6ml, 0.8ml, 1ml, 1.5ml, and 2ml of virus supernatant into seeded hek 293t cells (figure 2). *notes: thawing of virus supernatant from 40c can be done by keeping it in the biosafety cabinet to reach room temperature. be careful not to leave the tube containing virus supernatant in the water bath too long as high heat can reduce viral infectivity resulting in poor transduction. 4. incubate the cells at 37°c, 5% co2 incubator for 24 hours. 5. after 24 hours of incubation, transfer change media with complete fresh media. incubate cells at 37°c, 5% co2 incubator for another 48 hours. 6. at day three post-transduction (72 hours after transduction). observe cells for gfp expression using a fluorescence microscope and capture images to evaluate transduction efficiency. figure 2. a layout of 6 wellplate for optimization of transduction. a1: control 1: non-transduced, a2: 0.6ml viral supernatant, a3: 0.8ml viral supernatant, b1:1ml viral supernatant, b2: 1.5ml viral supernatant, b3: 2ml of viral supernatant. generation of stable clones expressing irf5v4 materials • serological pipettes, pipettor negative 0.8ml0.6ml 1ml 2ml 1.5ml generation of stably... 5 • pipettes, tips • multichannel pipettor • 1.5 ml microcentrifuge tubes and rack • 15 ml falcon tubes and holder • rpmi medium (cellgro mediatech, us) • fbs (sigma aldrich) • penicillin/ streptomycin (gibco, thermo fisher scientific, us) • hexadimethrine bromide is known as polybrene (sigma-aldrich). • 6 well plate • t-25 flask procedure for transduction in jurkat cells 1. prepare recombinant lentiviral particles harboring irf5v4 coding sequence following steps 1a in parallel with lentiviral particle harboring gfp to monitor experimental conditions. 2. prepare two sets of 6 well plates. seed 2x105 jurkat cells into 6 wells of 6 well plate cell culture flask containing 2ml of growth media and polybrene. in one 6 well plate label (irf5v4) and another one as (gfp). 3. bring virus supernatant to room temperature and mixed gently. transfer 2 ml of virus supernatant into each well. one plate containing lentiviral particles harboring gfp and another plate for lentiviral harboring irf5v4. 4. seal the plate with paraffin film and spin the cells* at 1,200 x g for 90 minutes at 250c without a break. incubate the cells at 37°c, 5% co2 incubator for 24 hours. *notes: spinoculation can be performed in a falcon tube if a plate adapter for centrifugation is not available. if the falcon tube is used, resuspend the mixture gently and transfer it into the 6 well plates. 5. the next day, change the media by centrifugation at 1000 x g for 10 minutes at 250c and replace it with fresh 2ml of complete media into each well. incubate the cells at 37°c, 5% co2 incubator for another 48 hours. 6. at day 3 post-transduction (72 hours after transduction), observe cells for gfp expression using a fluorescence microscope, and capture images to evaluate transduction efficiency. procedure for selection of transduced cells and isolation for monoclonal 1. collect the transduced cells from each well and pool them, and split into four t-25 flasks, with each flask containing 3ml of cells suspension. 2. add 6ml (1:3 ratio) of complete media containing 5 µg/ml puromycin into each well and culture the transduced cells * for two weeks to generate polyclonal cells. 3. after two weeks, split the cells and freeze down some polyclonal cells to have early passage cells that serve as storage and back up. use the remaining cells to check for the presence of the transgene. 4. using the early passage cells, perform limiting dilution to isolate a single cell (monoclonal) to generate a homogenous population of cells. 5. to do so, dilute 1 x104 transduced cells in 20 ml media. 6. transfer 5 µl of cells into a tube containing 5ml of complete media supplemented with 5 µg/ml puromycin and 5 ml of self-conditioned media*. *notes: the self-conditioned media is isolated from healthy growing jurkat cells (non-transduced cells). to spin down the cells and collect the supernatant into a sterile tube and filter using 0.22 µm polyvinylidene fluoride (pvdf) filter. 7. transfer 100 µl of the diluted cells suspension into each well using a multichannel pipettor. 8. incubate cells undisturbed at 370c in 5% co2 for 2 hours. 9. inspect wells and label wells containing only a single cell. 10. incubate plate undisturbed at 370c in 5% co2 for three weeks with regular inspection for confluency. 11. once cells reach about 30-50% confluency, transfer the cells to 24 wells and expand to 6 wells and later to t-25 flask. 12. check the expression level* and freeze down early passage cells to be used in subsequent experiments. *notes: routinely check expression level by reverse transcription pcr (rt-pcr) to ensure the transgene is not lost over the time of culture, a common gene silencing phenomenon associated with transgene under cmv promoter. if transgene exhibit toxic to cells resulting in extended lag phase cell growth, an alternate viral vector under inducible promoter can be used. method validation confirmation of successful transient transfection for recombinant lentivirus since gfp fragments were removed in order to construct experimental transfer vectors plenti-irf5v4; by inserting irf5v4 dna fragment in replacement of gfp dna fragment, there was no reporter marker to track these constructed experimental vectors during transient transfection for recombinant lentivirus production. therefore, the production of recombinant lentivirus harboring irf5v4 was conducted in parallel with plenti-gfp in separate t-75 flasks to ensure the setting for successful transfection for virus production. as seen in figure 3(a), co-transfection of plenti-gfp along with the two helper plasmids (pspax2 and pmd2g) in hek 293t cells exhibited about 60-70% of green fluorescence at day 2 of post-transfection, similar to the results obtained in the preliminary experiment conducted as described above. also, the morphology of the transfected cells was examined under the bright-field view. the photo bright-field of transfected cells shows the presence of syncytia represented by the multi-nucleated cells (in black arrow) that were kaur a et al. *notes: selection of transduced jurkat cells in puromycin containing medium, typically takes two weeks to achieve the population of cells that contain only integrated transgene as any unintegrated cells would have died. these bulk cells that survived the antibiotic selection are referred to as polyclonal cells. 6 caused by the translation of glycoprotein of vsv-g (envelope protein), indicating successful transfection and sign for lentivirus production [35, 40]. the third day of post-transfection, fluorescent microscopy showed gfp was expressed about 80-90%, as seen in figure 3 (b). based on these results, it was deduced that the production of lentivirus was successful and ready to be used for the transduction experiment. determination of the optimal amount of viral supernatant for transduction the presence of gfp expression visualized by a fluorescence microscope after transfection reveals the efficiency of transfection, but it does not display the lentivirus’s infectivity. it was necessary to ensure the recombinant lentivirus harvested was infectious for proper gene delivery to target cells [41]. it was also essential to make sure that there was no cell toxicity from a viral infection that could cause massive cell figure 3. detection of gfp expressions from recombinant lentivirus expressing gfp from post-transfection days.(a) on day 2, post-transfection. (b) on day 3, post-transfection. photos were taken under nikon inverted microscope eclipse ts2r under bright field or fluorescent light (x200 magnification). the exposure time of fluorescent images were 600 milliseconds. the black arrowhead shows the presence of syncytia in transfected cells. death, resulting in a lower transduction rate. based on the results, the minimal volume of lentiviral supernatant inoculum that showed some gfp expression was 0.6ml (figure 4 (b)), and the highest volume, 2 ml tested showed greater transduction efficiency of more than 90% gfp expression seen (figure 4(f)). moreover, about 95% of viable cells were observed by evaluating trypan blue, indicating no cell toxicity from a viral infection with the amount of virus supernatant used for transduction. this data provided the evidence that the recombinant lentivirus produced was infectious and good transduction efficiency was achieved with crude viral supernatant. figure 4. titers of lentivirus expressing gfp on hek 293t cells. (a) non-transfected cells, (b) 0.6 ml of lentivirus, (c) 0.8ml of lentivirus, (d) 1ml of lentivirus, (e) 1.5ml and (f) 2ml of lentivirus. photos were taken under nikon upright microscope eclipse ei under fluorescent light (x100 magnification). generation of stably... 7 kaur a et al. confirmation of successful transduction in jurkat cells transduction is more permissible in the adherent cell line. thus, spinoculation is not required transduction in hek 293t cells. but this is not the same for suspension cells like jurkat cells. numerous researchers have conducted transduction of jurkat cells by spinoculation and the presence of polybrene, which have shown to enhance the transduction of lentiviral vector by virus binding to the cells [32, 36-38]. the experiment was conducted in parallel with the control transfer vector, plenti-gfp in a separate culture flask to track the successful transduction experiment. after 72 hours of transduction, gfp expression was accessed by a fluorescence microscope. as seen in figure 5, the bright field photo of transduced cells revealed that infection with lentivirus did not alter jurkat cells’ morphology, implying no cell toxicity from the lentiviral transduction. moreover, jurkat cells’ transduction with virus supernatant obtained from the transfection of plenti vector expressing gfp displayed about 70-80% of green fluorescence. as expected, no gfp expression was observed in non-transduced cells. meanwhile, the transduced cells showed an excellent transduction efficiency of more than 70% of gfp expression accessed by a fluorescence microscope. furthermore, no cell toxicity from viral infection was noticed as the evaluation of trypan blue observed about 95% of viable cells. from these data, it was deduced that the infectious lentiviral constructs efficiently transduced jurkat cells. confirmation of irf5v4 overexpression in polyclonal jurkat cells puromycin is an aminonucleoside antibiotic produced by streptomyces alboniger. it inhibits peptidyl transfer on both prokaryotic and eukaryotic ribosomes, which cause premature chain termination during translation [42, 43]. the plenti-gfp and the engineered plenti-irf-v4 encode puromycin-n-acetyl transferase gene (puro) in their plasmid backbone constitutively express it. therefore, during the antibiotic selection, those cells with stably integrated transgene and constitutively expressed the resistant gene, puro, will survive when grown in a medium containing puromycin. those non-transduced cells and cells that uptake plasmid transiently will not survive the figure 5. detection of gfp expressions from transduced jurkat cells with lentivirus harbouring gfp at day 3 post-transduction. photos were taken under nikon inverted microscope eclipse ts2r under bright field or fluorescent light (x200 magnification). antibiotic selection. the presence of irf5v4 in the polyclonal cells generated from the experimental lentiviral constructs was checked western blot analysis. as seen in figure 6, no irf5 was detected in non-transduced jurkat cells. similarly, no irf5 was detected in jurkat cells transduced with plenti-gfp, which indicates that infection with lentiviral without irf5 spliced variant in the backbone did not lead to an upregulation of endogenous irf5. therefore, detecting irf5 in stably expressing cells indicates the de novo synthesis of integrated irf5v4 in jurkat cells. in line with this, approximately 60 kda bands corresponding to the irf5v4 protein were detected from polyclonal cells, as shown in figure 6. from these results, it was deduced that experimental lentiviral constructs had successfully integrated the transgene of irf5v4 in jurkat cells. 8 generation of stably... figure 6. confirmation of overexpression of irf5v4 in polyclonal cells. western blot analysis for detection of irf5v4 (approximately 60kda). housekeeping protein actin act as the loading control (43kda) was included as part of the analysis. the chemiluminescence signal was captured by gel doc imager. confirmation of irf5v4 in monoclonal cells polyclonal cells constitute of heterogeneous transgene expression level, which means the population of cells containing both high and low expression levels of the transgene [35]. over time, the transgene population of polyclonal cells may drop because cells with high transgene levels may have a slower growth rate. therefore the rapidly growing low-level transgene expression may take over the culture. to avoid the drift effect towards low transgene expression and obtain a homogenously higher level of expression of the irf5v4, the early passage of polyclonal cells was used to isolate single cells (monoclonal) limiting dilution method. limiting dilution is a traditional method for obtaining clonally identical cells (monoclonal) of stable expressing transgene derived from single isolated cells cultured in multi-well plates [44]. several monoclonal cells were isolated and expanded in culture with a complete medium containing 5 µg/ml of puromycin. the clones were screened for the presence of overexpression of irf5v4 using western blot analysis. from the result seen in figure 7, two clones expressing irf5v4 (referred to as b8, f9) were found to have stable high irf5 expression. while two clones, d7 and e6, showed a substantial level of irf5v4. another clone e4 had a low expression of irf5v4. . figure 7. confirmation of overexpression of irf5v4 in monoclonal cells. west ern blot analysis for detection of irf5v4 (approximately 60kda). housekeeping protein actin act as the loading control (43kda) was included as part of the analysis. the chemiluminescence signal was captured by gel doc imager. conclusion in conclusion, the present study shows the optimization protocol for generating irf5v4 stable clones using recombinant lentiviral transduction. stably overexpressing irf5v4 was successful achieved through lentiviral transduction. five monoclonal of irf5v4 expressing jurkat cells were obtained. the gfp expression from the control lentiviral vector helped evaluate whether the experiments carried out following the protocol designed were ideal and successful. authors contributions ak and c-mf performed the literature review and manuscript writing. c-mf conceptualizes the research project. conflict of interest the authors declare that there is no conflict of interest in this work. acknowledgement this project is funded by ministry of higher education, malaysia, fundamental research grant scheme (frgs/1/2013/skk01/musm/03/03). the schematic diagram was created using biorender.com. references 1. ryzhakov g, eames hl, and udalova ia, activation and function of interferon regulatory factor 5. j interferon cytokine res 2015; 35(2): 71-78. 2. eames hl, corbin al, and udalova ia, interferon regulatory factor 5 in human autoimmunity and murine models of autoimmune disease. transl res 2016; 167(1): 167-182. 3. kaur a, lee l-hlh, chow s-csc, et al., irf5-mediated immune responses and its 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159-172. 40. ching ngai s, ramasamy r, and abdullah s, production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer. pertanika j sci technol 2012; 20(2): 269-281. 41. khalil m and gout i, overexpression or downregulation of mtor in mammalian cells. methods in molecular biology 2012; 821: 87-103. 42. de la luna s and ortín j, pac gene as efficient dominant marker and reporter gene in mammalian cells. methods enzymol 1992; 216: 376-385. 43. vara j, perez-gonzalez ja, and jimenez a, biosynthesis of puromycin by streptomyces alboniger: characterization of puromycin n-acetyltransferase. biochemistry 1985; 24(27): 8074-81. 44. hunter m, yuan p, vavilala d, et al., optimization of protein expression in mammalian cells. curr protoc protein sci 2019; 95(1): e77. pmmb 2021, 4, 1; a0000195. doi: a0000195 http://journals.hh-publisher.com/index.php/pmmb genome report whole-genome sequence of bioactive streptomycete derived from mangrove forest in malaysia, streptomyces sp. musc 14 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; ser.hooileng@monash.edu (h-ls) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) 3illumina singapore pte ltd, woodlands industrial park e1, singapore; tmarilyn36@gmail.com (w-st) 4division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia; yinwaifong@yahoo.com (w-fy) 5vice chancellor office, jiangsu university, zhenjiang 212013, pr china *corresponding author: kok-gan chan, kokgan@um.edu.my (k-gc) received: 8 february 2021; received in revised form: 10 april 2021; accepted: 12 april 2021; available online: 22 april 2021 abstract: the contribution of streptomycetes to human health is undeniably important and significant, given that these filamentous microbes can produce interesting compounds that can be used to cure deadly infections and even cancer. isolated from the east coast of peninsular malaysia, streptomyces sp. musc 14 has shown significant antioxidant capacity. the current study explores the genomic potential of musc 14 via a genome mining approach. the genome size of musc 14 is 10,274,825 bp with g + c content of 71.3 %. antismash analysis revealed a total of nine biosynthetic gene clusters (with more than 80 % similarities to known gene clusters). this information serves as an important foundation for subsequent studies, particularly the purification and isolation of bioactive compounds by genetic manipulation techniques. keywords: streptomyces, antioxidant, mangrove, genome, musc 14, actinobacteria 1. short introduction gifted with the ability to form spores, streptomycetes are ubiquitous in nature and produce a diverse array of secondary metabolites that can be exploited for the benefit of humanity[1-16]. one of the significant breakthroughs in streptomycetes research is the discovery of streptomycin (from the soil bacterium, streptomyces griseus) by professor article history hooi-leng ser1, loh teng hern tan1,2, wen-si tan3,4, wai-fong yin4, kok-gan chan4,5* pmmb 2021, 4, 1; a0000195 2 of 8 waksman and his team – which subsequently led him to the nobel award in medicine later in 1960[13, 17-21]. as a matter of fact, the continuous search for pharmaceutically important compounds led to another (one half jointly) nobel prize in physiology or medicine in 2015 awarded to professor william c. campbell and professor satoshi ōmura for the discovery of avermectin, which was isolated from a soil streptomycete, streptomyces avermitilis[22-27]. the search for bioactive compounds from this prolific genus seems like a never-ending journey, with researchers now hunt for them in unique environments such as hot springs, volcanic soils, deserts, deep-sea, and the mangrove forest[28-42]. the hypothesis behind searching these special habitats is that microbes may come up with adaptation strategies to ensure their survival in the harsh condition like drastic temperature changes, salinity, and oxygen availability, which in turn may lead to the production of interesting compounds and drive the emergence of a novel bacterium [43-50]. streptomyces sp. musc 14 was isolated from the mangrove forest on the west coast of peninsular malaysia during a screening program for bioactive actinobacteria[4, 51, 52]. a thorough investigation conducted on the strain has reflected the antioxidant potential of musc 14, attributed to its production of bioactive secondary metabolites, including pyrrolopyrazines and fatty acid esters[51, 53-55]. the current study aims to obtain the genome sequence of streptomyces sp. musc 14 before exploring the genomic potential of this strain. 2. data description the whole genomic dna extraction of musc 14 was carried out using masterpure™ dna purification kit (epicentre, illumina inc., madison, wi, usa), preceding rnase treatment (qiagen, usa)[56-58]. subsequently, the dna library was constructed with nextera™ dna sample preparation kit (nextera, usa). the library quality was evaluated by bioanalyzer 2100 high sensitivity dna kit (agilent technologies, palo alto, ca)[59, 60]. paired-end sequencing was performed on the illumina miseq platform with miseq reagent kit 2 (2 × 250 bp; illumina inc., madison, wi, usa)[16, 61, 62]. post-trimming step, de novo assembly of the paired-end reads on clc genomics workbench version 7 (clc bio, denmark) resulted in 174 contigs and an n50 contig size of 162,032 bp. the genome size of musc 14 is 10,274,825 bp, with an average coverage of 211.0-fold and g + c content of 71.3% (table 1). the genome sequence of musc 14 has been deposited at ddbj/embl/genbank under accession of mlyn00000000. the version described in this paper is the first version. following genome assembly, musc 14 genome was annotated on rapid annotation using subsystem technology (rast) and ncbi prokaryotic genome annotation pipeline (pgap)[63-66]. gene prediction was performed using prodigal (version 2.6)[67], while ribosomal rna (rrna) and transfer rna (trna) were predicted using rnammer[68] and trnascan se version 1.21[69], respectively. the whole genome of musc 14 consists of 8,799 protein-coding genes and 81 rna genes (trna: 74, rrna:4). table 1. general genomic features of streptomyces sp. musc 14. properties streptomyces sp. musc 14 genome size (bp) 10,274,825 contigs 174 contigs n50 (bp) 162,032 g + c content % 71.3 pmmb 2021, 4, 1; a0000195 3 of 8 genome coverage 211.0 protein coding genes 8,799 trna 74 rrna (5s, 16s, 23s) 4 (2, 1, 1) figure 1. annotation of musc 14t genome using rapid annotation using subsystem technology (rast). the annotation on rast showed that most of the protein-coding genes were involved in metabolic processes, with the highest number of genes involved in carbohydrates metabolism (7.60%) (figure 1). subsequent analysis to investigate the genomic potential of musc 14 on antibiotics & secondary metabolite analysis shell (antismash)[70-74] revealed a total of nine biosynthetic gene clusters displaying more than 80% similarities to known biosynthetic gene clusters. besides detecting the gene cluster responsible for the production of the earthy odorant geosmin (100% gene similarities), there is one biosynthetic gene cluster related to nonribosomal peptides production and reflects 100% gene similarities with thioholgamide a/thioholgamide b biosynthesis. thioholgamide a and b were initially isolated from a mangrove-derived streptomycete, streptomyces malaysiense musc 136t, and possess cytotoxic activities against cancer cell lines[75, 76]. recently, a research group in germany has published an article studying the mechanisms involved behind the cytotoxic and anti-proliferative activities of thioholgamide a[75, 77]. the team showed that thioholgamide a induces apoptosis via caspase 3 and parp cleavage (at concentrations comparable to staurosporine)[77]. furthermore, it appears that thioholgamide a inhibits oxidative phosphorylation in tumor cells without displaying much toxicity towards nontumorigenic cells and zebrafish embryos, thus making the compound an appealing candidate that to be developed as anti-cancer therapeutics. harnessing the genomic potential of microbes, including streptomyces sp. is indeed one way forward in drug discovery[78-80]. combined with the conventional methods, including optimization of fermentation media and improvement in extraction processes, the activation of biosynthetic gene clusters via heterologous expression or introduction of promoters in the native host can potentially yield bioactive compound(s) of interest in high purity and quantity, allowing its large-scale production for pharmaceutical use[81-85]. pmmb 2021, 4, 1; a0000195 4 of 8 author contributions: hls, ltht, and wst carried out the experiments and analyzed the data. wfy and kgc provided vital guidance, technical support, and proofreading for the work. all authors approved the final draft. funding: this work was supported by the university of malaya for high impact research grant (um-mohe hir nature microbiome grant no. h-50001-a000027 and no. a000001-5001) and ppp grant (pg090-2015b) awarded to k-gc. conflicts of interest: the authors declare no conflict of interest. references 1. law jw-f, letchumanan v, tan lt-h, et al., the rising of “modern actinobacteria” era. prog microbes mol biol 2020; 3(1). 2. chang t-l, huang t-w, wang y-x, et al., an actinobacterial isolate, streptomyces sp. yx44, produces broadspectrum antibiotics that strongly inhibit staphylococcus aureus. microorganisms 2021; 9(3): 630. 3. law jw-f, pusparajah p, ab mutalib n-s, et al., a review on mangrove actinobacterial diversity: 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expression of secondary metabolism. proc natl acad sci 2010; 107(6): 2646-2651. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2023, 6, 1; a0000325. doi: 10.36877/pmmb.0000325 http://journals.hh-publisher.com/index.php/pmmb original research article bioactivity of walnut: investigating the triterpenoid saponin extracts of juglans regia kernels for antioxidant, anti-diabetic, and antimicrobial properties youssef elouafy1, salma mortada2, adil el yadini1, mohamed hnini3, youssra aalilou2, hicham harhar1, asaad khalid4,5, ashraf n. abdalla6, abdelhakim bouyahya7,*, my el abbes faouzi2 and mohamed tabyaoui1 article history 1laboratory of materials, nanotechnology and environment lmne, faculty of sciences, mohammed v university in rabat, bp 1014, rabat, morocco; youssef.elouafy@um5r.ac.ma (ye); a.elyadini@um5r.ac.ma (aey); h.harhar@um5r.ac.ma (hh); h.tabyaoui@um5r.ac.ma (mt) 2laboratories of pharmacology and toxicology, pharmaceutical and toxicological analysis research team, faculty of medicine and pharmacy, mohammed v university in rabat, bp 1014, rabat, morocco; salma.mortada95@gmail.com (sm); aalilouyoussra@gmail.com (ya); myfaouzi@yahoo.com (meaf) 3center of plant and microbial biotechnology, biodiversity and environment, microbiology and molecular biology team, faculty of sciences, mohammed v university in rabat, bp 1014, rabat, morocco; hnini007@gmail.com (mh) 4substance abuse and toxicology research center, jazan university, p.o. box: 114, jazan 45142, saudi arabia; akahmed@jazanu.edu.sa (ak) 5medicinal and aromatic plants and traditional medicine research institute, national center for research, p. o. box 2404, khartoum, sudan 6department of pharmacology and toxicology, college of pharmacy, umm al-qura university, makkah 21955, saudi arabia; anabdrabo@uqu.edu.sa (ana) 7laboratory of human pathologies biology, faculty of sciences, mohammed v university in rabat, bp 1014, rabat, morocco *corresponding author: abdelhakim bouyahya, laboratory of human pathologies biology, faculty of sciences, mohammed v university in rabat, bp 1014, rabat, morocco; a.bouyahya@um5r.ac.ma (ab) received: 18 december 2022; received in revised form: 16 january 2023; accepted: 20 january 2023; available online: 21 january 2023 abstract: the increasing demand for plant-based medicines makes plant research attractive to find natural alternatives that can replace synthetic products or at least reduce their use. saponins are a group of natural phytochemical compounds in most herbs, vegetables, and legumes. there are two different types of saponin compounds, which can be classified based on the nature of the aglycone bound to the oligosaccharide fragments, whether it is a steroid or triterpenoid. this paper is devoted to saponins with triterpenoid aglycone and aims to study the relationship between their content and several biological activities in different juglans regia kernel extracts, including antioxidant, antimicrobial and antibacterial activities. antioxidant activity was assessed against two free radicals: dpph and abts. oleanolic acid pmmb 2023, 6, 1; a0000325 2 of 22 was used as a standard to quantify triterpenoid saponins, measured by the vanillin method, whereas acarbose and tetracycline were used as positive controls to evaluate the αglucosidase inhibitory and antimicrobial activities, respectively, of walnut kernel extracts. findings show that hydroalcoholic extract (etoh 70%) has a considerable content of phenolic compounds, with 59.51 ± 0.26 mg gae/g in total polyphenol contents, while the content of total flavonoids and condensed tannins were 3.14 ± 0.27 mg qe/g and 85.65 ± 0.37 mg ce/g of crude extract, respectively. in terms of antioxidant activity, the butanolic extract proved to be the most effective against dpph with ic50 = 7.74 ± 1.49 µg/ml, while the precipitated extract showed the highest scavenging activity against the abts free radical with ic50 = 33.14 ± 2.96 µg/ml. in addition, butanolic and hydroalcoholic extracts of juglans regia kernels showed an α-glucosidase inhibitory activity even more efficiently than synthetic drugs such as acarbose, with ic50 values of 11.66 µg/ml and 16.17 µg/ml, respectively, versus 18.01 µg/ml in acarbose. the evaluation of the antimicrobial activity against bacillus subtilis, escherichia coli and klebsiella pneumoniae strains revealed that some extracts could inhibit these bacteria. this study successfully assessed the bioactivities of the extracts, suggesting that the extracts of medicinal plants can demonstrate stronger bioactivity than pure synthetic compounds. keywords: juglans regia kernels; triterpenoid saponin; antioxidant activity; dpph; abts; antidiabetic activity; α-glucosidase; antimicrobial activity; agar well diffusion method 1. introduction nowadays, walnut consumption is considered a key element that can improve human health [1,2]. several clinical trials have been devoted to highlighting the benefits of walnut seed consumption on human health and, in particular, on the brain and cardiovascular health [3–7], blood lipids and pressure [1,8–11], along with its effect on improving insulin resistance [12]. all these properties related to the consumption of walnut seeds are related to their composition in unsaturated fatty acids and bioactive compounds. currently, infectious diseases caused by multidrug-resistant strains (mdr) constitute a major problem for health. some predictions show the major future health damage that humanity could have due to the re-emergence of these infectious diseases. moreover, the search for antimicrobial molecules has become an essential approach to replace antibiotics which are undergoing elimination because of the inefficiency rendered by resistance [13–15]. among the most sought-after products, natural substances occupy the first place because they have already shown promising antibacterial effects [16–18]. on the other hand, oxidative stress and its related diseases, such as diabetes and cancer also constitute another group of complex pathologies which affect human health massively [19]. the treatment of these pathologies is based on the use of chemical and biochemical compounds targeting different pathways involved. however, these therapeutical approaches always remain inefficient because of the multiple risk factors associated with pmmb 2023, 6, 1; a0000325 3 of 22 these pathologies as well as the stochasticity of mechanistic events leading to their major phenotypes. therefore, the identification and evaluation of natural compounds as drugs candidate [20] for these diseases is based on, particularly, the evaluation of anti-oxidative properties of these bioactive compounds [21,22]. in fact, walnuts (juglans regia) are a great source of unsaturated fatty acids [23–25], fiber, minerals (potassium, calcium, and magnesium) [26,27], amino acids and proteins [28,29], phenolic compounds and saponins [30–32], as well as a variety of other compounds that make j. regia has a vast diversity of medicinal properties [33]. in addition, the phytochemicals present in plants like j. regia have been shown to have therapeutic effects, including anticancer, anti-inflammatory, and anti-bacterial properties [34]. on the other hand, saponin compounds have attracted huge interest over the past few decades, as they have proven their ability to treat several health problems [35]. various studies have been devoted to investigating the anticancer activity of saponin molecules isolated from different species of plants such as platycodon grandiflorum, kalopanax pictus, glochidion eriocarpum, entada phaseoloides, dipsacus asperoides, bupleurum falcatum, and acacia victoriae [36–43]. other studies have proven the anti-inflammatory activity of glycyrrhizin [44–46], which is a natural triterpenoid saponin isolated from glycyrrhiza glabra root [47]. furthermore, other authors have focused on hepatoprotective activity [48–50], and the possibility of employing saponins as anti-diabetic agents in type ii diabetes [51]. considering all these reasons and in order to contribute to the valorization of walnut kernels, and also to see whether plant extracts can possess biological activities better than pure synthetic compounds, this paper will present an assessment of the antioxidant, antimicrobial and anti-diabetic activities of triterpenoid saponins extracted from walnut kernels, through the quantification of phenolics, polysaccharides and saponins, as well as the examination of the free radical scavenging activity against dpph and abts. 2. materials and methods 2.1. plant materials walnut juglans regia fruits were harvested in june 2022, in demnate azilal province (31° 43′ 52″ n, 7° 02′ 10″ w), morocco, peeled and then manually dehulled to obtain only the kernels, which were ground to a fine powder. 2.2. triterpenoid saponins fractionation process. figure 1 shows the extraction protocol of triterpenoid saponins from juglans regia kernels which was performed according to chua's et al. protocol with some modifications [52]. pmmb 2023, 6, 1; a0000325 4 of 22 figure 1. diagram of triterpenoid saponins fractionation process from juglans regia kernels. the ground kernel powder was firstly defatted in a soxhlet cartridge using n-hexane as a delipidating solvent to delimit the non-polar fraction of our kernels. after defatting, the cartridge was dried overnight in an oven (t = 25°c) to remove traces of hexane. on the following day, the residue was removed from the cartridge and underwent reflux extraction with etoh/water solution (70:30) for 2 h at a temperature of 80°c. after that, the solution was filtered and concentrated using a rotary evaporator (heidolph hei-vap precision motor, germany) to obtain a crude extract of 70% etoh, which was used afterward in the fractionation procedure. the crude hydroalcoholic extract (etoh 70%) was diluted in distilled water which was then fractionated using the liquid-liquid extraction technique with butanol, and this process was repeated three times. after the liquid-liquid extraction and completion of the separation process, the aqueous phase was lyophilized using a freeze-dryer (vaco 2, zirbus technology gmbh, germany), while the butanol phase was concentrated using a rotary evaporator (heidolph hei-vap precision motor, germany). the crude butanol extract was reconstituted in 99.8% methanol, and upon addition of diethyl ether, a precipitate was formed carrying all compounds non-soluble in diethyl ether, this precipitated fraction was filtered using a fritted glass funnel, and then rotavaporized to remove any trace of solvent. pmmb 2023, 6, 1; a0000325 5 of 22 2.3. total polyphenols quantification quantification of the total phenols content (tpc) of the crude ethanolic 70% extract was performed by the folin-ciocalteu method according to the protocol described by sotomaldonado in 2022, with minor modifications [53]. shortly, 2500 µl of 10% folin-ciocalteu in distilled water was added to 2000 µl of na2co3 (7.5%) and 500 µl of the ethanolic extract prepared previously at a concentration of 1000 µg/ml. after 15 min of incubation at 45°c, the absorbance was measured at 756 nm against a blank solution, using a uv-visible spectrophotometer (model uv-5800pc uv/vis spectrophotometer, manufactured by shanghai metash instruments co., ltd). the blank contains the same volume of folinciocalteu and na2co3 and we replace the volume of ethanolic extract of j. regia kernels with 500 µl of etoh/h2o (70:30). the results were expressed as mg equivalent of gallic acid per gram of crude extract 2.4. total flavonoids quantification quantification of the total flavonoid content (tfc) of the crude ethanolic 70% extract was performed by the aluminium trichloride method according to the protocol described by el-guezzane et al., in 2021, with minor modifications [54]. 1 ml of the ethanolic extract (70%) at a concentration of 1000 µg/ml was diluted with 6.4 ml of distilled water, and then 0.3 ml of nano2 solution (5%) was added. afterward, 0.3 ml of alcl3 (10%) was added to the mixture after 5 min, then 2 ml of naoh (1m) was also added after another 5 min. the absorbance was measured at 510 nm against a blank solution and the results were expressed as mg equivalent of quercetin per gram of crude extract. 2.5. total condensed tannin quantification the transformation of condensed tannin into anthocyanidols using hydrochloric acid and vanillin is used to quantify the total condensed tannin concentration (tct) according to cesprini et al. protocol [55]. briefly, 25 μl of our 70% ethanolic extract solution (1000/ml) was added to 1.5 ml of 4% methanol vanillin solution and 750 μl of concentrated hydrochloric acid. thereafter, the absorbance was measured at 500 nm after 15 min against a blank solution, and the results were presented in mg equivalent of catechin per gram of crude extract. 2.6. total polysaccharides quantification quantification of the total polysaccharides content of the five extracts was performed by the phenolic sulfuric acid method according to the protocol described by zeng et al., in 2019 [56]. briefly, 1000 µl of each sample (1000 µg/ml) was added to 1000 µl of phenol (5%) and 5000 µl of concentrated sulfuric acid. the mixture was left for 10 min and then incubated for 20 min in a water bath at 30°c. the results were presented in mg glucose equivalent per gram of crude extract. pmmb 2023, 6, 1; a0000325 6 of 22 2.7. triterpenoid saponin quantification the content of triterpenoid saponins was measured by the vanillin method [52]. 250 µl of each sample was added to 250 µl of 8% ethanolic vanillin solution and 2500 µl of concentrated sulfuric acid. the mixture was incubated for 10 min in a water bath at 60°c and then placed in ice water for 5 min in order to stop the reaction. thereafter, the mixture absorbances were measured at 544 nm, and the results were presented in mg of oleanolic acid equivalent per gram of extract. 2.8. dpph free radical scavenging activity assessment of antioxidant activity by dpph (1,1-diphenyl-2-picrylhydrazyl) was performed according to the nounah protocol described in 2017 [57]. 2500 µl of different concentrations of each extract (5-100 µg/ml) were added to 500 µl of 0.2 mm dpph ethanolic solution, vortexed, and incubated in the dark at room temperature for 30 min, and then the absorbance values were measured at 517 nm against a blank containing 500 µl of our dpph solution and 2500 µl of pure ethanol. the results were expressed as the amount of concentration required (µg/ml) to reduce 50% of the free dpph radical (ic50). 2.9. abts free radical scavenging activity equal volumes of abts (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), and potassium persulfate solutions at the concentration of 7mm and 2.4mm, respectively, were vortexed and left in the dark for 16h at room temperature. the resulting solution was adjusted with ethanol absolute to achieve an optical density of 0.700 ± 0.02 at 734nm. afterward, 1800 µl of the adjusted solution was then added to 200 µl of different concentrations (5100 µg/ml) of each extract and the absorbances were measured after 30 min of incubation at 734 nm [58]. the results were expressed as the amount of concentration required (µg/ml) to reduce 50% of the free abts radical (ic50). 2.10. α-glucosidase inhibitory activity the assessment of α-glucosidase inhibitory activity was carried out following a protocol previously described [59–61]. 100 µl of 0.1m sodium phosphate buffer solution (ph = 6.4) that contains the α-glucosidase enzyme solution, was incubated along with 150 µl of each sample at 37°c for 10 min, afterwards, 200 µl of 4-nitrophenyl-α-d-glucopyranoside (pnpg) 1mm, prepared in the same buffer solution, the mixture was incubated at 37°c for 30min. 1000 µl of na2co3 was added to stop the reaction and the optical densities were measured at 405 nm. acarbose has been used as a positive control. 2.11. antimicrobial activity evaluation the antimicrobial activity of juglans regia kernel extracts is assessed using the agar well diffusion method [16,62,63]. similar to the procedure used in the disk diffusion method, the mueller hinton agar plate surface was inoculated by spreading a volume of the microbial inoculum over the whole agar surface (20-100 µl), depending on the growth of each strain. then, a well with a diameter of 6 to 8 mm is aseptically drilled with a sterile tip, and a volume pmmb 2023, 6, 1; a0000325 7 of 22 (10 µl) of the antimicrobial agent (juglans regia kernel extract) is introduced into the well. the plates were allowed to diffuse and incubated for 24 hours at 37°c. antimicrobial agent extracts from juglans regia kernels will diffuse in mueller hinton agar medium, and the size of the inhibition zone was measured in millimeters. the assay was performed in triplicate, whereas tetracycline (tet 30 µg) control disks (sigma) were used as the reference antibiotic. microbial susceptibility tests using agar dilution and minimum inhibitory concentration (mic) were performed to evaluate the antibacterial activity of the prepared extracts of juglans regia kernels against a gram-positive bacterium bacillus subtilis (mw471619) and two gram-negative bacteria, escherichia coli atcc 11775, klebsiella pneumoniae (mw524112). all materials were steam-sterilized at 120°c for 20 min. a 20 ml sterile culture medium was inoculated with tested bacteria. 3. results and discussion 3.1. total polyphenol, flavonoid, and condensed tannin content juglans regia kernels contain a considerable content of phenolic compounds such as polyphenols, flavonoids, and tannins [64–67], which may contribute to the prevention of a wide range of oxidative stresses [68], leading to reduce the risk of cardiovascular and degenerative illnesses [69–71]. those secondary metabolites exhibit many other pharmacological properties, including antioxidant, antimicrobial, and anticancer activities [72–74]. figure 2 represents the tpc, tfc and tct contents exhibited in the ethanolic extract of 70% of the walnut kernels (before the fractionation process). the results show that the walnut kernels have a total polyphenol content of 59.51 ± 0.26 mg gae/g, while the content of total flavonoids and condensed tannins were 3.14 ± 0.27 mg qe/g and 85.65 ± 0.37 mg ce/g crude extract, respectively, (p < 0.0001). these results are higher than those found in several polish walnut cultivars, which did not exceed 20.9 mg gae/g in terms of tpt [75]. however, these results are generally lower than the levels found in the leaves and flowers of walnut, which are considered an excellent source of phenolic compounds when compared to other parts of this plant [76,77]. pmmb 2023, 6, 1; a0000325 8 of 22 0 20 40 60 80 100 tpt (mg gae/g) tfc (mg qe/g) tct (mg ce/g) 85.65 3.14 59.51 ✱ ✱ ✱ ✱ ✱ ✱ ✱ ✱ ✱ ✱ ✱ ✱ figure 2. polyphenols, flavonoids, and condensed tannins content of 70% ethanolic extract of juglans regia kernels. tpt: total polyphenol content. tft: total flavonoid content. tct: total condensed tannin. ****p < 0.0001. 3.2. polysaccharides quantification polysaccharides are natural polymers which generally are made up of more than 10 monosaccharides linked by o-glycosidic bonds [78], which have an essential and primary role in the growth and development of organisms [79], and also in the reinforcement of the immune function [80,81], as well as having numerous pharmacological properties such as antioxidant [82,83], and anti-inflammatory activities [84,85]. the polysaccharide contents of the different juglans regia kernel extracts are summarized in table 1. the results show that all the contents are significantly different at p < 0.0001, indicating that the fractionation solvent may have an impact on the polysaccharide contents. table 1. polysaccharides and triterpenoid saponin contents of juglans regia kernel extracts. etoh 70% buoh aqueous supernate precipitate psc (mg ge/g) 95.02 ± 1.35 a 68.58 ± 0.80 b 79.02 ± 1.02 c 103.47 ± 1.13 d 170.13 ± 2.09 e tsc (mg oa/g) 94.94 ± 1.14 a 106.66 ± 1.98 b 10.09 ± 0.98 c 94.66 ± 1.47 a 140.94 ± 2.15 d psc: polysaccharides content; tsc: triterpenoid saponin content data with the same superscript numbers in the same row are not significantly different (p > 0.05). the highest polysaccharide content was recorded in the precipitate extract with 170.13 ± 2.09 mg ge/g, indicating that this fraction may contain a significant amount of saponins, since polysaccharides constitute the hydrophilic part of saponin molecules [86]. second, we found the supernate and hydroalcoholic extract with 103.47 ± 1.13 mg ge/g and 95.02 ± 1.35 mg ge/g, respectively (p < 0.0001). concerning the aqueous and butanolic pmmb 2023, 6, 1; a0000325 9 of 22 extracts showed the lowest contents compared to the other walnut kernel extracts, with 79.02 ± 1.02 mg ge/g in the aqueous extract and 68.58 ± 0.80 mg ge/g. overall, it can be seen that walnut kernels have respectable levels of polysaccharide compounds that give them the possibility of being valued in many pharmaceutical and nutratherapeutic fields [80,83,84]. 3.3. triterpenoid saponin quantification as previously mentioned, this study aimed to evaluate the impact of solvent fractionation on triterpenoid saponins and quantify their content in juglans regia kernels. we were interested in this group of compounds (triterpenoid saponin) because of their ability to provide a variety of pharmacological properties, which help to prevent a wide range of cancers and cardiovascular diseases [87–92], and also have potential anti-inflammatory activity as evidenced by numerous research [93–96]. the quantification results (table 1) showed that all investigated extracts had significantly different contents (p < 0.05) except for the hydroalcoholic and aqueous extracts which contained similar concentrations of triterpenoid saponins with 95.16 ± 1.14 mg oa/g and 94.88 ± 1.47 mg oa/g, respectively (p > 0.05). these significant differences mean that the triterpenoid contents have been influenced by polarity and type of fractionation solvent. the highest triterpenoid saponin content was found in the precipitated extract with 141.16 ± 2.15 mg oa/g, followed by the butanolic extract with 106.81 ± 1.98 mg oa/g (p < 0.0001), and these results indicate that the fractionation protocol applied in this study was effective in extracting the maximum content of the target compound in the last extraction step as a precipitate fraction. the fractionation process is based on the fact that the highest polar compound transfers to the aqueous phase while the less polar compounds (such as triterpenoid saponin in this case) remain in the organic phase, which may explain the lower saponin concentration in the aqueous extract 10.14 ± 0.98 mg oa/g. however, it is important to note that these results are similar to those reported by chen et al. in 2022, who quantified the saponin content of camellia sinensis seeds and reported concentrations above 140 mg/g in different collection periods [97]. furthermore, juglans regia kernel extracts show considerable saponin content compared to xanthoceras sorbifolium and tetracarpidium ionophore (black walnut) seeds with 6.7 mg/100g and 0.08 mg/100g, respectively [98,99]. this increases the possibility of being exploited for their pharmacological properties. 3.4. dpph and abts radical scavenging activity it is recommended to use several radicals to evaluate the antioxidant activity, since the bioactive compounds have varying sensitivity and activity towards different free radicals, and for this reason the antioxidant activity of our extract has been evaluated using two free radicals : dpph and abts. according to the results presented in table 2, it can be clearly seen that the hydroalcoholic and butanolic extracts showed stronger activity against dpph compared to pmmb 2023, 6, 1; a0000325 10 of 22 abts, opposite to the other extracts that showed more activity against the abts free radical than dpph. table 2. ic50 values (µg/ml) of the dpph, abts, and α-glucosidase assays of j. regia kernel extracts. data with the same superscript numbers in the same row are not significantly different (p > 0.05). α-glu: α-glucosidase. • acid ascorbic ic50 value; •• acarbose ic50 value. starting with dpph, our findings showed that the butanolic extract has the strongest antioxidant activity compared to the other extracts studied, with an ic50 value equal to 7.74 ± 1.49 µg/ml, indicating that juglans regia has potent antioxidant activity. this is well supported by the study of zhang et al, that also reported a higher activity in the butanolic fraction compared to ethyl acetate and aqueous fractions [100]. the 70% ethanolic extract also showed strong antioxidant activity similar to the butanolic extract (p > 0.05) with 9.16 ± 0.56 mg/ml in the ic50 value, which may be related to the considerable content of triterpenoid saponins and phenolic compounds in this fraction. all remaining fractions showed considerable antioxidant activity against dpph with ic50 values of 43.63 ± 2.33 mg/ml in the aqueous fraction, 63.93 ± 1.39 mg/ml in the precipitated fraction, and 88.51 ± 2.68 mg/ml in the supernatant fraction, which indicates that the fractionation protocol applied in this study was able to extract the target compounds and preserve their biological activity. as mentioned above, the bioactive compounds had different activity against different free radicals, and this was demonstrated in the present study. the precipitate extract which came third on antioxidant activity against dpph, was the most effective extract against abts free radical compared to other fractions of juglans regia, with ic50 = 33.14 ± 2.96 mg/ml. the aqueous extract showed a very similar result to that of the precipitate with an ic50 of 36.42 ± 1.79 mg/ml (p > 0.05), and both fractions were recorded the highest contents of triterpenoid saponins, which may justify their effectiveness as a source of antioxidant molecules. moreover, all other extracts showed significant antioxidant activity with ic50 values of 51.36 ± 1.10 mg/ml, 43.73 ± 0.61 mg/ml, and 61.37 ± 1.29 mg/ml, in hydroalcoholic (etoh 70%), butanolic and supernate extracts, respectively (p < 0.05). these findings support and confirm the potent antioxidant activity of juglans regia kernels reported in several studies [75,101–104]. etoh 70% buoh aqueous supernate precipitate std dpph 9.16 ± 0.56a 7.74 ± 1.49a 43.63 ± 2.33b 88.51 ± 2.68c 63.93 ± 0.23d 1.75• ± 0.05e abts 51.36 ± 1.10a 43.73 ± 0.61b 36.42 ± 1.79c 61.37 ± 1.29d 33.14 ± 2.96c 2.32• ± 0.01e α-glu 16.17 ± 0.24a 11.66 ± 1.21b 42.08 ± 0.92c 55.89 ± 1.04d 29.63 ± 1.31e 18.01••± 2.00a pmmb 2023, 6, 1; a0000325 11 of 22 3.5. α-glucosidase inhibitory activity diabetes is a chronic illness that happens when a patient's body can't produce sufficient insulin or can't use it [105]. the international diabetes federation (idf) ranks hyperglycemia as the third most severe risk that leads to death worldwide, after high blood pressure and smoking [106,107]. in africa, 14.2 million people were diagnosed with diabetes in 2015, and that number is expected to jump to 34.2 million by 2040, according to the idf [106], which makes diabetes one of the most prevalent diseases in africa and the world. on the other hand, herbal medicines are largely used in morocco to treat various diseases, including diabetes [108], and this is due to the fact that these medicinal plants have a strong ability to inhibit enzymes that are involved in carbohydrate digestion. α-amylase and α-glucosidase are important enzymes involved in carbohydrate metabolism [109], the first one is responsible for the hydrolysis of long-chain carbohydrates into oligosaccharides and then, α-glucosidase intervenes to hydrolyze oligosaccharides into glucose which is then absorbed by the small intestine [110], and increase blood glucose levels [111,112]. the glucosidase assay is essentially based on the inhibition of the enzyme glucosidase prepared in a sodium phosphate buffer solution (ph = 6.4), which is able to hydrolyze pnpg. the formation of a yellow solution is a sign of the hydrolysis reaction. it is inversely proportional to the inhibition capacity. the appearance of a light yellow or transparent mixture indicates a higher efficiency of the inhibition capacity of the extract. table 2 showed that all our extracts significantly inhibited α-glucosidase enzyme, in vitro, with all ic50 values not exceeding 60 µg/ml. the butanolic and hydroalcoholic extracts with the most potent antioxidant activity against dpph also show the strongest inhibition of α-glucosidase better than acarbose, with ic50 values equal to 11.66 ± 1.21 µg/ml and 16.17 ± 0.24 µg/ml, respectively, (p < 0.05), and this means that the antioxidant activity can impact the anti-diabetic property of our extract. in their study, tan et al. report that the compound isolated from etoh 70% walnut extract, especially flavonoids, shows a significant αglucosidase inhibitory activity (α-gia), which can justify and support our results [113]. the precipitated extract also showed significant inhibition of α-glucosidase enzyme with ic50 = 29.63 ± 1.31 µg/ml, this extract is previously reported with the highest triterpenoid saponin content, which may correlate and impact the anti-diabetic property of this extract. several triterpenoid saponins were isolated from gypsophila oldhamiana roots and the results shows that all active compounds exhibited better α-gia than acarbose with ic50s between 15.2 and 98.2 µm, whereas the ic50 of acarbose was 388.0 µm [114]. furthermore, in 2015, a study by nguyena et al. revealed that triterpenoid saponins isolated from schefflera sessiliflora leaves showed greater α-gia (ic50 = 5.99 76.58 µm) than acarbose (ic50 = 214.5 µm) [115], and this may support our hypothesis of saponin content are compounds responsible for α-gia from the precipitate extract. pmmb 2023, 6, 1; a0000325 12 of 22 the remaining extracts (aqueous and supernate) showed the lowest α-gia compared to other studied extracts, but still have significant anti-diabetic activity with ic50 values of 42.08 ± 0.92 µg/ml and 55.89 ± 1.04 µg/ml, respectively (p < 0.0001). overall, juglans regia kernels showed enhanced anti-diabetic activity, which was positively correlated with the dpph results (r = 0.900) (figure 3) and this result affirms that walnut kernels are a great source of bioactive compounds for various pharmacological purposes. 1.00 0.62 0.52 -0.32 0.15 0.36 -0.16 0.19 0.62 1.00 0.07 0.08 -0.35 0.89 0.57 0.85 0.52 0.07 1.00 0.20 0.90 -0.36 0.05 -0.05 -0.32 0.08 0.20 1.00 0.31 0.08 0.70 0.54 0.15 -0.35 0.90 0.31 1.00 -0.69 -0.08 -0.32 0.36 0.89 -0.36 0.08 -0.69 1.00 0.49 0.83 -0.16 0.57 0.05 0.70 -0.08 0.49 1.00 0.87 0.19 0.85 -0.05 0.54 -0.32 0.83 0.87 1.00 psc tsc dpph abts α-gia zoi (b) zoi (e) zoi (k) psc tsc dpph abts α-gia zoi (b) zoi (e) zoi (k) (ic50) (ic50) -1.0 -0.5 0 0.5 1.0 figure 3. pearson's matrix correlation coefficient between antioxidant, anti-diabetic and antimicrobial activities and contents of saponin and sugar. psc: polysaccharides contents. tsc: triterpenoids saponin contents. zoi (b) : zone of inhibition of b. subtilis. zoi (e) : zone of inhibition of e. coli. zoi (k) : zone of inhibition of k. pneumoniae. 3.6. antimicrobial activity the results of the antimicrobial activity of the studied walnut kernel extracts (zone of inhibition and mic in µg/ml) are summarized in table 3. the results showed that all extracts (except the aqueous extract) exhibited antimicrobial activity against the bacteria tested. discussing the mics based on table 3, it can be seen that our active extracts (etoh 70%, buoh, supernate and precipitate) inhibits gram-negative bacteria (mics ≤ 1000 µg/ml) more effectively than gram-positive bacteria, (5000 µg/ml ≤ mics ≤ 1000 µg/ml). the pmmb 2023, 6, 1; a0000325 13 of 22 butanol extract has the lowest mics, which is to say the highest antimicrobial activity against the three bacteria, with mic = 1000 µg/ml in b. subtilis and 500 µg/ml in both e. coli and k. pneumoniae. table 3. inhibition zone halos corresponding to the mics (µg/ml) of juglans regia kernel extracts. tet: tetracycline. nd: no antimicrobial activity detected, (-) inhibition zone < 1 mm, (+) inhibition zone 2-3 mm, (+ +) inhibition zone 3-4 mm, (+ + +) inhibition zone 4-6 mm, (+ + + +) inhibition zone > 6 mm. standard error of the mean of triplicate readings of inhibition zones ± 0.33. these results were stronger compared to the finding in pereira et al. study [116], which was dedicated to evaluating the bioactive properties (including antimicrobial activity) of six different cultivars of juglans regia l. the authors report mics of 100 mg/ml against the same bacteria studied in our investigation [116], suggesting that the fractionation protocol used in this paper can preserve the bioactive compound and retain its activity. regarding the zones of inhibition, figure 4 shows that both butanolic and supernate extracts inhibit e. coli better than the control with zio = 6.33 mm and 6.67 mm respectively, versus 6.00 mm in the control. similarly, the etoh 70%, butanolic and precipitated extracts inhibit b better than the control with zio = 6.00 mm, 5.33 mm, and 5.67 mm respectively, while tet have a zio = 5.00 mm. to explain these results, we adhere to the authors' interpretation which states that antimicrobial activity is strongly related to the content of phenolic compounds [117–119]. it is noteworthy that figure 4 shows that zones of inhibition of b. subtilis and k. pneumoniae are highly correlated with triterpenoid content (r = 0.890 and 0.850, respectively) which may explain the efficacy of our extracts against this bacterium and may also explain the inactivity of the aqueous extract (with the lowest triterpenoid saponin content 10.09 ± 0.98 mg oa/g) against the studied bacterium. etoh 70% buoh aqueous supernate precipitate tet b. subtilis 5000 (+ + + +) 1000 (+ + +) nd (-) 2000 (+ +) 5000 (+ + +) (+ + +) e. coli 500 (+ +) 500 (+ + + +) nd (-) 500 (+ + + +) 500 (+ + +) (+ + + +) k. pneumoniae 500 (+ + +) 500 (+ + +) nd (-) 1000 (+ + +) 500 (+ + +) (+ + + +) pmmb 2023, 6, 1; a0000325 14 of 22 5.00 6.00 8.00 6.00 3.67 5.33 5.33 6.33 5.67 0 0 0 3.00 6.67 5.67 5.67 2.67 4.67 bacillus subtillis esherchia coli kliebsiella pneumoniae tet etoh 70% buoh aqueous supernate presipitate 0 1 2 3 4 5 6 7 8 figure 4. heatmap diagrams representing zones of inhibition diameter (mm) of different extracts of juglans regia kernels compared to tetracycline (tet) as reference. 4. conclusions this study has provided an evaluation of the antioxidant, antimicrobial, and αglucosidase inhibitory activities of crude walnut extracts fractionated by a specific protocol in which triterpenoid molecules were precipitated in the last phase. the precipitated extract was the richest in polysaccharides, which was a positive indication of its richness in triterpenoid saponin compounds. indeed, the precipitated extract recorded the highest content of triterpenoid saponins compared to all the extracts investigated, which may explain its potent antioxidant activity against abts free radical that had the lowest ic50 value of 33.14 ± 2.96 µg/ml. the dpph radical scavenging activity reveals different activity results of the extracts compared to abts assay. the butanolic and ethanolic extracts showed the highest activity against dpph free radical, and this may also explain their α-gia potency which were better than acarbose. all extracts (excluding the aqueous extract) demonstrated antimicrobial activity against the studied bacteria. the most potent extract was the buoh extract, with a minimum inhibitory concentration (mic) of 1000 µg/ml against b. subtilis, and 500 µg/ml against e. coli and k. pneumoniae. these results indicate that crude extract may reveal more activity than pure compounds. author contributions: conceptualization, mt; aey; mef; methodology, literature search, ye; sm; mt; meaf.; aey.; validation, hh; ab; data, ye; mh; collection, ye; ye; sm; mh, hh; ab; ya; writing original draft preparation, ye, ya; reviewed, editing, proofread, ye, hh, ab, ak, ana. funding: this research has no funding. conflicts of interest: the authors declare no conflict of interest. pmmb 2023, 6, 1; a0000325 15 of 22 references 1. guasch-ferré m, li j, hu fb, et al. effects of walnut consumption on blood lipids and other cardiovascular risk factors: an updated meta-analysis and systematic review of controlled trials. am j clin nutr 2018; 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teng-hern tan1,3, vengadesh letchumanan1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; iong0002@student.monash.edu (ijo); ke.loo@monash.edu (k-yl); jodi.law1@monash.edu (jw-fl) 2monash credentialed pharmacy clinical educator, faculty of pharmacy and pharmaceutical sciences, monash university, 381 royal parade, parkville 3052, vic, australia; lydia19595@gmail.com (ln-sl) 3 clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) *corresponding author: vengadesh letchumanan, novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; vengadesh.letchumanan1@monash.edu (vl) received: 08 november 2022; received in revised form: 21 december 2022; accepted: 30 december 2022; available online: 04 january 2023 abstract: helicobacter pylori is a highly prevalent bacteria that can harm humans due to its major involvement in developing gastrointestinal diseases, particularly gastric cancer. therefore, eradicating h. pylori is one of the most important strategies for preventing gastric cancer. antibiotic treatment has always been the gold standard treatment for h. pylori infection. however, the decreasing efficacy of antibiotic therapy due to the rising antibiotic resistance and high incidence of dysbiosis-related adverse effects resulted in eradication failure. to enhance the effectiveness of antibiotic therapy, strategies that modulate the gut microbiome were proposed to play a positive role. generally, the integration of probiotics or symbiotic into antibiotic therapy was shown to enhance the eradication rate and reduce the incidence of adverse effects. this review aims to discuss the role and effect of h. pylori in gastric carcinogenesis and gut microbiome modulation in eradicating h. pylori infection. keywords: helicobacter pylori; gut microbiome; gastric cancer; carcinogenesis 1. introduction the gut microbiome is composed of trillions of microorganisms that live within the human gastrointestinal tract (git) [1]. the gut microbiome has been extensively researched for its association with human health and diseases. research has established the gut pmmb 2023, 6, 1; a0000273 2 of 19 microbiome connection in human digestion, nutrition, immune functions, and physiology. any dysbiosis of the gut microbiome has been linked to various diseases, such as irritable bowel syndrome (ibs) [2], obesity [3], type 2 diabetes [4, 5], autoimmune diseases [6-8], cancer [9], psoriasis [10, 11], obsessive-compulsive disorder [12], and autism spectrum disorder [13]. hence, modulating the gut microbiome has been proven to manage disease outcomes [14-18]. the git is very vulnerable to pathogens, such as vibrio sp. [19-22], salmonella sp. [23], listeria monocytogenes [24, 25], escherichia coli [26], staphylococcus aureus [27], campylobacter sp. [28], helicobacter pylori [29-31], and many more. upon invasion, these pathogens release their toxins and colonize the host gut, leading to a dysbiosis of the gut microbiome [32]. of the mentioned pathogens above, helicobacter pylori research has been reviewed extensively due to its impact on human health. helicobacter pylori is a gram-negative, spiral-shaped, microaerophilic bacteria in the gastric mucosa. it is highly prevalent among the world population affecting over half of them [33]. most people acquire this pathogen in early childhood and can remain asymptomatic throughout their life [34, 35]. h. pylori is among the well-known bacteria due to its role in developing gastrointestinal diseases such as gastritis, gastric ulcer, and gastric cancer [36]. the pathogen was shown to be the primary etiology for gastric cancer and was listed as a type 1 carcinogen in 1997 by the world health organization [37]. due to its association with gastric cancer, h. pylori remains a high burden to healthcare systems worldwide. according to global cancer statistics 2020, gastric cancer is the sixth most common cancer, with an estimated 1089,103 cases, and the fourth leading cause of death due to cancer, with 768,793 deaths in 2020 [38]. most gastric cancers are adenocarcinoma, which is then classified into two morphological types: intestinal type and diffuse-type gastric cancer. for intestinal-type cancer: tumour cells form adhesions, arranging in glandular or tubular formation, and a welldefined sequence has been proposed to be the carcinogenesis model, progressing from healthy gastric mucosa, chronic atrophic gastritis, intestinal metaplasia, dysplasia, and gastric cancer [39, 40]. as for diffuse-type cancer, tumour cells are more scattered and non-cohesive. carcinogenesis is a direct consequence of chronic active inflammation without any similar defined sequence [39]. the human stomach has always been a sterile organ due to its low ph [41]. however, the significant advancement of the technology in microbiome investigation led to the discovery of a diversified microbial community consisting of multiple commensal microorganisms forming a distinct ecological niche where complex interplay is present among h. pylori and the commensal microorganism [42, 43]. a delicate balance is maintained among the microbial community in the stomach, in which any alterations in the balance may lead to dysbiosis and induction of gastric diseases, particularly gastric cancer [44]. meanwhile, h. pylori suppress acid production and destroy the parietal cells, resulting in increased stomach ph and enabling the colonization of new microorganisms [40, 45, 46]. hence, eradicating h. pylori is essential for preventing gastric cancer [47]. pmmb 2023, 6, 1; a0000273 3 of 19 for the past few decades, standard triple antibiotic therapy was the gold standard treatment to eradicate h. pylori. recently, there has been a decreasing eradication rate by antimicrobial therapy due to the increasing prevalence of antibiotic resistance [48, 49]. despite developing new antibiotic regimens, they are still associated with adverse effects, leading to poor patient compliance and eradication failure [50]. antibiotic therapy causes perturbation of the gastric microbiome leading to dysbiosis-induced gastrointestinal side effects [51]. to reduce the incidence of adverse effects from antibiotic-induced dysbiosis, strategies and therapies that can modulate the gut microbiota are being extensively explored [52]. hence, this review aims to discuss the role and effect of h. pylori in gastric carcinogenesis and potential gut microbiome modulation in eradicating h. pylori infection. 2. pathogenesis of h. pylori the fact that not every individual infected with h. pylori develops gastric cancer shows that the pathogenesis of the variable clinical outcomes of h. pylori infection is multifactorial, including the virulence factors of the bacteria strain, host gene polymorphism, and environmental influences. gene polymorphism of pro-inflammatory cytokines such as il-1b increased gastric cancer risk through different mechanisms, including inflammatory injury, gastric acid secretion inhibition, and angiogenesis promotion [53-55]. besides il-1b, genetic variation in another pro-inflammatory cytokine, tnf-α, increased expression is also associated with enhancing gastric cancer risk [56]. with inflammation playing a vital role in cancer progression, polymorphism in inflammatory cytokines, which regulates the intensity of immune response, may augment gastric cancer risk [57]. environmental factors, including dietary habits, cigarette smoking, and alcohol consumption, also affect gastric cancer risk. the dietary habits consisting of high salt content, red meat, processed meat, and low fiber enhanced gastric cancer risk [58-60]. interestingly, former and current smokers demonstrated higher rates of gastric cancer than never-smokers, and the risk intensifies with the number of cigarettes per day for current smokers [61]. as for alcohol consumption, heavy drinkers with more than four drinks per day have significantly increased odds of developing gastric cancers compared to abstainers [62]. besides host genetic vulnerability and environmental influences, virulence factors associated with the bacteria's pathogenicity were involved in gastric carcinogenesis. the human stomach contains gastric fluid with a ph of around 2.0. this highly acidic environment remains a huge challenge for colonizing most invading pathogens, which enables the bacteria to survive in the highly acidic environment, colonize the gastric mucosa, and evade the immune cells. h. pylori possesses urease, one of the main virulence factors that neutralize acidity and enable it to survive and colonize in such a harsh environment [63]. the urease produced by h. pylori plays an alkalizing role as it hydrolyses urea in the stomach and produces ammonia plus carbon dioxide, which neutralizes the stomach's acidity and provides a favorable environment for the survival of h. pylori [63]. aside from acid neutralization, urease was also involved in the carcinogenesis of gastric cancer. recently, an in vitro study uncovered that ureases can trigger a specific pathway to initiate angiogenesis in the gastric epithelial cell. this can potentially lead to the carcinogenesis of gastric cancer [64]. additionally, urease displayed pro-inflammatory pmmb 2023, 6, 1; a0000273 4 of 19 activity in the gastric endothelial cells, inducing alterations in the oxidative profile of the cells and resulting in differentiation which may contribute to gastric carcinogenesis [63]. two main virulence factors affect the pathogenicity of the host cell, caga and vaca virulence protein. caga is the first bacterial oncoprotein shown to be associated with cancer in humans [65]. it is transported into the host gastric epithelial cells by a syringe-like structure called type 4 secretion system [66-68]. in the host cells, there is phosphorylation of caga by tyrosine kinase protein. then, caga binds to signaling proteins that perturb multiple intracellular signaling pathways leading to the host cells' malignant changes [69]. the expression of caga leads to morphological changes in the cells, specifically elongation and increased motility, known as the “hummingbird” phenotype [70]. this oncoprotein also causes disruption of the intercellular junction and the polarity of the epithelial cells [71-73]. in addition, this molecule induces anti-apoptotic activity in the gastric epithelial cells leading to decreased turnover of cells. also, it triggers instability in the genome which both are classic traits of cancer cells [74, 75]. recently, choi et al noted that caga upregulates the cdx1 expression involved in regulation of intestinal function, which may lead to gastric carcinogenesis [76]. another virulence factor vaca is a pore-forming cytotoxin that can induce vacuolation and promote apoptosis in gastric epithelial cells [77, 78]. vaca exerts its effects on cellular activity through the mitochondrial pathway, triggering the release of cytochrome c leading to autophagy and cell death [79, 80]. a study proposed that vaca induces cell apoptosis by activating the endoplasmic reticulum stress cascade [81]. moreover, vaca modulates the host immune reaction by preventing the recruitment of immune cells, including t cells and b cells, to ensure the longevity of the infection [82, 83]. vaca also targets macrophages, the first line of defense, by interfering with the maturation of the phagosome and blocking the activation of cytokines induced by macrophages [84, 85]. recent studies also showed the interplay between vaca and caga, where the disruption of autophagy by vaca promotes caga accumulation in gastric cells resulting in the persistence of h. pylori in the gastric mucosa [86]. 3. h. pylori and gastric microbiome the dogma that the human stomach is a sterile organ has been shattered since the revelation of h. pylori. since then, there has been a hypothesis that the human gut harbors a diversified bacterial community [87]. bik et al. conducted a critical study that explored the composition of the gastric microbiome through a biopsy of the gastric mucosa and pcr of the samples. this study shows that the gastric microbiota of healthy individuals comprises five major phyla, including proteobacteria, firmicutes, bacteroidetes, actinobacteria, and fusobacteria, which were then supported by newer studies [88, 89]. despite the variation of the microbiota composition among individuals, the human stomach contains a diversified microbiome [87, 90]. in the gastric microbiome consisting of a highly diversified bacterial community, h. pylori seemed to be the most dominant bacteria with the highest abundance in the gastric microbiome when present [42, 91]. schulz et al. noticed that the abundance of h. pylori pmmb 2023, 6, 1; a0000273 5 of 19 comprises more than 50% among h. pylori-positive subjects, and the abundance of the other major species decreased [42]. a recent study in 2019 obtained a more significant result where the bacterial communities of the stomach among h. pylori-positive children were notably dominated by 95.43% of h. pylori in the genus [91]. as for h. pylori-negative individuals, studies have shown that streptococcus was commonly the prominent genera with the highest abundance in the gastric microbiome [42, 91, 92]. with the dominance of h. pylori in the gastric microbiome, h. pylori were shown to alter the composition and species diversity. studies showed that the gastric microbiome was significantly less diversified in h. pylori-positive patients than in h. pylori-negative patients. in a study by anders et al., they identified 262 bacterial phylotypes compared to 33 in h. pylori-positive individuals. the result shows that for individuals with h. pylori infection, their gastric microbiota was significantly less diversified than h. pylori-negative individuals [93]. a recent study in indonesia supported the findings that bacterial diversity decreased in the h. pylori-positive group [94]. several other studies further emphasized that the alpha diversity of h. pylori-positive patients is significantly lower than healthy individuals [42, 89]. 3.1. h. pylori and gastric microbiome in gastric carcinogenesis according to correa’s model of gastric carcinogenesis, persistent colonization of h. pylori triggers an inflammatory process that then initiates the carcinogenesis cascade, starting from atrophic gastritis, intestinal metaplasia, dysplasia, and gastric cancer. long-term colonization of h. pylori leads to atrophy of the parietal cells, whose main function is the secretion of acid, leading to increased ph of the gastric fluid and atrophic gastritis [40]. as most bacteria cannot live in highly acidic conditions, h. pylori-induced achlorhydria disrupts the acid barrier, colonizing new microbes, and leading to the gastric microbiome alterations. meanwhile, the proliferation of new microbes may further promote inflammatory reactions, leading to cancer progression [40]. multiple studies attempted to discover the role of non-h. pylori in the carcinogenesis of gastric cancer. one hypothesis proposed that some of these microbes can reduce nitrate into nitrite, forming n-nitroso compounds that are potentially carcinogenic [95]. ferreira et al. analyzed the functional composition of the gastric microbiome in individuals with chronic gastritis and gastric cancer. they noticed that the gastric cancer microbiome has a higher nitrate reductase function, promoting the formation of carcinogenic compounds [45]. jo et al. reported twice as much nitrosating bacteria in individuals with gastric cancer compared to the control groups [96]. similarly, there was an enrichment of bacteria involved in nitrate metabolisms, such as escherichia coli, lactobacillus, and nitrospirae, in patients with gastric cancer [46, 97]. another hypothesis stated that oral microbiota plays a role in gastric carcinogenesis. coker et al. demonstrated that microbiome from the oral origin, including slackia exigua, peptostreptococcus stomatis, streptococcus anginosus, parvimonas micra and dialister pneumosintes were significantly enriched and formed strong interactions in the gastric microbiome of gastric cancer [98]. in another study among gastric cancer patients from malaysia and singapore, there is an increased abundance of oral bacterial taxa such as pmmb 2023, 6, 1; a0000273 6 of 19 leptotrichia, fusobacterium, haemophilus, veillonella, and campylobacter [99]. in a few other studies, there is reportedly a higher abundance of oral bacteria such as lactobacillus, streptococcus, and neisseria in gastric cancer patients [100-102]. sun et al. developed a scoring system to screen for patients with high suspicion of gastric cancer by detecting oral bacteria. the sensitivity was as high as 97.3%, with a 7.7% false-positive rate. however, this study consists of a small sample size of 50 subjects, and more extensive studies are required to validate the results [103]. some studies also examined the changes in gastric microbiota diversity along correa’s carcinogenesis cascade. most studies reported significant differences in gastric microbiome diversity in gastric cancer compared to precancerous lesions, demonstrating gastric dysbiosis's role in gastric carcinogenesis [45, 98]. jimenez et al. conducted a study to explore gastric microbiota changes along the carcinogenesis cascade and reported a decreased diversity in gastric cancer [104]. wang et al. supported these findings, showing that the gastric microbiome diversity decreases along the gastric carcinogenesis cascade [98]. however, studies have proposed contradictory results stating that gastric microbiome diversity increase in gastric cancer. rodriguez et al. and eun et al. reported that gastric microbiome diversity increases in subjects with gastric cancer [99, 100]. wang et al. further supported the findings in a study involving 315 patients showing that gastric microbiota diversity increases in gastric cancer [46]. the discrepancy in the results from different studies could be due to factors influencing the gastric microbiome, such as the variation of gender, ethnic group, and dietary habits among the subjects [87, 98]. 4. antibiotic therapy in h. pylori infections various antibiotic therapies have been evaluated to eradicate h. pylori. however, few were highly effective, with consistently high eradication rates and low incidence of adverse effects [105]. the reasons for eradication failure are mostly due to the increasing antibiotic resistance and non-compliance to treatment. according to clinical guidelines, bismuth quadruple therapy replaced standard triple therapy as the first line in areas with known high macrolide resistance. besides that, multiple alternative drug regimens are recommended as the first-line treatment, including levofloxacin, sequential therapy, concomitant therapy and hybrid therapy, depending on antibiotic resistance and penicillin allergies [49]. recently, vonoprazan emerged as one of the most promising drugs for h. pylori infection [106]. vonoprazan is a highly potent acid-inhibitory drug with rapid onset and longer duration of action than conventional proton pump inhibitors [107, 108]. studies have reported that antibiotic therapy reduces the diversity and composition of the gut microbiome [109, 110]. in a multicentre randomized trial, liou et al. explored the effect of standard triple therapy, bismuth quadruple therapy, and concomitant therapy on the gut microbiome. after antibiotic treatment, faecal microbiota analysis was done at 2, 8, and 10 weeks. the results demonstrated significant perturbation of gut microbiota with reduced microbiome diversity at week 2 for all three antibiotic regimens. after one year, the bacterial diversity was fully restored in standard triple therapy but not in bismuth quadruple and pmmb 2023, 6, 1; a0000273 7 of 19 concomitant therapy. there was an increased abundance of proteobacteria and decreased firmicutes and bacteroidetes in concomitant therapy and quadruple therapy groups [109]. in line with the previous study, chen et al. noticed a decreased microbial diversity, increased proteobacteria, and decreased firmicutes and bacteroidetes, which persisted for more than six weeks after treatment, suggesting the antibiotic-induced dysbiosis may persist without complete restoration after a long period [110]. the abundance of butyrate-producing bacteria, lachnospiraceae, decreased after antibiotic treatment, increasing the risk of clostridium difficile infection [110, 111]. few other studies supported the findings that gut microbiome diversity decreased after eradication therapy with increased proteobacteria and decreased firmicutes, bacteroidetes and actinobacteria after antibiotic therapy [112, 113]. interestingly, there was a reduced abundance of bifidobacterium in a few studies [114-116]. bifidobacterium prevents colonization of commensal pathogens and assists in the metabolism of carbohydrates, suggesting that decreased bifidobacterium may be linked to antibioticinduced adverse effects [115, 117]. 5. modulation of the gut microbiome probiotics are live microorganisms that may provide health benefits if given in optimum quantity [118]. today, probiotics have gained people's interest as an effective therapeutic option for managing digestive and immune health [119]. probiotics have proven effective in treating various diseases, from gastrointestinal issues to recent covid-19 infections [120-122]. lactobacillus, bifidobacterium, and other lactic acid-producing bacteria (lab) are common probiotics used in dairy products and yogurt. as human microbiome research expands, a range of potential probiotics with good benefits to the host has been discovered. among those new potential probiotic candidates is streptomyces sp. recent studies have revealed that streptomyces sp. has strong antibacterial, anti-vibrio activity and probiotic properties for aquaculture usage [123-127]. further studies should be conducted to strengthen these findings before they can be administrated to humans. based on the properties of different probiotics, several proposed mechanisms of action against h. pylori include immunological and non-immunological mechanisms. the first line of defense against foreign pathogens is its high acidity and intact stomach mucosa. probiotics can synthesize antimicrobial substances, including lactic acids, short-chain fatty acids, and bacteriocins. certain probiotic strains, such as bifidobacterium longum and lactobacillus casei were found to inhibit the activity urease in h. pylori, hindering its ability to colonize the stomach [128, 129]. in a study by kim et al., mice model gastric ph decreases significantly after administering lactic acid bacteria, eliminating h. pylori [129]. bacteriocins are proteinaceous toxins that possess anti-h. pylori activity and its antimicrobial activities vary with different probiotic strains. bacteriocin and lactic acid secreted by lactobacillus bulgaricus and lactobacillus pentosus strains were shown to inhibit antibiotic sensitivity and antibiotic resistance h. pylori strains [130, 131]. other than lactobacilli, lim et al. reported that the bacteriocins produced by enterococcus faecalis bk61 exhibit anti-h. pylori activity [132]. a recent in vitro study by sacacino et al. reported several probiotic strains, including lactobacillus casei, lactobacillus paracasei, lactobacillus acidophilus, bifidobacterium pmmb 2023, 6, 1; a0000273 8 of 19 lactis and streptococcus thermophilus strains possess both bactericidal and bacteriostatic against h. pylori [133]. additionally, reuterin, a nonpeptide antimicrobial substance secreted by lactobacillus reuteri was seen to exert effect through downregulation of h. pylori virulence factors, vaca and flaa [134]. probiotics can adhere to binding sites of gastric epithelial cells and provide competition for the adhesion of h. pylori. mukai et al. reported that l. reuteri strains have a specific affinity for two glycolipid binding sites on epithelial cells thus competing and occupying the adhesion site h. pylori, preventing the pathogen from colonizing the gastric mucosa [135]. a newer study by holz et al. added that l.reuteri decreases the mobility of h. pylori by entangling and forming aggregate with h. pylori which will eventually be flushed out from the gut [136]. additionally, lactobacillus gasseri and lactobacillus brevis strain was noted to reduce the adherence of h. pylori to the host cell by inhibiting the expression of saba gene [137]. a probiotic yeast named saccharomyces boulardii was noted to inhibit adherence of h. pylori to the host cell by removing α‐2,3‐linked sialic acid, the ligand for h. pylori adhesin with its neuraminidase activity [138]. probiotics reduce the inflammation in the gastric cells through modulation of the immune reaction and inhibit the production of pro-inflammatory cytokines. studies have shown that l. bulgaricus, l. acidophilus and l. rhamnosus inhibit the production of inflammatory cytokine, particularly il-8, in the cells infected with h. pylori [139, 140]. besides, yu et al. demonstrated that a probiotic cocktail consisting of e.faecalis, l.acidophilus and b.longum plays a significant role in the downregulation of inflammatory chemokines and cytokines, including tnf-α, il-10, il-1β, il-6, mip-2 and g-csf which eventually leading to improvement of h. pylori-induced gastritis [141]. lactobacillus strain probiotics have been widely studied in terms of their role in enhancing the eradication rate and improving gastrointestinal symptoms. a randomized controlled trial by poonyam et al. evaluated the effect of probiotic supplementation on antibiotic therapy by prescribing l.reuteri during bismuth-based triple therapy to eradicate h. pylori. results showed no significant difference in eradication rates but a significantly lower incidence of side effects, including nausea, diarrhea, black stools, and abdominal discomfort [142]. francavilla et al. assigned 100 h. pylori-positive individuals into two groups: one group received standard triple therapy plus two l.reuteri strain, and another group received the same antibiotic treatment and placebo. in contrast to the previous study, the probiotic group's eradication rate was significantly increased compared to the placebo group (75% vs 65.9%). similarly, lower side effects were reported in the probiotic group compared to the placebo (40.9% vs 62.8%) [143]. the probiotic yogurt containing l. gasseri was seen to reverse the dysbiosis and restore the balance in the gastric microbiome [144]. besides lactobacillus, bifidobacterium was also commonly used as supplementation for anti-h. pylori antibiotic therapy. in a study by jiang et al., 232 patients were randomly prescribed bismuth quadruple therapy plus live combined bifidobacterium probiotic or bismuth quadruple therapy without probiotic. significantly higher eradication rates and lower pmmb 2023, 6, 1; a0000273 9 of 19 incidences of side effects were noted in the probiotic group [145]. cekin et al. compared the eradication rate between one group receiving sequential eradication therapy with probiotics containing bifidobacterium animalis subsp. lactis b94 and one group without probiotics. the eradication rate was significantly higher with probiotics than with the controls (86.8% vs 70.8%). the probiotic group also experienced a lower incidence of diarrhea (1.88% vs 12.26%), leading to better compliance with treatment [146]. several studies have explored the role of yeast probiotics such as s. boulardii. seddik et al. reported that adding s.boulardii to anti-h. pylori sequential therapy resulted in an increased eradication rate compared to sole sequential therapy (86.0% vs 76.7%). the probiotic group also reported a significantly lower incidence of antibiotic-induced diarrhea (2.0% vs 46.4%) and overall side effects (17.0% vs 55.7%). this led to a significantly higher compliance rate in the probiotic group (95.0% vs 91.2%) [147]. in 2019, he et al. administered s.boulardii during bismuth-based quadruple therapy. no significant difference was noted in the eradication rate. still, the incidence of nausea, diarrhea, and side effects was significantly lower compared to the control group. this study also noted that supplementation of probiotics simultaneously with quadruple therapy recorded a lower incidence of adverse effects than administration on the 14th day after commencing quadruple treatment, suggesting the best timing for probiotic supplementation [148]. a recent study by cardenas et al. revealed that s. boulardii supplementation increased bacterial diversity in the gastric microbiome [149]. additionally, clostridium was also considered a potential candidate for probiotic supplementation. mukai et al. demonstrated that anti-h. pylori therapy regimen consisting of probiotic c. butyricum and standard triple therapy recorded a higher eradication rate of 87.1% compared to 70.1% in standard triple therapy without probiotics [150]. chen et al. also noted that c.butyricum supplementation improves gastrointestinal symptoms by restoring gut microbiota composition [139]. furthermore, there are also studies exploring the role of other probiotics. tang et al. prescribed bismuth quadruple therapy to 162 patients, with one group receiving probiotics consisting of enterococcus faecium plus bacillus subtilis and another group receiving a placebo. the result was consistent with the previous study, as the probiotic group's eradication rates were higher at 87.01% compared to 82.43% in the placebo group [151]. furthermore, beneficial bacteria were enriched with probiotic supplementation, including oscillospira and lactobacillales, known for producing short fatty acid chain butyrate, regulating immune reactions, and alleviating gastrointestinal symptoms [152, 153]. inflammation-linked harmful bacteria, such as collinsella, dialistera and sutterella were also reduced in the probiotic group [154]. jung et al. compared the eradication rate and side effects of one group receiving concomitant therapy and another group receiving standard triple therapy plus probiotics containing either freeze-dried l. casei rhamnosus or b.subtilis combined with e. faecium. in contrast to previous studies, there were no significant differences in the eradication rates between groups, but the probiotic group reported a lower incidence of adverse effects [155]. aside from probiotics, prebiotics and synbiotics also play a role in gut modulation. prebiotics are compounds that are non-digestible but can be metabolized by gut microbiota pmmb 2023, 6, 1; a0000273 10 of 19 to allow benefits for the host [156]. in contrast to probiotics, limited studies were done to investigate the role of prebiotics on anti-h. pylori therapy. instead, more studies researched synbiotics, a combination of probiotics and prebiotics. a study by sirvan et al. explored the impact of bifidobacterium lactis based synbiotics on standard triple therapy. results showed a higher eradication rate in the triple therapy plus synbiotics group compared to triple therapy alone (88% vs. 72%). in terms of the adverse effects, the synbiotics group experienced a significantly lower incidence of adverse effects, including nausea, vomiting, diarrhea, and abdominal pain [157]. contrary to this study, two other trials used the same b.lactis strain and inulin during standard triple therapy. there was no significant difference in the eradication rate compared to the control group. ustundag et al. reported no significant difference in the side effects in both groups. in contrast, a study by islek et al. reported significantly lesser side effects in the symbiotic group than in the triple therapy group (63% vs. 17%) [158, 159]. fecal microbiota transplant (fmt), which is the transplantation of fecal material from a healthy donor into the patient’s intestines, has been proven to restore the gut microbiota and treat gastrointestinal disorders, including irritable bowel syndrome, inflammatory bowel disease, and clostridium difficile infection [160]. additionally, fmt was shown to be more cost-effective than antimicrobial therapy [33]. therefore, researchers hypothesized that fecal microbiota transplantation could play a role in eradicating h. pylori through the restoration of gut microbiota. to verify this hypothesis, ye et al. conducted a study in 2020 where they administered washed microbiota transplant (wmt) to 32 patients. wmt was performed in this study due to its lesser adverse effects and higher efficacy as compared to conventional fmt [161, 162]. 32 patients diagnosed with h. pylori infection for the past year and not on eradication therapy were administered wmt once a day for three consecutive days. the result showed an eradication rate of 40.6% four weeks post-wmt [162]. this positive result warrants future randomized controlled trials with a larger sample size to determine the optimal dosage and frequency of wmt and to confirm the safety and efficacy of wmt. 6. conclusion antibiotic therapy has been the gold standard for the eradication of h. pylori. nevertheless, antibiotic therapies have limitations, including increasing antibiotic resistance and antibiotic-induced dysbiosis, leading to a high incidence of adverse effects and poor patient compliance. the few factors combined lead to decreased efficacy of antibiotic therapy and reduced eradication of h. pylori, contributing to a higher prevalence of gastrointestinal diseases, particularly gastric cancer. this review shows that integrating probiotics into antibiotic therapy enhances antibiotics' efficacy and restores the gastric microbiome's homeostasis. probiotics extensively studied and shown to be effective include lactobacillus, bifidobacterium, saccharomyces, and clostridium strains. however, there is insufficient evidence to determine the best probiotic strain for eradicating h. pylori. therefore, large sample trials should be conducted to determine the selection, optimum dosage, and duration of antibiotics and probiotics to improve the efficacy of antibiotic therapy. besides probiotics, there is also an increased eradication rate and reduced adverse effects with symbiotic supplementation. however, relatively fewer trials were done on symbiotic, prompting more pmmb 2023, 6, 1; a0000273 11 of 19 studies to investigate the efficacy of symbiotic supplementation and the possible combination of probiotics and prebiotics for the best effect. lastly, fecal microbiota transplant is potentially a highly cost-effective therapy for h. pylori. further studies should be conducted to evaluate its safety, effectiveness, and synergistic effect when 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original creator is stated. adaptation and remixing are also permitted. pmmb 2021, 4, 1; a0000193. doi: a0000193 http://journals.hh-publisher.com/index.php/pmmb review article legionella pneumophila — the causative agent of legionnaires’ disease loh teng hern tan1,2†, wei yu tee2†, tahir mehmood khan3,4, long chiau ming5, vengadesh letchumanan2* article history 1clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) 2novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia; weiyuwy1@gmail.com (w-yt) 3institute of pharmaceutical sciences (ips), university of veterinary & animal sciences (uvas), pakistan; tahir.khan@uvas.edu.pk (tmk) 4school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 5pap rashidah sa’adatul bolkiah institute of health sciences, universiti brunei darussalam, gadong, brunei darussalam; long.ming@ubd.edu.bn (lcm) *corresponding author: vengadesh letchumanan, vengadesh.letchumanan1@monash.edu received: 3rd march 2021; received in revised form: 3rd april 2021; accepted: 5th april 2021; available online: 8th april 2021 † these authors contributed equally to the writing. abstract: over the years, legionella pneumophila has increasingly become a public health threat that causes sporadic and epidemic community-acquired and nosocomial-acquired pneumonia. thus, this review aims to discuss the current knowledge of l. pneumophila, focusing on the global epidemiology, clinical features, diagnosis and treatment of legionnaires’ disease (ld). legionella bacteria are gram-negative rod-shaped bacteria that are ubiquitous in aquatic environments. l. pneumophila was first discovered in 1976 and recognized as the causative agent of ld. l. pneumophila is a facultative intracellular pathogen that infects and replicates within eukaryotic host cells such as macrophages and protozoan. diagnosis of ld remains a significant challenge as the clinical manifestation of ld is hardly distinguishable from pneumonia caused by other respiratory pathogens. therefore, early testing and appropriate treatment are keys to alleviating the rising morbidity and mortality caused by ld. keywords: legionella; pneumonia; amoeba; legionnaires’ disease; respiratory disease pmmb 2020, 4, 1; a0000193 2 of 12 1. introduction throughout history, human populations have encountered several serious epidemics caused by pathogens[1–3]. pathogens are various organisms that cause severe illnesses to their hosts, including bacteria, viruses, fungi and protozoa[4–11]. among the various illnesses, respiratory infections represent the largest category of human disease and the leading cause of mortality globally[12–16]. apart from the pneumonic plague caused by yersinia pestis as one of the most significant outbreaks in human history, legionella is another pathogenic bacteria that were discovered from investigations of an epidemic of a mysterious pneumonia outbreak in philadelphia in 1976[17]. at the time of writing, there are more than 60 species and serogroups under the genus legionella, a great variety of them can inflict human disease or legionellosis. legionellosis is defined as an infection caused by bacteria under the genus legionella, of which there are two distinct clinical manifestations in humans — pontiac fever (pf) and legionnaires’ disease (ld)[18]. legionella pneumophila serogroup 1 is the clinically most relevant species that cause ld in human, accounting for 90% of the total reported human infections. meanwhile, l. bozemanii, l. longbeachae, l. dumoffii are the several species also known to cause ld[19]. primarily, ld is caused by inhalation of aerosols or aspiration of water containing legionella bacteria. given the continued increasing incidence of ld globally, legionella bacteria present a significant threat to human health. there was a 286% increase in legionellosis cases reported in the united states from the year 2000 to 2014[20]. furthermore, a steady increase of ld cases was also observed in european countries, whereby more than 80% of the infections contributed by l. pneumophila serogroup 1[21]. therefore, this review aims to update the current knowledge of legionella bacteria prevalence, clinical manifestations, pathogenesis, detection and treatment strategies (figure 1). 2. sources of legionella bacteria legionella is a gram-negative, facultative aerobic and intracellular waterborne bacterium present in natural aquatic environments, such as lakes, rivers and groundwater, but at low concentrations[22]. the widespread distribution of legionella bacteria is attributed to their ability to proliferate within diverse types of niches, including live in planktonic form, infect and replicate within protozoan hosts as well as coexist within multi-organismal biofilms developed on the surfaces of water systems[23,24]. given the ubiquitous nature of legionella bacteria in aquatic habitats, legionella bacteria can easily enter and proliferate in man-manufactured water systems, including cooling towers and water distribution system of large buildings[25]. a relatively high prevalence of legionella bacteria has also been found in public facilities such as hospitals, showers, hotels and hot water systems[26–29]. several factors that enhance the colonization of legionella bacteria in the water systems include lukewarm water temperature, obstruction and stagnation of water flow, plumbing materials, biofilm formation and presence of amoeba, which support the growth of legionella sp. these risk factors are commonly present in the water distribution system of antiquated buildings, pmmb 2020, 4, 1; a0000193 3 of 12 especially in old hospitals[30]. in fact, half of the sporadic cases of hospital-acquired pneumonia are attributed to legionella bacteria, where patients are infected via inhalation of legionella sp. contaminated aerosols from medical devices like respiratory equipment, misting devices, cooling towers and hot tubs[31]. figure 1. the transmission sources, life cycle within water systems and human macrophage, the diagnostic tools and antibiotic treatment for legionella pneumonia. besides living in the planktonic phase, l. pneumophila utilizes amoebae as a host for survival and replication. currently, there have been more than 40 species of legionella sp. identified to possess the ability to parasitize amoebae, specifically acanthamoeba and naegleria[32]. thus, these protozoans act as a vector of various pathogenic microorganisms[33], including legionella, and facilitates pathogen dissemination. being an intracellular pathogen, legionella sp. also can infect and thrive within human macrophage [34]. a recent study demonstrated that the infection processes and the intracellular life cycle of legionella within amoeba are remarkably similar to that occurred within human macrophages[35], although both are evolutionary distant hosts. researchers believe that the l. pneumophila is primed for subsequent infection in the protist host and becoming more virulent and infectious after escaping from its environmental host, resulting in a more robust disease in humans[35,36]. in fact, a previous study indicated that intracellularly pre-grown l. pneumophila acquired higher resistance to disinfectants and antimicrobials, suggesting to confer increased infectivity to mammalian cells[37]. 3. epidemiology of legionnaires’ disease legionella pneumophila is a significant bacterial pathogen that causes severe sporadic and epidemic community-acquired and nosocomial acquired pneumonia[38]. an pmmb 2020, 4, 1; a0000193 4 of 12 estimate of 12 to 15% of overall mortality rate among hospitalized patients[39], the number may go up further to 27% for those who failed to receive adequate antibiotic treatment. furthermore, ld is also commonly associated with poorer outcomes with the increasing length of hospitalization[40]. dated back to july 1976 in philadelphia, the first-ever reported ld outbreak due to l. pneumophila resulted in 29 deaths out of the total 180 persons attending the 56th annual american legion convention[41]. over the years, the burden of ld is increasing each year in the united states and european countries. in the united states, there was an incidence of 1.89 cases of ld per 100,000 populations in 2015. similarly, 1.4 cases of ld per 100,000 populations were reported by the european centre for disease prevention and control in 2016. recently, a study in italy reported an incidence rate of 48.9 cases per 100,000 populations for legionellosis in 2018, of which 3.4% was of nosocomially acquired cases[42]. in hong kong, the incidence of ld was increasing from 0.16 cases/100,000 population in 2005 to 0.91 cases/100,000 population in 2015[43]. among all the legionella sp. identified to date, l. pneumophila has responsible for 80–90% of the ld cases reported in the united states and europe. within the similar legionella species, l. pneumophila serogroup 1 accounts for approximately 90% of cases. generally, the sources of outbreaks for nosocomial acquired ld are attributed to the contaminated water in the hospital plumbing system and the exposure to contaminated cooling towers. from 2006 to 2017, cooling waters, air conditions and evaporative condensers were reported to have contributed a major portion (60%) of outbreak-associated deaths due to ld or pf. moreover, building water systems contributed to approximately 13% and 17% of the outbreak-related cases and deaths, respectively[44]. besides that, the european working group for legionella infections and the united states centers for disease control and prevention (cdc) have identified numerous cases of travel-associated ld where the most common source is the contaminated water in hotels. travel-associated ld has also been linked to cruise ships[45]. the mode of transmission of ld to human is mainly by the inhalation of contaminated aerosols droplets where the source can disseminate water droplets such as the cooling towers. due to the small sizes of water droplets containing the legionella sp., which can be deeply inhaled into the respiratory airways, it has increased the ease of transmission of ld. besides that, there is limited evidence to indicate the person-to-person transmission of ld. not until recently, the first strong evidence of person-to-person transmission of ld was reported in vila franca de xira, portugal, in 2014[46]. the host-related risk factors for ld infection may be associated with factors such as old age, cigarette smoking, organ transplantation, the use of immunosuppressive medications, obstructive pulmonary disease as well as patients with acquired immune deficiency syndrome. moreover, the elevated risk of nosocomial infection may attribute to general anaesthesia and endotracheal intubation. besides that, there are reports of infection in children and premature neonates[45]. in addition, those workers who maintain the water pmmb 2020, 4, 1; a0000193 5 of 12 cooling towers and air-conditioning system are the most at-risk group to be infected with ld where the circulated air that contaminated with water droplets containing legionella sp. can be inhaled easily into the respiratory system[45,47]. 4. pathogenesis of legionella pneumophila predominantly, human exposure occurs when l. pneumophila are inhaled into the lungs through inhalation of contaminated aerosols which results in pneumonic respiratory disease. a combination of bacterial virulence factors and host immunity determines the outcome of legionella infection, whether leading to a self-limiting and mild respiratory disease (pf) or severe pneumonia (ld)[48]. once l. pneumophila gets into the lung, the bacteria are phagocytosed by the alveolar macrophages, where they can multiply intracellularly. this is due to the ineffective killing of l. pneumophila, which has been demonstrated through several in vitro models of infection by specific antibody and neutrophils[49]. meanwhile, the pathogenesis of pf may involve a host response to endotoxin from the lipopolysaccharide of legionella without replication of the bacteria in the host[50]. the infection process of l. pneumophila on protozoa and mammalian phagocytic cells are shown to be similar where legionella uses common gene and gene products to infect mammalian and protozoan cells[35]. the uptake of l. pneumophila by monocytes and macrophages is by the conventional and coiling phagocytosis which is an important feature in the pathogenesis of the bacteria[51]. after internalization of the pathogen by phagocytosis, l. pneumophila evades endocytic degradation, controls the innate immune response, and triggers the legionella-containing vacuole (lcv) biogenesis. the lcv biogenesis involves the recruitment of the rough endoplasmic reticulum and mitochondria result in formation of a specialized vacuole for the intracellular replication of l. pneumophila. this mechanism is directed by the type iv secretion system encoded by the dot/icm genes. the dot/icm type iv secretion system is the main virulence system of l. pneumophila which consists of bacterial proteins that promote the secretion of bacterial virulence factors suggested to be responsible for inhibition of phagosome-lyzosome fusion[52]. hence, l. pneumophila can multiply intracellularly in human macrophages by avoiding phagosome-lyzosome fusion[53]. as legionella becomes vigorously motile in the massive intracellular vesicle, it causes the host cell to become overwhelmed by the infection resulting in lysis of the host cell. this has led to the death of the host cell macrophage and the release of the progeny of the pathogen from the cell to the environment where new host cells are found. 5. detection and diagnosis of legionella pneumonia today, various diagnostic techniques are available for legionella pneumonia, including urinary antigen tests, respiratory specimen cultures, molecular detection tests, and serum antibody tests[54]. despite being the diagnostic gold standard, culture of legionella bacteria isolated from the clinical respiratory sample is limited by the requirement of a specific culture medium[55] and long laboratory turnaround time, which requires at least 3 to 5 days to identify culture positivity[56]. pmmb 2020, 4, 1; a0000193 6 of 12 serological test for antibodies against legionella bacteria was the principal diagnostic tool for ld in the early 1980s. however, serological test has been replaced by more rapid and definitive analyses in the modern laboratory, such as the urinary antigen test (uat) and molecular methods. the use of serology was reported to have declined from 61% to 6% from 1995 to 2010[57], due to the widespread adoption of the less technically demanding uat. the uat has been widely utilized globally due to its straight-forward procedure and rapid turnaround time. in europe and the united states, uat represents more than 80% to 90% of the diagnostic tools used for ld confirmation. the specific urinary antigens of legionella can be detected in most of the l. pneumophila infections in the first few days of clinical symptom onset and up to several days to more than ten months. typically, the specific urinary legionella antigen is no longer detected in most cases after 1 to 2 months of antibiotic therapy. however, the current commercially available uat for non-l. pneumophila serogroup 1 exhibit lower sensitivity and highly variable than those for l. pneumophila associated disease when tested with urine from confirmed ld patients[58,59]. remarkably, a recent study demonstrated a novel urinary antigen test kit (lac-116) that can detect non-l. pneumophila serogroup 1 pneumonia on top of showing comparable accuracy with the other existing kits for legionella pneumonia[54]. with the advances of molecular biology and the development of new technologies, the field of diagnostic and detection of pathogens has been improved tremendously, with many new molecular methods being developed to generate rapid, sensitive and accurate results[60]. currently, real-time pcr is regarded as the molecular method of choice for the detection of legionella bacteria. targeting the macrophage inhibitor protein mip gene, highly conserved in all l. pneumophila isolates, has been widely employed by numerous published studies, showing a 15% increased yield in detecting any l. pneumophila serogroup than that of culture method[61]. some studies performed pcr targeting 16s rdna gene of legionella sp. which was suggested to offer greater sensitivity. however, the capnetz study demonstrated a low detection rate of legionellosis (10%) with legionella sp. pcr targeting 16s rdna and only 60% of these samples were confirmed by dna sequencing. although legionella nucleic acid-based detection methods offer higher sensitivity and rapid turnaround time, there are several notable disadvantages and limitations. for instance, nonlower respiratory tract samples, such as urine and serum, are not suitable for the pcr test, and the inherent limitation of pcr which cannot assess bacterial viability. furthermore, nucleic acid amplification technologies also require highly trained personnel to handle sophisticated equipment to perform the diagnostic test[62] but are increasingly accessible to many laboratories on a moderate budget[61]. nevertheless, nucleic acid-based methods are valuable additions to ld diagnostic and detection methods and complement well with the use of other testing modalities to enhance the rate of successful detection[61]. in the united states, a clinical case of legionella pneumonia is considered when laboratory results show positive uat, bacterial culture and serology test for l. pneumophila serogroup 1. meanwhile, a suspected or probable case is considered when legionella pmmb 2020, 4, 1; a0000193 7 of 12 antigens in respiratory specimens are detected by serology test, detection of seroconversion to non-serogroup 1 or non-l. pneumophila, and/or detection of legionella nucleic acid[63]. 6. clinical manifestation and management of legionella pneumonia legionellosis presents two distinct clinical manifestations in humans: milder respiratory disease pf and severe pneumonia ld[64]. pf manifests as a milder form of ld that involves respiratory illness without pneumonia, which resembles the self-limiting flulike illness with symptoms lasting for approximately a week. meanwhile, ld is a more severe form of pneumonia that could also affect other organs, including the liver and kidneys[65,66]. typically, ld is a severe pneumonia that often occurs in susceptible persons, including old ages, smokers and those with comorbid conditions or immunosuppression[67]. ld does not present any specific or defining clinical features but shows a range of clinical manifestations and symptoms. ld basically begins with a headache, muscle pain and general feeling of sickness. then, these typical symptoms are followed by severe high fever, dry cough, shaking chills, nausea, vomiting and diarrhoea. the infected person also might experience chest pain and difficulty in breathing. in some rare incidences, a persistent or relapsing ld may develop into slow or nonresolving pneumonia which is characterized by the persistence of pulmonary infiltrates for more than 30 days since the disease onset[68]. majority of the previously reported cases occurred in immunocompromised patients as a result of intracellular survival of legionella pathogens due to ineffective antibiotic treatment[69,70] rather than de novo reinfection via exposure to contaminated sources. these slowly or non-resolving ld patients experience severe pulmonary complications, including pleural effusion, abscess formation and empyema, over the course of the infection[71]. considering the high mortality and morbidity associated with untreated ld, early diagnosis and prompt treatment with effective antibiotics are extremely crucial, together with appropriate clinical management of complications and underlying comorbidities should be prioritized for ld patients[72]. given that ld does not present any defining clinical features, empiric antibiotic therapy is well justifiable for the initial management of all moderate-tosevere communityand nosocomial-acquired pneumonia prior to ld confirmation made from microbiological diagnosis. primarily, therapy for ld is the antibiotic treatment of the infection and management of complications. in general, all legionella sp. tested are found to be susceptible to commonly prescribed macrolides (erythromycin, azithromycin) and fluoroquinolones (levofloxacin)[73]. antibiotics, such as macrolides and fluoroquinolones, are also known to achieve therapeutic intracellular concentration within tissue and alveolar macrophages where l. pneumophila resides. since 1976, erythromycin had been the treatment of choice for l. pneumonia but only until 1990s, because of its side effects when given intravenously. to date, azithromycin and levofloxacin have become the mainstay ld treatment in both healthy and immunocompromised patients[18]. a recent meta-analysis revealed that levofloxacin pmmb 2020, 4, 1; a0000193 8 of 12 resulted in a significant reduction in the length of hospitalization, lowered mortality, shorter time to apyrexia, and reduced risks of adverse effects compared to macrolides[74]. world health organization (who) has developed a framework that can be used to assess and manage the risks posed by legionella[31]. the framework includes health-based targets, water safety plans and surveillance. in addition to the framework proposed by who, many approaches of disinfection have been tried over the past 13 years with variable success. superheating of water up to 70°c and 80°c, hyperchlorination[75] and copper-silver oxidation method were also implemented to halt the ld outbreak. 7. conclusion legionella pathogen is one of the top causative agents of severe respiratory disease, which requires swift treatment and rapid diagnosis to achieve successful disease resolution with appropriate antibiotic therapy. legionella pathogen replicates intracellularly in the host cells, especially amoeba and macrophages. the process of invasion, development and replication of legionella sp. in the environment has been well studied. ld occurs as outbreaks or as sporadic cases in the community and hospital. the transmission of this disease is through inhalation of contaminated aerosols or aspiration of contaminated water. furthermore, the diagnosis and treatment of ld have changed over the years, where the urinary antigen detection test and molecular methods are commonly used nowadays. early testing and appropriate treatment are keys to mortality and morbidity caused 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right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology original research article 1 characterization of somatic mutations in malaysian luminal breast cancer yock ping chow1, ryia illani mohd yunos1, isa mohd rose1,3, norlia abdullah2, rohaizak muhammad2, shahrun niza abdullah suhaimi2, saladina jaszle jasmin2, norshahidah mahamad nadzir1, nurul-syakima ab mutalib1, rahman jamal1* 1 ukm medical molecular biology institute, kuala lumpur, malaysia 2department of surgery, faculty of medicine, universiti kebangsaan malaysia, kuala lumpur, malaysia 3department of pathology, faculty of medicine, universiti kebangsaan malaysia, kuala lumpur, malaysia abstract : luminal breast cancer subtype (er/pr+, her2+/-) represents about two-thirds of all breast cancers, and better understanding of the genetic alterations underpinning the disease pathogenesis is desirable to develop improved treatment plans and to increase patients’ survival. to date, other than hormonal and anti-her2 therapies (e.g. tamoxifen, aromatase inhibitor, trastuzumab), no other targeted therapies have been approved by fda for luminal breast cancer. despite thousands of breast tumour samples have been sequenced, there is no data yet on malaysian patients. therefore, it is clinically important to identify actionable mutated genes or pathways implicated in our local breast cancer patients to establish a more defined framework for precision medicine and clinical trials. in this discovery study, a total of nine pairs of newly diagnosed luminal breast cancer cases (>80% tumour content) and their matched normal samples were subjected to exome sequencing. we detected a total of 491 somatic from nine pairs of breast tumour-normal samples. pik3ca is the most frequently mutated gene in our discovery cohort of patients (n=4/9). kinases and phosphatases were found as the most significantly enriched mutated genes (enrichment score = 3.12), with all nine luminal breast cancer samples harboring at least one non-synonymous mutation in this cluster of genes. this profile suggested that alteration in protein phosphorylation processes is among the key drivers in luminal breast cancer pathogenesis. interestingly, genes involved in four key druggable cancer pathways, i.e. pi3k/ akt/mtor, mapk/erk, nf-kβ and vegf signalling pathways were found commonly mutated and require validation in a larger cohort. in conclusion, we have successfully profiled the somatic mutations in malaysian luminal breast cancer and this is the first study conducted in malaysian patients. these findings revealed the role of multiple gene testing in discovering luminal breast cancer mutational landscape. keywords: luminal breast cancer; exome sequencing; somatic mutation; actionable mutation; druggability received: 30th october 2018 accepted: 29th november 2018 published online: 19th december 2018 *correspondence to: rahman jamal, ukm medical molecular biology institute, ukm medical centre, jalan yaacob latiff, bandar tun razak, cheras, 56000, kuala lumpur; rahmanj@ppukm.ukm.edu.my citation: chow yp, mohd yunos ri, rose im, et al. characterization of somatic mutations in malaysian luminal breast cancer. prog microbes mol biol 2018; 1(1): a0000014. background breast cancer is the most common malignancy among malaysian women (national cancer registry report 2007) and remains as the leading cause of cancer-related mortality in women worldwide[1]. it is a heterogeneous disease and is broadly classified into three major subtypes based upon the expression of oestrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor 2 (her2) status[2]. luminal breast cancer subtype represents about two-thirds of all breast cancers and is the most heterogeneous subtype comprising of tumours with six different profiles, namely, er/pr/her2 (er+/ pr+/her2+, er+/pr-/her2+, er+/pr+/her2-, er-/pr+/her2+, er+/pr-/her2-, er-/pr+/her2-). even though er positive breast cancer patients could be treated with anti-oestrogen (e.g. tamoxifen, fulvestrant) and er/her2 positive breast cancer patients with antioestrogen and/or anti-her2 therapies (e.g. nolvadex, tamoxifen, herceptin, letrozole, anastrozole, exemestane), the lack of response in a subset of patients remains a major clinical issue. it has been reported that er positive breast cancers exhibited de novo (~30%) and acquired resistance (~40%) to anti-oestrogen therapies[3]. also, nearly 20% of her2 positive early breast cancers were resistant to anti-her2 therapies, whilst 70% metastatic breast cancer cases were not responsive[4]. given that there is a significant number of patients who precopyright 2018 by chow yp et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) 2 sented with de novo or acquired resistance to these therapies, it is therefore, imperative to obtain a better understanding of the molecular genetic changes underpinning the disease pathogenesis and to recommend additional treatment plans to improve patients’ survival rates. in the past decades, precision oncology with matching treatment plans based on the patient’s tumour molecular profiles have yielded remarkable success in several malignancies. for instance, stratification of metastatic melanoma patients who harboured braf v600e to vemurafenib treatment has increased patient’s survival rates[5]. additionally, stratification of lung cancer patients who harboured egfr mutations to receive anti-egfr therapies has achieved 70% response rate[6]. hence, incorporation of the mutational screening program to identify actionable therapeutic targets in cancer patients holds a great promise to transform clinical care and to allow implementation of personalised therapies. in recent years, the advent of next generation sequencing technologies have reshaped the paradigm of clinical trials from drug-centric to patient-centric[7]. accumulated evidence has shown that the individual tumour is unique and only harboured a low prevalence of common mutations across the different tumour types[8]. in breast cancer, a comprehensive list of somatic mutations has been identified through several large-scale international sequencing projects[9], and many somatic variants were found unique to individual patients. in fact, other than pik3ca and tp53 which are found in 36% and 37% breast cancer respectively, the majority of the mutations were found in less than 5% patients[10]. the lack of common mutations across cancer patients (prevalence of <5%) has increased the demand to implement genomic-driven clinical trials globally, and has strongly suggested that the concept “one drug fits all” may not be conducive anymore. armed with the evidence that the individual tumour genetic profile is distinct; it is believed that exome sequencing could be a more cost-effective approach to identify potential actionable candidate genes in an unbiased manner, whereby nearly 85% of the disease-causing variants were found in protein-coding regions[11]. materials and methods patient sample a total of nine malaysian female patients who were diagnosed with primary luminal breast cancer (grades ii-iv) at the ukm medical centre in kuala lumpur were included into this study. fresh frozen tissue samples and matched blood samples were collected from patients with written informed consent. this study has been approved by the research ethics committee of the ukm medical centre (approval number: ukm 1.5.3.5/244/umbi-2015003). approximately 6 ml of peripheral blood from the subject was collected into an edta blood tube, whereas the removed surgical tumour samples were immediately snap-frozen in liquid nitrogen prior to nucleic acid extraction. three patients presented with metastasis (lung, liver or spinal) at the initial diagnosis whereas the other six patients did not have distant metastasis. all the patients were radiotherapy-naive and chemotherapy-naive at the time their samples were collected. tumour samples were analyzed by immunohistochemistry for the status of oestrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor 2 (her2) at the histopathology unit, pathology department. the summary of the patients’ clinicopathological information is shown in table 1. dna extraction exome sequencing of malaysian luminal breast cancer... table 1. patient clinicopathological information and sequencing metrics. for tissue samples, a reference slide was taken from a 5 µm thickness cryosection, followed by staining with haemotoxylin and eosin. the slides were then reviewed by a pathologist. only samples with more than 80% tumour content were selected for dna extraction using allprep dna/rna/mirna universal kit (qiagen). for germline samples, dna was extracted from edta-tube collected blood samples using salt precipitation protocol. the dna purity was assessed by nanodrop spectrophotometer (thermo scientific) whereas quantity was determined by qubit dsdna hs sample age ethnicity richardson grade er/pr/ her2 distant metastasis sample type on target mean depth (x) 20x coverage uniformity c1 36 malay 3 +/-/lung, liver, spinal tumour 46.65% 51 82.82% 91.93% normal 61.93% 52 86.87% 93.03% c2 43 chinese 3 +/-/bone tumour 79.00% 62 85.38% 90.81% normal 81.25% 55 85.82% 92.01% c3 40 malay 2 +/+/+ spinal tumour 75.57% 82 88.94% 90.49% normal 76.08% 71 89.12% 91.75% c4 62 malay 2 +/+/absent tumour 75.64% 64 77.80% 82.87% normal 74.37% 70 80.20% 84.02% c5 58 chinese 2 +/+/+ absent tumour 75.89% 51 81.97% 89.60% normal 70.45% 50 81.50% 89.58% c6 47 malay 2 +/-/+ absent tumour 78.97% 58 85.12% 91.04% normal 75.70% 50 81.23% 90.12% c7 77 chinese 3 +/-/+ absent tumour 75.73% 60 85.24% 90.94% normal 77.04% 53 86.84% 92.57% c8 56 malay 3 +/-/absent tumour 78.96% 58 87.20% 92.67% normal 75.39% 52 85.22% 92.50% c9 76 chinese 3 +/+/ absent tumour 73.17% 65 88.63% 92.46% normal 81.95% 51 87.42% 93.52% average 74.71% 60x 85.20% 90.72% 3 assay kit (thermo scientific). preparation of libraries for targetseq exome capture briefly, 1 μg of total genomic dna extracted from the tumour and matched blood samples (except for sample c1 which had matched normal tissue) was randomly sheared by focused acoustic shearing (covaris). the sheared dna was purified using the ampure xp reagent (agencourt) with a target size peak of ~250 bp. the purified dna was then subjected for library construction according to the ion xpress™ fragment library kit manual (thermo scientific). the dna was end repaired, ligated with the p1 adapter and barcoded (ion xpress™ barcode adapters kit; thermo scientific), followed by size selection (~280 bp) using e-gel® ex agarose gels, 2% (thermo scientific). the library was then amplified with platinum pcr supermix high fidelity (thermo scientific), and purified with ampure xp reagent (agencourt) prior to exome capture. exome capture and sequencing with ion proton sequencer pre-capture library dna was hybridized to exome capture probes using the ion targetseq exome enrichment kit (thermo scientific) according to the manufacturer’s specifications. the probe-hybridized library dna was then captured with streptavidin-coated m-270 beads (dynal) the probe-hybridized dna was then subjected for 12 cycles of pcr amplification at 95°c for 15 s, 58°c for 15 s, and 72°c for 60 s. the amplified exome library was purified twice with a 1.5-fold volume of ampure xp reagent, and eluted in 25 μl ddh2o. the barcoded exome libraries were pooled and subjected to emulsion pcr using the onetouch 2 instrument (thermo scientific) with an ion pi template ot2 200 kit v2 following the manufacturer’s instructions. the enrichment of template-positive ion sphere particles (isp) was performed using the ion onetouch es enrichment system (thermo scientific). the enriched ion sphere™ particles were then loaded onto an ion p1 chip v2 and sequenced on the ion proton using sequencing v3 kit. bioinformatics analysis briefly, the reads were aligned to the hg19 reference sequence, followed by removal of duplicates and variant detection by torrent variant caller (tvc) version 4.4.2.1. the coverage of each amplicon was obtained by the coverage analysis plugin software v4.4.0.20 (thermo scientific). called variants with phred quality scores of <30 were then filtered out using snpsift, followed by annotation using annovar[12]. the variants with the following criteria were included in further analysis: (a) variants in coding region and splice site; (b) a minimum coverage of 8x was achieved for both tumour and germline samples; and (c) a minimum of 2x variant reads in tumour samples and absent or with a variant threshold less than 1% in matched normal. variants detected in the homopolymer region were excluded. the detected variants were then compared against public databases, including dbsnp142, 1000 genomes project, and 6500 exomes of the national heart, lung, and blood institute exome sequencing project to filter out polymorphisms with allele frequency >1%, except for those variants reported in the cosmic database (cosmic 70). somatic mutations were determined by subtracting against matched germline variants and manually verified by viewing through integrative genomics viewer (igv)[13]. the deleterious effect of the mutations was predicted using sift[14] and polyphen2[15]. the variants were annotated based on the ensembl gene database for hg19. furthermore, david analysis (http:// david.abcc.ncifcrf.gov/) was performed to investigate the gene ontology and enriched biological process or pathways implicated in the breast cancer pathogenesis. validation of mutations by sanger sequencing a total of 22 candidate druggable mutations were selected for validation by sanger sequencing to confirm the somatic mutation status and to determine the accuracy of exome sequencing. primers specific to the regions of interest were designed using primer-blast (additional file 1: table s2). pcr amplification was conducted using amplitaq gold® dna polymerase (thermo scientific) using the following cycling conditions: one cycle at 95 °c for 5 min, 35 cycles at 95 °c for 1 min, 56°c or 65°c for 1 min and 72 °c for 1 min, and one cycle at 72 °c for 5 min. sequencing was performed with abi bigdye terminator v3.1 (thermo scientific). the sequence chromatograms were visually inspected with bioedit software. results breast cancer patients this study involved nine malaysian patients with luminal breast cancer with various combinations of oestrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor 2 (her2) status. all the tumours expressed the er marker. the median age of the patients was 56 years, with a range of 36-77 years (table 1). there were five patients who presented with grade iii tumours while the other four patients had grade ii tumours. three patients (c1, c2, and c3) presented with synchronous distant metastasis whereas the others (c4, c5, c6, c7, c8, and c9) were not diagnosed with distant metastasis. identification of somatic mutation by exome sequencing both tumour and matched normal samples were sequenced to a mean coverage of 56x (range, 51x–82x) and 61x (range, 50x–71x) respectively, with an average of 85% of targeted bases covered by at least 20x across the sample set (table 1). by using the described filtration strategy (see materials and methods), we identified a total of 491 somatic mutations in the exonic and splice site regions. of these mutations, 368 were protein-altering (288 missense, 50 indels, 17 nonsense, 12 splicing), whereas the remaining 124 were synonymous (figure 1). patients who were >50 years or with distant metastatic disease did not have significantly more mutations as compared to patients with age <50 years (p-value=0.38) or without distant metastatic disease (p chow yp et al. 4 value=0.47). interestingly, patients with grade iii tumour harbour (figure 3)significantly more mutations (range 58-109) as compared to those with grade ii tumour (range 11-39) (p-value = 0.002). to understand the impact of the mutations on gene functions further, we applied sift [14] and polyphen2 [15], and found that ~76% (n=280/367) of protein-altering mutations were likely to have functional consequences (damaging or probably damaging) according to at least one of the two methods (additional file 1: table s1). in addition, consistent with previous data on breast cancer, analysis on the nucleotide substitution pattern in all our nine breast cancer samples confirmed that the c>t transitions were predominant (accounting for 5060% of all substitutions) (figure 2), a mutational signature associated with deamination of 5-methyl-cytosine to uracil by apobec enzyme[16]. recurrently mutated genes figure 1. number and type of somatic mutations identified in nine pairs of matched tumour-normal malaysian luminal breast cancers. our analysis identified a total of 491 somatic mutations, in which 367 were non-synonymous and 124 were synonymous. figure 2. mutation spectra of malaysian luminal breast exomes. c>t transition was predominant, a mutation signature associated with apobec expression. one way to identify driver mutations is to look for those that are recurrent across multiple samples. in our study, we detected a total of eight recurrently mutated genes, with pik3ca as the most frequently mutated gene (p.e545k, n=2; p.h1047r, n=1; p.r38c, n=1), followed by kif27 (p. r860x, p.g461a), map3k3 (p.i504n, p.r502w), sf1 (p.g572fs, p.g572r), syne1 (p.f7302fs, p.d4267y), tchh (p.a101d, p.e294x), utp20 (p.e1986d, p.i1927v), and hpse (c.1325+2t>a, p.k307m). majority of the mutations were novel and not listed in cosmic. of these candidate mutations, pik3ca mutations are hotspot mutations which have previously been classified as driver mutations in various types of cancer, including breast cancer[9]. enriched molecular functions and pathways functional enrichment analysis using david (database for annotation, visualization and integrated discovery) (http:// david.abcc.ncifcrf.gov) revealed that genes involved in protein phosphorylation was the most significantly enriched (enrichment score =3.12, p-value=0.008; additional file 1: table s3), in which 21 were kinases (aak1, akt, cdc42bpa, smg1, adrbk2, csk, cdkl5, ddr2, galk2 , ikbkb, irak2, irak3, kdr, lmtk2, mast4, map3k3, obscn, pik3ca, prkar1b, stk31, ttn) and 7 were phosphatases (enpp3, inppl1, pten, ptp4a2, ptpn11, ptpro, slc20a1) (figure 3). beyond that, our analysis also identified genes involved in cell adhesion processes (ppfia2, tnxb, magi1, col13a1, inppl1, nell1, lrrn2, fermt3, tnc, nell2, nlgn1, sdk1, pcdhgb6, vtn, ddr2, cdh5, podxl2, nphp4, frem2, exome sequencing of malaysian luminal breast cancer... 5 lama5, dsg1, susd5, lamc2, muc5b) (enrichment score=1.7, p-value = 0.02; additional file 1: table s3), and chromatin modification (ing4, epc1, csrp2bp, setdb2, huwe1, c11orf30, whsc1l1, smarcal1, actl6b, cbx2, rb1, irf4) (enrichment score = 1.4, p-value = 0.03; additional file 1: table s3) were commonly mutated, and may play significant roles in driving breast cancer pathogenesis. druggable alterations our analysis also identified the somatically mutated genes which are druggable, and for which approved drugs are available or in clinical trials. as depicted in figure 4, among those pathways affected include the pi3k/akt/ mtor signalling pathway (ago3, akk1, creb5, col4a4, ddr2, esr1, grap, gnaz, inppl1, lama5, lmtk2, lamc2, pdgfrb, pik3ca, ppp2r2b, pten, ptpn11, rb1, tenc1, tnc, tnxb, tp53), mapk/ erk signalling pathway (adcy2, atf7, cacna2d3, col13a1, fgf13, ksr1, map3k3, mapk7, nf1, prkar1b), nf-kβ signalling pathway (ikβkβ, irak2, irak3, irf2, irf4, malt1, pawr), and vegf signalling pathway (cdh5, csk, kdr, nell1, nell2, vtn). to evaluate the performance of exome sequencing on ion semiconductor chemistry, we randomly selected 22 druggable alterations for sanger validation. a total of 21/22 of the selected mutations were confirmed, gave rise to 95% specificity. however, one mutation in ikβkβ gene (ikβkβ g. 42162746c>t) ) was failed to be confirmed by sanger sequencing and identified as false positive. figure 3. the distribution of mutated kinases and phosphatases. our analysis found that dysregulation of protein phosphorylation processes is the key driver of luminal breast cancer pathogenesis. discussion it has been estimated that about 30-40% of women diagnosed with invasive breast cancer will eventually develop metastatic disease[17], in which there is little or no cure. even though primary luminal breast tumour responds well to existing chemotherapy, anti-her2 or hormonal therapy, the high recurrence rate indicates that current treatment strategies are not effective to eradicate residual disease which will later transform into a more aggressive and relatively resistant tumour subpopulation. therefore, a broader range of therapeutic options is needed to allow the tumour cells to be eliminated through different druggable pathways during initial treatment. in this preliminary study, we sought to uncover the mutational landscape of malaysian luminal breast cancers and to identify potentially actionable therapeutic targets for the disease. in agreement with many other studies[9], our findings indicated that mutations are rarely shared across patients, and each tumour harboured a distinct set of mutated genes or mutations which affected multiple druggable pathways, and may be amenable to broader therapeutic options. other than pik3ca which was found to be recurrently mutated in 4/9 samples, other genes were mutated in only 2/9 samples (map3k3, kif27, sf1, syne1, tchh, utp20, hpse). in contrast to other findings which reported tp53 as the second most common mutated gene implicated in luminal breast cancer, we only detected one protein-altering mutation (p.m246v). the low prevalence of tp53 mutation in our local samples could be attributed to the small sample size and warrants further validation in additional samples sets. despite individual breast tumour sample showing a distinct mutational profile, genes involved in protein phosphorylation processes appeared as the most significantly enriched biological process across all nine patients (additional file 1: table s3). we postulated that these non-recurrent mutated kinases and phosphatases may be interacting cooperatively leading to constitutive activation of signalling pathways essential in conferring growth advantage to breast cancer cells. our analysis found that at least four key druggable pathways were commonly mutated in luminal breast cancers (pi3k-akt-mtor, mapk/erk, nf-kβ, vegf) with some cases harbouring numerous mutated genes involved in similar pathways (e.g. c9 had mutations in pik3ca and pten which involve in pi3k-akt-mtor pathway; figure 4), whereas others had several mutated genes affected multiple pathways (e.g. c2 had mutations in genes involved in pi3k-akt-mtor (creb5, col4a4, esr1, grap, inppl1, ppp2r2b), nf-kβ (irak2, pawr), mapk/erk (adcy2, map3k3, prkar1b) and vegf (vtn); figure 4). genes involved in regulating the pi3k/akt/mtor pathway were found mutated in 7/9) of cases. activation of pi3k-aktmtor pathway plays pivotal roles in promoting cancer growth by mediating cell proliferation, angiogenesis, migration[18], and associated with the development of resistance to hormonal treatment in breast cancer[3]. over the past decades, inhibition of the pi3k-akt-mtor pathway has increasingly gained interest as a potential treatment modality for breast cancer patients. numerous clinical trials on pi3k inhibitors, mtor inhibitors, and akt inhibitors are actively ongoing, and preliminary findings supported that patients administered with combination therapies (aromatase inhibitor & pi3k/akt/ mtor inhibitor) have a better outcome as compared to patients treated with aromatase inhibitor alone[19]. given chow yp et al. 6 that somatic mutations in pik3ca and pi3k/akt/mtor signalling pathways were observed in four and seven of our luminal breast tumour samples respectively, it is believed that a substantial number of our local breast patients could benefit from this class of targeted therapy. it has been reported that genes regulating mapk/erk signalling pathway were recurrently mutated in nearly 2-10% of breast cancer[10], and involved in promoting breast cancer proliferation, invasion, migration, and resistance to tamoxifen[20]. in our study, mutations involving the mapk signalling pathway were seen in five out nine cases (figure 4). we also detected recurrent mutations in map3k3 (n=2/9). constitutive activation of mapk pathway is one of the key molecular alterations underpinning breast cancer oncogenesis, and increased mapk activity was found to be associated with lymph nodes metastasis[21], and shorter disease-free survival[22]. the aggressiveness of the disease also has been attributed to the presence of cancer stem cells. earlier study has shown that mek inhibitors was effective in eliminating breast cancer stem cells and prevented lung metastasis[23]. moreover, the effectiveness of mek inhibitor selumetinib in reversing resistance of er-positive high grade serous ovarian cancer towards anti-oestrogen therapy[24] also strengthened the fact that inhibition of mapk/ erk pathway may be a potential therapeutic strategy for improving treatment outcome in breast cancer. genes involved in nf-kβ pathways were found mutated in 5/9 cases (figure 4). earlier studies have shown that constitutive activation of nf-kβ pathway is frequently observed in breast cancer[25] and is associated with breast cancer oncogenesis and progression[26]. notably, nf-kβ activation has been reported to play an important role in erpositive endocrine-resistant breast cancer and the acquisition of anti-oestrogen (specifically tamoxifen) resistance, which correlates with earlier relapse, metastasis and a reduced overall survival[27]. similarly, nf-kβ activation and their involvement in chemoresistance were also observed in breast tumour cells treated with paclitaxel[28], cisplatin, 5-fluorouracil and gemcitabine[29]. earlier studies have shown the chemoresistance mechanism may be partially mediated by expansion of breast cancer stem cells which have self-renewal capacity and capable to drive tumour growth and recurrence[30]. the efficacy of nf-kβ inhibition in selectively eliminate breast cancer stem cells[31] shall be considered among the strategic therapeutic options to overcome chemoresistance in breast cancer treatment. beyond that, it has been well recognized that cancer pathways are highly complex and inhibition of a pathway may lead to augmentation of another pathway which promotes the disease progression. for instance, inhibition of pi3k signalling pathway alone led to augmented mek/erk signalling, and impaired anti-tumoural activity[32]. given that numerous evidence has shown deregulation of pi3k/akt/ mtor, mapk/erk and nf-kβ pathways are associated with breast cancer pathogenesis and disease recurrence[33], targeting these signalling pathways therefore represents among the most attractive and promising therapeutic option to overcome chemoresistance and to realizing personalized targeted therapies. moreover, it is increasingly recognized that combination regimens are more effective than mono-agent therapy to eliminate tumour cells which are genetically heterogeneous. for instance, a previous preclinical study showed that dual inhibition of pi3k/ akt/mtor and mapk/erk pathways is more efficient than single pathway inhibition[34]. also, blockade of the erk and nf-kβ pathways has been shown to actively suppress the emt, migration, invasion, and stem-like features in breast cancer cells[35]. interestingly, clinical trials on the dual-targeting strategy involving pi3k/ akt/mtor and ras/mek/erk pathways were particularly effective in tumours with genetic alterations in both pathways[36], and abrogated mechanisms of resistance in solid tumours, including lung cancer[37] and melanoma[38]. our analysis demonstrated the prominent co-existence of mutations across pi3k/mapk/nf-kβ pathways, hence suggested that a combination of drugs targeting oestrogen, pi3k, nf-kβ and mapk pathways simultaneously or sequentially is a rational therapeutic strategy for breast cancer patients. the association of the mutational status and their actionability warrants further investigation. figure 4. the landscape of potentially clinically actionable somatic alterations identified in 9 pairs of malaysian luminal breast cancer. conclusion in summary, pik3ca appeared as the most frequently mutated gene in malaysian luminal breast tumours. our analysis revealed that each of the tumour harboured exome sequencing of malaysian luminal breast cancer... 7 distinct mutation profiles which affected numerous druggable pathways, notably pi3k, mapk, nf-kβ, and vegf signalling pathways. these findings highlight the role of multiple gene testing in discovering luminal breast cancer mutational landscape. notwithstanding breast cancer is genetically well-characterized in caucasian populations, little is known about the mutational profile in malaysian patients. hence, this study was initiated to uncover the mutational landscape implicated in our malaysian luminal breast cancer cases, and to identify potentially actionable therapeutic targets or pathways to establish a more define clinical trial framework for our local patients. in this study, a total of nine pairs of matched tumour-normal luminal breast cancer samples were subjected to whole exome sequencing using the benchtop ion proton sequencer to an average coverage of 60-fold. our findings from the discovery set of data, demonstrated that exome sequencing on ion proton platform is clinically useful to identify somatic mutations in an unbiased manner, with an accuracy of 95% validated by sanger sequencing approach. however, further study in larger series of samples will be required to confirm the identified variants and how they implicated in treatment, especially in our local population. acknowledgment the present study was funded by a grant from the ministry of science, technology and innovation (07-05-mgigmb016). conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. reference 1. cornejo km, kandil d, khan a, et al. theranostic and molecular classification of breast cancer. arch pathol lab med 2014; 138(1): 44-56. 2. 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in patients with advanced cancer. clin cancer res 2012; 18(8): 2316-2325. 37. engelman ja, chen l, tan x, et al. effective use of pi3k and mek inhibitors to treat mutant kras g12d and pik3ca h1047r murine lung cancers. nat med 2008; 14(12): 1351-1356. 38. posch c, moslehi h, feeney l, et al. combined targeting of mek and pi3k/mtor effector pathways is necessary to effectively inhibit nras mutant melanoma in vitro and in vivo. proc natl acad sci u s a 2013; 110(10): 4015-4020. chow yp et al. pmmb 2021, 4, 1; a0000204. doi: 10.36877/pmmb.a0000204 http://journals.hh-publisher.com/index.php/pmmb review article covid-19: malaysia's fight against this deadly virus article history ke-yan loo1, vengadesh letchumanan1* 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia; ke.loo@monash.edu (k-yl) *corresponding author: vengadesh letchumanan; vengadesh.letchumanan1@monash.edu (vl) received: 19 may 2021; received in revised form: 29 may 2021; accepted: 31 may 2021; available online: 2 june 2021 abstract: malaysians are facing the biggest challenge in 2021 the rise in covid-19 cases and its variant of concern (voc) strains. the scale and impact of this coronavirus disease are unimaginable, scary, and full of sorrows. many lost their loved ones, relatives, friends, and children are becoming orphaned overnight. now our only hope is on the available vaccines to control this deadly virus infection. when this review article was in press, over 170 million confirmed cases with 3.5 million deaths were reported worldwide. malaysia is dealing with spikes in the number and severity of new cases. a record toll of new cases and fatalities for consecutive weeks has pushed malaysia's total cases to a soaring 500,000 confirmed cases with 2300 deaths – the third highest in southeast asia behind indonesia and the philippines. the healthcare system in malaysia is currently under heavy pressure to control the disease as the number of confirmed cases is rising exponentially. a national covid-19 immunization program was launched in february 2021 in the hope of providing immunity to 80% of the population in malaysia and achieve herd immunity. this review discusses the alarming covid-19 situation in malaysia, the management strategies, and the vaccinations program. keywords: covid-19; novel coronavirus; malaysia; vaccination; herd immunity 1. introduction it has been over a year since the world health organization (who) declared covid-19 as a global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2)[1–5]. this virus has vastly spread worldwide, with new variants becoming a source of concern as the variants are associated with higher infectivity linked to high fatality rates[6,7]. the total number of confirmed cases has surpassed 170 million, and 3.5 million deaths reported globally[8]. in 2020, vaccines were developed pharmaceuticals at warp speed to overcome the burden of covid-19[9]. in november 2020, pfizer officially announced that their vaccine has a 95% efficacy against this virus and subsequently rolled out the vaccine for use in december. since then, various other vaccines with considerable efficiency against covid-19 were produced by pharmaceutical companies; for example, moderna, astrazeneca, sinovac, etc. have also been distributed worldwide to fight against pmmb 2021, 4, 1; a0000204 2 of 11 the pandemic[9,10]. cumulatively, more than 1.4 billion vaccine doses have been administered internationally[8]. malaysia's fight against covid-19 started in early 2020. soon after the covid-19 outbreak in china, the first cases of covid-19 were detected in malaysia on 25th january 2020[11]. the cases involved three chinese nationals who were close contacts of an elderly covid-19 patient receiving treatment in singapore. the chinese nationals were part of a group of eight placed under quarantine in johor bahru after traveling into malaysia via singapore on the 24th of january 2020[12]. the three chinese nationals were then moved to the sungai buloh hospital in selangor for treatment[13]. the ministry of health malaysia (moh) quickly released a statement notifying the public about the confirmed covid-19 cases. it urged the public to practice good personal hygiene i.e., hand washing, wearing face masks, social distancing, and avoid large social gatherings or crowded areas to prevent the spread of the virus. individuals who displayed symptoms of difficulty in breathing and fevers should also seek treatment immediately. on the 4th of february 2020, a 41-year-old man became the first malaysian to be tested positive for the coronavirus. he had just returned to malaysia from singapore and had developed symptoms of fever and cough. he was referred to sungai buloh hospital and admitted to an isolation ward[14]. just two days later, his younger sister was tested positive for the virus, and this became the first case of local transmission of the sars-cov-2 virus in malaysia[15]. the malaysian government took various precaution measures and imposed restrictions throughout the country to control the spread of this virus. malaysians started to live and adapt to the "new normal" life, which caused a significant impact on the economic, social, and livelihoods[16]. a national covid-19 immunization program was launched in february 2021, in the hope of providing immunity to 80% of the population in malaysia and achieve herd immunity. as of 22 may 2021, around 3% of the nation's population is fully vaccinated. this review aims to provide insights into the alarming covid-19 situation, management strategies, and vaccination status in malaysia. 2. covid-19 cases in malaysia initially, the number of confirmed cases in malaysia was growing slowly, but in march 2020, there was a sudden exponential increase in the cases. the cases were traced back to a religious event held in sri petaling, kuala lumpur, which amassed 16,000 local participants and 1,500 foreign participants[17]. the abrupt spike in cases pushed the government to close its borders to the world as a preventative measure. prompted by the rapidly increasing number of cases in malaysia, the prime minister of malaysia also announced the first movement control order (mco) on 16th march 2020[18]. during this period, all mass movements and gatherings were prohibited, and only essential services could operate across the country. the government completely prohibited all interstate, inter-district, or international traveling during this time. the mco, which was scheduled to end on the 31st of march, got extended until 4th may 2020 to further prevent the spread of the virus. a conditional movement control order (cmco) was then implemented from 4th may to 9th pmmb 2021, 4, 1; a0000204 3 of 11 june 2020, whereby the lockdown restrictions were slightly loosened, and certain business sectors were allowed to resume operations[19]. the prime minister of malaysia then announced that the cmco would end and be replaced by the recovery movement control order (rmco) on 9th june as the covid-19 cases were well under control. interstate travel was now allowed, and almost all businesses, educational, religious, and social activities could resume under strict standard of procedures during the rmco. malaysians were still reminded to wear face masks when in public areas, practice social distancing, avoid crowded areas, and practice good hygiene to prevent a new wave of virus spread. however, amidst the rmco, positive cases hit an all-time high in october, reaching over 800 cases due to many individuals traveling interstate in september to vote in the state election in sabah[20]. this contributed to the third wave of infections in malaysia as the number of confirmed positive cases in malaysia continued to soar, hitting record high numbers and grew in an upwards trend up until 29th january 2021 where cases peaked at 5,725 cases[21]. there was a steady decrease in the daily cases until early april 2021; then, cases began to increase rapidly once more going into may 2021. the cases were increasing at an alarming rate in less than two months, and 9,020 cases were reported on 29th may 2021, surpassing the previous record (figure 1). the daily number of deaths due to covid-19 has also been rising, reaching 98 deaths a day on 29th may 2021. the spike in cases further overwhelmed the public healthcare system in malaysia, which was already under heavy pressure from handling covid-19 cases since 2020. figure 1. illustration of daily confirmed covid-19 cases, cumulative covid-19 cases, cumulative deaths due to covid19, and a brief timeline of the events in malaysia since the beginning of the covid-19 pandemic in 2020. pmmb 2021, 4, 1; a0000204 4 of 11 3. vaccination in malaysia despite the rising number of cases, the government has drawn up various strategies and continues to control coronavirus spread in malaysia. the earliest efforts were to enforce health screenings at all points of entry to detect fever among individuals. the number of hospitals that could treat covid-19 was also increased to accommodate the rising numbers of confirmed cases of infection. strict rules and regulations devised during the mcos aimed to prevent the further spread of the virus into local communities[22]. standard operating procedures (sop) were implemented in workplaces to reduce staff capacity, social distancing, prohibition of mass gatherings, face mask-wearing, etc., to prevent disseminating the virus into local communities[23]. furthermore, at-risk individuals within identified clusters were tracked down via contact tracing to undergo covid-19 screening and would serve as a mandatory home surveillance order[24]. the ministry of health (moh) also raised awareness for covid-19 via mass media infographics to continuously remind the public of the preventative measures that can be taken to prevent covid-19 infections. in addition, the moh would update the people on the number of confirmed cases, total deaths, and total recoveries daily on their website[25]. the press briefings, conference recordings, and relevant news on covid-19 are also frequently updated by the malaysian government and the moh. as the roll-out of vaccines developed by pfizer, astrazeneca, and sinovac began worldwide, the malaysian government has been placing orders for these vaccines since november 2020[26–28]. additional orders for the vaccines were placed in 2021 to speed up the vaccination process in malaysia to achieve the target of providing immunity to 80% of its 32 million population by february 2022. the first batch of 312,390 doses of the pfizer vaccine arrived on the 21st february 2021, while 268,000 doses of the astrazeneca vaccine were received on 23rd april 2021[29–31]. as for the vaccine doses from sinovac, 200 litres of bulk vaccine arrived from china on 27th february and were filled and finished at the pharmaniaga lifescience sdn bhd (pls) plant. as of 10th may, pls has prepared 1.2 million doses of sinovac vaccines, and 290,480 doses have been approved by the national pharmaceutical regulatory agency (npra) for distribution[31]. more vaccine shipments have since arrived in malaysia in the months following the first batches. these vaccines would be distributed freely upon registration to malaysians and foreigners residing in malaysia, with malaysians receiving priority. the vaccines would also be distributed through the covid-19 immunization programme in three phases, with the first phase completed in april 2021. while the target for phase 1 was to administer 500,000 doses of vaccines, 552,682 doses were distributed with an average of 23,410 daily doses. the first phase prioritized front liners, whereas phase two prioritizes individuals in high-risk groups such as senior citizens and those with chronic disease. phase two is scheduled to complete in august 2021, while phase three would prioritize the adult population over 18 years old[32]. pmmb 2021, 4, 1; a0000204 5 of 11 the covid-19 vaccination programme in malaysia has been distributing vaccines from pfizer (comirnaty), astrazeneca (vaxzevria), and sinovac (coronavac) to the public since february 2021[33]. a study was done in 2020 that studied the intent of malaysian nationals to receive the covid-19 vaccine revealed a high overall acceptance of a vaccine, with almost half of the participants expressing a definite intention to take the vaccine and 45% of participants expressing a possible and probable intention. the study also showed that participants were more likely to have a definite intention of receiving the vaccine if they had a high perception of benefits and low perception barriers in receiving the vaccine, along with high perceived susceptibility of getting a covid-19 infection[34]. thus, the government and moh have been continuously promoting the benefits of receiving the vaccination while reducing the barriers of receiving the vaccines, such as distributing the vaccines free of charge and setting up many vaccination centers countrywide to expand the outreach of the vaccines. since the global distribution of the covid-19 vaccines, many have been concerned about the adverse events that may occur upon receiving vaccines of specific brands. the vaccines used in malaysia have shown little to no side effects detrimental to the individual's health. the most common side effects are local pain at the injection site, sore muscles, fever, and fatigue post-vaccination. these side effects are generally well tolerated and subside in 12 days[35–37]. to note, only pfizer vaccine is recommended for pregnant or breastfeeding mothers as there is insufficient data to show the safety of the astrazeneca and sinovac vaccines in these demographics. although severe adverse events may occur, these instances are extremely rare. the moh has set up the vaccination system so that upon receiving the vaccine, recipients are to be observed for a period to enable swift detection of serious adverse events on the off chance that they occur. these measures ensure that the adverse events following immunization can be treated and monitored at an earlier stage[33]. however, the astrazeneca vaccines raised concerns from the public in malaysia. this was due to incidents that occurred abroad whereby individuals developed thrombosis with thrombocytopenia syndrome (tts) post vaccination with the astrazeneca vaccine[38,39]. further analyses done by researchers have shown that the occurrence of blood clotting combined with low platelet counts caused by the vaccine is relatively low. the risks of tts have been shown to occur approximately 4 cases per 1 million, further indicating that the benefits of taking the vaccine greatly outweigh the risks[40]. considering the public's concerns, the malaysian government opened bookings to the public for the astrazeneca covid-19 vaccines in may voluntarily. in addition, the vaccine would not be included in the mainstream national vaccination program and will be distributed only to those who want it[41]. the first round of registration for voluntary vaccination with the united kingdom developed vaccine opened on 2nd may and are to be distributed within klang valley. all 268,000 doses were booked within the first three hours, and the doses were administered in may. to date, no severe side effects from the astrazeneca vaccine have been reported in malaysia[42]. the second round of registration for 1.1 million doses of the vaccine opened from 23rd may to 26th may. high-risk groups and individuals over 60 years of age were prioritized during this pmmb 2021, 4, 1; a0000204 6 of 11 round of voluntary opt-ins. the supply of vaccines will be extended to penang, sarawak, and johor, currently in the covid-19 red zones[43]. according to data released by the special committee on covid-19 vaccine supply (jkjav), now, over 2 million individuals have received the covid-19 vaccine, and over 12.2 million have registered to receive the vaccine. to date, approximately 6.2% of the population has received at least one dose of the vaccine (figure 2)[44,45]. figure 2. an illustration of malaysian who shared their experience receiving at least one dose of covid19 vaccine on social media. 4. the emergence of voc strains when this review writing went into press, the number of covid-19 positive cases and deaths in malaysia rose to historic heights. both the public and private healthcare sectors are greatly overwhelmed by the number of confirmed cases. intensive care units in many hospitals are fully occupied with covid-19 patients in severe conditions, and quarantine centers are filled with covid-19 patients. at the beginning of the outbreak of covid-19 in malaysia, researchers analyzed 58 sars-cov-2 genome sequences from covid-19 patients in malaysia. they determined that the major contributor of the disease transmission in malaysia was the sars-cov-2 lineage b.6[46]. as the outbreak progressed and more confirmed cases were reported, different variants of the coronavirus were isolated in malaysia. for instance, in pahang, sars-cov-2 with d614g mutation was found in an asymptomatic patient and this mutation resulted in higher infectivity of the coronavirus[47]. other variants of concern (voc ) strains include the sars-cov-2 lineage b.1.351 (south african), b.117 (united kingdom), and b.1.617.1 (indian). to note, these three variants have raised the concern of the moh as individuals infected with this variant typically appear pmmb 2021, 4, 1; a0000204 7 of 11 asymptomatic and would present negative test results during covid-19 screenings. those infected would not experience any typical covid-19 symptoms such as fevers or loss of smell or taste, but signs of pneumonia would be shown through x-ray scans[6,48]. these variants were also linked to high fatality rates. the emergence of these variants raises concerns of many regarding the efficacy of the vaccines currently available to the public. data from qatar suggested that the pfizer vaccine was effective against infection and disease within qatar's population, where the b.1.1.7 and b.1.351 variants were the major causes of infection in the country[49]. meanwhile, in a study conducted in south africa, it was found that the astrazeneca vaccine was not effective in protecting against the south african variant[50]. a study by public health england produced data revealing two doses of the pfizer vaccine was 88% while two doses of the astrazeneca vaccine were 60% effective against symptomatic diseases from the indian variant of the coronavirus[51]. as for the vaccine from sinovac, there is insufficient data to confirm whether the vaccine is effective against the variants. as the situation in malaysia progresses with the rise of more covid-19 variants, there is a dire need for a rapid vaccine rollout in malaysia to protect the population’s health. the rate of vaccine distribution is influenced by the supply of vaccines from the manufacturers. as aforementioned, malaysia will be receiving more shipments of vaccines in the coming months. henceforth, individuals are highly encouraged to register for the vaccine to achieve immunity against covid-19 within the nation as soon as possible. while waiting for more vaccines to arrive, the public should play their responsibility in flattening the curve by practicing good hygiene, wearing face masks in public areas, avoiding social gatherings, and obeying all the sop implemented by the government. recently, the health director general (moh) shared effective ways of double masking; by using a surgical mask on the inside and a cloth mask on the outside. according to america’s center for disease control (cdc), this method provides up to 85.4% protection compared to 56.1% when wearing a single 3-ply surgical mask. if the cases continue to rise, the malaysian healthcare system may collapse, and the disease's burden can no longer be controlled, ultimately crippling the lives of millions in the physical, mental, and economic aspects. it is crucial to note that the betterment of the covid-19 situation in malaysia ultimately depends on the combined efforts of the malaysian government, the malaysian healthcare system, and the nation. author contributions: k-y.l performed the literature search, critical data analysis as well as manuscript writing. vl proofread the review writing and conceptualize this review writing project. funding: the seed funding funded this work from microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, (vote number: mbrs/jcsmhs/02/2020) and jeffrey cheah school of medicine and health sciences (jcsmhs) strategic grant 2021 (grant code: stg-000051). conflicts of interest: the authors declare no conflict of interest. acknowledgments: the authors would like to acknowledge professor shajahan yasin, head of school, jeffrey cheah school of medicine and health sciences, monash university malaysia. pmmb 2021, 4, 1; a0000204 8 of 11 references 1. world health organization (who). timeline: who’s covid-19 response 2021 [accessed 2021 may 20]; available from: https://www.who.int/emergencies/diseases/novel-coronavirus2019/interactive-timeline#event-7. 2. goh hp, mahari wi, ahad ni, et al., risk factors affecting covid-19 case fatality rate: a quantitative analysis of top 50 affected countries. prog microbes mol biol 2020; 3: 1–7. 3. tan lt-h, letchumanan v, ser h-l, et al., pmmb covid-19 bulletin: united kingdom (22nd april 2020). prog microbes mol biol 2020; 3(1). 4. ser h-l, letchumanan v, law jw-f, et al., pmmb covid-19 bulletin: spain (18th april 2020). prog microbes mol biol 2020; 3(1). 5. lee vs, chong wl, sukumaran sd, et al., computational screening and identifying binding interaction of anti-viral 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a0000204 11 of 11 48. emergence of new covid-19 variants worrying, says health dg. 2021 [accessed 2021 may 21]; available from: https://www.theedgemarkets.com/article/emergence-new-covid19-variantsworrying-says-health-dg. 49. abu-raddad lj, chemaitelly h, and butt aa, effectiveness of the bnt162b2 covid-19 vaccine against the b. 1.1. 7 and b. 1.351 variants. n engl j med 2021. 50. madhi sa, baillie v, cutland cl, et al., efficacy of the chadox1 ncov-19 covid-19 vaccine against the b. 1.351 variant. n engl j med 2021. 51. two covid-19 shots effective against b16172 variant from india: english health body. 2021 [accessed 2021 may 23]; available from: https://www.channelnewsasia.com/news/world/two-covid19-shots-effective-against-india-variant-14867452. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. https://www.theedgemarkets.com/article/emergence-new-covid19-variants-worrying-says-health-dg https://www.theedgemarkets.com/article/emergence-new-covid19-variants-worrying-says-health-dg https://www.channelnewsasia.com/news/world/two-covid-19-shots-effective-against-india-variant-14867452 https://www.channelnewsasia.com/news/world/two-covid-19-shots-effective-against-india-variant-14867452 abstract: malaysians are facing the biggest challenge in 2021 the rise in covid-19 cases and its variant of concern (voc) strains. the scale and impact of this coronavirus disease are unimaginable, scary, and full of sorrows. many lost their loved o... keywords: covid-19; novel coronavirus; malaysia; vaccination; herd immunity 1. introduction it has been over a year since the world health organization (who) declared covid-19 as a global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2)[1–5]. this virus has vastly spread worldwide, with new variants b... malaysia's fight against covid-19 started in early 2020. soon after the covid-19 outbreak in china, the first cases of covid-19 were detected in malaysia on 25th january 2020[11]. the cases involved three chinese nationals who were close contacts of a... the malaysian government took various precaution measures and imposed restrictions throughout the country to control the spread of this virus. malaysians started to live and adapt to the "new normal" life, which caused a significant impact on the econ... 2. covid-19 cases in malaysia initially, the number of confirmed cases in malaysia was growing slowly, but in march 2020, there was a sudden exponential increase in the cases. the cases were traced back to a religious event held in sri petaling, kuala lumpur, which amassed 16,000 ... 3. vaccination in malaysia despite the rising number of cases, the government has drawn up various strategies and continues to control coronavirus spread in malaysia. the earliest efforts were to enforce health screenings at all points of entry to detect fever among individuals... furthermore, at-risk individuals within identified clusters were tracked down via contact tracing to undergo covid-19 screening and would serve as a mandatory home surveillance order[24]. the ministry of health (moh) also raised awareness for covid-19... as the roll-out of vaccines developed by pfizer, astrazeneca, and sinovac began worldwide, the malaysian government has been placing orders for these vaccines since november 2020[26–28]. additional orders for the vaccines were placed in 2021 to speed ... the covid-19 vaccination programme in malaysia has been distributing vaccines from pfizer (comirnaty), astrazeneca (vaxzevria), and sinovac (coronavac) to the public since february 2021[33]. a study was done in 2020 that studied the intent of malaysia... since the global distribution of the covid-19 vaccines, many have been concerned about the adverse events that may occur upon receiving vaccines of specific brands. the vaccines used in malaysia have shown little to no side effects detrimental to the ... further analyses done by researchers have shown that the occurrence of blood clotting combined with low platelet counts caused by the vaccine is relatively low. the risks of tts have been shown to occur approximately 4 cases per 1 million, further ind... 4. the emergence of voc strains when this review writing went into press, the number of covid-19 positive cases and deaths in malaysia rose to historic heights. both the public and private healthcare sectors are greatly overwhelmed by the number of confirmed cases. intensive care un... as the situation in malaysia progresses with the rise of more covid-19 variants, there is a dire need for a rapid vaccine rollout in malaysia to protect the population’s health. the rate of vaccine distribution is influenced by the supply of vaccines ... references pmmb 2021, 4, 1; a0000203. doi: 10.36877/pmmb.a0000203 http://journals.hh-publisher.com/index.php/pmmb review article the covid-19 pandemic and diet change hong chuan loh1*, yin kar seah1, irene looi1,2 article history 1clinical research centre, hospital seberang jaya, ministry of health malaysia, seberang jaya 13700, penang, malaysia; lohhongchuan@gmail.com (hcl); seahyinkar@hotmail.com (yks) 2medical department, hospital seberang jaya, ministry of health malaysia, seberang jaya 13700, penang, malaysia; irenelooi@yahoo.com (il) *corresponding author: hong chuan loh, lohhongchuan@gmail.com (hcl) received: 25 march 2021; received in revised form: 20 may 2021; accepted: 25 may 2021; available online: 1 june 2021 abstract: covid-19 is now considered one of the world’s greatest challenges. even today, health experts and scientists are still seeking a conclusive answer regarding the source of this zoonotic disease. during the covid-19 pandemic, plant-based diets became a preferred diet choice for many people. in this paper, we discussed the trend toward a plant-based diet across the globe and some of the reasons for the shift. we note that there was a rise in plant-based food sales and a simultaneous decline in animal-based meat sales. sales of meat and seafood plummeted for many reasons, including distrust in meat due to fear of virus contamination, price increases, and ethical reasons. marketing strategies used by meat-alternative companies may have also played a role. while there has been an ongoing trend toward plant-based diets in recent years, that trend seemed to accelerate during the pandemic with more available vegan venues and places with vegan options. another reason that some people may have started exploring plant-based eating during the pandemic is because of the belief that such healthy eating will boost immunity or provide some other health-related benefit. plant-based diets are also more cost-effective than diets containing meat, fish, and dairy. we conclude that significant changes need to be made regarding the use of wild animals and livestock in order to prevent future pandemics of zoonotic origin. as the world’s population grows, zoonoses may occur with greater frequency. encouraging the adoption of healthy plant-based diets around the world with a simultaneous reduction in the use of animals as a food source is necessary and vital steps to prevent future pandemics due to zoonotic disease. keywords: covid-19, pandemic, plant-based diet, vegan, meat pmmb 2021, 4, 1; a0000203 2 of 14 1. introduction coronavirus disease-2019 (covid-19) was declared a pandemic by the world health organization (who) on march 11, 2020[1]. as of 17th may 2021, over 160 million people are infected with the virus and more than three million deaths worldwide. unfortunately, these numbers are still on the rise. when this disease first broke out in wuhan, china, it was initially identified as pneumonia, cause unknown[2–5]. in only a few months, it spread to the rest of the world. many of the original cases of covid-19 seemed to have connected to the huanan seafood wholesale market. it was subsequently determined that covid-19 is caused by the virus severe acute respiratory syndrome coronavirus-2 (sars-cov-2). sars-cov-2, from the family coronaviridae, and is a 96% identical match at the whole-genome level to a bat coronavirus[6–8]. although bats are a probable reservoir host for the virus, scientists had suggested that malayan pangolins (manis javanica) may have served as a possible intermediate host for sars-cov-2 before it was transmitted to humans[9]. the strain of the disease that affected humans evolved to become transmissible by human-to-human contact[10]. there was also some debate about whether swine may have been the intermediate host for the virus, either instead of or in addition to the pangolins[11]. as numerous zoonotic outbreaks emerged from livestock farms in the past, it is possible that there was a bat-swinehuman relationship with the spread of covid-19 due to the extensive swine factory farms in wuhan, china[11]. there was also some speculation about a relationship between the spread of covid-19 and the location of swine factory farms in santa catarina, brazil[11]. the world’s current food production and consumption paradigm has created the conditions for repeated zoonosis and is the root of the problem[12]. according to the world health organization, zoonotic disease, or zoonosis, is an infectious disease that has jumped from animals to humans. zoonotic pathogens may be bacterial, viral, parasitic, or may involve unconventional agents and can spread to humans through direct contact or food, water, or the environment[13]. outbreaks of zoonotic disease in humans have occurred many times over thousands of years of human evolution, but the rate of zoonotic transmission has been rising over time[14,15]. for example, zoonotic pathogens such as the severe acute respiratory syndrome coronavirus and the middle east respiratory syndrome coronavirus both caused pandemics in the past two decades[16]. in order to prevent or at least limit future outbreaks of zoonotic disease, it is crucial to identify and address the causes of the zoonotic links (in both wild animals and livestock) and the “spillover events” that give rise to the transmission of novel infectious viruses[17]. the sale of wild animals at the seafood market in wuhan appears to be the most likely source of pmmb 2021, 4, 1; a0000203 3 of 14 covid-19[18]. therefore, experts urged that authorities put in place a permanent global ban on wildlife consumption and the operation of wildlife markets. these steps are generally regarded as critical in preventing future pandemics[19]. there were also numerous mammalian and non-aquatic species, such as rabbits and birds, that were available at that market for purchase before the outbreak[20]. these animals could also serve as hosts for different species and strains of coronaviruses. the shedding of viral particles in the faeces of these animals may play a role in infectious spread, including in cross-species transmissions[21]. in april 2020, the acting head of biodiversity at the united nations said that countries could avoid pandemics by eliminating wildlife markets that trade live and dead wild animals for human consumption[22]. wildlife markets and illicit trade in wild animals have been identified by public health authorities in the united states (us) and around the world as conduits for the transmission of diseases. the transfer of diseases from animals to humans can happen by human consumption of live or dead animals or even by the close proximity of humans to live or dead animals. in addition, livestock animals can be infected by contact with wild animals and wet markets provide a particularly conducive environment for this kind of spread. strict laws against both the sale of live animals and the exotic meats trade should be enforced[23]. because of the covid-19 outbreak, the chinese government has now imposed a national ban on the use of wild animals for food[24]. it is at least a step in the right direction. the current zoonotic pandemic has caused health professionals around the world to address issues such as wet markets and livestock, and some of these comments have been highlighted in the media. this level of attention may cause the eating pattern of many to change as a result of the covid-19 crisis. this paper addresses the change in food choice by many people, the reasons for such change, and the lessons we could learn for the future as a result of the covid-19 pandemic. 2. global diet trends figure 1 is a graphical abstract to illustrate the main findings of this review. pmmb 2021, 4, 1; a0000203 4 of 14 figure 1. summary of the review (the covid-19 pandemic and diet change). 2.1. plant-based food sales and animal-based food sales during the covid-19 pandemic, there was an interesting shift in diet among many as plant-based food consumption, including meat alternatives, increased significantly. in this paper, we use the terms “vegan” and “plant-based” interchangeably but recognize that veganism relates to the rights of non-human species, whereas plant-based refers to only dietary choice. the us retail sales of plant-based foods greatly outpaced overall food sales suggesting that more customers turned to plant-based foods during the pandemic. march 2020 was the peak of food buying during the pandemic, and it is noteworthy that plant-based food sales grew by 90% compared to march 2019. when broken down by segment, vegan meat sales rose by 148% and continued to rise by 61% in the following four weeks, reflecting a growth rate two times that of animal-based meat[25]. while plant-based meat sales continued to soar, animal-based meat sales continued to decline. the united kingdom (uk) department for environment, food and rural affairs compared animal slaughter data from october 2020 to october 2019 and found a decrease of 7.3% in prime cattle, 4.9% in clean sheep, and 2.1% in clean pig slaughtering[26]. according to a report from the food and agriculture organization of the united nations, it is expected that the numbers from 2020, when finally tallied, will show that meat consumption per capita decreased by nearly 3% globally and that this reduced consumption was the biggest annual decline since 2000[27]. the covid-19 pandemic also negatively affected the meat products market in southeast asia. due to the major hit to the livestock industry, revenue losses, and pmmb 2021, 4, 1; a0000203 5 of 14 supply shortages, it was estimated that there was a lower growth rate during the outbreak. meat producers in southeast asia faced drastically reduced meat consumption as well as a scarcity of raw materials and were severely hampered by these factors[28]. seafood sales also plummeted during the pandemic. fishing communities and ports were seen as possible hubs for transmission of covid-19 infection because of the migratory nature of fishermen and the frequency of international visitors[29]. many fisheries experienced partial or complete shutdown resulting in a reduction of fishing activities globally. furthermore, there was a drastic reduction in demand for seafood, restriction to access cold storage, and cessation of shipping and air cargo[30]. activity on the fish exchange in portland, maine, us, declined from 60,000 to about 20,000 pounds per week during the pandemic[31]. the closure of many restaurants in the us significantly decreased demand and the nation's fisheries across geography, species, gear types, and management reported sales nosedives of up to 95%[32]. 2.2. covid-19 and consumer perceptions of meat a particularly significant concern with the covid-19 outbreak is the high infection rate among workers at slaughterhouses and meat processing plants. the environment in such facilities is conducive for zoonotic spread. there are structural and operational practices that hinder the practice of personal safety protection and the proper cleaning and disinfection of worksites. the facilities also have lower temperatures and extreme relative humidity, large quantities of droplets and aerosols, and use many stainless-steel surfaces that retain live viruses longer than cloth or paper surfaces. these conditions, along with the sociocultural and economic challenges facing the workers, facilitate the transmission of sars-cov2[33,34]. it is therefore not surprising that a series of sars-cov-2 outbreaks that occurred between march and june 2020 in meat factories led to plant closures, especially in europe and north america, including some in germany, france, spain, the uk, the us, and canada[35,36]. gütersloh and warendorf in the western german state of north rhinewestphalia, with a total of 640,000 residents, were returned to lockdown conditions after more than 1,400 employees at a meat-packing plant tested positive for covid-19[37]. meat processing factories in anglesey, merthyr tydfil, wrexham, and kirklees in the uk also became hot spots with over 450 covid-19 cases[38]. among 115 meat or poultry processing facilities in 19 states in the us, 4,913 employees were diagnosed with covid-19 and 20 deaths related to covid-19 were reported[34]. specifically, the largest reported cluster was 1,550 cases linked to a single meat-packing plant in alberta, canada[39]. with meat processing plant closures due to covid-19 outbreaks, consumers may have had concerns about meat shortages and meat safety (due to its possible contamination pmmb 2021, 4, 1; a0000203 6 of 14 with the virus), causing higher demand for plant-based alternatives. it is also possible that consumers may have been concerned about meat prices since the decline in the availability of meat could result in price increases. a higher cost for meat would further reduce the demand for meat. there is also the possibility that consumers may have deliberately protested against the meat to stand in solidarity with workers from slaughterhouses and meat processing plants[40]. a study conducted in china during the pandemic showed an increased demand for a plant-based diet. apart from dieting to lose weight, the study participants considered a plant-based diet safer for consumption, possibly implying distrust in meat[41]. a study by forsa on the eating habits of germans between 2015 and 2020 commissioned by the federal minister of food, julia klöckner, showed that the country's daily meat intake as a percentage of the average german meal declined among the respondents from 34% to 26%[42]. 2.3. plant-based food alternatives and venues as meat and seafood supply chains face disruption during the pandemic, plant-based companies that offer meat alternatives like “beyond meat” and “impossible foods” are seizing the chance to secure new customers with more competitive marketing strategies[43]. according to grubhub’s “year in food” trend report in which the delivery platform analysed 30 million orders, plant-based meat alternatives spiked in popularity by 463% in 2020. across the us, new york, california, oregon, massachusetts, and illinois took the top five spots. in addition, delivery platform doordash, with a newly released “doordash deep dish” report, also showed an increase in plant-based alternatives, specifically 433% in the six-month period from january 2020 to the end of june 2020[44]. other research by an online restaurant resource (the happycow platform) focused on how restaurants have been affected by covid-19. it found that since the onset of covid19, there were 517 newly opened vegan restaurants while 413 were closed, with a total of 104 more vegan restaurants available now worldwide[45]. as the results only represent purely vegan venues, the number of restaurants that are “vegan-friendly” or that have plant-based options available may have increased. even fast-food restaurant chains are offering more plant-based options. the largest chain in the world, subway, launched a meatless meatball marinara sub sandwich in all of its uk stores in early 2020 with a successful sell-out of the product. it won best vegan sandwich at the 2020 vegan food awards hosted by people for the ethical treatment of animals[46]. starbucks, the third-largest chain store, also added more plant-based items to its menu worldwide, such as impossible rendang pie in singapore, beyond meat dishes, and oatly milk substitute in china, as well as many others in various locations[47]. pmmb 2021, 4, 1; a0000203 7 of 14 2.4. a plant-based diet and its health-related effects the surge in demand for a plant-based diet could also be due to greater awareness of the benefits of leading a healthier lifestyle and the belief that it could result in stronger immunity. according to a current pan-india serosurvey conducted by the council of scientific and industrial research in its almost 40 institutes, vegetarians were found to have lower covid-19 seropositivity, suggesting that they might be at a lower risk of coronavirus infection[48]. with a strong impact on host homeostasis and immunostasis, it is known that the gut microbiota that reside in the gastrointestinal tract provide their host with important health benefits[49]. hong kong researchers have identified three significant findings in patients with covid-19: a) the composition of the gut microbiota is consistent with disease severity and magnitude of plasma concentrations of several inflammatory cytokines, chemokines, and blood markers of tissue damage; b) gut bacteria such as faecalibacterium prausnitzii, eubacterium rectale, and many species of bifidobacteria, with known immunomodulatory capacity, have been depleted; c) the dysbiotic gut microbiota composition remains after virus clearance[50]. a recent study showed favourable association between healthy plant-based food consumption and "good" microbes in the body[51]. this was due to the fact that a plant-based diet replenishes the host intestinal microbiota with beneficial microbes while maintaining a state of symbiosis with balanced microflora within the host that results in multiple health benefits, including improved immunity[52]. for instance, fibre, which is widely available in plants, has been found to consistently increase lactic acid bacteria, such as ruminococcus, e. rectale, and roseburia, and limit the growth of the clostridium and enterococcus species. polyphenols, also found exclusively in plants, tend to increase bifidobacterium and lactobacillus[53]. many of the risk factors reported to date that are associated with covid-19 infection and mortality relate to nutritional status and specific essential nutrients. a balanced diet with an abundance of vegetables, fruits, whole grains, and nuts is associated with many substantial health benefits owing to the high amounts of fibre, polyphenols, vitamins, unsaturated fats, and minerals[54]. there was level i and ii evidence that supports the use of vitamins b, c, and d in the treatment of coronavirus-like respiratory diseases, acute respiratory distress syndrome, and sepsis[55,56]. vitamin d, in particular, has been found to play a role in reducing the risk of covid-19 infections and deaths[57]. while many food sources only contain low amounts of vitamin d naturally, certain plant sources, such as algae, fungi, and yeasts, contain abundant amounts of vitamin d as well as provitamin d[58]. as mushrooms, fungi, and seaweed are commonly found in asian cuisine, predominantly among the chinese, japanese, and korean communities, it is interesting to note that some asian countries have managed to keep the outbreak of covid-19 under control and have experienced lower pmmb 2021, 4, 1; a0000203 8 of 14 mortality rates. this observation suggests further study on immunonutrition to determine any links that may exist. previous research also demonstrated that many naturally occurring compounds with anti-coronavirus infection properties could be found in common fruits. a recent study regarding tannic acid, which is abundant in fruits like grapes, berries, and apples, demonstrated high potential as part of developing anti-covid-19 therapeutics. tannic acid acts as a potent dual inhibitor of two independent enzymes (the main viral protease and the cellular tmprss2 protease), resulting in some degree of sars-cov-2 infection suppression[59]. 2.5. the trend and cost of plant-based diets a study using google trends data was carried out to examine the popularity of “vegan” search terms around the world in a variety of languages. it showed that the popularity of veganism was at an all-time high in the midst of the pandemic in 2020 and surpassed the previous all-time high reported in 2019. among all the countries in the world, the uk, australia, and israel hold the top three spots for being most inquisitive about plant-based diets. moreover, the vegan trend is now twice as popular as it was five years ago[60]. the vegan diet has also received the highest quality score for any diet as represented by the healthy eating index 2010 and the mediterranean diet score in a study that included 1,475 participants[61]. in addition, a recent recommendation by the eat-lancet commission on healthy diets from sustainable food systems supports a 50% or more reduction in the global consumption of unhealthy foods, including meat, by 2050 in order to meet global nutritional needs while preserving local and global ecosystems[62]. with greater opportunity to prepare meals at home and have more free time to learn about a healthier diet during home confinement compared to pre-quarantine days, there is probably a greater likelihood that people will choose plant-based food. a recent study showed that a plant-based meal prepared at home costs 40% less than meat or fish meals (£1.77 per person for a meat/fish meal versus £1.06 per person for a plantbased meal), and it takes one-third less time to prepare. these calculations are based on weekly meal diaries that were captured online from around 11,000 people in britain[63]. the covid-19 pandemic has affected 55 million domestic workers worldwide, many of whom were in fear of or actually experienced reduced working hours, lost income, and even loss of employment altogether[64]. the lower cost of plant foods versus meat or fish could also have contributed to the increase in the number of people adopting a plant-based diet during the crisis. covid-19 may have added momentum to an already rapidly growing trend toward the consumption of a healthier plant-based diet. as a result of increased awareness about the pmmb 2021, 4, 1; a0000203 9 of 14 detrimental effects of meat consumption on the environment and human health, and for ethical reasons relating to animal rights, plant-based diets are increasingly popular and are often promoted by celebrities and athletes. the acumen research and consulting report projects that the global vegan food market will reach around us$24.3 billion by 2026 and will grow at a noteworthy compound annual growth rate of around 9.1% throughout the forecast period from 2019 to 2026[65]. 3. lessons to learn from the covid-19 pandemic acknowledging the need for multidisciplinary collaboration to resolve health threats at the human-animal-ecosystem interface, the who, the food and agriculture organization, and the world organisation for animal health formalized a partnership in 2010 and identified three priority areas of collaborative work, two of which relate to zoonotic diseases[66]. in addition to giving rise to global infectious disease threats, an analysis performed by the world bank estimated that the economic losses from six major outbreaks of zoonoses were at least us$80 billion between 1997 and 2009[67]. in view of the deleterious effect of zoonotic disease, all countries should focus on ways to prevent future pandemics. since the covid-19 pandemic began, consumers have become better informed about the latest health-related information. consumers should demand greater transparency about how their food is produced and be able to base their dietary choices on options that result in less burden to the planet and less opportunity for zoonotic diseases to arise. this would include adopting a healthy plant-based diet, banning all wildlife trade at wet markets, or enforcing stricter regulations, and prohibiting most human activities that involve close contact with wild animals, such as sport hunting. these steps would help to minimize the risk of human exposure to novel viral pathogens, improve food security, and reduce the effect of food production on the environment. the beneficial changes in diet that we observed during the pandemic may not be sustained if they are not entrenched into the daily habits of consumers. established and well-regarded government agencies, such as the us food and drug administration could play an important role in encouraging and incentivizing restaurants, schools, and hospitals to offer plant-based food options, engage on social media to promote a healthy vegan lifestyle, and provide plant-based nutritional guidance to all health professionals. 4. conclusion the covid-19 pandemic has presented an exceptional situation in which many people around the world, aware of the causes of zoonoses and the health benefits of eating a plant-based diet, have changed their eating patterns. apart from the numerous beneficial health, environmental, economic, and ethical effects of a plant-based diet, the prospect of pmmb 2021, 4, 1; a0000203 10 of 14 lessening the risk of future pandemics is perhaps the most important to many people. multidisciplinary health experts and authorities across the globe should come together to produce and implement common sense and data-driven solutions. the emphasis should be on eliminating, or at least reducing, the use of wild animals and livestock for any purpose, particularly as a source of food, in order to mitigate future pandemics due to zoonotic disease. author contributions: hcl and yks drafted the manuscript. hcl and il provided review and editing for this manuscript. hcl, yks and il conceptualized this review writing project. funding: no external funding was provided for this research. acknowledgments: we would like to thank the director-general of health malaysia for his permission to publish this article. conflicts of interest: the authors declare no conflict of interest. references 1. mitra p, misra s, and sharma p, covid-19 pandemic in india: what lies ahead. indian j clin biochem 2020: 1–3. 2. loo ky, letchumanan v, ser hl, et al., covid-19: insights into potential vaccines. microorganisms 2021; 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a0000194. doi: a0000194 http://journals.hh-publisher.com/index.php/pmmb review article an overview of breast cancer: classification and related signaling pathways pit foong chan*, roslida abd hamid article history department of biomedical science, faculty of medicine and health sciences, universiti putra malaysia, 43400 serdang, selangor, malaysia; roslida@upm.edu.my (rah) *corresponding author: pit foong chan, pitfoong_chan@hotmail.com (p-fc) received: 21 january 2021; received in revised form: 31 march 2021; accepted: 1 april 2021; available online: 12 april 2021 abstract: the burden of cancer continues to grow in developed and developing countries, with about 70% of all cancer mortality in lowand middle-income countries. among different cancer types, breast cancer is recognized as one of the five most common causes of death in cancer in women worldwide, right after lung cancer. histological classification divides breast tumors into different categories based on their behavior and clinical outcome. however, the histological classification system has some limitations, thus molecular subtype classification has been studied extensively to improve the classification system for breast cancer. like any other cancers, several signaling pathways that enhance the proliferation, survival, invasion, and metastasis capability of tumor cells have been observed in breast cancer. these crucial signaling pathways contributing to the etiology of breast cancer include breast tumor kinase (brk) pathway, notch signaling, nuclear factor-kappab (nf-κb) pathway, and human epidermal growth factor receptor (her) pathway. in the present review article, we summarize our current understanding of breast cancer and its signaling pathways, which serve as basic information on tumor formation, maintenance, and expansion that could help form better breast cancer management in patients. keywords: breast cancer, signaling pathway, brk, nf-κb, her 1. introduction cancer has become one of the top causes of morbidity and mortality, with approximately 18.1 million new cases and about 9.6 million deaths in 2018 based on the global cancer observatory[1]. in other words, cancer is responsible for nearly one of the six deaths, leading to its recognition as one of the world’s most prominent “killers”[1,2]. in addition to that, it has been suggested that nearly 65% of the increase in deaths related to all cancer types will occur in less developed regions of the world by the year 2030 while developing countries are anticipated to face an estimated burden of 23.6 million new cases per year[3–6]. pmmb 2021, 4, 1; a0000194 1 of 14 as the leading malignancy in females, breast cancer cases continue to increase over the past few years, with a distinctive age-specific pattern reported by bray and the team[1,7] by the same token, the disease may have an early onset age, occurring in younger women below 40 years of age and statistics showed a sharp increase in incidence rate right before menopause[1,8]. a recent report by sharma, which extracted information from the globocan 2018, stated that breast cancer had claimed an estimation of 626679 lives at an age-standardized rate of 13/100000[9]. in the same report, the highest incidence of breast cancer was noted in east asia with 476509 cases (range: 474656–478370 cases), but the highest death counts due to breast cancer were observed south-central asia at 123,060 deaths (range:119,256–126,986) in 2018. in malaysia, the health facts 2013[10] released by the ministry of health (moh) malaysia highlighted cancer as one of the top ten causes of hospitalization and one of the top five causes of mortality (approximately 30000 cases annually). based on the report released by the malaysian national cancer registry report (2016)[11], 103507 new cancer cases were diagnosed between 2007 and 2011 in malaysia, and breast cancer appeared to be the most common cancer among female residents (32.1%) in malaysia. along with the advancement of detection technology, the survival rate of breast cancer patient seems to have improved through surgery, radiotherapy, chemotherapy, endocrine therapy, and targeted therapy[12]. while chemotherapy remained the broadest approach and first choice applied in inhibiting cancer cells' proliferation, it is often militated by numerous factors comprised of chemo-resistance, non-selectivity actions of chemotherapeutic agents, and high proliferation rates in breast cancer cells[13]. in fact, these prominent characteristics of cancer cells are often the result of abnormal activation of signaling pathways that granted these cells increased proliferation ability, along with invasion and metastasis capability. therefore, a better understanding of underlying signaling pathways is essential, particularly in ensuring an excellent therapeutic plan and achieving the best clinical outcome. 2. histological classification of breast cancer subtypes the breast is comprised of two main types of tissue: (a) glandular tissues that house lobules (i.e., milk-producing glands) and ducts, and (b) stromal tissues, which provide the “supporting framework” of the breast (i.e., fatty and connective tissues) (figure 1)[14]. however, due to the lack of biomarkers, the definition of breast cancer advancement remains challenging compared to other cancer types like colon cancer[15,16]. through histological investigations, breast cancer can be differentiated based on its site and invasiveness. the invasive form of breast cancer is seen to breach the duct and lobular wall, invading the breast's supporting tissues. despite that, this does not imply its metastatic capability, as some breast cancer can be invasive but do not spread further to other organs or lymph nodes. the other form of breast cancer is carcinoma in situ or non-invasive breast cancer, which can be subcategorized into two types: either ductal carcinoma in situ (dcis) or lobular carcinoma in situ (lcis). generally, dcis is considered as pre-invasive or non-invasive breast cancer and constitutes one in five new breast cancer cases reported. however, untreated dcis may spread pmmb 2021, 4, 1; a0000194 2 of 14 over time by invading into adjacent breast tissue and progress into invasive cancer. the detection of dcis has increased significantly following the use of mammography screening. in the united states, 90% of dcis cases detected through mammography are observed as suspicious calcifications. dcis is further categorized into several subtypes, primarily based on the morphological appearances: comedo, cribiform, micropapillary, papillary and solid[17]. on the contrary, lcsi is rather a rare condition in which abnormal cells develop in the lobules at the terminal end of the duct. it is also displaying a worrying trend, given that the number of lcsi cases has multiplied over the past few years, with the highest rate observed among women aged between 40 to 50 years[18]. figure 1. classification of breast cancer subtypes and examples of signaling pathways involved in the etiology of breast cancer[19,20]. on the other hand, the major invasive tumor types are infiltrating ductal, invasive lobular, ductal/lobular, mucinous (colloid), tubular, medullary, and papillary carcinomas. being the most prevalent type of invasive breast cancer, infiltrating ductal carcinoma (idc) accounts for approximately 80% of all invasive breast cancers. as indicated by its naming, idc arises in the breast's ductal region, invading through the duct wall, and subsequently expands into the supporting tissue of the breast. there are three grades of idc: well-differentiated (grade 1), moderately differentiated (grade 2), or poorly differentiated (grade 3). the grading of idc is carried on based on the rate of nuclear pleomorphism, glandular/tubule formation, and mitotic index, which aids in its prognosis[21]. 3. molecular classification of breast cancer subtypes originally assumed as a single disease, more evidence is emerging on breast cancer's complexity, suggesting it to be a group of diseases expressing distinguishable anatomical characteristics, reactive towards treatment and survival result. current histological classifications greatly limit the ability to completely identify the clinical results of this pmmb 2021, 4, 1; a0000194 3 of 14 disease[22]. with the availability of more affordable molecular techniques, including genome sequencing technologies, researchers are now getting more insights into breast cancer subtyping via gene expression and genomic profiling studies. while the current model for breast cancer classification may offer some levels of prognostic value, the molecular classification of breast cancer offers more tremendous advantages in predicting the clinical outcome and, more importantly, assessing potential (distinctive) responses to specific therapies that assist clinicians in choosing the best therapeutic options for breast cancer patients[23,24]. perou and team studied the gene expression of different breast cancer types using cdna microarray profiling and sorted them into few intrinsic gene subtypes: basal-like breast cancer (blbc) (estrogen receptor (er) negative, progesterone-receptor (pr) negative, and human epidermal growth factor receptor-2 (her2) negative), her2-enriched, normal breast-like, luminal subtype a (er+ and low grade) and luminal subtype b (er+ and often high grade)[25– 27] and the poorly described triple-negative subtype or “claudin-low” subtype [25–27] (figure 1). each of these molecular subgroups has a distinctive prognosis and chemotherapy sensitivity. additionally, the growth of breast cancer cells can be induced by hormones, estrogen, and progesterone. thus, selecting specific therapy for breast cancer varies depending on these hormone receptors' expression[28,29]. generally, 70% of breast cancer patients express er and receive hormonal therapy treatment[28,30,31]. studies also indicated that those with luminal-like cancers seem to have better long-term survival compared with other cancers, but those with basal-like and her-2-positive tumors show higher susceptibility and sensitivity towards chemotherapy[26,32]. in contrast, patients diagnosed with triple-negative cancer have exceptionally shorter survival in first metastatic occurrence than other breast cancer types[33] . by molecular classification, er-positive luminal breast cancer is the most common type of invasive breast cancer. the er-positive disease can be further classified into subtypes with different outcomes (i.e., luminal a and luminal b) using gene expression profiling with microarrays[34]. luminal a tumors tend to have high expression of er-activated genes, lowgrade histological appearance, and slower growth, resulting in a better prognosis than luminal b tumors[35]. furthermore, luminal a tumors are associated with the expression of luminal epithelial cytokeratins (ck) 8 and 18, other genes such as hepatocyte nuclear factor 3 alpha (foxa1), b cell lymphoma 2 (bcl2), erbb3, and erbb4[36]. in comparison, luminal b tumors typically display a high proliferate rate and tumor aggressiveness, which is subsequently explained by its high relapse rate and lower survival rates than luminal-a breast cancer[37–40]. the main difference between these two luminal subtypes is the greater level of proliferationrelated genes expressions such as avian myeloblastosis viral oncogene homolog (v-myb), gamma glutamyl hydrolase (ggh), lysosome-associated transmembrane protein 4-beta (laptmb4), nuclease sensitive element binding protein 1 (nsep1) and cyclin e1 (ccne1) in luminal-b tumors[41]. it should be noted that luminal tumors form a continuum, thus the determination of these tumors into two subtypes based on proliferation may be arbitrary[42]. pmmb 2021, 4, 1; a0000194 4 of 14 the her2 is overexpressed in 15–30% of invasive breast cancer, mainly due to the overexpression of her2/her2 signaling-related genes and genes located in the her2 amplicon on chromosome 17q12[25]. even though her2-enriched breast cancer can proliferate faster than luminal cancers, they are often highly responsive to targeted therapies aimed at the her2 protein, resulting in remarkably improved outcomes[43]. the normal cell-like subgroup, comprising 5–10% of breast cancer patients[44]. some researchers defined that this subtype was featured by similar gene expression to normal breast epithelium. its proliferation rate is often low and responds to adjuvant chemotherapy[44,45]. the blbc is an aggressive subtype that mainly occurs in young women and displays exceptionally high metastasis rates to the brain and lung[46,47]. this subtype is characterized by high histological grade, poor tubule formation, central necrotic zones, pushing borders, high mitotic indices, and proliferation rates[47]. furthermore, the blcl does not benefit from antiestrogen hormonal treatment or trastuzumab as estrogen receptor (er), progesterone receptor (pr), and her2 were not expressed in this subtype. therefore, a better understanding of the molecular background of blbc to develop promising therapeutic regimes is necessary[48]. for instance, a study conducted by hallett et al.[49] distinguished blbc into two subgroups based on 14-gene. they assumed that this categorization might provide aggressive therapeutic regimes to the poor prognosis subgroup while avoiding such treatment in low-risk patients[50]. 4. genetic and hormonal risk factors in breast cancer in general, cell dna impairment can cause mutations or chromosome rearrangements, which leads to carcinogenesis. undoubtedly, breast cancer is a complex and multifactorial disease due to hormonal or genetic factors, or even a combination of both factors, as seen in many cases. hormonal factors, main estrogen, play a vital role in breast cancer development[51]. researchers hypothesized that excessive production of estrogen could stimulate the aberrant growth and development of organs affected by hormonal levels[52]. by this theory, hyperplasia tissue may pose as a “preview” before neoplasia development. however, while this is true, breast cancer risk appears to be predictable by the exposure to estrogen[53]. brca1 (breast cancer gene one) and brca2 (breast cancer gene two) are two of the most important breast cancer susceptibility genes[54]. the brca genes play a critical role in cell damage repair and induce cell death to those cells if the damage is beyond rescue[55]. brca mutation leads to abnormal breast tissue proliferation and increases breast cancer risk[55,56]. it has been estimated that 5–10% of breast cancers diagnosed in women are associated with hereditary susceptibility, attributed to mutations in autosomal dominant genes, such as brca1 and brca2[20,57]. aside from that, 15–20% of female breast cancers occurred due to family inheritance but without a plausible autosomal dominant genes type[58]. there have been more than 500 sequence variations were identified since the isolation of brca1. despite most are frameshift mutations, several missense mutations are reported that may alter the specific function of a protein. furthermore, splice donor or acceptor site mutations are commonly reported[59]. the mutation spectrum of bcra2 may not as established as that of brca1. pmmb 2021, 4, 1; a0000194 5 of 14 however, the mutations reported in brca2 mainly happen in exons 10 and 11 and always include insertions or deletions; these unwanted changes then cause missense alterations and premature stop codon seen in truncated and completely nonfunctional protein[60]. 5. signaling pathways involved in breast cancer knowing the exact mechanisms involved in any disease, including breast cancer, is essential before deciding on appropriate treatments. lately, newer approaches are designed to target cell cycle regulatory pathways, proto-oncogenic signaling pathways targeted agents such as notch, wnt, shh (sonic hedgehog), er (estrogen receptor), pi3k/akt/mtor, and her2 (human epidermal growth receptor 2)[61]. besides that, researchers also investigate the potential of focusing on the breast tumor microenvironment as a therapeutic target in breast cancer[62]. 5.1. cyclin dependent kinase there are three main key families of molecules involved in cell cycle regulation, namely cyclins, cyclin dependent kinase (cdks), and cyclin dependent kinase inhibitors (cdkis)[63]. dysregulation of the interplay between cyclins and their related cdk partners contributes to one of the hallmark features of cancer, a sustained proliferation of tumor cells[64]. cdks could be either overactive or cdk-inhibiting proteins that are not in function in most cancers[61]. a study suggests that remarkable overexpression of cdk4 and cyclin d1 occurred in breast tumors. consequently, it has been postulated that cdk4 is dispensable for the development of the normal mammary gland and poses as a suitable therapeutic target, specifically promote breast cancer cells inhibition while sparing other healthy cells[65,66], 5.2. breast tumor kinase the overexpression of breast tumor kinase (brk) has been implicated in several malignancies such as prostate, ovarian, colon, and metastatic melanoma[67–70]. brk is a nonreceptor tyrosine kinase that is overexpressed in 60% of human breast tumors. nevertheless, it is not expressed in normal human mammary gland and benign tumors[71,72]. a high brk expression has been shown in invasive carcinoma, but it is also significantly expressed in her2 and her4[73,74]. a previous study demonstrated that brk contributes to upstream of p38 mitogen-activated protein (map) kinases and extracellular signal-regulated kinase 5 (erk5) as well as downstream of epidermal growth factor receptor (erbb) expression[75]. the overexpression and constitutive activation of brk breast cancer cells subsequently induced elevated cell survival and anchorage-independent growth, respectively[76,77]. similarly, constitutive brk expression also induces the egfr tyrosine kinase pathway and upregulates breast tumor cell migration via paxillin and mitogen-activated protein kinase (mapk) activation, and enhances cancer cell proliferation through phosphatidylinositol 3-kinase (pi3k) and akt expression[13,76–78]. even though the exact cellular roles of brk in breast cancer have yet to be fully delineated, recent data strongly imply that the deficiency of brk in breast tumor cells can pmmb 2021, 4, 1; a0000194 6 of 14 activate egfr-regulated signaling molecules, preceding breast cancer cell proliferation, migration, and elevated mapk activity[79,80]. hence, further investigation on the brk role in breast cancer is needed to achieve the optimum treatment outcome. 5.3. notch signaling notch signaling has been identified for more than two decades in developing the human mammary gland, governing a plethora of cellular activities such as stem cell maintenance, regulation of cell fate, differentiation, and proliferation, motility, and survival. alterations of these processes are known to enhance human breast cancer progression[81]. while mammals have four notch homologues, but solid tumors such as breast cancers may co-express some notch homologues that grant them resistance to highly selective therapeutic agents. the oncogenic role of notch has been shown in breast cancer tumorigenesis through cross-talk with some other signaling pathways, including estrogen, human epidermal growth factor receptor 2 (her2), ras, and wnt signaling pathway[82]. for example, approximately 80% of breast malignancies treated with anti-estrogens develop treatment resistance, and it is believed to be due to the involvement of the notch pathway[83]. therefore, targeting in both signaling pathways concurrently may help to overcome or delay this undesired resistance. despite that, some notch homologues may confer inhibitory action against cancer cells. as described by o’ neill et al.[84], pro-oncogenic effects of notch-1 and notch-4 are counteracted by notch-2 in human breast cancer cells. notch-1 expression was found to be increased in poorly differentiated breast tumors, whereas expression of notch-2 was increased in well-differentiated breast tumors. additionally, a study suggested that notch-1 may exert tumor-promoting properties while notch-2 may possess tumor-suppressing functions[85]. the interactions between different notch homologues could mean different cancer treatment outcomes, so taking a deeper look into these homologs' role could essentially assist the development of a promising treatment plan. 5.4. nuclear factor-kappab (nf-κb) the nuclear factor-kappab (nf-κb) superfamily comprises transcription factors that take on a crucial role in regulating processes such as angiogenesis, cell proliferation, cell invasion, cell migration, metastasis, and apoptosis[86–89]. in normal cells, the nf-κb signalling pathway is tightly regulated. it can only be activated upon divergent stimulation of epidermal growth factor (egf), bacteria and lipopolysaccharides (lps), chemical and physical stresses, inflammatory cytokines such as interleukin (il)-1 α and β as well as tumor necrosis factor-α (tnf-α)[90]. upon degradation of iκb by iκb kinase (ikk) phosphorylation, the activation of nf-κb signaling pathways commences with free p50 (nf-κb1) and p65 (rela) subunit release into the cytoplasm before further activation of downstream pathways that lead to the physiological responses[91,92]. dysregulated activation of nf-κb signaling pathways, which drives abnormal expression, has been observed in various types of human cancers[93,94], including enhancing pmmb 2021, 4, 1; a0000194 7 of 14 tumor’s metastatic and angiogenic potential, increasing proliferation of cancer cells, and allowing cells to escape apoptotic processes[93,95]. in reality, various antiapoptotic factors comprise of the bcl2 family (such as bcl-xl and bcl-2), cellular inhibitors of apoptosis (ciaps) and caspase-8/fadd (fas-associated death domain)-like il-1beta-converting enzyme (flice) inhibitory protein (c-flip) can be activated by nf-κb signaling pathways[91,95]. increased nf-κb activity can lead to abnormal chemokines expression and elevates cell migratory activity[92]. in line with that, several matrix metalloproteinases (mmps) identified at κb sites can enhance cell invasion of surrounding tissue[96]. active nf-κb also regulates the expression of specific molecules such as vascular cell adhesion molecule-1 (vcam-1), intercellular adhesion molecule-1 (icam-1), and endothelial leukocyte adhesion molecule-1 (elam-1). these molecules are vital players in cancer metastasis, allowing vessel wall penetration to transport the cancer cells to distant parts of the body[86,95]. likewise, high expression of nf-κb protein stimulates extracellular matrix destruction by tumor cells, boosting the tumor’s metastatic ability[97,98]. all in all, inhibiting aberrant activation of the nfκb pathway illuminates potential therapeutic options in suppressing human cancer[99]. 5.5. human epidermal growth factor receptor (her) falling within the tyrosine kinase receptors family, the human epidermal growth factor receptor (her) consists of four subfamilies and is usually expressed in normal tissues[100]. amongst the subfamilies of her, only human epidermal growth factor receptor 2 (her2) is correlated to various breast cancers[101]. her2 is found to be amplified in 20-30% of invasive breast tumors; its overexpression is associated with cancer cell proliferation, cancer development, and cancer cell metastasis, thus resulting in poor prognosis and a low survival rate in breast cancer patient[29,102]. the fda has approved therapeutic her2-targeted drugs against metastatic breast cancer. these drugs include trastuzumab, lapatinib, and pertuzumab[103,104]. as reviewed by vranic and the team recently, these anti-her2 drugs can improve the outcome of patients with her2-positive breast cancer when used alone or in combination with other conventional chemotherapeutics[105]. however, trastuzumab resistance was reported to be developed in some patients, although a portion of them may show substantially improved outcomes[102]. 6. conclusions breast cancer is the most common multifactorial disease occurred in women and brought the highest rate of death. a general model of breast cancer carcinogenesis postulates that a normal cell achieves several new capabilities, including genome instability, proliferate infinitely, resistance to cell-death signaling, angiogenesis, and escape from immune surveillance. these pathways may either work independently or inter-signaling to each other, contributing to multi-drug resistance (mdr), which is now a major challenge reported for breast cancer chemotherapy. a better understanding of breast cancer signaling pathways could pmmb 2021, 4, 1; a0000194 8 of 14 help improve tumor treatment and prevent recurrence and metastasis. therefore, therapeutic targeting in a specific signaling pathway would allow tailored treatments with more appropriate and effective individual responses, resulting in more personalized and conservative breast cancer interventions. author contributions: the literature search and manuscript writing were performed by p-fc. the manuscript was proofread and edited by rah. rah conceptualized this project. funding: this research was funded by ministry of higher education, malaysia, grant number um.c/hirmohe/sc/03 and um.c/hir-mohe/sc/12. conflicts of interest: the authors declare no conflict of interest. references 1. bray f, ferlay j, soerjomataram i, et al., global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin 2018; 68(6): 394– 424. 2. stewart bw, and wild, cw., world cancer report 2014. 2014, international agency for research on cancer. 3. national cancer institute. cancer statistics. 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22(1): 1–12. 105. vranić s, bešlija s, and gatalica z, targeting her2 expression in cancer: new drugs and new indications. bosn j basic med sci 2021; 21(1): 1. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2021, 4, 1; a0000182. doi: a0000182 http://journals.hh-publisher.com/index.php/pmmb review article modulation of gut microbiota by dietary macronutrients in type 2 diabetes: a review hong jing wang, oumaima battousse, amutha ramadas* article history jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; hwan0087@student.monash.edu (h-jw); obat0001@student.monash.edu (ob) *corresponding author: amutha ramadas, amutha.ramadas@monash.edu (ar) received: 21 january 2021; received in revised form: 12 february 2021; accepted: 16 february 2021; available online: 25 february 2021 abstract: dietary interventions have been a first-line strategy in the long-term management and prevention of type 2 diabetes mellitus (t2dm). recently, low carbohydrate diets and ketogenic diets emerged as common dietary approaches in managing t2dm. literature suggests that dietary macronutrients result in significant impacts on the gut microbiome related to glucose tolerance, insulin resistance and inflammation. although there is insufficient literature on gut microbiota modulation via macronutrient management, this area of study has been gradually gaining interest amongst researchers. this review describes the current evidence and summarizes specific macronutrients effects in sculpting the diverse gut microbiome. potential crucial research concepts could help develop macronutrient-specific diets to modulate gut microbiota beneficial to t2dm management and pathogenesis. keywords: type 2 diabetes mellitus; gut microbiota; macronutrients; modulation 1. introduction type 2 diabetes mellitus (t2dm) is defined as a metabolic disorder identified by the presence of hyperglycemia and other hemostatic control systems such as insulin secretion defects, disturbance in insulin action, and increased hepatic glucose production[1]. as a result, the metabolism of macronutrients becomes disrupted within the body. the world health organization has defined the diabetes diagnostic criteria by four main measurements, 2-hour (2-h) post-load plasma glucose after a 75 g oral glucose tolerance test (ogtt); fasting plasma glucose; random blood glucose in the presence of diabetes signs and symptoms and hemoglobin a1c (hba1c), with respective cut-off values of ≥11.1 mmol/l (200 mg/ dl); ≥ 7.0 mmol/l (126 mg/dl); ≥ 11.1 mmol/l (200 mg/dl); ≥ 6.5% (48 mmol/mol)[1]. t2dm is estimated to be the most common type of diabetes, accounting for 90%–95% of its patients, pmmb 2021, 4, 1; a0000182 2 of 16 with an expected significant quadruple increase since 1980 from 422 million to 693 million by 2045[2]. the etiology of t2dm can largely be credited to one’s alterable characteristics, especially nutrition, body weight, and various metabolic profiles[3]. henceforth, dietary strategies focus on energy restriction and dietary quality to improve glycemic control by helping individuals with t2dm adopt healthy eating habits[3]. recent studies have attributed the effect of dietary factors, specifically macronutrient intake, on diabetes parameters and their ability to manage diabetes. gut microbiota in healthy individuals conveys multiple beneficial functions within the body. for instance, it aids in host nutrient metabolism, immunomodulation and crucially maintains gut mucosal barrier structure and integrity. therefore, a slight disrupt in the microbiome’s ecosystem may lead to the pathogenesis of major diseases including irritable bowel syndrome, osteoporosis, myasthenia gravis, colon cancer, spleen deficiency syndrome, and hives[4–9]. as such, the gut microbiota’s potential role in one’s health and the development of diabetes will be further explored in this review. a summary of the narrative review can be found in figure 1. figure 1. effects on dietary macronutrients on gut microbiota that have potential impact on diabetes. pmmb 2021, 4, 1; a0000182 3 of 16 2. role of macronutrients in diabetes carbohydrate intake has been sparking interest amongst researchers as a critical macronutrient in diabetes management. diets enriched with high carbohydrate composition have often been associated with heightened glycemic index and t2dm risks[10]. increased insulin secretion required to reduce dietary carbohydrate-induced hyperglycemia may ultimately lead to glucose intolerance and t2dm[11]. however, certain non-digestible carbohydrates like dietary fibers in the diet have been associated with protective effects against t2dm[12]. thus, while many studies have discussed the beneficial effects of low carbohydrate diets (lcds) in t2dm, more studies are now focused on specific carbohydrate types, especially non-digestible carbohydrates. dietary fat is another macronutrient heavily focused on due to the rising popularity of the ketogenic diet (kd). like carbohydrates, the diverse sources of dietary fats instead of the amount of fats consumed can significantly impact diabetes parameters. many papers have discussed that saturated fats derived from animal sources could negatively affect people with t2dm and should be minimized in diets[13]. in contrast, recent studies have pointed out the beneficial effects of consuming unsaturated vegetable fats by improving t2dm patients’ lipid profiles and glycemic control[14]. recent studies have also observed a possible association between protein intake and the success of t2dm management. while the effects of total protein intake on diabetes are inconclusive, differing protein sources have displayed different outcomes in t2dm patients. for example, animal proteins have been indicated to result in inconclusive effects on diabetes parameters, while plant-based protein sources, specifically from legumes, may positively affect the parameters[15–17]. however, the limited number of studies on dietary protein’s effects on t2dm indicates that detailed studies should further establish the potential associations. 3. role of gut microbiota in diabetes recently, studies on t2dm have begun to speculate the diverse effects that specific gut microbiota populations may have on improving or worsening diabetes parameters. studies have shown that individuals with t2dm present with lower gut microbial diversity than healthy individuals[18]. although substantial research has yet to be conducted, gut microbiota modulation could become a potential avenue for diabetes management. specific gut microbiota species have been found to have a negative correlation with t2dm development. bifidobacterium spp., roseburia spp., akkermansia spp., faecalibacterium spp. and distinct strains of bacteroides spp. and lactobacillus spp. are negatively associated with t2dm with varying degrees of significance[19-23]. for example, bifidobacterium spp., specifically bifidobacterium bifidum, bifidobacterium longum, bifidobacterium adolescentis, and bifidobacterium pseudocatenulatum may have the most protective potential towards diabetes[20–22]. strains of lactobacillus spp. such as l. amylovorus, l. plantarum, l. reuteri, l. casei, l. curvatus, l. gasseri, l. paracasei, l. rhamnosus and l. sakei indicated negative correlations with t2dm[19]. a majority (21 out of pmmb 2021, 4, 1; a0000182 4 of 16 23) of operational taxonomic units bacteroides spp. were also indicated to correlate with diabetes[23] negatively. studies have also suggested that bifidobacterium spp. and lactobacillus could potentially complement each other to improve diabetes parameters[24,25]. on the other hand, several gut microbiota species like ruminococcus spp., fusobacterium spp., blautia spp. and certain lactobacillus strains have shown positive correlations with t2dm development and worsening t2dm symptoms[19]. while results remained inconsistent due to varied t2dm treatments, ruminococcus spp. and blautia spp. were observed to be elevated in t2dm patients. surprisingly, the association between firmicutes/bacteroidetes (f/b) ratio, a standard marker used to identify the metabolic disease, and t2dm was inconsistent, with 6 out of 14 studies indicating no association between both factors[19]. despite this lack of consistency, this ratio may still be used in many studies as one of the means of suggesting t2dm development. 4. macronutrients and gut microbiota specific macronutrient-based diets help reduce blood glucose levels, prevent any long-term diabetes-related complications, and contribute to shaping and restoring healthy human gut microbiota. multiple human and experimental-animal studies claim that carbohydrates enriched diets are proven to have a positive effect on health-beneficial gut microbes[26]. for instance, high-fiber diets increase the abundance of bifidobacterium population, reduce the f/b ratio, and improve gut microbiota diversity[27–31]. besides, increased lactobacillus spp., akkermansia spp., faecalibacterium spp., roseburia spp., bacteroides spp., and prevotella post-high carbohydrate diets (hcd) illustrates their potential prebiotic effect. similarly, arabinoxylan, resistant starch, and inulin-type fructans, other types of dietary fibers modulate health-beneficial bacteria such as bifidobacterium, faecalibacterium, and lactobacillus by increasing their pool size[32]. further studies on other dietary fibers, such as oligofructose and polydextrose, were also found to modulate many health beneficial bacteria such as ruminococcus intestinalis, clostridium leptum, and roseburia[26]. therefore, such carbohydrates discussed in multiple studies have provided rigorous evidence on their potential ability to be used as a therapeutic intervention for metabolic diseases such as t2dm according to their multiple beneficial outcomes. clinical and preclinical studies on protein-enriched diets suggest that the quality and quantity of proteins have a varying effect on gut microbiota. for example, plant protein consumption such as mung beans in a high-fat diet (hfd)-fed mice reduced the hfd-induced f/b ratio[26]. it was also evident that animal-based protein could increase detrimental gut microbiota compared to plant-based protein, increasing intestinal inflammation sensitivity[33]. similar to protein, types, and quantities of dietary fats may have substantial effects on either beneficial or detrimental gut microbiota in individuals with diabetes. unlike pmmb 2021, 4, 1; a0000182 5 of 16 carbohydrates, the results of fifteen studies examining the effect of hfd on gut “microbiota” diversity and richness suggest that both total and saturated fats have negatively affected microbiota[26]. for instance, studies show that saturated fats cause a consistent decrease of health-beneficial microbes such as faecalibacterium and bifidobacterium and increase f/b ratio. multiple studies supported these findings where the diet consisted of 44% to 72% of fat[34]. on the contrary, 20%–40% of dietary fat consumption was evident to reduce f/b ratio[35]. consumption of unsaturated fats also decreased the f/b ratio, alongside detrimental bacteria such as escherichia spp. and streptococcus spp.[26]. in summary, hfd and saturated fat-enriched diet are mainly detrimental to gut health, as they reduce the population of beneficial microbes. however, this can be reversed if t2dm patients consume an unsaturated fat diet or low-fat diet (lfd). the subsequent parts of this narrative review assess scientific studies that evaluated the effect of dietary macronutrients on the gut microbiome composition using in vitro and in vivo models, human and animal clinical trials. the f/b ratio, the shift in potential detrimental and beneficial gut microbiota species, and gut microbial diversity are the significant findings discussed in this review. 5. dietary carbohydrates 5.1. low carbohydrate diet in type 2 diabetes a recommended dietary pattern for people with diabetes includes carbohydrates from various sources for good health, and monitoring carbohydrate intake is a critical strategy in achieving glycemic control[36]. low carbohydrate diets (lcds) are common among people with diabetes. for example, ma and colleagues reported the adoption of low-carbohydrate, low-fiber and highfat diet in patients with t2dm enrolled in a dietary intervention[37]. however, the authors warned that though patients may find reducing weight and controlling blood glucose appealing, such nutritional patterns may have severe cardiovascular implications. several studies have shown the effectiveness of lcds in controlling blood glucose. one such trial by haimoto et al. reported a remarkable reduction in hba1c levels after a 30% carbohydrate diet over six months[38]. the researchers suggested the lcds effectiveness to be comparable to insulin therapy. this finding is supported by similar studies[39–41]. most of the reported trials were, however, short-term in nature. dyson et al. and colleagues reviewed six studies investigating the effects of hypocaloric reduced carbohydrate diets in patients with t2dm and found all studies reported reductions in body weight and hba1c[42]. although studies were few and small in sample sizes, lcds are safe and effective over the short term for patients with t2dm. metaregression of 13 studies showed that hba1c and fasting blood glucose (fbg), improved with lcds in patients with t2dm[43]. a more recent systematic review of 9 randomizedcontrolled trials (rcts) by meng et al. (2017) showed lcds were beneficial in controlling blood glucose levels of patients with t2dm, assessed by significant reduction on hba1c level (weighted mean difference (wmd): -0.44; 95% ci: -0.61, -0.26)[44]. a similar finding pmmb 2021, 4, 1; a0000182 6 of 16 was reported by sainsbury et al. (2018)’s meta-analysis of 25 rcts, where a significant reduction in hba1c level was noted at three months (wmd: -0.47%, 95% ci: -0.71, -0.23) and six months (wmd: -0.36%, 95% ci: -0.62, -0.09)[45]. however, lcds were not associated with a significant effect on long-term weight loss in t2dm[43,45]. a meta-analysis has shown an improvement in patients’ lipid profile with t2dm after restricting their carbohydrate intake. triglyceride levels (tg) of these patients have been shown to decrease after lcd trials (wmd: -0.33; 95% ci: -0.45, -0.21)[44]. the analysis also showed that lcds with calorie limitation effectively increased high-density lipoprotein (hdl)-c levels (wmd: 0.07; 95% ci: 0.03, 0.11). patients with t2dm are regarded as an ideal target group for lcds. however, during the diet, insulin requirement needs to be reviewed closely because the lcds can be very effective at lowering blood glucose. patients on diabetes medication who use this diet should be under close medical supervision or capable of adjusting their medication[46]. 5.2. gut microbiota and dietary carbohydrate dietary carbohydrates can be differentiated into digestible and non-digestible carbohydrates. this review focuses on the effects of non-digestible carbohydrates, referred to as dietary fibers, on gut microbiota. non-digestible carbohydrates (plant fiber and resistant starch) are the main form of energy to the large intestinal microbiota mainly because enzymes found in the upper gastrointestinal tract cannot digest resistant starch and are fermented by the colonic microbiome[47]. under macronutrients, carbohydrates form the major modulator for health-beneficial microbes. dietary fiber, arabinoxylan, galactooligosaccharides (gos), inulin-type fructan, resistant starch, and polydextran have significant bifidogenic effects (increase the growth of bifidobacteria) and positively modulate health-beneficial microbes in the gut[12]. significant health-beneficial microbes modulated by these major carbohydrates are bifidobacterium spp., lactobacillus spp., akkermansia spp., fecalibacterium spp., roseburia spp., bacteroides spp. and prevotella, roseburia, clostridium leptum and ruminococcus intestinalis[12,26]. diets involving non-digestible carbohydrates like whole grains, traditional chinese medicine food plants, and foods rich in dietary fibres increased b.pseudocatenulatum c1 levels, b. pseudocatenulatum c62, and mostly b.pseudocatenulatum c15 (accounted for more than 50% of bifidobacterium population), while b.pseudocatenulatum c55 and b.pseudocatenulatum c95 were unresponsive. b. pseudocatenulatum c15 correlates with improved inflammatory markers, where leptin levels are decreased, and adiponectin levels are increased[48]. human clinical trials showed that increased resistant starch consumption positively correlated with increasing bacterial species like bifidobacterium spp., fecalibacterium spp., eubacterium spp., ruminococcus spp. and parabacteroides distasonis, which improves t2dm. when combined with arabinoxylan, resistant starch could alter gut microbiota by pmmb 2021, 4, 1; a0000182 7 of 16 increasing bifidobacterium spp. concentration and reducing dysbiotic bacterial species while concurrently increasing short-chain fatty acids availability, thus improving metabolic syndromes and colonic health[12,26]. oligosaccharides, which include fructans, raffinoseoligosaccharides, and gos, has shown positive impacts on enhancing gut microbiota, mainly due to its bifidogenic effects[26]. fructans alter gut microdiversity by increasing bifidobacterium spp. and faecalibacterium spp, while simultaneously decreasing detrimental microbes like bacteroides and clostridium[12]. clinical trials in vitro fermentation of galactooligosaccharides, a potential prebiotic, have shown an increase in bifidobacterium spp. and specific lactobacillus strains[26]. these bacteria promote and positively modulate a healthy gut microbiome, indicating gos’s bifidogenic potential. moreover, gos simultaneously reduces gut inflammation by increasing gut mucosa immunoglobulin a and plasma c-reactive proteins[12]. butyrate, a complex carbohydrate digestion product, is responsible for modulating inflammation, immune cell function, and migration. butyrate can also be produced by specific gut microbiomes (clostridiales spp. ss3/4, f. prausnitzii, eubacterium rectale, roseburia intestinalis and certain lactobacillus species) within healthy individuals[46]. in contrast, t2dm patients have a reduced population of butyrate-producing bacteria[46]. therefore, a high non-digestible carbohydrate diet aids in modulating butyrate-producing bacteria, roseburia spp. and fecalibacterium prausnitzii, associated with improving insulin sensitivity[12]. consumption of foods high in polyphenols such as flavonoids, stilbenes, and lignans from vegetables, fruits, coffee, wine, tea, and cereals has indicated a rise in mucopolysaccharide-degrading a.muciniphila populations, which plays a potentially protective role in t2dm development[46]. a. muciniphila maintains gut mucosa integrity, reduces inflammation, improves glucose tolerance, and reduces insulin resistance[19]. furthermore, polyphenols can modulate microbial diversity and gut microbes by inhibiting potential pathogenic organisms. overall, most non-digestible dietary carbohydrates are positively related to the beneficial gut microbe species in t2dm patients. 6. dietary protein 6.1. dietary protein in type 2 diabetes observation of t2dm patients’ blood amino acid content suggests increased circulation of high branched-chain amino acids and aromatic amino acids [49]. according to the american diabetes association, an optimum amount of protein consumption should be personalized on a case-by-case basis due to the lack of compelling evidence-based conclusions derived from the latest research[50]. currently, available literature on the effects of animal and plant protein on diabetes parameters are inconsistent. a systematic review and meta-analysis of 13 rcts investigating the impact of substituting animal protein with plant-based proteins, a majority of its soy, indicated a drastic reduction in hba1c levels (md: -0.15%; 95% ci: -0.26, -0.05%), fbg (md: -0.53 mmol/l; 95%-ci: -0.92, -0.13 mmol/l), and fasting insulin (md: -10.09 pmol/l; pmmb 2021, 4, 1; a0000182 8 of 16 95%-ci: -17.31, -2.86 pmol/l) compared to control arms[51]. this evidence suggests plant proteins to be more beneficial than animal proteins in t2dm. plant proteins’ potentially protective effects on t2dm have been recorded in several studies, specifically pulses. for example, tree nuts consumption indicated improvements in fbg (md: -0.15 mmol/l, 95% ci: -0.27, -0.02 mmol/l) and hba1c levels (md: -0.07%, 95% ci: -0.10, -0.03%)[52]. however, the beneficial effect of soy protein on glycemic endpoints is not entirely supported. while some human and animal studies on soy protein’s effects on t2dm suggest that soy protein consumption can improve insulin resistance, reduce serum very-low-density-lipoprotein and low-density-lipoprotein cholesterol, and triacylglycerol studies are indicating that soy protein’s effects on hba1c, fbg, and insulin are insignificant[17,53,54]. similarly, dairy protein is another type of protein reported to improve insulin resistance and reduce inflammation in overweight patients with t2dm[55]. more specifically, insulin sensitivity enhancement accompanied by a 55% increase in adiponectin, as well as reductions in oxidative stress and several inflammatory markers were discovered to be associated with the dairy-based protein consumption. whey protein, a milk product, has also been shown to lower blood glucose concentration by stimulating insulin secretion postprandial. it is hypothesized that glucose-regulation is possible due to gut hormones and incretins[55]. on the contrary, animal protein, including red meat, poultry, and fish, have been widely reported to increase the risk of t2dm development upon inclusion into the diet[17]. yet, some studies report that its overall effects on glycemic and metabolic control in t2dm patients remain relatively insignificant, despite having a statistically negligible negative association with hba1c levels[15,56]. for instance, trials conducted on animal and plant protein found improved glycemic and metabolic control of t2dm patients. in this trial, the hba1c levels of both patient groups subjected to high plant and animal protein diets after six weeks were reduced by ~0.5%, although the plant protein diet group had a more significant association with reduced hb1ac levels. also, there was an improvement in the whole-body insulin sensitivity after following animal and protein diets, without substantial differences[16]. the findings were supported by another trial that reported high animal and plant protein diets to decrease hba1c and fbg, while animal protein diets improved whole-body insulin sensitivity[57]. hence, it seems that while animal protein may worsen t2dm management and plant proteins may have protective effects against t2dm, studies specifically detailing the results of each protein type and quantity could entail a much more valid conclusion. in summary, the different qualities rather than quantities of proteins play different roles in t2dm management and development. pmmb 2021, 4, 1; a0000182 9 of 16 6.2. gut microbiota and dietary protein as mentioned earlier, plant-based proteins have illustrated a positive association in t2dm management. this is because specific plant-based proteins have produced promising results in altering gut microbiota to benefit t2dm patients. different types of protein (animal, plant bases, and dairy) and the quantities have varied effects on gut microbes. during amino acid catabolism, enterocytes play a role in modulating intestinal barrier function, such as the type of bacteria present[58]. for instance, an animal-based protein diet displayed an increase in bacteroidales and clostridiales population within the gut[26]. similarly, an increased trimethylamine n-oxide level, a proatherogenic microbial metabolite, was associated with high red meat intake, which was proven to be a precursor for t2dm risk[59]. furthermore, a 70-day supplementary study of blend whey isolate and beef hydrolysate was conducted to examine gut microbiota’s effect. the results suggested a decreased health-beneficial microbiota level of b. longum, blautia and roseburia, e. rectale, as well as firmicutes[26]. further human studies on dietary protein intake indicate a positive relationship between animal protein consumption and reduced bacteroidetes, increasing f/b ratio, which is positively related to t2dm[60-61]. conversely, consumption of vegetable proteins in individuals increased the growth of bifidobacterium spp. and lactobacillus spp. while decreasing bacteroides and clostridium spp.[61]. mung bean proteins were evident to increase the ruminococcacea family abundance in hfd mice models. this aid in mediating bile acid metabolism is hypothesized to provide some health benefits[26]. overall, plant-based protein can potentially shift the gut microbiome to benefit t2dm patients. in contrast to plant based-diet, dairy protein consumption such as casein in piglets was found to increase the fecal enterobacteriaceae and decrease beneficial fecal lactobacillus spp. some clinical studies suggested that caution must be taken while exposed to a casein protein diet [26]. this is because it appears to affect overweight ”individuals’ rectal mucosa by disturbing the regular gene expression of bacteria. on the other hand, cheese whey protein, another form of dairy protein, potentiates the fecal counts of beneficial lactobacillus and bifidobacterium spp. while reducing the clostridium spp. population[26]. therefore, both beneficial and detrimental metabolites were produced by specific microbes, stimulating different gut microbiome effects in t2dm patients. overall, most studies have evidenced that plant proteins display beneficial effects on gut microbiota modulation, while studies on animal protein’s impact on diabetes have conflicting results. 7. dietary fats 7.1. high fat ketogenic diet and type 2 diabetes while low-carbohydrate ketogenic diets (kd) have risen in prominence among t2dm patients, the debatably beneficial effects of reducing carbohydrates intake in these patients are yet to be firmly proven. a kd is defined as a very low carbohydrate (vlc) diet pmmb 2021, 4, 1; a0000182 10 of 16 consisting of less than 50 g of daily carbohydrates from non-starchy vegetables, resulting in ketosis and reduced insulin levels[62]. typically, kd consists of 5% carbohydrates, 80% fats, and 15% protein[63]. multiple studies have illustrated a positive weight loss outcome due to kd. sahama st al. study suggests that kd likely to result in weight loss three times more than lfd[64]. this is due to the excessive fatty acid metabolism resulting in high ketone bodies. moreover, hba1c levels were also proven to be significantly reduced in multiple studies due to the possibility of ketone bodies’ ability to decrease glucose metabolism[64–65]. a prospective cohort study suggested a statistically significant association between consumption of a lowcarbohydrate ketogenic diet with a decrease in ldl, tg, and cholesterol levels and an increase in hdl level[65]. a trial comparing high carbohydrate lfd or very low carbohydrate, high-unsaturated fat or low-saturated-fat diet among obese t2dm patients showed a significant decrease in body weight and hba1c levels in both groups[14]. on the other hand, the lipid profile in a vlc diet improved at a higher level than hcd due to the possible fat quality. a similar study also noted that both diets were clinically significant in reducing hba1c (-0.7%; 95% ci: 1.0, -0.5%)[66]. a more significant improvement was also marked by diabetes medication reduction, which could reach up to half the amount (anti glycemic medication effect score (mes): -0.5; 95% ci: -0.6, -0.3), hc: -0.2; 95% ci: -0.4, -0.02units), as well as blood glucose variability[66]. while there are concerns on long-term kds increasing the risk of diabetic ketoacidosis, a trial with follow up after two years indicated its beneficial weight loss effects void of potentially adverse renal effects[14]. 7.2. gut microbiota and dietary fat dietary fat plays a significant role in modulating the gut microbiome in t2dm patients. specifically, dietary fats (saturated and unsaturated) and the quantity consumed may have distinctly different gut microbiota effects. a human study suggested that a diet rich in polyunsaturated fat protects against t2dm development by increasing bacterial populations of roseburia and f.prausnitzii[26]. similarly, a mice study indicated that saturated fatty acids like lard could promote harmful bacteria like bilophila and increase the f/b ratio. in contrast, polyunsaturated fatty acids increased the beneficial bacteria genus of bifidobacterium, lactobacillus and a. muciniphila[26]. a systematic review of fifteen studies reported hfd or saturated fat diet negatively associated with gut microbiota’s diversity[26,67]. according to qi et al., hfds increase the f/b ratio indicating an opposite effect on kd[63]. this could be attributed to ketone bodies’ presence in kd compared to hfd. elevated ketone bodies like βhb can increase the bacteroides population, reducing the f/b ratio[63]. a trial also indicated that a 40% fat diet caused a rise in the detrimental bacteroides and alistipes genus while simultaneously reducing the beneficial faecalibacterium genus[26]. pmmb 2021, 4, 1; a0000182 11 of 16 moreover, the stimulation of ketone body production in kds inhibits bifidobacterium spp’s growth, resulting in decreasing the levels of pro-inflammatory th17 cells and adipose tissues. the reduced levels of th17 cells in both adipose tissue and gut could improve metabolic syndrome aspects such as glycemic control[63]. similarly, mice studies have indicated that kd reduces desulfovibrio and turicibacter, potential pro-inflammatory bacteria genus, and increases the abundance of a. muciniphila and lactobacillus spp. a. muciniphila results in low blood glucose concentration caused by the increase in insulin sensitivity[68]. interestingly, kd’s potential benefits to t2dm patients may be overshadowed by a few possible adverse effects. kd stimulates ketogenesis, reverting the body’s primary energy source from glucose to ketone bodies. as a result, the serum concentration of ketone bodies like beta-hydroxybutyrate (βhb) becomes elevated. according to a human study, βhb displays bacteriostatic effects and a negative correlation with bifidobacterium spp. through a ph-dependent mechanism[63]. bifidobacterium spp. has a role in the metabolism of nondigestible carbohydrates; hence a reduction in carbohydrate intake explains the reduced bifidobacterium population[48]. since bifidobacterium spp. are beneficial to t2dm patients, kd’s overall benefits are potentially reduced. also, several human studies concluded that a reduction in dietary fat to 35% of one’s diet aids in normalizing the gut microbiome. this suggests that the gut microbiome’s adverse changes may result from diets with a dietary fat composition of more than 35%[26]. 8. conclusions this current research review summarized multiple studies on the different macronutrients and their role in modulating t2dm gut microbiota. however, further research is required to provide evidence and supporting data to the questions in doubt. for instance, it is unclear why macronutrients affect individuals differently and their mechanism of action within the gut to alter the microbiome. some studies have suggested gnotobiotic or humanized mouse models, as they could be used to overcome these issues despite their several limitations. this is because they enable careful and close control of dietary nutrients and evaluate gut microbiome changes[70]. additionally, a diverse range of individuals can undergo a well-defined, closely monitored dietary intervention to understand better how their microbiome responds to specific macronutrients according to their intraand inter-variability. moreover, a long-term dietary analysis must be integrated into different macronutrient dietary interventions with intensive longitudinal data collection to improve research results. although extensive research has already been conducted, dietary macronutrients’ role in gut microbiota modulation as an effective management mechanism for t2dm remains uncertain. however, gut microbiota modulation by dietary macronutrients should still be thoroughly evaluated as a means of improving t2dm complications. in this review, we have identified and analyzed each dietary macronutrient’s effects on specific strains of gut microbes and the management of t2dm. thus far, our discoveries indicate that the integration of non-digestible carbohydrates and polyunsaturated fats in kds best modulate pmmb 2021, 4, 1; 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52: 1383–1396. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology review article 1 insights into quorum sensing (qs): qs-regulated biofilm and inhibitors wen-si tan1, jodi woan-fei law2, lydia ngiik-shiew law3, vengadesh letchumanan2, kok-gan chan4,5* 1illumina singapore pte ltd, woodlands industrial park e1, singapore 2novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 3faculty of pharmacy and pharmaceutical sciences, monash university, 381 royal parade, parkville vic 3052, australia 4division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia 5international genome centre, jiangsu university, zhenjiang, china abstract: in the environment, bacteria can communicate with a known mechanism called quorum sensing (qs). these bacteria will communicate in a group for social interactions like a multi-cellular organism. it provides significant benefits to the bacteria in host colonization, the formation of biofilms, defense against competitors, and adaptation to environmental changes. the bacteria that organize in biofilms are difficult to control and manage, resulting in a higher dosage of antibiotics to clear the infectious biofilms. also, many qs-controlled activities are involved in virulence and pathogenicity. hence, understanding the details of quorum sensing mechanisms, its phenotype regulation (biofilm), and qs inhibitors (which attenuate virulence/pathogenicity) may open a new avenue for controlling bacterial infections. keywords: quorum sensing; biofilm; inhibitors; virulence; infections received: 19th october 2020 accepted: 19th november 2020 published online: 28th november 2020 citation: tan w-s, law jw-f, law ln-s, et al. insights into quorum sensing (qs): qs-regulated biofilm and inhibitors. prog microbes mol biol 2020; 3(1): a0000141. https://doi.org/10.36877/pmmb.a0000141. introduction bacteria are a group of microorganisms that can interact with each other and their surroundings via quorum sensing. quorum sensing is a bacterial cell-to-cell communication process that depends on the release and response to extracellular chemical signaling molecules known as autoinducers[1]. these autoinducers will increase in concentration in a synchronized manner with the density of the bacterial cell population. thus, detecting a minimum threshold concentration of signaling molecules could stimulate an alteration in gene expression[2, 3]. in other words, qs regulates gene expression as a result of changes in the cell population density. several types of autoinducers have been identified, and they consist of small peptides, quinolones, and acyl homoserine lactones (ahl). the ahl-based qs system is the most studied among other methods, and ahl molecules are the primary qs signals utilized by gram-negative bacteria[4]. briefly, a typical ahl-based qs system comprises two primary proteins: luxi-type protein (cytoplasmic ahl synthase) and luxr-type protein (ahl-responsive dnabinding transcriptional regulator)[5,6]. the bacterial cells generate ahl signals (synthesized by luxi-type ahl synthase) at a low basal rate, which can penetrate the cell membrane without using a receptor. once the threshold concentration of ahl signals is achieved, the signs are sensed by luxr-type transcriptional regulator protein and thereby produce a luxr/ahl complex that alters gene expression upon binding to lux box dna a conserved site in the promoter region[6–8]. the ahl is known to be involved in regulating different phenotypes, which is strain-dependent through qs[9,10]. in the natural environment, bacteria can sense the cell population density and regulate a wide range of physiological processes, including expressing essential phenotypes such as bioluminescence, biofilm formation, virulence factor production, swarming motility, chemotaxis, toxin secretion, and antibiotic resistance[1,2,9]. these qs-regulated phenotypes are also essential for bacteria to successfully establish a symbiotic (beneficial or pathogenic) relationship with higher organisms. this review aims to provide insights copyright @ 2020 by tan w-s and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: kok-gan chan, division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia; kokgan@um.edu.my. 2 insights into quorummm seeensing... into the quorum sensing mechanisms, phenotype regulation (biofilm), and the qs inhibitors in which attenuate bacterial virulence/pathogenicity. bacterial quorum sensing and biofilm development the attachment to surfaces is the first step for bacteria forming communities (known as biofilm) that enmeshed in a self-produced polymeric matrix[11,12]. the majority of bacterial infections in humans (more than 80%) involve biofilm development[13]. notably, biofilm formation is one of the phenotypes which is closely related to qs. the development of biofilm in vitro involves five stages. first, the reversible attachment of bacterial cells to the surface will turn into irreversible attachment mediated by exopolymeric material[14,15]. fibrinogen and fibronectinbinding proteins are usually found to play a role in this attachment process. next, microcolonies are formed, and this indicates the beginning of biofilm maturation. the mature biofilms engineered varies, from flat, homogenous biofilms to highly structured 3-dimensional biofilms. the matured biofilm contains cells that are packed in clusters with channels in between to allow water and nutrient transportation and waste removal. the architecture of developed biofilm is often influenced by motility, rhamnolipid production, and extracellular polymeric substance matrix production. ahl-based qs has been shown to affect biofilm formation at the maturation stage. labbate and colleagues (2004)[16] proved that a mutation in s. liquefaciens acyl-synthase gene, swri results in thin biofilms that lacked aggregates and filaments as compared to its wildtype’s biofilm, which is heterogenous that consist of an aggregation of long filaments of cells. this is further substantiated by work on burkholderia cepacia h111 with mutations in either cepi or cepr[17]. both mutants showed defective in biofilm maturation and were only arrested at the microcolony stage of growth compared to the robust biofilms covered with attachment surface formed by the wildtype. additionally, the maturation of biofilm is influenced by the luxs-based qs other than the ahl-dependent pathway[18,19]. in streptococcus mutans, the mutation in luxs resulted in a mature biofilm with decreased biomass as compared with its wildtype. the final stage of biofilm involved aggregation and detachment, dissolution or dispersal of cells from the biofilm to initiate a new biofilm formation. the dispersed cells showed similarity with planktonic cells, which is non-adherent. this dispersal process allows bacteria to colonize new surfaces and spread its virulence effectively within a closed environment. in this final stage of biofilm formation, the cell dispersal was also found to be qs controlled. in rhodobacter sphaeroides, the mutation in its ahl synthase resulted in hyper-aggregation of cells; but qs’s role in this bacteria still remains unknown[20]. other than that, yspr mutant of yersinia pseudotuberculosis resulted in increased swimming motility[15]. the complex formation of biofilm provides a “room” with a hydrated matrix of microbially produced proteins, nucleic acids, and polysaccharides that allows the cells to act less as individual entities but more as collective living systems[14]. biofilm shields the bacteria by significantly increased in resistance to environmental stresses (ph fluctuation, high salt, and nutrient fluctuation) or microbially harmful particles (antibiotics and biocides). the exciting point arises the criteria for determining the role of qs in biofilm formation[15]. perhaps it is not surprising that qs indeed plays a major role in biofilm formation, evident by increasing the study of mutant construction experiments that produce pleiotropic phenotypes that affect motility, surface attachment expression, or cell chemistry surface, which is later translated into biofilm formation. however, it would be best if the role of qs could be evaluated by monitoring the signaling process in situ in a developing biofilm in the parental strain and determine if the onset of qs corresponds to any observable transition in bacterial biofilm development that relates with other phenotypes such as incline of antimicrobial tolerance. use of quorum sensing inhibitors as potential antipathogens the pathogenesis portrayed by bacteria is a multi-factorial process regulated by the production of virulence factors, which causes a variety of bacterial infectious diseases[21]. qs could regulate many of the bacterial infectious diseases in humans, animals, and plants. consequently, qs-regulated biofilm formation plays a vital role in bacterial pathogenesis. this has raised the level of concern in clinical settings and other industrial settings where biofilms pose a significant issue, such as aquaculture, agriculture, wastewater treatment plants, and drinking water processing[22]. the dedication of antibiotics in the early 20th century initiated a new era in treating microbial infections, and they were the most rewarding drug that saves myriad lives[23]. however, antibiotics usage over a long time could cause substantial evolutionary stress on the bacterial population and lead to the emergence of multidrug-resistant strains that possessed defensive mechanisms against these antibiotics[24,25]. methicillin-resistant staphylococcus aureus (mrsa) [26,27], vancomycin-resistant enterococci (vre)[28], multidrug resistant salmonella enterica subsp. enterica[29–31], multidrug-resistant mycobacterium tuberculosis[32], multidrug-resistant vibrio parahaemolyticus[33–42] are some dangerous bacterial species that have emerged due to over usage of antibiotic. the emergence of multidrug-resistant bacteria has caught medical attention, and various approaches are now taken to investigate alternative antimicrobials from different sources (e.g., plants and microorganisms)[24,43–49]. interestingly, scientists have also considered another approach in recent years by exploring into qs linking to bacterial pathogenicity. the findings into the association of qs and bacterial pathogenicity have been evidently strong as virulence has been greatly reduced in mutants that are defective in qs [21,50,51]. in addition, researchers are actively venture into the investigation of different approaches to interrupt or inhibit qs for the control of bacterial diseases. this inhibition process is generally known as “quorum quenching”. quorum quenching (qq) can be carried out by the application of enzymatic degradation of autoinducers, 3 tan w-s et al. pathway could serve as a potential new strategy to attenuate bacterial pathogenicity and inhibition of biofilm formation (figure 1). blockage of autoinducer compounds synthesis, and utilization of inhibitor compounds to block the signal detection[52–54]. therefore, techniques that target the qs halogenated furanone compounds or known as fimbrolides are intensively studied as a group of qs inhibitors[23]. they are isolated from red microalga delisea pulchra, an alga that can produce secondary metabolites that are made up of more than 30 types of furanones. previous studies had shown that these secondary metabolites could interfere with the ahl-based qs communication circuit. a study performed by janssens and colleagues (2008)[55] showed that brominated furanones could prevent biofilm formation of salmonella serovar typhimurium at nongrowth inhibiting concentrations. brominated furanones were also found to meddle with the biofilm formation of several other bacterial species including e. coli, b. subtilis, p. aeruginosa and streptococcus species. another study performed by givskov and colleagues (1996)[56] evident that 100 µg/ml of furanone extracted from d. pulchra could inhibit swarming abilities of serratia liquefaciens. moreover, defoirdt et al. (2006)[57] also showed that furanone can inhibit bioluminescence of vibrio harveyi strain jmh597 at a concentration of 100 mg/l. however, drawbacks of halogenated furanones are too reactive and could cause toxicity towards human cells. thus, researchers exert into finding potential quorum sensing inhibitors (qsis) from various natural sources. it has been proposed that a potential qsi should fulfill specific criteria[22]: (i) small molecule with high efficiency in reducing qs regulated genes, (ii) high degree of specificity with no adverse effect, (iii) chemically stable and resist to host metabolic system, (iv) longer than ahls to prevent bacteria resistance, (v) do not affect the host microbiome, and (vi) show no toxicity effects towards the host. to date, numerous naturally occurring qsi is presently well established and grouped into various categories. besides, several qq enzymes have been discovered from prokaryotes and animal sources. one of the qq enzymes is ahl-acylase that cleaves acyl side chain. acylase produces by streptomyces sp. is similar to acylase i produce by porcine kidney, where both cleaves the acyl chain longer than six carbons[58,59]. some other qq enzymes are ahl lactonases that produced by bacillus spp.[60] and mammalian paraoxonases[61] that function to hydrolyze ahl lactone ring. however, researchers have been focusing on exploring potential qsis from plant extract because it has been anticipated that plant sources are safer for human consumption. these natural compounds are known as secondary metabolites (or phytochemicals), and many classes of these phytochemicals demonstrated their potential as antimicrobials or synergists of other products[62]. recent studies have promoted the potential of these phytochemicals as potential qsis. as a result, the active compounds have been extracted from plants and their qs inhibition activity has been evaluated by numerous studies (table 1). further toxicology study should be performed on these extracted compounds to validate their safety as biopharmaceutical agents. one of the qsis consists of phenolic products or polyphenols, which constitute one of the most abundant and omnipresent as plant secondary metabolites (phytochemicals)[63]. phenolics are considered potential qsis because they are used to treat ailments such as diabetes, cancer, or inflammatory diseases besides having antimicrobial properties. jagani and colleagues (2009)[64] proved that naturally occurring phenolics could act against biofouling of p. aeruginosa. another study conducted by vandeputte and colleagues (2010)[65] showed that catechin extracted from combretam albiflorum reduces elastase, pyocyanin, and biofilm formation p. aeruginosa pao1. they had selected eight types of phenolics, anarcadic acid, polyanarcadic acid, salicyclic acid, polysalicyclic acid, polyphenol, catechin, epigallocatechin, and tannic acid; all eight compounds showed significant reduction towards p. aeruginosa biofilm formation. flavonoids extracted from citrus species such as quercetin and naringenin hinder the biofilm formation of e. coli o157:h7 and v. harveyi bb120[66,67]. another subclass of phenolics, furocoumarins, shows qsi abilities in which purified furocoumarins — dihydroxybergamottin and figure 1. application of qs inhibitors against biofilm formation. 4 berggamottin inhibit autoinducer activities v. harveyi[68]. girennavar and colleagues (2008)[69] further substantiated that furocoumarins from grapefruit juice inhibited more than 95% of autoinducer-1 and autoinducer-2 activities in v. harveyi. other than that, ferulic acid and gallic acid (grouped under subclass of phenolic acids) were found to block bacterial motility, adhesion, and biofilm formation of e. coli, p. aeruginosa, s. aureus, and listeria monocytogens[70]. a study carried out by plyuta and colleagues (2013)[71] showed that the usage of 200 µg/ml of gallic acid reduced the biofilm formation of p. aeruginosa pao1 to 30 %. gallic acid has been proven as a potential qsi. gallic acid at a concentration of 1mm resulted in an 80% reduction of biofilm formation by eikenella corrodens as demonstrated in the experiment matsunaga et al. (2010)[72]. as for ferulic acid, application at a concentration lower than eight µg/ml found to forbid s. aureus’ biofilm formation[73]. other groups of phytochemicals such as isothiocyanates and essential oils could serve as potential qsis. isothiocyanates are products formed during glucosinolate hydrolysis, and they are considered the most critical biological active products in plants[74]. one of the aliphatic isothiocyanates, allylisothiocyanate, interfered with the adhesion-related genes in s. aureus in work done by lee et al. (2013)[75]. this compound demonstrated to reduce the pseudomonas sp. planktonic cell growth and the number of cells adhered to the brassica nigra. likewise, essential oils have proven to be potential qsis as they are complex mixtures of volatile compounds synthesized from several plant organs[76]. the qs activities of p. aeruginosa, proteus mirabilis, and s. marcescens — swarming, production of extracellular polymeric substances and biofilm formation were inhibited upon exposing to methanolic extracts of cuminum cynimum, where one of the components is methyl eugenol — an essential oil with an aromatic ring[77]. this plant-based qsis may not function as bactericidal compounds; however, the infection process could be interrupted by interfering the bacterial qs and this eventually leads to elimination of pathogens by the host immune system. biotechnological implications of studying qs as the number of bacteria that employ qs systems continues to bloom, the research into qs could span a wide variety of potential applications, mostly controlling bacteria growth and activities by interfering with the signaling pathways[78]. qs cross talk is also another exciting implication as bacteria always exist in the mixedspecies population, such as biofilms in nature. this could cause an outbreak of infectious diseases or further health complications[79]. the study into qs paved the way for discovering various qsis that is feasible as a treatment for bacterial infections in all living organisms. given the growing numbers of multidrug-resistant strains, the rational strategy is to control these bacteria’s outbreak by manipulating qs properties. nowadays, scientists are exploiting the possible benefits of understanding the bacterial qs system. ultimately, this could significantly contribute to many fields, such as improving the water treatment process, preventing bacterial diseases in aquaculture systems, and treating human infections[80]. another interesting fact of qs is that eukaryotes can recognize bacterial qs molecules. this cross-kingdom interaction alters the physiological adaptation in colonized eukaryotes that modify their defense system, immune responses, hormonal responses, or growth responses[81]. besides creating a pathogenic relationship with higher organisms, the interesting interaction is signaling molecules (ahls); reported to mediate root growth through biosynthesis of phytohormones. indole-3-acetic acid (iaa), or known as auxin, is a crucial phytohormone that enhances different developmental processes in plants. iaa production is widely spread among plant-associated bacteria. they can play a critical role in promoting plants’ growth and development, especially root elongation[82]. plant growth-promoting bacteria (pgpb) have been extensively studied as potential bio-fertilizers due to increasing pollution by over-usage of chemical fertilizers[83]. biosynthesis of iaa by microbial strain is considered one of the essential criteria to be selected as an efficient pgpb. to date, there is an increasing number of reports stating that qs facilitates the pgpb in enhancing plant growth. as previously reported, treatment of arabidopsis thaliana roots with 1–10 µm of c4and c6-hsl increased the ratio of iaa/cytosine that led to promoted root growth[84]. in their study, they found out that the introduction of c6hsl did not induce the systemic resistance and priming effect of a. thaliana. they further stated that shortchain ahls might play a better role in promoting plant growth due to the hydrophobicity of long-chain ahls. a study substantiates this fact revealed that c6-hsl was transported to the leaves of yam beans and barley leaves but not the c10-hsl[85]. various studies also showed that rhizobium mutants that were unable to produce ahls were unable to nodulate legume plants compared to the wildtype strain[86]. these also further support the idea that ahls could be participating in beneficial plant-bacteria interactions. thus far, the qs studies could lead us into a different dimension in searching the potential of qs bacteria to contribute beneficially. conclusion it is undeniable that qs plays an essential part in the physiological processes of bacteria. nonetheless, further studies are still required to characterize the function and pathway-related to qs fully. the occurrence of antibiotic resistance and infections reflects the downside of utilizing antibiotics to treat biofilm linked continual bacterial infections. the application of qsis has exhibited promising results against biofilm formation; however, the utilization of qsis as an approach to the battle against multidrugresistant bacteria entails additional investigation. future work needs to reveal if qsi compounds can be developed as antipathogenic treatment and their successful bacterial eradication mechanisms. insights into quorum seeensing... table 1. examples of qs inhibitors (qsis) from plant origin and its effect on qs activity. phytochemical group qsis (phytochemicals) effect on qs activity references phenolics ascorbic acids reduction in autoinducer-2 activities, spore production, and enterotoxin production in clostridium perfringens [87] baicalein, hamamelitannin inhibition of biofilm formation increased permeability of vancomycin and reduced production of staphylococcal enterotoxin in s. aureus [88, 89] curcumin attenuation of virulence in p. aeruginosa [90] ellagic acids inhibit biofilm production in e. corrodens; reduction of ahls production in e. carotovora. [91] epigallocatechin gallate, catechin interference with biofilm formation of e. coli and p. putida. reduction in extracellular polymeric substance of staphylococcus sp. [65, 92, 93] ferulic acids inhibition of biofilm in p. aeruginosa, interference to the motility of p. fluorescens and b. cereus [94, 95] gallic acids inhibition of biofilm in s. mutans [96] giganteone a reduction of qs-related activity in e. coli biosensors [97] qs activity references tan w-s et al. phytochemical group qsis (phytochemicals) 5 phenolics gingerone reduction in swarming and biofilm-forming capacity in p. aeruginosa pao1 [98] glycyrrhiza glabra flavonoids interference of motility and reduction in biofilm formation in acinetobacter baumannii [99] malabaricone c reduction in pyocyanin production and biofilm formation in p. aeruginosa [100] rosamarinic acid influence the protease and elastase production, biofilm formation, and virulence factors of p. aeruginosa [101] salicylic acids reduction of ahl production, interference towards twitching and swimming motility of p. aeruginosa [102] tea polyphenols (camellia sinensis l.) reduction of proteolytic activity, elastase, swarming motility, and biofilm formation in p. aeruginosa [103] pyrizine-2-carboxylic acid inhibition of biofilm formation in multidrug-resistant v. cholerae [104] proanthocyanidins reduction in production of qs-regulated virulence determinants in p. aeruginosa [105] phytochemical group qsis (phytochemicals) qs activity references essential oils cinnamon oil, ferula oil, dorema oil interference of qs related phenotypes; production of pyocyanin, alginate, and rhamnolipid in p. aeruginosa [106, 107] cinnamon bark oil modification of permeability of outer membrane and inhibition of bacterial qsactivity in e. coli [108] clove oil reduction of violacein production in c. violaceum and interference of swarming ability of p. aeruginosa [109] coriander oil inhibition of biofilm formation and lipid peroxidation in campylobacter coli and c. jejuni [110] linalool inhibition of biofilm formation and alteration of the adhesion of a. baumannii [111] oregano oil inhibition of violacein production by c. violaceum [112] rose oil, geranium oil, lavender oil, rosemary oil reduction in violacein pigmentation in c. violaceum and ahls production in e. coli [113] thyme oil reduction of flagella gene expression in c. violaceum and interference of biofilm formation in p. fluorescens km121 [114, 115] phytochemical group qsis (phytochemicals) qs activity references isothiocyanates allicin, ajoene renders p. aeruginosa sensitive towards tobramycin; inhibition of biofilm [116, 117] allyl isothiocyanate interference of adhesion and motility, inhibition of biofilm formation in e. coli, s. aureus, l. monocytogenes, and p. aeruginosa [75, 118-120] iberin interference of rhamnolipid production and gene expression of lasb and rhla in p. aeruginosa [121, 122] sulforaphane, erucin antagonists of transcriptional activator of lasr and inhibition of biofilm formation in p. aeruginosa [123] stilbenoids resveratrol, piceatannol, oxyresveratrol reduction of violacein in c. violaceum cv026; decreased in 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effects of stilbenoids and their potential structure–activity relationship. bioorg med chem lett, 2015; 25(22): 5217–5220. insights into quorum seeensing......... progress in microbes and molecular biology review article 1 a review on the characteristics, taxanomy and prevalence of listeria monocytogenes vengadesh letchumanan1,2,3,9, peh-chee wong3,9, bey-hing goh1,2,3,4, long chiau ming5,6, priyia pusparajah3, sunny hei wong7, nurul-syakima ab mutalib8, learn-han lee1,2,3,4* 1 novel bacteria and drug discovery research group (nbdd), biomedicine research advancement centre (brac), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2 biofunctional molecule exploratory research group (bmex), biomedicine research advancement centre (brac), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 3 biomedical research laboratory, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 4 center of health outcomes research and therapeutic safety (cohorts), school of pharmaceutical sciences, university of phayao, phayao, thailand 5 faculty of pharmacy, quest international university perak, ipoh, perak, malaysia 6 pharmacy, school of medicine, university of tasmania, hobart, tasmania, australia 7 li ka shing institute of health sciences, department of medicine and therapeutics, the chinese university of hong kong, shatin, hong kong 8 ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia 9 authors contributed equally in the writing abstract : listeria monocytogenes is a gram-positive bacterium that is commonly isolated from food sources. this opportunistic human pathogen is the causative agent of listeriosis, an illness that mainly effects immunocompromised, the elderly, infants, and pregnant women. ever since its detection, listeriosis cases has been very prevalent and commonly associated with food sources such as dairy products, ready-to-eat food, and poultry. the prevalence of listeriosis is of public health concern and proper management is required to control this illness from spreading widely. thus, this review seeks to elucidate a better understanding on the characteristics, taxanomy, prevalence and their associated food sources, as well as management methods in order to devise proper control actions to reduce listeriosis incidence worldwide. keywords: listeria monocytogenes; gram-positive; listeriosis; dairy products; characteristics; prevalence received: 8th october 2018 accepted: 15th november 2018 published online: 9th december 2018 *correspondence to: learn-han lee, novel bacteria and drug discovery research group (nbdd), biomedicine research advancement centre (brac), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; lee.learn.han@monash.edu; leelearnhan@yahoo.com citation: letchumanan v, wong pc, goh bh, et al. a review on the characteristics, taxanomy and prevalence of listeria monocytogenes. prog microbes mol biol 2018; 1(1): a0000007. introduction the bacterium listeria monocytogenes is an ubiquitous facultative saprotroph which present widely in the soil, plants, ground water and vegetation(1;2). animals, such as cattles, sheeps, goats and poultry which fed on these silages are also identified as a host of this pathogen. as the causative agent of listeriosis, l. monocytogenes has been identified as animal pathogen since 1920s (3). over the last two decades, l. monocytogenes has been associated with many foodborne diseseas in humans(3;4;5). humans who consume contaminated food, especially ready-toeat (rte) food, dairy products, meat and poultry, will later develop listeriosis(6;7;8). despite the advancements in food preparations, healthcare system, and public awareness, this pathogen remains as a major cause of foodborne illness. this bacterium was first identified by murray in 1926 from unusual sudden death of laboratory rabbits in department of pathology of cambridge. due to its striking characteristic of mononuclear leukocytosis, the isolated microorganism was named bacterium monocytogenes(9;10). prior to 1926, this pathogen was actually copyright 2018 by letchumanan v et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) 2 encountered in 1891 by hayem in france, 1893 by henle in germany and 1911 by hulphers (which he named the organism as bacillus hepatis)(11). in 1927, pirie discovered a unique microorganism which was responsive for “tiger river disease” (later also known as listeriosis) in south africa from gerbilles and named it listerella hepatolytica, devoting it in honour of lord lister. both the strains discovered by murray and pirie were found to be similar by the national type collection at the lister institute in london, and it was named listeria monocytogenes(10). initially, l. monocytogenes was known to cause veterinary illness till nyfeldt isolated this bacterium from 3 patients who succumbed from infectious mononucleosislike disease in 1929. nonetheless, its route of transmission was obscured until 1980 when a series of outbreaks associated with food occurred(11;12). listeriosis first welldocumented foodborne outbreak was reported in canada in 1983 after consumption of contaiminated coleslaw (4). ever since then, many reported outbreaks happened in high-income countries including united states of america, japan and europe, predominantly attributed to meat and poultry products, dairy products, seafood and vegetables(13). fish and fish products are known to be frequently contaminated with l. monocytogenes. for instance, this microorganism was isolated from 44% of fresh prawns in malaysia(14), 10.5% of fish patties in malaysia(15), 7.5% and 13.6% of crab and smoked fish samples in the us, respectively(1), and 5.5 to 12.1% of minced tuna and fish roe products in japan. in 2011, a severe and deadliest listeriosis outbreak occurred in united states of america (usa). the outbreak was associated with cantaloupe and caused 146 illneses with 30 deaths and one miscarriage(16). the high case fatality rate of listeriosis has demanded a need for scrutined identification and surveillance of l. monocytogenes. this bacterium is able to infect the human food chain directly or via animal farms(17). a contaimination level above 104 cfu/g is professed as a high level of contaimination and responsible for listeriosis(18;19). anything below 102 cfu/g of l. monocytogenes contaimination in food unlikely to cause clinical illness(20), however there is reported levels of 102-104 cfu/g of l. monocytogenes causing illness in immunocomromised people(19;21). according to who, pregnant women, the elderly or individuals with a weakened immune system, such as people with immuno-compromised status due to hiv/aids, leukaemia, cancer, kidney transplant and steroid therapy, are at greatest risk of severe listeriosis and should avoid high risk foods(22). l. monocytogenes has spread widely in the environments and management of listeria in food production sector requires constant surveillance. hence, better understanding on the characteristics, taxanomy, prevalence and their associated food sources, as well as management methods is necessary in order to devise proper control actions to reduce listeriosis incidence worldwide. 2. characteristic of listeria monocytogenes l. monocytogenes is characterized as a short, gram positive, non-spore forming and facultative anaerobe. it is a bacillus that may sometimes appear singly or in short chains resembling streptococci(1;6;23). it is a non-fastidious bacillus with low g+c % dna content(1;24). in addition, it is a facultative intracellular microorganism in which its expression of flagellum-based motility is dependent on the growth temperature, with the desirable temperatures being less than 30 oc(25). this bacterium occurs naturally in environments and often reported to be isolated from human, animals, food products and food processing plants including abattoirs and smokehouses(26). l. monocytogenes grows rapidly on most of the commonly used bacteriological media. on nutrient agar, bluish grey, smooth and slightly raised colonies of the diameter 0.2-0.8mm are generally observed after 24 hours of incubation(6). a characteristic blue-green iridescence is demonstrated under obliquely transmitted light, making the colonies readily discriminated, even among high numbers of contaminants(6;27). furthermore, it displays a characteristic of end-over-end tumbling motility at room temperature and this can be seen by an umbrella-shaped pattern that develops overnight incubation in a semisolid agar(27;28). as a psychotropic foodborne pathogen, l. monocytogenes is capable of multiplying at a range of temperatures 1-45oc and remains moderately inactivated at temperatures below 0 oc, thus making rte foods with a relatively long shelf-life of particular concern(1;6). temperatures above 50oc can completely inactivate the microorganism(1). this thermo-related characteristic allowed who to ensure high-temperature short-time pasteurization (71.7oc/15 sec) is sufficient to completely inactivate normally populating l. monocytogenes in raw milk (6). besides, its resiliency is proven by the ability to tolerant acid (ph range 4.4 to 9.6) and high salt concentration (up to 10% nacl), and the propensity of forming biofilms in order to thrive in food-processing environments(1;6;29). 3. taxanomy listeria genus consists of 17 species: l. monocytogenes, l. ivanovii, l. seeligeri, l. innocua, l. welshimeri, l. martii, l. rocourtiae, l. grayi, and recently discovered l. weihenstephanensis(30), l. fleischmannii(31), l. floridensis, l. aquatica, l. cornellensis, l. riparia, l. grandensis(32), l. booriae and l. newyorkensis(33). among these species, l. grayi is the most distantly related species(1;23). both l. ivanovii and l. monocytogenes are known to cause listeriosis primarily in ruminants and human, respectively(1;34). although all species are phenotypically very similar, they can be easily differentiated by the following 5 tests: acid production from mannitol, d-xylose, l-rhamnose and α-methly-d-mannoside, and hemolysin production(6). l. monocytogenes is catalase positive, urease test positive, voges-proskauer test positive and oxidase test negative(6;27;28). it hydrolyses esculin and sodium hippurate, and shows beta-hemolysis on blood agar. other characteristics include camp test positive with staphylococcus aureus and negative with rhodococcus equi, production a review on the characteristics, taxanomy... a of acid but not gas from d-glucose, production of acid from d-salicin, l-rhamnose and α-methyl-d-mannoside, no production of acid from d-mannitol or d-xylose, no reduction of nitrates to nitrites, methyl red positive, negative reactions for hydrogen sulphide production and ammonia production from arginine(6;28). however, since identification using biochemical methods are laborious, time-consuming and of greater chance inaccuracy, molecular typing procedures are commonly employed(35). the high sensitive and discriminatory genotypic approach, such as pulsed-field gel electrophoresis (pfge) and multi-virulence-locus sequence typing (mvlst) are frequently used recently to identify an outbreak. l. monocytogenes is one of the human foodborne pathogens of great public concern. it can be classified into 12 serotypes (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e and 7), which is designated based on the immunoreactivity of the cell surfaces antigens, o and h. 1/2a, 1/2b and 4b were identified to cause most (95%) human listeriosis, with 4b serotype most frequently (98%) associated with outbreaks(23;34). 1/2a and 1/2b serotypes were commonly related to sporadic cases(23). l. monocytogenes isolates can also be categorized into at least 4 evolutionary lineages, with different but overlapping ecological niches(26). lineage i which consists of serotypes 1/2b, 4b, 3b and 3c, are associated with most human listeriosis outbreak due to their high pathogenecity. it contains 2 epidemic clones (ecs) of serotype 4b (eci and ecii) that have been associated to multiple listeriosis epidemics globally and also significantly overrepresented among sporadic cases(36). isolates in this subgroup carry listeriolysin s hemolysin that is not present in others(26). evidences of low prevelance of plasmids and insertion sequence elements in these isolates, suggesting the existence of mechanisms that limit the acquisition of foreign dna by horizontal gene transfer. lineage ii which consists of serotypes 1/2a, 1/2c and 3c, are commonly isolated from food, nature and farms. it is often associated to animal listeriosis and sporadic human cases. most are virulenceattenuated due to premature stop codon mutations (pmsc) in inla and prfa. the isolate are more resistant to bacteriocins and have higher genetic recombination rates than that from lineage i, thus probably confers an advantage for their survival in environment. they have better ability to be recovered in certain enrichment media, indicating their enhanced ability to survive environmental stress and hence related to their genuine overrepresentation in foods. lineage iii and lineage iv are less clinically important as they had low incidence among human listeriosis, suggesting of reduced human virulence. 4. prevalence of listeria monocytogenes the world health organization (who) has estimated around 600 million people are infected with foodborne disease yearly(37). foodborne diseases effect the socioeconomic development by straining the healthcare system, as well as harms the country’s economic, tourism and trade(37). the identified foodborne pathogens are such as salmonella sp.(38;39;40;41), listeria sp.(42;43) and vibrio sp.(44;45;46) are often associated with foodborne gastroenteritis cases worldwide. l. monocytogenes, the causative agent of listeriosis is known to cause sporadic cases, which a specific food source is rarely identified. the occurrence of ≥ 3 listeriosis cases of the same pulsovar strain over a period is defined as cluster(47). an outbreak is considered when the clusters of cases caused by a source strain are greater than expected during a specified period of time and place. it is challenging to investigate the origin of an outbreak as listeriosis has long and variable incubation period (3 to 70 days), which can lead to recall bias and difficulty in establishing an appropriate exposure period for food histories, and it will be much difficulty to identify rapidly foods that are not typically known to be a source of contamination in humans(48;49). 4.1 united states the us centers for disease control and prevention (cdc) has reported listeriosis was responsible for an approximately 1,600 illnesses and 260 deaths annually in the us. in 2013, the annual incidence was 0.26 cases per 100,000 individuals, with a total number of 123 cases, 112 hospitalizations and 24 deaths were reported(50;51). the incidence had declined by approximately 42% in 2012 as compared to that of in 1996-1998. the signification declines in the number of outbreaks in ready-to-eat (rte) red meats and poultry is due to the regulatory initiatives and industry actions that was implemented between 1998 and 2008. in contrast, listeriosis outbreaks is more prevalent from dairy products(52). the us has identified a number of listeriosis attributed food sources including fruit and vegetables (e.g., celery, lettuce, cantaloupe, sprouts, stone fruit and caramel apples), as well as ice cream. there were no significant changes observed in incidence between 2006-2008 and 2012. in texas, machine cut diced celery which was served in 5 different hospitals caused listeriosis(53). the infected patients were over 55 years old, with mean age of 80 and had underlying health illnesses. 5/10 of the patients died and listeriosis was attributed as the cause of death. all 10 patients had one or more immunocompromising conditions or were receiving corticosteroid or acid-reducing treatments that could have increased their susceptibility to invasive listeriosis. the largest multistate outbreaks in the us history took place in 2011 since 1998, associated with cantaloupe consumption from jensen farms in colorado(50;54). 147 cases were reported among residents of 28 states, resulting in 33 deaths and 1 miscarriage. the median patient age was 78 years; most ill people were over 60, and 99% of the patients were hospitalized. seven of the cases were related to pregnancy or were newborns. with the nationwide implementation of listeria initiative (li) since 2005, the strong association of outbreaks to cantaloupe was rapidly identified and its transmission was efficiently halted in less than 2 weeks as compared to the one-month delay between outbreak detection and product recall during 1985 outbreak associated with mexican-style cheese(50;55). this outbreak is extraordinary because it is associated with melon and comprising of widely differing pfge pattern combinations and 2 serotypes (1/2a and 1/2b). letchumanan v et al. 3 4 in 2011, another multistate outbreak associated with the consumption of mexican-style cheese among the hispanic, pregnant women was reported(56). 7 pregnancy-related cases were identified and all reported to have consumed mexican-style cheese during the month before the onset of illness. 2 out of 7 cases experienced stillbirths and 5 neonates were affected. investigation had suggested post-pasteurization contamination as the pasteurization procedure of the milk was properly done. recently, five significant outbreaks were announced by cdc. during the period of september 2013 to october 2014, 5 cases related to hispanic-style soft cheese (quesito casero) consumption were reported. 4 were hospitalized and 1 death was announced, and among these cases, 3 were identified to be pregnancy-related and a newborn was diagnosed with listeriosis(57). in february 2014, 8 people were infected with 1 succumbed from the listeriosis, due to consumption of fresh cheese curd (cuajada en terron) produced by roos food(58). in june and august 2014, 5 cases suggestive of consumption of mung bean sprouts from wholesome soy products, inc, were reported from two us states. all were hospitalized and 2 deaths had occurred(59). a multistate outbreak associated to commercially produced, prepackaged caramel apples was later reported in december 2014. as of january 2015, 32 cases were reported from 11 states 31 hospitalization cases and 7 deaths cases have been reported. 31 were hospitalized and 7 deaths have been reported. among these deaths, at least 3 were resulted from listeriosis. 10 cases were pregnancyrelated, with resulting in abortion. 3 cases of meningitis were diagnosed among otherwise healthy children aged 5-15 years. 25 of 28 (89%) patients have a history of eating commercially produced, prepackaged caramel apples. the outbreak strain was identified in the californian packing plants. on 6th january 2015, bidart bros of bakersfield voluntarily recalled all granny smith and gala from this processing plant(60). lately in february 2015, l. monocytogenes was isolated from single-serving ice-cream during a routine sampling, and it was believed to cause 10 cases and 3 deaths(61). 4.2 europe in the european countries, there is an increase in incidence from year 2000 (4.7 cases/million persons) to 2006 (6.3 cases/millions person)(47;62). total of 1554 cases were confirmed in 26 eu members in 2007, with an average notification rate of 0.3 per 100, 000 population(62). a increasing pattern of incidences were seen in france, finland, denmark, england, belgium and wales, whereas a decrease pattern was observed in sweden. however, the reason of the increase number of incidence cases remains unclear. in france, it was proposed that the reduction of salt content in food products as a preventive measure for hypertension recommended by the french food safety agency in 2002, has result in foods, such as rte meat products, to be more frequently and heavily contaminated in 2006-2007(47;62). the trend continues to demonstrate increment statistically, along with seasonal pattern over the period 2008-2012. in 2012, a total of 1642 confirmed cases were reported with a high fatality rate of 17.8% (198 deaths), which was the highest number of fatal cases reported since 2006. most of these cases were believed to be contributed from fishery products as most often l. monocytogenes sampling from these products were found beyond the safety limit(63). during january 2013-february 2014, 27 cases were reported which was significantly higher than the reported annual incidence of 7-12 cases during 2009-2012 in northern spain. 11 cases were pregnancy-related and a total of 6 deaths had occurred. sequence type (st) 87 serotype 1/2b strain, a rare l. monocytogenes type that had not been shown to cause human illnesses, was isolated from 15 cases in two epidemiologically unrelated outbreaks. first cluster involving 5 cases was identified during august-september 2013 and out of 3 were pregnancy-related. specific food as source of outbreak was failed to be identified. in the early november 2013 till late february 2014, 10 cases were reported to be possibly linked to the consumption of foie gras(64). it is also important to note that the prevalence of l. monocytogenes was much lower in fish products caught from tropical water as compared to that of in the western countries(65). interestingly, serotype 4b isolates were commonly associated to summer and their existence in surface water was corresponded to higher air temperatures and the existence of bacterial indicators of fecal contamination such as escherichia coli and enterococci(26). conversely, serotype 1/2a strains were more prevalent in water during fall and winter. it is difficult to deduce the effect of natural environment on the l. monocytogenes strains due to limited studies available; however, 1/2a strains may be typical inhabitants of natural environment while 4b strains may be from the cattle’s excreta into water during summer. thus, it somehow explained why listeriosis outbreaks are more prevalent in summer(7). in a short, l. monocytogenes was commonly isolated from dairy and poultry products. due to various factors like dietary habits, methods of processing food and health surveillance system, listeriosis was more prevalent in the us and europe (as shown in figure 1). it is important to keep in mind that the uncommon food sources can also be a source of transmission as shown in the recent outbreaks, thus with rapid detection allow effective interruption of the route of transmission. 4.3 asia unlike united states and europe where health surveillance system was efficient, data collection of listeriosis was not appropriately done in asia. perhaps, also because of different diet habits and prevalence of infectious illnesses, detailed investigations were focused on the commonly affected illnesses such as tuberculosis and dengue fever. in japan, an estimation of 83 cases occurred annually (equivalent to 0.65 per million individuals) and it was deduced that raw rte seafood products might have contributed to some of the cases, though the most probable number (mpn) of the l. monocytogenes detected was low(66). in china, the average recovery rate of l. monocytogenes was deduced to be 3.7% in all food categories, with raw meat as the leading source, in 13 a review on the characteristics, taxanomy... provinces. however, there was no surveillance data due to limited identification techniques and insufficient attention to l. monocytogenes contamination. the only record was about the veterinary and human outbreaks happened in yunan province in 1997(13). 4.4 antibiotic resistant l.monocytogenes statistical analysis showed that l. monocytogenes was responsible for 23150 illnesses (0.337 per 100,000 population incidence rate) with 5463 death globally in 2010. among all the cases, septicemia was the most common outcome, followed by central nervous system (cns) infection. however, data from only 52% of world population was available and also due to differences in health surveillance system, the incidence and disease burden could not be extrapolated confidently(67). over the years, the food sector has witnessed the occurrence of antimicrobial resistant bacteria such as salmonella, stapylococcus aureus, escherichia coli and l. monocytogenes(68;69;70;71). in 1988, the first antibiotic resistant l. monocytogenes was identified in france. since then, more and more strains from food sources are identified as antimicrobial resistant and human sporadic listeriosis cases(72;73;74). as a precaution measure and to control the increasing number of antibiotic resistant strains, the european union has banned the usage of antibiotic as animal feed additives as of january 2006(75). the indiscriminate use of antimicrobials in community and farms has led to increasing cases of antimicrobial resistant l. monocytogenes in the environments. a study reported l. monocytogenes strains from dairy farm exhibiting multidrug resistance towards phenotype towards cephalosporin c, streptomycin, and trimethoprim(76). in addition in 2008, a another study found that l. monocytogenes strains from a turkey processing plant were multidrug resistant namely to ceftriaxone, ciprofloxacin, and oxacillin(77). antimicrobial resistant l. monocytogenes strains has also been isolated from ready-to-eat foods. the strains exhibited antibiotic resistance towards ampicillin, gentamycin and methicillin(78). given the increasing number of antibiotic-resistant l. monocytogenes strains being isolated around the world, it is imperative that we gain a better understanding of the extent of antibiotic resistance in l. monocytogenes, the antibiotic resistance gene patterns of this pathogen, and the ability of this pathogen to acquire resistance from other bacterial species. in addition, the range of antibiotics to which l. monocytogenes has acquired resistance is broad. within this range of antibiotics are several that are traditionally used to treat listeriosis, such as penicillin and gentamicin. multi-resistant strains are not common, but evidence for emergence is available(76). overall, the use of antibiotic in the aquaculture and agriculture should be control and managed in order to reduce the emergence of antibiotic resistant strains of l. monocytogenes. conclusion listeriosis is a rare but serious illness that predominantly affects the high risk population. especially in recent years where there was increase in outbreaks and cases especially in the developed countries eg. the united states, finland and denmark, which mostly related to consumption of contaminated processed food. majority of the affected individuals are pregnant or immunocompromised, and develop severe illnesses which result in abortion, long-term neurological complications and figure 1. incidence rate and outbreak of listeriosis in different regions global. listerosis have effected around the world from usa to the asia counties letchumanan v et al. even death. the severity of this illness alarmed the community about the importance of surveillance and prevention of l. monocytogenes. besides the “zero tolerance” policy for rte foods in us, most of the countries do not take any essential actions to counteract this problem. it is yet unclear about the causes of the significant low prevalence of cases in the developing countries which might perhaps due to dietary habit or inefficient health surveillance system. to understand the listeriosis better and future therapeutic regimen development, it is crucial to have a detailed understanding about the pathogenesis of l. monocytogenes. prfa and the sigma factors are the main regulators of the expression of downstream virulence genes and also for the bacteria adaptation to the stress conditions. for mammalian cells invasion, inla and inlb play critical for adhesion and internalization. once within the cell, llo and plc aid in escaping from the phagosome, and subsequently together with actin assembly, allow intercellular spread. to treat the infection, the first-line treatment is penicillin with or without aminoglycosides, and tmp-smz is used in patients who are allergic to penicillin. in the current situation, antibiotics should be administrated wisely in aquaculture, agriculture and health care sectors to control the occurrence of antimicrobial resistance among pathogens. a non-antibiotic approach of utilizing bacteriophages in the aquaculture and agriculture fields may help to manage the increasing antimicrobial resistance problem. bacteriophages have great advantages such as having host specificity, environmental friendly, readily discovered and isolated from the environment, and cost effective compared to antibiotics(79). in addition, hactery farmers may adapt the method of switching antibiotics used in the aquatic field from time to time in order to allow withdrawal of antibiotic resistance profile in strains(80). in summary, antibiotic resistance is a public health worry that effects millions of healthcare treatment worldwide. it is very important to educate the public on this issue through awareness campaigns or electronic media to prevent the over use of antibiotics and ensure the effectiveness of clinical antibiotics for treatments. in line with the campaigns, the food and agriculture organization (fao) had campaigns worldwide to increase awareness and promote the use of antimicrobials among the public (90; 91). acknowledgement this work was supported by external industry grant (biotek 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(2018) discovery on antibiotic resistance patterns of vibrio parahaemolyticus in selangor reveals carbapenemase producing vibrio parahaemolyticus in marine and freshwater fish. front. microbiol. 9, 1-13. a review on the characteristics, taxanomy... pmmb 2021, 4, 1; a0000213. doi: 10.36877/pmmb.a0000213 http://journals.hh-publisher.com/index.php/pmmb review article stories from the east: covid-19 situation in india rebecca jane joseph 1, hooi-leng ser 1* article history 1novel bacteria and drug discovery research group (nbdd), microbes and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor, malaysia; rebecca.jane@monash.edu (rjj) *corresponding author: hooi-leng ser, ser.hooileng@monash.edu (h-ls) received: 8 april 2021; received in revised form: 3 june 2021; accepted: 8 june 2021; available online: 18 june 2021 abstract: the war against covid-19 is still ongoing since the first report in december 2019. on 1st may 2021, india has reported over 400,000 new covid-19 cases within 24 hours. the situation in the country continued to progress, which was associated with the reporting of the variant-of-concern and the variant-of-interest, including the b1.617 viruses. the government has taken public health measures, including campaigns to increase awareness about mask-wearing and physical distancing as well as lockdown plans to prevent further spread of the disease in the densely populated country. furthermore, the countrywide vaccination program has begun in early 2021, while the government of india continues to monitor the covid-19 situation in the country. the current review aims to provide a brief situation report in india before discussing management strategies taken and different phases of the vaccination program. keywords: sars-cov-2; covid-19; situation; india; variants 1. introduction since the ongoing covid-19 pandemic, which was first reported in china around december 2019, there have been 172,630,637 confirmed cases and 3,718,683 deaths worldwide on the 7th june 2021[1−7]. this has taken a significant toll both socially and economically throughout the world. currently, india is a densely populated country severely affected by covid-19 and experiencing one of the worst crises [8−10]. on 1st may 2021, there were over 400,000 new covid-19 cases for the first time within 24 hours in india. based on data retrieved from the ministry of health and family welfare (mohfw) on the 15th may 2021, there was a total of 3,673,802 active cases, with 20,432,898 discharged and 266,207 deaths in total[11]. while many countries (including australia, united kingdom, italy) have suspended traveling to and from india, the government of india has taken the approach to strictly enforce the test-track-treat protocol throughout the country, emphasizing the public to adhere to covid-appropriate behavior and to boost the vaccination drive[12,13]. several steps are taken using the test-track-treat protocol, such as increasing the number of testing using real time-pcr (rt-pcr) to more than 70%, quarantine new positive cases as https://en.wikipedia.org/wiki/wuhan pmmb 2021, 4, 1; a0000213 2 of 11 soon as possible, and tracing their contacts sooner. the containment zones are demarcated meticulously updated on the network page by each district collectors and by the states, and this information is regularly shared with the mohfw[14]. these measurements ensure close monitoring for any influenza-like illness (ili) or severe acute respiratory infections (sari) cases, house-to-house surveillance, and strict perimeter control. in addition, covid appropriate behavior such as wearing a face mask, physical distancing, and hand hygiene have been strictly enforced; failure to comply with the law will result in imposition of fines and administrative actions. 2. important events since the first covid-19 case reported in 2020 the first confirmed case on the 30th january 2020 was a patient who returned from wuhan to kerala[15,16]. until 3rd february 2020, only three confirmed cases were reported in kerala who were all stable and isolated in the hospital. the government of india ensured those who have returned from china since 15th january 2020 undergo a mandatory quarantine for 14 days and issued travel advisories requesting the public to refrain from traveling to the specified country. at the same time, the ministry of tourism has also taken initiatives to coordinate with the hotels association in india to encourage the public to report themselves if they have visited any tourists of religious areas. the world health organization (who) declared the covid-19 outbreak as a pandemic on 11th march 2020, and the government of india announced this as a notified disaster under the disaster management act 2005[17]. travelers from china, iran, italy, france, germany, spain and the republic of korea after 15th february 2020 were mandated to quarantine for at least 14 days. there were 84 cases of covid-19 reported in india, and two deaths were reported on the 14th march 2020. cases were from 13 states in india, mainly originating in kerala (19 cases) followed by maharashtra (14 cases), haryana (14 cases), uttar pradesh (12 cases), delhi (7 cases), karnataka (6 cases), ladakh (3 cases), rajasthan (3 cases), jammu & kashmir (2 cases), andhra pradesh (1 case), punjab (1 case), telangana (1 case) and tamil nadu (1 case). a 76 years old male from karnataka and 68 years old female from delhi with underlying comorbidities passed away. due to the rise in cases, “janata curfew” for 14 hours from 7.00 a.m. to 9.00 p.m. was announced by the prime minister of india, narendra modi on 22nd march 2020, pleading to the public to stay at home and to observe social distancing[18]. mohfw had also taken precautionary steps to ensure sufficient isolation wards, personal protective equipment (ppes), medicines, testing kits and workers. as thousands of cases continue to spike per day, the prime minister of india issued a lockdown throughout the country starting on the 25th march 2020. however, the lockdown was further extended thrice till 31st may 2020[19−21]. international flights were suspended from 25th march till 14th april 2020, and any interstate travel was restricted[22]. during the lockdown period, the government of india has taken the opportunity to make advancements in the healthcare system and infrastructures. there were 930 dedicated covid hospitals with 158,747 isolation beds, 20,355 icu beds, 69,076 oxygen supported beds, and 2,362 dedicated covid health centres with 132,593 isolation beds; 10,903 icu beds and 45,562 oxygen supported beds operationalized as reported by mofhw on 27th may 2020[23,24]. pmmb 2021, 4, 1; a0000213 3 of 11 furthermore, during the global vaccine summit 2020, the prime minister of india emphasized the ability of india to produce quality vaccines and medicines with minimal costs, and 15 million usd was pledged to the vaccine alliance, gavi[25]. the indian council of medical research and state authorities also ensured that no individual was restricted from being tested. although rt-pcr is the gold standard to diagnose covid-19, the government of india has decided to increase more rapid antigen tests because it is safe, simple, and provides quicker results. there were also 788 in the government sector and 317 private labs, making it up to a total of 1,105 labs[26]. along with this, the government of india launched the “aarogya setu” mobile application that uses bluetooth technology to aid in surveillance and contact tracing[27]. the application is available in 11 languages and can be installed into smartphones, allowing users to determine the risk of catching infection based on their interaction with others[28]. besides that, a video consultation program called e-icu was started by new delhi-based all india institute of medical sciences (aiims) across the country on the 8th july to allow front liners to share their experiences in treating covid-19 patients and have discussions with other experts in this field using this platform[29]. ministry of home affairs issued guidelines on the 30th september on re-opening some states, but the lockdown continued in containment zones until the 30th november 2020[30]. on the 8th october 2020, ‘jan andolan’ was launched to closely observe these three appropriate behaviors: social distancing of six feet, wearing a mask and washing hands often[31]. in november 2020, daily new cases of covid-19 and the number of deaths per million started to decline and was much lesser compared to some parts of the world. ever since mid-september 2020, the lowest case number of 9,110 was reported on 9th february 2021. however, a surge of cases returned in some states with 14,264 cases on the 21st february 2021 and continues to spike within two weeks with an almost 43% increase of covid-19 cases and 37% new deaths on a weekly basis. the national capital of india, new delhi was heavily affected during the second wave and has been under lockdown since the 19th april 2021[32]. based on the situation report of 28th april 2021, india has the highest daily cases[33]. at that point in time, india was ranked fourth highest in terms of the number of deaths with a case fatality ratio of 1.12% in the world. the leading state with more than four million cases is maharashtra followed by a million over cases in kerala, karnataka, uttar pradesh, and tamil nadu[34]. on 1st may 2021, india has become the first country to record over 400,000 new covid-19 cases within 24 hours since the pandemic. there were 401,993 new infections with a total of 19.1 million cases and 3,523 deaths, adding up the total number of deaths to 211,853[35,36]. two weeks later, these figures continue to rise, resulting in a total of 3,673,802 active cases, with 20,432,898 discharged and 266,207 deaths[11]. as of 1st june 2021, the data retrieved from who coronavirus (covid-19) dashboard (https://covid19.who.int/) recorded a total of 28,208,619 confirmed cases and 331,899 total deaths in india. https://covid19.who.int/ pmmb 2021, 4, 1; a0000213 4 of 11 figure 1. illustration of daily confirmed covid-19 cases, cumulative covid-19 cases, cumulative deaths due to covid-19, and a brief timeline of the events in india since the first case reported in the country. 3. combating covid-19: medications and vaccination program in india along with directorate general of health services, mohfw, and indian council of medical research's icmr-covid-19 national task force, aiims has recently published an updated comprehensive guideline for the management of covid-19 adult patients, removing recommendations on hydroxychloroquine and convalescent plasma use[37−39]. based on the guidelines, the use of medications like remdesivir (under emergency use authorisation) and tocillizumab (off-label use) should be considered carefully and not to be used in mild covid-19 patients. additionally, the exportation of remdesivir injection has been prohibited since the 11th april 2021. remdesivir manufacturers also had to ramp up their production to 7,400,000 of vials every month. twenty more manufacturing sites have been approved to meet the demands of hospitalized patients on oxygen support in 19 states[40]. as for the case of steroids, the guideline emphasized that they should be used at “right time, right dose and for the right duration”, only in hospitalized moderately severe and critically ill covid-19 patients. on the 9th january 2021, the prime minister of india has shared on his social media account and issued a press release that 16th january represents a “landmark step” for the country in the fight of covid-19, as the national-wide vaccination drive begins, intending to vaccinate 300 million people by july 2021[41]. phase i of the national covid-19 vaccination strategy targeted healthcare and frontline workers with more than 60% vaccinated within a month[42]. the registrations and booking for vaccination appointment for phase 2 using the cowin portal or via the “aarogya setu” mobile application started on 1st march 2021, for those 60 years old and above as well as those aged between 45−59 with comorbidities that accounted for more than 80% of mortality in the country. subsequently, the government announced the “liberalised and accelerated phase 3 strategy of covid-19 vaccination” to include individuals above 18 years old with registration opening from 28th april 2021 and pmmb 2021, 4, 1; a0000213 5 of 11 vaccination begun from the 1st may 2021[43]. vaccination is provided free of charge at the government vaccination centres[43−49]. at the time of writing, the national regulator (drugs controller general of india) has granted emergency use authorisation (eua) of the covishield® (astrazeneca's vaccine), manufactured by serum institute of india (sii) with an efficacy of 62−90%, and covaxin manufactured by icmr in collaboration with bharat biotech international limited (bbil). covaxin phase 1 and 2 clinical trials conducted around july till october 2020 on 755 participants showed promising results and safety profiles. the largest clinical trial conducted on 25,800 participants also showed 81% efficacy against the sars-cov-2 virus in early march. the drugs controller of india (dcgi) has already approved covaxin for the restricted use in emergency situation. russia's sputnik v, the third vaccine, was approved in india in mid-april. within merely 26 days, india becomes the fastest country to reach a milestone of vaccinating 7,000,000 citizens[50]. on top of that, the union minister for health and family welfare, dr. harsh vardhan has also reassured the safety of both the vaccines and efficacy against current strains, stating that the percentage of severe adverse events following immunisation is 0.0002% of the total number of beneficiaries vaccinated[51]. as of 1st may 2021, 156,816,031 vaccines have been administered, and 28,644,878 have already received their second dose[52]. 4. emergence of variant-of-concern (voc) and variant-of-interest (voi): b.1.617 viruses the genomic changes sars-cov-2 through whole-genome sequencing (wgs) is continuously being monitored by the indian sars-cov-2 genomics consortium (insacog) since december 2020. out of 13,614 wgs samples processed in a network of 10 laboratories, 1,189 samples tested positive for variants of concern which are 1,109 uk variants; 79 variants which were firstly described in south africa, and 1 of them was the variant firstly described in brazil as of 15th april 2021[53]. for the past few weeks, the steep rise of cases across india has spread to about 40 nations, including singapore, united kingdom, and fiji was associated with the b.1.617 variant[54,55]. the b.1.617 viruses are categories into three lineages: b.1.617.1, b.1.617.2 and b.1.617.3[56]. on the 1st june 2021, the who released a public statement to facilitate communication regarding the naming of voc and voi to avoid country (or area) stigma[57]. in the same report, they have revised the status of voc and voi, labeling b.1.617.2 or variant delta as voc and b.1.617.1 as variant kappa as voi. b.1.617.3 is no longer listed as voc or voi due to few reports of the variants as pointed out by the who. while some studies are investigating the spread of different sars-cov-2 variants around the world, there are ongoing discussion revolving the efficacy of vaccines[58−64]. several teams believed that the double mutation results in a more virulent variant. till then, more studies are necessary to fully understand how these genomic changes contribute to the virulence and “behaviour” of these variants, particularly whether these changes would affect the efficacy of vaccines[65−68]. 5. conclusions the constant updates on covid-19 situations from official authorities have allowed citizens to access the latest information quickly. the statistics on vaccination in india pmmb 2021, 4, 1; a0000213 6 of 11 continue to rise, enabling the community to achieve immunity against covid-19 in due time. the next hurdle for universal control of the pandemic would involve successful immunization programs globally[69]. some countries are struck with vaccine shortages as well as economic and logistic issues[32,69−72]. for india, several organizations and governments have started delivering critical lifesaving supplies and medicines to support the country’s healthcare system[73−75]. the war against covid-19 is not over yet and has taught the world not to take this pandemic lightly. previous collaborative efforts between countries have yielded success in developing vaccines following the availability of genome sequences of sars-cov-2 viruses. moving forward, everyone needs to join hands to break the transmission chain and curb the spread of this notorious coronavirus. author contributions: r.j.j performed the literature search, critical data analysis as well as manuscript writing. h.-l.s. proofread the review writing and conceptualise this review writing project. funding: the seed funding funded this work from microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, (vote number: mbrs/jcsmhs/02/2020) and jeffrey cheah school of medicine and health sciences (jcsmhs) strategic grant 2021 (grant code: stg-000051). conflicts of interest: the authors declare no conflict of interest. references 1. world health organisation. who coronavirus (covid-19) dashboard. 2021 [accessed 15 may 2021; 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a0000246. doi: 10.36877/pmmb.a0000246 http://journals.hh-publisher.com/index.php/pmmb review article covid-19: an updated situation from singapore ke-yan loo1, angel yun-kuan thye1, lydia ngiik-shiew law2, jodi woan-fei law1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; ke.loo@monash.edu (k-yl); angelthye.yunkuan@monash.edu (ay-kt) 2monash credentialed pharmacy clinical educator, faculty of pharmacy and pharmaceutical sciences, monash university, 381 royal parade, parkville 3052, vic, australia; lydia19595@gmail.com (ln-sl) *corresponding author: jodi woan-fei law; 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; jodi.law1@monash.edu (jw-fl) received: 30 september 2021; received in revised form: 30 october 2021; accepted: 1 november 2021; available online: 9 november 2021 abstract: since the first reports of covid-19 in 2019, the viral respiratory disease has spread across nations, sending the world into a global pandemic. the pandemic has heavily impacted the public health of the global community. over 237 million confirmed cases have been reported, and more than 4.8 million lives have been lost due to the novel coronavirus. in singapore, the government quickly took action in the early stages of the pandemic to limit the spread of the virus to protect the local communities from the disease. singapore has been able to keep their confirmed covid-19 cases and deaths at low numbers by implementing movement restrictions, raising public awareness, mask mandates, social distancing, providing free vaccinations for the public, and utilizing advancements in technology for contact tracing. the public has also upheld their social responsibility in cooperating with the singaporean government to control the disease spread. covid-19 is now moving into an endemic phase in singapore as the vaccination rates are at an all-time high resulting in lower death rates, and the confirmed cases are primarily mild to asymptomatic. singapore has set a precedent for how pandemics can be handled in the future to minimize mortality rates and protect public health. keywords: covid-19; sars-cov-2; coronavirus; endemic; singapore 1. introduction two years have passed since discovering the novel coronavirus, sars-cov-2, which causes covid-19, a highly contagious respiratory disease that sent the world into a global pandemic. the virus has since spread across countries across the globe, upending the livelihoods of many worldwide[1–4]. to date, more than 237 million confirmed cases have been documented globally, and the virus has claimed more than 4.8 million lives[5]. covid19 spread at an alarming rate from china to other countries within a short period. the severity of the disease led the world health organization to declare covid-19 as a global pandemic mailto:lydia19595@gmail.com mailto:jodi.law1@monash.edu pmmb 2021, 4, 1; a0000246 2 of 8 in march 2020[6]. the severity and fatality of the viral illness prompted the development of vaccines at lightning speed, and finally, in december 2020, vaccines effective in controlling covid-19 were available for use. at the time of writing, over 6.3 billion individuals worldwide have received at least one dose of the covid-19 vaccines[5]. in the early stages of the pandemic, there were no cures or vaccines that could effectively treat and prevent the transmission of the coronavirus across national borders[7]. thus, leaders of countries such as singapore, china, japan, south korea, and taiwan acted quickly and implemented safety measures to prevent the rampant spread of the disease. these countries closed their borders, upscaled the production of face masks and covid-19 test kits, increased illness testing, and enforced mask mandates to protect their civilians[8]. singapore, a sunny tropical country in southeast asia with a population of 5.7 million, has often been cited as exemplary for other countries in handling the covid-19 pandemic[9,10]. singapore has been able to control the spread of the virus within the country, with the total number of confirmed cases sitting at 187,851 cases, while 364 deaths have been reported as of october 28, 2021. these numbers are significantly lower when compared to other first-world countries[11]. the singaporean government quickly developed a successful framework to manage disease outbreaks earlier on in the pandemic. the government enforced stringent measures necessary to protect the people from the virus by implementing a national lockdown, extensive testing, contact tracing, mask mandates, social distancing, and raising awareness for covid-19 among the public[12]. 2. covid-19 situation in singapore the first incidence of covid-19 in singapore was an imported case involving a 66year-old chinese national on january 23, 2020[13]. the public was still at ease until a local transmission was reported in early february, which set the whole nation on high alert. singapore then raised its national assessment level, the disease outbreak response system condition (dorscon) level from “yellow” to “orange” to alert the public of a possible local pandemic. during this time, the daily number of confirmed cases in singapore was still less than 10 cases a day. in singapore, technological advancements were greatly utilized to handle the pandemic. the government technology of singapore (govtech) had already developed a mobile phone-based tracing application known as tracetogether once confirmed cases were reported in the country. tracetogether was launched in march 2020 to track the interactions between those diagnosed with the coronavirus and their close contacts. those who may have contracted the virus unknowingly from the community could be traced and put under quarantine to prevent further dissemination of the virus. an “exposure notification” would be provided to affected individuals to protect public health. moreover, the source code for tracetogether is open, so any country that wishes to develop its contact tracing app quickly could do so based on tracetogether’s development[14]. however, a surge in cases was reported in march due to singaporeans returning from abroad, followed by a massive outbreak in the foreign workers’ dormitories (figure 1). the cases increased from 102 cases a day at the end of february to more than 1000 cases a day in early april. the situation was further exacerbated by the rapidly increasing number of new pmmb 2021, 4, 1; a0000246 3 of 8 and unlinked cases within the community. thus, a “circuit breaker” was implemented with a stringent set of lockdown measures from april 7 to june 1 to better control the spread of the infection. schools and non-essential services were closed during the circuit breaker, and visitations or gatherings with family members or friends who did not reside together were prohibited. furthermore, the government also introduced safeentry, a national free-to-use cloudbased visitor registration system that logs individuals’ visits to businesses or venues providing essential services. before entering the premises, individuals are required to provide personal information such as their name, national registration identity card (nric) number, and mobile phone number. they could do so by scanning the safeentry quick response (qr) code, commonly displayed at the entrance of businesses or premises. they are also required to log the time when they leave the premises within the system. over 40,000 locations in the country have utilized safeentry for contact tracing purposes, enabling the officials to quickly track down possible outbreak clusters and close contacts of infected persons[15]. the circuit breaker successfully reduced the daily number of confirmed cases, resulting in the resumption of economic and community activities in phases with less strict rules. in may 2021, breathalyzers used to detect covid-19 which gives accurate results within 1-2 minutes were provisionally approved for use in singapore[16]. the breathalyzers were developed locally by the national university of singapore’s spin-off start-up breathonix (brefence go covid-19 breath test system) and silver factory technology (traciex beathalyser). every exhalation contains thousands of volatile organic compounds (voc), which can indicate the presence of different diseases or infections in the host. when an individual breathes into the breathalyzer, the exhaled air is collected and analyzed by a portable reader, which results within two minutes[17,18]. brefence go utilizes mass spectrometry while raman spectrometry is used in traciex to analyze the voc. moreover, brefence go has a sensitivity of 85.3% and a specificity of 97%, while traciex has a sensitivity of 95% with a specificity of 97.8% in identifying the voc in individuals with covid-19[16]. the breathalyzers are cost-effective screening options for covid-19 as they are relatively inexpensive (sgd5-sgd20 per device) and can deliver results at high specificity and sensitivity[19]. after june 1, singapore was in phase 1 of reopening, whereby some activities beyond essential services were allowed to reopen. phase 2 of reopening commenced shortly after that on june 17, and the public was allowed to dine-in at restaurants with a 5-person limit, and retail operations were able to resume. six months later, phase 3 of reopening commenced with the limitation of public gatherings and home visitations increased from 5 to 8 people. free vaccinations were also introduced to all singaporeans and long-term residents. the priority would be given to healthcare and frontline workers, followed by the elderly aged 70 and above[12]. pmmb 2021, 4, 1; a0000246 4 of 8 figure 1. total number of confirmed cases and deaths in singapore. in 2021, travelers or singaporeans from abroad were required to take a polymerase chain reaction (pcr) test to detect the covid-19 infection upon arrival in singapore as an effort to prevent the spread of the virus from imported cases. an extra 7-day self-isolation is also mandatory for those who have traveled from the united kingdom and south africa in addition to their 14-day stay home notice (shn) at dedicated facilities[20]. the number of daily confirmed cases was decreasing. henceforth by late march, the singapore ministry of health (moh) announced that from april 5, 2021, onwards, more employees would be allowed to return to their workplaces to boost the central business district and the industrial park sector[21]. mass vaccination had begun at this time, and approximately 800,000 individuals had received at least one dose of the vaccine, and the vaccination program was set to roll out to younger age groups[21]. currently, the pfizer-biontech (comirnaty) and moderna covid-19 vaccines are authorized for use by the health sciences authority in singapore under the pandemic special access route (psar). both of these vaccines are mrna vaccines, and they have been found to be effective against various variants of the sars-cov-2 coronavirus[22-24]. these vaccines work by delivering mrna to the host cells to make copies of the spike proteins of sars-cov-2. the host immune system recognizes these spike proteins and elicits an immune response, ultimately activating memory b cells for adaptive immunity. in the event of future exposure to the actual virus, the host immune system will be able to recognize the virus, and immune responses will be triggered to fight against it[24]. in may 2021, there was an increase in reports of dangerous variants of the coronavirus worldwide[25–27] and the detection of several clusters of infection within the community in singapore. these situations prompted the singaporean government to revert to phase 2 (heightened alert) to prevent the spread of these variants and avoid enforcing another circuit breaker that could be detrimental to the economy and negatively affect the citizens[28]. as vaccination rates continued to rise to achieve herd immunity in singapore, the pfizer-biontech covid-19 vaccine was authorized for use in those aged 12 to 15 on may pmmb 2021, 4, 1; a0000246 5 of 8 18[29]. once herd immunity is achieved, working from home and home-based learning would cease. at the same time, contact tracing and border restrictions will be retained to hinder the dissemination of dangerous variants of the virus into the country[30]. in june 2021, moh announced the movement into phase 3 in two steps on june 14, with the increase in the limit for social gathering and distinct visitors allowed per household and the possibility of more restrictions lifted on june 21[31]. nevertheless, with the cases spike again shortly after the announcement, singapore was again in phase 2 from july 22 to august 18[32]. the singaporean government then gave out diy antigen rapid test (art) kits to every household in singapore and pre-school staff and students to encourage more covid-19 testing within the community. these initiatives gave the public the social responsibility in making singapore a covid-19 resilient nation[33]. throughout the fight against the pandemic, the singaporean government has been consistently transparent in updating the public on the covid-19 situation in the country through the moh’s website. the latest restrictions and safety measures are constantly posted to remind the public to stay safe and protect themselves during these trying times[34]. social media platforms such as whatsapp, twitter, telegram, and facebook were used to inform the public on the latest updates of covid-19 in singapore and circulate information on reducing the risk of infections[13]. at the time of writing, the daily number of confirmed cases in singapore has steadily increased to over 3500 cases a day. interestingly enough, 84% of the residents in singapore have already been fully vaccinated against covid-19[11]. the driving force for the increase in cases is likely due to the government’s four-step strategy announced in august, where the focus would be put on higher vaccinations and severe cases of infections. the government aims to see covid-19 as endemic and for the people to carry on with their lives as the virus remains in the community[35]. rules and restrictions have begun to loosen for those fully vaccinated, allowing the residents to move forward to a new normal in their lives. in addition, the presence of the more infectious delta variant of the coronavirus in singapore also contributes to the spike in confirmed cases[36]. currently, over 98% of the reported cases had mild to no symptoms, while only 1.1% of cases require oxygen supplementation, and 0.1% of cases require intensive care[34]. the government is not overly fixated on the increasing daily number of confirmed cases due to its mild nature. they remain optimistic about the situation in singapore as serious infections are rising at a slower rate[36]. although high vaccination rates can minimize the effects of the disease, non-pharmacological interventions such as mask-wearing and social distancing should not be abandoned as the population remains vulnerable to the airborne virus, especially in the presence of dangerous variants[37]. testing for the virus should also be continuously upheld within the community to detect any infections earlier on to prevent outbreaks and protect individuals who are more susceptible to the illness, such as those who are ineligible for the vaccines. these efforts could also prevent the healthcare system from becoming overwhelmed and ensure that covid-19 and non-covid-19 patients would still receive timely care from healthcare professionals. pmmb 2021, 4, 1; a0000246 6 of 8 3. conclusion the cooperation between the local government and the public, which upholds their social responsibility, has successfully controlled the covid-19 pandemic in singapore. high vaccination rates resulting in significantly lower covid-related death rates, coupled with the majority of confirmed cases being mild or asymptomatic in the country, the disease is moving towards an endemic phase in singapore. the governance, transparency, utilization of technological advancements, and enforcement of social responsibility in tackling the pandemic in singapore set a good precedent for other countries that aim to achieve a new normal with covid-19. author contributions: k-yl performed the literature search, critical data analysis, and manuscript writing. ay-kt and ln-sl performed proofreading and editing. jw-fl conceptualized this review writing project. funding: no external funding was provided for this research. acknowledgments: the authors would like to acknowledge professor shajahan yasin, head of school, jeffrey cheah school of medicine and health sciences, monash university malaysia conflicts of interest: the authors declare no conflict of interest. references 1. ser h-l, letchumanan v, law jw-f, et al. pmmb covid-19 bulletin: spain (18th april 2020). prog microbes mol bio 2020; 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a0000249. doi: a10.36877/pmmb.0000249 http://journals.hh-publisher.com/index.php/pmmb original research article circular rna-ephb4 as a potential biotarget in colorectal cancer: a preliminary analysis siti nurmi nasir1, muhiddin ishak1, ismail sagap2, isa rose3, nurul syakima ab mutalib1, rahman jamal1, nadiah abu1* article history 1ukm medical molecular biology institute (umbi), universiti kebangsaan malaysia, 56000 kuala lumpur, malaysia; ctnurminasir@ppukm.ukm.edu.my (snn); muhiddin@ppukm.ukm.edu.my (mi); syakima@ppukm.ukm.edu.my (ns-am); rahmanj@ppukm.ukm.edu.my (rj) 2department of surgery, faculty of medicine, universiti kebangsaan malaysia, 56000 kuala lumpur, malaysia; ismalsagap@ppukm.ukm.edu.my (is) 3department of pathology, faculty of medicine, universiti kebangsaan malaysia; isa@ppukm.ukm.edu.my (ir) *correspondence: nadiah abu; ukm medical molecular biology institute (umbi), universiti kebangsaan malaysia, 56000 kuala lumpur, malaysia; nadiah.abu@ppukm.ukm.edu.my (na) received: 3 november 2021; received in revised form: 4 december 2021; accepted: 3 january 2021; available online: 20 january 2022 abstract: colorectal cancer (crc) is among the top causes of cancer-related deaths. with the advent of new biomedical technologies, new therapeutic targets are being actively discovered. circular rnas (circrnas) are characterized by the absence of covalently closed-loop structures at the 3′ and 5′ ends. recent studies have shown that circrnas are abnormally expressed in tumor tissues. certain circrnas have been reported to have potential as cancer biomarkers. among all the circrnas previously discovered, circephb4 seemed promising. therefore, this study is aimed to determine the function of circephb4 in crc. by utilizing in-silico prediction tools, qpcr and cell-based assays, we were able to provide preliminary insights into the role of circephb4. the expression of both circephb4 and linear ephb4 were upregulated in our malaysian crc tissues and commercial cell lines as compared to the non-cancerous counterparts. reduction of both circephb4 and ephb4 had effects on wound healing, drug response and clonogenic abilities of sw-480 and hct116 cells. our initial findings suggest that circephb4 may be involved in the regulation of crc though further in-depth studies are needed. keywords: colorectal cancer; circular rna; ephb4 1. introduction colorectal cancer (crc) is ranked worldwide as the third most prevalent cancer[1]. colorectal cancer incidence and mortality rates are still on the rise in most countries including highly developed nations[1]. in malaysia, crc is the second most diagnosed cancer in males pmmb 2022, 5, 1; a0000249 2 of 14 and third in females[2]. most patients are diagnosed at a late stage; therefore, it may not be effective in eradicating cancer through the treatment offered. there is a need to find biomarkers or targeted therapies that can identify crc earlier, understand disease progression and improve the clinical outcome. multiple potential molecular markers include proteins, mrnas, micro rnas (mirnas), and long noncoding rnas (lncrnas) have been reported. apart from the standard biomarkers, a newly discovered type of non-coding rna, the circular rnas have been shown to have advantageous properties as biomarkers compared to the other types of rna[3]. circular rnas (circrnas) are a class of closed rna loops that lack the termination 5’ cap and 3’ polyadenylated tail structure of linear rnas[4,5]. apart from that, circrnas are known to be more stable and are have longer half-lives[6,7], thus making them ideal targets for biomarker identification. functionally, circrnas have been reported to be involved in multiple processes including chemoresistance[8] and migration processes in multiple cancers such as crc[9,10], prostate cancer[11,12], hepatocellular carcinoma[13] and others. on top of that, dysregulation of circrnas have been widely reported in crc[14]. erythropoietin-producing hepatocellular (eph) type-b receptor 4 (ephb4) is a member the largest family of receptor tyrosine kinases (rtks)[10,15] that consists of 14 types of receptors in human[16]. ephb4 is reported to be involved in a number of cancer-promoting effects including angiogenesis, invasion, and metastasis[17]. from previous studies, it has been suggested that high ephb4 expression enhances migratory abilities in crc cells, leading to increase rate of metastasis[18]. ephb4 was also be found in a circular rna form [19]. a recent study by tan et al., has shown that circephb4 could have functional effects in hepatocellular carcinoma[19]. previously, our group has identified several potential circrnas in chemoresistant hct-116 cells[9], as well as in the corresponding extracellular vesicles[20], nevertheless, we did not perform any assessment on circephb4. therefore, in this study, we aimed at determining the level of circephb4 in our malaysian crc patients as well as perform preliminary assays to assess its potential as a biotarget. 2. materials and methods 2.1. cell culture six human colorectal cancer cell lines (sw480, ccd-112-con, ccd-18-con, colo-205, hct-116, and ht-29) were purchased from american type culture collection (atcc). these cells were maintained in rpmi-1640 medium (invitrogen, usa) except for ccd-112-con and ccd-18-con, which were cultured in dulbecco's modified eagle medium, dmem (invitrogen, usa). all media were supplemented with 10% fetal bovine pmmb 2022, 5, 1; a0000249 3 of 14 serum (gibco, usa). the cultures were grown in an incubator at 37°c in a 5% co2containing humidified atmosphere. 2.2. collection of the tissue specimens all the crc tissues and normal colonic tissues were obtained from the ukm medical molecular biology institute (umbi) biobank, this study was conducted following the 1964 helsinki declaration and its later amendments. prior to sample collection and storage, written informed consent was obtained from all patients. all of the tumor tissue was diagnosed histopathologically by an experienced pathologist. the study was conducted using only tumor tissues containing >80% cancerous cells and normal tissues (non-tumor tissue) containing <20% necrotic cells. tumor and non-tumor tissue samples were obtained from crc patients during surgical resection of the tumor. a subset of tissues from these patients was previously used to detect a specific long non-coding rna in a different study[21]. demographic data of the patients is shown in table 1. table 1. demographic information of the malaysian crc specimens used in this study. crc specimens (%) overall 26 (100) age <70 17 (65.4) >70 9 (34.6) gender male 15 (57.7) female 11 (42.3) race malay 16 (61.5) chinese 10 (38.5) stage dukes a 3 (11.5) dukes b 11 (42.3) dukes c 12 (46.2) 2.3. total rna isolation and quantitative real-time rt-pcr (qrt-pcr) total rna from cells was extracted using the rneasy mini kit (qiagen, germany) as described. while, for total rna from crc tissue was isolated allprep dna/rna/mirna isolation kit (qiagen, germany) according to the manufacturer’s protocol. the amount and purity of the rna were measured with nanodrop 1000 (thermo fisher scientific, usa). the mirna-specific primer sequences and circular rna levels of pmmb 2022, 5, 1; a0000249 4 of 14 ephb4 were designed independently. the primer sequence for mrna ephb4 was designed using the primerbank database[22] other primer sequences for circephb4 were designed using the web tool circinteractome[23]. the complementary dna (cdna) was synthesized using the rt2 first strand kit (qiagen, germany) based on the manufacturer’s instructions. the expression level of circephb4 and ephb4 in primary crc tissue and secondary crc cell lines were measured using the quantinova qpcr mastermix (qiagen, germany) on a cfx96 touch™ real-time pcr detection system (biorad, usa). the qrt-pcr results were analyzed and the relative rna expression of ct (threshold cycle) value, which was then converted to fold changes. fold change of gene was calculated by the equation 2−δδct. 2.4. prediction of the circrna-mirna-mrna expression network and function analysis the corresponding mirna response elements (mres) were predicted using the webbased interface, circinteractome[23]. target mirnas were obtained from the starbase database, using default settings[24]. the expression of the mirnas was obtained from the mir-tvdatabase[25]. the target genes targeting the selected mirna were obtained from the funrich tool[26] where the further biological pathway was determined. furthermore, the enrichment network (ora) was also determined using the webgestalt platform[27] and the kegg database[28]. 2.5. transfection of colorectal cancer cell two types of sirnas (si-linear ephb4 and si-circephb4) were designed and synthesized by dharmacon (thermo fisher, usa). the cells were seeded in 24-well plates at a density of 5×104/well and cultured to a confluency of 80-90%. cells were transfected with control or gene-specific sirnas using jetprime® (polyplus-transfection sa, usa) as described by the manufacturer. 2.6. wound healing assay cells were transiently transfected with the sirnas and harvested at 48 hours after transfection. the ibidi culture-inserts (ibidi gmbh, germany) were placed on a cell culture surface and cells were seeded at 5×105 cells/ml in each of the ibidi culture-insert well (70 µl into each well) according to the manufacturer protocol. the dish was viewed under the inverted microscope (nikon, japan) and the image was captured. the observation process was started by taking images several times throughout the following hours (0 h, 24 h, 48 h and 72 h). the percentage of wound healing (%) was measured using the t scratch software[29]. pmmb 2022, 5, 1; a0000249 5 of 14 2.7. clonogenic analysis after transfection, 5000 cells were plated in a 6-well plate with complete media. the cells were grown in ~10 days until cells in control plates have formed colonies with substantially good size (50 cells per colony is the minimum for scoring) with media replacement every 3 days. after that, the media was removed, and cells were washed twice with pbs. the colonies were fixed with acetic acid/methanol 1:7 (vol/vol) for 10 min, dried and stained with 0.5% crystal violet solution for 2 hours at room temperature. images of the stained plates were captured, and each treatment was performed in triplicates. 2.8. statistical analysis statistical analysis was performed using graphpad prism v7 (graphpad software, usa). the quantitative data were presented as the mean ± standard deviations (sd). differences were considered statistically significant at values of p <0.05. 3. results 3.1. both circephb4 and linear ephb4 were up regulated in crc tumor tissue and crc cell lines we examined the expression of both circephb4 and linear ephb4 in our cohort of crc tissues and cell lines. as shown in figure 1a, the average expression of both circephb4 and linear ephb4 was upregulated in crc tissues as compared to non-cancerous colonic tissues. nevertheless, within the crc group, linear ephb4 had a significantly higher expression than circephb4 (p=0.03). interestingly, for circephb4, from five of our normal colon samples, only two samples had detectable expression (cq<38). similarly, in the cell lines, the expression of both circephb4 and linear ephb4 was upregulated in crc cell lines (sw480, hct116 and colo205) as compared to the non-cancerous colon cell lines (ccd112-con and ccd-18-con) as displayed in figure 1b. pmmb 2022, 5, 1; a0000249 6 of 14 figure 1. relative expression of both linear ephb4 and circephb4 in a) our cohort of crc tissues and normal colonic tissues, and b) a panel of commercially available crc cell lines and normal colon cells. gene ontology and pathway enrichment analyses of the predicted target genes for circephb4. 3.2 circrna-mirna-mrna co-expression network for circephb4 since circrnas have been reported to sponge to mirnas[3], we decided to identify potential target mirnas for circephb4. the prediction of mirna target sites was conducted using the web tool starbase[24] using the default parameters. there were 7 mirnas predicted to bind to circephb4, as shown in table 2. due to our limited number of clinical samples, we were unable to verify the expression of these mirnas. nevertheless, we used the mir-tv database[25], from the tcga cohort to get an overview of the expression of selected mirnas. out of the 7 mirnas, only 4 were expressed in the coad dataset, as shown in figure 2. from the four mirnas, three mirnas had a lower expression in crc tissues as compared to the normal tissues, which are hsa-mir-193a-5p, hsa-mir-3605-5p and hsa-mir-193b-3p. afterward, the functional analysis for the target genes was further conducted using the funrich web tool[26]. we performed target prediction to three of the identified mirnas. the funrich tool identified 252 possible target genes; however, no significant biological pathways identified using this set of genes, as shown in figure 3a. when the same set of genes were subjected to an enrichment network (ora) using the webgestalt[27] platform, some of the most statistically enriched pathways include micrornas in cancer and the glioma pathway as shown in figure 3b. figure 3c shows the distribution of the target genes based on the gene ontologymolecular function, biological process and cellular components. pmmb 2022, 5, 1; a0000249 7 of 14 table 2. predicted target mirnas to hsa_circ_001730 (circephb4) based on the miranda database. the table was obtained from the starbase database[24]. mirnaid mirnaname geneid chromosome clipexpn um rbp merclass mirseq mimat001 9761 hsa-mir4677-3p hsa_cir c_0001 730 chr7 1 ago1-4 8mer ucaucaagaa accagagu gucu mimat000 3245 hsa-mir580-3p hsa_cir c_0001 730 chr7 1 ago1-4 7mer-m8 ggauuacuaag uaguaaga guu mimat000 0459 hsa-mir193a-3p hsa_cir c_0001 730 chr7 2 ago1-4 7mer-m8 ugacccugaa acauccgg ucaa mimat000 2819 hsa-mir193b-3p hsa_cir c_0001 730 chr7 2 ago1-4 7mer-m8 ucgcccuga a---acucccggucaa mimat001 7982 hsa-mir3605-3p hsa_cir c_0001 730 chr7 1 ago1-4 7mer-m8 gaucuccugu ccauugug ccucc mimat000 4614 hsa-mir193a-5p hsa_cir c_0001 730 chr7 1 ago1-4 7mer-m8 aguagagcg ggcguuuc ugggu mimat000 4909 hsa-mir450b-5p hsa_cir c_0001 730 chr7 1 ago2 8mer auaaguccu uguauaac guuuu figure 2. expression of 4 mirnas that were predicted to bind to circephb4. data obtained using the mirtv[25] database utilizing the tcga (coad) dataset. pmmb 2022, 5, 1; a0000249 8 of 14 figure 3. a) biological pathways that were predicted from the target genes of three selected mirnas using the funrich tool. b) enriched network of the same target genes using the webgestaltweb-based tool. c) gene ontology analysis of the target genes from the webgestaltweb. pmmb 2022, 5, 1; a0000249 9 of 14 3.3 circephb4 and linear ephb4 affect the wound healing process in hct-116 and sw480 cells now that we have shown that circephb4 may be involved in cancer-related processes, we performed two preliminary cell-based assays. firstly, we performed the wound healing analysis to determine the migration ability of the cells upon the reduction of circephb4 and ephb4. as shown in figure 4a, that knockdown of circephb4 significantly reduced the wound healing efficiencies in both sw480 and hct116 cell lines. however, the knockdown of linephb4 only significantly reduced the closure of wound in the sw480 cell line. the second assay was to determine the clonogenic ability of the cells upon knockdown of our target. as shown in figure 4b, there was no statistically significant difference in the number of colonies between si-circephb4 and si-ephb4 in both cell lines. 4. discussion ephb4 is a subfamily of receptor tyrosine kinase (rtks) that regulates key biological processes including cell differentiation, proliferation and motility. the expression of ephb4 in crc has been conflicting, for instance an earlier study by stephenson et al. showed that ephb4 was up-regulated in crc[30]. on the contrary, a study by davalos et al., showed the opposite regulation in crc[31]. in this study, we validated the expression of both circephb4 and ephb4 in primary crc tissues and secondary cell lines. we showed that the expression of both transcripts was upregulated in diseased samples as compared to the non-cancerous controls. it was also observed that the linear ephb4 had a higher expression than the circephb4. interestingly, a study by bachmayr-heyda showed that there is a global reduction of circrnas in crc[32]. nevertheless, this study showed that expression of a circular rna located at the chr7:100410369-100410830 region was up-regulated in crc tissues[32]. this circrna corresponds to the same location as circephb4 (hsa_circ_0001730). interestingly, a study by tan et al., showed that circephb4 is downregulated in hepatocellular carcinomas[19]. this shows that the expression of circephb4 is heterogenous and is likely cancer-dependent, nevertheless a larger sample size is needed to further confirm this observation. it is known that circrnas regulate the cellular machinery by sponging to multiple mirnas, hence we identified the predicted mirna targets for circephb4 using the web tool circinteractome and starbase. we postulate that there should be a negative correlation between the expression of circephb4 and the selected mirnas. we then selected 2 mirnas with the highest probability of binding to our circrna of interest, which ishas-mir-193a and has-mir-193b.through the tcga dataset in the oncomir database, both of these mirnas were downregulated in crc tissues. these mirnas have previously been reported as being involved in the progression of cancer. for instance, hsa-mir-193a was shown to be deregulated in several cancers including crc. although from our analysis using the tcga dataset showed that hsa-mir-193a-3p was upregulated in crc, several other studies showed pmmb 2022, 5, 1; a0000249 10 of 14 an opposite pattern. for instance, in a study by mamoori et al., the expression of hsa-mir193a-3p was significantly downregulated in 70% of the tested crc tissues[33]. figure 4. a) wound healing analysis of hct-116 and sw-480 cells and b) clonogenic assay upon knockdown of circephb4 and linear ephb4. (upper left: control, upper middle: si-circephb4, upper right: si-ephb4, lower left: si-circephb4+si-ephb4). pmmb 2022, 5, 1; a0000249 11 of 14 the authors show that the up-regulation of hsa-mir-193a-3p was able to impede cell proliferation and induce cell death in crc cells in vitro[33]. apart from that, a study by zhang et al., has shown that the expression of hsa-mir-193a-5p was downregulated in crc tissues, and even more reduced in samples with metastasis[34]. this shows that the expression of this mirna family is heterogeneous and needs further validation. similarly for hsa-mir-193b, the relative expression was reduced in crc tissues as compared to normal mucosa[35]. the expression of hsa-mir-193b was significantly associated with tnm stages and lymph node invasion[35]. a similar study by guo et al., also showed that hsa-mir-193b was significantly downregulated in crc[36]. the authors also show that this mirna targets stmn1 in crc[36]. for hsa-mir-3605-5p, there were limited studies correlating this mirna to cancers, thus making this an interesting target to further explore on. although we did not find any significant biological pathway from the funrich tool, we were able to obtain two significant pathways from the kegg database. the mirnas in cancer pathway had an enrichment ratio of 6.2546 with the target genes. most of the detected genes are well-known cancer-related genes such as kras, pten and pak4. therefore, we postulate that circephb4 could be involved in the tumorigenesis process in crc. to ascertain this, we performed two basic cell-based assays, the wound healing assay and clonogenic assay. from our data, we showed that circephb4 could indeed affect the migration process of two crc cell lines. further validation using other methods is needed to confirm this effect. nevertheless, circephb4 did not affect the clonogenic ability of the cells, showing a selective effect of circephb4. 5. conclusion in summary, we have shown that circephb4 and linear ephb4 were indeed upregulated in crc tissues and crc cell lines. through in-silico analysis, we showed that circephb4 could interact with three possible mirnas and downstream pathways include cancer-related pathways. apart from that, we were able to show that circephb4 could affect the migration process in crc cell lines, though more assays are needed to confirm this. in the future, it will be necessary to explore the molecular mechanism of circephb4 involving the development and progression of crc and more work is needed to understand the mechanism underlying the regulatory effects. additionally, this study is mainly based on invitro experiments, and the results require to be further verified by in-vivo experiments. overall, circephb4 is a promising biotarget in crc that warrants further attention. author contributions: na conceptualize the study, snn, na, mi performed the experiments and data analysis, is, mir, nsam provided materials, na, nsam ,rj provided critical input and feedback. pmmb 2022, 5, 1; 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20(34): 12241. 35. fang z, li c, and li s. microrna‑193b acts as a tumor suppressor in colon cancer progression via targeting rab22a. exp ther med 2019; 17(5): 3921–3928. 36. guo f, luo y, mu y-f, et al. mir-193b directly targets stmn1 and inhibits the malignant phenotype in colorectal cancer. am j cancer res 2016; 6(11): 2463. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. 1. introduction 2. materials and methods 3. results 4. discussion 5. conclusion references pmmb 2021, 4, 1; a0000250. doi: 10.36877/pmmb.a0000250 http://journals.hh-publisher.com/index.php/pmmb review article updated covid-19 condition in australia ke-yan loo1, vengadesh letchumanan1, loh teng-hern tan2, jodi woan-fei law1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; ke.loo@monash.edu (k-yl); vengadesh.letchumanan1@monash.edu (vl) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) *corresponding author: jodi woan-fei law; 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; jodi.law1@monash.edu (jw-fl) received: 28 november 2021; received in revised form: 12 december 2021; accepted: 15 december 2021; available online: 16 december 2021 abstract: the covid-19 pandemic has had detrimental effects on the lives of citizens worldwide in the past two years. the highly infectious respiratory disease discovered in late 2019 was a public health emergency that prompted the rapid development of vaccines to prevent the further spread of the virus. in late 2020, vaccines were finally ready to be distributed globally to reduce the severity of the disease and fatalities. the early stages of the pandemic were crucial as detecting the virus earlier on was a critical step in protecting the community from the coronavirus. in australia, the local government quickly closed national borders, implemented lockdowns and restrictions, increased testing within the community, established mask mandates and developed vaccination programs. the strategies used by the australian government have proven to be effective and efficient in managing outbreaks amidst the pandemic. the authorities were able to control the virus spread and maintained a low fatality rate within the country. keywords: covid-19; coronavirus; australia; endemic; vaccines 1. introduction since the discovery of the novel coronavirus, sars-cov-2, the virus has spread worldwide as the coronavirus plummeted the world into a covid-19 pandemic for nearly two years[1]. those infected with this airborne respiratory disease would often present symptoms of fever, cough, shortness of breath, muscle aches, fatigue and loss of taste or smell[2]. during the late stages of the infection, patients may require intensive care such as supplemental oxygen via tracheal intubation[3]. in addition, covid-19 patients with other underlying medical conditions or are immunocompromised, the prognosis for the disease is generally poor[4]. even when covid-19 patients recover from the illness, they may still encounter post-covid conditions which can affect their daily lives. these conditions include multiorgan system effects involving the immune, hematological, pulmonary, pmmb 2021, 4, 1; a0000250 2 of 9 cardiovascular, gastrointestinal, hepatic and renal systems[5]. other reported symptoms which may persist after recovery from covid-19 include fatigue, cognitive impairment, myalgia, fever, impaired daily function and mobility and poor endurance[6]. throughout the pandemic, this public health emergency took millions of lives and jeopardised the livelihoods of many as covid-19 also plunged the world economy into a recession[7-9]. during the initial stages of the pandemic, leaders of countries were quick to act by closing national borders, enforcing mask mandates, restricting travels within local communities in hopes of breaking the chain of disease spread[10, 11]. the government implemented travel restrictions in australia as national borders were closed to all nonresidents. moreover, individuals returning to australia from a higher-risk country were not allowed to attend public gatherings for 14 days. those undergoing testing for covid-19 were also prohibited from attending public gatherings[12]. the local government also implemented lockdown restrictions progressively to restrict citizens’ movement and reduce gatherings. australia prioritises testing on active cases and close contacts to prevent the disease spread, and close contacts of covid-19 patients would also be required to undergo quarantine[12]. nevertheless, the virus is highly transmissible through air, and once it infiltrated communities or spaces with high occupancy, outbreaks were frequently reported[13-16]. nevertheless, since the discovery of covid-19 vaccines and their rapid distribution worldwide[17], things started to look up, and life was expected to be back to normal as the daily total of confirmed cases was better stabilised. 2. covid-19 cases in australia the first case of covid-19 in australia was isolated from a 58-year-old chinese national who arrived in melbourne on 19 january 2020. he was admitted to the monash medical centre in melbourne on 24 january 2020 with fever, cough and progressive dyspnea[18]. in the following week, 11 more cases were confirmed whereby all were acquired in china, similarly to the aforementioned first case. the first local case was subsequently reported when the patient contacted a chinese tourist in the first week of february[19]. the disease spread quickly, and by the end of march 2020, the total number of confirmed cases in australia had skyrocketed to over 4000 cases, with 19 deaths peaking at 458 cases a day on 28 march 2020[20]. at this point, 2129 cases were hospitalised, with 176 cases in intensive care units (icu). among the cases in icu, five required ventilator intubations. when april 2020 came along, the rate of increase in cases reduced steadily, possibly due to the closure of national borders, lockdown implementations and social distancing measures introduced by the government[21]. in late april 2020, a contact tracing app called covidsafe was developed based on the singaporean version to quickly and effectively trace any close contacts of covid-19 patients[22]. this app will notify individuals of their status, and if there are any close contacts, they can self-isolate and undergo covid-19 tests for further confirmation. following its release, the app has been downloaded by 6.3 million australian adults with smartphones as of 16 june 2020[19]. however, there was a spike in confirmed cases in july 2020, which could be attributed to outbreaks across multiple settings and locations in victoria. more than half of the infections pmmb 2021, 4, 1; a0000250 3 of 9 were locally acquired from known clusters. at the same time, the overseas-acquired cases were related to cruise-ship travel or travel originating from europe or the americas[23]. the sudden increase in cases led to another lockdown in victoria, metropolitan melbourne and mitchell shire. residents were only allowed to leave their homes to shop for food and essentials, provide care or seek medical treatment, exercise, study, and work if working from home was not possible. non-essential services and businesses such as physical recreation facilities and entertainment facilities were also required to close during this time. the local authorities rapidly conducted covid tests nationally to detect the disease in the early stages, and overall positivity rates in the country was found to be less than 0.5%. on the other hand, in victoria, where the most significant number of cases were reported, the positivity rate was 0.84. of all the cases in australia at this time, most of the individuals presented with mild symptoms, with only 12% of the cases requiring hospitalisation[23]. in august 2020, daily reported cases of covid-19 were still increasing despite the mitigation measures enforced by the australian government. thus, a nightly curfew was imposed along with mandatory face coverings in public, and schools and businesses were required to close. outbreaks were determined to be from aged care facilities where the residents were at higher risk of covid-19 infection due to communal living. they were also at risk of severe complications if they contracted the disease. on 2 august 2020, 1436 cases were associated with 148 residential aged care facilities, with 800 cases involving the residents, while the remaining cases occurred in the facilities' staff members[24]. the restrictions implemented by the australian government proved to be effective as the daily confirmed cases and deaths began to decrease throughout the country. in october 2020, the restrictions were eased as case numbers continued to dwindle. finally, it was announced on 26 october that the 112-day lockdown in victoria would end, and various sectors were allowed to reopen on 28 october. shortly after, on 1 november 2020, australia recorded zero locally acquired covid cases for the first time since 9 june 2020[25]. on 19 november 2020, a short lockdown was reimplemented for four days in response to a cluster of cases in south australia, and restrictions were eased once the outbreak was speedily controlled[26]. towards the end of 2020, australia continues to report low numbers of confirmed covid-19 cases, and restrictions were further eased across the different jurisdictions in the country, except for new south wales. border restrictions were implemented in new south wales from 17 december 2020 due to outbreaks, and heightened restrictions such as gatherings limits, travel and stay at home requirements were also imposed. border restrictions were also implemented between new south wales and other jurisdictions such as tasmania and the northern territory to prevent disease spread[27]. as of 31 december 2020, australia recorded 28,408 covid-19 cases with 909 deaths (figure 1). pmmb 2021, 4, 1; a0000250 4 of 9 figure 1. total number of confirmed covid-19 cases and covid-19 deaths in australia. australia’s decreasing trend of confirmed covid-19 cases continued into 2021, with most cases being from overseas-acquired cases, and most of them did not require hospitalisation. although multiple variants of the coronavirus that were highly infectious[28] had been detected in australia, the number of confirmed cases remained low with low fatality rates[29, 30]. vaccinations finally began in late february 2021, and the first people to receive the vaccines were quarantine and border workers, frontline healthcare workers, aged and disability care residents, and staff at a significantly higher risk of covid-19[31]. the vaccine would later be distributed to the mass public as more vaccines arrived and were made available in australia. the three vaccines approved for use in australia are comirnaty (pfizer), spikevax (moderna) and vaxzevria (astrazeneca)[32]. comirnaty and spikevax are mrna vaccines that deliver mrna encoding for the spike protein of sars-cov-2 to the host. upon vaccination, the host cells would produce the viral spike proteins, and the immune system will be triggered to produce antibodies to neutralise the virus. as for vaxzevria, the vaccine utilises non-replicating chimpanzee viral vectors to deliver genetic information of the coronavirus to the host cells to produce viral spike proteins that will trigger an immune response to produce antibodies[16, 17]. studies have shown that these vaccines are also effective towards variants of the novel coronavirus such as the b.1.1.7 (alpha), b.1.351 (beta) and b.1.617.2 (delta) variants[33, 34]. although an individual is fully vaccinated, they may still get infected by the virus as the vaccines are not 100% effective. despite that, these vaccines will help prevent severe symptoms and illness in individuals with covid-19 vaccine breakthrough cases[35]. the vaccines were quickly distributed to the public, and by june 2021, over 6.5 million doses of covid-19 vaccines were administered nationwide. furthermore, reported cases of covid19 and related deaths remained low, and fatality rates were stable at around 3% in australia throughout the first half of 2021. whenever outbreaks occurred, the local government would pmmb 2021, 4, 1; a0000250 5 of 9 quickly impose restrictions or lockdown measures in the areas involved to prevent the disease from spreading like wildfire within the community[36, 37]. in spite of the continuous efforts by the australian government and the cooperation of the public, covid-19 cases began to rise again in july 2021. the upwards trend was attributed to an ongoing outbreak in the bondi area of sydney, where the delta variant of the coronavirus was detected in a driver transporting international flight crew. meanwhile, in queensland, locally-acquired cases were linked to a hotel quarantine transmission event where an international aircrew contracted the alpha variant of the virus[38]. on 1 july 2020, australia moved into phase one of the national plan to transition australia’s covid-19 response, known as “vaccinate, prepare and pilot”. in phase one, the aim was to vaccinate as many individuals as possible and impose early, stringent and short lockdowns where outbreaks occurred. the cap for international travellers entering the country was also halved[39]. as cases continue to increase despite the high vaccination rates, strict lockdown measures were imposed on areas with high positivity rates, such as sydney and melbourne[40, 41]. the daily toll of confirmed covid-19 cases peaked in october 2021 when cases reached over 2000 cases per day[42]. on 20 october 2021, phase two, the vaccination transition phase had occurred where lockdowns were less likely, and a target of 70% vaccination rates was in place. moreover, travel bans were lifted for vaccinated residents, and quarantine was imposed for international arrivals[39, 43]. although the positivity rates were high, the government remains optimistic as their vaccination rates were high, with 73.4% of people aged 16 and over were fully vaccinated as of 24 october 2021. cities like melbourne and sydney that were under lockdown were prepared to ease restrictions to begin living life with the coronavirus. the main focus of the australian government was to keep up with the vaccination rates, as restrictions would be progressively lifted once the target for vaccination rates is met[44]. at the time of writing, australia has amassed 214,708 confirmed covid-19 cases, with 2,020 deaths in the country. they have also achieved the target (80%) of fully vaccinated individuals as 87.9% of the population was fully vaccinated on 3 december 2021[32]. this target was set as part of phase three, the vaccine consolidation phase in the national plan to transition australia’s covid-19 response. during this phase, vaccinated residents were exempt from domestic restrictions, and restrictions in travelling outbound for these individuals were lifted. lockdowns would also be infrequent and, if implemented, would be highly targeted to specific areas of outbreaks only[43]. like the singapore society, the australians were prepping to move into an endemic phase with covid-19. however, a new variant (b.1.1.529) of the coronavirus known as the omicron variant was detected in south africa on 24 november 2021[45]. an update given by the local government on 30 november 2021 notified the public that six cases of the omicron strain had been reported in australia, with all six cases in quarantine and the individuals had mild to no symptoms. the australian government continues to reassure the people that they are well prepared to manage the new variant as they begin to implement travel restrictions, revised quarantine and home isolation requirements. for instance, non-citizens traveling from south africa will be temporarily denied entry into australia, while australians from south africa must undertake a 14-day quarantine under state and territory public health requirements. moreover, the government noted that they are utilising a suppression approach pmmb 2021, 4, 1; a0000250 6 of 9 in handling the omicron variant to limit the rate of incursions into australia. it was also noted that there is a sufficient supply of booster doses of the covid-19 vaccines for australians[46]. the country is still looking forward to moving into phase four, the post-vaccination phase of the national plan, but plans are placed on hold due to the emergence of the omicron variant in australia. with phase four, individuals who are 18 and older will be able to receive a booster dose of vaccine if they have received the second dose of their covid-19 vaccination at least six months ago. the booster dose will help increase protection against severe disease due to covid-19, and currently only comirnaty is approved and preferred as a covid-19 booster dose in australia[47]. in addition, phase four is when the community will live with covid-19, international borders will be opened, and both vaccinated and non-vaccinated will be allowed entry into the country following specific guidelines[39, 43]. 3. conclusion as of 3 december 2021, the daily total for covid-19 cases in australia has been decreasing since the peak in october 2021, and vaccination rates have achieved 87.9%, with over 17 million residents fully vaccinated. australia is rapidly moving into an endemic phase with covid-19, hoping that life will return to normal soon. the australian government has successfully controlled the pandemic despite the outbreaks as they could maintain a low fatality rate. early actions by the government, such as the closure of national borders, increasing community-wide testing, establishing lockdowns for areas with high positivity rates, and mask mandates indoors, have effectively controlled the spread of the virus[19]. the government also released an app to identify covid-19 positive individuals and their close contacts effectively and efficiently. these individuals could be easily traced and put into isolation and treatment. moving into 2021, the australian government focused on distributing the covid-19 vaccines as soon as possible to reduce the spread of the virus and prevent severe illnesses within those tested positive. in addition, the authorities were not lax with restrictions even when vaccination rates were high, as lockdowns were still being enforced when outbreaks occurred. in short, the strategies used in australia to handle the pandemic can be modeled by other countries that wish to move into an endemic phase with covid19. author contributions: k-yl performed the literature search, critical data analysis, and manuscript writing. lt-ht performed proofreading and editing. vl and jw-fl performed proofreading and conceptualised this review writing project. funding: no external funding was provided for this research. acknowledgements: the authors would like to acknowledge professor shajahan yasin, head of school, jeffrey cheah school of medicine and health sciences, monash university malaysia. conflicts of interest: the authors declare no conflict of interest. references 1. letchumanan v, ab mutalib n-s, goh b-h, et al. novel coronavirus 2019-ncov: could this virus become a possible global pandemic. prog microbes mol biol 2020; 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[accessed 2021 nov 5]; available from: https://www.australia.gov.au/national-plan. 44. australia's melbourne set for covid-19 lockdown exit despite record cases as vaccinations spike, in the straits times. 2021. 45. australian government department of health. omicron variant. 2021 [accessed 2021 dec 2]; available from: https://www.health.gov.au/news/health-alerts/novel-coronavirus-2019-ncov-healthalert/omicron-variant. 46. prime minister of australia, national cabinet statement 58. 2021. 47. australian government department of health. covid-19 booster vaccine advice. 2021 [accessed 2021 nov 10]; available from: https://www.health.gov.au/initiatives-and-programs/covid-19vaccines/getting-your-vaccination/booster-doses. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. https://www.australia.gov.au/national-plan https://www.health.gov.au/news/health-alerts/novel-coronavirus-2019-ncov-health-alert/omicron-variant https://www.health.gov.au/news/health-alerts/novel-coronavirus-2019-ncov-health-alert/omicron-variant https://www.health.gov.au/initiatives-and-programs/covid-19-vaccines/getting-your-vaccination/booster-doses https://www.health.gov.au/initiatives-and-programs/covid-19-vaccines/getting-your-vaccination/booster-doses pmmb 2021, 4, 1; a0000243. doi: 10.36877/pmmb.a0000243 http://journals.hh-publisher.com/index.php/pmmb review article insights into covid-19 delta variant (b.1.617.2) angel yun-kuan thye1, ke-yan loo1, kyle bond chene tan2, jenny may-sim lau3, vengadesh letchumanan1* article history 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, selangor darul ehsan 47500, malaysia; angelthye.yunkuan@monash.edu (aykt); ke.loo@monash.edu (kyl) 2northern ireland centre for stratified medicine (nicsm), biomedical sciences research institute, ulster university, c-tric building, londonderry bt47 6sb, united kingdom; tan-bc@ulster.ac.uk (kbct) 3queen’s university belfast, wellcome-wolfson institute for experimental medicine, 97 lisburn road, belfast bt9 7bl, united kingdom; jennymf.lau@hotmail.com (jmsl) *corresponding author: vengadesh letchumanan; novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, selangor darul ehsan 47500; vengadesh.letchumanan1@monash.edu (vl) received: 22 september 2021; received in revised form: 20 october 2021; accepted: 24 october 2021; available online: 4 november 2021 abstract: since beginning of the severe acute respiratory syndrome coronavirus 2 (sarscov-2), different variants of concern (voc) have been discovered. one of the variants that stood out was the delta variant (b.1.617.2), first found in india. it caught worldwide attention due to its greater transmissibility than the progenitor strain and the first variant of concern (voc)alpha variant (b.1.1.7). b.1.617.2 spread rapidly across the globe and became a voc due to its high transmissibility, clinical implications, and impact on vaccine efficacy. this review discusses the background and prevalence of b.1.617.2 and its sensitivity to convalescent sera and vaccinated individuals. we will provide an insight into the impact b.1.617.2 has on vaccine efficacy and discuss the level and type of protection an individual could get by being vaccinated. we will also discuss briefly on the covid-19 vaccine booster doses and whether it is needed. keywords: sars-cov-2, delta (b.1.617.2), variants of concern (voc), transmissibility, vaccine, booster 1. introduction the covid-19 global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that took place in 2020 has yet to come to an end[1–4]. throughout the pandemic, the sars-cov-2 spike protein is continuously mutating, leading to the discovery of more and more new variants. four of the many variants discovered have pmmb 2021, 4, 1; a0000243 2 of 10 been listed as variants of concern (voc). this includes variants b.1.1.7 (alpha), b.1.351 (beta), b.1.617.2 (delta) and p.1 (gamma)[5]. b.1.617.2 was initially given the designation as a variant of interest (voi) by the world health organization (who) in april 2021, which quickly turned into a voc in may 2021[6]. interestingly, as the delta variant became the predominant lineage in many countries, its sub-lineages are given the alias ay. this separates b.1.617.2 into smaller related clusters, allowing for better monitoring of diversity and enhancing the identification of newly emerging clades. new ay lineages are phylogenetically defined and treated as delta variants. their designation to a sub-lineage does not necessarily imply any functional biological differences compared to b.1.617.2 unless stated otherwise by other health bodies or the who[7]. on a side note, in recent months, a new variant called the delta plus (b.1.617.2.1 and ay.1) has emerged from india and has spread to a few other countries, including the united states (us). an analysis showed that this variant traveled to the us via england and japan, with the first case reported in washington state on the 3rd may 2021. research, however, found ay.1 to be more than just a delta variant with an additional k417n mutation, and compared to b.1.617.2, ay.1 has a distinct mutation profile[8]. this review focuses only on b.1.617.2 which gained widespread attention due to its sudden emergence, rapid transmission, and severe clinical implications. in comparison to previous circulating variants, b.1.617.2 seems to be a more virulent variant. it is deemed to have greater transmissibility, increased severity risk, and is more resistant to current vaccines. based on the weekly epidemiological update of covid-19 on 31st august 2021 published by the who, b.1.617.2 has been reported in 170 countries[9]. hence, we aim to discuss the background and prevalence of b.1.617.2 and provide valuable insights into how this variant caused the crisis, planting fear in people worldwide. we will also discuss concerns regarding covid vaccines against the b.1.617.2. 2. background and prevalence of b.1.617.2 b.1.617.2 first emerged in india in december 2020[5], causing the second wave of covid-19 in india, which resulted in a health crisis affecting millions of people[10]. the confirmed daily new cases in india spiked from 53 per million population (up to march 2021) to >200 per million population (after mid-march 2021). on the contrary, the ratio of daily recoveries to daily new cases from the beginning of march declined sharply, indicating the severe burden on india’s healthcare system. shortages in essential supplies were seen in leading hospitals and the number of deaths of covid-19 patients in intensive care increased in >130 cities as a result of oxygen and medicine shortage[11]. on the 1st of may 2021, india became the first country to record >400,000 new covid-19 cases within 24 hours since the pandemic[12]. b.1.617.2 quickly became the most prevalent variant in many other countries, including the united kingdom (uk) and the us. in the uk, b.1.617.2 emerged in mid-april and later accounted for 95% of all new cases[13], in the us, it accounted for 99% of new cases[14], while in portugal, it accounted for 70% of cases in lisbon[13]. it also leads to new pmmb 2021, 4, 1; a0000243 3 of 10 record highs for covid-19 cases and deaths in many southeast asian countries, including malaysia, myanmar, and indonesia[15]. b.1.617.2 is one of the three sub-lineage of the b.1.617 lineage[5]. it has several spike mutations, including d614g, p681r, l452r, t19r, t478k, a222v, r158g, g142d; 156– 157 deletion in the nterminal domain (ntd) and s2 substitution d950n[16]. regarding some of the mutations, a study found d614g is linked to higher viral loads in the respiratory tract of infected individuals, increased human host infectivity, and possibly a higher transmission efficiency to sars-cov-2[17,18]. it has also been shown to strengthen cleavage efficiency due to substitution on spike conformational diversity[19,20]. p681r is in the s1-s2 furin cleavage site[21]. similarly, mutation at the p681 position also contributes to sarscov-2 transmission and infection[22,23]. on the other hand, l452r and t478k present in b.1.617.2 have been associated with vaccine escape and increased transmissibility[24]. several studies showed that the l452r increases infectivity by stabilizing the sars-cov-2 spike glycoprotein and human angiotensin-converting enzyme 2 (ace2) receptor interaction[25-27]. the stronger cell-virus attachment and increased infectivity result as the l452r mutation causes huge increments in free energy at the receptor-binding domain (rbd) and ace2 binding complex[26,28]. other studies also demonstrated that l452r mutant could evade the human leukocyte antigen (hla)-24 limited cellular immunity, boost viral infectivity, and possibly increase viral replication[29]. together, these mutations contribute to the increased transmissibility, infectivity and immune evasion characteristics of b.1.617.2. research indicated that b.1.617.2 is 60% more transmissible than the b.1.1.7 which is already more transmissible than the progenitor strain[30]. in addition, when most countries enforced lockdown measures, the b.1.617.2 was found to have a mean basic reproductive number (r0) value of 5.08 while the ancestral strain’s r0 is 2.79[31]. a china study has proposed that b.1.617.2 has a higher viral replication rate, resulting in their infections having a 1000 times higher viral load than the 19a/19b strains (during the initial 2020 epidemic) infections when the day testing turns positive. hence, greater infectivity during the early stages[32]. a singapore study also associated b.1.617.2 with a lower polymerase chain reaction (pcr) cycle threshold (ct) value and a longer viral shedding, possibly providing a mechanism for increased transmissibility[33]. hence, suggesting the need for a greater vaccination rate. furthermore, b.1.617.2 has a higher risk of hospitalization and greater disease severity than b.1.1.7[34–36]. studies from both england and scotland were consistent that those infected with the delta variant have twice the risk of hospitalization than those infected with the alpha variant. based on a cohort study in england where 74% of covid-19 patients were unvaccinated, they found that within 14 days of testing, those infected with b.1.617.2 have a higher risk of hospitalization and greater disease severity than those infected with the b.1.1.7 variant[36]. a singapore study was comparing patients with b.1.1.7, b.1.351, and b.1.617.2 also found b.1.617.2 was associated with higher odds of oxygen requirement, intensive unit care (icu) admission, or death[33]. a uk report also stated b.1.617.2 has a higher secondary attack rate compared to b.1.1.7 in both household and non-household pmmb 2021, 4, 1; a0000243 4 of 10 contacts and traveler and non-traveler cases. in contrast, the 28-day case fatality for b.1.617.2 stays low at 0.1% compared to b.1.1.7, which is at 1.9%[37]. recently, there are also concerns about whether children have an increased risk of getting infected with b.1.617.2. it seems that the increased cases in children were most likely because they are not yet fully vaccinated. moreover, in a press conference, the centers for disease control and prevention (cdc) head rochelle walensky said although there are increased pediatric cases and increased overall cases, there was no increased severity in pediatric cases[38]. (table 1) table 1. summary of the background and prevalence of b.1.617.2 delta/ b.1.617.2 references country first detected india [5] month+year detected december 2020 [5] confirmed daily new cases in india (up to march 2021) 53 per million population increased to >200 per million population [11] ratio of daily recoveries to daily new cases in india (from midmarch 2021) significant reduction [11] total countries affected 170 [9] mutations d614g, p681r, l452r, t19r, t478k, a222v, r158g, g142d; 156–157 deletion in the n terminal domain (ntd) and s2 substitution d950n [16] transmissibility 60% more transmissible than b.1.1.7 [30] basic reproductive number (r0) 5.08 while ancestral strain’s r0 is 2.79 [31] risk of hospitalization and disease severity twice the risk of hospitalization and a greater disease severity compared to b.1.1.7 [36] secondary attack rate higher than b.1.1.7 in both household and nonhousehold contacts and traveller and non-traveller cases [37] are children at an increased risk? unlikely. probably due to being unvaccinated [38] 3. sensitivity of b.1.617.2 to convalescent sera and vaccinated individuals in a month 12 cohort study in france, individuals vaccinated with a single dose of either pfizer, astrazeneca, and moderna vaccines demonstrated greater neutralizing antibody (nab) titers against alpha, beta, and delta variants than those convalescents of unvaccinated individuals. hence, a single dose of vaccine boosts cross-neutralizing antibody responses to the delta variant. however, those vaccinated with a single dose of pfizer or astrazeneca showed low efficiency against delta variant with the percentage of sera of individuals vaccinated with pfizer or astrazeneca neutralizing b.1.617.2 to be 13% and 9% respectively. an efficient neutralizing response was generated only after the second dose[39]. an india study also showed that relative to the prototype strain (d614g), there is a 3.2 (two dose)-4.5 (one dose) fold reduction in nab titer in the sera of covishield vaccinees against the b.1.617.2, indicating the significance of a second dose. moreover, it also showed that the nabs in breakthrough participants and covid-19 recovered individuals with one or two pmmb 2021, 4, 1; a0000243 5 of 10 vaccine doses showed higher protection against b.1.617.2 than vaccinees who received one or two vaccine doses[10]. another study also evaluated the neutralization of sera collected from covid-19 recovered patients (post 5-20 weeks of infection) and individuals (post 28 days) vaccinated with two doses of bharat biotech’s covaxin (bbv152) against b.1.617.2. findings demonstrated a 4.6-fold and 2.7-fold reduction in neutralization titer compared to the b.1 strain (d614g) with sera of covid-19 recovered patients and covaxin vaccinees. studies have shown that against b.1.617.2, although there is a decrease in neutralization titer with covaxin vaccinees sera, the neutralization potential is yet to be established. the broad epitope coverage in an inactivated vaccine (bbv152) reduces the magnitude of decreased neutralization against emerging variants by inducing an immune response against whole virion[40]. besides that, studies have demonstrated the effectiveness of two shots of pfizer vaccine against b.1.617.2 infection[41,42]. one study showed two shots of pfizer vaccine demonstrated 88% efficacy two weeks after the second dose, while two shots of astrazeneca vaccine could provide 60% protection against symptomatic disease from b.1.617.2[43]. therefore, although b.1.617.2 has demonstrated immune evasion characteristics, vaccines still offer protections against b.1.617.2 to some extent. 4. understanding the b.1.617.2 variant there is an association between the emergence of new variants and epidemic severity and sars-cov-2 transmission. the high transmission rate is a prognostic factor for genomic variations. as a consequence, outbreaks happen following large gatherings[5,44]. the emergence of the b.1.617.2 has been raising concerns globally due to its high transmissibility, increased disease severity, and immune evasion ability. with the emergence of the covid-19 pandemic, several types of vaccines have been developed, consisting of inactivated virus vaccines, a protein subunit vaccine, mrna vaccines, and viral vector recombinant vaccines[45]. the covid-19 vaccines enable the activation of the immune response upon binding to the spike protein[5]. however, mutations on the rbd that are present in many new emerging variants are responsible for most of the escape from vaccineinduced neutralization[46]. thus, concerns on vaccine efficacy arise as new variants with mutations on the spike protein emerge. in addition, the durability of antibody response in semi and fully vaccinated individuals are not yet fully understood. the toll b.1.617.2 takes depends mainly on the population’s vaccination status and the number of people having immunity from previous infection[13]. for instance, in the uk with a high vaccination rate, fatality did not increase with increased cases. however, in countries with low vaccination rates such as thailand and indonesia, the covid-19 cases are more serious. new cases in the us also were focused on unvaccinated individuals[47]. another example is in israel, the estimated pfizer vaccine effectiveness against sars-cov2 infection was 95.3% ≥ 7 days after the second dose[48]. however, the israeli ministry of health stated that the efficacy of the pfizer vaccine dropped to 39% due to b.1.617.2. even so, there is still 88% protection against infection, progressing to hospitalization, and 94.1% pmmb 2021, 4, 1; a0000243 6 of 10 against severe illness[47]. thus, demonstrating vaccination still plays a role in alleviating the disease burden. according to data from the coronavirus resource center, john hopkins university of medicine, many countries have initiated a national vaccination process. many have indeed had a high percentage of the population vaccinated. however, there are still a handful of countries whose vaccination rate is still far below the world average[49]. not to forget, as mentioned earlier, b.1.617.2 has a much higher reproduction number compared to the ancestral strain, which means a much higher vaccine coverage needs to be achieved compared to the assumed initially 60–70% vaccine coverage for the ancestral strain. a reproductive number of 5 would mean a vaccine coverage of 80% will be needed based on the equation q=1-1/r0, assuming a 100% vaccine efficacy[31]. b.1.617.2, having high infectivity means more people need to be vaccinated to reduce its spread and disease burden. hence, it is crucial to increase vaccination coverage to control the spread b.1.617.2. now, for some countries where most of the population had been fully vaccinated with two doses of covid-19 vaccine, this brings us to the issue of covid-19 vaccine booster doses. currently, the cdc and the food and drug administration (fda) have approved the booster shot of the pfizer-biontech covid-19 vaccine in specific populations and those in high-risk occupational and institutional settings, while booster shots of covid-19 vaccines by moderna and johnson & johnson will be evaluated in the coming weeks. according to the statement released by the cdc on 24th september 2021, with the dominance of b.1.617.2 and the increase in covid-19 cases in the us, booster shots can strengthen protection against severe disease in those with high-risk exposure to covid-19 or complications for severe disease[50]. with that, israel is the first country in the world to administer covid-19 vaccine (pfizer) booster doses to adults with weak immune systems following the announcement by the israeli ministry of health on the 11th july 2021[47]. however, some believe booster doses should not be prioritized over primary vaccination. the clinical trials lead for the vaccine and director of the oxford vaccine group, andrew pollard emphasized “urgent priority” should be given to those who have not got their first dose before initiating a third booster dose[51]. in line with that, a statement released by the who on the 4th august stated that 4 billion vaccine doses had been administered globally, in which >80% went to upper-middleand high-income countries even though they accounted for <50% of the world’s population. although booster doses may help combat the delta variant, people from low-income countries should not be left unprotected[52,53]. moreover, current evidence is limited and inconclusive on the widespread need for booster doses after receiving primary vaccination[54]. 5. conclusion despite the concerns mentioned regarding the b.1.617.2, national and international vaccination is still crucial for containing this global sars-cov-2 pandemic, as vaccines induce neutralizing antibodies and block the viral rbd from binding to the ace2 receptor. although there is a reduction in vaccine efficacy against the b.1.617.2, vaccines can still pmmb 2021, 4, 1; a0000243 7 of 10 provide a considerable amount of protection, decreasing the risk of hospitalization and moderating the severity[5]. nonetheless, the government should implement testing campaigns, mass vaccination, efficient contact-tracing, restricting gatherings, have strict quarantine of international travelers and strict traveling bans[55–58]. furthermore, reminders and awareness within the community could increase compliance to preventive measures[59]. with the continuous emergence of new variants, there is a need for constant surveillance of the evolutionary changes of sars-cov-2 and how mutations of the spike protein contribute to immune escape[60]. this could allow for better definition and implementation of countermeasures. last but not least, we must continue practicing safety measures to protect ourselves and others from covid-19 infection and slow its transmission[5,61]. author contributions: ay-kt performed the literature search, critical data analysis as well as manuscript writing. k-yl, kbct, jm-sl, and vl performed technical support and proofreading of this writing. vl provided conceptualization and set up this review writing project. all authors have read and agreed to the published version of the manuscript. funding: this work is supported by early career research grant, jeffrey cheah school of medicine and health sciences ecr-000021, awarded to vl. conflicts of interest: the authors declare no conflict of interest. references 1. goh hp, mahari wi, ahad ni, et al., risk factors affecting covid-19 case fatality rate: a quantitative analysis of top 50 affected countries. prog microbes mol biol 2020; 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3(1). 57. tan lt-h, letchumanan v, ser h-l, et al., pmmb covid-19 bulletin: united kingdom (22nd april 2020). prog microbes mol biol 2020; 3(1). 58. loo k-y and letchumanan v, covid-19: malaysia's fight against this deadly virus. prog microbes mol biol 2021; 4(1). 59. johnson d, ren sec, johnson hd, et al., covid-19: are malaysians embracing or suffering the new normality? prog microbes mol biol; 3(1). 60. ser h-l, tan lt-h, law jw-f, et al., genomic analysis of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) strains isolated in malaysia. prog microbes mol biol 2020; 3(1). https://coronavirus.jhu.edu/vaccines/international https://www.cdc.gov/media/releases/2021/p0924-booster-recommendations-.html https://www.who.int/director-general/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-4-august-2021 https://www.who.int/director-general/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-4-august-2021 https://www.who.int/news/item/04-10-2021-interim-statement-on-booster-doses-for-covid-19-vaccination https://www.who.int/news/item/04-10-2021-interim-statement-on-booster-doses-for-covid-19-vaccination pmmb 2021, 4, 1; a0000243 10 of 10 61. hoo he, loh hc, ch’ng ash, et al., positive impacts of the covid-19 pandemic and public health measures on healthcare. prog microbes mol biol 2021; 4(1). author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2023, 6, 1; a0000329. doi: 10.36877/pmmb.0000329 http://journals.hh-publisher.com/index.php/pmmb review article covid-19: an update on the latest therapeutic agents ke-yan loo 1,2 , loh teng-hern tan 1,3 , jodi woan-fei law 1,4 , kar-wai hong 1 , kok-gan chan 5,6 , vengadesh letchumanan 1,2 * article history 1 novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia; ke.loo@monash.edu (k-yl); hong.karwai@monash.edu (k-wh) 2 pathogen resistome virulome and diagnostic research group (pathrid), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia 3 innovative bioprospection development research group (inbiod), clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) 4 next-generation precision medicine & therapeutics research group (nmet), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia; jodi.law1@monash.edu (jw-fl) 5 division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia; kokgan@um.edu.my (k-gc) 6 international genome centre, jiangsu university, zhenjiang, china *corresponding author: vengadesh letchumanan, novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway 47500, selangor darul ehsan, malaysia; vengadesh.letchumanan1@monash.edu (vl) received: 13 march 2023; received in revised form: 11 april 2023; accepted: 14 april 2023; available online: 17 april 2023 abstract: the covid-19 pandemic has plagued the world for over three years since discovering the causative virus, sars-cov-2, in china. the rampant spread of the virus led to the loss of livelihoods of millions across the globe. this public health emergency prompted the rapid development of vaccines and treatments to fight against viral infection. vaccines against the viral infection started rolling out in late 2020, and the distribution of the vaccines worldwide managed to reduce the symptoms of covid-19 and prevent outbreaks in local communities. however, covid-19 infections are still prevalent, with patients suffering from severe symptoms which require oxygen support or mechanical ventilation. thus, therapeutic agents for covid-19 play a significant role in reducing the risk of disease progression into severe disease and improving hospitalized patients' clinical outcomes. existing drugs such as remdesivir, molnupiravir, baricitinib, anakinra, and tocilizumab have been repurposed to treat covid-19 earlier during the pandemic to meet the urgent demand for treatment. there are also novel antiviral and immunomodulating treatments (nirmatrelvir pmmb 2023, 6, 1; a0000329 2 of 21 plus ritonavir, ensitrelvir, regdanvimab, sotrovimab, and vilobelimab) that were developed during the pandemic to fight against covid-19 infections. these therapeutic agents have been reported to be effective and safe for use to treat covid-19 infections of different severity. nevertheless, continuous surveillance is imperative in ensuring that these treatment methods maintain efficacy and safety profiles in treating covid-19 caused by different variants of the virus. keywords: covid-19; antiviral; immunomodulator; hyperinflammation; therapeutic agents 1. introduction the coronavirus disease 2019 (covid-19) has plunged the world into a public health and economic crisis since its discovery in late 2019, when it was first discovered during an outbreak of pneumonia in china [1] . patients infected presented symptoms such as fever, dry cough, fatigue, and occasional gastrointestinal symptoms [2,3] . upon further genomic characterization, the pathogen from the outbreak was identified as the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [4,5] . by february 2020, the coronavirus had spread to other countries and confirmed cases outside china were steadily increasing [6,7] . the virus is highly infectious as it is easily transmitted from an infected individual to close contacts via respiratory droplets [8] . the population at a higher risk of getting infected by the coronavirus are the elderly, male sex, immunocompromised, and individuals with existing comorbidities such as diabetes, cardiovascular disease, hypertension, kidney failure, and chronic respiratory disease [9,10] . as the virus continued to spread to every corner of the world, the world health organization (who) declared covid-19 a global pandemic in march 2020, eleven years after the previous h1n1 pandemic in 2009 [11] . as of april 2023, approximately 762 million confirmed cases and over 6.8 million deaths due to covid-19 have been reported [12] . the covid-19 pandemic has put immense pressure on the global healthcare system, particularly in resource-limited countries. in addition to managing many patients with covid-19, healthcare facilities struggled to provide essential services to the public, e.g., preventive and curative services for communicable and non-communicable diseases [13–16] . at the beginning of the pandemic, world leaders quickly implemented strategies to prevent the spread of the disease, such as travel restrictions, quarantine, wearing of masks in public, contact tracing, and frequent screening for the coronavirus within local communities [17,18] . meanwhile, researchers were working towards developing a safe and effective vaccine to fight against sars-cov-2. in december 2020, the first vaccine against covid-19, comirnaty by biontech and pfizer, received approvals and authorizations for use in several countries [19,20] , thus kickstarting the distribution of covid-19 vaccines to meet global demands. following comirnaty, other vaccines such as covishield by astrazeneca, spikevax by moderna, coronavac by sinovac, covaxin by bharat biotech, covovax by novavax, and jcovden by johnson & johnson were also approved for pmmb 2023, 6, 1; a0000329 3 of 21 emergency use by who [21] . vaccines were deployed to reduce the risk of developing severe symptoms when infected with covid-19 and to prevent the rampant spread of the virus in the community. moreover, herd immunity can be achieved with mass immunizations with the covid-19 vaccines to protect individuals who are ineligible to get the vaccines. the increase in vaccine production has resulted in over 13 billion vaccine doses administered worldwide as of april 2023 [12] . as the vaccines continue to be distributed and the virus is under better control than at the beginning of the pandemic, stringent rules regarding travel restrictions, social distancing, and quarantine have been loosened worldwide. the world was slowly recovering from covid-19, and society was trying to resume a sense of normality pre-pandemic. although 69.9% of the world has received at least one dose of a covid-19 vaccine at the time of writing [22] , outbreaks are still being reported, and the number of confirmed cases and deaths due to covid-19 are still fluctuating across continents [23–33] . the available covid-19 vaccines are not 100% effective at preventing sars-cov-2 infections, i.e., individuals who have received their vaccinations may still get infected [34] . they can be asymptomatic or develop common symptoms such as fever, cough, fatigue, sore throat, and sputum production [35] . in moderate cases, the individual may have difficulty breathing or mild pneumonia, while severe pneumonia, acute respiratory distress syndrome, and organ failure can occur in severe covid-19 infections [36] . these symptoms have a negative impact on the quality of life of those infected. hence, studies have been done to explore the potential treatment options for covid-19 infections. in addition, the emergence of new variants of sars-cov-2 raises concern as the efficacy of the vaccines and treatments may be altered due to the ability of the virus to evade host immunity [21,37–40] . therefore, by understanding the currently available treatments for covid-19, and their safety and efficacy profiles, healthcare professionals can determine the best option to manage the viral infection based on the patient demographic and disease severity. 2. mechanism of action of sars-cov-2 coronaviruses are pleomorphic, enveloped particles with a single-stranded positive-sense rna as their nuclear material. they belong to the family of coronaviridae and are under the order nidovirales [41] . the sars-cov-2 genome encodes for four structural proteins, nucleocapsid (n) protein, membrane (m) protein, spike (s) protein, and envelop (e) protein and several non-structural proteins. the virus targets the multiciliated cells in the nasopharynx or trachea, or sustentacular cells in the nasal olfactory mucosa, and replicates mainly in the respiratory system of humans [42,43] . it is suggested that covid-19-related anosmia is because of the virus targeting the sustentacular cells of the host. upon exposure to sars-cov-2, the s-protein attaches to angiotensin-converting enzyme 2 (ace2) receptors on the surface of human cells. the s-protein is made up of the s1 subunit, which is responsible for binding to ace2 receptors, and the s2 subunit mediates membrane fusion [44] . the s2 subunit of the s-protein then undergoes proteolytic cleavages by transmembrane serine protease (tmprss2) to facilitate virus entry into the host cells [45,46] . after entry into the host cell, there is uncoating of the n-protein from the viral rna genome, fully releasing the viral rna into the cell cytoplasm [47] . replication and transcription processes mediated pmmb 2023, 6, 1; a0000329 4 of 21 by replication/transcription complex (rtc) made up of non-structural proteins can occur within the cell. structural proteins of the virus m, s, and e are subsequently synthesized in the cytoplasm, and nucleocapsids also form in the cytoplasm from the encapsulation of replicated genomes by the n protein. the e-proteins are small membrane proteins that are important in virus assembly, membrane permeability of the virus particle, and virus-host cell interaction [48] . after the assembly of the virions, they are transported out of the infected cells via exocytosis. stress from viral production on the endoplasmic reticulum within the host cells typically leads to cell death. viral exposure of host epithelial cells in the upper respiratory tract is detected by pattern recognition receptors (prrs) in the cytoplasm, triggering a signaling cascade for the transcription of type i and type iii interferons. toll-like receptors (tlrs) of bystander epithelial cells and local immune cells also detect sars-cov-2, thus initiating the production of chemokines and interferons. the paracrine effect of locally produced interferons further enhances the production of chemokines and interferons [49,50] . the autocrine and paracrine effects of interferons induce an antiviral cellular state by attracting immune cells to the infected site. the development of adaptive b cell and t cell responses is also promoted via the production of cytokines. when the innate or adaptive immune responses fail to clear the viral infection, the virus particles can travel to the lower respiratory tract via inhalation or dissemination along the tracheobronchial tree. the lower respiratory tract can also be the initial site of infection in covid-19 which can result in infections in the alveoli. inflammation of the alveoli limits its functionality in terms of gas exchange as sars-cov-2 primarily attacks the alveolar type 2 (at2) cells during infection [51–53] . at2 cells are responsible for lubrication in the lung by secreting pulmonary surfactants to reduce surface tension in the alveoli during respiration. moreover, they are the progenitor cells for alveolar type (at1) that mediate gas exchange [54] . individuals infected with sars-cov-2 can present with no symptoms, but a majority of covid-19 patients present with mild to moderate symptoms such as cough, fatigue, myalgia, sore throat, and gastrointestinal symptoms [2] . however, as the disease severity progresses, hypoxemia occurs, resulting in dyspnea [55] which can quickly lead to progressive respiratory failure. in severe covid-19 cases, there is a potential development of acute respiratory distress syndrome (ards) [56] , characterized as severe hypoxemia and lung infiltration with an acute onset of 7 days upon exposure. in ards, inflammation, apoptosis, necrosis, and increased alveolar-capillary permeability damage the pulmonary epithelial and endothelial cells. this is followed by pulmonary vascular leakage, which ultimately results in alveolar oedema and proteinosis, a protein build-up in the alveoli that reduces gaseous exchange, causing difficulty in breathing [57] . systemic hyperinflammation is often reported in severe covid-19 with hypoxic respiratory failure and it is characterized by the release of pro-inflammatory cytokines such as interleukin-1 (il-1), il-6, il-8, and tumor necrosis factor (tnf). during hospitalization due to covid-19, il-6 and il-8 serum concentrations are predictors of disease prognosis. there is also an increased concentration of inflammatory markers such as d-dimer, ferritin, and c-reactive protein (crp). severe covid-19 can also pmmb 2023, 6, 1; a0000329 5 of 21 cause organ damage to the heart, liver, and kidneys which can lead to multiorgan failure, shock, and finally death [58] . the mechanism of action of sars-cov-2 has been studied extensively since its discovery to aid in the development of therapeutic agents which can target specific pathways in the pathogenesis of the virus to reverse the negative effects of the infection. however, developing a novel therapeutic agent to prevent or treat covid-19 can be costly and time-consuming. therefore, clinical trials have been done since 2020 to repurpose existing drugs for covid-19. the interventions used in the clinical trials include, antivirals, antimalarials, immune modulators, inhaled gas, antifibrotics, and antioxidants [59] . as of april 2023, there have been approved treatment options that are currently being administered for the treatment of covid-19 based on the disease severity. this review explores the approved therapeutic agents and provides insights into the safety and efficacy of these interventions. 3. current therapeutic agents for covid-19 extensive research has been done to develop effective treatments with good safety profiles for covid-19. as the process of developing a novel drug can be challenging and time-consuming, researchers looked to existing drugs for answers given the urgency of the current circumstance. antivirals such as remdesivir and molnupiravir were found to have antiviral properties which could be advantageous in treating sars-cov-2 infections, thus, they have been repurposed to do so. in addition, immunomodulators such as baricitinib and anakinra were previously used in treating rheumatoid arthritis. by studying their mechanism of action and their targeted pathways in the immune system, these drugs were able to reduce inflammation in host cells, making them suitable for the treatment of covid-19. this review provides an overview of the latest therapeutic agents available to treat covid-19 (table 1). and their respective mechanisms of action during treatment (figure 1). 3.1. antivirals 3.1.1. remdesivir the first drug approved for covid-19 is remdesivir (veklury®), which received emergency use authorization in the united states on 1 may 2020 [60] . this was followed by approval for emergency use in other countries, including japan [61] , taiwan [62] , singapore [63] , and australia [64] in the following months. remdesivir is a nucleotide analogue prodrug that elicits its activity when metabolizes in the host cell to form a pharmacologically active nucleoside triphosphate [65] . it was first developed to target emerging pathogenic rna viruses and has broad antiviral activity. it has demonstrated antiviral activity against filoviruses, coronaviruses, paramyxoviruses, and pneumoviridae [66] . remdesivir targets the viral rna dependent rna polymerase (rdrp) of sars-cov-2 by competitively inhibiting the binding of adenosine triphosphate (atp) during the formation of new strands of rna. this prevents the elongation step in rna synthesis, ultimately putting a halt to further transcriptional and translational processes for the synthesis of new virions [65] . in addition, it has been suggested that the vehicle used for veklury®, namely pmmb 2023, 6, 1; a0000329 6 of 21 sulfobutylether-β-cyclodextrin (sbecd), can inhibit the binding of spike proteins to ace2 receptors, thereby preventing the entry of the virus into host cells [67] . in a clinical study to evaluate the efficacy of remdesivir, it was reported that the antiviral drug could reduce the recovery time from a median of 15 to 11 days in patients hospitalized for covid-19. the study also found that the intervention was associated with fewer days of subsequent oxygen use for patients who required supplemental oxygen during their hospitalization [68] . in moderate to severe cases of covid-19, remdesivir can be useful in reducing recovery time, mortality, adverse events, and the need for oxygen support [69] . in adults, remdesivir is given as a single 200 mg dose on the first day via the intravenous route, followed by 100 mg once daily for the rest of the treatment. remdesivir can also be given to infants who weigh more than 3kg, children, and adolescents. the duration of treatment and dose of veklury® should also be adjusted based on disease severity, comorbidities, or ongoing concurrent therapies of the patients [70] . remdesivir is generally well tolerated by patients; however, the most reported adverse events involved the hepatic and renal systems, which included elevated liver enzymes, acute kidney injury, and increased blood creatinine levels [71–73] . 3.1.2. molnupiravir molnupiravir, sold under the brand name lagevrio, was first approved for use in the united kingdom in november 2021 to treat mild to moderate covid-19 infections [74] . molnupiravir has also been authorized for use in other countries, such as the united states [75] , japan [76] , singapore [77] , and china [78] . lagevrio is an oral antiviral prodrug that is rapidly converted to ribonucleoside analogue n-hydroxycytidine (nhc) by host esterases [79] . nhc is then phosphorylated into the pharmacologically active compound, ribonucleoside triphosphate (nhc-tp) which is used by rdrp as a substrate instead of cytidine-triphosphate and uridine-triphosphate during rna synthesis. incorporating nhc-tp into the viral rna induces errors in the viral genome, thus inhibiting further replication [79,80] . prior to repurposing molnupiravir for covid-19, this antiviral has been used to treat infections caused by a range of viruses, including chikungunya virus, venezuelan equine encephalitis virus, respiratory syncytial virus, norovirus, and influenza a and b viruses, ebola virus, and human coronaviruses [80] . jayk bernal et al. reported that oral treatment of molnupiravir successfully reduced the risk of hospitalizations and deaths in at-risk, unvaccinated adults with covid-19 [81] . moreover, a study by fischer et al. found that treatment of 800mg of molnupiravir twice daily for five days resulted in significantly lower isolation of sars-cov-2 by the end of the course of treatment, thus indicating rapid viral rna clearance by molnupiravir [82] . in addition, multiple studies reported intervention with molnupiravir was well tolerated among participants, with similar incidences of adverse events across all groups [81–83] . among the most reported adverse events during molnupiravir intervention were diarrhea, nausea, headache, and dizziness [81,82,84] . however, it has been shown that administration of molnupiravir in hospitalized covid-19 patients did not yield clinical benefit [83] , thus pmmb 2023, 6, 1; a0000329 7 of 21 indicating that treatment with lagevrio should be given during early symptom onset to elicit its pharmacological effects effectively. molnupiravir is given to patients aged 18 years old and above with mild to moderate covid-19, at 800 mg twice daily for five days via the oral route [85] . 3.1.3. nirmatrelvir and ritonavir nirmatlrelvir is a novel antiviral drug developed by pfizer that acts as an orally active sars-cov-2 3c-like (3clpro) protease inhibitor [86,87] . in the generation of new virions, enzymes such as 3clpro, otherwise known as major protease (mpro), play a role in the cleavage and maturation of proteins to facilitate the viral replication process for the generation of new virions. nirmatrelvir binds directly to the active site of mpro, thereby hindering the enzyme from processing the proteins needed for viral replication [86,87] . however, nirmatrelvir is metabolized by the enzyme cytochrome p450 (cyp) 3a, hence ritonavir is added to the formulation to inhibit this process, thereby extending the half-life of nirmatrelvir in the host [88] . the formulation which constitutes nirmatrelvir and ritonavir is known as paxlovid®, with nirmatrelvir as the antiviral and ritonavir as the pharmacological enhancer. since december 2021, paxlovid® has been authorized in countries such as the united states, south korea, the united kingdom, canada, australia, europe, and japan [88] . paxlovid® is given orally as 300mg of nirmatrelvir with 100mg of ritonavir twice daily for five days for individuals with mild to moderate covid-19 [89] . it has been reported to effectively reduce the risk of progression to severe covid-19 in non-hospitalized, symptomatic adults. by day 5 of treatment, the viral load was lower with nirmatrelvir plus ritonavir compared to the placebo group [90,91] . adverse events commonly reported with the use of paxlovid® were dysgeusia, diarrhea, nausea, and myalgia, with no serious adverse events reported [88,90,92] . however, ritonavir as a cyp3a inhibitor raises concern for potential drug-drug interactions that may occur with other drugs metabolized through this pathway. for example, the conversion of clopidogrel to its active metabolite can be reduced by ritonavir which leads to insufficient inhibition of platelet aggregation. this increases the risk of blood clot formation, strokes, and heart attacks [93] . therefore, healthcare professionals need to identify any potential drug-drug interactions between paxlovid® and the current medications taken by patients to avoid causing harm during treatment. 3.1.4. ensitrelvir enstirelvir (xocova®) is a novel non-covalent, non-peptidic oral antiviral that targets the mpro in sars-cov-2 developed in japan by shionogi in collaboration with hokkaido university [94] . by targeting mpro, corresponding inhibitory effects on rdrp can occur, thus hindering viral replication. the discovery of ensitrelvir could overcome issues of low bioavailability due to low cell permeability, low metabolic stability, and low stability in the blood of its predecessors due to its non-covalent, non-peptidic features. in animal studies, xocova was found to have a long elimination half-life, good oral bioavailability, and it was effective in inhibiting intrapulmonary replication of sars-cov-2 in mice [94] . moreover, pmmb 2023, 6, 1; a0000329 8 of 21 ensitrelvir has is highly selective towards coronavirus proteases, as it did not show any inhibitory effects on human cell proteases even at high concentrations [95] . as the discovery of xocova is fairly recent at the time of writing, it has only received authorization for use in japan [96] , and it remains an investigational drug outside of japan. clinical trials for xocova® have yielded promising results, with multiple studies reporting that ensitrelvir treatment has a favorable antiviral efficacy with an acceptable safety profile. hiroshi et al. showed that intervention with ensitrelvir in mild to moderate cases significantly improved respiratory symptoms and pyrexia which are common symptoms of covid-19 [97] . ensitrelvir also decreased the median time for viral clearance by approximately 50 hours with minimal adverse events [98] . a study by shimizu et al. that included healthy japanese and white participants also showed that ensitrelvir is well-tolerated as it only elicited mild adverse events that resolved without treatment [99] . xocova® aims to treat mild to moderate covid-19 in adults and children above 12 years of age. it is to be given in a 5-day course, starting with a loading dose of 375mg (3 tablets) on the first day, subsequently followed by 125mg (1 tablet) daily [100] . given the similarities in their mechanisms, xocova® can be a potential treatment alternative for patients who are allergic or cannot tolerate paxlovid®. 3.2. immunomodulators 3.2.1. baricitinib baricitinib (olumiant®), by lilly, first gained approval for the treatment of rheumatoid arthritis. still, its ability to control inflammatory responses via inhibition of janus-associated kinases (jak) renders it a potential candidate for treating covid-19 [101] . during inflammation, cytokines bind to their respective receptors on the cells' surface, triggering the activation of jak enzymes to phosphorylate recruited signal transducers and activators of transcription (stat) molecules. the phosphorylated stat molecules translocate to the cell nucleus for gene transcription, enhancing immune responses [102,103] . inhibition of jak by baricitinib blocks the subsequent jak/stat pathway and reduces cytokine signaling, thereby preventing the progression of cytokine storm in covid-19. in addition, it has been reported that baricitinib also interrupts the passage and intracellular assembly of sars-cov-2 into target cells mediated by ace2 receptors. baricitinib binds to numb-associated kinases (nak), ap2-associated protein kinase 1 and cyclin g-associated kinase to prevent viral propagation in host epithelial cells [104,105] . to support the use of baricitinib in covid-19, a randomized controlled trial done to determine the efficacy of baricitinib in hospitalized covid-19 found that treatment with baricitinib reduced the risk of mortality in these patients by approximately 20% [106] . a separate study by marconi et al, reported similar findings and that treatment with baricitinib had a similar safety profile with standard care alone [107] . it has also been shown that combining baricitinib and other covid-19 treatments can yield favorable results. a covid-19 patient, aged 50 with non-hodgkin lymphoma successfully recovered from pmmb 2023, 6, 1; a0000329 9 of 21 covid-19 after receiving a combination treatment of remdesivir, baricitinib, tocilizumab, hydroxycholoroquine, and broad-spectrum antibiotics [108] . a separate study showed that combination therapy with baricitinib, remdesivir, and dexamethasone reduced hospitalization and recovery time, with minimum adverse events [109] . given its efficacy in treating covid-19, the rheumatoid drug has since been approved for use in japan [110] , the u.s. [111] , and singapore [112] . baricitinib is currently administered to hospitalized patients of covid-19 at 4mg once daily as part of an appropriate combination regimen for 14 days or until discharge [113] . 3.2.2. anakinra like baricitinib, anakinra (kineret®) treats rheumatoid arthritis and has been repurposed for covid-19 in the u.s. and europe [114,115] . it acts as an il-1 receptor antagonist to block the activity of il-1α and il-1β to reduce inflammation in the host. in severe cases of covid-19 where ards occurs, il-1α and il-1β are the major proinflammatory cytokines that facilitate the recruitment of immune cells and induce the production of secondary cytokines. in ards, the damaged epithelial cells release il-1α causing the recruitment of immune cells and the production of il-1β. consequently, a cascade of proinflammatory cytokines secretion is initiated, resulting in hyperinflammation, further exacerbating the infection [116] . from a pharmacokinetics perspective, anakinra has a short half-life and has rapid clearance after drug continuation. this allows for flexible dosing without excessive immunosuppression, and adverse effects such as hepatotoxicity can be better managed [117] . in patients requiring supplemental oxygen and are at risk of developing ards, anakinra is given as a subcutaneous injection of 100mg/0.67ml for 10 days [118] . this il-1 receptor antagonist has been shown to reduce the need for mechanical ventilation, which was evident as 20% out of 15 patients receiving required endotracheal intubation compared to the control group with 66.7%, indicating that the intervention was able to improve the respiratory symptoms of the patients [119] . the improvement in oxygenation in patients allows for earlier recovery, thus shortening the length of hospitalization. moreover, treatment with anakinra has been shown to reduce the mortality risk in patients with moderate to severe covid-19 compared to standard care, further supporting drug use [120] . 3.2.3. regdanvimab regdanvimab (regkirona®) is a recombinant human monoclonal antibody (mab) that targets the receptor binding domain (rbd) of the spike protein of sars-cov-2. this neutralizing antibody effectively prevents the binding of sars-cov-2 to ace2 receptors on host epithelial cells, thus inhibiting the virus's entry and stopping viral replication [121] . regdanvimab is given as a single intravenous infusion of 40mg/kg. first approved in south korea for treating covid-19 in 2021, it is now available in europe and australia for mild to moderate sars-cov-2 infections [122] . the mab has been indicated for use in mild to moderate covid-19 as it was shown to significantly reduce the risk of disease progression pmmb 2023, 6, 1; a0000329 10 of 21 to severe infection during hospitalization [123] . furthermore, a study done in south korea showed consistent results with previous studies. in the earlier stages of covid-19, treatment with regdanvimab can reduce the need for additional therapeutic options such as remdesivir, dexamethasone, and supplemental oxygen. treatment with regdanvimab was generally well-tolerated, and no serious adverse events occurred throughout the study [124] . regdanvimab has also been reported to be effective against mild to moderate covid-19 caused by sars-cov-2 of the delta variant [125] . besides, a retrospective study showed that administering regdanvimab and remdesivir improved clinical outcomes of patients with severe covid-19. the duration of oxygen supplementation was shortened with the combination of regdanvimab and remdesivir instead of remdesivir alone [126] . on that account, regdanvimab has the potential to be used in conjunction with other covid-19 treatments to greatly improve the clinical outcomes in severe covid-19. 3.2.4. sotrovimab sotrovimab, marketed under the brand name xevudy®, is a recombinant human mab developed by glaxosmithkline that has been authorized for use in mild to moderate covid-19 in australia, europe, the united kingdom, and the u.s [127] . it binds to a highly conserved epitope in the s protein of sars-cov-2, thereby inhibiting the virus from binding to ace2 receptors. targeting the highly conserved epitope lowers the risk of developing viral resistance towards sotrovimab [128] . binding of sotrovimab with high affinity to the ace2 receptors prevents virus entry into host cells at the first stages of viral exposure. the subsequent viral replication and signaling cascades are prevented after which inflammation can be minimized to avoid developing severe respiratory symptoms and cellular damage in the respiratory system. to achieve effective treatment, sotrovimab is given as 500mg in a single intravenous infusion over 15 minutes in mild to moderate infections. in a study involving 1057 participants, a single dose of sotrovimab was shown to be effective in reducing the risk for hospitalization in high-risk mild to moderate covid-19 patients and deaths compared to the placebo group [128] . a separate study involving 583 participants had consistent results, with sotrovimab reducing the risk of progression in high-risk patients with mild-moderate covid-19 and a lowered risk of adverse events with sotrovimab compared to the placebo group [129] . in a rapid review and meta-analysis done by amani et al., sotrovimab was also found to reduce mortality rates and hospitalizations in patients infected with the omicron or delta variants of the coronavirus [130] . all in all, sotrovimab is an effective treatment for mild to moderate covid-19 which can prevent disease progression and its efficacy is broad enough to cover the newer variants of sars-cov-2. 3.2.5. tocilizumab tocilizumab is a recombinant humanized mab marketed under the brand name actemra® which has been approved to treat covid-19 in the u.s. [131] , japan [132] , and australia [133] . the pan american health organization has also made the mab available to 15 countries in latin america and the carribean [134] . tocilizumab is an il-6 receptor antagonist as it competitively inhibits the binding of il-6 to its receptor. this inhibits further signal pmmb 2023, 6, 1; a0000329 11 of 21 transduction by il-6, reducing subsequent signaling cascades that induce inflammatory responses [135,136] . it has been shown that there is a significant increase in pro-inflammatory cytokines, such as il-6, in patients with covid-19. therefore, the inhibitory effects by tocilizumab can prevent the initiation of hyperinflammatory responses to improve the symptoms of covid-19. this can be useful in treating severe covid-19 as the state of hyperinflammation can be reduced to mitigate the progression to ards. the recommended dose for tocilizumab in the treatment of moderate to severe covid-19 is 8 mg/kg as a single intravenous infusion. various studies on the efficacy of tocilizumab in patients with severe covid-19 found a reduction in the likelihood of progression to mechanical ventilation [137,138] . the time needed for clinical improvement was also reduced, along with a shorter duration of invasive ventilation [139] . however, one study showed that tocilizumab effectively improved symptoms of severe covid-19 until day 14. further, using up to 28 days did not produce significantly better clinical status or lower mortality than a placebo [140] . therefore, in cases where tocilizumab is administrated, should the symptoms not improve by day 14, an alternative treatment should be considered. on the flip side, a systematic review and meta-analysis by hariyanto et al. in 2021 found that tocilizumab reduced mortality rates. still, the severity of the disease and length of hospital stay was not altered [141] . nonetheless, the review did not standardize the dosing, route of administration, and timing of administration which might have affected the outcomes of their study. given that tocilizumab had only recently been used to treat covid-19, further research and clinical trials need to confirm its efficacy and safety. 3.2.6. vilobelimab vilobelimab, a novel anti-c5a mab is the latest addition to the list of approved covid-19 treatments by the food and drug administration (fda) [142] . marketed under the brand name gohibic®, it targets c5a, a component in the complement system of patients with severe covid-19 requiring invasive oxygen support ventilation. during severe sars-cov-2 infection, there is an activation of the complement system in the lungs, resulting in the proteolytic cleavage of the complement factor c5. this process generates c5a, an anaphylatoxin, which facilitates the recruitment of myeloid cells to the lungs. these immune cells then secrete pro-inflammatory cytokines, causing a cytokine storm. prolonged inflammation caused by c5a can lead to neutrophil-mediated viral lung damage [143,144] . vilobelimab binds with high affinity to c5a to prevent binding to c5a receptors, thus inhibiting the subsequent signaling cascades, ultimately reducing inflammation and lung damage. a multicentre, double-blind, randomized, placebo-controlled, phase 3 trial showed that vilobelimab could improve the survival rates of patients receiving invasive ventilation. the intervention with intravenous vilobelimab at 800mg effectively decreased c5a concentrations in the vilobelimab group, significantly reducing mortality rates [144] . the clinical trials took place across multiple countries, and vilobelimab was given as an additional therapy to standard care based on the treatment guidelines in various countries. on that account, it is vital to ensure that standard care is already given to the infected patients pmmb 2023, 6, 1; a0000329 12 of 21 prior to administrating vilobelimab. the safety of vilobelimab was also evaluated by bauer et al. in which the treatment was well tolerated with zero vilobelimab-specific adverse events reported [143] . nevertheless, as vilobelimab suppresses the activity of the immune system to a certain extent, patients can be vulnerable to bacterial, fungal or viral infections when hospitalized. table 1. the various therapeutic agents for covid-19. therapeutic agent mechanism of action route of administration and dosage indication (severity of covid-19) antivirals ensitrelvir (xocova®) protease (3clpro/mpro) inhibitor po, 375mg on day 1, 125mg once daily on subsequent days for 15 days mild to moderate molnupiravir (lagevrio®) viral genome disruption via integration into viral rna genome po, 800mg once daily for 5 days mild to moderate nirmatrelvir and ritonavir (paxlovid®) protease (3clpro/mpro) inhibitor po, 300mg nirmatrelvir with 100mg ritonavir for 5 days mild to moderate remdesivir (veklury®) rdrp inhibitor iv, 200mg on day 1, 100mg once daily on subsequent days moderate to severe immunomodulators regdanvimab (regkirona®) neutralising antibody against s protein of sars-cov-2 iv, single infusioin of 40mg/kg mild to moderate sotrovimab (xevudy®) neutralising antibody against s protein of sars-cov-2 iv, single infusion of 500mg mild to moderate anakinra (kineret®) il-1 receptor antagonist sc, 100mg/0.67ml for 10 days moderate to severe baricitinib (olumiant®) jak inhibitor po, 4mg once daily for 14 days moderate to severe tocilizumab (actemra®) il-6 receptor antagonist iv, infusion of 8mg/kg moderate to severe vilobelimab (gohibic®) c5a inhibitor iv, 800mg for maximum 6 doses on day 1, 2, 4, 8, 15, 22 moderate to severe pmmb 2023, 6, 1; a0000329 13 of 21 * iv: intravenous infusion, po: oral, sc: subcutaneous injection. figure 1. illustration of the mode of action of sars-cov-2 and active site of covid-19 treatments. this figure was partly generated using servier medical art. 4. conclusion covid-19 is the first documented coronavirus pandemic in the history of humankind. it has negatively affected the global economy and the physical and mental health of the public [145,146] . the fluctuating numbers of confirmed cases and deaths caused by sars-cov-2 remain a concern as there is an underestimation and under-reporting of cases [147,148] . developing therapeutic agents such as antivirals and immunomodulators to manage covid-19 infections provides a positive outlook for society in recovering from the pandemic. covid-19 treatment should be administered during early symptom onset to reduce viral load, prevent disease progression into hyperinflammatory state, reduce respiratory symptoms, and ultimately prevent death. further studies can be done to explore the potential advantages of combining these treatments to achieve significantly beneficial results in covid-19. since the initial discovery of sars-cov-2, the virus has evolved into several variants: alpha, beta, gamma, delta, and omicron. with each evolution, the virus has increased transmissibility and evasion of the host immune system, enabling it to continuously spread worldwide [21] . the increase in selective pressures on the coronavirus can potentially result in the generation of new variants which are resistant to the treatments for covid-19 that are readily available. moghadasi et al. found that mutations in mpro of sars-cov-2 confer resistance towards nirmatrelvir and ensitrelvir [149] . in addition, the efficacy of the antivirals towards different variants of the coronavirus varies. for instance, antiviral drugs such as remdesivir and molnupiravir target the rdrp of sars-cov-2, but the mutation of rdrp in the omicron pmmb 2023, 6, 1; a0000329 14 of 21 variant potentially decreases the efficacy of these drugs against the variant [150] . it is currently unclear what magnitude of resistance in the virus will render treatment failure. therefore, genetic surveillance on emerging variants of sars-cov-2 and strategies to minimize the spread of viral resistance should be implemented to manage covid-19 effectively. moreover, research is ongoing to develop new therapeutic agents to treat covid-19 at different stages of disease. with the equal distribution and effective use of vaccines and treatment for covid-19 worldwide, there is emerging hope that society can transition into the endemic phase of covid-19 at a global scale in the foreseeable future. author contributions: literature search, data analysis, writing – original draft preparation, writing – final manuscript preparation, k-yl; writing – review and editing, proofreading, lt-ht, jw-fl, k-wh, and k-gc. writing – review and editing, conceptualization, vl. funding: no external funding was provided for this research. acknowledgment: parts of the figure were drawn by using pictures from servier medical art, licensed under a creative commons attribution 3.0 unported license (https://creativecommons.org/licenses/by/3.0/) conflicts of interest: the authors declare no conflict of interest. references 1. letchumanan v, ab mutalib n-s, goh b-h, et al. novel coronavirus 2019-ncov: could this virus become a possible global pandemic. prog microbes mol biol 2020; 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omicron variant. new engl j med 2022; 386(10): 995-998. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2021, 4, 1; a0000236. doi: 10.36877/pmmb.a0000236 http://journals.hh-publisher.com/index.php/pmmb original research article oral dietary supplements use among healthcare workers during the covid-19 pandemic in malaysia heoy teng lee1, hong chuan loh1*, siti nur liyana ramlee1, irene looi1,2 article history 1clinical research centre, hospital seberang jaya, ministry of health malaysia, seberang jaya 13700, penang, malaysia; leeheoyteng@gmail.com (htl); lohhongchuan@gmail.com (hcl); ctyana.ramlee@yahoo.com (snlr) 2medical department, hospital seberang jaya, ministry of health malaysia, seberang jaya 13700, penang, malaysia; irenelooi@yahoo.com (il) *corresponding author: hong chuan loh; clinical research centre, hospital seberang jaya, ministry of health malaysia, seberang jaya 13700, penang, malaysia; lohhongchuan@gmail.com (hcl) received: 9 august 2021; received in revised form: 9 september 2021; accepted: 10 september 2021; available online: 20 september 2021 abstract: healthcare workers (hcws) must be aware of the latest data supporting or refuting the use of oral dietary supplements (ods) in order to disseminate evidence-based health information and help patients make informed decisions. nevertheless, there is relatively scant data on the prevalence of dietary supplement use among hcws, the types of dietary supplements recommended to patients by hcws, and their reasons for recommending these products, particularly during the covid-19 outbreak. this study examined the prevalence of ods use among surveyed hcws, considered the types of ods used and recommended by those hcws, identified the reasons given by those hcws for using or not using ods, and determined whether those hcws were recommending or not recommending ods to their patients during the covid-19 pandemic. this cross-sectional study targeted all hcws working at a district hospital in malaysia and was conducted via a self-administered online survey. the survey revealed that 67.3% of hcws did not recommend ods for patient use during the pandemic, despite 55.3% of hcws reported personal use of ods during the pandemic. type of hcws (p=0.001), monthly household income (p=0.019), prior ods use (p<0.001) and recommendation of ods to patients (p<0.001) were significantly associated with personal ods use during the pandemic. vitamin c was the most commonly used (81.3%) and recommended (95.0%) ods. “to maintain overall health and wellness” was the most common reason for personal ods use and recommendation to patients (83.3% & 79.2%). given the increasing rate of ods use during the pandemic, further research should be done so that evidence-based recommendations can be formulated to ensure patient safety. keywords: oral dietary supplements; healthcare workers; personal use; recommendation; covid-19; pandemic 1. introduction individuals exposed to risks and uncertainties tend to seek affordable self-protective measures such as complementary and alternative medicine (cam), including taking oral mailto:lohhongchuan@gmail.com pmmb 2021, 4, 1; a0000236 2 of 16 dietary supplements (ods), to maintain good health, mitigate risk, and make an unpredictable condition more manageable[1,2]. ods are products that can be taken by mouth (such as a tablet, capsule, powder, or liquid), and include vitamins, minerals, herbs or other botanicals, amino acids, enzymes, tissues from organs or glands, or extracts of these. ods are a common type of cam[3,4]. malaysia has undergone rapid urbanization in recent years resulting in the transformation of dietary intake patterns, including the consumption of dietary supplements. the most common ods consumed by malaysian adults was vitamin c[3, 5]. since the onset of the covid-19 outbreak, some online media platforms have been promoting dietary supplements such as vitamins c and d for the treatment and prevention of coronavirus infection, although there is still a lack of evidence and guidance for the use of micronutrient supplements[6–8]. recent scientific literature on the use of vitamins and minerals as prevention and treatment for critically ill patients with covid-19 appear to support the idea of high-dose of vitamin c and other micronutrient intervention due to their excellent safety profile, low-cost and immediate availability[9–11]. the personal use of ods by healthcare workers (hcws) and the recommendation by hcws for others to use ods is of interest for several reasons. hcws are important sources of dietary information for patients and are expected to provide sound advice about the use of various dietary supplements. some hcws provide information and dietary advice based on their personal experiences and eating habits. therefore, we explored personal ods use among hcws themselves since their personal health habits could affect their decision to recommend a dietary supplement[12-14]. hcws must be aware of the latest data supporting or refuting ods use in order to disseminate evidence-based health information and help patients make informed decisions. previous studies have shown that dietary supplements are used by a large proportion of the general public to treat and prevent diseases. nevertheless, there is relatively scant data on the prevalence of dietary supplement use among hcws. there is even less available up-to-date information regarding the types of dietary supplements recommended to patients by hcws and their reasons for recommending these products, particularly during the covid-19 outbreak[15–17]. therefore, this study was done to (a) examine the prevalence of ods use among surveyed hcws, (b) consider the types of ods used and recommended by those hcws, (c) identify the reasons given by those hcws for using or not using ods, and (d) determine whether those hcws were recommending or not recommending ods to their patients during the covid-19 pandemic. 2. materials and methods 2.1. ethics statement this study was registered with the malaysian national medical research register (nmrr-20-2275-56853) and received ethical approval from the medical research and ethics committee, ministry of health malaysia. pmmb 2021, 4, 1; a0000236 3 of 16 2.2. study design and participants this descriptive cross-sectional study was conducted via an anonymous, selfadministered online survey using a convenient sampling method. the targeted study population comprised all of the hcws (including doctors, nurses, pharmacists, allied health professionals, and assistant medical officers) working at hospital seberang jaya (hsj), malaysia. the time period for data collection was between 4th november 2020 and 4th december 2020. the survey was conducted using a self-administered online survey method in order to comply with the standard operating procedures imposed by the government during the covid-19 pandemic. hcws aged 18 years old and above were eligible to participate in the survey and were required to complete the electronic informed consent form. the study excluded non-malaysian people and those who were unable to read or understand the questionnaire. 2.3. sample size cochran’s formula was used to calculate the minimum recommended sampling size and an estimated proportion of dietary supplements users of 0.760 was calculated from a previous study[15]. the sample size required for this study with a confidence level of 95% and ± 5% precision was 280 study participants. a further 30% (n=84) was added to compensate for nonresponse, resulting in a final sample size of 365[18]. 2.4. survey instrument because previous research tools on ods use among hcws during the covid-19 outbreak were unavailable, a semi-structured survey questionnaire instrument was developed for this study after doing a thorough literature review[3,16,17,19–25] and seeking opinions from experts. to assess the content and face validation of the survey instrument, the questionnaire was reviewed by five researchers to evaluate appropriateness, relevancy, clarity, adequacy, and organization of the questions[26]. the questionnaire was pilot-tested to examine length, clarity, and difficulty of the questions, and items were revised based on the results. responses obtained from the pilot study were not included in the final data analysis of this study. the study questionnaire was made available in the english language. the finalised survey included 24 items with both multiple-choice and open-ended questions and consisted of four major sections. the participants were asked to complete their socio-demographic information, such as age, gender, marital status, ethnicity, religion, education level, type of hcws, and monthly income, in the first section. the second section consisted of four questions on health-related characteristics. the third part had six questions about participants’ personal ods use before and during the covid-19 pandemic (11th march 2020 onwards); types of ods consumed; reasons for taking or not taking ods; and average expenditure per month for ods. available choices for types of ods included vitamins a, b complex, b12, c, d, and e, as well as zinc, selenium, magnesium, multivitamin/multimineral (mvmm), omega-3 and probiotics, due to their potential utility pmmb 2021, 4, 1; a0000236 4 of 16 in covid-19 as had been highlighted in different studies[24,27–31]. herbal products were excluded because they are not taught in traditional health care professional education programs. the fourth section contained five questions regarding the recommendation by hcws that patients should or should not use ods during the covid-19 pandemic. the last section asked questions relating to participants’ educational background regarding ods and their interest in receiving continuing education regarding the subject of ods. 2.5. statistical analysis methods the collected data of 367 participants was analysed statistically using the statistical package for social sciences (spss) version 20.0. descriptive statistics were used to analyse the prevalence of ods use and the socio-demographic and health-related characteristics of all hcws involved in the study. data on types of ods used and recommended by hcws to patients; reasons for using or not using ods; and reasons for recommending or not recommending ods to patients during the covid-19 pandemic were summarized using frequency counts (n) and percentages (%). mean and standard deviation were calculated for the normally distributed continuous data whereas continuous data that were not normally distributed were reported using median and interquartile range (iqr). the ods users and non-ods users were compared by using the mann-whitney test. pearson chi-square or fisher's exact tests were performed to determine the categorical variables related to ods use and any associations between ods use and recommendation of ods to patients. tests were 2-tailed and p-value of less than 0.05 was considered statistically significant for all analyses. 3. results a total of 367 hcws participated in this study. table 1 summarised the sociodemographic and health-related characteristics of all participants. the median age of participants was 32.00 years (iqr = 9.00). most of the participants were female (87.2%), married (69.8%), malays (64.9%) and muslims (66.2%). more than 90% of the participants received tertiary education, did not smoke, and did not consume alcohol. among the participants surveyed, 52.6% were nurses, 21% were pharmacists, 15.3% were doctors, and 11.2% were allied health professionals. half of the participants (51.0%) had monthly household income ranging from rm 4 850 to rm 10 959. about 80% of the participants had no underlying co-morbidities. a majority (99.5%) of them were not infected with covid19. however, there were 2 unconfirmed cases at the time the survey was conducted. the percentage of participants who had used ods before the covid-19 pandemic was 47.7% but during the pandemic 55.3% of participants used ods. despite that, the survey revealed that 67.3% of hcws did not recommend ods for patient use during the pandemic. only 120 of the hcws (32.7%) recommended ods to patients during the covid-19 pandemic. pmmb 2021, 4, 1; a0000236 5 of 16 table 1. sociodemographic and health characteristics of all participants (n=367). characteristics n (%) age (years) a 32 (9.0) gender female 320 (87.2) male 47 (12.8) marital status married 256 (69.8) single 104 (28.3) widowed 4(1.1) divorced 3(0.8) ethnicity malay 238 (64.9) chinese 78 (21.3) indian 48 (13.1) othersb 3(0.8) religion islam 243 (66.2) buddhism 68 (18.5) hinduism 40 (10.9) christianity 16 (4.4) educational level secondary school 14 (3.8) pre-university 165 (45.0) undergraduate 132 (36.0) postgraduate 56 (15.3) type allied health professions 41 (11.2) doctors 56 (15.3) pharmacist 77 (21.0) nurse 193 (52.6) monthly household income < rm 4850 per month 133 (36.2) ≥ rm 4850 – rm 10 959 per month 187 (51.0) > rm 10 959 per month 47 (12.8) smoking status no 359 (97.8) yes 8 (2.2) alcohol consumption no 341 (92.9) yes 26 (7.1) co-morbid(s) present no 295 (80.4) yes 72 (19.6) covid-19 status no 365 (99.5) possibly infected 2 (0.5) infected 0 (0.0) pmmb 2021, 4, 1; a0000236 6 of 16 characteristics n (%) prior ods use no 192 (52.3) yes 175 (47.7) use ods during covid-19 pandemic no 164 (44.7) yes 203 (55.3) recommendation of ods to patients no 247 (67.3) yes 120 (32.7) covid-19 = coronavirus disease 2019, ods = oral dietary supplements, rm = ringgit malaysia. a median (iqr), b including kadazan, dusun, and siamese table 2 summarizes all types of ods used and recommended by hcws to their patients. among 367 participants, 203 (55.3%) used ods and 120 (32.7%) recommended ods to patients during the covid-19 pandemic. it was found that the use of vitamin c was the highest (81.3%), followed by multivitamin/ multimineral (mvmm) supplement (17.7%), vitamin b complex (17.7%), omega-3 (10.3%), and probiotics (9.9%). both vitamin c (95.0%) and mvmm (32.5%) were also the most common ods recommended to patients during the pandemic, followed by omega-3 (12.5%), vitamin b complex (10.0%), and probiotics (10.0%). hcws were also asked how much on average they spent on ods per month during the covid-19 pandemic. the majority of the respondents (96.6%) reported their monthly expenses on ods in median (iqr) was rm100.00 (110.00). table 2. types of ods used and recommended by hcws during the covid-19 pandemic. types of ods used personally (n = 203) recommended to patients (n = 120) n % n % vitamin a 6 3.0 3 2.5 b complex vitamin 36 17.7 12 10.0 vitamin b12 12 5.9 3 2.5 vitamin c 165 81.3 114 95.0 vitamin d 7 3.4 3 2.5 vitamin e 18 8.9 6 5.0 zinc 14 6.9 4 3.3 selenium 1 0.5 0 0 magnesium 6 3.0 1 0.8 multivitamin/multimineral 36 17.7 39 32.5 omega-3 21 10.3 15 12.5 probiotics 20 9.9 12 10.0 ods = oral dietary supplements, hcw = healthcare workers, covid-19 = coronavirus disease 2019 our survey questions on the reasons for using and recommending ods during the covid-19 pandemic are summarized in table 3. the most frequently reported reason for taking ods was “to maintain overall health and wellness” (83.3%). the second highest reason was “to prevent getting infection” (58.6%) followed by “to strengthen the immune system” (24.6%). similarly, the most commonly used reasons for recommending ods to pmmb 2021, 4, 1; a0000236 7 of 16 patients were “to maintain overall health and wellness” (79.2%), “to prevent getting infection” (74.2%), and “to strengthen the immune system” (37.5%). table 3. reasons for using and recommending ods during the covid-19 pandemic. reasons using ods recommending ods n = 203 n = 120 n % n % to maintain overall health and wellness 169 83.3 95 79.2 inadequate dietary intake and nutritional deficiency 20 9.9 17 14.2 to prevent getting an infection 50 24.6 45 37.5 to strengthen the immune system 119 58.6 89 74.2 to help cope with the adverse effects of conventional treatment 3 1.5 4 3.3 to help in the treatment of a specific disease 12 5.9 8 6.7 conventional treatment/modern medicine is not effective 1 0.5 0 0.0 ods = oral dietary supplements, covid-19 = coronavirus disease 2019. meanwhile, table 4 presented the feedback of the hcws on the reasons for not using and not recommending ods during the covid-19 pandemic. nearly 44% of them reported “dietary intake is adequate” as the main reason for not using ods. other reasons for not using ods included “worried about its safety and adverse effects” (31.1%) which was second, followed by “do not know much about dietary supplements” (17.1%) and “worried about its potential drug-dietary supplement interactions” (11.6%). in contrast, the most common reasons for not recommending ods to patients were “worried about its safety and adverse effects” (34.8%) and “worried about its potential drug-dietary supplement interactions” (31.6%), followed by “lack of scientific evidence on its effectiveness in disease treatment” (23.5%), “dietary intake is adequate” (22.3%), and “do not know much about dietary supplements” (18.6%). table 4. reasons for not using and not recommending ods during the covid-19 pandemic. reasons not using ods n = 164 not recommending ods n = 247 n % n % lack of scientific evidence on its effectiveness in disease treatment 13 7.9 58 23.5 worried about its potential drug-dietary supplement interactions 19 11.6 78 31.6 worried about its safety and adverse effects 51 31.1 86 34.8 dietary supplements are expensive 11 6.7 28 11.3 do not know much about dietary supplements 28 17.1 46 18.6 satisfied with conventional treatment/modern medicine 17 10.4 18 7.3 dietary intake is adequate 72 43.9 55 22.3 ods = oral dietary supplements, covid-19 = coronavirus disease 2019. the association among socio-demographic, health-related characteristics, and ods use during the covid-19 pandemic is depicted in table 5. we observed that personal ods use during the covid-19 pandemic was not associated with age, gender, marital status, ethnicity, religion, educational level, smoking status, alcohol consumption, or underlying non-communicable diseases. however, type of hcws (p=0.001), monthly household pmmb 2021, 4, 1; a0000236 8 of 16 income (p=0.019), prior ods use (p<0.001), and recommendation of ods to patients (p<0.001) were significantly associated with personal ods use during the covid-19 pandemic, tested using pearson chi-square test. table 5. association between socio-demographic, health-related characteristics, and ods use during the covid-19 pandemic. characteristics n ods users n = 203 n (%) non-users n = 164 n (%) p-value age in years, median (iqr) 367 32.0 (8.0) 31.5 (11.0) 0.811a gender 0.060b female 320 183 (90.1) 137 (83.5) male 47 20 (9.9) 27 (16.5) marital status 0.471c single 104 55 (27.1) 49 (29.9) married 256 146 (71.9) 110 (67.1) widowed 4 1 (0.5) 3 (1.8) divorced 3 1 (0.5) 2 (1.2) ethnicity 0.662c malay 238 128 (63.1) 110 (67.1) chinese 78 47 (23.2) 31 (18.3) indian 48 27 (13.3) 21 (12.8) othersd 3 1 (0.5) 2 (1.2) religion 0.164b islam 243 129 (63.5) 114 (69.5) buddhism 68 43 (21.2) 25 (15.2) christianity 16 6 (3.0) 10 (6.1) hinduism 40 15 (7.4) 25 (15.2) educational level 0.232b secondary school 14 5 (2.5) 9 (5.5) pre-university 165 88 (43.3) 77 (47.0) undergraduate 132 74 (36.5) 58 (35.4) postgraduate 56 36 (17.7) 20 (12.2) type 0.001b allied health professions 41 12 (6.0) 29 (17.7) doctors 56 28 (13.8) 28 (17.1) pharmacist 77 51 (25.1) 26 (15.9) nurse 193 112 (55.2) 81 (49.4) monthly household income 0.019b < rm 4850 per month 133 63 (31.0) 70 (42.7) ≥ rm 4850 – rm 10 959 per month 187 107 (52.7) 80 (48.8) > rm 10 959 per month 47 33 (16.3) 14 (8.5) smoking status 0.736c no 359 198 (97.5) 161 (98.2) yes 8 5 (2.5) 3 (1.8) alcohol consumption 0.284b no 341 186 (91.6) 155 (94.5) yes 26 17 (8.4) 9 (5.5) co-morbid(s) present 0.827b no 295 164 (80.8) 131 (79.9) yes 72 39 (19.2) 33 (20.1) pmmb 2021, 4, 1; a0000236 9 of 16 characteristics n ods users n = 203 n (%) non-users n = 164 n (%) p-value prior ods use <0.001b no 192 31 (15.3) 161 (98.2) yes 175 172 (84.7) 3 (1.8) recommendation of ods to patients <0.001b no 247 104 (51.2) 143 (87.2) yes 120 99 (48.8) 21 (12.8) covid-19 = coronavirus disease 2019, ods = oral dietary supplements, rm = ringgit malaysia. a mannwhitney test, b pearson chi-square test, c fisher’s exact test, d including kadazan, dusun, and siamese. table 6 shows that most of the hcws involved in this study did not receive any formal education or training about ods (77.9%) but were interested in receiving continuing education about ods (86.9%). table 6. hcws’ education background and interest in receiving continuing education about ods (n = 367). variables n (%) received formal education or training about ods no 286 (77.9) yes 81 (2.1) interested in receiving continuing education about ods no 48 (13.1) yes 319 (86.9) hcw = healthcare workers, ods = oral dietary supplements, tv = television. a including google search and nurse. 4. discussion to our knowledge, this study is the first in the country to examine the use of ods among hcws during the covid-19 pandemic. socio-demographic and health-related characteristics were explored to understand the association of these factors with ods use among hcws. as such, the findings of this study will hopefully help support the development of nutritional guidelines and policies, education programs, and future studies related to dietary supplements in order to guide hcws in providing the best advice to patients. according to our survey, almost half of hcws had used ods before the covid-19 pandemic. the malaysian adults nutrition survey 2014 (mans 2014) conducted during a non-pandemic period among malaysian adults showed that the prevalence of ods use among adults in the general population was 59.1%[3]. notably, the data showed that the use of ods was less common among hcws as compared to the general population during the nonpandemic period. nevertheless, the consumption of ods among hcws increased during the covid-19 pandemic as indicated by our study. the recent plifecovid-19 online study conducted in poland revealed that ods was consumed more often during the first wave of the pandemic than in the second[32]. this trend might be due to the belief that ods can give benefits and potentially prevent and help manage a variety of health conditions, including covid-19 and other infections, although evidence to prove their efficacy with covid-19 pmmb 2021, 4, 1; a0000236 10 of 16 is still lacking[8,24,27,31,33–35]. similarly, a study in korea also demonstrated an overall rise in cam use by 21.4% during the middle east respiratory syndrome outbreak among outpatients[16]. another study also found that dietary supplements were one of the most commonly used cam by patients with dengue fever[21]. therefore, like other members of the public, hcws could be expected to consume dietary supplements for their health benefits during the outbreak of disease. our results suggest that the type of hcw (e.g., doctor, nurse, etc.) influenced the personal use of ods during the covid-19 pandemic. concerning each type of hcw, ods use was higher among nurses (55.2%), followed by pharmacists (25.1%), doctors (13.8%) and allied health professionals (6.0%). for comparison, the healthcare professionals impact study found that 72% of physicians and 89% of nurses used dietary supplements regularly, occasionally, or seasonally, whereas 51% of physicians and 59% of nurses used dietary supplements regularly[15]. it is challenging to obtain reliable estimates of the prevalence of ods use due to differences in definitions, frequency of use, diversity of dietary supplement formulations, and availability of dietary supplements[5,36]. overall, previous studies showed the prevalence of ods use varying among hcws, ranging from 21% to 88%[12,37–39]. this study also found that monthly household income was significantly associated with the use of ods. those with a monthly income of more than rm 4 850 showed higher ods use than those with a monthly income of less than rm 4 850. interestingly, those with a monthly income of more than rm 10 959 showed the least use of ods despite having a higher income. nevertheless, our findings were consistent with previous studies suggesting that the relationship between ods use among hcws and monthly income remains unclear[3,40]. apart from that, our study revealed a positive association between prior ods use, recommendation of ods to patients, and the use of ods during the covid-19 pandemic. among ods users during the pandemic, most of them had used ods before the pandemic. our study found that the majority of hcws did not recommend ods to patients. however, among those who did recommend ods to patients, a large proportion of them used ods during the covid-19 pandemic themselves, which result was similar to other studies [17,41,42]. one previous study revealed that personal use of dietary supplements was associated with a twofold increase in the likelihood that a pharmacist would recommend a dietary supplement to others[43]. our results showed that the most commonly used and recommended ods during the pandemic was vitamin c, followed by mvmm, and these results concurred with previous studies conducted in malaysia and other countries before the covid-19 outbreak[3,15,16,38,44]. another recent study conducted in saudi arabia during the covid-19 pandemic discovered that almost all of the ods users reported taking vitamin c[45]. the high use of vitamin c could be related to recent studies that promoted vitamin c's potential benefits as a prophylactic intervention to combat viral infection and as an immune modulator to mitigate the risk of contracting severe covid-19[10,46–49]. selenium appeared to be the least consumed and recommended among hcws which could be due to limited knowledge about this pmmb 2021, 4, 1; a0000236 11 of 16 supplement among hcws[50]. nevertheless, previous studies suggest that it could contribute to the prevention and management of covid-19 and other viral infections[50,51]. our survey also revealed that hcws commonly used ods to maintain their overall health and wellness, strengthen their immune system, and prevent getting an infection during the covid-19 pandemic. these findings were similar to the reasons reported in previous surveys of other hcws and the general population during the non-pandemic period[15,17,52,53]. most of them used ods as a complementary therapy and a primary prevention intervention rather than a substitute for conventional treatment. ods should not replace a healthy and balanced diet. promoting the health effects of ods in a generally healthy population and, in particular, their effectiveness during a pandemic warrant further investigation[34, 54, 55]. since ods, especially vitamin c and mvmm, are thought to boost immunity against infection during the pandemic, more studies or clinical trials should be conducted to explore their possible beneficial effects in prevention or management of disease. it is crucial to explore the exact intracellular mechanisms and anti-inflammatory and anti-oxidative activities of ods[49,56]. while the majority of participants in this study used ods during the pandemic, there were still almost half of them (44.7%) who did not. most of those who did not use ods believed that their dietary intake is adequate. therefore, dietary guidelines provided by governing bodies play a crucial role in ensuring proper nutritional practices, such as eating a balanced diet and maintaining optimal health[57]. most of the hcws who did not recommend the use of ods to patients had concerns about their safety and possible adverse effects as well as potential drug-dietary supplement interactions. indeed, many marketed ods or nutraceutical products are not strictly regulated, thus their safety and efficacy can be questioned[58]. it is important to evaluate the evidence regarding the use of such products especially relating to their safety, adverse effects, and drug interactions. data from ongoing large randomized trials will be necessary to establish the role of ods during a disease outbreak or pandemic. in addition, our survey also revealed that most of the hcws had not received any formal education or training regarding ods but were interested to learn about it. in agreement with prior studies, most of the hcws who were likely to recommend that their patients consume ods would be interested in continuing medical education courses and training on various ods therapies[59,60]. the majority of hcws expressed a marked preference and interest in integrative medicine modalities in the management of certain types of infectious diseases and their complications[60]. accordingly, it is hoped that our findings will help government, policymakers, and international organizations in efforts to provide effective guidelines and evidence-based recommendations for patient safety. 5. limitation several limitations were encountered throughout this study. this was a questionnairebased cross-sectional study in which we relied completely on information provided by the participants, and their responses could have been biased or based on a misunderstanding of pmmb 2021, 4, 1; a0000236 12 of 16 the questions. the results obtained are based on self-reported data rather than on direct observation or by interview which could over or underestimate the true use of ods. our study was conducted at only one district hospital and it is possible that the results may not be generalized to the majority of hcw in our country. in addition, the survey sample was not selected with true randomization methods but rather relied on a convenience sample. these limitations may affect the generalizability of the results. nevertheless, it provides useful information and may serve as a benchmark for further study. 6. conclusion in summary, further research is required before evidence-based recommendations can be formulated. hcws still share responsibility for providing correct information about ods use. given the weak evidence relating to the use of many ods, there is a need to strengthen the health science curriculum to produce better informed future professionals. lastly, the use of ods should be evidence-based to ensure patient safety. author contributions: conceptualization by htl, hcl; methodology prepared by htl, hcl; validation by htl, hcl, il; formal analysis by htl, hcl; investigation by htl, hcl; resources by htl, hcl, il; writing original draft preparation by htl; writing review and editing by hcl, snlr, and il; supervision by hcl, il; project administration by htl, hcl, snlr. all authors read and approved the final manuscript. funding: no external funding was provided for this research. acknowledgments: the authors would like to honour all the hcws around the world who dedicated themselves bravely and tirelessly to patient care in the fight against covid-19. also, the authors would like to express gratitude to all the participants for their cooperation in completing the survey questionnaire, all of whom directly and indirectly assisted in the success of this study. we thank the director-general of health malaysia for permission to present these findings. conflicts of interest: the authors declare no conflict of interest. references 1. mitchell, m, mcclean, s. pregnancy, risk perception and use of complementary and alternative medicine. health risk soc 2014; 16(1): 101–116. 2. omar, uh, putit, l. consumer behavioral intention to use 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the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2021, 4, 1; a0000223. doi: 10.36877/pmmb.a0000223 http://journals.hh-publisher.com/index.php/pmmb review article covid-19 vaccination during pregnancy in southeast asia jia ni kwan1, hong chuan loh1*, irene looi1,2 article history 1clinical research centre, hospital seberang jaya, ministry of health malaysia, 13700 seberang jaya, penang, malaysia; kwan.jia.ni@gmail.com (jnk) 2medical department, hospital seberang jaya, ministry of health malaysia, 13700 seberang jaya, penang, malaysia; irenelooi@yahoo.com (il) *corresponding author: hong chuan loh, lohhongchuan@gmail.com (hcl) received: 5 july 2021; received in revised form: 6 august 2021; accepted: 9 august 2021; available online: 19 august 2021 abstract: southeast asia is rapidly becoming the region hit hardest by coronavirus disease (covid-19), as evidenced by the surging daily number of new confirmed cases and deaths. the covid-19 crisis continues to worsen with the entry of the more transmissible variants of concern, primarily the delta variant of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which was first identified in india. pregnant women are among the vulnerable population groups at risk of suffering from severe covid-19 and may experience poor pregnancy and neonatal outcomes due to the infection. vaccination seems to be the most effective strategy to curb the pandemic and secondarily by social distancing, wearing face masks and practising hand hygiene. there has been limited yet reassuring evidence in support of vaccinating pregnant women against covid-19. we sought to review the latest evidence regarding the safety, immunogenicity and reactogenicity of covid-19 vaccines in pregnant women as well as the recommendations and guidance provided by the public health authorities in the countries in southeast asia. keywords: covid-19; vaccination; pregnancy; southeast asia; pandemic 1. introduction the coronavirus disease (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has caused a pandemic worldwide. globally, as of the 24th of july 2021, there have been more than 190 million cases and greater than four million deaths as reported by the world health organization (who). southeast asia ranked third in the total number of confirmed cases and deaths after america and europe, having over 37 million cases and more than 500,000 deaths attributable to covid-19[1]. the virus was first spread from wuhan, china to southeast asia on the 13th of january 2020 when the first covid-19 case outside china was detected in thailand[2–4]. entry of the more virulent and highly transmissible delta variant of sars-cov-2 which was first detected in india in the late 2020 pmmb 2021, 4, 1; a0000223 2 of 18 has worsened the covid-19 crisis across most of southeast asia[5], leading to new record highs for covid-19 cases and deaths in many countries in southeast asia such as indonesia, myanmar and malaysia. indonesia has become the new epicenter of the covid-19 pandemic in asia. being the largest country in southeast asia, it was hit worst by the pandemic, having the highest number of cases and deaths as compared to other countries in the region. from the beginning of the pandemic until july 2021, indonesia recorded more than three million confirmed cases of covid-19 and 84,766 deaths[6]. according to data from the indonesian obstetrics and gynaecology association (pogi) reproductive tract infection working group, there were 536 cases of covid-19 in pregnant women from april 2020 to april 2021. up to 72% of them were at above 37 weeks of gestation, 4.5% needed treatment in the intensive care unit (icu) and approximately 3% succumbed to covid-19[7]. evidence has shown that pregnant women infected with covid-19, especially in the late second or third trimester have a higher likelihood of getting admitted to the icu and require invasive ventilation[8]. they are especially more vulnerable to severe covid-19 which may lead to maternal death if they have pre-existing comorbidities such as hypertension, diabetes and obesity[9,10]. in addition, complications like pre-eclampsia, emergency caesarean section and preterm delivery are more likely to occur in pregnancy with covid-19[11]. besides the association of covid-19 in pregnancy with adverse maternal outcomes, it is also reported to impact negatively on the foetus. increased incidences of stillbirth, neonatal deaths, admission to the neonatal icu, foetal distress and poor apgar score were reported in babies born to mothers with covid-19[9]. given the emerging evidence of the association between covid-19 and negative maternal-foetal outcomes, it is reasonable and justifiable to vaccinate pregnant women against an infection that could potentially harm the mother and foetus. a variety of vaccine candidates have been developed and authorised for emergency use by the who, comprising inactivated virus vaccines, protein subunit vaccines, mrna vaccines and recombinant viral vector vaccines[12]. many guidelines and recommendations were published by governmental public health agencies as well as healthcare organisations such as the who and the centers for disease control and prevention (cdc) regarding the administration of covid-19 vaccines in pregnant women[13]. the cdc and the advisory committee on immunization practices have released clinical guidance stating that pregnant women are eligible for and can receive covid-19 vaccination[14]. in this study, we aim to review the evidence regarding covid-19 vaccination in pregnant women as well as current covid-19 vaccination guidance for pregnant women in southeast asia. pmmb 2021, 4, 1; a0000223 3 of 18 2. covid-19 vaccination in pregnant women 2.1. immunogenicity there have been studies looking into the immunogenicity of covid-19 vaccination in pregnant women. results showed that the vaccine-induced antibody titres in pregnant women who had received covid-19 mrna vaccines were equivalent to non-pregnant women. covid-19 mrna vaccines were shown to be able to produce robust humoral immunity in pregnant women. the immunogenicity and reactogenicity of the vaccines were comparable to non-pregnant populations as evidenced by the cd4 and cd8 t-cell responses as well as the presence of binding, neutralising and functional non-neutralising antibodies seen in vaccinated pregnant women[15,16]. maternal antibodies were detected as early as five days after receiving the first dose of vaccine, with the igg levels increasing significantly over time[17]. vaccination against covid-19 was also shown to elicit a greater antibody response than natural sars-cov-2 infection[15]. 2.2. safety a recent study reported on the safety of mrna covid-19 vaccines, bnt162b2 (pfizer-biontech) and mrna-1273 (moderna) in pregnant women using the data obtained from the v-safe surveillance system and pregnancy registry in the united states (us). this study involved 35,691 pregnant women; 54% of them received the pfizer-biontech vaccine while 46% received the moderna vaccine. it was found that the mrna vaccines did not cause an increase in the incidence of adverse pregnancy and neonatal outcomes such as spontaneous abortion, preterm birth, congenital anomalies and neonatal death[18]. another observational case-control study conducted in israel also reported similar results in terms of obstetric and neonatal outcomes in pregnant women who had received pfizer’s bnt162b2 covid-19 vaccine[19]. besides, there was no reported increase in the incidence of abnormal placental findings such as decidual arteriopathy, chronic villitis, foetal vascular malperfusion and chronic histiocytic intervillositis in vaccinated pregnant women[20]. overall, no obvious safety concerns with regard to pregnancy or neonatal outcomes were found to be associated with covid-19 vaccination in pregnant women. 2.3. reactogenicity v-safe survey data reported that the overall reactogenicity profile was similar between the pregnant and non-pregnant groups. pain at the injection site, headache, fatigue and myalgia were the most commonly reported local and systemic reactions for both the pfizer-biontech vaccine and the moderna vaccine[18]. another cross-sectional study also compared the side effect profile of mrna covid-19 vaccines reported by pregnant and pmmb 2021, 4, 1; a0000223 4 of 18 non-pregnant healthcare workers. among 1,029 healthcare workers enrolled in the study, 38 were pregnant, 20 of them received the pfizer-biontech vaccine while 18 received the moderna vaccine. the side effects profile seemed to be similar in both groups immediately after and in the early post-vaccination period as there was no statistically significant difference observed[21]. a similar result was seen in another study, with myalgia, arthralgia and headache significantly less commonly reported by the pregnant women receiving pfizer’s bnt162b2 covid-19 vaccine[19]. 3. covid-19 vaccine recommendations for pregnant women in southeast asia the latest guidelines and covid-19 vaccination practices adopted by each country in southeast asia for pregnant women are shown in table 1. our search regarding the most updated recommendations was up to the 24th of july 2021. 3.1. malaysia on the 24th of may 2021, the special committee on ensuring access to covid-19 vaccine supply (jkjav) released an advisory on vaccination for pregnant and lactating women. among the three vaccines available in malaysia at that point in time, pfizerbiontech vaccine was the only vaccine deemed to be suitable for pregnant women if administered between 14 to 33 weeks of pregnancy or post-delivery[22]. azd1222 (oxfordastrazeneca) and coronavac (sinovac), the other two vaccines available in malaysia at the time, were not recommended for pregnant women. nonetheless, a month later on 25th june 2021, the advisory was updated to include the pfizer-biontech, oxford-astrazeneca and sinovac vaccines as suitable for pregnant women between 14 to 33 weeks of pregnancy[23]. according to the covid-19 vaccination guideline regarding pregnancy published by the ministry of health (moh), since the oxford-astrazeneca vaccine is not a live vaccine, it is not contraindicated in pregnancy. however, there is a rare but potential risk of vaccineinduced immune thrombotic thrombocytopaenia (viit) which the pregnant women would want to discuss with their obstetricians. meanwhile, sinovac vaccine was recommended based on who’s interim guidelines since the benefits outweighed the risks. both oxfordastrazeneca and sinovac vaccines can be administered if the woman conceives after getting first dose of the respective vaccine. the guideline also mentioned that there was no contraindication for the use of ad5-ncov (cansinobio) and ad26.cov2.s (janssen) vaccines which were granted conditional approval for emergency use on 15th june 2021. however, the availability of safety data for these two vector-based vaccines was limited, hence pfizer-biontech vaccine remained the preferred option for pregnant women[24]. the recommended window for vaccination of pregnant women was between 14 and 33 weeks as the period before 14 weeks is the period of organogenesis. given the unknowns, pmmb 2021, 4, 1; a0000223 5 of 18 healthcare professionals would like to minimise exposure to vaccines during organogenesis since it is possible that vaccination might affect organ formation during embryonic development. meanwhile, the upper limit was set at 33 weeks to ensure that pregnant women gained sufficient immunity and protection before the late second trimester to prevent significant implications if they contract covid-19 during the vulnerable late second and third trimester. however, pregnant women can still get the vaccine after 33 weeks of gestation upon consultation with their doctor[25]. up to 25th june 2021, a total of 109,607 pregnant women had voluntarily registered for the covid-19 vaccination via mysejahtera, a mobile application that allows the citizens to register for vaccination[26]. 3.2. singapore the expert committee on covid-19 vaccination (ec19v) published vaccination guidelines for subgroups of individuals, including pregnant women on the 31st of may 2021. it reported that there was no evidence to suggest pfizer-biontech vaccine or moderna vaccine may bring negative consequences to the health of the mother or child. however, pregnant women were advised to discuss and receive endorsement from their obstetrician before getting vaccinated. following updates on the vaccination programme, the registration for pregnant women commenced on 4th june 2021[27]. in june 2021, the college of obstetricians and gynaecologists singapore (cogs) and obstetrical & gynaecological society of singapore (ogss) published a joint guideline on covid-19 vaccination for pregnant women. it was recommended that pregnant women might get their vaccination after 12 weeks of pregnancy[28]. the local availability of vaccines will determine the choice of vaccine for pregnant women. at the time of this writing, vaccines available in singapore are pfizer-biontech vaccine, moderna vaccine and sinovac vaccine. however, sinovac vaccine was only started being administered on 15th june 2021 under the special access route in private clinics. it was not being included in singapore’s national vaccination programme as the moh claimed that the evaluation of sinovac vaccine’s safety and efficacy had yet to be completed[29]. hence, only the mrna vaccines, pfizer-biontech vaccine and moderna vaccine were administered to pregnant women[28]. 3.3. thailand the royal thai college of obstetricians and gynaecologists and the perinatal society of thailand recommended that every pregnant mother should be vaccinated against covid-19 after the 12th week of pregnancy[30]. thailand’s food and drug administration pmmb 2021, 4, 1; a0000223 6 of 18 (fda) had approved six covid-19 vaccines for emergency use which were oxfordastrazeneca vaccine, sinovac vaccine, janssen vaccine, moderna vaccine, pfizer-biontech vaccine and bbibp-corv (sinopharm)[31]. the ministry of public health recommended sinovac vaccine as the choice of vaccine for pregnant women since it is an inactivated vaccine. meanwhile, oxford-astrazeneca vaccine, being a viral vector vaccine raised concern since there is a high likelihood of developing maternal pyrexia post vaccination[32]. 3.4. philippines the department of health reported that pregnant women are not contraindicated to get their covid-19 vaccine. they were advised to get vaccinated only after the first trimester of pregnancy[33]. regarding the choice of covid-19 vaccines, among the eight vaccines, namely sinopharm vaccine, pfizer-biontech vaccine, sinovac vaccine, janssen vaccine, moderna vaccine, astrazeneca vaccine, covaxin (bharat biotech) and sputnik v (gamaleya) that had been authorised by the philippines fda for an emergency use authorisation, gamaleya vaccine was the only vaccine not recommended for pregnant women[34,35]. 3.5. indonesia on the 22nd of june 2021, pogi urged vulnerable pregnant women to get their covid-19 vaccination following spikes in severe covid-19 cases in pregnant women and the entry of the more contagious delta variant of sars-cov-2 into indonesia. pregnant women, especially those with high-risk conditions such as advanced maternal age (more than 35 years), body mass index (bmi) above 40 kg/m2, comorbidities of diabetes and hypertension as well as those working in healthcare settings, were strongly recommended for vaccination. pogi also recommended that pregnant women should get their vaccines between 12 to 33 weeks of pregnancy[7]. however, the type of vaccines to be administered to pregnant women was not specified. 3.6. brunei there was no specific guideline for covid-19 vaccination in pregnant women in brunei. however, in a document titled “frequently asked questions on covid-19 vaccines” published by the moh, pregnant women were advised to discuss vaccination with their doctors before getting vaccinated[36]. there were five covid-19 vaccine candidates for brunei citizens, namely pfizer-biontech vaccine, moderna vaccine, oxford-astrazeneca vaccine, sinopharm vaccine and janssen vaccine[37]. the moh had published health advisories for two of the covid-19 vaccines, sinopharm vaccine and oxford-astrazeneca vaccine. neither sinopharm vaccine nor oxford-astrazeneca vaccine was recommended for pmmb 2021, 4, 1; a0000223 7 of 18 pregnant women due to a lack of clinical trial data at the time of publication of the advisories[38,39]. 3.7. vietnam, laos, cambodia the who’s representative office for vietnam, laos and cambodia did not recommend the administration of covid-19 vaccines to pregnant women unless the benefits outweighed the risks. it was recommended that pregnant healthcare workers who have a high risk of exposure and pregnant women with comorbidities who have a higher risk of getting severe covid-19 should be vaccinated after discussing with their doctors. further information about vaccine choices and the appropriate time for vaccination was not provided[40–42]. 3.8. myanmar, timor-leste data regarding covid-19 vaccination for pregnant women in myanmar and timorleste are unavailable, hence they are not included in our review. p m m b 2 0 2 1 , 4 , 1 ; a 0 0 0 0 2 2 3 8 o f 1 8 table 1. covid-19 vaccine recommendations for pregnant women in southeast asia. country vaccines available time of vaccination guidelines last updated recommendations malaysia[43] pfizer-biontech* oxfordastrazeneca* sinovac* cansinobio janssen 14–33 weeks of pregnancy guidelines on covid-19 vaccination in pregnancy and breastfeeding[24] 23 june 2021  “covid-19 vaccination should be advocated in pre-pregnancy care, especially for front liners and mothers with identifiable risk factors and those seeking infertility treatment.”  “based on virology principles, mrna, vectorbased and inactivated vaccines are not contraindicated among pregnant or breastfeeding mothers. although evidence continues to emerge as more pregnant mothers are included in the study cohort, current data suggest that mrna vaccines are the preferred option. live vaccines are contraindicated in pregnancy.” singapore[29] pfizer-biontech* moderna* sinovac after 12 weeks of pregnancy expanding singapore’s vaccination programme: updated vaccination guidelines for sub-groups of individuals and enhancing access for seniors[27] 31 may 2021  “there is currently no evidence to suggest that the pfizer-biontech or moderna vaccines may cause harm to the mother or child.”  “as there is less data available for pregnant women, pregnant women should discuss with their doctors to make an informed decision.” covid-19 vaccination for pregnant women, breastfeeding women and women planning to conceive[28] june 2021  “all pregnant and lactating women should be provided information and offered covid-19 vaccination.”  “pregnant women should be offered vaccination at the same time as the rest of the population, based on their age and clinical risk group, if no contraindications exist. as covid-19 is associated with more severe complications in the later part of pregnancy, some women may choose to delay their vaccine until after the first p m m b 2 0 2 1 , 4 , 1 ; a 0 0 0 0 2 2 3 9 o f 1 8 12 weeks which is the most crucial period for foetal development.”  “the choice of vaccine administered is largely based on local availability. currently, mrna vaccines (pfizer-biontech’s bnt162b2 and moderna’s mrna-1273) are preferable to adenovirus vector vaccines for pregnant women and women younger than 50 years of age.” thailand[31] oxford-astrazeneca sinovac* janssen moderna sinopharm pfizer-biontech after 12 weeks of pregnancy the coronavirus disease 2019 situation[32] 1 may 2021  “the department of health, ministry of public health suggested that pregnant women should receive covid-19 vaccines.”  “the appropriate time for pregnant women should be vaccinated after 12-week pregnancy.”  “sinovac vaccine is recommended for pregnant women because it is an inactivated vaccine.”  “while astrazeneca vaccine is a viral vector vaccine, it has a high probability of developing a fever after vaccination.”  “women who plan to become pregnant and lactating mothers can be vaccinated like general persons.” philippines[34] sinopharm pfizer-biontech sinovac bharat biotech janssen moderna oxford-astrazeneca after first trimester of pregnancy can pregnant women get the covid-19 vaccine?[33] 30 march 2021  “pregnancy is not a contraindication to getting the covid-19 vaccine (except for the gamaleya vaccine, which shall not be administered to the pregnant and breastfeeding populations).”  “pregnant women can get the vaccine with precaution, given that there is limited data on pregnant women from clinical studies.” p m m b 2 0 2 1 , 4 , 1 ; a 0 0 0 0 2 2 3 1 0 o f 1 8 gamaleya  “if a pregnant woman is part of a group recommended for vaccination, vaccination can be offered.” indonesia sinopharm sinovac moderna oxford-astrazeneca pfizer-biontech 12 to 33 weeks of pregnancy pogi's recommendation regarding the spike in cases of pregnant women with covid19[7] 22 june 2021  “the pogi has recommended vaccination for pregnant women, especially high-risk pregnant women, namely those aged over 35 years, pregnant women with a bmi 1 above 40 and comorbidities of diabetes and hypertension, as well as for pregnant health workers.”  “injecting covid-19 vaccine to women with pregnancy ages of over 12 weeks to avoid risks.”  “pogi recommended pregnant women be vaccinated 33 weeks into their pregnancy, which was meant to create protection for their foetuses.” brunei[37] moderna oxford-astrazeneca sinopharm pfizer-biontech janssen information on the sinopharm covid-19 vaccine[38]  “moh does not recommend pregnant group to receive the sinopharm vaccine due to lack of clinical trial data at present. this may be revised as more evidence becomes available.” information on the oxfordastrazeneca covid-19 vaccine[39]  “the astrazeneca vaccine is not currently recommended for pregnant women.” vietnam oxford-astrazeneca gamaleya sinopharm pfizer-biontech moderna viet nam covid-19 situation report 50[40] 11 july 2021  “while pregnancy puts women at higher risk of severe covid-19, very little data are available to assess vaccine safety in pregnancy.”  “pregnant women may receive the vaccine if the benefit of vaccinating a pregnant woman outweighs the potential vaccine risks.”  “for this reason, pregnant women at high risk of exposure to sars-cov-2 (eg. health workers) or who have comorbidities which add to their risk p m m b 2 0 2 1 , 4 , 1 ; a 0 0 0 0 2 2 3 1 1 o f 1 8 of severe disease, may be vaccinated in consultation with their health care provider.” laos pfizer-biontech oxford-astrazeneca gamaleya sinopharm sinovac covid-19 vaccines frequently asked questions (faqs)[41] 7 april 2021  “the available data on covid-19 vaccines are insufficient to assess vaccine efficacy or vaccineassociated risks in pregnancy. as data from studies become available, recommendations on vaccination will be updated accordingly.”  “in the interim, who recommends not to use covid-19 vaccines in pregnant women unless the benefit of vaccinating a pregnant woman outweighs the potential vaccine risks, such as in health workers at high risk of exposure and pregnant women with comorbidities placing them in a high-risk group for severe covid-19.” cambodia oxford-astrazeneca sinopharm sinovac frequently asked questions: safety of covid-19 vaccines[42] 25 march 2021  “given the current lack of data on safety and efficacy regarding covid-19 vaccines for pregnant women, they are not prioritised or recommended for covid-19 vaccination at this point in time. studies are ongoing.” * vaccines recommended for pregnant women pmmb 2021, 4, 1; a0000223 12 of 18 4. ongoing vaccine trials and observational studies on pregnant women since pregnant women are at high risk of getting severe covid-19, vaccination is undoubtedly a crucial prevention strategy. nevertheless, there are limited data regarding the efficacy and safety of covid-19 vaccines in pregnancy as pregnant women were being excluded from the first wave of vaccine trials due to safety concerns. this caused pregnant women to be denied vaccines that would have offered protection for themselves and their babies. initially, there were inconsistent advice and recommendations from the public health, governmental and professional authorities globally regarding covid-19 vaccination during pregnancy, leading to confusion and hesitancy among pregnant women and healthcare providers[44]. pregnant women should be considered candidates to receive covid-19 vaccination and the safety of vaccine products to be administered during pregnancy is of utmost importance. pregnant women should have the opportunity to be enrolled into appropriately designed covid-19 vaccine trials whenever the prospect of benefit prevails over the risks[45]. at the time of writing this paper, there are a few ongoing clinical trials and observational studies registered at clinicaltrials.gov which focus on the safety, immunogenicity and reactogenicity of different covid-19 vaccines in pregnant women. there are two vaccine trials that are currently underway. the first trial sponsored by biontech se in collaboration with pfizer aims to evaluate the safety, immunogenicity and tolerability of the bnt162b2 vaccine which will be administered in two doses 21 days apart, in pregnant women between 24 to 34 weeks of gestation[46]. another trial sponsored by janssen vaccines & prevention b.v. is being conducted to evaluate the safety and reactogenicity of the ad26.cov2.s vaccine, a recombinant, replication-incompetent human adenovirus type 26 vector given to pregnant women during the second and third trimester of pregnancy[47]. these two clinical trials are estimated to be completed only in mid-2022 and mid-2023, respectively. besides clinical trials, there are six prospective cohort studies and one case-control study that involve pregnant women. these observational studies mainly intend to assess the safety of covid-19 vaccination in pregnant women in terms of obstetric and neonatal outcomes[48–54]. furthermore, the cdc has also established the v-safe covid-19 vaccine pregnancy registry to recruit pregnant women who had received their covid-19 vaccination during the periconception period or during pregnancy. information regarding pregnancy outcomes, complications and neonatal complications will be investigated to better understand the potential effects of covid-19 vaccination on pregnant women and foetuses which will guide pmmb 2021, 4, 1; a0000223 13 of 18 future recommendations on covid-19 vaccination for pregnant women. up to 19th july 2021, 5,103 pregnant women in the us have enrolled themselves in the registry[55]. 5. covid-19 vaccine acceptance among pregnant women despite the various covid-19 vaccination recommendations and guidelines published by governmental authorities in various countries, the pandemic cannot be curbed without widespread acceptance of covid-19 vaccination by the public and in our context, by pregnant women. an online cross-sectional survey was conducted in 16 countries, including the philippines, to gauge the level of acceptance of covid-19 vaccination among pregnant women. among 5,282 pregnant women who participated in the survey, 52% wished to get vaccinated against covid-19 if there was a 90% vaccine efficacy. however, the responses varied considerably by country. mexico and india had the highest covid-19 vaccine acceptance level where more than 80% of the pregnant women expressed that they were either “somewhat likely”, “fairly likely” or “very likely” to get vaccinated. for the philippines, the acceptance level was approximately 70%. in contrast, the covid-19 vaccine was least accepted in russia and australia, where close to 70% expressed their reluctance about getting vaccinated during pregnancy. the main reasons for vaccine hesitancy were concerns about potential exposure of their baby to side effects, concerns regarding the vaccine approval process as well as insufficient data about the safety and effectiveness of the vaccines in pregnant women[56]. two other similar studies conducted in turkey and new york also reported a low acceptance rate of 37%[57] and 44.3%[58], respectively. on the other hand, relatively high acceptance rates of covid-19 vaccination among pregnant women were reported in china[59] (77.4%) and ethiopia[60] (70.7%). significant geographical variation was observed in the covid-19 acceptance among pregnant women. this might be due to the difference in women’s risk perception of covid19, their lack of trust towards science and public health agencies[56]. hence, vaccination campaigns and targeted efforts should be carried out to boost covid-19 vaccine confidence among pregnant women. 6. conclusion in general, most of the countries in southeast asia recommended covid-19 vaccination in pregnant women after taking into consideration that the benefits far outweigh the risks. a few countries have published their covid-19 vaccination guidelines for pregnant women according to the latest evidence available. for instance, the moh in malaysia just released the third edition of its clinical guidelines on covid-19 vaccination in july 2021 which included detailed guidelines for the pregnant population. singapore also pmmb 2021, 4, 1; a0000223 14 of 18 published a joint guideline on covid-19 vaccination in june 2021, focusing on pregnant and breastfeeding women and on women who are planning to conceive. these guidelines are extremely useful in providing evidence-informed recommendations about covid-19 vaccination in pregnancy. countries like myanmar and timor leste have not published local vaccination guidelines, hence we are unsure about their vaccination program for pregnant women. there are a few limitations to our study. first, there were official documents and guidelines written in foreign languages that we experienced difficulty in interpreting. this may have resulted in a less comprehensive presentation of related information in this review. data related to covid-19 vaccines are rapidly evolving and information contained herein might change in times to come. considering the ongoing efforts of researchers around the world, we believe that there will be more evidence regarding covid-19 vaccination in pregnancy in the future. this will help pregnant women to make the best decision possible and will help healthcare providers to give clearer guidance and advice based on the available evidence. author contributions: jnk and hcl drafted the manuscript. hcl and il provided review and editing for this manuscript. jnk, hcl and il conceptualized this review writing project. funding: no external funding was provided for this research. acknowledgments: we would like to thank the director-general of health malaysia for his permission to publish this article. conflicts of interest: the authors declare no conflict of interest. references 1. who coronavirus (covid-19) dashboard. 2021. available at: https://covid19.who.int [accessed on 24 july 2021]. 2. thailand: how a strong health system fights a pandemic. 2020. available at: https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&ved=2ahukewi-vkk8_3xahvazdgghq_ddeuqfjalegqibrad&url=https%3a%2f%2fwww.who.int%2fdocs%2f default-source%2fcoronaviruse%2fcountry-case-studies%2fthailand-c19-case-study-20september.pdf%3fsfvrsn%3dd5534183_2%26download%3dtrue&usg=aovvaw13r7cfafavfrrg kn02-50w 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pregnant, breastfeeding, and nonpregnant reproductive-aged women. am j obstet gynecol 2021; 3(5): 100403. 59. tao l, wang r, han n, et al., acceptance of a covid-19 vaccine and associated factors among pregnant women in china: a multi-center cross-sectional study based on health belief model. hum vaccin immunother 2021: 1–10. 60. mose a and yeshaneh a, covid-19 vaccine acceptance and its associated factors among pregnant women attending antenatal care clinic in southwest ethiopia: institutional-based cross-sectional study. int j gen med 2021; 14: 2385–2395. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. https://clinicaltrials.gov/ct2/show/nct04659759 https://clinicaltrials.gov/ct2/show/nct04845048 https://clinicaltrials.gov/ct2/show/nct04418557 https://clinicaltrials.gov/ct2/show/nct04958304 https://www.cdc.gov/coronavirus/2019-ncov/vaccines/safety/vsafepregnancyregistry.html pmmb 2021, 4, 1; a0000261. doi: 10.36877/pmmb.a0000261 http://journals.hh-publisher.com/index.php/pmmb review article omicron: the rising fear for another wave in malaysia lydia ngiik-shiew law1, ke-yan loo2, joanna xuan hui goh2, priyia pusparajah2* article history 1 monash credentialed pharmacy clinical educator, faculty of pharmacy and pharmaceutical sciences, monash university, 381 royal parade, parkville 3052, vic, australia; lydia19595@gmail.com (ln-sl) 2 novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, subang jaya, selangor, 47500, malaysia; ke.loo@monash.edu (kyl); joanna.goh@monash.edu (jxhg) *corresponding author: priyia pusparajah, novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, subang jaya, selangor, 47500, malaysia; priyia.pusparajah@monash.edu(pp) received: 25 december 2021; received in revised form: 28 december 2021; accepted: 28 december 2021; available online: 31 december 2021 abstract: the past two years have been a turmoil for the world and people due to the covid-19 pandemic. it was the first time in history that the whole world was on a standstill after many countries-imposed movement control orders and restrictions. many innocent lives were lost, and the spread of infection is still ongoing. pharmaceutical companies raced against the time to develop vaccines, believing that it would be sufficient to control covid19. nevertheless, the recent emergence of the sars-cov-2 variant as omicron has become a global concern. this new variant of concern (voc) spreads faster than other voc strains and poses a high risk, mounting fears of new waves of infections in many countries, including malaysia. this review discussed characteristics of omicron, the emergence of omicron cases in malaysia, and preventive measures to control the spread of covid-19. keywords: covid-19; omicron; variant of concern (voc); malaysia; preventive 1. introduction the world has been affected by the profound coronavirus disease 2019 (covid-19) pandemic for two years since the first discovery of sars-cov-2 in december 2019. in these two years of the covid-19 pandemic, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus has been evolving and emerging as different variants that have been classified as the variants of concern (vocs), variant of interest (vois), and variants under monitoring (vums) according to the world health organization (who). the well-known vocs are alpha (b.1.1.7), beta (b.1.351), gamma (p.1), and delta (b.1.617.2) that had caused a significant increase in covid-19 cases and deaths in several countries. these variants carry mutations on the spike protein, which cause them to be more transmissible and pmmb 2021, 4, 1; a0000261 2 of 8 prone to immune evasion and vaccine escape[1]. the current vois include lambda (c.37) and mu (b.1.621), while there are several vums such as az.5, c.1.2., b.1.617.1, b.1.630, and b.1.640[2]. recently, the world was uproared by the emergence of a new variant known as the omicron (b.1.1.529). the omicron was designated as one of the vocs by who on 26th november 2021. the world health organization (who) states that the omicron variant spreads faster than the original sars-cov-2 virus and other concern variants. it poses a very high risk worldwide, leading to a surge of infection cases. some countries, including the united kingdom (uk), the united states of america (usa), and eu countries, have imposed travel restrictions from african countries. israel and japan took drastic precautions by closing their borders to all foreign visitors[3]. nevertheless, early studies have given hope that omicron clinical manifestation is milder than previous variants[4]. it is reported that researchers in england, scotland, and south africa have found that the hospital admission risks are between 15% and 80% lower with omicron compared to the delta variant[4]. the emergence of predecessor covid-19 voc; the alpha, beta, and delta were linked with new waves of infections across the countries[5]. the most predominant voc is the delta variant. the delta voc was detected from india and associated with a higher viral load, a more extended infectious period, and higher rates of reinfections[6-8]. the delta variant became a fast spreader and dominant variant in a short duration across the globe, causing waves of new infections. as countries fight against the waves of new infections, there have been mounting concerns about the efficacy of covid-19 vaccines. it is noted that covid19 vaccines have reduced vaccine efficacy over time. as new variants like omicron emerged, the situation has changed the perception of healthcare professionals, scientists/researchers, and pharmaceuticals that vaccination would be sufficient to control covid-19. the omicron variant is causing fear to the people of another new wave of infections in many countries, including malaysia. hence, this review discusses the characteristics of omicron, the covid19 cases and omicron cases in malaysia. the preventive measures to control the spread of omicron in malaysia will also be reviewed. 2. the new variant of concern – omicron (b.1.1.529) the first confirmed omicron covid-19 was detected on 11 november 2021 in botswana and on 14 november 2021 in south africa, where its emergence later coincides with the steep increase in covid-19 cases in south africa[9]. the omicron was classified as voc by who within the shortest period, two days after it was reported to who on 24 november 2021. it has triggered significant concerns from the public. omicron appears to spread rapidly after a few days of its identification. it is currently reported in 57 countries, including malaysia, singapore, hong kong, italy, israel, belgium, the united states of america, and many more. the origin of the omicron variant warrants further investigation; however, phylogenetic analyses suggested that this variant did not evolve from the previously known vocs (e.g., alpha or delta); it instead appears to evolve parallel to them and has diverged early from pmmb 2021, 4, 1; a0000261 3 of 8 them[10]. researchers have hypothesized that the omicron variant might have emerged and circulated in the neglected population, gestated in chronically ill covid-19 patients or immunocompromised patients. it could be initially evolved from non-human species[10-12]. genomic studies have revealed that the omicron variant possesses the most mutation sites than that of other sars-cov-2 variants as it contains over 60 substitutions/ deletions/insertions occurring along the viral genome within orf1a, orf1b, orf9b, and structural proteins including envelope, membrane, and nucleocapsid proteins. it should be noted that more than 50% of the mutations occur in the spike of omicron: 30 substitutions (a67v, t95i, y145d, l212i, g339d, s371l, s373p, s375f, k417n, n440k, g446s, s477n, t478k, e484a, q493r, g496s, q498r, n501y, y505h, t547k, d614g, h655y, n679k, p681h, n764k, d796y, n856k, q954h, n969k, and l981f), three deletions (h69/v70, g142/v143/y144, and n211), and one insertion (three amino acids (epe) at position 214)[12]. thus far, omicron has the highest number of spike mutations (3 to 4 times higher) than other vocs. despite the comprehensive genomic information of the omicron variant, there is still insufficient data on its transmissibility and severity of the disease. nevertheless, there are some similarities between the spike mutations of omicron and other vocs, for instance, d614g, n501y, k417n, p681h, and e484. these mutations have been previously reported to increase transmissibility, binding affinity with angiotensin converting enzyme 2 (ace2), and resistant to neutralization by antibodies[12,13]. in addition, the study offered preliminary findings regarding the higher risk of covid-19 reinfection by the omicron variant, in which omicron variant may acquire substantial ability to evade immunity from prior covid-19 infection[14]. further studies are required to investigate whether omicron infection is more transmissible or causes more severe symptoms than other vocs. the omicron variant is detectable by covid-19 diagnostic tests such as real-time rtpcr and rapid antigen tests[13,15]. the real-time rt-pcr targeting the s gene can detect the omicron variant at a higher rate by showing the s-gene drop out of the variant, followed by sanger sequencing to confirm the identity of the variant[15,16]. as for the rapid antigen diagnostic test, bekliz et al. revealed that the rapid antigen test showed lower sensitivity in the detection of omicron than other vocs (alpha, beta, gamma, and delta)[17]. 3. the covid-19 pandemic in malaysia malaysia was one of the southeast asian countries with the lowest covid-19 daily cases at the initial stage. after a religious outbreak at sri petaling, the cases slowly started to creep up. the religious gathering was composed of 16,000 participants, with 1,500 being foreigners. by 18th march 2020, 673 people tested positive for covid-19 in malaysia, the highest number in southeast asia. around two-thirds of the confirmed cases were linked to the event at sri petaling[18,19]. this event contributed to the biggest covid-19 cluster in malaysia, with 3375 individuals infected and 34 deaths[20]. pmmb 2021, 4, 1; a0000261 4 of 8 ever since then, the malaysian government has imposed different phases of movement control order (mco), conditional movement control order (cmco), and recovery movement control order (rmco) to curb the rise of covid-19 in the country. the nation has bravely fought against three covid-19 waves from march 2020 to december 2021. at one point, the number of cases rose exponentially to a scary daily case of 24,599 on 26th august 2021[21]. it was the highest number of new daily cases since the onset of the pandemic. on the same day, the country recorded the highest number of deaths of 393, including 100 brought-in dead cases. the healthcare sector was on the verge of collapsing as doctors worked around the clock to cope with the increasing number of hospitalization cases and deaths due to covid19 infections. the worst was not over yet for malaysians. the new daily covd-19 cases started to fall gradually but the death cases remained high. on 11th september 2021, malaysia recorded 592 daily death cases within 24 hours[21]. the following ten days in a row, the daily death cases were between 350–500 each day. the fear was mounting on the people, as funeral parlors could not cope with the burial or crematorium of so many dead bodies. people were in despair and sorrow of losing their loved ones, which affected their livelihood by the pandemic. nevertheless, with adequate measures and strict covid-19 sop, the government managed to bring down the infection rate and control the spread. as of 15th december 2021, the daily new covid-19 cases were 3900, with 33 deaths (figure 1). figure 1. illustration of covid-19 cases in malaysia pmmb 2021, 4, 1; a0000261 5 of 8 4. omicron cases in malaysia in malaysia, the first covid-19 omicron case was reported on 2nd december 2021, which involved a 19-year-old south african student returning to ipoh, perak[22]. by 25th december 2021, there were 62 confirmed omicron cases in malaysia. out of the 62 cases, 61 cases were imported infections (international arrivals from saudi arabia, united kingdom, qatar, nigeria, italy, turkey, united arab emirates, and the united states of america), and 1 case was suspected to be local transmission[23,24]. the suspected local transmission was reported on 24th december 2021, involving a 38-year-old chinese national without travel history in sarawak. it is anticipated that there will be a spike in covid-19 omicron cases in malaysia as the virus has started to spread within the community. the main reason driving the spread of omicron is the breach of the quarantine order[24]. the nation has been warned of the possibility of a fourth wave due to the spread of omicron. when this article went to press, many malaysians were aboard either for a holiday or religious purposes. 5. preventive measures the malaysian government has been proactively drawing up various strategies to control the covid-19 spread in malaysia. among the initial efforts by the government were enforcing health screening at all the country entry points to detect individuals with suspected covid-19. many government hospitals in every state were designated as covid-19 hospitals to treat patients. in addition, strict rules were implemented in different movement control order phases to prevent the further spread of the virus into the local communities [25]. strict standard operating procedures (sop) were enforced at workplaces, social distancing, prohibiting mass gatherings, compulsory from wearing a double face mask and a face shield, to prevent the spread of covid-19[26,27]. these restrictions and sops did cost a significant impact on the livelihood of the public; however, it was effective in bringing down the number of cases to a manageable level. gradually, the malaysian government announced the national recovery plan (nrp) on 15th june 2021 to ease the burden of movement control order on the livelihood and revive the nation economic sector[28]. as of december 2021, all the states in malaysia have entered the 4th phase of nrp; achieving the set target of over 60% vaccinated individuals, reduced usage of beds in icu, reduced hospitalization cases and death cases. the introduction of the covid-19 vaccine differs across the countries, but malaysia currently accounts for the largest proportion of the vaccinated population in southeast asia[29]. the national covid-19 vaccination program in malaysia has been distributing vaccines from pfizer, astrazeneca, and sinovac to the public since february 2021, over three phases: phase 1 targeting the frontline workers; phase 2 targeting individuals aged 60 years and above, disabled, and high-risk groups; phase 3 individuals/groups/locations with high disease burden, and the remaining adult population[30]. then the government approved the use and rolled out moderna, cansino, janssen, and sinopharm for the vaccination program. malaysia aims to have nearly 80% of its population fully vaccinated by 4th august 2022. ever since the emergence of the omicron variant in other countries, the health ministry has proactively taken precautionary measures to ensure the spread of the omicron variant in pmmb 2021, 4, 1; a0000261 6 of 8 the country. malaysia announced new covid-19 restrictions, banning large gatherings and encouraging booster vaccinations for high-risk groups. the health ministry announced that all malaysian over 60 and adults’ recipients of sinovac vaccines must get either pfizer or sinovac booster dose by february 2022 to keep their status fully vaccinated on mysejahtera app[31]. as the fear of omicron mounting high, the government encourages all individuals who have completed the two doses of any covid-19 vaccines to get a booster dose to protect themselves. 6. conclusion in face of omicron emergence in malaysia, the government and healthcare proactiveness in imposing preventive measures has helped to control a wide spread of omicron infection in the community. the public should exercise their responsibility in ensuring all standard operating procedures (sop) are strictly abided and adapt to the new normal culture of social distancing and wearing a face mask. getting vaccinated would-be best option for now, especially those who are vulnerable to viral infections. as we know the omicron would not be the last sars-cov-2 variant to emergence, thus making the eradication process more complicated. nevertheless, with rapid genome data sharing enable all countries to impose precaution measures to face new variant of concerns in future. author contributions: the literature searches, data collection and manuscript writing were performed by lnsl, k-yl and jxhg. the manuscript was critically reviewed and edited by pp. the project was conceptualized by pp. funding: no external funding was provided for this writing project. acknowledgments: the authors would like to acknowledge professor shajahan yasin, head of school, jeffrey cheah school of medicine and health sciences, monash university malaysia. conflicts of interest: the authors declare no conflict of interest. references 1. thye ay-k, law jw-f, pusparajah 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25 december 2021]; available from: https://www.thestar.com.my/news/nation/2021/12/25/covid-19-watch-62omicron-variant-cases-detected-in-malaysia-so-far. 24. tee k, khairy: 62 omicron cases detected in malaysia so far, surge in cases possible as variant already within community, in malay mail. 2021. 25. shah aum, safri sna, thevadas r, et al. covid-19 outbreak in malaysia: actions taken by the malaysian government. int j infect dis 2020; 97: 108–116. 26. letchumanan v, ab mutalib n-s, goh b-h, et al. novel coronavirus 2019-ncov: could this virus become a possible global pandemic. prog microbes mol biol 2020; 3(1): a0000068. https://www.cdc.gov/coronavirus/2019-ncov/variants/omicron-variant.html https://www.cdc.gov/coronavirus/2019-ncov/variants/omicron-variant.html https://www.who.int/publications/m/item/update-70-update-on-sars-cov-2-variant-of-concern-omicron https://www.who.int/publications/m/item/update-70-update-on-sars-cov-2-variant-of-concern-omicron https://www.abc.net.au/news/2020-03-19/coronavirus-spread-from-malaysian-event-to-multiple-countries/12066092 https://www.abc.net.au/news/2020-03-19/coronavirus-spread-from-malaysian-event-to-multiple-countries/12066092 https://www.worldometers.info/coronavirus/country/malaysia/ https://www.theedgemarkets.com/article/covid19-malaysia-detects-first-omicron-case-khairy https://www.theedgemarkets.com/article/covid19-malaysia-detects-first-omicron-case-khairy https://www.thestar.com.my/news/nation/2021/12/25/covid-19-watch-62-omicron-variant-cases-detected-in-malaysia-so-far https://www.thestar.com.my/news/nation/2021/12/25/covid-19-watch-62-omicron-variant-cases-detected-in-malaysia-so-far pmmb 2021, 4, 1; a0000261 8 of 8 27. nawawi mh. for safety wear double masks and face shields, says health dg. new straits times 2021 [accessed 23 august 2021]; available from: https://www.nst.com.my/news/nation/2021/08/720588/safety-weardouble-masks-and-face-shields-says-health-dg. 28. tang khd. from movement control to national recovery plan: malaysia’s strategy to live with covid-19. int j sci healthcare res 2021; 6(4): 286–292. 29. pazim k, mahmud r, yee blf, et al. the impact of covid-19 pandemic on malaysian senior citizens: a review. int j aquatic sci 2021; 12(01). 30. suah jl, tok psk, ong sm, et al. pick-ing malaysia’s epidemic apart: effectiveness of a diverse covid-19 vaccine portfolio. vaccines 2021; 9(12): 1381. 31. latiff r. malaysia imposes stricter rules, booster requirements over omicron threat. 2021 [accessed 18 decemebr 2021]; available from: https://www.reuters.com/world/asia-pacific/malaysia-imposes-stricter-rulesbooster-requirements-over-omicron-threat-2021-12-16/. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. https://www.nst.com.my/news/nation/2021/08/720588/safety-wear-double-masks-and-face-shields-says-health-dg https://www.nst.com.my/news/nation/2021/08/720588/safety-wear-double-masks-and-face-shields-says-health-dg https://www.reuters.com/world/asia-pacific/malaysia-imposes-stricter-rules-booster-requirements-over-omicron-threat-2021-12-16/ https://www.reuters.com/world/asia-pacific/malaysia-imposes-stricter-rules-booster-requirements-over-omicron-threat-2021-12-16/ 1. introduction 2. the new variant of concern – omicron (b.1.1.529) 3. the covid-19 pandemic in malaysia malaysia was one of the southeast asian countries with the lowest covid-19 daily cases at the initial stage. after a religious outbreak at sri petaling, the cases slowly started to creep up. the religious gathering was composed of 16,000 participants... ever since then, the malaysian government has imposed different phases of movement control order (mco), conditional movement control order (cmco), and recovery movement control order (rmco) to curb the rise of covid-19 in the country. the nation has b... the worst was not over yet for malaysians. the new daily covd-19 cases started to fall gradually but the death cases remained high. on 11th september 2021, malaysia recorded 592 daily death cases within 24 hours[21]. the following ten days in a row, ... figure 1. illustration of covid-19 cases in malaysia 4. omicron cases in malaysia 5. preventive measures 6. conclusion references pmmb 2021, 4, 1; a0000260. doi: 10.36877/pmmb.a0000260 http://journals.hh-publisher.com/index.php/pmmb review article covid-19 situation in thailand yi-he kuai1, hooi-leng ser1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; yi-he.kuai@monash.edu (yhk) *corresponding author: hooi-leng ser; novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; ser.hooileng@monash.edu (hls) received: 3 december 2021; received in revised form: 17 december 2021; accepted: 24 december 2021; available online: 28 december 2021 abstract: the number of covid-19 cases continues to surge globally, with millions of people affected and many innocent lives lost. thailand reported the first confirmed covid19 case on 13th january 2020. among the three waves of the covid-19 pandemic in thailand, the third wave is more severe than the previous waves, indicating a lack of strong public health interventions. the health authorities in thailand are constantly promoting the covid-19 vaccination plan to achieve herd immunity against the coronavirus. facing economic pressure, the government decided to open up border tourism from 1st november 2021 and continue monitoring the covid-19 transmission among the community. the war against covid-19 is still ongoing. in order to win this challenging war, unified collaborations between public, governmental agencies, and industries are essential for longterm success for all humanity. keywords: covid-19; sars-cov-2; thailand; situation update; coronavirus 1. introduction coronavirus disease 2019 (covid-19) is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2)[1]. thailand reported its first confirmed covid-19 case in january 2020 (figure 1). by deploying multiple strategies, including communitybased contact tracing, isolation, quarantine, and border control, thailand’s covid-19 pandemic was brought under control during december 2020[2]. by mid-2021, a new wave of covid-19 outbreaks began to sweep across the region, which further pushed the highest daily records of covid-19 cases in southeast asia, with most associated with a more “aggressive” variant, the delta variant (b.1.617.2)[3]. despite the global effort in raising awareness for vaccination against sars-cov-2, the unresting situation continues to progress as another new variant emerges omicron or sars-cov-2 (b.1.1.529), as reported to the world health organization (who) on 24th november 2021. in fact, this new variant was pmmb 2021, 4, 1; a0000260 2 of 8 reported from clinical specimens in two countries in the south africa continent on separate occasions in botswana on 11th november 2021 and in south africa on 14th november 2021. while there is still a lack of understanding regarding the virulence and transmissibility of the omicron variant, thailand announced the country’s first case of the omicron variant of the coronavirus, involving a traveler from spain on 6th december 2021. from january 2020 to october 2021, thailand experienced three waves of covid-19 pandemic within 22 months. the highest daily new cases numbered 188 in late march 2020[4]. by mid-december 2020, the entire coronavirus case was 4246[5]. the accumulative covid-19 case reached 2,156,587 around mid-december 2021[6]. the current writing aims to summarize the situation of different stages during the covid-19 pandemic in thailand, along with measurements that have been taken to curb the spread of this notorious coronavirus. figure 1. illustrations of confirmed covid-19 cases and deaths. the stacked bar graph shows the daily confirmed cases and deaths since the first case reported in thailand while the line graph shows the cumulative confirmed cases based on who reports[7]. a heat map shows the distribution of confirmed cases across thailand as of 20th december 2021[8]. 2. under the spotlight: situation in thailand within two weeks after the who branch in china received the notification of sarscov-2, thailand was the first country to report a covid-19 case outside of china on 13th january 2020. prior to the increase in domestic infections in mid-march 2020, the number of covid-19 cases in the country remained relatively low. based on the reports from the local government, the critical event that contributed to the spike was a boxing match on 6th march 2020, despite a ban on large gatherings that was put in place days before[9]. the number of pmmb 2021, 4, 1; a0000260 3 of 8 new infections continued to increase to over a hundred per day following the event. a total of 143 covid-19 cases were directly linked to the boxing match and hastened the community spread of the virus to other provinces, with some attendees traveling home or to business trips afterward[10]. compared to many countries, thailand seems to have controlled the initial pandemic relatively quickly, and the virus has been well controlled for most of 2020[11]. according to the who situation report for thailand dated as 7th february 2020, the country had sturdy capacities for case detection, danger assessment, case investigation, laboratory diagnosis, scientific management, contamination prevention, and control, as well as danger conversation[12]. however, many new cases in a wholesale shrimp market were reported in december 2020, with daily new case numbers as high as 7,284, which marked a sharp increase compared with march 2020. the government took intervention measures and strengthened social distancing policies to control the covid-19 pandemic effectively[13]. in march 2020, the number of daily injections in the community decreased and stabilized at around 1,000 people per day. this was marked as the first wave of covid-19 in thailand. fast-forwarding to 2021, the cases involving thonglor nightlife venues developed rapidly in bangkok in april[14]. fearing a further economic downturn, the thai government did not take the same severe measures as the subsequent outbreak. instead of a total blockade, the government allowed inter-provincial travel during the thai new year holiday in april 2021. additionally, the strain delta variant (b.1.617.2) was detected and found to spread much faster than other variants, leading to severe illnesses or even death. thailand subsequently reported more outbreaks in crowded, closed areas (such as institutions, prisons, markets, shopping centers, and communities)[15]. superspreading incidents were found in entertainment venues, pubs, bars, karaoke halls, and gambling establishments in different regions. the number of cases rose to more than the sum of the first and second waves. up to 17th november 2021, 2,037,224 confirmed covid-19 cases and 20,199 deaths had been recorded in thailand[16]. 3. managing covid-19 situation in thailand due to the highly contagious nature of covid-19, delayed intervention may lead to rapid spread. singapore learned from its encounter with sars in 2003, formulated a graded disease outbreak response system, and adopted timely and coordinated response measures to this covid-19 outbreak[17]. despite the shortcomings of a highly dense population and a large urban population due to globalization[18,19], a series of public health intervention measures adopted by singapore in the early stage effectively slowed down the spread of the pandemic, which can be reflected in its relatively flat epidemic. on the other hand, in thailand, the government adopted a series of strict measures to control the spread during the pmmb 2021, 4, 1; a0000260 4 of 8 first wave of covid-19, including site closures, travel restrictions, entry point screening, quarantine, contract tracking, and comprehensive infection prevention and control measures. these actions were very effective, and there were relatively few cases in thailand until the second wave of more giant waves in mid-december 2020[20]. early detection strategies can cover up the actual severity of the pandemic and minimize hidden dangers for outbreaks in the community environment. therefore, based on the lessons learned in the early stages of the pandemic, travel restrictions (including border controls) are still a necessary strategy to deal with the emerging sars-cov-2 variant. however, multiple studies have confirmed the effectiveness of current vaccines against specific variants[21,22]. the grim reality is that when the pandemic began, thailand imposed a blockade in its capital bangkok. unfortunately, the policy was designed to reduce new infections, but the virus continued to spread across the country. the approach ignored the dynamic interaction between people’s motivation to slow the spread of covid-19 and allow their economic ability to survive. in order to control the covid-19 pandemic on a global scale, it is imperative to call for epidemiologically informed, evidence-based public health response measures (including surveillance, outbreak investigation, training, and intervention standards) while reducing and mitigating the spread of public facilities measures[23-24]. these must be integrated into a comprehensive, resilient, and publicly funded health system to provide universal health coverage and prepare for the next pandemic[25,26]. in response to the large-scale movement of people across the country, the ministry of public health in thailand provides online courses, and local health officials offer face-toface training for volunteers[27]. the training content includes basic knowledge of covid-19, educating people on how to stay safe, identifying and monitoring high-risk community members (the elderly or people with chronic diseases such as cardiovascular disease, diabetes, or hypertension), and data collection and reporting[28]. although volunteers can lodge records in paper form (or in-person), the ministry of public health strongly encourages specially designed smartphones or internet applications to report data online (via phone applications). the covid-19 pandemic has caused a dilemma between economic stimulus and public health control[29]. before the covid-19 pandemic, tourism accounted for 22% of thailand’s gdp, and in 2018, one-sixth of thailand’s jobs were in tourism[30]. the economic costs of thailand’s border and business closures and strict restrictions are enormous. companies in the service and tourism industries have been hit hardest. therefore, thailand has developed a covid-19 vaccination plan for individuals with severe symptoms and the general population in outbreak areas to improve the community’s immunity. the thai pmmb 2021, 4, 1; a0000260 5 of 8 government aims to achieve herd immunity against covid-19 through vaccination, and the goal is to reach 70% of the population by the end of 2021[31]. in addition, thailand has permitted vaccinated people with at least one dose to engage in socio-economic activities such as public gatherings and public transport[32]. the vaccination program's rollout is considered a great success, with a drop in the number of new cases[33]. thailand opened up phuket’s tourism industry on 1st july 2021. despite the third wave of coronavirus outbreaks in thailand, economic pressures urged the restoration of its tourism industry[34]. as a matter of fact, thailand has announced to welcome fully vaccinated foreign tourists, including returning thais and foreign residents, who enter the country by plane from an approved country/region without quarantine requirements from 1st november 2021, by the tourism authority of thailand (tat)[35]. 4. going beyond: what is known and wait in the future thailand’s ministry of public health, on 6th december 2021, announced the country’s first case of the omicron variant related to a u.s. citizen (visiting from spain) who entered thailand nearly a week ago. the 35-year-old american who received vaccination in june of the same year was first tested for the virus on 30th november 2021 and subsequently received a positive result the following day[36]. since 1st november 2021, thailand became one of the first southeast asian countries to reopen its borders to fully vaccinated tourists, allowing arrivals by air from 63 countries, including the united states, china, india, southeast asia, and much of europe. fully vaccinated tourists from these regions should undergo pcr tests upon arrival and stay at an approved hotel for one night while waiting for the results[37]. the prime minister of thailand responded quickly on this matter with advice to the public not to panic over the emergence of the omicron covid-19 variant in the country[38]. the prime minister also urged the unvaccinated individuals to get the vaccination as soon as possible. meanwhile, thailand’s tourism minister announced that there would be no going back for the country’s reopening plans, despite the emergence of the omicron variant[39]. in reality, about one-fifth of thailand’s economy depends on tourism. before the pandemic, thailand was one of the world’s top tourist destinations, drawing nearly 40 million visitors in 2019. the government projects that the country will complete the 110 – 120 million doses target to the entire population by december 2021[40]. as of 19th december 2021, 934 omicron cases had been confirmed in thailand[41]. at the time of writing, the who recommended that countries continue to implement effective public health measures to reduce covid-19 circulation overall. in addition, inequities in access to covid-19 vaccines should be addressed urgently to ensure that vulnerable groups everywhere, including health workers and older persons, receive their first pmmb 2021, 4, 1; a0000260 6 of 8 and second doses, alongside equitable access to treatment and diagnostics[42]. by the same token, with the discovery of the covid-19 variant with highly transmissibility rate, omicron further emphasizes the need to develop viral detection methods following the discussion on the ability of molecular kits to detect this specific variant accurately[43]. the war against covid-19 is neither lost nor ending; there is still hope in curbing its spread with tight collaborations between public, governmental agencies, and industries. this is the time where we need exceptional unity to lead and spearhead unabashed joint efforts to forge a resilient future as we push for long-term success for all humanity. author contributions: yk performed the literature search, critical data analysis as well as manuscript writing. hls provided technical support, conceptualization and set up this review writing project. all authors have read and agreed to the published version of the manuscript. funding: this work is supported by the early career research grant, jeffrey cheah school of medicine and health sciences (ecr-000015) awarded to hls. conflicts of interest: the authors declare no conflict of interest. references 1. world health organization who director-general’s opening remarks at the media briefing on covid19—11 march 2020. [accessed on 10 july 2021]. available online: https://www.who.int/directorgeneral/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19--11-march-2020. 2. oxford policy management. social impact assessment of covid-19 in thailand. oxford policy management limited; oxford, uk: 2020. 3. thye ayk, law jwf, pusparajah p, et al. emerging sars-cov-2 variants of concern (vocs): an impending global crisis. biomedicines 2021; 9(10):1303. 4. who thailand coronavirus disease 2019 (covid-19) who thailand situation report—22 march 2020. available online: https://reliefweb.int/sites/reliefweb.int/files/resources/2021_03_22_eng_sitrep163-covid19.pdf 5. rebecca r. how have thailand and cambodia kept covid cases so low? 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(2021). bangkok nightlife clusters expose thailand's virus stumbles. ap news. retrieved may 12, 2021, from https://apnews.com/article/world-news-coronavirus-pandemic-prayuthchan-ocha-bangkok-thailand-fdd0bc6333d378172aa36a4593ff0c97. 15. iftimie s, lópez-azcona af, vallverdú i, et al. first and second waves of coronavirus disease-19: a comparative study in hospitalized patients in reus, spain. plos one 2021;16(3). 16. the coronavirus disease 2019 situation-thailand situation update on 17 november 2021. available online: https://ddc.moph.go.th/viralpneumonia/eng/file/situation/situation-no678-171164.pdf. 17. ser hl, letchumanan v, law jw, et al. pmmb covid-19 bulletin: spain (18th april 2020). prog microbes mol biol 2020;3(1). 18. wong d, li y. spreading of covid-19: density matters. plos one 2020;15:e242398. 19. department of disease control, m.o.p.h. covid-19 situation reports. 2021. 20. loo ky, letchumanan v, ser hl, et al. covid-19: insights into potential vaccines. microorganisms 2021; 9(3):605. 21. bedford j, enria d, giesecke j, et al. living with the covid-19 pandemic: act now with the tools we have. lancet 2020. 22. dostal jm. governing under pressure: german policy making during the coronavirus crisis. polit q 2020;91:542–552. 23. forman r, atun r, mckee m, et al. 12 lessons learned from the management of the coronavirus pandemic. health policy 2020;124:577–580. 24. tan lt, letchumanan v, ser hl, et al. pmmb covid-19 bulletin: united kingdom (22nd april 2020). prog microbes mol biol 2020;3(1). 25. [village health volunteer system management for local quarantine and home quarantine]. nonthaburi: department of health service support; 2020. thai. 26. [video conference: emergency medical and public health center for covid-19]. nonthaburi: ministry of public health; 2020. thai. https://pher.moph.go.th/wordpress/02-03-2020-1/. 27. dubé e, vivion m, macdonald ne. vaccine hesitancy, vaccine refusal and the anti-vaccine movement: influence, impact and implications. expert rev vaccines 2015;14(1):99–117. 28. godman b, haque m, islam s, et al. rapid assessment of price instability and paucity of medicines and protection for covid-19 across asia: findings and public health implications for the future. front public health 2020;8. 29. suthar s, das s, nagpure a, et al. epidemiology and diagnosis, environmental resources quality and socio-economic perspectives for covid-19 pandemic, j environ manage 2021; 280. 30. fitch solutions virus imperils thai economy—knock-on impact from sharp tourism slowdown could be broad and last for many months. 31. national news bureau of thailand. (2021). 1 million doses of sinovac vaccine arrive in thailand. retrieved may 11, 2021, from https://thainews.prd.go.th/en/news/detail/tcatg210410190955642. 32. hindustan times. (2021). southeast asia reopening borders and living with covid-19. retrieved dec 10, 2021, from https://www.hindustantimes.com/ht-insight/international-affairs/southeast-asia-reopeningborders-and-living-with-covid19-101639122051684.html 33. suphanchaimat r, tuangratananon t, rajatanavin n, et al. prioritization of the target population for coronavirus disease 2019 (covid-19) vaccination program in thailand. int j environ res public health 2021;18(20):10803. 34. tourism authority of thailand (tat). (2021). thailand confirms reopening the country plan from 1 july 2021. retrieved jun 21, 2021 from https://news.cision.com/tourism-authority-of-thailand/r/thailandconfirms-reopening-the-country-plan-from-1-july-2021,c3370827. https://apnews.com/article/world-news-coronavirus-pandemic-prayuth-chan-ocha-bangkok-thailand-fdd0bc6333d378172aa36a4593ff0c97 https://apnews.com/article/world-news-coronavirus-pandemic-prayuth-chan-ocha-bangkok-thailand-fdd0bc6333d378172aa36a4593ff0c97 https://ddc.moph.go.th/viralpneumonia/eng/file/situation/situation-no678-171164.pdf https://pher.moph.go.th/wordpress/02-03-2020-1/ https://thainews.prd.go.th/en/news/detail/tcatg210410190955642 https://www.hindustantimes.com/ht-insight/international-affairs/southeast-asia-reopening-borders-and-living-with-covid19-101639122051684.html https://www.hindustantimes.com/ht-insight/international-affairs/southeast-asia-reopening-borders-and-living-with-covid19-101639122051684.html https://news.cision.com/tourism-authority-of-thailand/r/thailand-confirms-reopening-the-country-plan-from-1-july-2021,c3370827 https://news.cision.com/tourism-authority-of-thailand/r/thailand-confirms-reopening-the-country-plan-from-1-july-2021,c3370827 pmmb 2021, 4, 1; a0000260 8 of 8 35. tourism authority of thailand (tat). (2021). “quarantine-free” holiday in thailand from 1 november 2021. retrieved oct 22, 2021 from https://news.cision.com/tourism-authority-of-thailand/r/quarantinefree--holiday-in-thailand-from-1-november-2021,c3428974. 36. the new york times (2021). thailand reports its first case of the omicron variant, in a u.s. citizen visiting from spain.dec. 6, 2021 from https://www.nytimes.com/2021/12/06/world/asia/thailandomicron-covid.html. 37. thai pbs world (2021). pm urges public not to panic over first case of omicron variant in thailand. dec. 6, 2021 from https://www.thaipbsworld.com/pm-urges-public-not-to-be-panic-over-first-case-ofomicron-variant-in-thailand/. 38. thai pbs world (2021). thailand records 194 more covid-19 omicron cases on wednesday. dec.30, 2021 from https://www.thaipbsworld.com/thailand-records-194-more-covid-19-omicron-cases-onwednesday/. 39. phucharoen c, sangkaew n, stosic k. the characteristics of covid-19 transmission from case to highrisk contact, a statistical analysis from contact tracing data. eclinicalmedicine 2020;27: 100543. 40. bangkokpost (2021). omicron will not affect reopening, says phiphat nov. 30, 2021 from https://www.bangkokpost.com/business/2223767/omicron-will-not-affect-reopening-says-phiphat. 41. boon-itt s, rompho n, jiarnkamolchurn s, et al. interaction between age and health conditions in the intention to be vaccinated against covid-19 in thailand. hum vaccin immunother 2021; 6:1–7. 42. bangkokpost (2021). bma mulling distribution of fourth booster shots dec 28, 2021 from bma mulling distribution of fourth booster shots (bangkokpost.com). 43. guardian journalism (2021). scientists find ‘stealth’ version of omicron that may be harder to track dec. 7, 2021 from scientists find ‘stealth’ version of omicron that may be harder to track | coronavirus | the guardian. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. https://news.cision.com/tourism-authority-of-thailand/r/quarantine-free--holiday-in-thailand-from-1-november-2021,c3428974 https://news.cision.com/tourism-authority-of-thailand/r/quarantine-free--holiday-in-thailand-from-1-november-2021,c3428974 https://www.nytimes.com/2021/12/06/world/asia/thailand-omicron-covid.html https://www.nytimes.com/2021/12/06/world/asia/thailand-omicron-covid.html https://www.thaipbsworld.com/pm-urges-public-not-to-be-panic-over-first-case-of-omicron-variant-in-thailand/ https://www.thaipbsworld.com/pm-urges-public-not-to-be-panic-over-first-case-of-omicron-variant-in-thailand/ https://www.thaipbsworld.com/thailand-records-194-more-covid-19-omicron-cases-on-wednesday/ https://www.thaipbsworld.com/thailand-records-194-more-covid-19-omicron-cases-on-wednesday/ https://www.bangkokpost.com/business/2223767/omicron-will-not-affect-reopening-says-phiphat https://www.bangkokpost.com/thailand/general/2238643/bma-mulling-distribution-of-fourth-booster-shots https://www.bangkokpost.com/thailand/general/2238643/bma-mulling-distribution-of-fourth-booster-shots https://www.theguardian.com/world/2021/dec/07/scientists-find-stealth-version-of-omicron-not-identifiable-with-pcr-test-covid-variant https://www.theguardian.com/world/2021/dec/07/scientists-find-stealth-version-of-omicron-not-identifiable-with-pcr-test-covid-variant 1. introduction 2. under the spotlight: situation in thailand 3. managing covid-19 situation in thailand 4. going beyond: what is known and wait in the future references pmmb 2021, 4, 1; a0000245. doi: 10.36877/pmmb.a0000245 http://journals.hh-publisher.com/index.php/pmmb original research article biofilm formation and blaoxa genes detection among acinetobacter baumannii from clinical isolates in a tertiary care kirtipur hospital, nepal upasana ghimire1,2*, rupesh kandel2, mary neupane3, sanjit shrestha4, sudeep k c1, santosh khanal1, dev raj joshi1* article history 1central department of microbiology, tribhuvan university, kathmandu, 44600, nepal; santoshkhanal007@gmail.com (sk); sudeep.765710@cdm.tu.edu.np (skc) 2department of bionanotechnology & bionanoconvergence engineering, graduate school, jeonbuk national university, jeonju, 561-756, republic of korea; rupkandel87@jbnu.ac.kr (rk) 3department of microbiology, national college, kathmandu, 44600, nepal; maryneupane@gmail.com (mn) 4phect-nepal kirtipur hospital, kathmandu, 44600, nepal; shrestha.s@hotmail.com (ss) *corresponding author: upasana ghimire, dev raj joshi; central department of microbiology, tribhuvan university, kathmandu, 44600, nepal; upughi1@gmail.com (ug), dev.joshi@cdmi.tu.edu.np (drj) received: 30 september 2021; received in revised form: 30 october 2021; accepted: 1 november 2021; available online: 9 november 2021 abstract: (1) background: acinetobacter baumannii has emerged as a leading cause of nosocomial infections as they are capable of evolving resistance to various classes of antibiotics. the ability of a. baumannii to form biofilm might also be associated with increased antibiotic resistance and hence treatment failure. this study was carried to associate the biofilm formation with the drug resistance pattern of a. baumannii and to detect blaoxa23, blaoxa-24, and blaoxa-51 from carbapenem resistance isolates. (2) methods: among different clinical samples, a total of 19 acinetobacter spp. were identified with conventional microbiological procedures. the biofilm production was determined by a quantitative adherence assay. the antimicrobial susceptibility test was carried out by the kirby-bauer disc diffusion method, carbapenemase production detection was confirmed by modified hodge test, and target resistant genes were detected by polymerase chain reaction. (3) results: out of 90 clinical specimens, 64.44% (58/90) showed bacterial growth, whereas 32.75% (19/58) isolates were identified as a. baumannii. among all a. baumannii isolates, 84.21% (16/19) were multidrug-resistance and 63.16% (12/19) carbapenem resistance phenotypically. blaoxa-51 was detected in all the isolates and blaoxa-23 was detected only in 63.16% (12/19) isolates. however, blaoxa-24 was not detected in any of the isolates. among a. baumannii, 89.47% (17/19) isolates produced biofilm with 47.37% (9/19) strong biofilm producers. (4) conclusions: in the majority of mdr a. baumannii, blaoxa-51 and blaoxa23 were detected as the determinant factor for carbapenem resistance having a direct relation with biofilm formation. this study provided a valuable clue for the management of a. baumannii infections in clinical settings. pmmb 2021, 4, 1; a0000245 2 of 17 keywords: a. baumannii; mdr; biofilm; carbapenem resistance; blaoxa genes 1. introduction acinetobacter, a ubiquitous, non-fermentative, gram-negative bacteria, persisting in the hospital environment, is a nosocomial pathogen responsible for a wide range of infections[1,2]. it is an opportunistic pathogen causing wound infections, peritonitis, endocarditis, cholangitis, ventilator-associated pneumonia (vap), bacteremia, icu infections, and urinary tract infections (utis), particularly in patients with a severe health condition[3]. the emergence of acinetobacter as a significant nosocomial and opportunistic pathogen is influenced by its survival ability, ubiquitous presence, and instant progress of resistance to the commonly used antimicrobials[4,5]. the high level of a. baumannii resistance to antibiotics is a result of its capacity to produce carbapenemase and to form biofilms[6]. carbapenemase-producing bacteria receive particular consideration as it is linked with multidrug-resistance (mdr) having a limited antibiotic choice. however, resistance to these agents is now considered as world health organization’s number one critical priority pathogen[7]. the underlying antibiotic resistance mechanisms may be related to efflux pump overexpression, decreased permeability, and carbapenemase production. among the carbapenemases, carbapenem-hydrolyzing class d β-lactamases (chdls) are considered the most prevalent cause of carbapenem resistance in a. baumannii[8]. the chdls in a. baumannii can be grouped into six subclasses: intrinsic chromosomal oxa-51-like, acquired oxa-23-like, oxa-24/40-like, oxa-58-like, oxa-143-like, and oxa235-like βlactamases[9]. the intrinsic blaoxa-51 gene is characteristic of this species and is usually weakly expressed[10]. though, it can illustrate that carbapenem resistance increases its expression when an insertion sequence isaba1 precedes the gene by providing promoter sequences[11]. the blaoxa-23 gene was first categorized in scotland which has been progressively reported worldwide. acinetobacter radioresistens was recently recognized as the progenitor of the blaoxa-23-like genes. sequence comparisons from different groups of oxa-type enzymes, oxa-23, oxa-24, and oxa-58, which can be plasmid or chromosomally encoded, have been most often linked to carbapenem-resistant clinical strains of a. baumannii, but oxa-23 has the highest distribution worldwide[8]. various studies conducted in nepal have confirmed and reported different groups of oxa-type enzymes in a. baumannii. a university hospital, kathmandu, reported 80.3% carbapenem-resistant mdr a. baumannii where all isolates harbored blaoxa51-like, most of them had blaoxa-23 and blaoxa-420 (a novel blaoxa-58 variant), and 36% isolates had blaoxa-104 [12] . similarly, in another study conducted at a tertiary hospital, nepal, 98% carbapenem-resistant mdr a. baumannii were reported. this study showed that all the isolates contained blaoxa-23 whereas blaoxa-24 pmmb 2021, 4, 1; a0000245 3 of 17 and blaoxa-58 were not detected in any of the analyzed isolates [13]. besides, previous studies have reported varying prevalence of carbapenem resistance in bacteria in different settings, including 9.09% in shahid gangalal national heart centre, kathmandu[14], 25.7% in nepal medical college, kathmandu[15], 56% in shree birendra hospital, kathmandu[16], and 28% in paropakar maternity and women’s hospital, kathmandu[17]. the prevalence of carbapenem-resistant strains varies in different hospitals, laboratories, and research centers among the samples used for the isolation of bacteria. a. baumannii has been identified as a “red-alert” human pathogen due to the rapid progress of multiple antibiotic resistance contributed by biofilm formation as well[18]. biofilm is a structured community of microorganisms enclosed in a self-produced polymeric matrix adherent to an inert or living surface[19,20]. biofilm formation is phenotypically related to exopolysaccharide (eps) production, nutrient availability, bacterial surface components (outer membrane proteins, adhesins), quorum sensing, and pilus formation[21,22]. formation of biofilm helps acinetobacter easily survive and transfer in hospital environments such as attachment to various biotic and abiotic surfaces, e.g., vascular catheters, cerebrospinal fluid shunts, or foley catheters[23]. biofilm is a relevant process for bacterial survival due to its mechanism for antibiotic resistance, transfer of resistance plasmids, and a medium for intercellular communication[24]. among acinetobacter spp., biofilm-producing strains are immensely resistant to antimicrobial agents than those that do not produce biofilm[24]. the mechanism responsible for antimicrobial resistance in biofilm-producing organisms may be delayed penetration of antimicrobial agents through the biofilm matrix, trapping of the antibiotics in the exopolysaccharide matrix, an altered growth rate of biofilm organisms, and other physiological changes due to the biofilm mode of growth[25]. biofilm formation by these bacteria has been their protective weapon for continuing their survival in a wide range of environmental conditions. this increases a prominently therapeutic question for the treatment of nosocomial infections throughout the globe. in nepal, previous studies have reported varying prevalence of biofilm-producing a. baumannii in different settings, including 14% in shree birendra hospital, kathmandu[16], 53.97% in bpkihs, dharan[26], and 51.11% in annapurna neurological institute and allied science, kathmandu[27]. the rising incidence of antibiotic resistance in a. baumannii is a matter of great concern to mankind. this study is crucial for understanding the occurrence and distribution of the biofilm-producing multidrug-resistant a. baumannii in the nepalese clinical environment and the association between carbapenem resistance and the presence of blaoxa genes in a. baumannii. pmmb 2021, 4, 1; a0000245 4 of 17 2. materials and methods 2.1. study design, study site, and sample population this was a hospital-based cross-sectional study conducted from march to september 2019. the clinical specimens were collected and processed at phect-nepal kirtipur hospital, kathmandu, and dna extraction and polymerase chain reaction were carried out at the central department of microbiology, tribhuvan university. the study population included patients of all age groups and both genders. 2.2. sample collection and transportation all the clinical specimens, including wound swab, pus, blood, urine, sputum, catheter tips, tissue, and body fluids, were collected by experienced medical personnel in a clean, leak-proof, sterile container according to the specimen type as per the guidelines of the hospital. the collected specimens were immediately transferred without any delay to the microbiology laboratory for routine culture and antibiotic sensitivity testing[28,29]. 2.3. culture and identification of bacterial isolates each clinical specimen was inoculated onto blood agar and macconkey's agar plates and incubated aerobically for 24 hours at 35˚c. individual colonies were identified by standards microbiological tests as per the morphology, gram staining, and various biochemical tests which were catalase test, oxidase test, voges proskauer test, methyl red test (mr-vp), citrate test, urease test, oxidative-fermentative test, tsi (triple sugar iron agar) test[28,29,30]. 2.4. antibiotic susceptibility test of a. baumannii bacterial isolates were analyzed for antibiotic susceptibility tests using the modified kirby-bauer disc diffusion method as stated by clinical laboratory standards institute guidelines (28). the antibiotics used in the study were amoxicillin/clavulanic acid (30/10 µg), amikacin (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefepime (30 µg), cotrimoxazole (25 µg), ciprofloxacin (5 µg), gentamicin (10 µg), imipenem (10 µg), meropenem (10 µg), piperacillin-tazobactam (100 µg), tetracycline (30 µg), polymyxin b (300 µg), colistin (10 µg), doxycycline (30 µg). multidrug resistance was defined as resistance to three or more classes of the antimicrobials tested[31]. 2.5. screening for carbapenemase production for screening of carbapenemase production, bacterial isolates resistant to imipenem and meropenem were selected. if the inhibition zone was ≤19 mm for imipenem and ≤19 mm for meropenem, the isolates were subjected to tests for confirmation of carbapenemase[31]. pmmb 2021, 4, 1; a0000245 5 of 17 2.6. phenotypic confirmation of carbapenemase producers modified hodge test (mht) was used for phenotypic confirmation of the carbapenemase producers[31]. for the test, e. coli atcc 25922 in 5 ml mueller-hinton broth (mhb) was prepared corresponding to 0.5 mcfarland standard. it was then diluted 1:10 by adding 0.5 ml of broth culture to 4.5 ml of mhb. a lawn culture of diluent was prepared on mha and allowed to dry for 3-5 minutes. a 10 µg meropenem disc was placed on the center of the test area. in a straight line, a. baumannii was streaked from the edge of the disc to the edge of the plate at 3 different places. the plate was incubated overnight at 35℃ in ambient air for 16-24 hours. they were examined for a clover leaf-type indentation or flattening at the intersection of the a. baumannii and the e. coli 25922 within the zone of inhibition of the carbapenem susceptibility disc. 2.7. detection of blaoxa the genomic dna was extracted from a. baumannii by using the protocol as described by pu et al[32]. for the detection of blaoxa-23, blaoxa-24, and blaoxa-51 the following primers were used; oxa-23f (5’-gat cgg att gga gaa cca ga-3’), oxa-23r (5’att tct gac cgc att tcc at-3’) with expected pcr product of 501 bp; oxa 24f (5’-ggt tag ttg gcc ccc tta aa-3’), oxa-24r (5’-agt tga gcg aaaagg gga tt-3’) with expected pcr product of 246 bp; oxa-51f (5’-taa tgc ttt gat cgg cct tg-3’), oxa-51r (5’-tgg att gca ctt cat ctt gg-3’) with expected pcr product of 353 bp[33]. the conditions used were as follows: 5 min at 94°c, followed by 30 cycles of 45 s at 94°c, 1 min at 52°c, and 1 min at 72°c, and a final extension of 5 min at 72°c. the amplified products were visualized on 1% agarose gel, containing ethidium bromide. 2.8. biofilm formation assay the biofilm-forming ability of the bacterial isolates was determined by using a quantitative adherence assay[34]. each isolate was cultured overnight in trypticase soy broth (tsb) at 37℃. 2 µl of cell suspension was inoculated in sterile 96 well polystyrene microtiter plates with 198 µl of tsb. after 24 h of incubation at 37℃, the wells were gently washed three times with 200 µl of phosphate-buffered saline (pbs), then dried in an inverted position and stained with 50 µl of 0.1% crystal violet. subsequently, the wells were gently washed three times with 200 µl of distilled water and dried in an inverted position. the wells were added with 200 µl of 5% isopropanol to solubilize the residual crystal violet. the optical density (od) at 570 nm was determined using a microtiter plate reader. each isolate was tested by using several wells (repeated 8-12 wells), and the average optical density was pmmb 2021, 4, 1; a0000245 6 of 17 obtained. optical density cut-off (odc) was defined as an average od of negative control + (3 × standard deviations (sd) of negative control). the interpretation of biofilm production was done according to the following criteria[35]. • non-biofilm producer: od ≤ odc • weak biofilm producer: 2×odc < od ≤4×odc • medium biofilm producer: 2×odc < od ≤4×odc • strong biofilm producer: od > 4×odc 2.9. data analysis all the data obtained were analyzed using the statistical programming statistical package of social sciences (spss version 21.0). a chi-square test was used to determine the association of independent variables and a p-value ≤ 0.05 was considered statistically significant. 2.10. ethics statement ethical approval for this study was obtained from the institutional review committee (irc), phect-nepal (004-2019). written informed consent was obtained from each patient for their voluntary participation in the study. 3. results 3.1. distribution of a. baumannii isolates a. baumannii was detected in 19 of 90 non-duplicate clinical isolates (21.11%). the majority of a. baumannii was identified in wound swabs (n=6, 31.58%), tissue (n=4, 21.05%), sputum (n=3, 15.79%), and pus (n=2, 10.52%), followed by catheter tips (n=1, 5.26%), blood (n=1, 5.26%), urine (n=1, 5.26%), and body fluid (n=1, 5.26%). among 19 isolates of a. baumannii, 84.21% were mdr of which 26.31% isolated from wound swabs followed by tissue (21.05%). similarly, 89.47% of the samples produced carbapenemase, with the greatest levels detected in wound swab (n=6, 31.58%), tissue (n=3, 15.78%), sputum (n=3, 15.78%), and pus (n=2, 15.78%) (figure 1). pmmb 2021, 4, 1; a0000245 7 of 17 figure 1. distribution of a. baumannii, mdr, and carbapenem among different clinical specimen 3.2. antibiotic susceptibility pattern of a. baumannii all isolates were susceptible to colistin and tetracycline but resistant to cefotaxime and ceftazidime. most of them were resistant to amoxicillin/clavulanic acid (94.74%), cefepime (73.68%), imipenem (89.48%), meropenem (89.48%), cotrimoxazole (89.48%), ciprofloxacin (83.6%), amikacin (84.21%), gentamicin (89.48%), and doxycycline (89.48%) (figure 2). figure 2. antibiotic susceptibility pattern of a. baumannii pmmb 2021, 4, 1; a0000245 8 of 17 3.3. association between biofilm formation and mdr acinetobacter isolates the microtiter plate technique was used to assess biofilm formation. nine strains (47.37%) of a. baumannii isolates are strong biofilm producers, five strains (26.31%) are moderate biofilm producers, three strains (15.79%) are weak biofilm producers, and two strains (10.53%) are non-biofilm producers. mdr was detected in 84.21% of a. baumannii isolates. a statistically significant relationship between mdr a. baumannii and biofilm generation capability was discovered (p ≤ 0.005) (table 1). table 1. association between biofilm formation and mdr acinetobacter isolates biofilm production mdr n (%) non-mdr n (%) total n (%) p-value* strong moderate weak non-producer 9 (47.37) 0 9 (47.37) 0.005 4 (21.05) 1 (5.26) 5 (26.31) 3 (15.79) 0 3 (15.79) 0 2 (10.53) 2 (10.53) total 16 (84.21) 3 (15.79) 19 (100) *chi-square test 3.4. association between biofilm formation and carbapenemase-producing acinetobacter isolates among 12 carbapenemase producers, (n=8, 42.10%) were strong biofilm producers and (n=3, 21.05%) were moderate biofilm producers. a significant statistical association (p<0.043) had been observed between biofilm formation ability and carbapenemase production (table 2). table 2. association between biofilm formation and phenotypic carbapenemase-producing acinetobacter isolates biofilm production carbapenemase producers n (%) carbapenemase non-producers n (%) total n (%) p-value* strong moderate weak non-producer 8 (42.10) 1 (5.26) 9 (47.37) 0.0043 3 (21.05) 2 (10.53) 5 (26.31) 1 (5.26) 2(10.53) 3 (15.79) 0 2 (10.53) 2 (10.53) total 12(63.15) 7 (36.83) 19 (100) *chi-square test pmmb 2021, 4, 1; a0000245 9 of 17 3.5. prevalence of blaoxa genes in a. baumannii isolates of the 19 a. baumannii, all isolates harbored blaoxa-51, none of the isolates carried blaoxa-24 and 63.2% isolates had blaoxa-23. the blaoxa-51 and blaoxa-23 co-occurred in 63.2% of a. baumannii isolates. however, all 3 genes did not co-occur in a single isolate of a. baumannii (figure 3). figure 3. venn diagram showing concurrence of oxa genes in a. baumannii isolates 4. discussion acinetobacter species are ubiquitous organisms and prevail in natural environments. transmission of an isolate is usually through the hands of staff, contaminated equipment, or the overall hospital environment. the prevalence of a. baumannii was 21.11% (19/90) in this study which is similar to some previous studies reported from nepal[36,37]. as the pathogen can endure dry conditions for a long time, they persist in the hospital environment and are responsible for opportunistic infections[17]. hence, the highest number of isolates were isolated from in-patients (84.2%) which is following other studies[38,49]. all isolates of a. baumannii were resistant to cefotaxime and ceftazidime, nearly 90% of the isolates were resistant to imipenem and meropenem, and more than 80% were resistant to cotrimoxazole, amikacin, and gentamicin. however, all the isolates were susceptible to colistin and tetracycline. these are the deliberated narrow alternatives for the treatment of carbapenem-resistance a. baumannii. also, nearly 85% of a. baumannii isolates were found to be multidrug-resistance. other studies conducted in nepal have reported 97.9%[13] and 82.8%[15] a. baumannii to be mdr. the increased antibiotic resistance of a. baumannii is most probably due to the extensive misuse of antimicrobial agents in nepal[40]. the pmmb 2021, 4, 1; a0000245 10 of 17 prevalence of carbapenem resistance in acinetobacter in uk, usa, singapore, chile, korea, portugal, india, and pakistan was 47-77%, 48%, 50%, 70%, 92%, 85%, 40% and 62-100%, respectively[41-46]. the development of resistance in a. baumannii may be due to the presence of wide array 𝛽 -lactamases that hydrolyze and confer resistance to penicillins, cephalosporins, and carbapenems[17]. resistance to carbapenem is facilitated by resistance mechanisms including enzymatic inactivation, active efflux of drugs, and modification of target sites[47]. among meropenem-resistance a. baumannii isolates, 63.16% were carbapenemase producers and all phenotypic carbapenemase producers carried at least one or more carbapenemase genes as shown by pcr. similarly, the study carried out in morocco in 2019 showed that 69.6% were positive for carbapenemase production by mht[48]. following clsi 2018, mht is no longer recommended as a phenotypic test for carbapenemase detection presumably because of the poor sensitivity of the test when detecting certain extended-spectrum β-lactamase synthesis associated with porin loss[49]. in the current investigation, however, all isolates identified as carbapenemase producers by mht also had one or more carbapenemase genes, as shown by pcr. results of the current study (blaoxa-51 (100%), blaoxa-23 (63.15%)), are consistent with those of other studies, which had reported the most prevalent carbapenem hydrolyzing β-lactamases genes in a. baumannii to include blaoxa-51 (83–100%) and blaoxa-23 (59–96%) [50-52]. therefore, the most prevalent mechanism underlying the resistance of a. baumannii to carbapenem antibiotics is the production of oxa-type β-lactamases which can hydrolyze carbapenem antibiotics and also be considered as the main factor that causes multidrug resistance in a. baumannii. furthermore, blaoxa-24 was not detected in any isolates in this study. the blaoxa-24 gene is common in a. baumannii isolated from european countries[53]. this result is similar to the study conducted in brazil[54] and the united states of america[55]. the previous studies from nepal did not detect this gene too[12,13]. however, the blaoxa-24 gene was reported in some studies in taiwan[56], iran[57], poland[58], and france[59]. altogether, these results suggest a specific distribution of blaoxa gene variants in different regions. to the best knowledge, this study revealed the highest co-occurrence of blaoxa-51/23 in a. baumannii isolates from nepal and demonstrated the increase of co-occurrence of blaoxa-51/23 overtime in nepal. the results of this study are consistent with the previous studies that report high carbapenem resistance in a. baumannii strains from nepal with oxatype carbapenemase predominancy[60,61]. it seems that the increasing co-occurrence of these genes and elements may lead to an increase in the resistance rate against carbapenems among a. baumannii isolates. pmmb 2021, 4, 1; a0000245 11 of 17 biofilm formation is suspected of being one of the key pathogenic features of a. baumannii, particularly with device-related infections. in this study, 89.47% of isolates of a. baumannii were biofilm producers. out of 16 isolates, 9 (47.37%) strong biofilm producers and 5 (21.05%) moderate biofilm producers were mdr. there was a statistically significant relationship between biofilm formation and antibiotic resistance. similar results of 100%, 77% 100%, and 98.5%, respectively in india[62], south korea[63,64], algeria[65], and egypt[66] showed a higher prevalence of biofilm formation. the strong ability to form biofilms by a. baumannii is associated with a significant increase in antibiotic resistance of the bacteria as biofilms limit the diffusion of antibiotics to the site of action due to its components or alter the phenotypes or genotypes of the strains[67]. in this study, 84.21% of the a. baumannii strains were mdr, having the ability to form biofilms. this finding is in line with a previous study from tehran where 92% biofilmforming a. baumannii was reported to be mdr and 86% of isolates were extensively drugresistance[68]. impaired drug diffusion due to microbial aggregations, overexpression of the exopolymeric substance (eps) matrix, alterations in microbial phenotypic and genotypic features due to stress responses, and physiological heterogeneity due to physicochemical gradients and persisters enhance antibiotic resistance in the biofilm phenotype[69]. biofilm formation which is associated with long-term persistence in the hospital environment[64] depends on an interaction between three main components: the bacterial cells, the attachment surface, and the surrounding medium[65]. thus, interventions such as contact precautions, environmental cleaning, active surveillance, and restrictions on administering broadspectrum antibiotics can be used for controlling a. baumannii[64]. the study also evaluated the correlation between biofilm formation and resistance to carbapenem among isolates of a. baumannii and reported a significant statistical association. although among carbapenem sensitive isolates, a strong biofilm producer was seen, the majority of carbapenem-resistance isolates were strong biofilm producers which explains some association exists between biofilm formation and antibiotic resistance among a. baumannii strains. the ability of a. baumannii to attach and form biofilms in abiotic surfaces has increased the opportunistic infections in hospitalized patients with the increased antibiotic resistance rate. other factors attributing to the increasing rate of a. baumannii infection in the hospital environment are inappropriate or inadequate management of infections, poor sanitation, inappropriate machinery handling practices by hospital personnel, misuse and pmmb 2021, 4, 1; a0000245 12 of 17 overuse of antibiotics, and lack of national guidelines and regulations for the use of antibiotics[66]. hence, for reducing the incidence of antibiotic resistance, regular surveillance of hospital-associated infections, prescription of antibiotics based on antibiogram obtained from the microbiological analysis, and formulation of definitive antibiotic policy may be helpful. as biofilm production by acinetobacter was associated with antibiotic resistance in this study, the screening tests for biofilm production in the laboratory may be useful to predict the drug resistance among the isolates. however, it is not clear whether any underlying mechanisms correlate biofilm production and antibiotic resistance. therefore, future studies are recommended to understand the co-expression of biofilm and resistance-related genes. this study might be helpful to carry out future studies capable of covering a wider range of healthcare settings and community surveillance to establish the actual burden of disease and the prevalence rates of multidrug-resistant strains. 5. conclusion this study reported the carbapenemase-producing a. baumannii to be sensitive towards colistin and tetracycline, hence these are recommended as potential drugs to treat the infections caused by carbapenem resistance acinetobacter species. in most of the isolates of multidrug-resistance a. baumannii, both blaoxa-51 and blaoxa-23 were detected as the determinant factors for carbapenem resistance having a direct relation with biofilm formation. author contributions: ug is the primary author who has developed the study methodology, carried out laboratory investigations, and prepared the manuscript. the ss planned the experiments and guided the project. rk, sk, skc, and mn contributed to the critical revision, data analysis, and editing of the manuscript. drj has contributed to the overall editing, review, and supervision of the project. the final version of the manuscript was read and accepted by all authors. funding: this work was funded by the university grant commission (ugc), nepal through master thesis grant program (mrs-75/76-s &t-47). acknowledgments: we would like to acknowledge central department of microbiology, tribhuvan university, phect-nepal kirtipur hospital and all the staff of the research department for guiding the study. we would also like to thank university grants commission (ugc), nepal for funding this project. conflicts of interest: the authors declare no conflict of interest. references 1. bazmara h, rasooli i, jahangiri a, et al. antigenic properties of iron regulated proteins in acinetobacter baumannii: an in-silico approach. int j pept res ther 2019; 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6(6): 528–533. 69. yang ys, lee y, tseng kc, et al. in vivo and in vitro efficacy of minocycline-based combination therapy for minocycline-resistant acinetobacter baumannii. antimicrob agents chemother 2016;60(7): pmmb 2021, 4, 1; a0000245 17 of 17 4047–54. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2021, 4, 1; a0000247. doi: 10.36877/pmmb.a0000247 http://journals.hh-publisher.com/index.php/pmmb review article covid-19: gastrointestinal manifestations and complications angel yun-kuan thye1, priyia pusparajah1, loh teng-hern tan1,2, jodi woan-fei law1, vengadesh letchumanan1*, learn-han lee1* article history 1 novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, subang jaya, selangor, 47500, malaysia; angelthye.yunkuan@monash.edu (ay-kt); priyia.pusparajah@monash.edu (pp); jodi.law1@monash.edu (jwfl) 2 clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) *corresponding author: vengadesh letchumanan, learn-han lee; novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, subang jaya, selangor, 47500, malaysia; vengadesh.letchumanan1@monash.edu (vl), lee.learn.han@monash.edu (l-hl) received: 15 october 2021; received in revised form: 14 november 2021; accepted: 15 november 2021; available online: 22 november 2021 abstract: the virus responsible for the covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which belongs to the genus betacoronavirus. this genus also includes the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov). the common symptoms of covid-19 infection are fever and respiratory symptoms, but it can also involve the gastrointestinal tract (git), resulting in manifestations such as diarrhea, nausea and/or vomiting and abdominal pain. the emergence of covid-19 led to public health emphasis on droplet transmission and precautions of contact with respiratory secretions. however, mounting evidence demonstrates detection of sars-cov-2 rna in stool samples of covid-19 patients. it has also been demonstrated that the host receptor angiotensinconverting-enzyme-2 (ace-2) is highly expressed not just in respiratory cells but also in gastrointestinal sites involving the glandular cells of gastric, duodenal, and rectal epithelium. this suggests that sars-cov-2 can infect the digestive system, serving as another route of transmission. this review aims to study the prevalence of some of the gastrointestinal manifestations following covid-19 infection and findings of positive sars-cov-2 rna in stool specimens while making parallels to the severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) infection. we will also discuss the possible pathophysiology of covid-19 related gastrointestinal involvement. keywords: covid-19; sars-cov-2 rna; gastrointestinal; angiotensin converting enzyme 2 (ace-2); stool 1. introduction the coronavirus disease 2019 (covid-19) was declared a pandemic by the world health organization (who) in early march 2020[1-3]. this global pandemic started with an pmmb 2021, 4, 1; a0000247 2 of 13 outbreak of pneumonia of unknown aetiology in wuhan, hubei province of china[4-8]. the virus responsible for causing covid-19 was named the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) by the coronavirus study group of the international committee on taxonomy of viruses (ictv)[9]. it is a positive sense, single-stranded rna virus belonging to the genus betacoronavirus that originated from bats[4, 10]. interestingly, sars-cov-2 has a 79% overlap of genomic sequence identity with the severe acute respiratory syndrome coronavirus (sars-cov) and 50% with the middle east respiratory syndrome coronavirus (mers-cov)[11]. at the time this paper went to press, the sarscov-2 had infected over 250,000,000 people and caused 5,072,046 deaths worldwide (as of 10th november 2021)[12]. the virus spread swiftly worldwide, with the emerging of new variants of concern (voc) with a higher infectivity linked to high fatality rates[13]. the data suggests that sars-cov-2 primarily infects respiratory epithelial cells and spreads via respiratory route from human to human, however, the exact viral target cells and organs are yet to be determined[14]. some of the most common symptoms of covid-19 infections include fever and respiratory symptoms such as dry cough and dyspnea, which is similar to the severe acute respiratory syndrome (sars) in 2003 and middle east respiratory syndrome (mers) in 2012. aside from respiratory symptoms, gastrointestinal symptoms including diarrhea, nausea, vomiting and abdominal pain may also be present[15]. there has been escalating evidence of sars-cov-2 rna being detected in stool specimens[14, 16, 17], and anal[18], or rectal swabs[19], of covid-19 patients despite the clearance of sars-cov-2 in the respiratory cells/upper respiratory tract[14, 18, 20]. besides that, the angiotensinconverting enzyme-2 (ace-2) receptor is highly expressed in gastrointestinal epithelial cells, particularly the ileum, duodenum, jejunum, caecum and colon[14, 21]. taken together, these suggest that sars-cov-2 may also demonstrate fecal-oral transmission, in addition to droplet transmission; which has implications on sars-cov-2 transmission, infection control and management. the aim of this review is to study the prevalence of some of the gastrointestinal symptoms and the positive detection of sars-cov-2 rna in stool specimens of covid-19 patients, while making parallels to sars and mers infection. we will also discuss some of the possible causes of gastrointestinal involvement following covid-19. 2. prevalence of gastrointestinal symptoms of covid-19 the well-established signs and symptoms of covid-19 patients are fever and respiratory symptoms. however, gastrointestinal symptoms such as diarrhea, vomiting and abdominal pain seem to frequently also be part of the manifestations of the disease. in the united states, the first confirmed covid-19 case was reported on the 20th january 2020 pmmb 2021, 4, 1; a0000247 3 of 13 involving a 35-year-old man who just returned from wuhan, china. upon admission, he presented with persistent dry cough and a 2-day history of nausea and vomiting. on the second day of hospitalization, he reported diarrhea and abdominal discomfort[16]. in a meta-analysis consisting of 4,243 covid-19 positive patients (60 studies) from 6 countries (china n=53, south korea and singapore n=2, vietnam, united states and united kingdom n=1) the pooled prevalence of gastrointestinal symptoms was 17.6%[22]. for individual gastrointestinal symptoms, 18 studies reported the prevalence as follows: lack of appetite (26.8%), diarrhea (12.5%), nausea/vomiting (10.2%), and abdominal pain (9.2%)[22]. a cross sectional multicenter study involving 204 hospitalized covid-19 positive patients in china’s hubei province found 103 (50.5%) patients had ≥ 1 digestive symptoms, including diarrhea (34%), vomiting (3.9%), abdominal pain (1.9%)[23]. furthermore, in a hong kong cohort with 59 covid-19 positive patients, 15 (25.4%) patients presented gastrointestinal symptoms of diarrhea (22%), abdominal pain (11.9%), vomiting (1.7%), and 9 (15.3%) had positive viral rna in stool specimens[22]. a few other cohorts also reported frequencies of diarrhea ranging 2.0–10.1% and nausea and/or vomiting ranging 1.0–10.1%[10, 24-32]. similar to adults, children also displayed gastrointestinal symptoms following positive covid-19 assays. however, in terms of covid-19 severity, children and/or adolescents were found to have less severe covid-19 infection, with milder symptoms and possibly better prognosis than adults[33, 34]. in a meta-analysis of 280 covid-19 positive children, the pooled prevalence of gastrointestinal symptom was 22.8%. the pooled prevalence of diarrhea, vomiting and abdominal pain was respectively 12.4%, 10.3%, and 5.4% respectively[35]. on the other hand, another meta-analysis consisting of 2855 children showed 4% had diarrhea and none had abdominal pain[33]. the variability in rates in different studies could be a result of variability in clinical presentation and study size. hence, more clinical data is needed. similar to covid-19, sars and mers outbreaks also demonstrated gastrointestinal (gi) manifestations during the course of the disease. a retrospective study in hong kong involving 138 sars patients revealed diarrhea to be the most common gi symptom with an average (±sd) duration of diarrhea of 3.7±2.7 days. out of 138 patients, 28 (20.3%) had diarrhea at presentation while 53 (38.4%) had diarrhea at the first 3 weeks of illness[36]. using reverse transcriptase-polymerase chain reaction (rt-pcr), it was also demonstrated that the stool specimens from sars patients had a 16% detection rate of severe acute respiratory syndrome coronavirus (sars-cov) rna, comparable to the detection rate in nasopharyngeal aspirates[36]. mers also presented with gastrointestinal symptom — in a study of 47 mers infected patients in saudi arabia, diarrhea (26%), vomiting (21%) and pmmb 2021, 4, 1; a0000247 4 of 13 abdominal pain (17%) were present at presentation[37]. a retrospective observational study from korea with 186 mers patients also showed gastrointestinal symptoms of diarrhea (19.4%), nausea and vomiting (14%) and abdominal pain (8.1%)[38]. it is clear that covid-19 has a similar gastrointestinal presentation as that seen in sars and mers. covid-19 positive patients whether children or adults can present with gastrointestinal symptoms early in the course and it should not be taken lightly. for instance, a study found nearly a quarter of covid-19 positive children have at least one gastrointestinal symptom with diarrhea being the most common symptom followed by vomiting and abdominal pain[35]. this finding was quite consistent with a meta-analysis in adult covid-19 patients that found the prevalence of gastrointestinal symptoms to be 18%[22, 35]. nonetheless, there could be variation in the rate of prevalence among different studies due to variability in study size and clinical presentation. 3. detection of sars-cov-2 rna in stool specimens of covid-19 patients in relation to sars-cov-2 rna detection in stool specimens, us’s first covid-19 positive patient presented with gastrointestinal symptoms and his stool sample together with respiratory specimens (nasopharyngeal and oropharyngeal) and serum were sent for real time rt-pcr testing in which both stool (illness day 7) and respiratory specimens (illness day 4 and day 7) detected sars-cov-2 rna[16]. it is also important to note that there is also positive live sars-cov-2 detection in stool samples of patients without diarrhea[17]. a study showed among children tested for sars-cov-2 by stool pcr or rectal swab, 89% had positive results even though 82.6% of them had no gastrointestinal symptoms[19]. this finding is consistent with a hong kong cohort where 15.3% of covid-19 patients have sars-cov2 rna detected in their stool specimen on presentation regardless of whether or not they have gastrointestinal symptoms, and when compared to those without diarrhea, those with diarrhea have higher stool rna positivity and viral load[22]. this suggests, with or without gastrointestinal symptoms, sars-cov-2 could be present in stool samples. even if viral nucleotides cannot be detected in oral swabs, they can be found in anal swabs or blood[18]. furthermore, there is a rising number of studies that have detected sarscov-2 rna in stool samples. worryingly, the constant positive detection of sars-cov-2 rna from feces, indicates virions are secreted from the virus-infected gastrointestinal cells. for instance, in a china study consisting of 73 covid-19 patients, 39 (53.42%) patients were found to have sars-cov-2 rna in stool, whereas 17 (23.29%) patients remained positive for sars-cov-2 rna in stool with a duration of positive stool ranging from 1–12 days despite negative results in respiratory samples[14]. a study from china also showed an asymptomatic 10 year old child who had persistently positive stool samples for 26 days even pmmb 2021, 4, 1; a0000247 5 of 13 though respiratory specimens were persistently negative[39]. taken together, these indicate that the conventional testing using rt-pcr of nasopharyngeal swabs alone may not be an accurate diagnostic test to detect the presence or clearance of sars-cov-2 rna. importantly, a study based on patients from wuhan also found that there could be a possible shift from oral positive during early infection to anal swab positive during late infection[18]. sars-cov-2 can be detected in the intestine of covid-19 infected patients at early or late stages, whereas sars-cov and mers-cov infected individuals, intestinal infections were detected at later stages[18, 40-42]. this may be related to the cycle threshold (ct) value. a lower ct value corresponds to a higher viral load and ct value <40 indicates positive sars-cov-2 rna. wang et al showed there is a similar mean cycle threshold between feces (31.4), sputum (31.1) and pharyngeal (32.1) swabs of sars-cov-2 rna, while nasal swab had the lowest mean cycle threshold of 24.3[17]. in the us's first covid19 case, despite initial mild symptoms at presentation, the nasopharyngeal and oropharyngeal specimens obtained on illness day 4 showed ct values of 18-20 and 21-22 respectively. on illness day 7, the ct value of nasopharyngeal specimens increased to 23-24 while stool specimens showed greater ct value of 36–38. but, on illness day 11 and 12, there is a trend of decreasing viral levels observed for nasopharyngeal and oropharyngeal specimens[16]. furthermore, a study found that in 70.3% of patients, prolonged shedding of sars-cov-2 rna was detected in stool rather than respiratory samples, which could be up to ≥33 days from illness onset[22]. similarly, in a sars patient, even postmortem examination (a few days after death of patients) of biopsy specimens of the small intestine yielded viable sarscov when all other organ tissues collected during autopsy had no viable virus recovered. in addition, non-viable sars-cov can be detected in stool specimens by pcr for up to 73 days from symptom onset[36]. thus, it seems like that the shift from oral positive to anal positive can be seen via the trend in ct value throughout the course of illness and the prolonged shedding of sars-cov-2. 4. implications of covid-19 on the liver covid-19 infection may have an effect on liver enzymes and other indices of hepatic function. one study hypothesized that liver tissue injury is caused by the upregulation of ace-2 expression in liver tissue as a result of compensatory proliferation of hepatocytes derived from bile duct epithelial cells[43]. various studies have reported various rates of prevalence of liver injury in covid-19, ranging between 15% and 78%[44]. in a single-center study consisting of 148 covid-19 positive patients, 55 (37.2%) patients had abnormal liver function upon hospital admission and this cohort were hospitalized longer[45]. besides that, in the first reported case in the us, the patient had raised levels of creatine kinase and changes pmmb 2021, 4, 1; a0000247 6 of 13 in hepatic function measures whereby on day 5 of hospitalization, levels of alkaline phosphatase (alp)(68 u per liter), alanine aminotransferase (alt)(105 u per liter), aspartate aminotransferase (ast)(77 u per liter), and lactate dehydrogenase (ldh)(465 u per liter) were all raised[16]. another meta-analysis also showed a mild increase in alp, alt and ast in 4.6%, 20.6%, and 22.8% covid-19 patients and a mild reduction in serum bilirubin (39.8%)[44]. the implications of covid-19 on the liver seems to follow a pattern as seen in mers, as the concentrations of alt, ast and ldh were elevated in five (11%), seven (15%) and 23 (49%) mers patients, respectively[37]. nonetheless, findings linking liver injury with covid-19 is still debatable. it is unsure why there are dissimilarities among different studies. for instance, pan et al and wu et al found covid-19 patients had no significant liver injury[23, 46], however 2 other studies stated otherwise[47, 48]. there is also study on the pathological analysis of liver tissue from a deceased covid-19 patient that showed no viral inclusion in the liver[49, 50]. hence, more studies must be conducted to fully understand the association of covid-19 and hepatic function abnormalities. 5. possible reasons for gastrointestinal symptoms following covid-19 there are several reasons gastrointestinal symptoms manifest quite frequently following covid-19 infection, and they seem to be associated with the human host receptor ace-2[23], the intestinal microflora[23], the use of antibiotics and antiviral medicines[51, 52], and the direct or indirect inflammatory response as sars-cov-2 damages the digestive system[53] (figure 1). figure 1. the possible explanation for gastrointestinal manifestations following sars-cov-2 infection. firstly, sars-cov-2 entry into host cells is mediated by the binding of viral surface spike protein to the host receptor ace-2[54, 55]. ace-2 is expressed in type ii alveolar cells pmmb 2021, 4, 1; a0000247 7 of 13 in the lungs and goblet cells in the nasal mucosa[54, 56]. however, ace-2 is also abundantly present in the glandular cells of gastric, duodenal, and rectal epithelium. there is marked variation throughout the gi tract as ace-2 staining is seldom found in esophageal mucosa, probably because ace-2 is less expressed in squamous epithelial cells (esophageal epithelium) than glandular epithelial cells[14]. another study showed ace-2 is expressed primarily on the luminal surface of differentiated small intestinal epithelial cells, while crypt cells and the colon have lower expressions[57]. given that the infectivity of sars-cov-2 is determined by the binding affinity of ace-2 receptors[22], the high levels of these receptors indicate that the virus could possibly infect and replicate within the gastrointestinal tract[10]. furthermore, intestinal inflammation is modulated by ace-2[57], so it may be possible that the disruption of ace-2 function by the sars-cov-2 results in diarrhea[22]. ace-2 also has a ras independent function, it regulates the intestinal amino acid homeostasis, is involved in the expression of antimicrobial peptides and is associated with the gut microbial ecology[57]. amino acid malnutrition can result in intestinal inflammation and diarrhea; ace2 mutants demonstrated reduced expression of antimicrobial peptides and an altered gut microbial composition. this leads to the speculation of an association between covid-19 and the gut microbiota[57, 58]. secondly, the intestinal microbiota. the human intestine houses a diverse and huge amount of gut microbiota. for instance, comparing sites in the gastrointestinal system, the colon has the most microbes, consisting of 33% of total bacteria cells in the human body[59]. the gut microbiota plays vital roles including supporting the host’s metabolism, defending the host against harmful pathogens through habitat colonization and immunoregulatory responses, and regulating the development and maturation of the body’s immune system[23, 59-64]. reasons for changes in gut flora include the disease itself, concomitant infections, the use of antimicrobials, and an increase in proinflammatory mediators as a result of viralinduced inflammation[52]. alterations to the composition and function of the gastrointestinal tract microbiota may affect the respiratory tract through the common mucosal immune system, and respiratory tract flora disorders also influence the digestive tract via immune regulation. this is the gut-lung axis effect[23, 65, 66], which possibly explains why digestive symptoms occur following covid-19 infection. hence, probiotics may be a new treatment option or an adjuvant treatment. in the treatment of severe covid-19 infections, probiotics is recommended by the guidance (version 5) established by the china’s national health commission and national administration of traditional chinese medicine, to maintain the balance of intestinal microecology and the prevention of secondary bacterial infection, indicating that first-line medical staffs and the chinese government trust the importance of the role of gut microbiota in covid-19 infection[58, 67]. pmmb 2021, 4, 1; a0000247 8 of 13 thirdly, the use of antibiotics and antiviral drugs could result in gastrointestinal symptoms in some covid-19 patients. patients suspected with secondary bacterial infection are commonly treated with antibiotics in which some antibiotics (fluoroquinolones and cephalosporins) can cause antibiotic-associated diarrhea. furthermore, antimicrobial use can have serious effects on the gut microbiota, antibody production, and immune system which could prolong the clearance of sars-cov-2 from the gut[52]. in patients being treated with antiviral drugs such as remdesivir, chloroquine phosphate, hydroxychloroquine, and lopinavir-ritonavir, the medications themselves can also cause diarrhea[51, 52]. fourthly, sars-cov-2 can cause direct or indirect damage to the digestive system via the inflammatory response[9]. sars-cov-2 infection could cause an “inflammatory storm”, in which overactivated cytokines, inflammatory storms, and immune dysregulation causes inflammatory damage to the intestine resulting in diarrhea[9, 68]. with mounting evidence showing the presence of sars-cov-2 rna in stool specimens of covid-19 patients, it might suggest sars-cov-2 directly damages the intestinal mucosa, causing digestive symptoms such as diarrhea. 6. conclusions gastrointestinal manifestations seem to be part of the course of covid-19 infection and presentation with symptoms including — but not limited to — diarrhea, nausea and/ or vomiting and abdominal pain, should definitely not be taken lightly. some of the possible causes of gastrointestinal manifestations seems to be associated with direct viral invasion utilizing the ace-2 receptor[23], alterations in the intestinal microflora[23], the use of antibiotics and antiviral medicines[51, 52], and the direct or indirect inflammatory response as sars-cov-2 damaging the digestive system[9]. as covid-19 severity increases, the digestive symptoms are more distinct. clinicians should be alert to patients presenting with gastrointestinal symptoms due to the important implications as this cohort of patients have much longer duration from symptom onset to admission resulting in late diagnosis and treatment, compared to patients without digestive symptoms. it is possible that the increased viral load and replication in the gastrointestinal tract (git) results in more severe disease and the delay in reporting by patients with extrapulmonary symptoms but without the typical respiratory symptoms led to a later and less curable stage[23]. this is consistent with a metaanalysis that showed a higher prevalence of gastrointestinal symptoms in those with severe covid-19 than non-severe covid-19 (17.1% vs 11.8%)[22]. importantly, with the detection of sars-cov-2 in specimens from other sites other than the nasopharyngeal site, it is likely that sars-cov-2 can be transmitted in ways other than respiratory droplets. it is also likely that the virus can infect and replicate in other sites, in this case, the gastrointestinal tract of the human host. this suggests possible tissue tropism of sars-cov-2 in the intestinal cells and a possible fecal-oral transmission. pmmb 2021, 4, 1; a0000247 9 of 13 with these findings, there will be implications on the disease transmission, viral detection, infection control and management. hence, changes must be made to efficiently combat the worsening of sars-cov-2. clinicians and health care workers should be more cautious if patients present with initial gastrointestinal symptoms and take precautionary measures including droplet and fomite precautions, proper handling of covid-19 patients’ excreta, and have barrier precautions when performing colonoscopy[36]. furthermore, testing of stool specimens for sars-cov-2 rna should be made in addition to respiratory specimens. efforts should also be made to alert all people on the gastrointestinal manifestations of covid-19 so there can be early detection, diagnosis, isolation, and intervention, and reduce sars-cov-2 transmission within the family and in the community. author contributions: ay-kt performed the literature search, critical data analysis as well as manuscript writing. pp, lt-ht, jw-fl, vl, and l-hl provided review, editing, and proofreading for the manuscript. vl and l-hl conceptualize the project. funding: the seed funding funded this work from microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, (vote number: mbrs/jcsmhs/02/2020) awarded to vl. acknowledgments: professor dr. shajahan yasin, professor, and head of school, jeffrey cheah school of medicine and health sciences, monash university malaysia. conflicts of interest: the authors declare no conflict of interest. references 1. johnson d, ren sec, johnson hd, et al. covid-19: are malaysians embracing or suffering the new normality? prog microbes mol biol 2020; 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55: 102763. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. http://www.nhc.gov.cn/yzygj/s7653p/202002/d4b895337e19445f8d728fcaf1e3e13a/files/ab6bec7f93e64e7f998d802991203cd6.pdf http://www.nhc.gov.cn/yzygj/s7653p/202002/d4b895337e19445f8d728fcaf1e3e13a/files/ab6bec7f93e64e7f998d802991203cd6.pdf progress in microbes and molecular biology 1 original research article determination of antibiotic resistance patterns of vibrio parahaemolyticus from shrimp and shellfish in selangor, malaysia vengadesh letchumanan1,2, nurul-syakima ab mutalib3, sunny hei wong4, kok-gan chan5,6*, learnhan lee1,2,7 1novel bacteria and drug discovery research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, selangor darul ehsan, malaysia 2institute of biomedical and pharmaceutical sciences, guangdong university of technology, guangzhou 510006, pr china. 3ukm medical molecular biology institute (umbi), ukm medical centre, universiti kebangsaan malaysia, kuala lumpur, malaysia 4li ka shing institute of health sciences, department of medicine and therapeutics, the chinese university of hong kong, shatin, hong kong 5division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia 6international genome centre, jiangsu university, zhenjiang 212013, pr china 7center of health outcomes research and therapeutic safety (cohorts), school of pharmaceutical sciences, university of phayao, phayao, thailand abstract : high consumer demand for seafood has led to the need for large-scale, reliable supply through aquaculture farming. however, bacterial infections which can spread rapidly among the dense farming area pose a major threat to this industry. the farmers therefore often resort to extensive use of antibiotics, both prophylactically and therapeutically, in order to protect their stocks. the extensive use of antibiotics in aquaculture has been postulated to represent a major contributing factor in the rising incidence of antimicrobial resistant pathogenic bacteria in seafood; which may then lead to the spread of antimicrobial resistant bacteria in the environment as well as posing a significant threat to human health. this study aimed to characterize antibiotic resistance of vibrio parahaemolyticus from shrimp and shellfish in selangor, malaysia. the antibiotic susceptibility of 385 v. parahaemolyticus isolates was investigated against 14 antibiotics followed by plasmid profiling and plasmid curing to determine the antibiotic mediation. a large number of isolates showed resistance to ampicillin (85%), amikacin (66.8%), and kanamycin (50.1%). a notable resistance pattern was also observed to the third generation cephalosporins (cefotaxime 55.8% and ceftazidime 34%). only 338 v. parahaemolyticus isolates had 1-7 different plasmids and could be categorized into 27 patterns based on the number and pattern of plasmid present. interestingly, there was no correlation between the number of plasmids and antibiotic resistant patterns seen in the isolates. the antibiotic resistance was mediated by both chromosomal and plasmid mediation among the resistant isolates. in summary, our results demonstrate that incidence of pathogenic v. parahaemolyticus in seafood in selangor remains in relatively assuring levels, however the identification of antibiotic resistance among the isolates does rises a public health concern and warrants for continuous surveillance. keywords: consumers; aquaculture; vibrio parahaemolyticus; antibiotic resistance; malaysia received: 22nd january 2019 accepted: 25th february 2019 published online: 15th march 2019 *correspondence to: kok-gan chan; institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia. kokgan@um.edu.my. citation: letchumanan v, ab mutalib ns, wong sh, et al. determination of antibiotic resistance patterns of vibrio parahaemolyticus from shrimp and shellfish in selangor, malaysia. prog microbes mol biol 2019; 2(1): a0000019. introduction the global public health is endlessly challenged by the threat of foodborne diseases and the latter restraints the socioeconomic development by affecting the healthcare system, country’s economic, tourism and trade (1,2). since decades ago, there has been increasing number of foodborne diseases related with consumption of raw or undercooked foods in developed and developing countries (3). among the identified foodborne pathogens are salmonella sp. (4,5,6,7), listeria sp. (8,9) and vibrio sp. (10,11,12,13) that are often associated with gastroenteritis cases copyright 2018 by letchumanan v, et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) 2 worldwide. vibrio parahaemolyticus – a member of the vibrionaceae family is a gram-negative, rod-shaped halophilic bacterium that naturally lives in aquatic environments (13,14). v. parahaemolyticus is widely distributed in marine and estuarine environments thus causing gastrointestinal illnesses after being eaten raw or undercooked seafood (15,16). during the 2016, the centers for disease control and prevention (cdc), united states reported that v. parahaemolyticus was acclaimed as the major foodborne bacterium compared to other vibrio sp. this pathogen is accounted for nearly 34,664 foodborne cases annually in the united states (17,18). in malaysia, v. parahaemolyticus is naturally identified in the marine coastal region of malaysia in all seasons and known to cause foodborne gastroenteritis (19). in the early 1980s, a study revealed the incidence of v. parahaemolyticus in malaysian shrimp processing industry. it is of interest to note that 21 different serotypes were isolated from malaysian shrimp, with type 01:k38 and 01:k32 were predominates (20). in addition, the eu countries has rejected the import of frozen black tiger shrimp from malaysia due the presences of v. parahaemolyticus which subsequently affected the malaysian economic (21). the virulent v. parahaemolyticus carrying tdh and/ or trh genes was also identified from the frozen shrimps in malaysia, prompting a possible health risk for people consuming raw shrimp (22). the food safety in malaysia is further declining due to the rising cases of the detection of antibiotic resistant v. parahaemolyticus strains in seafood and environmental samples. there has been many reported cases of antibiotic resistant v. parahaemolyticus strains isolated from seafood namely shellfish, fish, and shrimps from malaysia (10,11,19,23,24,25,26). antibiotics and other chemotherapeutic agents are often incorporated as feed additives or immersion baths in aquaculture farms to control bacterial infections (27,28,29). though antibiotics are effective in controlling bacterial infections, the misuse of antibiotics has caused the occurrence of multidrug resistant bacteria in the environments (25). henceforth, there is a need for appropriate management and control of the use of antibiotics in the aquaculture sectors. the increase in bacterial resistance to many clinical antibiotics effects many country’s healthcare and food production sectors (30). in agreement with previous reports and the expected severity of infections, constant investigation on antimicrobial resistance of v. parahaemolyticus is needed for epidemiological purpose and guidance in healthcare treatment. for this reason, our study aimed to characterize antibiotic resistance of v. parahaemolyticus from shrimp and shellfish in selangor, malaysia. materials and method bacterial strains v. parahaemolyticus isolates from previous study was used for the present study (10,11). a total of 385 v. parahaemolyticus isolates were from shrimp and shellfish – red prawn (solenocera subnuda), banana prawn (penaeus indicus), mud crab (scylla serrate), flower crab (portunus pelagicus), carpet clam (paphia texile), hard shell clam (meretrix meretrix), and mud creeper (cerithidea obtuse) was collected from wetmarket and supermarket in selangor, malaysia. all these isolates were confirmed v. parahaemolyticus by toxr-pcr assay and thermostablerelated direct haemolysin (trh) gene was detected in the isolates (10,11). antibiotic susceptibility test (ast) the antibiotic susceptibility of v. parahaemolyticus isolates was determined using kirby-bauer disc diffusion method (31). fourteen different types of antibiotics discs (oxoid, uk) was tested: ampicillin (10µg), ampicillin/ sulbactam (30µg), amikacin (30µg), cefotaxime (30µg), ceftazidime (30µg), chloramphenicol (30µg), gentamicin (30µg), imipenem (10µg), kanamycin (30µg), levofloxacin (5µg), nalidixic acid (30µg), oxytetracycline (30µg), suphamethox/trimethoprim (25µg), and tetracycline (30µg). e. coli atcc 25922 with known sensitivity pattern was included as a positive control in each test. v. parahaemolyticus isolates was grown in tryptic soy broth (tsb) (himedia, india) supplemented with 2% w/v sodium chloride (nacl) (vivantis, usa) at 37oc for 18 hours under constant agitation (13). the bacteria cultures are lawn onto mueller hilton agar (himedia, india) with 2% w/v sodium chloride (nacl) (vivantis, usa), placed with antibiotic discs and incubated at 37°c for 18 hours. the zone of inhibition was measured and interpreted following the guidelines of clinical and laboratory standards institute (clsi) m45-a2 (32). the multiple antibiotic resistance (mar) index was calculated based on the ratio isolate’s resistance to the total number of tested antibiotics (33). plasmid profiling plasmid profiling was carried out following the method adapted from previous study with slight modification (27). v. parahaemolyticus cell were grown in tryptic soy broth (tsb) containing 2% w/v sodium chloride and incubated at 37oc in a shaker incubator (220rpm) for 18 hours. about 1.5 ml of the culture was transferred into a micro-centrifuge tube followed by centrifugation (10,000 rpm for 2 minutes at 4oc). the supernatant was removed by aspiration leaving the cell pellet as dry as possible. the pellet was resuspended in ice-cold 100ul alkaline lysis solution i (glucose 50mm; tris cl 25mm; edta 10mm) by vigorous vortexing followed by addition of freshly prepared 200ul alkaline lysis solution ii (naoh 2n; sds 2% w/v). the contents were mixed by vortexing rapidly after which 150ul ice-cold solution iii (potassium acetate 5m: 60ml; glacial acetic acid 11.5ml; dissolved in 28.5m sterile distilled water) was added to it. the tube was closed and gently vortexed for 10 seconds to disperse solution iii through the viscous bacterial lysate. then the tubes were stored in ice for 5 minutes before being centrifuged at 12,000 rpm for 2 minutes at 4oc. an equal volume of phenol-chloroform (1:1, w/v) was added to the supernatant in a fresh tube, by vortexing. the contents in the micro-centrifuge tube were centrifuged determination of antibiotic... 3 at 8,000 rpm for 3 minutes at 4oc and the supernatant was transferred into a fresh tube. this was repeated with chloroform: isoamyl-alcohol (24:1, v/v) for removing the phenol. the double stranded dna was precipitated with 2 volumes of ethanol at room temperature, followed by vortexing before it was allowed to stand for 5 minutes at room temperature. the aliquot was centrifuged at 12,000 rpm for 12 minutes at 4oc and the supernatant was removed by gentle aspiration. the pellet of double stranded dna was rinsed with ethanol (1ml, 70% v/v) at 4oc and centrifuged. the supernatant was removed leaving the pellet dry as possible. the pellet was air-dried before it was re-dissolved in 30ul ultrapure water. electrophoresis was performed using 1% agarose gel. plasmid curing the antibiotic resistance mediation of v. parahaemolyticus isolate was determined by plasmid curing method using two different intercalating agent, acridine orange (ao) and ethidium bromide (eb) (10,34). the isolates were revived in freshly prepared tryptic soy broth (tsb) supplemented with 0.2 mg/ml of respective curing agent and the tubes were incubated at 37°c for 18 hours under constant agitation. the treated culture was subjected to antibiotic susceptibility test as described in section 2.2 to re-examine the antibiotic resistance profiles. the phenotype results were compared with the antibiotic phenotype of non-treated isolate. the plasmid profiling as described in section 2.3 was performed with the treated culture in order to determine and compared the presences of plasmids before and after treatment. statistical analysis the data analysis was performed using ibm spss statistical analysis software version 20. statistical analysis was performed to determine whether there is any significant difference in type of samples and mar index of resistant v. parahaemolyticus isolates. a one-way analysis of variance (anova) followed by suitable post-hoc test (turkey) was used and p < 0.05 is considered as significant. results antibiotic resistance of v. parahaemolyticus strains fourteen antibiotics belonging to β-lactams, aminoglycosides, carbapenems, quinolones, tetracycline, sulphonamides, and chloramphenicol were used for the determination of antibiotic susceptibility of v. parahaemolyticus isolates. as shown in table 1, a large number of isolates showed resistance to ampicillin (85%), amikacin (66.8%), and kanamycin (50.1%). a notable resistance pattern could be observed to the third generation cephalosporins (cefotaxime 55.8% and ceftazidime 34%). in contrast, high susceptibility rate was seen to imipenem (94%), chloramphenicol (92.5%), tetracycline (83.1%), ampicillin-sulbactam (81%), levofloxacin (76.1%), trimethoprim-sulfamethoxazole (75.8%), nalidixic acid (73.85), and gentamicin (70.6%). a high percentage (68%) of isolates have a significant mar index more than 0.2. the value of mar index ranged from 0.00 to 0.79, with the highest mar index attributed from two isolates respectively (vp152 from supermarket banana prawn and svp129 from supermarket carpet clam) exhibiting resistance profile towards 11/14 antibiotics tested. letchumanan v et al. antibiotics no. of resistant isolates (%) no. of intermediate isolates (%) no. of susceptible isolates (%) ampicillin (10ug) 327 (85) 29 (7.5) 29 (7.5) ampicillin-sulbactam (30ug) 41 (10.6) 32 (8.3) 312 (81) cefotaxime (30ug) 215 (55.8) 51 (13.2) 119 (30.9) ceftazidime (30ug) 131 (34) 98 (25.5) 156 (40.5) imipenem (10ug) 5 (1.3) 18 (4.7) 362 (94) amikacin (30ug) 257 (66.8) 90 (23.4) 38 (9.9) gentamicin (30ug) 28 (7.3) 85 (22.1) 272 (70.6) kanamycin (30ug) 193 (50.1) 161 (41.8) 31 (8.1) tetracycline (30ug) 57 (14.8) 8 (2.1) 320 (83.1) oxytetracycline (30ug) 67 (17.4) 108 (28.1) 210 (54.5) nalidixic acid (30ug) 38 (9.9) 63 (16.4) 284 (73.8) levofloxacin (5ug) 31 (8.1) 61 (15.8) 293 (76.1) trimethoprim-sulfamethoxazole (25ug) 18 (4.7) 75 (19.5) 292 (75.8) chloramphenicol (30ug) 22 (5.7) 7 (1.8) 356 (92.5) table 1: the percentage of antibiotic resistant v. parahaemolyticus isolates isolated from shrimp and shellfish samples. 4 this study revealed a high percentage of susceptibility towards imipenem, however it should be noted that five of the isolates (vp71, svp90, vp114, vp145, and vp146) exhibited resistance to imipenem. although the resistance to imipenem is only 1.3% of the total isolates, it still warrants a concern on the use of antibiotics as carbapenems are among the beta-lactams that are the last line of antibiotic used for bacterial treatment (35). these five isolates had mar index of 0.21 to 0.64, and resistant to more than two different type of antibiotic tested. imipenem resistance profiles was observed among isolates isolated from both shrimp and shellfish samples, demonstrating that the resistance occurred in different seafood samples regardless the habitat of marine organism. the vp114, vp145 and vp146 isolates was isolated from the banana prawn samples whereas vp71 was isolated from the red prawn and svp90 was isolated from the flower crab sample. interestingly, the 32 trh-positive v. parahaemolyticus exhibited resistance to more than two different type of antibiotic tested (table 2). of the thirty-two isolates, 30 trhpositive isolates were seen resistant to ampicillin. isolate svp54 demonstrated resistance to six different antibiotics tested including ampicillin, amikacin, ceftazidime, cefotaxime, kanamycin, and levofloxacin. the 32 trh-positive isolates had mar index of 0.21 to 0.64, with 62.5% (20/32) isolates are resistance to three and more different types of antibiotics tested. the presence of multi-resistant trh-positive isolates in the marine environment may hamper clinical treatment if one gets infected with these strains. this emphasises the need for frequent monitoring of seafoods. based on the one-way anova analysis, there was a significant effect (p < 0.05) between type of samples and mar index of v. parahaemolyticus isolates. in line with tukey’s post hoc analysis, there was a significant difference in the mean mar index (p < 0.05) of v. parahaemolyticus isolates between red prawn and all the other type of seafood. there was a significant difference in the mean mar index between banana prawn with red prawn, p = 0.000 (p < 0.05). the mar index of swimming crab sample was significantly different with mar index of red prawn (p = 0.000) and carpet clam (p = 0.041) (p < 0.05). there was no significant difference in the mean mar index of v. parahaemolyticus isolates between hard shell clam and all the other type of samples, except for red prawn, p = 0.000 (p < 0.05). figure 1 illustrates the comparison of mean mar index of v. parahaemolyticus isolates from different type of seafood. figure 1: comparison of the mean mar index of v. parahaemolyticus isolates from different type of seafood. each bar represents mean mar index of isolates from type of seafood. the vertical lines associated with the bars represent two times the standard error of the mean. plasmid profiles of v. parahaemolyticus three hundred and eighty-five v. parahaemolyticus isolates were analyzed for the presence of plasmids. only 338 v. parahaemolyticus isolates have 1-7 different plasmids (figure 2) and could be categorized into 27 patterns based on the number and pattern of plasmid present. the sizes of plasmids ranged from 1.2kb to above 10kb. as shown in figure 2, from 27 plasmid profiles, the profile that forms the largest group was the plasmid profile 1.3 that consisted of 1 band above 10kb size plasmid. a total of 95 isolates (24.7%) have plasmid profile 1.3. additionally, in this profile, 22 isolates were from shellfish samples and 73 isolates were from shrimp samples. the isolates grouped in this plasmid profile were identified to be resistant to at least one typed of the antibiotic tested. the isolate vp152 from supermarket banana prawn and isolate svp129 from supermarket carpet clam which exhibited resistance profile towards 11/14 antibiotics tested respectively were grouped under plasmid profile 1.3. isolate vp183 that was resistant towards 5/14 antibiotic tested (ak/amp/c/ot/te) and svp61, a trh-positive isolate resistant towards 4/14 antibiotic tested (amp/ctx/ak/caz) respectively harboured seven plasmids each. overall, a total of 47/385 isolates (12%) did not express any plasmid profiles. the results demonstrated high discriminatory power of plasmid profiling conducted in this study. figure 2: bar chart on plasmid profile of 385 v. parahaemolyticus isolates. the y-axis represents different type of plasmid pattern while the x-axis represents the number of isolates that possess the particular pattern. determination of antibiotic... 5 table 2 shows an interesting relationship between the antibiotic resistance and plasmid profiles of the 32 trh-positive v. parahaemolyticus isolates. 21/32 trh-positive isolate contained 1-7 plasmids, where else another 11 isolates did not exhibit any plasmid profiles. all the trh-positive isolates were resistant to at least one type of antibiotic tested in study except isolate vp98 that was not resistant to any antibiotic and did not have any plasmid profile. isolates antibiogram mar indes svp54 amp/ak/caz/ctx/k/lev 0.43 svp55 amp/ak/caz/ctx/k 0.36 svp56 amp/ak/caz/ctx/k 0.36 svp70 amp/ak/caz/ctx/k 0.36 vp102 amp/ctx/ak/caz/k 0.36 vp103 amp/ctx/ak/caz/k 0.36 svp61 amp/ak/caz/ctx 0.29 svp66 amp/ak/caz/ctx 0.29 svp69 amp/ak/caz/ctx 0.29 svp72 amp/ak/caz/ctx 0.29 svp75 amp/ak/ctx/k 0.29 vp90 amp/ctx/ak/caz 0.29 vp95 amp/ctx/ak/k 0.29 svp73 ak/ctx/k 0.21 svp64 amp/ak/ctx 0.21 svp52 amp/ak/ctx 0.21 vp93 amp/ak/k 0.21 vp101 amp/ak/k 0.21 vp89 amp/ctx/ak 0.21 vp91 amp/ctx/ak 0.21 vp178 amp/ak 0.14 vp99 amp/ctx 0.14 vp175 amp/ctx 0.14 svp60 amp 0.07 vp92 amp 0.07 vp96 amp 0.07 vp97 amp 0.07 vp176 amp 0.07 vp177 amp 0.07 vp94 amp 0.07 vp100 amp 0.07 vp98 0.00 table 2: antibiotic resistant profile of trh-positive v. parahaemolyticus isolates ampicillin (amp), oxytetracycline (ot), nalidixic acid (na), chloramphenicol (c), cefotaxime (ctx), sulfamethoxazole/trimethoprim (sxt), imipenem (imp), amikacin (ak), ampicillin/sulbactam (sam), levofloxacin (lev), ceftazidime (caz), kanamycin (k), gentamicin (cn), tetracycline (te). plasmid curing in this study, two different intercalating agents – acridine orange (ao) and ethidium bromide (eb) were used to determine the antibiotic resistance mediation. the plasmid curing revealed that both intercalating agents ao and eb produced same curing profiles of isolate and the results is demonstrated in figure 3. all 338 v. parahaemolyticus isolates that harbour 1-7 different plasmid ranging of size 1.2kb to above 10kb in size lost their plasmids upon being subjected to curing agents. in figure 3, it could be observed that 327 v. parahaemolyticus isolates that were resistant towards ampicillin before plasmid curing showed the same phenotype resistance after plasmid curing. similar resistance pattern could be observed in a group of 57 tetracycline resistant isolates. the plasmid curing results revealed that 51/57 isolates (89%) were still resistant towards tetracycline. this suggests that the resistance phenotype to ampicillin and tetracycline expressed by the isolates could be chromosomally mediated. all the ampicillin/sulbactam resistant strains lost their plasmid after the curing assay and subsequently were susceptible to ampicillin/sulbactam suggesting it was plasmid mediated resistance. the antibiotic resistant patterns of ot/c/ctx/sxt/ak/caz/k/cn presented after plasmid curing had lower number of resistant isolates towards respective antibiotic. these results demonstrate that the phenotype resistance observed could be both plasmid and chromosomal mediated. figure 3: bar chart on antibiotic resistance profile of v. parahaemolyticus before and after plasmid curing. the y-axis represents different type of antibiotic agents while the x-axis represents the number of resistant isolates towards the antibiotic agents. ampicillin (amp), oxytetracycline (ot), nalidixic acid (na), chloramphenicol (c), cefotaxime (ctx), sulfamethoxazole/ trimethoprim (sxt), imipenem (imp), amikacin (ak), ampicillin/sulbactam (sam), levofloxacin (lev), ceftazidime (caz), kanamycin (k), gentamicin (cn), tetracycline (te). with reference to the 32 trh-positive v. parahaemolyti letchumanan v et al. 6 cus isolates (table 2), the antibiotic resistance profile of the 21 plasmid containing isolates changed after curing while the remaining 10 were unchanged. all 20/21 were ampicillin resistant initially, and after curing, the isolates (svp61, svp54, svp75, svp69, svp72, vp89, vp90, vp91, vp92, vp93, vp94, vp95, vp99, vp101, vp102, vp102, vp103, vp1175, vp176, vp177, vp178) remained resistant to ampicillin and cefotaxime, and became susceptible to the other antibiotics tested. one isolate (svp73) became susceptible to all antibiotic resistant after plasmid curing, suggesting the resistance phenotype observed was plasmid mediated. this suggests that while antibiotic resistance is mediated by both plasmid and chromosomes in pathogenic v. parahaemolyticus isolates, in plasmid containing strains aside from ampicillin and cefotaxime resistance, most of the remaining resistance phenotypes are plasmid mediated. svp129 isolate contained one plasmid profile with size more than 10kb and expressed antibiotics resistance towards 11/14 antibiotics tested. after plasmid curing, svp129 isolate lost its plasmid and changed its antibiotic resistance phenotype. svp129 isolate remained resistant to ampicillin, oxytetracycline, chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim; intermediate resistance to amikacin, ceftazidime, cefotaxime and kanamycin; and susceptible to gentamycin and ampicillin/sulbactam after plasmid curing assay. discussion in view of many reported cases of antibiotic resistance of v. parahaemolyticus from aquaculture, healthcare personals and members of the public should be caution in the application of antibiotics in healthcare sectors and aquaculture sectors. the rising number of antibiotic resistance as well as resistant genes within the v. parahaemolyticus population does causes a global health issue (36-44). hence, continuous monitoring is required to review the antibiotic resistance patterns and followed by controlling the use of antibiotics in the environments. the study’s susceptibility test placed the 1st generation antibiotic – ampicillin at the top of the v. parahaemolyticus resistance scope (85%) (table 1). this result is in close agreement with previous reports from india, indonesia, korea and malaysia that reported prevalent of ampicillin resistant v. parahaemolyticus strains isolated from seafood samples (10,19,27,45-50). the 1st generation antibiotics including ampicillin has a very low efficacy in treatment of infections due to the misuse of these antibiotics in aquaculture and agriculture which in turn led to a low susceptibility rate (25). these findings signify that ampicillin may longer be an effective antibiotic to treat vibrio sp. infections. the occurrence of high ampicillin resistance rate in the environment is still of great concern since the resistance phenotype seen could be chromosomally mediated in the bacteria thus require proper management method to control the resistance phenotype (13). interestingly, multi-resistance profile was observed among the 32 trh-positive v. parahaemolyticus. these pathogenic isolates were seen resistant to aminoglycosides, 3rd generation cephalosporins, and quinolone. the v. parahaemolyticus isolates exhibited high resistance rate towards the 3rd generation cephalosporins – cefotaxime (55.8%) and ceftazidime (34%) in this study. these findings are in line agreement with a study from terengganu, malaysia, who reported ceftazidime and cefuroxime resistant v. parahaemolyticus isolates from shellfish (51). in the neighboring country, korea, another similar study reported high percentage (70%-80%) of v. parahaemolyticus isolates from korean seafood to be resistant to the 3rd generation cephalosporin, cefotaxime and ceftazidime (52). in contrast, a study from the us reported low percentage of cefotaxime resistant v. parahaemolyticus isolates isolated from food (53). the inconsistencies in v. parahaemolyticus resistance rate to 3rd generation cephalosporin may be due to difference in sample, geographical variations, or difference in methodology test applied. it is reassuring to note that the isolates in this study are still susceptible to some antibiotics tested including imipenem (94%) (table 1). nevertheless, there were five isolates (svp90, vp71, vp114, vp145, vp146) exhibited resistance towards imipenem. the detection of imipenem resistant isolates raises concern as carbapenems are the most potent β-lactam antibiotic and is usually administrated in treatment of any serious bacterial infections (35). the results are in agreement with previous reports on the isolation of carbapenem-resistant vibrio sp. from environmental samples. walsh and colleagues reported carbapenem resistant v. cholerae isolated from drinking water and seepage in new delhi, india and further analysis revealed that the carbapenem gene blandm-1 was found in the chromosome of v. cholerae isolate (54). the occurrence of carbapenem resistance was also detected in a v. cholerae 01 el tor ogawa strain isolated from faecal specimen of a 2-year-old child in puducherry, india. another study reported an increasing trend of carbapenem resistance among v. cholerae 01 or 0139 isolates between 1986 to 2012 in southwest china (55). recently, bier and colleagues reported the isolation of four carbapenem resistant v. cholerae from different locations of the german coast line. these four isolates were not only resistant to carbapenem but also exhibited resistance to cefoxitin, aztreonam, and aminopenicillin (56). in addition, there have been reports on the emergence and spread of carbapenem-resistant enterobacteriaceae (cre) in the united states (us) (57,58). any infections with carbapenem resistant bacteria may cause higher mortality rates compared to those infections caused by carbapenem-susceptible bacteria. the wide incidence of carbapenem resistant vibrio sp. is an important emerging threat to public health, thus requires proper management action to limit the spread of this organism. in this study there was no significant difference between the sampling location and mar index of v. parahaemolyticus isolates. this result demonstrates that the isolates isolated from wetmarket and supermarkets are exposure to antibiotics. our results came to an agreement with many studies that reported high percentage of v. parahaemolyticus isolated from seafood are resistant to more than one antibiotic tested (28,29,48,59,60). according to the one-way anova analysis results, there was a significant difference between the groups of sampling location determination of antibiotic... 7 on the mar index of v. parahaemolyticus isolates (p < 0.05). the isolates from the supermarket sampling sites had a higher mean mar index compared to the isolates from wetmarket sampling sites. this situation could be attributed by the geographical difference in seafood samples that been sold in supermarket, thus causing a mar resistance profile. in addition, it could be suggested that seafood samples may have originated from similar environmental conditions in terms of antibiotic exposure or cross contamination may have occurred during the post-harvest, resulting in the isolates to have similar mar index. when compared the antibiotic resistance patterns and plasmid profiles, there was no correlation observed. even within the isolates with same resistance profiles, the plasmid profiles were different and a few isolates even did not exhibit any plasmids, which was similar to findings by lajnef and colleagues (61). hence, it could be concluded that the antibiotic resistance is not been influenced by the number of plasmids acquired by the isolates. the exposure of antibiotic in environment causes the bacteria to display a multidrug resistant characteristic. in some strains, the resistance observed could be plasmid mediated, and in some are chromosomally coded. further research could be done to confirm the origin of antibiotic resistance among the isolates. in this study, two different plasmid curing agent, acridine orange (ao) and ethidium bromide (eb), both are intercalating agents. two different intercalating agents were used because to observed the efficacy of each agent. intercalating agents such as ao and eb have been successfully used many studies of curing bacterial plasmids (31,37,62-66). the modes of action of intercalating agents are through preferential inhibition of plasmid replication. both the intercalating agents yield the similar curing profiles for each isolate (figure 4.8). the present results are closely in agreement with other studies that reported vibrio sp. isolates lost their plasmids when treated with concentration of 0.2mg/ml acridine orange (ao) and the isolates demonstrated changes in their resistance profile (64-67). a brazilian study reported ao agent was successfully used to cure multi-resistant vibrio isolates from marine shrimp and concluded the ampicillin resistance strains in study are plasmid mediated (37). in contrast, another study reported their isolates resistance was chromosomal mediated after ao curing agent treatment (66). likewise, another study reported the alteration in antibiotic resistance patterns and loss of plasmid among vibrio sp. isolates when treated with 0.3mg/ml eb. in that study 79% of the vibrio sp. isolates loss their plasmid profiles but showed phenotype resistance pattern to amoxicillin, ampicillin, furazolidone and tetracycline after curing assay, which indicate the resistance may be chromosomally borne (28). conclusion in conclusion, the current study provides an overview on the seafood contamination levels in selangor, malaysia and the distribution of v. parahaemolyticus in shrimp and shellfish samples. the shrimp and shellfish samples analyzed were contaminated with v. parahaemolyticus regardless their sampling locations. there was no correlation observed between the antibiotic resistance and plasmid profiles. yet, the antibiotic resistance mediation was studied via the plasmid curing assay. in some isolates, the resistance was plasmid mediated, while others were chromosomally borne resistance. the information derived from this curing assay is useful for public health personnel to understand better on the antibiotic resistance of v. parahaemolyticus in shrimp and shellfish from selangor, malaysia. the plasmid curing assay is fast, cost saving, provides fundamental knowledge, and may influence effective antibiotic management policies in the aquaculture industry. with this knowledge, the aquaculture farmers may alternate the antibiotics in their aquaculture fields from time to time in order to allow withdrawal of antibiotic resistance among the bacteria (34). in summary, the antibiotic resistance presented by v. parahaemolyticus isolates could be due to the excessive use of antibiotic in aquaculture to control bacterial infection and huge production loses (68,69). in addition, antimicrobial resistance is also caused by exposure of antibiotics via agriculture runoff, wastewater treatment plants, and thru mobile genetic elements or horizontal gene transfers among bacteria (70). there have been prevalent cases of multiple resistance reported among environmental pathogens such as salmonella sp. (71,72), v. vulnificus (73), listeria monocytogenes, escherichia coli and v. parahaemolyticus (74). hence, the present results would provide a baseline information on the severity of resistance among v. parahaemolyticus in shrimp and shellfish in selangor malaysia, then may allow management personal to overcome this problem with proper management strategies. conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. funding this work was supported by pvc award grant (project no. pvc-ecr-2016), external industry grant (biotek abadi – vote no. gba-808138 and gba-808813) awarded to l-hl. reference 1. who (world health organization). who fact sheets: food safety. retrieved on december 15, 2018 from http://www.who.int/newsroom/fact-sheets/detail/food-safety 2017. 2. letchumanan, v., wong, pc., goh, bh., et al. a review on the characteristics, taxanomy and prevalence of listeria monocytogenes. prog microbe mol biol 2018; 1(1): 1-8. 3. pumipuntu, n., indrawattana, n., vibrio parahaemolyticus: a seafood-borne pathogen. j trop med parasitol 2017; 40: 50-62. 4. khoo, ch., cheah, yk., lee, lh., et al. virulotyping of salmonella enterica subsp. enterica isolated from indigenous vegetables and poultry meat in malaysia using multiplex-pcr. antonie van leeuwenhoek 2009; 96(4): 441-457. 5. cheah, yk., lee, lh., noorzaleha, as., et al. characterization of multiple‐antimicrobial‐resistant salmonella enterica subsp. en letchumanan v et al. 8 terica isolated from indigenous vegetables and poultry in malaysia. lett appl microbiol 2008; 46(3): 318-324. 6. eng, sk., pusparajah, p., ab mutalib, ns., et al. salmonella: a review on pathogenesis, epidemiology and antibiotic resistance. front life sci 2015; 8(3): 284-293. 7. cheah, yk., salleh, na., lee, lh., et al. comparison of pcr fingerprinting techniques for the discrimination of salmonella enterica subsp. enterica serovar weltevreden isolated from indigenous vegetables in malaysia. world j microbiol biotech 2008; 24(3): 327-335. 8. law, jwf, ab mutalib, ns, chan, kg., et al. an insight into the isolation, enumeration, and molecular detection of listeria monocytogenes in food. front microbiol 2015a; 6: 1-15. 9. law, jwf, ab mutalib, ns, chan, kg., et al. rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations. front microbiol 2015b; 5: 1-19. 10. letchumanan, v., yin, wf., lee, lh., et al. prevalence and antimicrobial susceptibility of vibrio parahaemolyticus isolated from retail shrimps in malaysia. front microbiol 2015a; 6: 1-11. 11. letchumanan, v., pusparajah, p., tan, lth., et al. occurrence and antibiotic resistance of vibrio parahaemolyticus from shellfish in selangor, malaysia. front microbiol 2015b; 6: 1-11. 12. letchumanan, v., chan, kg., khan, tm., et al. 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16(1): 191-202. 72. learn-han, l., cheah, yk., salleh, na., et al. analysis of salmonella agona and salmonella weltevreden in malaysia by pcr fingerprinting and antibiotic resistance profiling. antonie van leeuwenhoek 2008; 94(3): 377-387. 73. heng, sp., letchumanan, v., deng, cy., et al. vibrio vulnificus: an environmental and clinical burden. front microbiol 2017; 8: 1-14. 74. law, jwf., letchumanan, v., chan, kg., et al. insights into detection and identification of foodborne pathogens. edited by om v. singh. food borne pathogens and antibiotic resistance. wiley blackwell 2016. letchumanan v et al. pmmb 2021, 4, 1; a0000256. doi: 10.36877/pmmb.a0000256 http://journals.hh-publisher.com/index.php/pmmb review article covid-19 booster vaccines administration in different countries angel yun-kuan thye1, loh teng-hern tan1,2, jodi woan-fei law1, vengadesh letchumanan1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; angelthye.yunkuan@monash.edu (ay-kt); jodi.law1@monash.edu (jw-fl) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) *corresponding author: vengadesh letchumanan; novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; vengadesh.letchumanan1@monash.edu (vl) received: 14 december 2021; received in revised form: 21 december 2021; accepted: 22 december 2021; available online: 31 december 2021 abstract: the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that resulted in the covid-19 global pandemic had consequently led to the development of different types of covid-19 vaccines, including the messenger rna (mrna) vaccines, inactivated virus vaccines, a protein subunit vaccine, and viral vector recombinant vaccines. countries worldwide started their national vaccination program as soon as the covid-19 vaccines got approved by the world health organization (who) under the emergency use listing. this includes covid-19 vaccines by pfizer-biontech, moderna, astrazeneca, janssen, sinovac, and sinopharm. findings suggested that protection against covid-19 provided by these vaccines may be waning or that the protection reduces against variants of concern (voc) or even inadequate protection of the primary vaccination for some risk groups. this led to the development of the covid-19 booster vaccine that aims to improve and prolong the protection against covid-19. this review aims to discuss the various covid-19 booster vaccines that are being authorized and administered, the eligibility criteria for the different booster vaccines, and the extent of protection these booster vaccines provide in the united states (us), israel, united kingdom (uk), singapore and chile. keywords: sars-cov-2; covid-19; vaccine; booster; waning 1. introduction covid-19 is caused by the severe acute respiratory syndrome coronavirus 2 (sarscov-2). this pandemic started in 2020 and has yet to end[1, 2]. as of 13 december 2021, there had been 270,218,553 confirmed cases, and 5,308,045 deaths reported globally[3]. covid19 has consequently led to the development of covid-19 vaccines at an unprecedented pace. pmmb 2021, 4, 1; a0000256 2 of 14 these vaccines activate the immune response upon binding to the spike protein[1, 4], and the primary goal of immunization against covid-19 is to protect against severe disease, hospitalization, and death[5]. therefore, our hope now lies in getting the world fully vaccinated to quell this global pandemic. the various covid-19 vaccines developed include messenger rna (mrna) vaccines, inactivated virus vaccines, a protein subunit vaccine, and viral vector recombinant vaccines[4, 6]. vaccines that are approved by world health organization (who) under the emergency use listing (eul) include mrna vaccines by pfizer-biontech (bnt162b2) and moderna (mrna-1273); viral vector vaccines by astrazeneca and janssen; and inactivated virus vaccines by sinopharm and sinovac[7]. when this review went to press, at least 56% of the world population had received at least one dose of the covid-19 vaccine, and 8.47 billion covid-19 vaccine doses have been administered globally (as of 12 december 2021) [8]. however, despite the development of vaccines and the global initiation of immunization, the pandemic is still ongoing. recently, there have been findings that the protection covid-19 vaccines provide against the infection may be waning[9-12]. hence, the further development of covid-19 booster vaccines comes into place. in fact, in recent months, several countries have begun administering covid-19 booster vaccines to eligible individuals that have completed their primary vaccine series. this review aims to discuss the covid-19 booster vaccination program in a few countries, including the united states (us), israel, united kingdom (uk), singapore, and chile. we will also discuss the eligibility criteria for the various booster vaccines and the extent of protection they provide against covid-19. 2. administration of covid-19 booster vaccine in various countries the rationale for the administration of covid-19 boosters includes (i) the waning protection against infection or disease, particularly severe disease, over time (i.e., waning immunity), (ii) reduced protection against variants of concern (voc), or (iii) inadequate protection from the currently recommended primary series for some risk groups for which evidence from the phase 3 clinical trials may have been lacking. nonetheless, its rationale may differ by vaccine product, epidemiological setting, risk group, and vaccine coverage rates[13]. covid-19 booster vaccines aim to restore antibody levels[14] and improve an individual's protection after their primary doses, allowing for longer-term protection[15]. they are administered when, with time, the immunity and clinical protection have dropped below a rate deemed sufficient[5]. in this review, some of the covid-19 booster vaccines that will pmmb 2021, 4, 1; a0000256 3 of 14 be discussed includes mrna vaccinespfizer-biontech/cominarty (bnt162b2)[16], and moderna (mrna-1273)[17], viral vector vaccines-janssen biotech inc. (jnj78436735)[18], and oxford astrazeneca (azd1222)[15], inactivated virus vaccinessinovac (coronavac)[10], and sinopharm (bbibp-corv)[19] (table 1) . table 1. covid-19 vaccines and their respective manufacturers. vaccine brands/ research name manufacturer references pfizer-biontech (before fda approval)/cominarty (after fda approval) (bnt162b2) pfizer, inc., and biontech [16] moderna (mrna-1273) modernatx, inc [17] jnj-78436735 janssen biotech, inc., a pharmaceutical company of johnson &johnson [18] coronavac sinovac biotech ltd. [20] bbibp-corv beijing bio-institute of biological products co., limited. (bibp), placed under the china national pharmaceutical group corporation (sinopharm) [19] 2.1. united states (us) the us's authorized and recommended covid-19 booster vaccines are from pfizerbiontech, moderna, and janssen[11, 21]. as of 22 november 2021, 196.4 million people have been fully vaccinated and 36.1 million have received a booster dose[22]. the centers for disease control prevention (cdc) has allowed for a mix and match dosing; thus, individuals can choose any covid-19 vaccines for their booster shot regardless of their previous vaccines[11, 17]. however, mixing products for an initial two-dose series or additional doses is not recommended[17]. on 12 august 2021, the us food and drug administration (fda) amended the authorizations for both mrna covid-19 vaccines (pfizer–biontech's bnt162b2 and moderna's mrna-1273) to allow for the use of an additional dose in immunocompromised patients[23, 24]. on 22 september 2021, the pfizer-biontech booster vaccine authorization was extended to include individuals aged 65 years or older and 18-64 with a high risk of severe covid-19 or whose frequent institutional or occupational exposure to sars-cov-2 puts them at high risk[23, 25]. the fda then made similar booster recommendations for moderna in october 2021[11] and the inclusion criteria are the same as those for pfizerpmmb 2021, 4, 1; a0000256 4 of 14 biontech[11, 21]. later, on 19 november 2021, the us fda amended the use of pfizerbiontech and moderna covid-19 vaccines, authorizing the single-use of covid-19 boosters for individuals aged 18 and above who completed their primary vaccination series with any covid-19 vaccines authorized or approved by the fda[21]. the administration of booster doses for both vaccines have to be at least six months after completing pfizerbiontech or moderna’s covid-19 primary vaccination series or at least two months after the completion of janssen’s covid-19 primary vaccination series[21]. regarding the covid-19 vaccine by janssen, the fda advisory panel is also recommending their single booster dose covid-19 vaccine of janssen or pfizer biontech or moderna to all adults 18 and above, 2 months after janssen’s primary vaccination[11]. in terms of booster vaccine effectiveness, on 21 october 2021, pfizer-biontech announced their phase 3 randomized, controlled covid-19 vaccine booster trial data, which consists of 10,000 participants 16 years and above. the results showed that the booster doses administered to individuals who received pfizer-biontech primary two-dose series had vaccine protection restored and showed relative vaccine efficacy of 95.6% compared to those who did not receive a booster dose[26]. besides that, the fda also analyzed the immune response data from around 200 participants aged 18 through 55 who received a single booster dose about 6 months after their second dose. the antibody response against the sars-cov2 virus in the same individual showed booster response 1 month after receiving a booster dose of pfizer-biontech, compared to the response 1 month after the two-dose primary series[21]. moderna’s booster vaccine is a reduced dose that is to be administered at least 6 months after its primary vaccination series[11]. this reduced dose is shown to reactivate the immune memory and increase the worldwide vaccine supply[27]. the fda analyzed the immune response data from 149 participants ≥18 years of age from the original clinical studies who received a booster dose at least 6 months after their second dose and compared it to the immune responses of 1,055 study participants after completing their two-dose series. results show that booster response was demonstrated from the antibody response against the sars-cov-2 virus 29 days after a booster dose of the vaccine[11]. in terms of janssen’s (a pharmaceutical company of johnson and johnson (j&j)) vaccine efficacy in the us, those who received a single dose of the janssen vaccine were only 73%, while those who received the booster shot vaccine efficacy increased to 94%[18]. in addition, j&j announced on 21 september 2021 that their real-world evidence and phase 3 studies confirmed the strong and long-lasting protection of the single-shot j&j vaccine against covid-19 related hospitalization. data from the phase 3 trial further ensures pmmb 2021, 4, 1; a0000256 5 of 14 protection against covid-19 related death. according to j&j, they have also generated evidence that a booster shot can further increase protection against covid-19 and is expected to significantly extend the protection duration of protection greatly[28]. according to j&j’s phase 3 study, the booster shot administered 56 days after the single shot provided 100% protection against severe/critical covid-19 (≥14 days post-vaccination), 75% protection against symptomatic moderate to severe/critical covid-19 globally and 94% protection against symptomatic moderate to severe/critical covid-19 in the us. furthermore, compared to the single vaccine shot, the booster shot administered after 2 months, showed antibody levels rose 4-6 times higher than after the single shot. when the booster shot was administered 6 months after the single shot, antibody levels rose 9-fold 1 week after the booster. they continued to increase to 12-fold higher 4 weeks after the booster, in which the increase was irrespective of age[18, 28]. hence, booster shots may induce the humoral immune response and possibly boost the immune response, further increasing the vaccine efficacy against symptomatic infection[18]. 2.2. israel israel is one of the countries that vaccinated their population very early and widely. by the end of march 2021, >50% of israel’s population had been fully vaccinated with two doses of pfizer-biontech covid-19 vaccine[8, 29], while other countries were still struggling for their first dose. even with 55% of the population completing two pfizer-biontech vaccination doses, israel is still struck with a fourth pandemic wave[23]. recently with covid-19 booster shots authorized for use, israel is the first country in the world to administer the covid-19 booster vaccine[4, 30]. the israeli ministry of health announced a campaign to administer the third dose pfizer-biontech covid-19 vaccine, which started with immunocompromised individuals on 13 july 2021. it then extended to individuals ≥60 years of age on 30 july 2021. after that, it extended to those 50 years of age (12 august), 40 years (19 august), 30 years (24 august), and the entire population above 12 years of age on the 30 august. within the first 2 weeks, more than half the population ≥ 60 years of age were vaccinated with the booster dose[23]. the effect of pfizer-biontech booster doses on covid-19 is demonstrated in a study involving 1,137,804 israelis that are ≥60 years old and had received two doses of pfizer-biontech at least five months earlier. they found that at least 12 days after the booster dose, the booster group showed a lower rate of infection than the non-booster group by 11.3 while the rate of severe illness was lower by a factor of 19.5[29]. similarly, another observational study using mass vaccination data in israel (1,158,269 israelis) also found that the pfizer-biontech booster vaccine effectively prevents severe covid-19 associated pmmb 2021, 4, 1; a0000256 6 of 14 outcome. compared to two doses of vaccines administered at least 5 months earlier, the vaccine effectiveness after at least 7 days of receiving the booster dose was estimated to be 93% effective in preventing covid-19 associated hospital admission, 92% in preventing severe disease, and 81% in preventing covid-19 associated death[23]. 2.3. united kingdom (uk) since the launch of the covid-19 vaccine program in december 2020, by midseptember 2021, 89.1% of the uk population had received their first dose, while 81% received both doses. the booster vaccine program started in september 2021[31], with the joint committee on vaccination and immunization (jcvi) stating that the decision was “precautionary” and that, on balance it was preferable to maintain a high level of protection in vulnerable adults throughout winter[32]. booster vaccines available in the uk include pfizer-biontech, moderna, and astrazeneca. they are given to high-risk individuals that are age ≥50, individuals who live or work in care homes, frontliners or social care workers, individuals aged 16 and above with underlying medical conditions or a carer of a high-risk individual or living in a high-risk setting. eligible individuals can receive their booster doses 6 months after their second vaccine dose[15]. the jcvi advised that the pfizer-biontech booster doses be preferred regardless of which vaccine brand an individual received for their primary doses. this follows data from covid-19 booster (cov-boost) trial that indicates the pfizer-biontech vaccine is well tolerated as a third dose and provides a strong booster response[32]. alternatively, it can be a half dose of the moderna vaccine. for individuals who cannot have the mrna vaccines, booster doses of oxford astrazeneca will be offered[15, 31]. as of 1 november 2021, the government reported that 8.1 million uk people had received their booster shot[33]. nonetheless, on the same day, john roberts from the covid-19 actuaries response group reported that approximately 5.8 million eligible individuals have not yet had their booster[33]. 2.4. singapore as part of singapore’s national vaccination program (nvp), the health sciences authority (hsa) under the pandemic special access route (psar) has authorized the pfizer-biontech, moderna, and sinovac covid-19 vaccines for use in singapore to prevent covid-19[34, 35]. it was only until 23 october 2021 that the multi-ministry taskforce (mtf) announced that the sinovac vaccine would be included in singapore's nvp for those ≥18 years of age and unable to be vaccinated with mrna vaccine. the administration for sinovac vaccination will start on 30 october 2021[34]. pmmb 2021, 4, 1; a0000256 7 of 14 on 14 september 2021, singapore started its vaccination booster program, including covid-19 booster mrna vaccines by pfizer-biontech, and moderna[34]. the eligibility criteria for booster vaccination are those ≥30 years of age or healthcare or frontliners ≥18 years of age who have received two doses 5 months ago[36]. singapore allows for a mix and match vaccine concept for booster doses as individuals need not receive the same vaccine as their previous two doses[34]. although sinovac-coronavac was later added into the nvp on 23 october 2021[34], it’s used as a booster vaccine for individuals who cannot receive the third dose of the psar-authorized mrna vaccines due to valid medical reasons[37]. as of 22 november 2021, 86% of singapore’s total population has received one dose of the covid-19 vaccine, while 85% of the total population had two doses of the covid-19 vaccine, and 24% of singapore’s total population has received the covid-19 booster shot[36]. to study the effectiveness of vaccination boosters, singapore’s ministry of health studied the covid-19 positive infection rates of individuals who have received their booster doses vis-à-vis fully vaccinated individuals who have not yet received their booster doses. the study included 685,083 fully vaccinated individuals above 60 years eligible for booster doses and had received their second vaccination dose >180 days before 15 september 2021. they found that with the administration of booster doses, the risk reduction against covid19 conferred roughly a further 70%, while against severe infections is 90% compared to those who did not receive a booster dose. there is indeed more protection with the addition of booster doses because two doses of mrna vaccines already provided 40-60% effectiveness against covid-19 infection when comparing those vaccinated versus unvaccinated individuals and >90% against severe illness. to sum up, based on estimates of combined data from different sources, individuals who are fully vaccinated and boosted benefit from vaccine effectiveness of about 80% or more against covid-19 infection and about 99% against severe illness[34]. 2.5. chile the authorized covid-19 vaccines in chile are from sinovac, pfizer-biontech, astrazeneca, cansino[38], janssen, and gamaleya[39]. based on chile's vaccination plan, healthcare personnel was vaccinated first, followed by an age-descending strategy, in addition to teachers and school staff, and essential workers. even individuals aged 12–17 years are eligible for vaccination[38]. chile’s mass covid-19 vaccination campaign started in february 2021[40]. by october 2021, 91.6% of individuals had received a single vaccine dose, and 88.84% of its target population had been fully vaccinated. the covid-19 booster vaccination started on 11 august 2021, which took into consideration recommendations from pmmb 2021, 4, 1; a0000256 8 of 14 the national immunization program, the vaccines and immunizations advisory committee (cavei), the covid-19 advisory council, national scientific societies, and international experts with whom the president and health ministry authorities have met during the past few months. the administration of booster doses was first given to individuals who received the sinovac vaccine, and during the first 8 weeks, 3,547,177 individuals received the booster dose [41]. in early october 2021, the president of chile, sebastián piñera stated that vaccines administered in the country are safe and effective, can decrease infections and hospitalizations significantly, and have saved many lives[41]. at the same time, the government of chile reported preliminary results on the effectiveness of booster doses based on some 2 million individuals who have received two doses of sinovac and the third dose of sinovac, oxford astrazeneca, or pfizer-biontech vaccines[10]. the vaccine effectiveness, sinovac, increased from 56% to 80.2%, while astrazeneca rose from 56% to 93%, and pfizer-biontech increased from 56% to 90%. overall, all three booster vaccines effectively reduced the need for hospitalization[41]. 3. conclusion with studies showing waning immunity against covid-19[9, 10], reduced protection against voc, and that primary vaccination itself may be inadequate to protect some against covid-19 infection, booster doses are introduced[13]. the additional administration of a booster dose will restore high antibody levels[14], further improving and prolonging the protection against covid-19[15]. booster vaccines mentioned in this review include pfizerbiontech, moderna, janssen, astrazeneca, sinovac, and sinopharm. based on the few countries discussed above that have started administering booster vaccines to their population, they seem to demonstrate positive results in protecting against covid-19. although the authorized brand of the booster vaccine and the eligibility criteria of booster vaccines may differ depending on countries, findings appear to be consistent that the administration of booster doses further increases vaccine effectiveness, decreases risk of infection, severity of disease, hospitalization, and death (table 2). pmmb 2021, 4, 1; a0000256 9 of 14 table 2. summary of booster vaccination programs in different countries country united states (us) israel united kingdom (uk) singapore chile population fully vaccinated (primary vaccination) 196.4 million (as of 22 november 2021)[22] >50% of the population (by end of march 2021)[8, 29] 81% of the population (by mid-september 2021 [31]. 85% of the total population (as of 22 november 2021[36] 88.84% of its target population (by october 2021)[41] population vaccinated with booster vaccine 36.1 million (as of 22 november 2021)[22] within first two weeks >50% of those ≥ 60 years of age [23] 8.1 million (as of 1st november 2021) [33] 24% of the total population (as of 22 november 2021)[36] 3,547,177 individuals (during the first 8 weeks)[41] type of booster vaccine pfizer-biontech, moderna and janssen[11, 21] pfizerbiontech [23] pfizerbiontech, moderna, astrazeneca[15] pfizerbiontech, moderna , sinovac[34, 37] sinovac, oxford astrazeneca, pfizerbiontech[10] eligibility criteria for booster vaccine ≥18 years old after primary vaccination (6 months after pfizer-biontech or moderna) (2 months after janssen’s)[11, 21] >12 years old after primary vaccination[23] high risk individuals age ≥50 -individuals living or working in care homes frontliners/ social care workers ≥16 years old with underlying medical conditions carer of a high-risk individual/ living in high risk setting above 30 years old, and healthcare or frontliners ≥18 years old after primary vaccination 5 months ago[36] n/a pmmb 2021, 4, 1; a0000256 10 of 14 nonetheless, there are also debates on the waning of immunity[12, 14]. the increase in disease severity could result from waning immunity or vulnerable individuals being vaccinated first. nevertheless, we believe that getting vaccinated with the additional booster dose could be one of the strategies to decrease the risk of infection, hospitalization, the *6 months after primary vaccination[15] findings on booster vaccine effectiveness pfizer-biontech vaccine efficacy of 95.6% [27]; positive booster response after 1 month boosted[21] moderna positive booster response 29 days after booster dose[11] janssen vaccine efficacy increased from 73% to 94% [18]; antibody levels rose 4–6 times higher than after the single shot (post 2 months); antibody levels rose 9-fold 1 week after the booster and continued to increase to 12fold higher 4 weeks after the booster (post 6 months)[18, 28] (≥12 days after booster) rate of infection lowered by a factor of 11.3 while rate of severe illness lowered by a factor of 19.5[29] ( ≥ 7 days after booster) 93% effective in preventing covid-19 associated admission to hospital, 92% in preventing severe disease and 81% in preventing covid-19 associated death[23] n/a vaccine effectiveness of about 80% or more against covid-19 infection, and about 99% against severe illness[34] sinovac increased from 56% to 80.2%; astrazeneca rose from 56% to 93%; pfizerbiontech increased from 56% to 90%[41] pmmb 2021, 4, 1; a0000256 11 of 14 severity of illness, and death, allowing for better control of the pandemic and a return to normalcy. it may even help improve people's quality of life, including mental health. nonetheless, even after being fully vaccinated or vaccinated with an additional booster dose, other preventive measures should continue to be practiced. this includes physical distancing, wearing a face mask, and handwashing, while the government should also continue with mass vaccination, testing campaigns, contact tracing, and restricting large gatherings[4, 42-47]. author contributions: ay-kt performed the literature search, critical data analysis, and manuscript writing. lt-ht, jw-fl and vl performed proofreading and editing. vl conceptualized this review writing project. funding: no external funding was provided for this research. acknowledgments: the authors would like to acknowledge professor shajahan yasin, head of school, jeffrey cheah school of medicine and health sciences, monash university malaysia. conflicts of interest: the authors declare no conflict of interest. references 1. thye ay-k, law jw-f, pusparajah p, et al. emerging sars-cov-2 variants of concern (vocs): an impending global crisis. biomedicines 2021; 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a0000256 14 of 14 https://www.gob.cl/en/news/president-pinera-presents-results-first-study-covid-19-booster-doserevealing-increased-effectiveness/. 42. tan lt-h, letchumanan v, ser h-l, et al. pmmb covid-19 bulletin: united kingdom (22nd april 2020). prog microbes mol biol 2020; 3(1): a0000078. 43. loo k-y and letchumanan v. covid-19: malaysia's fight against this deadly virus. prog microbes mol biol 2021; 4(1): a0000204. 44. johnson d, ren sec, johnson hd, et al. covid-19: are malaysians embracing or suffering the new normality? prog microbes mol biol 2020; 3(1): a0000102. 45. ser h-l, letchumanan v, law jw-f, et al. pmmb covid-19 bulletin: spain (18th april 2020). prog microbes mol biol 2020; 3(1): a0000074. 46. letchumanan v, ab mutalib n-s, goh b-h, et al. novel coronavirus 2019-ncov: could this virus become a possible global pandemic. prog microbes mol biol 2020; 3(1): a0000068. 47. hoo he, loh hc, ch’ng ash, et al. positive impacts of the covid-19 pandemic and public health measures on healthcare. prog microbes mol biol 2021; 4(1): a0000221. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. https://www.gob.cl/en/news/president-pinera-presents-results-first-study-covid-19-booster-dose-revealing-increased-effectiveness/ https://www.gob.cl/en/news/president-pinera-presents-results-first-study-covid-19-booster-dose-revealing-increased-effectiveness/ pmmb 2021, 4, 1; a0000221. doi: 10.36877/pmmb.a0000221 http://journals.hh-publisher.com/index.php/pmmb review article positive impacts of the covid-19 pandemic and public health measures on healthcare hung eun hoo1, hong chuan loh1*, alan swee hock ch’ng1,2, fan kee hoo3, irene looi1,2 article history 1clinical research centre, hospital seberang jaya, ministry of health malaysia, 13700 seberang jaya, pulau pinang, malaysia; hungeun11@gmail.com (heh) 2medical department, hospital seberang jaya, ministry of health malaysia, 13700 seberang jaya, pulau pinang, malaysia; alanchng@yahoo.com (a.s.h.c.); irenelooi@yahoo.com (il) 3department of medicine, faculty of medicine and health sciences, university putra malaysia, 43400 serdang, selangor, malaysia; hoofan@gmail.com (hkf) *corresponding author: hong chuan loh, lohhongchuan@gmail.com (hcl) received: 4 june 2021; received in revised form: 6 july 2021; accepted: 7 july 2021; available online: 27 july 2021 abstract: in the midst of the covid-19 pandemic, several unexpected positive outcomes have surfaced. the who public health measures have positively transformed people’s behaviour and lifestyles. the pandemic has prompted more focus on self-care and health awareness. hand hygiene practice has been greatly emphasised. the acceptance rate for the use of personal protective equipment, such as face masks, has been remarkable. people with co-morbid conditions are paying more attention to their primary illnesses by improving diets and exercise methods. people are more willing to accept and act on public health messages. the pandemic lockdowns have not only successfully mitigated the transmission of coronavirus, but they have also indirectly reduced the hospital admission rates for endemic community respiratory infections and trauma-related emergencies like motor vehicle accidents. fetomaternal health and wellness have significantly improved during the pandemic. the abrupt emergence of covid-19 has also led to a massive societal shift on tobacco smoking cessation. smokers are compelled to reflect on the harmful effects of cigarette smoking in relation to covid-19. issues of mental, relational and sexual health are put in the spotlight during the pandemic. people are investing more time in themselves, family and relationships. the world has seen an unprecedented global race in healthcare innovation and technology development in tackling the same global issue. artificial intelligence, including robots and drones, have been rapidly developed and employed for healthcare as well as food and delivery services in order to minimise human physical contact. this article discusses several unforeseen positive impacts on healthcare that emerged from the covid-19 public health measures that have been implemented. the positive impacts of the covid-19 pandemic should be highlighted in order to provide hope to our community. keywords: positive impacts; covid-19 pandemic; healthcare; public health measures pmmb 2021, 4, 1; a0000221 2 of 17 1. introduction the coronavirus disease 2019 (covid-19) was one of the most popular searched keywords on the internet in 2020. issues relating to the covid-19 pandemic are ubiquitous in our daily lives now. covid-19 is discussed constantly from social media to local news. this disease, caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first discovered in the hubei province of wuhan, china at the end of 2019 and was declared a global pandemic by the world health organisation (who) in march 2020[1–4]. it has affected over 195 countries[5]. according to the who, nearly a total of 117 million confirmed cases, including approximately 2.6 million deaths globally up to march 2021 were recorded[6]. the pandemic has had a disastrous impact on the lives of humans all over the world. the infectious disease mortality rate is alarming, our healthcare system is overwhelmed and medical resources are compromised. global economic development has taken a critical hit too, as many countries experienced a severe contraction of their gross domestic product[7]. coronavirus is spread from person to person via respiratory droplets, mainly through coughing or sneezing. other transmission routes of covid-19 include contact with contaminated fomites and inhalation of aerosols[8]. with a lack of knowledge regarding the long-term efficacy and safety of vaccines as well as the unavailability of absolute antiviral treatment for coronavirus, the prevention and control methods are currently the best approaches to control the pandemic[9]. the who introduced several public health measures, including hand hygiene, social distancing, self-isolation or quarantine, respiratory etiquette and avoiding touching eyes, nose and mouth[5]. many nations in the world also implemented nationwide lockdowns to minimise interpersonal contact among their citizens in order to reduce the virus transmission. schools were shut down, traffic was severely restricted, non-essential economic activities were suspended which could lead to loss of jobs, mass gatherings or events were cancelled and social distancing became the new norm[10, 11]. it imposes great challenges on the economic and social lives of people everywhere who faced difficulties and inconveniences on a daily basis[12]. covid-19 public health measures have become mandatory in our daily lives now. the lockdown or movement restriction that has been implemented in many places proved to be effective in bringing down the number of covid-19 cases[13–16]. numerous studies demonstrated that the pandemic also positively affected our lives. environmental quality was significantly improved due to large emission reductions of greenhouse gases[17–19]. the pandemic itself and the related measures taken by governments and individuals also resulted in some unprecedented health benefits to our society[10,20,21]. since healthcare is a major concern in the 21st century, the positive impacts of the covid-19 pandemic on health should be highlighted in the hope that we may retain some of the beneficial changes. we should explore every advantage brought by the pandemic in preparation for the next public health concern. this review presents the positive aspects during the covid-19 pandemic, such as the positive changes in behaviours and lifestyles of many people during the pandemic, the lower hospital admission rates for various kinds of illness and disease, pmmb 2021, 4, 1; a0000221 3 of 17 the large number of people who ended their tobacco smoking habit, the improvements that were experienced in mental, relational and sexual health, and the advances that were made in healthcare innovation. 2. positive impacts of the covid-19 pandemic and public health measures on healthcare figure 1 is a graphical abstract to illustrate the main findings of this review. figure 1. summary of the review (the positive impacts of the covid-19 pandemic and public health measures on healthcare). 2.1. positive changes in people’s behaviour and lifestyle the covid-19 pandemic is one of the greatest global health crises in human history. the lifestyles of many were fundamentally transformed — from wearing a face mask to physical distancing and practising hand sanitisation in public areas. although these novel practices might have seemed bizarre to our ancestors who never heard of the word ‘pandemic’, the majority of the population in this era perceived the pandemic as a serious threat and have accepted the new way of living as normal. self-hygiene and self-care awareness have largely spread across the globe, just like the virus itself. pmmb 2021, 4, 1; a0000221 4 of 17 for example, muto et al. showed that more than three-quarters of japanese people (75% out of 11,342 respondents) imposed preventive measures on themselves since the beginning of the pandemic[22]. there were sporadic covid-19 cases scattered in various regions of japan since february 2020. interestingly, most japanese (80% out of 2,400 participants from machida et al.) understood the deleterious effects of this infectious disease. they practised hand hygiene, self-isolation and cough etiquette, even before the who declared a pandemic emergency[23]. the practice of public health measures by japanese people highlighted their awareness of global health issues, perception of infectious disease risks and insight into self-care since the first sars crisis. the current pandemic prompted many to focus on self-care, particularly personal hygiene. indeed, hand hygiene is one of the important measures to curb virus or bacterial transmission[24,25]. in the early phase of the pandemic, prior to any recommendation about hand hygiene by the who, japanese people were washing their hands on average at least five times daily[23]. meanwhile, in hospitals, the average hand hygiene compliance rate dramatically increased up to approximately 90% among healthcare workers within the first quarter of the year[26,27]. hand hygiene has never received this level of attention as it was not taken as seriously as it should have been before the pandemic[28]. the public’s fear of and anxiety about coronavirus may have reinforced compliance with good hygiene practices[29]. the raised awareness about hygiene practice was not only observed in hand washing but also in the adoption of personal protective equipment, particularly face masks. with the implementation of mandatory wearing a face mask in public areas by many governments around the world, people nowadays travel with a face mask[23]. the face mask acceptance rate is as high as 80% in asia. it is rather impressive that the estimated number of face masks used daily across 49 asian countries is approximately 2.23 billion, with china holding the top place (around a billion masks used daily)[30]. turkey is another example of a country where people generally have positive attitudes toward public health measures. a survey study done in april 2020 showed that almost all study participants (97.3%) washed their hands more than usual. about two-thirds (63.7%) of participants went out with a mask and one-third (34.3%) wore gloves. almost all of them sanitised items brought in from outside (93%) and participants did not accept visitors to their homes (97%)[31]. the use of sanitising solutions increased during the pandemic, with almost 90% (out of 124 participants) declaring that they use it quite often[32]. in particular, many people with chronic diseases have faced great challenges during the pandemic lockdown. due to the inaccessibility of certain healthcare services during the pandemic, some patients have engaged in self-care or self-management strategies to deal with cardiometabolic diseases, including diabetes mellitus, hypertension, cardiovascular diseases and chronic kidney disease[33]. self-care programmes may improve a patient’s awareness, knowledge, attitude, self-care behaviour and efficacy of chronic disease management[34,35]. for example, in people with diabetes mellitus, self-care management was shown to significantly improve glycosylated haemoglobin type a1c (hba1c) status[36]. many individuals were able to optimise their glycaemic status during lockdown because pmmb 2021, 4, 1; a0000221 5 of 17 they had additional time to focus on diabetes self-management[37]. grabia et al.[32] stated that 60−65% of people with diabetes in poland had improved their diet by eating more regular, nutrient-dense meals during the pandemic. about 40% of the surveyed participants with diabetes started to monitor their disease more vigorously[32]. in malaysia, a local market survey showed that up to 72% of malaysians reduced sugar intake during the period of ‘movement control order’, as the lockdown was called. about 36% of them experienced weight loss during the pandemic[38]. meanwhile, kaye et al. [39] observed a relative increase of 14% in adherence to controller inhalers among 7578 people with bronchial asthma and chronic obstructive pulmonary disease (copd) during the pandemic. this may be due to the increased awareness in patients themselves in response to covid-19 guidelines that were published and their concern about controlling their primary respiratory illness with medicine[39]. the pandemic has empowered patients with co-morbidities to assume responsibility for their own conditions through dietary precaution, medication adherence, self-monitoring of blood glucose and blood pressure, as well as stress management[40]. with increased self-awareness of one’s own health, it is possible that the condition of non-communicable diseases may be optimised and controlled in the long run[41]. the attitude of the public towards the new health practices imposed during the pandemic was not as unfavourable as it might have been. the number of covid-19 cases may have risen daily, but awareness about hygiene practices and self-care were greatly stimulated by the pandemic. stringent measures such as ‘stay at home’ orders were implemented in countries across the globe. citizens were asked to stay home in an effort to flatten the covid-19 epidemic curve while lessening the burden on the healthcare system. it is a fair assumption that home confinement may have certain detrimental impacts on lifestyle, especially regarding physical activities. home confinement imposed difficult challenges for many people in their effort to maintain fitness since outdoor sports and physical activities were restricted or prohibited[42]. people began searching for innovative ways to stay active and healthy even while staying at home. since the pandemic started, there has been a growing body of literature regarding the use of new technologies and strategies for home-based exercises and fitness programmes[43]. plenty of fitness videos were advertised by exercise practitioners on social media sites, such as facebook, youtube, twitter, etc.[44]. people with adequate internet connections were able to easily access online electronic platforms in order to engage with others in various physical activities. besides social media channels, virtual based exercise gadgets such as the xbox 360 gaming console, your shape fitness evolved software, nintendo wii fit, oculus rift dk2 virtual reality (vr) headset, etc. have become popular as media for exercise performance[45]. these vr-based gadgets offer a large variety of home-based exercise programmes, including vr yoga, tai chi, kayaking, gym etc. and even mental exercises where users get to stay physically active as well as mentally healthy at home[46]. home-based exercise is not a new idea since it existed in the pre-pandemic era[44]. however, it is now in the spotlight as its usefulness and convenience have been helpful pmmb 2021, 4, 1; a0000221 6 of 17 during the lockdown. the pandemic provided an opportunity to explore internet resources for physical activities. people under home confinement have adequate time to explore their interests. the accessibility and availability of online fitness resources may help maintain people’s wellness but can also raise awareness of self-care and health concerns even during home confinement. the ‘stay at home’ order may restrain one socially, but it does not stop people from being physically and emotionally healthy[47]. 2.2. reduced hospital admissions public health measures have been successful in mitigating the transmission of coronavirus in certain countries where stringent standard operating procedures were implemented[20]. these approaches, which were favourable in the prevention and control of covid-19, had positive impacts on other diseases too. the number of hospital admissions for community-related communicable diseases scaled down drastically after public health measures against covid-19 were introduced. for instance, a study from singapore general hospital showed that the number of cases for the non-influenza respiratory virus in 2020 was 50% lower in comparison to preceding years[48]. similarly, the admission rate for influenza cases decreased up to 90% and the percentage of influenza positivity in singapore was reduced by up to 64% in 2020 when compared with each of the years from 2017 to 2019[49]. meanwhile, in finland, there was a decline in the number of paediatrics admissions caused by non-covid-19 respiratory viral infections. two hospitals revealed that the daily median rate of finnish paediatric visits decreased up to 70% after the national lockdown was implemented[50, 51]. it was not expected that the annual influenza season would end swiftly among the paediatric population. this phenomenon could be attributed to the closure of schools and nurseries which broke the transmission chain of the virus. moreover, frequent screening for covid-19 also proved to be beneficial for other non-covid-19 communicable diseases, such as tuberculosis (tb). clinicians were able to pick up an early diagnosis of pulmonary tb on suspected covid-19 patients as both diseases present similar signs and symptoms[52]. the covid-19 containment strategy may contribute to achieving the goal of a tb-free world by 2030[53,54]. covid-19 safe practices like hand hygiene, social distancing and wearing face masks are effective not only in containing coronavirus but all the communicable diseases transmitted through coughing, sneezing and touching. of note, the covid-19 lockdown seems to have had a significant positive impact on the health and wellness of pregnant women and also on preterm infants with very low birth weight (vlbw) and extremely low birth weight (elbw). philip et al. stated that there was a huge decline in the rates of vlbw infants (73% reduction) and elbw infants (100% reduction) in the first four months of the pandemic year in ireland when compared to the preceding 20 years[55]. denmark also reported a reduction of 90% in live births of premature infants after the nationwide lockdown as compared with the preceding five years[56]. similar findings were reported in the netherlands in relation to the reduction of preterm births[57]. these positive impacts on overall fetomaternal well-being could be pmmb 2021, 4, 1; a0000221 7 of 17 explained by the improvement of socio-environmental and behavioural modifiers induced by the lockdown. at home, mothers received family and partner support, adequate rest hours and exercise, reduced chances of infection and accidents, optimum nutrition as well as financial aid from governments. the lockdown also effectively reduced road traffic which eased access to maternity services during emergencies[55]. the covid-19 public health measures had a positive impact on trauma-related emergency cases too, particularly motor-vehicle accidents (mva). one of the main reasons for mva is driving under the influence of alcohol or illicit drugs[53]. with the covid-19 lockdown, a series of measures were introduced, including restriction of non-essential travel, prohibition of alcohol sales, closure of non-essential services and places, and bans on social gatherings. the number of mva cases was successfully brought down by the implementation of restriction measures. morris et al. reported that there was a reduction by almost half in trauma cases that presented to emergency departments at hospitals in south africa during the lockdown in comparison to two previous years. the majority of the cases were mva and pedestrian-vehicle accidents[58]. manyoni et al. observed that the most significant decline (around 90%) in trauma, particularly mva-related soft tissue injuries, happened in april 2020 after the south african government implemented a nationwide lockdown[59]. by eliminating the risk of driving under the influence of alcohol or illicit drugs and with fewer cars on the roads, there was a reduction in the number of mva cases that presented to hospitals and that lessened the burden on the currently overwhelmed healthcare system. the decline in the number of hospital admissions of non-covid-19 community respiratory diseases, preterm births and mva cases during the pandemic is a reflection of multiple contributing factors. raised awareness about hygiene practices, self-care and improvements in traffic situations are the primary reasons behind the reduction in the number of cases. additionally, this could be partially explained by patients balancing the urgency of an immediate healthcare need with concerns regarding the risk of contracting coronavirus infection in an emergency department or a hospital. the covid-19 public health measures that produced unintended positive effects may serve as a model for future public health concerns. 2.3. tobacco smoking reduction and cessation the continued popularity of tobacco smoking has been one of the major public health concerns since the pre-pandemic era. it is widely known that smoking has noxious health effects on our bodies, particularly with regard to its strong association with lung cancer[60]. smoking also suppresses immunity, increases risks of respiratory diseases (asthma and copd) and cardiovascular diseases such as myocardial infarction[61]. during the pandemic, the public was concerned about the link between smoking and susceptibility to covid-19 infection as well as the severity of the illness if contracted by a smoker. several meta-analyses reported that the percentage of severe covid-19 cases was higher in smokers (21.2%; 65/305) compared to non-smokers (10.7%; 978/9067). there has been a pmmb 2021, 4, 1; a0000221 8 of 17 clear association between severe covid-19 cases and those patients who are smokers or who have a past history of smoking[62,63]. many believe that smoking increases the risk of getting coronavirus infection and the severity of the disease[64]. fear and anxiety about the deadly virus may cause smokers to think twice before smoking another cigarette. numerous research papers identified a relationship between the covid-19 pandemic and the lower use of tobacco. elling et al. indicated that one-third of smokers in the netherlands found the motivation to quit smoking since the beginning of the pandemic. about one-fifth of them reduced the number of daily cigarettes since they perceived that coronavirus could cause severe illness[65]. more than half of american smokers were motivated to quit smoking during the pandemic. some even found that pandemic-related restrictions like the ‘stay-at-home’ order made it easier to quit smoking due to less accessibility to cigarettes[66]. kowitt et al. reported that more than 70% of smokers in the united states (us) had intentions to quit tobacco and almost half of them attempted to quit in the first six months of the pandemic[67]. the covid-19-induced lockdown also affected the us collegiate smokers and vapers. about 30% of us college students paused tobacco use after campuses were closed[68]. bommele et al. showed that the covid-19 lockdown in the united kingdom had positive impacts on the number of smokers who attempted to quit smoking (39.6% after vs 29.1% before the lockdown) and who were successful in quit smoking (21.3% after vs 13.9% before the lockdown)[69]. last but not least, a dutch online survey found that there are more current smokers (16.1% after vs 12.1% before the lockdown) who are motivated to quit smoking since the pandemic began[70]. it is probable that tobacco users with higher-level covid-19 risk perceptions are more likely to attempt to quit smoking and are more likely to be successful in quitting smoking[67]. the pandemic has the potential to impact multiple individual psychosocial factors particularly relevant to tobacco use, including daily behavioural patterns, living contexts, social contexts, mental health and perceived health risks. this is an opportunity for smokers to reflect on smoking and for public health regulators to revise tobacco control policies and tobacco cessation services. 2.4. mental, relational and sexual health regrettably, little attention has been devoted to the importance of maintaining mental health and relational health before the pandemic despite the fact that they are considered important parts of one’s overall health and quality of life. there is a boom in interesting research articles about the impact of the covid-19 pandemic on our mental health since the beginning of the pandemic. although the evidence regarding positive impacts from the pandemic on mental health is very limited, we can still note some positive trends[71]. children and adolescents in the twenty-first century face tremendous stress and pressure from scholastic performance. the pressure may result in mental health problems such as depression, anxiety, panic disorder, etc.[72]. since the implementation of national lockdowns, many schools closed temporarily and were forced to postpone examinations. pmmb 2021, 4, 1; a0000221 9 of 17 children obtained a temporary ‘break’ from their unavoidable stressors[73]. online classes also save time and energy for children since they did not have to travel. many children experienced more leisure time to explore their own interests. with school closures, peer bullying issues may have temporarily declined since children at home are more likely to be under parental supervision[73]. hawke et al. reported that the mental and relational health of almost half (47.3%) of the young people in canada benefited in a positive way from the covid-19 lockdown. some expressed that they had improved social relationships at home, better sleeping quality and rest, more personal time for hobbies and exercises and greater self-reflection and self-care[74]. this pandemic is also an opportunity for family bonding time. family relationships were strengthened during the lockdown as household members invested more time together doing household chores or exploring their creativity in culinary pursuits. in terms of culture, younger people were spending more time with their parents which inevitably connects them to their cultural roots[75,76]. moreover, the covid-19 pandemic forced some non-essential services employees to work from home (wfh). it is believed that wfh increases some aspects of productivity and efficiency. wfh helps people to avoid the stress of travelling. they get to work at a familiar place, dress comfortably and avoid direct contact with their employers or colleagues which may be one of their work stressors[19]. other than mental and relational health, sexual health, which is still a taboo subject in much of the world, also received public attention during the pandemic. safe sex practices were observed more often between cohabiting monogamous partners during the lockdown[77]. some reduction of the incidence of sexually transmitted diseases (std) and diagnoses of the human immunodeficiency virus (hiv) were reported after the travel restriction was imposed worldwide[78]. physical distancing, one of the recommended covid-19 precautions, minimises the amount of close contact with other people who do not live in the same household and therefore reduced sexual and physical intimacy generally. hammoud et al. reported that there was a relative reduction of approximately 85% in random sex with casual partners among australian gay and bisexual men in april 2020 shortly after the pandemic began. the reduction in sexual behaviour that was observed may be attributed to a higher perceived risk of contracting covid-19 along with the pressure of physical distancing, both of which diminished sexual desire overall[79]. an israeli study demonstrated that the covid-19 pandemic altered sexual behaviour among men who have sex with men by showing a significant decrease in the number of sexual partners and a more limited sexual repertoire (by avoiding kissing their partners) in comparison to their pre-social distancing sexual behaviour[80]. the dutch public health measures in response to the pandemic effectively changed risky behaviour, indirectly driving std and hiv transmission downward in a short period of time, with an 8% positivity rate of bacterial stds during covid-19 restrictions versus 19% before the pandemic. there were also no hiv infections diagnosed between march and june 2020 in amsterdam[81]. certainly, the ultimate impact of the pandemic on mental, relational and sexual health is yet to be determined. perhaps the biggest benefit we experienced was the opportunity to pmmb 2021, 4, 1; a0000221 10 of 17 spend more time together with our loved ones compared to the pre-pandemic time when many people were more focused on individual pursuits. 2.5. healthcare innovation and technology development the pace of healthcare innovation and technology development was rapid in the past year because of the pandemic. many people from around the world came together to fight covid-19. innumerable public health experts, clinicians, researchers, data analysts, pharmacists, medical device companies, engineers, designers, entrepreneurs, etc., collaborated as cross-functional teams to find solutions for real-world problems. the pandemic started a global race in healthcare innovation. one extraordinary example is the development of remote-controlled robots for covid-19 screening by scientists from tsinghua university, china. the robots are able to perform a throat swab and disinfect themselves after performing their duties[82]. similar 3d-printed robotic arms were invented in denmark to perform covid-19 screening on people who arrived at drive-thru facilities in their cars[83]. a taiwanese physician invented an “aerosol box” that covers a patient’s head but has arm holes for doctors so that they may perform intubation while being protected from infection[84]. the guangdong provincial people’s hospital in guangzhou, china used robots for autonomous transportation of drugs. the robots were loaded with medicines, given instructions to travel to particular hospitals, and were able to open and close doors and take lifts without human assistance[85, 86]. harvard medical school in the us used intelligent robots to measure the vital signs of patients. the americans have also developed a robot equipped with a camera, a microphone and a stethoscope that performed a consultation with a covid-19 patient who was held in an isolated room originally designed during the ebola crisis[87]. these technologies greatly minimise the risk of exposure of healthcare providers. the world of healthcare innovation is borderless, as anyone can innovate, regardless of age. a 13-year-old boy scout invented a pair of 3d-printed ear guards to help healthcare workers relieve pain caused by prolonged wearing of face masks[88]. the pandemic opened many minds and brought new perspectives, allowing innovation to blossom even during the most difficult time. innovation in healthcare ranges from artificial intelligence (ai) to medical services. when social distancing was implemented, physicians came up with an innovative yet effective solution to replace traditional consultations. telemedicine, also termed ‘telehealth’ or ‘teleconsultation’ is defined as the delivery of health care service by telecommunication irrespective of location via the means of electronic, digital, internet-based, or telephone-based systems or any sort of information technology[89]. the use of telemedicine by healthcare providers during the pre-pandemic time was scarce. implementing it was difficult due to a lack of demand for it, logistical issues, concerns about healthcare provider reimbursement, laws and regulations and a lack of universal access. however, the covid-19 crisis resulted in a surge of telemedicine due to an increased demand for virtual medical visits and a temporary lift on medicolegal barriers that had limited telehealth pmmb 2021, 4, 1; a0000221 11 of 17 expansion before the pandemic[90]. telemedicine services became very important during the pandemic. telesurgery, which is conducted by general surgeons onsite under the supervision of sub-specialised surgeons who may interact remotely via a virtual interface, has also been used during the covid-19 pandemic[87, 91]. advanced technology is not only seen in connection with the delivery of healthcare but also exists in our daily lives. implementation of physical distancing has been instrumental in mitigating the transmission of the virus. deliveries of food and other packages of all kinds were replaced in some instances by delivery robots and drones. a drone, also known as an ‘unmanned aerial vehicle’ (uav) is a flying device controlled remotely by a person or a computer. the chinese e-commerce company jd.com launched a group of drones to carry out numerous food distribution tasks. the drones could, for example, replace an hour of ground delivery with a 10-minute flight[92]. the use of robots was increased by fast-food chains, including mcdonald’s, and by warehouses, such as amazon, fedex, dhl and walmart[93,94]. singapore was also testing a four-legged robot named spot to enforce physical distancing in a public park[95]. using ai services during this pandemic such as robots and drones, not only saved manpower and time but also helped to reduce the spread of the virus[96]. under normal circumstances, innovation is a time-consuming process that can take months or even years. a primary example is the timeline for a new pharmaceutical drug which could take 12 years to develop from bench to market[97]. the covid-19 global health crisis greatly accelerated the pace of research and development. innovation is a learning progress. applying all these new innovations to healthcare services generally, even after the pandemic has waned, may provide many clinical benefits beyond simply reducing the infection risks of covid-19. 3. conclusions the covid-19 pandemic is certainly a tragedy of apocalyptic measure as the world has suffered serious, negative impacts ranging from socio-economic problems to the death of millions. the harm cannot be undone, but we can take some solace in the positive impacts that the pandemic had on our healthcare and other aspects of life: beneficial environmental effects on the planet; people have become more health-conscious; the number of hospital admissions for various kinds of illness and disease was drastically reduced; more people quit smoking; mental health, sexual health and family relationship issues were positively impacted in some ways; and there was a global movement toward healthcare innovation and technology development. highlighting the positive aspects of the current situation is not to downplay the harmful effects or the tragic losses, but this is to provide some hope and optimism during a challenging time. the abovementioned positive impacts may contribute to lessen public fear and give a different perspective to those who have undergone hardships during the pandemic. our daily activities in the future may incorporate some of the hard-learned lessons from the pmmb 2021, 4, 1; a0000221 12 of 17 pandemic. perhaps people will have a greater appreciation for our former ways of living in the time before the pandemic and will realise the great value of healthcare systems worldwide. nothing is more valuable than our own health and the pandemic may inspire people to give a greater priority to good health practices. public health policymakers such as the who may also emphasise the silver linings of the covid-19 pandemic and seek to promote greater use of some of the public health measures in order to prevent, or at least lessen the impact of, the next global health concern. global solidarity would ensure the survival of humanity even during the darkest time. as the covid-19 pandemic runs from a sprint to a marathon, we must consider how we can keep some of the positive impacts on a long-term basis to help us build a better future. author contributions: all authors have approved the final article and authorship is limited to those who have contributed substantially to the work reported. conceptualisation by h.e.h., h.c.l.; 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a0000264. doi: 10.36877/pmmb.a0000264 http://journals.hh-publisher.com/index.php/pmmb review article south africa’s battle against covid-19 pandemic ke yan loo1, jodi woan-fei law1, loh teng hern tan1,2, vengadesh letchumanan1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, subang jaya, selangor, 47500, malaysia; ke.loo@monash.edu (kyl); jodi.law1@monash.edu (jw-fl) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) *corresponding author: vengadesh letchumanan, novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, subang jaya, selangor, 47500, malaysia; vengadesh.letchumanan1@monash.edu (vl) received: 23 december 2021; received in revised form: 27 january 2022; accepted: 28 january 2022; available online: 4 february 2022 abstract: south africa is one of the countries heavily impacted by the covid-19 pandemic. as of 9 january 2022, over 3.5 million confirmed cases of covid-19 have been reported, and 93 551 deaths have been recorded in the country. the south african healthcare system faced a lack of essential resources and financial burdens by outbreaks and its new variant of concern (voc), the omicron. the local government has done as much as possible to control the spread of the virus in the local communities by quickly implementing lockdowns and enforcing movement restrictions. an eight-stage program to combat covid-19 and a national vaccination strategy was also developed soon to manage the coronavirus spread in the country better. as the country struggles to secure and administer covid-19 vaccines to its people, the coronavirus has been rapidly mutating and causing new waves of infections within the nation. the covid-19 experience in south africa demonstrates the great importance of equitable access to medicine, medical equipment, and vaccines globally. equitable access to these essential resources is critical to prevent the virus spread across borders and reduce mortality rates worldwide. keywords: south africa; covid-19; coronavirus; vaccination, pandemic 1. introduction in 2019, a deadly virus causing a debilitating respiratory disease was discovered in wuhan, china, causing the country to lockdown with strict rules and regulations[1,2]. the virus was later identified as the novel coronavirus, sars-cov-2, which is now widely known as covid-19[3]. when diagnosed with covid-19, the individual could present asymptomatic or present symptoms similar to the flu. for instance, the individual may experience mild to severe symptoms such as fever, cough, difficulty breathing, fatigue, myalgia, sore throat, and loss of taste or smell[4,5]. since the emergence of the coronavirus, countries across the globe have put in vigorous efforts to overcome the pandemic. however, pmmb 2022, 5, 1; a0000264 2 of 12 the rapid spread of the virus and its subsequent mutations has had detrimental effects on public health globally[6–12]. at the time of writing, over 296 million confirmed cases of covid-19 have been reported worldwide, with over 5.4 million deaths recorded[13]. nevertheless, effective vaccines against the coronavirus have been deployed internationally to reduce the severity of the disease if infected and prevent the further spread of the virus[14– 16]. although, to date, over 3.8 billion individuals are fully vaccinated globally, data shows that vaccination rates in the african continent are significantly lower than in its neighboring regions[13]. vaccine access is still a challenge in the continent as global equity in vaccine distribution is yet to be achieved[17]. in addition, there is a lack of capacity to manufacture large quantities of vaccines within the continent, even though it is essential to maintain public health. countries in africa currently heavily rely on external donors such as gavi or unicef to supply covid-19 vaccines[18]. these vaccines could only provide a limited number of vaccinations as a supply for inoculations was already scarce[19]. pharmaceutical companies were under public scrutiny as they prioritized richer countries to supply the vaccines, which indirectly led to the vaccination programs in poorer countries such as those in africa being delayed [17, 19]. among all the countries in the african continent, south africa is most heavily affected by the covid-19 pandemic as over 3.5 million individuals have been infected, and over 93,000 deaths have been recorded[20,21]. since the first case of covid-19 in south africa, the local government took quick action in closing its borders and implementing lockdowns with strict rules to break the chain of the virus spread. despite the challenges faced by the local government in controlling the disease, they continue to be transparent when keeping the people informed on the latest covid-19 situation within the country. testing for the sars-cov-2 virus has also increased to detect the virus at earlier stages. individuals are encouraged to register for the national vaccination plan to get the vaccine as soon as possible. the past two years have been tumultuous for the people in south africa as they have experienced multiple waves of the coronavirus. disease management has been complex due to the shortage of vaccines and new virus variants[22,23]. the consequential effects of covid-19 in south africa and the distribution of vaccines within the region show the importance of equitable access to vaccines for all global citizens. 2. combating covid-19 in south africa once covid-19 was announced a global pandemic, the south african government had planned to combat the pandemic in an overlapping eight-stage program[24] (figure 1) before any cases were detected within the country. during the preparation stage, the public was educated on the coronavirus disease and ways to prevent the spread. surveillance was also being done to detect any active cases within the country. the first reported case of covid-19 in south africa involved a citizen returning from italy to gauteng with his wife and eight others on 1 march 2020[25] (figure 2). the patient experienced the typical symptoms of covid-19 and was practicing self-isolation. a few days later, a woman from the same traveling group as the first patient also tested positive[26]. the local government quickly traced down any contacts of the traveling group, performed covid-19 swab tests, and implemented home-based pmmb 2022, 5, 1; a0000264 3 of 12 quarantine for these individuals[26]. ten days after the first incidence, stage two of the national covid-19 response began, focusing on primary prevention of the disease. figure 1. south africa eight-stage program to combat covid-19[24]. figure 2. covid-19 timeline in south africa [20]. the public was urged to practice social distancing and hand-washing, closed schools, and international travel was prohibited[24]. more confirmed cases unrelated to the traveling pmmb 2022, 5, 1; a0000264 4 of 12 group from italy began to emerge in other provinces in south africa, and these cases involved individuals returning from other countries[27]. confirmed cases of covid-19 continued to rise, and on 18 march 2020, the first local transmission of the respiratory disease was documented[28]. by the end of march 2020, the virus had spread across all nine provinces in south africa[20]. the authorities are working round the clock towards tracking and tracing the source of the infection within the community. international flights were suspended as a preventative measure to limit the entry of travelers from abroad into south africa. the government then implemented stage three of the covid-19 response, a strict level five (l5) lockdown in south africa to control the spread of the virus in the country[29]. every individual who was not an essential service worker was to stay at home, and they were only allowed to leave home to buy food or medicine, seek medical care, or collect a social grant. those who tested positive for the virus would be placed in quarantine, and all businesses were closed, with essential services being the exception[29]. the primary purpose of the lockdown was to flatten the transmission curve of covid-19 to minimize the negative impacts of the disease in terms of public health and the country’s economy. since the lockdown, testing for the sars-cov-2 virus was done in over 47,000 individuals, and mobile testing units have been organized for easier access to testing facilities. there was also an increase in army patrols to enforce the rules during the lockdown, and free hand sanitizer was being distributed to the citizens. in april 2020, south africa had commenced stage four of the program, which is active case-finding via contact tracing for contacts of covid-19 positive individuals. lockdown measures started to loosen to level four in may 2020, whereby curfews were implemented for each individual and permitted businesses were only allowed to open until 8 pm. interprovincial travel was still restricted, schools remained closed, and social gatherings were prohibited except for funerals, workplaces, or buying goods[30]. later in june 2020, the lockdown measures would be further eased to level 3, permitting more businesses to open within specific hours, and schools would be open. stringent social distancing measures were still in place socially and in workplaces to address the high risk of transmission in these settings[31,32]. the confirmed cases of covid-19 continued to rise and reached their first peak in july 2020, when more than 450,000 cases were reported, and the death toll had reached 8,005 (figure 2). at this point, stages five to eight of the covid-19 response started as hotspots were quickly identified, medical care was promptly given to those infected, and the nation was prepared to deal with deaths due to covid-19. the country was also on high vigilance to detect any new outbreaks or virus variants and ready to distribute vaccines whenever they became available[24]. the number of confirmed cases decreased with the lockdown measures in place, and the curve flattened in august 2020. thus, it was decided that the lockdown restrictions were to move to level two, but the national state of disaster was extended by another month[33,34]. during this phase, individuals had more mobility for family visitations, gatherings, and leisurely activities. more businesses, including bars and taverns, were allowed to open within pre-determined hours, and occupants of these premises were to maintain social distancing and mask-wearing practices. inter-provincial travel was now permitted, and national pmmb 2022, 5, 1; a0000264 5 of 12 borders were only partially open to those who complied with the specified protocols[34]. the covid alert sa app was also launched in august to trace individuals who tested positive for covid-19[35]. the restrictions were further lowered to level one when the covid-19 situation in south africa became more stabilized[36,37]. larger gatherings were now allowed as long as attendees were less than 50% of the venue capacity. venues for social or leisurely activities were also allowed to operate at 50% of the total capacity. in addition, international travel was now permitted for business purposes, and travelers would need to be covid-19 negative to cross country borders. towards 2020, yearend school parties were held frequently, and the festive season increased travelers between provinces[38,39]. the parties were later identified as super-spreaders events for the coronavirus, which subsequently led to a spike in covid-19 cases in south africa and initiated the second wave of infections. hotspots for the coronavirus were once again put under stricter restrictions, and although participants of the yearend school parties were immediately put in quarantine, the damage had already been done. the situation was worsened as a new variant of sars-cov-2, the b.1.351 (beta) variant with higher transmissibility, was detected in south africa[40]. covid-19 cases and deaths continued to climb, overwhelming the local healthcare facilities, and finally, over one million patients were recorded on 27 december 2020[20]. the south african president quickly announced a partial level three lockdown for two weeks to reduce the virus spread during the holiday season. a curfew was implemented between 9 pm to 6 am, sales and transport of alcohol were banned, public amenities were to be closed, and masks were to be worn at all times in public[41]. shortly after the temporary lockdown, a vaccine rollout strategy was announced in early january 2021, with vaccines sufficient only to be administered to 10% of the population in south africa. nevertheless, the government secured more vaccines to be delivered as soon as possible. frontline healthcare workers were prioritized for the vaccination during the first phase. in contrast, phase two would prioritize essential workers, persons in congregate settings, elderly over 60 years old, and adults above 18 years old with co-morbidities. lastly, phase three would distribute the vaccines to individuals above 18 years old. the vaccine rollout aimed to vaccinate approximately 67.25% (40.35 million) of the population by the end of phase three[42]. in february 2021, the first shipment of one million doses of the oxford-astrazeneca vaccine (vaxzevria) arrived in south africa. still, the rollout was put on hold as the vaccine only provided minimal protection against the beta variant of the virus, which accounted for most infections in the country[43,44]. the johnson and johnson (janssen) vaccine arrived in south africa. hence, the vaccine rollout commenced on 17 february as planned, with nine million more vaccine doses to arrive in the months to come[45]. as the second wave of covidpmmb 2022, 5, 1; a0000264 6 of 12 19 infections began to dwindle and more vaccines were administered, the government decided to scale back lockdown restrictions to level one[31]. by the end of march, the country had successfully vaccinated 100,000 people against covid-19, and the number of daily confirmed cases was not fluctuating wildly. this continued up until may 2021 when new variants, b.1.1.7 (alpha) and b.1.617.2 (delta) of the coronavirus were detected in south africa[46], and a spike in covid-19 infections soon followed. south africa was now facing its third wave of covid-19, and the government quickly implemented tighter lockdown restrictions at level two, beginning on 31 may 2021. in may, pfizer delivered its first covid-19 vaccines (comirnaty) to south africa. these vaccines were quickly administered to the public as per the vaccination plan, and over 960,000 people have received one vaccine dose by the end of may[47]. however, the lockdown restrictions escalated to level 4 in june as the virus spread and cases rapidly surged[48]. the level four lockdown continued into july as the virus spread within the local communities[49]. as of july 2021, over two million individuals tested positive for covid-19 in south africa, and over 70,000 deaths have been recorded since the pandemic in 2020[20]. by enforcing the lockdown restrictions, south africa managed to control the third wave of infections, and the number of daily cases gradually decreased. this prompted the government to ease the rules to level 1 in october[50]. the minister of health then announced a reduction in cases and hospitalizations, and there were only 5000 people in the hospital with covid-19 as of 15 october 2021. positivity rates and reported deaths also decreased across the country, putting the country on the right trajectory towards recovery from covid-19 outbreaks. the vaccination program had delivered 20 million doses of vaccines to individuals, with at least 13.8 million people receiving the first dose of vaccines. the vaccines currently used in south africa’s national vaccination program against covid-19 are janssen from johnsons and johnsons and comirnaty from pfizer. the janssen vaccine consists of adenovirus, which acts as the viral vector to deliver a gene coding for the spike protein of sars-cov-2 into the host cell. in contrast, comirnaty utilizes the mrna from sars-cov2 that codes for the vaccine’s spike protein, which is injected into the host. as the genetic information is delivered to the host cells, spike proteins of the coronavirus will be produced and detected by the host’s immune cells, triggering an immune response. during this process, the immune system recognizes the spike proteins, and in future infections, antibodies and immune cells will be deployed to protect the host from the virus[51]. these vaccines have been proven to reduce the severity of the disease and control the virus spread in south africa[52–54]. pmmb 2022, 5, 1; a0000264 7 of 12 the ministry of health was also ready to open up vaccinations for children between 12–17 of age to control the virus in schools. however, scientists in south africa discovered a new heavily mutated coronavirus, now known as the b.1.1529 (omicron) variant, in late november 2021[55]. president cyril ramaphosa of south africa later announced that despite the presence of the omicron variant, there would be no changes to the current alert level. however, everyone should still abide by the enforced restrictions to prevent the transmission of the virus. the country was now taking a new approach towards combating the coronavirus by increasing vaccination rates instead of reimplementing stricter restrictions[56]. 45.84% of the adult population have received at least one vaccine dose, and 40.52% of adult south africans are fully vaccinated against covid-19. even with the vaccine rollouts, the emergence of the omicron variant still led to the fourth wave of covid-19 infections in africa, with a record high of over 20,000 confirmed cases within a day in december[20,57]. a study done in south africa found that during the fourth wave of infections, there were significantly fewer hospital admissions for covid-19. the conditions were less severe than the cases from previous waves in the country[58,59]. by the end of december 2021, the peak of the fourth wave had passed and daily confirmed covid-19 cases were declining. thus the government lifted more restrictions, including removing the curfew[60]. as the fourth wave continues to die down into the new year, south africa has amassed over 3.5 million covid-19 cases and 93,551 deaths since the start of the pandemic[61]. 3. conclusion the south african government took the necessary measures to prevent the spread of the virus, such as implementing lockdowns and restrictions to the public. covid-19 tests were being done diligently at a mass scale to detect infections and new variants at an earlier stage. individuals who tested positive for the virus were also put in quarantine, and medical care was also given. moreover, contact tracing via the covid alert sa app was also influential in detecting the close contacts of infected individuals, and the connections were notified to get themselves tested for the virus at nearby testing facilities[35]. a national vaccination strategy was also quickly planned to achieve herd immunity within south africa as soon as possible. although continuous efforts have been put to control the virus, the emergence of new variants and super-spreader events still caused multiple outbreaks. now, almost a year has passed since the first covid-19 vaccine rollout in south africa, but only 40.52% of the adult population has been fully vaccinated[62]. this low percentage of people with immunity against the disease can be attributed to the issues surrounding procuring enough vaccines, logistical obstacles of delivering them, and vaccine hesitancy among the population[63–65]. besides, the lockdowns during the pandemic in south africa greatly affected the already weak public health care and further burdened the healthcare management systems with pre-existing problems. pmmb 2022, 5, 1; a0000264 8 of 12 the country was already dealing with other infectious diseases such as hiv and malaria pre-covid. hiv transmission has accelerated among the poor and young women during the lockdown. psychological problems have also been more frequently reported during the long-term lockdowns[66]. the government has also been scrutinized for easing restrictions because of the lack of available resources and financial means to keep the country in lockdown[66]. the negative impacts of covid-19 on the healthcare system in south africa depict the importance of having a good and well-prepared health system. with well-managed healthcare systems, it becomes less likely the system would get overwhelmed during an outbreak, and patients with other chronic or debilitating diseases can still receive necessary healthcare. the covid-19 situation in south africa also highlights the importance of sharing knowledge, technology, resources, medical equipment, and medication among countries worldwide. this global pandemic cannot be eradicated if any country is behind in vaccination rates. sars-cov-2 has been rapidly mutating, and the equal distribution of vaccines globally is the solution to prevent the emergence of new variants and outbreaks that could potentially cause severe disease and high mortality rates shortly. author contributions: the literature searches, data collection and manuscript writing were performed by kyl. the manuscript was critically reviewed, proofread, and edited by jw-fl, lt-hl, and vl. the project was conceptualized by vl. funding: no external funding was provided for this research. acknowledgments: the authors would like to acknowledge professor shajahan yasin, head of school, jeffrey cheah school of medicine and health sciences, monash university malaysia. conflicts of interest: the authors declare no conflict of interest. references 1. wang x, shi l, zhang y, et al. coping with covid-19: core elements of lockdown wuhan city policy. j health care poor underserved 2021; 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a0000264 12 of 12 64. kollamparambil u, oyenubi a, and nwosu c. covid19 vaccine intentions in south africa: health communication strategy to address vaccine hesitancy. bmc public health 2021; 21(1): 2113. 65. adepoju p. as covid-19 vaccines arrive in africa, omicron is reducing supply and increasing demand. nat med 2021. 66. hatefi s, smith f, abou-el-hossein k, et al. covid-19 in south africa: lockdown strategy and its effects on public health and other contagious diseases. public health 2020; 185: 159–160. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2021, 4, 1; a0000259. doi: 10.36877/pmmb.a0000259 http://journals.hh-publisher.com/index.php/pmmb review article sexual dysfunction in asian males with type 2 diabetes mellitus vurmila venggadasamy1*, loon shin ho2, badariah ahmad1, khalid abdul kadir1 article history 1jeffrey cheah school of medicine and health sciences, monash university malaysia, monash university malaysia, subang jaya 47500, malaysia; badariah.ahmad@monash.edu (ba); khalid.kadir@monash.edu (ka-k) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; ho.loon.shin@monash.edu (l-sh) *corresponding author: vurmila venggadasamy; jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; vurmilavenggadasamy@gmail.com (vv) received: 28 november 2021; received in revised form: 27 december 2021; accepted: 28 december 2021; available online: 31 december 2021 abstract: in 2021, 537 million people were diagnosed with diabetes globally, where over 90% of these cases were type 2 diabetes mellitus (t2dm). in the top 10 countries for many adults with diabetes in 2021, asia accounted for six spots in the rankings, with china (140.9 million), india (74.2 million), and pakistan (33 million) sitting at the top three. in addition, the prevalence of diabetes in asians are higher than in individuals from other regions. in asia, diabetes affects a younger age group resulting in more people suffering from the complications of diabetes at an earlier stage in life. one of the most serious diabetes mellitus (dm) complications is sexual dysfunction (sd). in men with diabetes, several issues may occur in sd, such as erectile dysfunction (ed), ejaculatory disorders, orgasmic disorders, and reduced libido. there are many tools available to assess sds. however, the sexual dysfunction in asian men with diabetes (sad-men) questionnaire is the only tool specific to asian males with diabetes. sd develops because of the interaction between several biopsychosocial factors. there is insufficient data on the genetic, psychological, and social factors contributing to sds in males with diabetes in asia. men with diabetes induced sds experience a poor quality of life (qol) due to the distress caused by sd. however, people from different cultural backgrounds and ethnicities perceive qol differently. therefore, future studies must be done on other predictors of life in a multiethnic population like southeast asians. there is still insufficient data on sds in asian males with diabetes. more studies are warranted to research different aspects of sds, such as its prevalence in other parts of asia, biopsychosocial factors contributing to sd, and the impact of sd on the quality of life of males with diabetes. keywords: sexual dysfunction; type 2 diabetes mellitus; males; gene; quality of life pmmb 2021, 4, 1; a0000259 2 of 16 1. introduction diabetes is a chronic health condition that affects the ability of the pancreas to produce sufficient insulin or when the produced insulin is not utilized effectively to digest glucose in the body for energy production[1]. approximately 537 million people worldwide are currently living with diabetes, which is a 400% increase from 108 million in 1980[2,3]. according to the international diabetic federation (idf), these values are predicted to reach a staggering number of 592 million by 2035[3]. over 90% of diabetes is attributed to type 2 diabetes mellitus (t2dm), and this chronic condition has been dubbed the ninth leading cause of death as it claimed 1.5 million lives in 2019[2]. the 20-year prospective cohort study done by iris shai et al. (2006) showed that the asian population is at a significantly higher risk (adjusted rr =1.43, 95% ci 1.08–1.90) of t2dm than caucasians, contributing to 60% of dm cases worldwide[4,5]. this can be attributed to the increased susceptibility to insulin resistance due to the higher percentage of body fat in asians compared to europeans of the same bmi values[6]. besides, a drastic increase in diabetic patients was also seen in the asian population. in the past 30 years, countries like korea, indonesia, and thailand have experienced a three to five-fold rise in the number of people suffering from this chronic disease. there was a three-fold rise in diabetic patients in an even shorter time in china. although there are an increase t2dm patients in other parts of the world, the changes were not as drastic as in asia[6]. the prevalence is higher in this region; people from a younger age group are affected by this condition. most people with t2dm in asia are from 45 to 64 years old compared to europeans who predominantly develop t2dm at the age of 65 and above[6]. soon, more younger people in asia will suffer from microvascular (e.g. retinopathy, neuropathy and nephropathy) and macrovascular (e.g. peripheral vascular, cardiovascular and cerebrovascular diseases) complications of t2dm[7]. therefore, this review aims to discuss on the sexual dysfunction among men with type 2 diabetes mellitus (t2dm). 2. sexual dysfunction, a serious complication of t2dm according to the world health organization (who), sexual dysfunction is described as the inability of a man or woman to have a sexually satisfying relationship with his or her partner[8]. in male diabetics, several issues may occur in the realm of sd. other than erectile dysfunction (ed), which has been extensively researched for the past decade, male diabetics also suffer from ejaculatory disorders, orgasmic disorders, and reduced libido, which can be equally if not more distressing to diabetic men[8–10]. not only is it distressing, but it also affects the fertility of these men[11]. men with diabetes mellitus (dm) were at a higher risk of suffering from sd than non-diabetic men[9]. thus, the primary focus of this paper will be on all sds experienced by men with t2dm. the different sds discussed in this paper are erectile dysfunction, ejaculatory disorders, orgasmic disorders, and reduced libido. 2.1. erectile dysfunction erectile dysfunction occurs when men frequently experience difficulty maintaining a penile erection during sexual intercourse leading to a dissatisfactory sexual experience[10]. in pmmb 2021, 4, 1; a0000259 3 of 16 diabetic males, the amalgamation of autonomic neuropathy decreased vascular nitric oxide levels, accelerated atherosclerosis, and increased oxidative stress due to endothelial dysfunction results in the development of ed[10,12]. this multistep mechanism makes diabetic men more vulnerable to ed than non-diabetic men[13]. it is still uncertain if hypogonadotropic hypogonadism, a common occurrence in diabetic men, contributes to ed[14,15]. current evidence shows a wide range of prevalence for diabetes-induced ed in different parts of the world (35 to 90%)[8,16]. asian countries have reported a higher incidence of ed compared to non-asian countries. sri lanka, china, and japan have documented a prevalence of 73.1%, 75.2%, and 80.9%, respectively, in contrast to the us and italy which reported a lower prevalence of ed (45.8% and 43.3% respectively)[17–21]. apart from western countries, even in the african countries, diabetic men did not experience ed as frequently as the asians[22]. some of the results of studies on the prevalence of ed among diabetic men may be skewed for several reasons. the inclusion of many older men compared to younger participants will explicitly increase the overall prevalence of ed as the development of ed is shown to increase with age[13,20]. furthermore, some studies did not exclude participants who have type 1 diabetes mellitus, leading to inaccurate results as this group of men may experience ed as well[17,23]. in southeast asia, there is still a lack of high-quality, large-scale studies done on the prevalence of ed among diabetic males. a cross-sectional study showed that 84.4% of indonesian diabetic men had ed. however, the sample size is too small to make a definite conclusion on the prevalence of ed[24]. malaysia has reported a 70.1% prevalence rate of ed among men aged 50 to 93 but did not stratify participants between diabetic and nondiabetics[25]. the prevalence of ed is predicted to increase drastically in all regions worldwide, especially in asia[17]. this can be attributed to the high prevalence of ed, as shown by the studies done in this region. however, the results of these studies do not reflect accurately on the prevalence of diabetes induced-ed in sa as this region is more diverse in terms of ethnicity[8]. in the general population, the prevalence of ed in sa is higher than in other parts of asia, warranting more research to determine the percentage of them experiencing ed due to t2dm[26]. 2.2. ejaculatory disorders ejaculatory disorders in diabetic men include premature ejaculation (pe), delayed ejaculation, anejaculation, and retrograde ejaculation. pe can be categorized into acquired premature ejaculation and lifelong premature ejaculation[27]. acquired pe is defined as ejaculation, which always or almost always occurs within three minutes of penetration and frequently struggles to delay ejaculation upon vaginal penetration. it is also associated with extreme distress and exasperation, resulting in the affected men being sexually intimate with their partners[10,27]. in comparison, delayed ejaculation is the complete absence of a delay in reaching orgasm after usual sexual excitement during sexual intercourse. it is often associated with extreme distress in the patient[10]. another form of the ejaculatory disorder is anejaculation which is the absence of ejaculation with or without an orgasm. lastly, retrograde ejaculation describes the retrograde movement of semen into the urinary bladder pmmb 2021, 4, 1; a0000259 4 of 16 due to the disruption in coordination between the internal urinary sphincter and external urinary sphincter during ejaculation[10,28]. retrograde ejaculation can either be partial or complete, leading to infertility[10,29]. it is vital to address ejaculatory disorders in t2dm patients due to their higher prevalence in diabetic males than in general. a cross-sectional study conducted in sri lanka and egypt showed that 40.2% of diabetic men had pe whereas, in italy, the prevalence rate was 28.3%[8,21,30,31]. in malaysia, a high prevalence rate was shown in both malay and english speaking participants (38% and 46% respectively)[8]. the studies were done in sri lanka, malaysia, and italy did not exclude participants who had lifelong pe, which may have contributed to a higher prevalence rate of pe. unlike ed, age is not a risk factor of pe[27]. data on the other ejaculatory disorders listed above are still scarce, as there are very few studies on these conditions. however, a small-scaled study done in denmark showed 34.6% of diabetic men had retrograde ejaculation[29]. small sample size is a limitation to this study, warranting more studies to be done on retrograde ejaculation. as of now, there is also no definite normal range to the number of spermatozoa that can be present in post-ejaculation urine, making the diagnosis of retrograde ejaculation inconsistent. ejaculatory disorders in diabetic males are still a poorly researched topic around the world. besides the study done in malaysia and sri lanka, there are no other studies conducted in the multiethnic groups that exist in the sa. 2.3. other sexual dysfunctions orgasmic disorders can manifest as the inability of having an orgasm, delay in reaching orgasm, and reduced intensity of orgasm[10]. the results of a large-scale crosssectional study done in the us found no significant difference between the prevalence of orgasmic disorders in men with and without diabetes[32]. without other studies about this disorder, it is impossible to determine the risk of orgasmic disorders in men with t2dm. in addition, men with t2dm can also experience reduced libido, resulting in a significant reduction in sexual fantasies and sexual desire. reduced libido in diabetic men may be caused by hypogonadism[10]. in the aforementioned study, men living with dm had a significantly higher risk of experiencing reduced libido (aor = 1.72, 95% ci 1.12– 2.63)[32]. in the asian population such as sri lanka, a 25.3% prevalence rate was reported among diabetic men[18]. over the years, very few studies have been conducted on decreased libido in diabetic men. one of the community-based studies on non-diabetic men showed not only did more japanese men experience reduced libido compared to americans, but libido was also found to decrease with age[33]. since t2dm increases with age, it is of utmost importance to investigate the relationship between t2dm and reduced libido in different populations. 2.4. coexisting sds different sds have been seen to coexist in patients with t2dm. for instance, pe is said to exist concomitantly with ed due to the development of performance anxiety that arises from ed. diabetic men with ed rush through their sexual intercourse, leading to faster pmmb 2021, 4, 1; a0000259 5 of 16 ejaculation[31]. in the cross-sectional study done by malavige et al., ed was significantly associated with both pe (or = 4.41, 95% ci 2.08–9.39) and reduced libido (or = 4.38, 95% ci 1.39–13.82). on top of that, the prevalence of pe was also found to increase as the severity of ed increases[30]. however, this study did not stratify males who had lifelong pe and acquired pe, which is a significant downside of this study. another study in egypt also showed a strong link between pe and ed[31]. future studies must explore different types of sds that may coexist in a diabetic male, as there is evidence of it occurring in other populations in the world. 2.5. diagnosing sexual dysfunctions in asian men with diabetes the diagnosis of sd should be made by taking a complete medical history, including the sexual history, examining the patient for other t2dm complications, and screening for androgen deficiency[10]. validated questionnaires play a pivotal role in assessing sds in patients because sexuality and sexual functioning are sensitive topics to discuss, even with doctors. therefore, self-administered questionnaires ease the history-taking process by allowing patients to self-report sds on paper and overcome barriers that may prevent them from being frank about their condition. the international index of erectile dysfunction (iief) is an internationally validated diagnostic tool used to assess the sexual functioning of men across five domains which are erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall satisfaction. nevertheless, iief should not be used as a primary measure for pe and reduced libido as it is only validated for ed[34]. the international index of erectile function-5 (iief-5) is a five-item questionnaire that is a simplified version of iief to assess ed. this highly sensitive and specific diagnostic tool includes four items from the erectile function domain and one from the intercourse satisfaction domain from iief[35]. this questionnaire has been used in many studies to assess ed in diabetic men, although it is not specific for diabetes-induced ed. the validated tools available for use in pe are premature ejaculation profile (pep), index of premature ejaculation (ipe), and the premature ejaculation diagnostic tool (pedt). however, the pep and ipe do not have validated cut-off scores to diagnose pe, and all of the tools are not specific for their use in the asian population[36]. a newly available tool called the sad-men questionnaire can assess sd among asian men with dm. this questionnaire consists of three parts; the first part assesses the individual's demographics, medical background, medications, and social history. the second part involves examining the history of existing sexual dysfunctions, while the last part includes inquiries about the sexual functioning of the individual. questions assessing sexual functioning were divided into two components which are sexual performance and sexual desire, both of which have cronbach’s α values of 0.949 and 0.775, respectively, indicating high internal consistency. sad-men also considers other factors contributing to sd other than t2dm, such as polypharmacy and side effects of medications. unlike the iief-5, this questionnaire can be used for ed, pe, and reduced libido[8]. therefore, the sad-men questionnaire is a stepping stone to conducting more studies on sd in diabetic men residing in asia, especially southeast asia. pmmb 2021, 4, 1; a0000259 6 of 16 3. modifiable and non-modifiable risk factors of sexual dysfunction to determine the susceptibility of men with dm to sd compared to men with other types of diabetes, studies have been extensively done across patients with different demographics and varying medical histories. studies have also been done to investigate the risk factors contributing to the development of diabetes-induced sd (table 1). table 1. current evidence of precipitating factors of sexual dysfunction. type of study reference results meta-analysis [37] • there is a significant association between depression and ed in diabetic men systematic review [10] • factors influencing the development of ed include duration of dm, age, blood glucose levels, smoking, hypertension, dyslipidemia, obesity, lack of physical activity, other dm complications, medications (eg. antidepressants, beta-blockers, thiazide, spironolactone, fibrates), and other medical conditions (hypogonadism, mood disorders such as depression, peyronie’s disease) • factors influencing the development of pe include longer duration of dm, poor control of blood glucose level, coexisting ed, and other complications of dm • reduced libido is associated with older men, coexisting ed, other comorbidities (eg. depression, hypogonadism, cardiovascular disease, kidney failure), and medications (eg. antidepressants, antihypertensives) prospective cohort [31] • the prevalence of pe doubled in diabetic men 50 years old and above compared to men younger than 50 • men with dm for more than 10 years, poor metabolic control, coexisting cardiovascular disease are at a higher risk of pe pmmb 2021, 4, 1; a0000259 7 of 16 crosssectional [38] • significantly increased risk of ed in men who have diabetes for more than five years, hypertensive, consumes more than two standard drinks of alcohol daily, bmi > 25 kg/m2, beta-blockers, and other microvascular complications of t2dm • no significant associations to household income, education, dyslipidemia, macrovascular complications of t2dm, good control of blood glucose levels (hba1c < 7) and smoking crosssectional [9] • no significant difference in the prevalence of ed among south asian men with dm as compared to the europeans crosssectional [22] • age more than 41, uneducated, divorced or widowed, dm comorbidities (nephropathy, retinopathy, diabetic neuropathy, diabetic foot ulcer, cardiovascular diseases, hypertension), practicing sedentary lifestyle, and depression were significantly associated with sd in men with dm • no significant associations were found between sd and sex, religion (orthodox, protestant, muslim, others), ethnicity, and duration of treatment for dm crosssectional [39] • the risk of ed is shown to increase in older men, living with extended family, high bmi, on combined treatment for dm, presence of other complications of dm, smoking, depression, poor marital relationship observational study [21] • the risk of ed is significantly increased with severe depression, beta-blockers, and at least one complication of t2dm • pe was significantly associated with severe depression • reduced libido was increased dramatically in men with severe depression, hyperprolactinemia, and obesity (bmi > 30 kg/m2). consumption of coffee, however significantly decreased the risk of reduced libido pmmb 2021, 4, 1; a0000259 8 of 16 when it comes to the southeast asian population, there are still many unanswered questions regarding the risk factors of sd in diabetic men due to the lack of studies being done in these populations. several studies done elsewhere have shown that the development of sd in men with t2dm is dependent on a range of biopsychosocial factors. unfortunately, the results of these studies cannot be applied to the population in southeast asia due to the cultural and ethnic variation in this region. moreover, due to the cross-sectional nature of most of the studies, a temporal causal-effect relationship between the independent factors and sd cannot be formed. for instance, in asian diabetic men with depression and ed, did the depression precipitate ed, or is ed a manifestation of depression? the only way to answer this is to conduct more prospective studies to determine factors that influence the development of ed. future studies should investigate unexplored psychosocial factors affecting the development of sd in men with t2dm. sexual behavior is influenced by the religion and religiosity of a person[40]. thus, this factor can potentially play an important role in the development of sd among males in southeast asia, the home of a religiously diverse population. a study on malaysians residing in urban areas showed a high prevalence of ed[25]. future studies should be done on the rural population to make comparisons as the different lifestyles of both urban and rural people may influence the prevalence of sd. it is essential to identify all of the risk factors of ed in this population because most asian men tend to attribute sd to aging and choose to suffer in silence[41]. knowing all of the predisposing factors of sd in a diabetic man can help physicians predict its development in a patient. 4. quality of life of diabetic men with sexual dysfunction quality of life (qol) is defined as an individual's perception of their position in life regarding their culture, value systems, goals, expectations, standards, and concerns[42]. in the context of t2dm, measuring only the biological outcome of dm is insufficient as dm is a chronic disease that affects the biopsychosocial well-being of a person. therefore, psychological and social factors affecting a person’s life should be considered to improve their qol[43]. one of the predictors of qol in diabetic men is sd as it is linked to the psychological derangement of these men. men with ed develop low self-esteem due to their inability to perform sexually, which importantly translates into poor self-care resulting in poor glycemic control[9,44]. ed was also shown to be emotionally distressing, as illustrated in the cross-sectional study by chen et al., using the symptom checklist 90-revised (scl-90r) questionnaire[45]. however, that was the only study of its kind, warranting more studies to be conducted on the association between emotional distress and sd in different populations. pmmb 2021, 4, 1; a0000259 9 of 16 a large-scale prospective study done by berardis et al. shows that the development of ed was significantly associated with the deterioration in the physical and mental wellbeing of these men, as measured by the 36-item short-form health survey (sf-36)[46]. the limitation of this study is the failure to use a validated questionnaire to diagnose ed. another study has explored other sds in diabetic men, such as pe and reduced libido, and found significant associations between these sds and qol. in this study, ed was significantly associated with poor qol on both the physical and mental scales of sf-36 and reduced libido on the physical scale. the strength of this study is the usage of the disease-specific psychological impact of erectile dysfunction (pied) questionnaire to measure the impact of ed on qol. ed reduced libido and pe were significantly associated with poor quality of sexual experience, whereas poor emotional life was associated with ed and reduced libido[18]. a comprehensive study was conducted in malaysia to evaluate different factors influencing qol in men with t2dm using the disease-specific asian diabetes quality of life (asiandqol) questionnaire. this study also showed the significant association between sds and qol of english-speaking men from different ethnicities[47]. the aforementioned study represents how people from various cultural backgrounds have different perceptions of qol. for instance, the qol of english-speaking chinese were only affected by sd compared to mandarin-speaking chinese men, who are concerned about hyperlipidemia and hba1c levels in addition to sd. this differed from malay and indian men who had other predictors of qol[47]. in the context of sds, it is important to find the association between different ethnicities in sa and how sds affect their qol to provide more holistic management of t2dm. 5. genetic factors associated with sexual dysfunction in t2dm several candidate genes are associated with the development of sd in males with t2dm. however, most animal and human studies conducted are much focused on ed in males with t2dm. one of the better-studied genes is the enos (endothelial nitric oxide synthase) gene. chromosome 7q35 codes for the enos gene. the size of this gene is 21 kb, and it consists of 26 exons[48]. one of the enos gene variants is g894t[49]. the singlenucleotide polymorphism at position 298 in exon 7 led to the transversion of guanine with thymine, resulting in the substitution of glutamate with aspartate in the mature protein[49–52]. this polymorphism increases the selective proteolytic cleavage of endothelial cells and vascular tissue leading to the reduction in the nos activity and subsequent decrease in the production of vascular no. the 786t-c polymorphism gives rise to another variant of the enos gene. this variant inhibits the activity of the gene promoter. as a result, there was a reduction in the expression of enos protein, thus decreasing the activity of the protein[50,53]. pmmb 2021, 4, 1; a0000259 10 of 16 a decrease in constitutive nitric oxide synthase (cnos) expression is significantly associated with ed in males with dm. as shown by an animal study conducted by ahn et al., a marked downregulation in the neuronal nitric oxide synthase (nnos) and enos mrna expression levels is seen in the corpus cavernosum tissue in rats induced with dm compared to controls[54]. a study conducted on 228 taiwanese males showed a significantly increased risk of ed in enos 894t allele carriers compared to enos 894g allele carriers (80.0 % vs. 63.3 %; p = 0.04). this suggests the association of g894t polymorphism to ed in the general population[52]. however, a study conducted in brazil showed that g894t polymorphism is not associated with ed development (or = 1.05, 95% ci 0.69–1.60; p = 0.86)[55]. another study on t-786c polymorphism in the promoter of the enos gene in the turkish population showed a significant increase in the prevalence of ed in cc alleles carriers (or = 3.2; 95% ci 1.4–7.6; p = 0.006). males with cc genotype were shown to develop earlier symptoms of ed compared to controls (51.7% vs. 10.7%; p < 0.001). however, the small sample size is a limitation of this study[56]. in males with t2dm, evidence on the association between enos gene polymorphisms and the development and/or progression of vasculogenic ed is replete with conflicting results. a study conducted in tunisia included 164 males with t2dm and 148 healthy males subjected to genotyping for enos gene polymorphism (g894t and t786c). the result of this study shows that g894t (cor = 1.66, 95% ci = 1.16–2.38; p = 0.005) and t-786c (cor = 1.46, 95% ci = 1.05–2.04, p = 0.02) polymorphisms are significantly associated to vasculogenic ed among males with t2dm[53]. another study conducted in taiwan showed a higher prevalence of 894t allele carriers in males with dm than males without dm, although not statistically significant (32.5% vs 13%; p = 0.13)[52]. enos polymorphisms occur at a different frequency among various ethnic groups. these differences lead to a lack of reproducibility of results across different populations[48]. furthermore, the lack of evidence on the variants of the enos gene concerning ed in males with t2dm makes it harder for a conclusion to be drawn. angiotensin-converting enzyme (ace) is another gene commonly associated with ed. the ace gene codes it, and it is responsible for converting angiotensin i (ang i) to angiotensin ii (ang ii)[48]. the insertion (i) or deletion (d) of a 287 bp region in the intron 16 of the ace gene is responsible for the alteration of serum ace levels[57]. these variants give three possible genotypes: dd, di, or ii[48]. the d allele increases ace activity in the body[57]. in a study conducted by mazo et al., dd alleles were significantly associated with ed development in russian males with metabolic syndrome (p < 0.001). the author hypothesizes that the increase in the ace activity leads to the dysfunction of the endothelial pmmb 2021, 4, 1; a0000259 11 of 16 cells, resulting in ed[58]. another study performed on 84 japanese males showed a significant relationship between the dd genotype and ed (p < 0.01)[59]. to date, there is still insufficient evidence to prove the association of the d allele and the development of ed in males with t2dm. the peptide ang ii plays a role in balancing the cavernous smooth muscle tone by inducing contraction, initiating detumescence in males[60]. ang ii gene plays a key role in the mechanism of ed in males with dm. in an animal study conducted by zhang et al., male sprague-dawley rats with dm and silenced ang ii gene demonstrated a significantly longer penile erection time (1.3 ± 0.6 min, p < 0.05) compared to rats in dm-induced ed and adnull groups. ang ii silencing was shown to inhibit the expression of the rhoa/rho-kinase signaling pathway, thus decreasing contraction and increasing relaxation of smooth muscles to improve erectile function[61]. the extracellular matrix of erectile tissue encompasses collagen, elastic fibers, and different types of proteoglycans[62]. in males with dm, these intracavernous components (i.e., elastic fibers) are significantly reduced compared to healthy counterparts. this alteration results in the deterioration of erectile function[63,64]. sullivan et al. demonstrated the downregulation of extracellular matrix genes (i.e., collagen and elastin) such as genes encoding collagen (i.e., type 1, 3, 5, 6, and 11), elastin, and fibrillin1 in dm[65]. lysyl oxidase mitigates the stabilization and covalent crosslinking of collagen and elastin fibers in the extracellular matrix[66]. the gene expression that codes for lysyl oxidase, lox, is also reduced in the erectile tissue of males with dm[65]. programmed cell death 4 (pdcd4) is a putative gene regulated by microrna-21-5p, associated with ed in dm. the animal study by huo et al. demonstrated the upregulation of the pdcd4 in the cavernous tissue of rats with dm-induced ed. this leads to apoptosis and a decrease in the proliferation of smooth muscle cells of the corpus cavernosum[67]. lipoprotein lipase (lpl) is another putative gene highly expressed in dm-induced ed. an in vitro study showed that the upregulation of long noncoding rna myocardial infarction was associated with transcript (lncrna-miat) in vascular smooth muscle cells of the corpus cavernosum combined with the downregulation microrna-328a-5p (mir-328a-5p), upregulates the lpl gene. this concert of events leads to the injury of endothelial cells, ultimately causing the inhibition of enos[68]. insulin-like growth factor-1 (igf-1) plays a vital role in the regeneration of nerve fibres with the nnos in dorsal and intracavernosal nerve[69]. in an animal study conducted by pu et al., rats that were transfected with adcmv-igf-1 demonstrated a significantly higher ratio of maximal intracavernous pressure-to-mean arterial pressure (icp/map) upon pmmb 2021, 4, 1; a0000259 12 of 16 cavernosal nerve stimulation compared to controls (p < 0.05). this proves the importance of the igf-1 gene in regulating penile erection[70]. more studies are required to highlight the relationship between genetic composition and ed in asian males with t2dm. to date, there is still a lack of evidence on the association between genetic factors and other types of sd, such as ejaculatory disorders, orgasmic disorders, and reduced libido. 6. conclusion the existing evidence shows that sd is a severe complication of t2dm that is more prevalent among asian men than men from other regions. as sd is one of the main predictors of qol in asian males with diabetes, efforts to improve their qol should include the sadmen questionnaire, a validated tool to assess sd. moreover, there is a range of biopsychosocial factors influencing the development of sd; hence, identifying each patient's risk factors could greatly help physicians develop an effective treatment plan. however, more work on diabetes-induced sds needs to be done in sa as there is still a lack of data of this issue in this region. studies should explore more on sds other than ed as they are proven to occur in males with diabetes. the prevalence and risk factors of orgasmic disorders, ejaculatory disorders, and reduced libido are still understudied, warranting more studies to be done in the future. we should also look into the psychosocial factors affecting the development of sds among men with t2dm in sa. future studies should focus on the degree of emotional distress caused by sds and how the qol of different populations with different cultural backgrounds, ethnicities, and religions are affected by sds. author contributions: vv performed the literature search and manuscript writing. l-sh, ba, and ka-k performed proofreading and editing. ka-k conceptualized this review writing project. funding: no external funding was provided for this research acknowledgments: the authors would like to acknowledge professor shajahan yasin, head of school, jeffrey cheah school of medicine and health sciences, monash university malaysia. conflicts of interest: the authors declare no conflict of interest. references 1. centers for disease control and prevention (cdc). diabetes. 2021; 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1865(6): 1226–1240. 69. jung gw, kwak jy, yoon s, et al. igf-i and tgf-beta2 have a key role on regeneration of nitric oxide synthase (nos)-containing nerves after cavernous neurotomy in rats. int j impot res 1999; 11(5): 247–259. 70. pu xy, hu lq, wang hp, et al. improvement in erectile dysfunction after insulin-like growth factor1 gene therapy in diabetic rats. asian j androl 2007; 9(1): 83–91. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2022, 5, 1; a0000276. doi: 10.36877/pmmb.a0000276 http://journals.hh-publisher.com/index.php/pmmb genome report whole-genome sequence of chelatococcus daeguensis strain m38t9, isolated from ulu slim hot spring in malaysia yi xian goh1, kok gan chan2,3,4, kar wai hong5* article history 1department of computational biology, high impact research building, university of malaya, kuala lumpur, 50603, malaysia; elizabel8683@gmail.com (yxg) 2department of biotechnology, faculty of applied sciences, ucsi university kuala lumpur, 56000, kuala lumpur, malaysia 3international genome centre, jiangsu university, zhenjiang, china. 4institute of biological sciences, faculty of science, university of malaya, 50603, kuala lumpur, malaysia; kokgan@um.edu.my (kgc) 5novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, jalan lagoon selatan, 47500 bandar sunway, selangor darul ehsan, malaysia *corresponding author: kar-wai hong; novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, jalan lagoon selatan, 47500 bandar sunway, selangor darul ehsan, malaysia; hong.karwai@monash.edu (kwh) received: 13 july 2022; received in revised form: 10 august 2022; accepted: 17 august 2022; available online: 23 august 2022 abstract: chelatococcus daeguensis strain m38t9 is a thermotolerant bacterium isolated from a hot-spring in malaysia. the draft genome of c. daeguensis strain m38t9 consists of 4,218,658 bp assembled into 50 scaffolds. the gc content of the genome is 67.91 %, and the sequencing coverage of 184×. there are 4,046 predicted genes, 3,962 protein-coding genes, and 53 rna-coding genes (trna: 45, rrna: 4). the draft genome has been deposited at ddbj/ena/genbank under the bioproject accession number prjna668056. the raw reads were deposited in the sequence read archive (sra) under accession number srr13805582. here we report the draft genome of this strain to expand our understanding of the genomic information available on the genus chelatococcus. keywords: chelatococcus daeguensis; nitrogen metabolism; denitrification; dissimilatory nitrate reduction; genome pmmb 2022, 5, 1; a0000276 2 of 5 1. introduction the genus chelatococcus falls within the class of alphaproteobacteria and was first reported as obligately aerobic, gram-negative bacteria by auling and colleagues in the year 1993[1]. according to the list of prokaryotic names with standing in nomenclature (lpsn)[2], this genus comprises six species at the time of writing, namely c. asaccharovorans, c. caeni, c. composti, c. daeguensis, c. reniformis, and c. sambhunathii [1, 3-7]. most notably, there has been a growing interest in c. daeguensis in the recent years for its capability in utilizing a variety of carbon sources and its capability in performing aerobic denitrification at high temperatures, as well as its capability in biodegrading crude oil, coal and toxic metals[5, 8-11]. to provide insights into the genomic basis for these mechanisms, we hereby present the draft genome of c. daeguensis m38t9, isolated from ulu slim hot spring, malaysia (3.8986 n 101.4847 e, 110°c, ph 7). 2. data description the genomic dna from m38t9 was extracted using a masterpuretm dna purification kit (epicentre, illumina inc., madison, wi, usa) upon growing the cells at 37°c on luria-bertani (lb) agar[12]. the quality and quantity of dna were quantified using nanodrop spectrophotometer (thermo scientific, waltham, ma, usa) and a qubit version 2.0 fluorometer (life technologies, carlsbad, ca, usa), respectively[13]. the sequencing library was constructed using the nextera dna library kit, followed by sequencing performed using illumina hiseq 2500 platform[14]. the quality of these raw reads was checked using fastqc (version 0.11.9)[15], and the low-quality reads were trimmed using trimmomatic (version 0.39)[16] with the default settings for paired-end reads. subsequently, the quality-filtered reads were de novo assembled by velvet (version 1.2.10)[17]. the quality of assembly was analyzed using quast (version 5.0.2)[18] and busco (version 3)[19]. the assembled gene was annotated using ncbi prokaryotic genome annotation pipeline (pgap)[20] as well as microscope[21]. default parameters were used for all software unless otherwise specified. the identity of strain m38t9 was first determined using microflex lt (bruker daltonics, bremen, germany), and the strain m38t9 was identified as chelatococcus species[22]. the 16s ribosomal rna (rrna) gene of strain m38t9 was compared with the ezbiocloud database[23]. interestingly, the 16s rrna gene of strain m38t9 showed the highest sequence identity with the 16s rrna genes of c. daeguensis and c. sambhunathii, both at 99.72% of identity. however, the estimated average nucleotide identity (ani) value determined by automlst[24] suggested the strain m38t9 is likely to be c. daeguensis as the estimated ani for strain m38t9 by referring to c. danguensis strain m3 is 100% (pvalue=0.000). the draft genome of c. daeguensis strain m38t9 consists of 4,218,658 bp assembled into 50 scaffolds, with n50 and l50 of 311,252 bp and 6 bp, respectively. the gc content of the genome is 67.91 %, and the sequencing coverage of 184×. there are 4,046 predicted genes, 3,962 protein-coding genes, and 53 rna-coding genes (trna: 45, rrna: 4). the pmmb 2022, 5, 1; a0000276 3 of 5 draft genome has been deposited at ddbj/ena/genbank under the bioproject accession number prjna668056. the raw reads were deposited in the sequence read archive (sra) under accession number srr13805582. the version described in this paper is the first version. figure 1. complete pathway of dissimilatory nitrate reduction and denitrification in c. daeguensis strain tad1, m3 and m38t9. by comparing the metabolic pathway of three c. daeguensis strains, namely tad1 (ncbi accession number: cp018095.1), m3 (ncbi accession number: lqqt01000000), and m38t9 (ncbi accession number: jafduy010000000), a complete metabolic pathway of denitrification has been identified (figure 1). the denitrification metabolic pathway comprises of periplasmic nitrate reductase complex napab which reduces nitrate (no3 -) to nitrite (no2 -), copper-containing nitrite reductase nirk which further reduces no2 to nitric oxide (no), nitric oxide reductase complex norbc which reduces no to nitrous oxide (n2o), and nitrous oxide reductase nosz which reduces n2o to nitrogen (n2). similarly, via comparative genomics, the complete pathway of dissimilatory nitrate reduction was identified in all three strains. the dissimilatory nitrate reduction comprises periplasmic nitrate reductase complex napab, which reduces nitrate (no3 -) to nitrite (no2 -), followed by nitrite reductase, which reduces nitrite (no2 -) to ammonia (nh3). this genome report provided vital insights into the genomic basis for these mechanisms. author contributions: yxg and kwh conducted the experiments and analyzed the data. kwh and kgc provided vital guidance, technical support, and proofreading for the work. all authors approved the final draft. funding: this work was supported by the university of malaya via ppp grant (pg085-2015b) awarded to kwh, and high impact research grants (um-mohe hir grant um.c/625/1/hir/mohe/chan/14/1, no. h-50001-a000027 and a-000001-50001) which are awarded to kok-gan chan. conflicts of interest: the authors declare no conflict of interest. pmmb 2022, 5, 1; 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47(w1): w276-w282. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology review article 1 a review on the potential of bruton’s tyrosine kinase (btk) inhibitor – ibrutinib for treatment of multiple myeloma (mm) sze-ting bong1*, lydia ngiik-shiew law2, jodi woan-fei law3 1department of biochemistry and molecular biology, bio21 molecular science and biotechnology institute, university of melbourne, parkville, victoria, australia 2monash credentialed pharmacy clinical educator, faculty of pharmacy and pharmaceutical sciences monash university, 381 royal parade, parkville vic 3052 3novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, selangor darul ehsan, malaysia abstract: multiple myeloma (mm) is characterized by the over-production of monoclonal plasma cells that eventually become malignant in the bone marrow. mm remains as an incurable cancer, but it can be treated through chemotherapy. nonetheless, research on novel therapies for effective treatment of mm is ongoing and in this case the involvement of bruton’s tyrosine kinase (btk) in b cell malignancies has made it one of the new therapeutic targets. in mm patients, it has been reported that the expression of btk was elevated and this could potentially contribute to chemoresistance indirectly via enhancement of cell proliferation and survival. ibrutinib is a highly selective irreversible btk inhibitor commonly used as treatment for b cell malignancies such as mantle cell lymphoma (mcl) and chronic lymphocytic leukemia (cll). with reference to the potential involvement of btk in mm and current treatment of mcl and cll using ibrutinib, researchers have begun to examine the effect of ibrutinib treatment on mm. this review provides information on the association of mm and btk in conjunction with the treatment using ibrutinib. to date, clinical trials of ibrutinib as therapeutic alternative for mm have produced promising results, particularly as combination therapy with other agents such as dexamethasone and carfilzomib. however, there is limited evidence on the btk mechanisms involved in mm, hence, it is important to further investigate the btk oncogenic signalling pathway(s) in mm cells in order to establish successful and improved treatment of mm. keywords: multiple myeloma; ibrutinib; cancer; blood; chemotherapy received: 15th may 2019 accepted: 22nd june 2019 published online: 30th may 2019 *correspondence to: sze-ting bong, department of biochemistry and molecular biology, bio21 molecular science and biotechnology institute, university of melbourne, parkville, victoria, australia; bong-123@ hotmail.com. citation: bong st, law lns, law jwf. a review on the potential of bruton’s tyrosine kinase (btk) inhibitor ibrutinib for treatment of multiple myeloma (mm). prog microbes mol biol 2019; 2(1): a0000028. introduction multiple myeloma (mm) or myeloma is a b cell malignancy initiated from the over-production and accumulation of malignant plasma cells in the bone marrow[1]. it is a progressive disease with increasing occurrence in elderly group. among the different types of blood cancers, 15% consisted of the disorder of bone marrow mm[2]. in mm, the over-proliferative malignant plasma cells outcompete the population of other blood cells in bone marrow which can cause some of the disease symptoms such as anaemia. as antibody-producing cells, the malignant plasma cells produce large quantities of a monoclonal antibody (also called m protein) in mm[3]. excessive m protein will interfere with the normal function of immune system. the protein could be accumulated in the kidney, thereby causing renal failure and patients become prone to infections[3]. also, the malignant plasma cells could over-activate osteoclasts but not osteoblasts in the bone marrow[4]. osteoclast is a type of bone cells responsible for bone resorption, while osteoblast directs bone formation. the over-activated osteoclasts could cause excessive bone resorption, resulting in bone lesion commonly encountered in mm patients[3]. hence, patients often suffer from back pain. moreover, the continuous release of calcium from bone resorption can cause hypercalcemia (excess calcium level in blood) and further contribute to kidney damage[4]. presently, treatments such as stem cell transplantation and chemotherapies involving drugs such as thalidomide, bortezomib, dexamethasone and lenalinomide are currently adopted for mm[5-8]. however, most of the drugs did not produce promising results as the complete copyright 2019 by bong st et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) 2 remission only last for a few months to a few years[3,4,9,10]. therefore, new treatment strategies are required for effective treatment of mm. as such, the search for new cell surface or molecular targets has been the aim of many researches to explore additional therapeutic options. bruton’s tyrosine kinase (btk) as novel target the origin, structure and functions of btk in recent years, a non-receptor tyrosine kinase known as bruton’s tyrosine kinase (btk) has been one of the most discussed promising novel targets that could play a role in the treatment of mm due to its involvement in many b cell malignancies[11,12]. btk is a predominant cytoplasmic protein consisting of 659 amino acid residues, which mainly expressed in the b cell lineage[13-15]. it is a member of tec family protein kinases, a subfamily of nonreceptor tyrosine kinases. other members of the family include tec, itk, bmx and txk[16]. tec family kinases are structurally conserved, consisting of plekstrin homology (ph) and tec homology (th) domains at the n terminal region, followed by src homology 3 (sh3), src homology 2 (sh2), and kinase domains (figure 1)[17]. figure 1. illustration of btk’s protein linear structure. the full-length protein consists of 659 amino acid residues, with molecular weight of about 76.3 kda. the labelled boxes represent the corresponding domains of btk. the auto-phosphorylation (y223) and transphosphorylation (y551) sites are the important activating phosphorylation sites of btk (image adapted from mohamed et al. (2009) [17]). the btk has acquired its name in the honor of dr. bruton c. ogden, a paediatrician, who described an immunodeficiency condition known as agammaglobulinemia in year 1952[18]. this condition was observed in a boy who was first admitted to the clinic at the age of 4.5 years old. prior to admission, the boy had encountered several infections including the development of recurrent pneumococcal sepsis. several attempts have been done to determine the cause of the patient’s recurrent infections. finally, dr. bruton had achieved a breakthrough in this research he discovered a complete absence of gamma globulins in the patient’s blood sample and thus the patient seemed to be unable to produce any antibodies to combat the infection[18,19]. few years later, studies revealed that the lack of antibody-producing cells is the reason of this observed humoral phenotype. the xlinked agammaglobulinemia (xla) is the most common agammaglobulinemias known; lost-of-function mutations had contributed to this inheritable xla, for which this disorder is associated with bruton’s name, hence, it is also known as bruton’s agammaglobulinemia[19,20]. the defects of btk gene (encoding btk) located at chromosome x long arm, xq. 21.3-22 is the cause of human xla (x-linked recessive disease)[15,19,21,22]. the btk plays critical roles in mediating signals from cell membrane to transcriptional events in the nucleus, regulating b cell development and/or maturation[20,22]. therefore, the mutation of btk gene will result in dysfunction of btk or lack of expression of btk which could prevent the transition of pre-b cell to mature b cell. consequently, there will be insufficient number of mature b cells to respond and produce antibodies or immunoglobulins to fight against the pathogenic bacterial infections[20]. biochemical studies had revealed that ph domain is crucial for btk to be recruited to plasma membrane by binding to phosphatidylinositol-3, 4, 5 triphosphate (pip3) [23-25]. this allows btk to be activated by src family kinases (sfks) and subsequently mediate transmission of signals to the downstream substrates[26]. mutations at ph domain that abolished the binding ability of btk to pip3 result in development of x-linked agammaglobulinemia (xla), indicating the importance of btk being recruited to plasma membrane for activation[27]. other than that, ph domain was shown to interact with other proteins like protein kinase c (pkc), heterotrimeric g protein subunits, transcription factor (tfii-i, also known as bap-135)[28-31]. btk also binds fas via its ph and kinase domains to prevent interaction between fas and fas-associated death domain (fadd), and inhibit fas-mediated apoptosis[32]. th domain next to ph domain is another characteristic structural domain of tec family kinases. it is made up of a btk motif (zinc finger motif) and a proline-rich region (prr)[33]. the btk motif contains one histidine and three cysteines that are critical for the binding of zinc ions and stabilize the protein. mutations of the conserved cysteine residues affect protein folding and stability, and hence, xla is developed[34]. the prr plays role in protein-protein interaction by binding to sh3 domain. it also binds to btk itself at sh3 domain, regulating its activity either by preventing its interaction with other cellular proteins or phosphorylates tyr223 to activate btk kinase activity[35,36]. besides that, the binding of btk’s sh3 domain to liar was reported to be important for its nucleocytoplasmic shuttling[37]. similar to the sh2 domain of other protein tyrosine kinase, it binds to specific tyrosine containing substrate. it was shown to preferentially bind phosphopeptides with the sequence of pyeei[38]. sh2 is important for btk to interact with spleen tyrosine kinase (syk) and b cell linker (blnk; a protein adaptor), and these interactions were essential for activation of phospholipase c (plcg) by btk[39,40]. in the protein structure of btk, there are two phosphorylation sites that regulate btk’s kinase activity: tyr223 in sh3 domain and tyr551 at the catalytic loop in the kinase domain. transphosphorylation at tyr551 increases the kinase activity of btk and permits further autophosphorylation on tyr223 for full activation[41-43]. biophysical analysis revealed that btk adopted an elongated conformation in solution, with minimal interactions among the functional domains[44]. sfks, another subfamily of nonreceptor tyrosine kinase that share the same arrangement of sh3, sh2 and kinase domains in their primary structures as btk. sfks are inhibited when their sh3 domain binds the linker between sh2 and kinase domains, and the sh2 domain binds their c-terminal tail at phosphotyrosine 527 to form a globular inactive conformation[45]. in contrast, the elongated structure adopted by btk suggests that the activity of btk is regulated by intermolecular interactions between btk and other cellular proteins a review on the potential of bruton... 3 and/or another btk molecule instead of forming a globular inactive conformation[17,46]. moreover, deletion of other domains did not affect btk kinase activity, further confirming that intramolecular interactions among the functional domains are not involved in regulating its kinase activity[47]. nevertheless, the regulatory mechanism remains unclear. btk’s central role in b-cell receptor (bcr) signalling pathways b cell is a type of white blood cells essential for humoral immune response[48]. b cell develops from pro-b cell in bone marrow up to mature b cell which then migrate into periphery tissue. upon antigen activation, these mature b cells differentiate into antibody-producing plasma cells or memory b cells. loss-of-function mutations in btk block the transition from pro-b cell to pre-b cell which causes defect in xla (figure 2)[15,22,49]. figure 2. the different stages of b cell development. the b cell development is governed by the pre-b cell receptor (pre-bcr) and b-cell receptor (bcr) which are formed by surface immunoglobulins and other cellular proteins. the signalling event is triggered when antigen binds to the membrane immunoglobulin; the receptors oligomerized and trigger downstream signalling pathways for cell growth, differentiation, adhesion and migration. this promotes sfks such as lyn and lck to phosphorylate immunoreceptor tyrosine-based activation motive (itam) on both igα and igβ. the phosphorylated itam motifs act as a docking site for sfks and syk[50,51]. the binding activates sfks and syk. the activated sfks and syk then phosphorylate and activate phosphoinositide 3-kinase (pi3k), resulting in the conversion of phosphatidylinositol-4,5-bisphosphate (pip2) to phosphatidylinositol-3,4,5-triphosphate (pip3) [52]. the resultant pip3 formed on the membrane recruits btk to the plasma membrane via interaction between pip3 and ph domain of btk[23,50,53]. syk also phosphorylates blnk to create a binding site for specific sh2-domain containing proteins, including btk and plcg[54]. the binding of btk to blnk brings btk in close proximity to syk and sfks, allowing them to transphosphorylate btk at tyr551[38]. as discussed earlier, phosphorylation of tyr551 facilitates btk to further autophosphorylate itself on tyr223. phosphorylation at tyr223 prevents the binding of inhibitory protein to btk, enabling btk to interact with its substrates[17]. the fully activated btk can then phosphorylates and activates plcg which cleaves pip2 to inositol-1,4,5-trisphosphate (ip3) and diacylglycerol (dag). ip3 and dag are secondary messengers that direct calcium mobilization and activation of mitogen-activated protein kinase (mapk) pathway through protein kinase c (pkc) and nuclear factor kappa-lightchain-enhancer of activated b cells (nf-κb) pathway (figure 3)[55, 56]. ever since btk was found highly expressed in b cells and constitutive activation of btk causes sustained signalling which contribute to prolonged cell survival and over-proliferation of malignant b cells, thus, btk has become the therapeutic target for b cell malignancies. bong st et al. figure 3. summary of early events of bcr signalling pathways and the consequences after btk’s activation. 4 the bruton’s tyrosine kinase specific inhibitor, ibrutinib ibrutinib the treatment of b cell malignancies ibrutinib is a specific btk inhibitor, mainly used for the treatment of mantle cell lymphoma (mcl) and chronic lymphocytic leukemia (cll) as approved in the year 2013 and 2014 respectively by food and drug administration (fda)[57]. this approval was driven by the promising results demonstrated in the phase 2 clinical trial that involved a total of 111 patients with relapsed or refractory mcl[57,58]. these patients, either with or without treatment of the therapeutic proteasome inhibitor, bortezomib (another type of chemotherapy drug for b cell malignancies) before, were treated with oral 560 mg per day of ibrutinib until undesirable events had occurred. the results revealed 68% of high response rate and 58% of estimated overall survival rate at 18 months. moreover, ibrutinib was found to be well-tolerated without critical toxic effects[58]. this outcome had eventually led to the clinical trials of ibrutinib in relapsed cll. the results revealed that the drug improved the chance of remissions in patients with 75% of progression-free survival rate and 83% of overall survival rate[59]. development and design of ibrutinib as a highly selective btk inhibitor ibrutinib (or imbruvica, formerly referred as pci-32765) was one of the several irreversible inhibitors designed by pan, z. et. al. (2007)[60] at celera genomics for the celera’s small molecule btk inhibitory discovery program. a series of irreversible inhibitors was generated during the program, with the focus to target btk and specifically inhibit its kinase activity. the compounds developed in the discovery program were later acquired by pharmacyclics and janssen, followed by clinical trials of ibrutinib for various haematological malignancies[57]. during the drug development, inhibitors were designed to target the following structural features of btk in order to have high selectivity against btk[60]: (i) atp binding cleft of btk kinase domain in the inactive form (ii) nucleophilic amino acid residue unique to btk kinase domain and (iii) the gatekeeper residue governing the accessibility of the active site to mg2+-atp/mn2+-atp. for inhibiting kinases, atp binding cleft (active site) in the kinase domain is a common targeted site. this is to block the binding of atp and inhibits activity of the kinase[61]. the inactive kinase domain is preferably targeted than active kinase domain. this is because active kinases share similar catalytic structure, but the inactive kinase domains adopt distinct conformation [62]. despite that, this strategy alone is not enough to provide high selectivity of the drug as the active site is still highly conserved among many kinases[61,63]. therefore, pan et. al. (2007)[60] focused on developing inhibitor with an electrophilic functional moiety capable of forming a covalent bond with btk to increase selectivity of the inhibitor. in order to increase the selectivity, the authors screened for nucleophilic amino acid residues that are unique to btk[60]. cysteine 481 (cys481) that resides in atp binding pocket of btk was postulated to be the suitable nucleophilic site that can covalently interact with the electrophilic inhibitors[60]. sequence alignments of the sequences of 491 protein kinases had shown that only btk and nine other protein kinases shared a cysteine residue at a homologous position in the kinase domain. the other nine kinases were: blk, bmk, egfr, erbb2, erbb4, itk, jak3, tec and txk as shown in figure 4[60,61]. among them, itk, tec and txk are members of the tec family tyrosine kinase. this had provided a good strategy to greatly increase the selectivity of the inhibitor. thus, a small molecule inhibitor with an enamide double bond group that could form covalent bond with the thiol group (-sh) of cys481 was created. figure 4. sequence alignment of the 10 protein kinases that consisted cysteine residue at position same as cys 481 of btk. protein sequences of the 10 protein kinases were obtained from uniprotkb and aligned using clustalw2. only the fragment of the multiple sequence alignment that contains the nucleophilic site (cys) and gatekeeper residue is shown here. btk’s cys 481 is highlighted as yellow and thr 474 as green. the cysteine and gatekeeper residues of the other 9 protein kinases were also highlighted as yellow and green respectively. all of them contained cys residue at the same position as cys 481 of btk, and thr residue as the gatekeeper residue except itk and jak3. a review on the potential of bruton... 5 besides cys481, the gatekeeper residue was also considered in the design of btk inhibitors. this special residue plays an important role as selectivity filter of kinase inhibitors. gatekeeper residue is the amino acid residue that lies behind the atp binding site[61,63]. in some of the protein kinases, the gatekeeper residue contains a bulky aromatic side chain restricting the accessibility of the active site to compounds with bulky moiety ibrutinib is one of these compounds[63]. in btk, the gatekeeper residue threonine 474 (thr474) consists of relatively small hydroxyl (-oh) and methyl groups (-ch3) in its side chain. only 20% of protein kinases in the kinome contain threonine as the gatekeeper residue. this approach can potentially exclude 80% of protein kinases in the kinome as targets of ibrutinib and further increases the selectivity of ibrutinib as btk inhibitor[63]. a series of btk’s irreversible inhibitors had been synthesized, followed by screening of selectivity and efficacy of the generated inhibitors in inhibiting a list of protein kinases’ activity (including btk). ibrutinib possesses highest selectivity against btk with ic50 of 0.5 nm (in vitro) [64] and only a few of protein kinase could be derivatized by ibrutinib at low concentration. for instance, it was reported that ibrutinib had ic50 of 1 nm for blk, bmk and tec while it had higher value of ic50 for the other suspected protein kinase (egfr, itk and jak3) that is able to be bound by ibrutinib[63]. as a result, ibrutinib (pci-32765) was selected and proceeded into clinical studies. the promising outcome of the clinical trials had enabled ibrutinib to be approved as drug for treatment of mcl and cll. btk’s pathological role in multiple myeloma (mm) owing to the remarkable result of using ibrutinib for the treatment of mcl and cll, several studies have started to explore its efficacy for the treatment of other types of b cell malignancies such as mm. as suggested earlier, btk is expressed in hematopoietic cells from b cell lineage and repressed in the terminally differentiated plasma cell[65,66]. however, it has been demonstrated that btk expression is elevated in mm cell lines and also in cancer cells obtained from mm patients[1,6,12]. besides, btk was able to amplify its gene expression by activating nf-κb[55,67]. the activated nf-κb then binds and activates transcription of btk gene[55,67]. the activation of nf-κb requires active btk and plcg[55,56]. the signalling pathways are not clear but btk was found to be involved in b cell activating factor (baff) mediated activation of nf-κb[56,68]. in addition, the expression of btk was significantly higher in dexamethasoneand also bortezomibresistant mm patients and cell lines, supporting the fact that increase in btk expression is correlated with increasing activity of nf-κb[69,70]. these findings may have suggested that malignant plasma cells could potentially utilise the upregulated btk as an alternative mechanism to promote cell growth and survival, contributing to chemoresistance. moreover, btk participates in maturation and differentiation of osteoclast (oc) in bone marrow[71,72]. oc is a type of bone cell in which its bone resorption function is upregulated in mm and contributes to the mm-related osteolysis. the function of btk in mm was also evaluated in a study conducted by tai et al. (2012)[12] through btk knockdown and the findings of this study have confirmed the pathogenic role of btk activation in promoting mm cell growth and survival, interaction with other bone marrow stromal components, and inducing mmrelated osteolytic bone disease. alternatively, inhibition of endogenous btk was found to negatively impact on myeloma cell growth, migration, and adhesion to microenvironment[1,12,73]. the inhibition was reported to induce caspase-dependent cell death, which overruled btk’s role in inhibiting fas-mediated apoptosis as mentioned earlier[6]. overall, there is increasing evidence on the involvement of btk in regulating mm cell growth, survival, migration and adhesion. therefore, ibrutinib can potentially be use for the treatment of mm or relapsed mm. ibrutinib for multiple myeloma (mm) given that many studies have suggested the involvement of btk in several signalling pathways that are related to cell survival and proliferation, as well as cell adhesion and migration in mm, thus, researchers are motivated to trial the drug as a therapeutic option for mm. it has been demonstrated that ibrutinib is cytotoxic to malignant plasma cells through in vitro experiments. according to cell titer glo assay conducted by rushworth et al. (2013)[6], ibrutinib (10µm) had induced significant plasma cell death of about 7 % – 46 % in mm cells. besides, the cytotoxicity against malignant plasma cells in mm was significantly increased when ibrutinib was utilized in combination with either bortezomib or lenalidomide, thus, suggesting the synergistic effect of ibrutinib with these drugs[6]. apart from in vitro experimentation, several clinical trials have been conducted to examine the efficacy of ibrutinib for treatment of mm, either as monotherapy or in combination with other drugs. for example, a phase 2 open-label dose escalation trial conducted by vij et al. (2014)[74] involving the use of ibrutinib as a single agent monotherapy or in combination therapy with dexamethasone was examined in patients with relapsed and relapsed/refractory mm. the preliminary results indicated that both treatments: ibrutinib as single agent and ibrutinib in combination with dexamethasone, have produced anti-tumor effect in the patients. it was also reported that there was a trend towards enhanced efficacy in treatment with 840 mg ibrutinib (daily) and 40 mg dexamethasone (weekly) with well tolerated and manageable toxicities. additionally, the treatment using ibrutinib in combination with carfilzomib ± dexamethasone was first evaluated in patients with relapsed or relapsed/refractory mm by chari et al. (2018)[75] in a phase 1 dose-finding trial. the outcomes demonstrated the combination therapy of ibrutinib (840 mg) and carfilzomib (36 mg/m2) with dexamethasone showed the most promising result as it has the shortest median duration of response for patients who achieved above partial response which was 7.2 months. hence, this dosage was recommended for the following phase 2 trial. bong st et al. 6 future directions and conclusion in light of the recent advances in the use of ibrutinib for treatment of mm, it is therefore important to chart the btk oncogenic signalling pathway in mm cells. other than the involvement of btk in regulating nf-κb and stromal cell-derived factor-1 (sdf-1) induced signalling pathways[1,12,76], not much is known about its actual role in mm cells. in myeloma cells, ibrutinib activity seems to be mediated through interfering nf-κb signalling pathway which subsequently promoting cell apoptosis[77]. several studies also proposed that the immunomodulatory effects of btk inhibitors on macrophages in tumor microenvironment and direct cytotoxic effects of btk inhibitors on malignant b cells may be contributing to the therapeutic efficacy of these medicines[78]. apparently, combination therapy of ibrutinib with other drugs is more effective than monotherapy for the treatment of mm. furthermore, ibrutinib is still more effective in treating cll than mm. this could be due to the reason that the relative protein expression of btk in mm cells is significantly lower than that in cll cells[6], hence, mm cells are less sensitive towards ibrutinib compared to cll cells. also, it is possible that mm cell survival and proliferation may be supported by other complementary signalling pathways which are yet to be determined in the future. thus, further studies should be conducted to investigate the downstream btk targets which can be utilized to improve the efficacy of ibrutinib and the overall treatment of mm. authors contributions the literature review and 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8(24): 3921839229. bong st et al. progress in microbes and molecular biology 1 genome report whole genome sequence of mum116, a bacillus species isolated from intertidal soil hooi-leng ser1, wen-si tan2, wai-fong yin3, kok-gan chan3,4, vengadesh letchumanan1* 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, bandar sunway, selangor darul ehsan, malaysia 2illumina singapore pte ltd, woodlands industrial park e1, singapore 3division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia 4vice chancellor office, jiangsu university, zhenjiang 212013, pr china abstract: over the past few years, mangrove-derived bacillus sp. have been characterized frequently for their bioactive potential. bacillus sp. mum 116 was isolated from mangrove forest in kuala selangor which is located on the west coast of peninsular malaysia. in order to obtain better understanding of the strain, the genome sequence of mum 116 was acquired through illumina miseq sequencing platform and yielded 5,720,395 bp along with 165 trna and 25 rrna genes. based on antismash and rast annotation, there was one cluster associated with production of bacteriocin. a deeper analysis into the genome sequence of mum 116 would be essential to exploit the strain for production of bioactive compounds, which could potentially be developed as potent antibacterial agent. keywords: bacillus; antibiotics; mangrove; secondary metabolite; genome received: 26th november 2019 accepted: 26th december 2019 published online: 20th january 2020 citation: ser h-l, tan w-s, yin w-f, et al. whole genome sequence of mum116, a bacillus species isolated from intertidal soil. prog microbes mol biol 2020; 3(1): a0000052. https://doi.org/10.36877/pmmb.a0000052 short introduction as a unique ecosystem, the mangrove forest are habitat for many plants as well as microbial populations that highly capable of adapting to fluctuations in temperatures, organic matter content, salinities and oxygen conditions[1,2]. owing to these factors, some strains came up with adaptation strategies to survive and persist in the environment; one of which is by modifying metabolic pathway by scavenging nutrients available in the environment before converting them into useful, bioactive compounds that improve their survivability (i.e. antibacterials and antifungal)[3–5]. with reference to mangrove forest, asia represents an ideal “hunting zone” for bioactive microbial strains as this continent has got the largest coverage of mangrove forests, contributing 42 % of the global total[6,7]. several studies have shown that bacillus sp. derived from mangrove forest have great potential in producing bioactive compounds[8–13]. bacillus sp. mum 116 was isolated from the west coast of peninsular malaysia during a screening program for bioactive microbes[14–19]. 16s rrna analysis showed that mum116 showed high similarities (<90%) to some bioactive type strains including bacillus ginsengisoli, bacillus niacini and bacillus mesonae[20]. given that mangrove-derived bacillus sp. have been demonstrated to possess potential bioactive potential and mum 116 displayed high 16s rrna gene similarities with bioactive type strains, the strain was subjected to genome sequencing to uncover its genomic potential. data description the genomic dna of mum 116 was extracted using masterpuretm dna purification kit (epicentre, illumina inc., madison, wi, usa) before subjected to rnase copyright © 2020 by ser h-l et al. and hh publisher. this work under licensed under the creative commons attributionnoncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: vengadesh letchumanan, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, bandar sunway, selangor darul ehsan, malaysia; vengadesh.letchumanan1@monash.edu 2 (qiagen, usa) treatment[21,22]. genomic dna quality was evaluated using nanodrop spectrophotometer (thermo scientific, waltham, ma, usa) and a qubit version 2.0 fluorometer (life technologies, carlsbad, ca, usa)[23,24]. nextera™ dna sample preparation kit (nextera, usa) was used to generate dna library and its quality was examined with bioanalyzer 2100 high sensitivity dna kit (agilent technologies, palo alto, ca) prior to sequencing[25,26]. whole genome sequence of mum 116 was obtained via paired-end sequencing on illumina miseq platform with miseq reagent kit 2 (2 × 250 bp; illumina inc., madison, wi, usa)[27]. the assembly of trimmed seqeuence was done with clc genomic workbench version 5.1 (clc bio, denmark), resulting in 208 contigs and an n50 contig size of approximately 52,003 bp. the assembled genome size of mum 116 consists 5,720,395 bp, with an average coverage of 74.0fold and g + c content of 38.4%. the genome sequence of bacillus sp. mum 116 has been deposited at ddbj/ embl/genbank under accession of mlyr00000000. table 1. general genomic features of bacillus sp. strain mum 116. genome size (bp) 5,720,395 contigs 208 contigs n50 (bp) 52,003 g + c content % 38.4 protein coding genes 5,273 trna 165 rrna 25 annotation of mum 116 genome was carried out using rapid annotation using subsystem technology (rast) [28] while gene prediction was performed using prodigal version 2.6. the detection of ribosomal rna (rrna) and transfer rna (trna) was done using rnammer and trnascan se version 1.21, respectively[29–31]. based on rast analysis, more than one-quarter of the proteincoding genes were associated with primary metabolism and highest number of genes were related with metabolism of amino acid and derivatives (12%). furthermore, both rast and another bioinformatics tools, antibiotics & secondary metabolite analysis shell (antismash) revealed potential of mum 116 in producing bacteriocin under the thiazole/oxazole-modified microcins (tomms) class[32,33]. several bacillus sp. have been described to have the potential of synthesizing tomms[34,35]. for instance, bacillus amyloliquefaciens fzb42 isolated from plant-pathogen-infested soil was capable of compounds producing not just plant-promoting activity, the strain produced a novel tomms — plantazolicin which can suppress growth of bacterial and fungal plant pathogens[35]. even though bacillus sp. isolated from terrestrial region showed great potential in producing bioactive compounds, several studies have hinted that genomes of bacillus sp. from special environment like mangrove area are generally more “enriched” than those from terrestrial area, as the dynamic environment imposes selective pressure on genomic region associated with adaptation which then promotes production of unique secondary metabolites[36,37]. altogether, the availability of mum 116 genome sequences enabled further investigation into its genomic potential, 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mangrove forest. prog microbes mol biol 2018; 1(1). ser h-l et al. progress in microbes and molecular biology review article 1 vibrio parahaemolyticus: the protagonist causing foodborne diseases vengadesh letchumanan1*, ke-yan loo1,2, jodi woan-fei law1, sunny hei wong3, bey-hing goh2, nurul-syakima ab mutalib4, learn-han lee1* 1novel bacteria and drug discovery research group (ndbb), microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2biofunctional molecule exploratory research group (bmex), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 3li ka shing institute of health sciences, department of medicine and therapeutics, the chinese university of hong kong, shatin, hong kong 4ukm medical molecular biology institute (umbi), ukm medical centre, universiti kebangsaan malaysia, kuala lumpur, malaysia abstract : food contamination is a worrying condition faced by us today. we often discuss on food safety and how to control food contamination. food products are easily tainted by bacteria at any level of food production to human consumption, subsequently developing gastroenteritis. the people from developed and developing countries are at high risk from harmful effects of unsafe food. of all the foodborne pathogens, vibrio parahaemolyticus has been accounted for many outbreaks globally and still at rise even with proper management methods. v. parahaemolyticus infection occurs as a result of improper food handling and preparation, ability of the bacterium to withstand human gut to launch virulence, antibiotic resistant bacterium, and failure of regulatory bodies to safe-guard food quality. this scenario poses a global health issue that warrants rapid control measures to ensure food safety from production to consumption by consumers. for that reason, this review aims to provide an overview of the epidemiology of v. parahaemolyticus as well as discuss the challenges faced to encounter this bacterium. keywords: food safety; contamination; foodborne; vibrio parahaemolyticus; epidemiology received: 18th may 2019 accepted: 30th june 2019 published online: 02nd july 2019 *correspondence to: learn-han lee; jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. email: lee.learn.han@monash.edu; leelearnhan@yahoo.com. vengadesh letchumanan; jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. email: lvengadesh@ yahoo.com. citation: letchumanan v, loo ky, law jwf, et al. vibrio parahaemolyticus: the protagonist causing foodborne diseases. prog microbes mol biol 2019; 2(1): a0000029. introduction food is an essential part of every individuals for survival. it provide us with all the energy and nutrients for our healthy and active life. however, many of us ponder on one matter – how safe is our food? the world health organization (who) claimed that food is unsafe as it contains harmful bacteria, viruses, parasites or chemical substance, causing various illness ranging from diarrhea to cancers. it is estimated that 1 in 10 people in the world fall ill after consumption of contaminated food and lead to 420,000 die each year[1,2,3]. food products are likely to be contaminated at any stages from production, processing, distribution, storage and preparation. these unsafe food poses a global health threat, increase hospitalization and healthcare cost, and constrains the national’s economic development. numerous studies revealed that marine and estuarine environments are the ecological niche of many bacteria. undeniably, these bacteria are often encountered by consumers through seafood products, including salmonella sp.[4-9], vibrio sp.[10,11,12,13,14], listeria monocytogenes[15,16], and escherichia coli, presented with foodborne outbreaks, pathogenicity, and clinical manifestations. despite the good benefits of seafood, health hazards associated to seafood consumption cannot be ignored[17]. copyright 2019 by letchumanan v et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) 2 vibrio species are common foodborne pathogen group that’s accounted for human gastroenteritis cases worldwide; the most human pathogenic species are vibrio cholerae, vibrio parahaemolyticus and vibrio vulnificus[14]. this gram-negative rod-shaped bacterium is a natural constituents of marine and estuarine environments, and often been associated with seafood[18]. the non-cholera vibrio sp., vibrio parahaemolyticus, causes vibriosis and readily isolated from seafood including shellfish, oysters, shrimps, cockles, and fish[11,12,19]. the outbreaks of v. parahaemolyticus are becoming increasingly common in developed and non-developed countries such as asia region, united states, europe, australia, and other nations[10]. according to data published by centers for disease control and prevention (cdc) in foodborne diseases active surveillance network (foodnet), and morbidity and mortality weekly report (mmwr), v. parahaemolyticus has accounted for approximately 34,664 incidents of domestically developed foodborne infection cases and known as the leading bacterium unlike to other vibrio sp. in the united states (us) in 2016[20,21].the consumption of contaminated food, undercooked or raw seafood leads to v. parahaemolyticus gastroenteritis with manifestation of watery diarrhea, stomach pains, nausea, and fever[22]. in rare cases, infections of v. parahaemolyticus can cause septicemia – leading to an increase in number of fatality cases[23]. by taking into consideration of past reports and possibility of severe infections, this review aims to provide an overview of the epidemiology of v. parahaemolyticus as well as the rising challenges faced to curb this bacterium. history of vibrio and vibrio parahaemolyticus vibrio genus was first described by an italian physician, filippo pacini in 1854. he discovered the first vibrio species, vibrio cholera, the causative agent of cholera while studying outbreaks of cholera disease in florence[24]. subsequently, this strain was renamed as vibrio cholerae, which is now the type of species of the genus. he further pointed out that cholerae is contagious but his discovery on vibrio was ignored by the scientific community around the world[24]. after nearly 30 years, robert koch successfully isolated vibrio from pure culture in calcutta, india. at that time vibrio epidemic was very active in calcutta, india. koch’s discovery had created an important social consequence and regarded as a public health triumph[25]. vibrio genus consist of 142 species that are marine originated and its taxonomy is continuously been revised due to the discovery and inclusion of new species[26]. vibrio sp. infects any living being including animals and humans[27]. it was reported that a few of the species from this genus have been identified and classified among the top 15 pathogens causing nearly 95% of the foodborne diseases, hospitalizations and even deaths in the united states[28]. recently, the worldwide ocean warming and climate changes have caused emerges of vibrio sp. including the foodborne pathogenic strains with several virulence factors in marine environments. as a member of the vibrionaceace family and vibrio genus, vibrio parahaemolyticus has been in limelight for the rising vibriosis and foodborne cases worldwide. v. parahaemolyticus was first identified in 1951 by tsunesaburi fujino from research institute of microbial diseases (rimd), osaka university from an acute gastroenteritis outbreak. the outbreak occurred in a southern suburb of osaka, japan due to consumption of ‘shirasu’, a type of dried sardine which resulted in 20 deaths and 272 infected patients[29,30]. after several bacteriological testing and analysis, fujino noticed that the strain exhibited hemolytic activity on blood agar and named the strain as pasteurella parahaemolytica, assigning it to genus pasteurella. the progression in taxonomy and various scientific discoveries led to the re-examination of pasteurella parahaemolytica by fujino. he reported that the genus of the isolate should be vibrio instead of pasteurella. in 1963, sakazaki investigated fujino’s isolates and confirmed it was the same species belonging to vibrio genus and propose to name the isolate as vibrio parahaemolyticus[30]. epidemiology of vibrio parahaemolyticus vibrio parahaemolyticus is largely present in the aquatic environments and often isolated from seafood[31]. since its discovery in 1950, v. parahaemolyticus has caused many foodborne outbreaks around the world including in japan[32-36], in taiwan[37], in china since early 1990s[38], bangladesh[39], laos[40], hong kong and indonesia[40] (figure 1). despite the advances in hygiene and food processing, this foodborne pathogen still represents a significant threat to human health worldwide. asia vibrio parahaemolyticus was initially identified as a seafood-associated disease in the eastern asia region. this bacterium was first isolated in 1951 from a foodborne outbreak in osaka, japan due to consumption of shirasu, which resulted in 272 infected patients and 20 deaths[29,30]. since then, many v. parahaemolyticus gastrointestinal cases have been reported in around japan are due to the habit of eating raw or undercooked seafood such as sushi, sashimi, shellfish, crabmeat, fish, squid and sea urchin[41,42]. there was an increasing trend of reported cases from 1993 (837 cases) to 1998 (12, 318 cases), nevertheless the figures drastically dropped to 14 cases in 1999 and 280 cases in 2009[42]. the decrease in number of v. parahaemolyticus foodborne cases from 1999-2000 is due to the implementation of regulatory actions to improve the hygiene conditions in all seafood production sites in japan[35]. even with appropriate regulatory measures, there are still many reported v. parahaemolyticus cases in japan. in the neighboring nation china, v. parahaemolyticus was identified as a major cause of foodborne disease since early 1990s. seafood such as crustaceans was the vehicle for v. parahaemolyticus to transmit vibriosis infection in china[43]. from 1991-2001, a total of 5770 foodborne cases was reported, which 31% of them was caused by v. parahaemolyticus[44]. the number of outbreaks decline to 322 cases between 2003 and 2008[43]. li and colleagues found that v. parahaemolyticus was the main cause of acute diarrhea during 2007-2012 in southern coastal region of china, with the most prevalent serotype o3:k6 followed by o4:k8 and o3:k29[37]. in taiwan, many foodborne gastroenteritis outbreaks were identified to be caused by v. parahaemolyticus[32,37,45]. vibrio parahaemolyticus:... 3 in southeast asia regions – laos, thailand, indonesia and cambodia, v. parahaemolyticus was accounted for several foodborne outbreaks[46]. v. parahaemolyticus first outbreak occurred in kampung speu, cambodia resulted in 49 cases of acute diarrhea[47]. in thailand, the pandemic o3:k6 serotype strains was accountable for most of the foodborne cases between 2006 and 2010[48]. thailand is known as the main producer and exporter of cultured shrimp to around the global. this industry is on alert due the occurrence of antimicrobial resistant v. parahaemolyticus isolated from white leg shrimp and black leg shrimp from inland ponds[49]. in malaysia, v. parahaemolyticus occurs naturally in the marine and coastal regions. it spreads in the tropical marine surroundings during all seasons and causes foodborne gastroenteritis[50]. in the early 1980s, a study revealed the detection of v. parahaemolyticus in malaysian shrimp. it is of interest to note that 21 different serotypes were isolated from malaysian shrimp, with type 01:k38 and 01:k32 were predominated[51]. in 2005, a study reported the isolation of v. parahaemolyticus from cockles (anadara granosa) at a harvesting area at tanjong karang, kuala selangor. the analysis revealed virulent v. parahaemolyticus isolates having the thermostable direct hemolysin (tdh) and tdhrelated hemolysin (trh) genes[52]. virulent v. parahaemolyticus carrying tdh genes and trh genes was also identified from frozen shrimp in malaysia, prompting a possible health risk for people consuming raw shrimp[53]. in 2011, a study reported high occurrence of vibrio sp. (98.6%) and v. parahaemolyticus (24%) in freshwater fish collected from hypermarket. this outcome indicates a potential source of unsafe food to consumers in malaysia[54]. in addition, recently there was report about european union (eu] countries rejected frozen black tiger shrimp from malaysia due to the presences of v. parahaemolyticus and this further affected the malaysian economic[55]. paydar and colleagues reported prevalence of v. parahaemolyticus in the seafood samples from retail and hypermarkets in malaysia. out of the 43/150 v. parahaemolyticus isolates detected, six isolates carried the trh genes and another two carried the tdh genes[55]. recently, a comparative study was done to detect the contamination level of v. parahaemolyticus in seafood marketed in thailand, vietnam, malaysia, and indonesia. interestingly, the study’s results revealed that all the four countries had a similar levels of v. parahaemolyticus contamination in fish, shrimp, squid, crab, and shellfish. the study did not detect any virulent strains among the seafood samples from malaysia[56]. the findings in agreement with other reports globally that mentioned virulent genes, the tdh and trh are very low number (17%) among environmental and seafood samples[57-61]. the food safety levels in malaysia further declined due to the prevalence of antibiotic resistant v. parahaemolyticus isolated from seafood[11,12,62]. in terengganu, malaysia, a study reported the detection of cefuroxime and ceftazidime-resistant v. parahaemolyticus isolates in shellfish samples[62]. in addition, ampicillin resistant profiles are often detected among seafood samples in malaysia[11,50,63,64,65]. elexson and colleagues reported in their study that all of the v. parahaemolyticus isolates from cultured seafood products were resistant to both penicillin and ampicillin [63]. in a recent study, high level of penicillin and ampicillin resistant isolates were obtained from short mackerels in malaysia[66]. the ampicillin resistance seen may be due to misappropriation of the first-generation antibiotic for pathogen management in aquaculture, thus reducing the efficacy of ampicillin in the treatment of vibrio infection[67]. hence, it is indeed vital to address and manage the antimicrobial resistance issue. in india, v. parahaemolyticus was detected and identified from both clinical and environmental samples. the first serotype o3:k6 v. parahaemolyticus was discov letchumanan v et al. figure 1: illustration of v. parahaemolyticus epidemiology around the world. the first identified case was in osaka, japan in 1951 and ever since then the occurrence has spread to whole of asia region, australia, europe, and the united states (us). 4 ered in an on-going surveillance in calcutta, india[68-70]. subsequently, the serotype o3:k6 v. parahaemolyticus has turned into a widespread around asia. in a clinical study, 178 v. parahaemolyticus strains was isolated from 13,607 diarrheal patients admitted in infectious diseases hospital, kolkata since 2001 to 2012[71]. v. parahaemolyticus diarrheal cases were also detected from around the urban slums of kolkata, india[72]. reyhanath and colleagues have reported detection and isolation of antimicrobial resistant v. parahaemolyticus strains from a fishing land in south india[72]. in cochin, a study reported the isolation of vibrio sp. including pathogenic and antimicrobial resistant v. parahaemolyticus strains from seafood. most of the isolates was seen resistant to ampicillin and multidrug resistance was prevalent among the isolates[73]. the prevalence of multidrug resistant v. parahaemolyticus isolates in environment and clinical setting is of public health concern, thus require continuous monitoring and management. europe in european countries, v. parahaemolyticus infections are seldom reported, unlike asia and us countries where v. parahaemolyticus infections are commonly reported[74]. however, there were several sporadic outbreaks reported over the last 20 years in countries such as france and spain[32,74]. v. parahaemolyticus was isolated from the baltic sea, the north sea, the mediterranean sea[75], and black sea[76]. in 1978, studies were conducted in coastal waters of guadeloupe and isolated v. parahaemolyticus from 53/100 water samples that was investigated [77]. over the years, many cases of v. parahaemolyticus gastroenteritis were detected and isolated in spain, greece, britain, turkey, denmark, yugoslavia, the scandinavian areas, and italy[78,79]. in 1989, v. parahaemolyticus was accounted for 8 acute gastroenteritis cases linked with intake of fish and shellfish in spain[80]. in 1997, a major outbreak of v. parahaemolyticus involving 44 patients had occurred in france and it was associated with the consumption of shrimps imported from asia[81]. in 1999, the first large outbreak of v. parahaemolyticus occurred in galicia, spain. this outbreak involved 64 illnesses and it was associated with consumption of raw oysters[82]. a more recent outbreak of v. parahaemolyticus was reported in spain in 2004, whereby it involved 80 illnesses among the guests who attended weddings in a restaurant. the investigation revealed that the outbreak was caused by consumption of boiled crab prepared under unsanitary conditions[83]. in 2004-2005, only 57 cases of v. parahaemolyticus infections was reported in united kingdom and most of the infections were obtained through travel to endemic areas[84]. in addition, serotype o3:k6 v. parahaemolyticus strains were isolated from patients of outbreak in spain and patients of gastrointestinal infection in italy[83,85,86]. united states (us) in 1971, v. parahaemolyticus was first identified as an etiological food borne pathogen in maryland, us after three outbreaks of 425 gastroenteritis cases associated with consumption of improperly cooked crabs[87]. ever since then, intermittent v. parahemolyticus outbreaks have been reported throughout the us coastal regions due to the consumption of raw shellfish or uncooked seafood. in 1973 to 1998, a total of 40 outbreaks of v. parahaemolyticus infection was reported by the cdc[88]. four out of 40 outbreaks involved over 700 cases of diseases linked with consumption of raw oyster in the gulf coast, pacific northwest, and atlantic northeast regions between the years 1997 to 1998. in 1997 summer, 209 (including one death) of v. parahaemolyticus infection cases was reported involving consumption of raw oyster in around the pacific northwest (oregon, washington, california and british columbia of canada)[89]. in 1998, there were two separate reports on v. parahaemolyticus infection cases in washington (43 cases) and texas (416 cases) [90]. in between july to september 1998, there was eight v. parahaemolyticus infection cases reported in around connecticut, new jersey, and new york as a result of eating oysters and clams harvested from long island sound of new york[89]. in summer 2004, 14 passengers on a cruise ship in alaska experienced gastroenteritis symptoms after ingestion raw oysters produced in alaska[91]. the o6:k18 isolates from the alaska outbreak were in differentiated by pfge from those isolated in the sporadic cases from pacific coast states over the previous decade. from july to october of 2004, 96 ecological samples were collected from 17 alaska oyster farms, and 32% of the samples were v. parahaemolyticus with the prevalent serotypes of o1:k9, o4:k63, and o6:k18[92]. in summer 2006, there was an outbreak involving 177 gastroenteritis cases resulted from ingestion of oysters contaminated with v. parahaemolyticus in washington and british columbia[93]. in summary, the prevalence of v. parahaemolyticus in both clinical and environmental samples potentially rises a serious food safety concern in the us. major challengers of vibrio parahaemolyticus the regulatory bodies and healthcare sector is continuously challenged by increasing numbers of antibiotic resistant v. parahaemolyticus strains in the environments. the occurrence of multidrug-resistant (mdr) towards clinically used antibiotics is a major health issue and deprives the global drug discovery programs[94]. each year, more and more pathogenic vibrio sp. have been reported to develop resistance towards most of the clinically used antibiotics (figure 2). drug resistance is an alarming issue worldwide and is spreading rapidly due to overuse, self-medication or the non-therapeutic use of antimicrobials[95]. the countries around the world have reported the detection of mdr v. parahaemolyticus in seafood, prompting the need for continuous surveillance and monitoring in the aquaculture industry[96-99]. it is reported that over 90% of the marine originated bacteria isolates display resistance towards more than one type of antibiotic. in addition, 20% of them exhibited resistance towards five types of antibiotics[100]. the marine environments are more prone to antibiotics and antibiotic resistant genes due to the misuse of antibiotic agents in hospital or veterinary treatment, aquaculture and agriculture locations, and their successive release into wastewater treatment plant[101]. the elevated levels of antibiotic agents in the aquatic could play a role as a vibrio parahaemolyticus:... 5 selective pressure contributing to the rise and distribution of resistant and pathogenic bacteria within the same aquatic environment[102]. additionally, bacteria in the environments are able to produce antimicrobial compounds, thus making them capable of acquiring or expressing antimicrobial resistant genes to protect themselves from the toxicity of antibiotics present in the environments[103]. therefore, the presences of aquatic bacterium may function as reservoirs for antibiotic resistance genes and plays a crucial role in the spread of antibiotic resistance in aquatic environments[101]. vibrio parahaemolyticus – both as a pathogenic strain carrying virulence genes (direct hemolysin (tdh) and/or tdhrelated hemolysin (trh) and as a mdr strain, is difficult to be controlled. recently, there is a discovery on the ability of v. parahaemolyticus to withstand the bile salts, then utilize bile as signaling cue to launch its virulence[104]. the human bile in gastrointestinal system is known as the first defense mechanism against bacteria invasion in human. bile salts in human not only aid during digestion of food but possess antimicrobial activities as they have the ability to inhibit the survival of bacteria in the human gastrointestinal tract. however, this usual defense is interrupted with the ability of v. parahaemolyticus to sense bile salts. the bacteria enters into the human gastrointestinal system, two major complex protein vtra and vtrc will interact and forms complex protein on host cell, binds with bile salts and triggers the cell to produce toxins. upon binding of bile salts to the vtra/vtrc complex, the cytoplasmic dna binding domain of vtra is activated which in turn induces vtrb to activate, resulting in the t3ss2 expression. t3ss2 virulence is secreted thus causing illness to human. this mode of mechanism ensures the survival of pathogenic v. parahaemolyticus in the environments and increase in the bacterial infections[104,105]. in addition, the t3ss2 is associated with tdhand/or trh-positive v. parahaemolyticus strains[106]. hence, this information is significant to all healthcare personnel in order to know the mechanism of v. parahemolyticus infections and able to decide the best treatment for the infection. conclusion v. parahaemolyticus infection is a predominant global health threat to both developed and developing countries. the pathogenesis of infection and symptoms are minor or self-limiting upon ingestion of unsafe food. however, the rising number of people falling ill with v. parahaemolyticus has constrained the socioeconomic and healthcare systems. there are various factors contributing for foodborne diseases to remain as a global public health challenge. although many foodborne diseases have been controlled with proper management methods, new threats do continuously emerge. the changes among microorganism leads to the emergence of new pathogens, increased antibiotic resistant strains in the environment, and alteration in pathogen’s virulence. in addition, people in many countries eat food prepared outside their homes which potentially exposing themselves to high risks of poor hygiene in retail food service surroundings. in many situations, foodborne diseases go unrecognized, underreported, unreported, or not investigated at all[107]. all these challenges involve a constant monitoring of foodborne pathogens and management food safety to ensure human wellbeing. letchumanan v et al. figure 2: illustration of vibrio parahaemolyticus transmission to humans and plasmid curing assay. (a) vibrio parahaemolyticus is found in the marine, estuarine, and aquaculture settings. antibiotics are incorporated in feed and water to control vibrio parahaemolyticus infections on aquatic animals such as shrimp, cockles, fish, and shellfish. this bacterium eventually develops antibiotic resistance and carries the resistance genes in them. 1) vibrio parahaemolyticus uses seafood as a vehicle to transmit the carries the antibiotic resistance and resistant genes. 2) the antibiotics resides in water and transmitted to agriculture area thru irrigation. 3) undercooked and raw seafood potentially carry pathogenic vibrio parahaemolyticus and resistant strains. 4) consumers will eat contaminated seafood and vegetable, thus exposing themselves to gastroenteritis. (b) vibrio parahaemolyticus is isolated from seafood. antibiotic susceptibility test is performed to determine the resistance profile. the antibiotic resistance could be either in the plasmid or chromosomal of the bacterium. the strain would be subject to plasmid curing assay to determine the antibiotic resistance mediation. intercalating agents such as ethidium bromide or acridine orange can be used to cure the bacteria plasmid. after the assay, antibiotic resistance mediation could be determine either it is plasmidial or chromosomal mediated. 6 hence, adequate management of v. parahaemolyticus is required to control the widespread of this bacterium. aquatic products are one of the main reservoirs for pathogenic and multidrug resistant v. parahaemolyticus. therefore, there is an urgent prerequisite for the expansion of non-antibiotic technique to manage multidrug resistance (mdr) among pathogens due to declining efficacy of antibiotics and deficiency of new antibiotic in development pipeline[108-110]. hence, further research can be done using bacteriophages and exploring the usefulness of it the management of v. parahaemolyticus. phages are approved and recognized by the us regulatory bodies as a potential bio-control agent to control and prevent pathogens including vibrio sp[111-117]. in addition, it can be utilized in the agriculture and aquaculture industries instead of antibiotics to control bacterial infections that occur in the farms. this will sooner or later reduce the dependency towards antibiotics that leads to resistant genes profile in the environment[118]. in addition, the antibiotic resistance mediation of v. parahaemolyticus could be detected by plasmid curing assay (figure 2). this fast, reliable and inexpensive method uses curing agent to eliminate bacteria plasmids and determine antibiotic resistance mediation. mostly food safety studies involve a huge number of sample thus hindering the use of costly ngs. hence, many researchers have utilized and reported the use of plasmid curing assay to determine antibiotic resistance mediation among environmental isolates[119-124]. the results from this curing assay may influence an effective antibiotic management policy in the aquaculture sector. with this valuable knowledge, farmers could alternate the antibiotics used in their farms occasionally which will allow the bacteria to lose its resistance to a specific antibiotic[11,12]. furthermore, the study of v. parahaemolyticus genome could provide vital information on the particular strain and further strengthen the management strategies[125,126]. in summary, the public should be given adequate information on v. parahaemolyticus thru awareness campaigns in order to ensure food safety thru out the whole food industry from production to consumption[127]. authors contribution the literature review and manuscript writing were performed by vl and k-yl. jw-fl, n-sam, shw, b-hg and l-hl provided vital guidance and support as content expert and proofread of the writing. l-hl and vl founded the research project. conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. acknowledgements this work was supported by pvc award grant (project no. pvc-ecr-2016), external industry grant (biotek abadi – vote no. gba-808138 and gba-808813) awarded to l-hl. reference 1. world health organization. who estimates of the global burden of foodborne diseases. foodborne diseases burden epidemiology reference group 2007-2015. 2015. accessed on 4 july 2019; 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7: 1410. 127. letchumanan v, wong pc, goh bh, et al. a review on the characteristics, taxanomy and prevalence of listeria monocytogenes. prog microbes mol biol 2018; 1(1). vibrio parahaemolyticus:... pmmb 2023, 6, 1; a0000328. doi: a10.36877/pmmb.0000328 http://journals.hh-publisher.com/index.php/pmmb review article toxicological review of anticancer plants used in traditional medicine in morocco soufiane drioua1, abha cherkani-hassani1, otman el-guourrami1, mouna ameggouz1, ahmed zahidi2, abdelhakim bouyahaya3*, sayyed ibrahim shah4, yaser mohammed al-worafi5,6, long chiau ming7, hanane benzeid1, anass doukkali1 article history 1laboratory of analytical chemistry, faculty of medicine and pharmacy, mohammed v university in rabat, morocco; driouasoufiane92@gmail.com (sd); abha.cher@gmail.com (ac-h); elgourrami29@gmail.com (oe-g); mouna.ameggouz6@gmail.com (ma); benzeid_hanane@yahoo.fr (hb); doukkali73@gmail.com (ad) 2department of drug sciences, laboratory of medicinal chemistry, faculty of medicine and pharmacy, mohammed v university in rabat, morocco; a.zahidi@um5r.ac.ma (az) 3laboratory of human pathologies biology, faculty of sciences, mohammed v university in rabat, bp 1014, rabat, morocco 4department of pharmacy, abdul wali khan university mardan, mardan 23200, pakistan; ibrahimshah@awkum.edu.pk (sis) 5college of medical sciences, azal university for human development, amran 447, yemen; yworafi@yahoo.com (ymaw) 6college of pharmacy, university of science and technology of fujairah, fujairah 2202, united arab emirates 7school of medical and life sciences, sunway university, bandar sunway 47500, selangor, malaysia; longchiauming@gmail.com (lcm) *corresponding author: abdelhakim bouyahya; laboratory of human pathologies biology, faculty of sciences, mohammed v university in rabat, bp 1014, rabat, morocco; a.bouyahya@um5r.ac.ma (ab) received: 14 january 2023; received in revised form: 24 march 2023; accepted: 13 april 2023; available online: 2 may 2023 abstract: in morocco, traditional medicine utilizes many toxic plants for cancer treatment, despite a lack of scientific evidence supporting their effectiveness. further research may be able to explore and discover the potential therapeutic effects of these plants' bioactive molecules with antioxidant and anticancer properties. based on our review, we have determined that the 13 plants under examination possess various pharmacological and biological activities due to their diverse phytochemical composition. despite their toxicity, these plants have a history of traditional use in morocco for treating multiple diseases. further research, including preclinical and clinical trials, should be conducted to investigate the potential therapeutic benefits of these plants. moroccan cuisine commonly incorporates gruels, herbal drinks, and spicy beverages, which possess significant health benefits, including chemo-preventive properties and natural inhibitors against certain infections. mailto:mouna.ameggouz6@gmail.com mailto:a.bouyahya@um5r.ac.ma pmmb 2023, 6, 1; a0000328 2 of 48 these properties may aid in reducing the incidence of cancer and potentially have therapeutic effects in various human pathologies when consumed in appropriate amounts and in combination with a healthy lifestyle. keywords: toxic plants; anticancer activity; traditional medicine, morocco. 1. introduction toxic and poisonous plants are characterized by their ability to accumulate one or more toxic chemical compounds. these compounds may include alkaloids, terpenes, phenolic compounds, and proteins. these toxic substances can be present in specific parts of the plant or the entire organism[1]. from a toxicological perspective, the toxins mentioned above have the potential to impact the central nervous systems of both humans and animals, resulting in chronic, acute, and occasionally fatal intoxications. alkaloids, which constitute approximately 45–47% of the total toxins found in most plant families, is considered to be the most prevalent toxic compound. terpenes, representing 27–31% of the toxins, are the second most prevalent. phenolic compounds, accounting for 10–12%, are the third most prevalent. the remaining toxins, including proteins, amino acid derivatives, thioglucosides, oxalic acid, and nitrates, constitute 15–17%[2]. toxins can be present in various plant parts or the entire organism. a study of 13 organs of plants revealed that the whole plant, seeds, leaves, latex, and fruits could accumulate large quantities of toxins. the percentage of toxicity among these different parts indicates that the entire plant accounts for 27.45% of cases. seeds and leaves occupy the second position with respective percentages of 13.7% and 15%. latex and fruits show rates of 11.7% and 11%. the remaining toxic parts, including the root, essential oil, rhizome, bulb, flower, pollen, stem, and resin, are represented by a cumulative rate of 24.83%. secondary metabolism in plants refers to a series of biochemical reactions or transformations that occur within the plant and can contribute to the accumulation of toxins[2,3]. medicinal plants have been found to possess various biological properties with potential therapeutic effects against diseases such as microbial infections, urinary lithiasis, cancer, and other oxidative stress-related diseases[4–6]. the pharmacological properties of medicinal plants, such as cancer chemoprevention and cytotoxic effects, are believed to be attributed to various phytochemicals, including pmmb 2023, 6, 1; a0000328 3 of 48 flavonoids, alkaloids, and terpenes in the plant species[7,8]. therefore, it is vital to screen some commonly used toxic plants in morocco to identify and isolate the bioactive molecules that may be responsible for their traditionally reported therapeutic effects[9–11]. this review aims to highlight toxic plants with the reported anticancer activity used in morocco's traditional medicine to treat various diseases. to achieve this objective, 13 toxic plants with potential anticancer properties were selected from the traditional moroccan pharmacopeia and investigated for their use in diverse therapeutic pathways and the treatment of various pathologies in morocco. a summary of the phytochemical composition, anticancer activity, toxicological properties, and the traditional use of the included plants is presented in supplementary table s1. 2. the toxic anticancer plants 2.1. geographical location of aristolochia longa (a. longa) a. longa, or turmeric, is a medicinal plant from the aristolochiaceae family. it is widely distributed in asia, africa, and north and south america[12]. this plant is commonly found in tropical, subtropical, and mediterranean regions[13]. 2.2. phytochemical composition according to a study by m. dhouiouia et al., the essential oil of a. longa is characterized by a high content of oxygenated sesquiterpenes (50.2–81.1%), followed by oxygenated monoterpenes (5.9–28.0%), sesquiterpene hydrocarbons (0.7–18.4%), and monoterpene hydrocarbons (0.0–0.8%). regardless of the season, the significant component of the essential oil was found to be maaliol. reciprocal trends were observed for aromadendrene and its oxygenated derivatives[14]. another study by b. benarba et al. reported the presence of polyphenols, flavonoids, tannins, heterosides, carbohydrates, and saponins but did not detect the presence of alkaloids and coumarins[15]. 2.3. anticancer activity a. longa has been shown to exert antitumor activity through its immunostimulatory effects, induction of an inflammatory response, and high cytotoxicity, resulting in the development of tissue necrosis. according to a study by g. benzakour et al., treatment with a. longa extract was found to amplify 4-nitroquinoline1-oxide (4nqo)-induced hyperplasia compared to controls. many mononuclear cells were observed in filtrates, lymphoid follicles, and lymph nodes in animals treated with a. longa. additionally, many pmmb 2023, 6, 1; a0000328 4 of 48 eosinophils were observed in the different tissues examined. the inflammatory reaction and cytotoxicity mechanisms can be explained by the release of reactive oxygen species (ros) by activated eosinophils, which then leads to the catalyzation of the reduction of oxygen to superoxide by nicotinamide adenine dinucleotide phosphate oxidase (nadph)[16]. a study by b. benarba et al. investigated the effects of an aqueous extract of a. longa roots on cell viability in vitro by incubating human breast epithelial cell line hbl100 and mda-mb-231 breast cancer cells with different concentrations of the aqueous extract. cell viability was determined after 48h and 72h using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium (mtt) bromide assay. the results showed that both cell lines were inhibited dose-dependent by the aqueous extract of a. longa after 72h of incubation. the concentration of 500μg/ml of aqueous extract of a. longa induced cell death in 91.99% and 96.97% of hbl 100 and mda-mb-231 cells, respectively[15]. 2.4. toxicological properties a study by g. benzakour et al. found that the aqueous extract of a. longa l. at a dose of 1.25 g/kg did not produce toxic effects in mice and did not induce histopathological changes in kidney, liver, and intestine tissues. however, at a dose of 2.5 g/kg, it produced toxic effects in mice after six weeks of treatment by oral gavage, such as atypical locomotion, anorexia, asthenia, ataxia, diarrhea, and increased urination. these symptoms were observed immediately after oral administration of the extract and persisted until the end of the experiment[16]. another study by cherif h.s et al. found subacute oral toxicity of the aqueous extract of a. longa l. (1.5 g/kg) resulted in a slight decrease in body weight and diarrhea in treated mice. however, more severe toxic effects, such as diarrhea, fatigue, and weight loss, were observed in the 2.5 g/kg and 3.5 g/kg dose groups[17]. 2.5. traditional use the species a. longa, known as "barraztam" locally, is frequently used in moroccan traditional medicine. traditional healers often prepare a small amount of rhizome powder mixed with honey or salted butter to treat upper respiratory infections and abdominal pain[18]. currently, the a. longa roots are commonly used to treat digestive disorders, constipation, and aortic palpitations (bûmezwi) in traditional medicine. it is also used in marrakech to treat skin conditions, particularly mycosis[19]. the species a. longa, known as "barraztam" locally, is frequently used in moroccan traditional medicine. traditional pmmb 2023, 6, 1; a0000328 5 of 48 healers often prepare a small amount of rhizome powder mixed with honey or salted butter to treat upper respiratory infections and abdominal pain[18]. currently, the a. longa roots are commonly used to treat digestive disorders, constipation, and aortic palpitations (bûmezwi) in traditional medicine. it is also used in marrakech to treat skin conditions, particularly mycosis[19]. 3. artemisia herba-alba (a. herba-alba) 3.1. geographical location a. herba-alba, also known as artemisia herba-alba, is a species of the asteraceae family native to north africa, ranging from morocco to egypt. the plant is a shrubby undergrowth found in various habitats, including plateaus, steppe areas, and the sahara. a. herba-alba typically forms clumps of 0.3 to 0.8 meters and is a salty and whitish appearance. its leaves are finely divided and give off a strong aroma. the yellow flowers are small[20,21]. 3.2. phytochemical composition a. herba-alba contains diverse secondary metabolites, the most prominent being sesquiterpene lactones[22] ; esquiterpene lactones are a prevalent class of natural products found in artemisia species and are considered to be responsible for the medicinal and pharmaceutical importance of these plants. different structural types of sesquiterpene lactones have been identified in the aerial parts of a. herba alba[23] (figure 1). a. herba-alba has been found to contain a wide range of flavonoids, which vary in structure from common flavones and flavonol glycosides to highly methylated flavonoids. some studies on a. herba-alba leaves collected from sinai (egypt) have resulted in the isolation and identification of a total of eight oand c-glycoside flavonoids[24,25]. additionally, phenolic compounds and waxes of chlorogenic acid have also been observed in a. herba-alba. a chemical study of 49 moroccan drugs was performed by esr (electron spin resonance) spectroscopy, which identified the presence of these compounds in the plant species[26]. pmmb 2023, 6, 1; a0000328 6 of 48 figure 1. some sesquiterpene lactones from a. herba-alba. an investigation of the antiulcerogenic properties of a. herba-alba led to the isolation of eight polyphenols and their related constituents. these include chlorogenic acid, 4,5-o-dicaffeoylquinic acid, isofraxidine 7-o-β-d-glucopyranoside, 4-o-β-dglucopyranosylcaffeic acid, rutin, schaftoside, isoschaftoside, and vicenin-2. additionally, a study of the components of the wax of a. herba-alba, obtained by extracting the dry plant with ether, revealed that it contains 32.1% saturated acids, 23.2% hydrocarbons, 27.1% esters, and 16.96% saturated alcohols (figure 2)[27]. the essential oil of a. herba-alba mainly comprises oxygenated monoterpenoids, such as 1,8-cineole, chrysanthenone, chrysanthenol (and its acetate), α/β-thujones, and camphor[28]. r=come, herbolide a herbolide b herbolide c herbolide d herbolide e herbolide f ch3 oh h h o o ch3 ch2 herbolide g dihydroreynosinvachanic acid pmmb 2023, 6, 1; a0000328 7 of 48 figure 2. some flavonoids of artemisia herba-alba. 3.3. anticancer activity an evaluation of the cytotoxicity of the hydroalcoholic extract (methanol/water; 3/1) of a. herba-alba was conducted against several cell types, including rt112 cells, hep2 cells, k562 cells, and pbmc (peripheral blood mononuclear cells) cultured with pha (phytohemagglutinin). the results indicated that all extracts exhibited dose-dependent cytotoxic activity, suggesting that a. herba-alba extracts target carcinoma cell lines and induce their destruction. this type of response has been previously observed for the natural phenolic compound thymol, which induces dose-dependent cytotoxicity on hl-60 cells. these findings highlight the potential of a. herba-alba as a source of new chemopreventive agents against cancer progression[29]. 3.4. toxicological properties a study on the effects of a. herba-alba on maternal organ weights and embryo weights in female rats showed a slight but non-significant reduction in body and uterine o o oh oh oh oh o o oh oh oh oh oh o o oh oh o o oh oh oh oh oh oh oh oh schaftoside isoschaftoside o o o oh oh o ch3 ch3 och3 oh o o oh oh och3 cirsilineol hispidulin oho o oh oh oh o o oh oh oh o o oh oh oh oh oho o o oh oh o oh oh oh oh ch3 quercétine-3-rutinoside pultetin-3-glucoside pmmb 2023, 6, 1; a0000328 8 of 48 weights. however, compared to controls, a statistically significant decrease in the relative weight of the ovary and embryo was observed in the treated group[30]. 3.5. traditional use the traditional uses of a. herba-alba in moroccan medicine includes being prescribed as a venom-repellent, emmenagogue, stomachic, intestinal antiseptic, tonic, depurative, cholagogue, and antidiabetic[31]. 4. calotropis procera (c. procera) 4.1. geographical location c. procera is a species of flowering plant in the apocynaceae family, native to north africa, tropical africa, western asia, south asia, and indochina. this species, known as sahara-sindiana, is frequently found in the central and southern sahara and extends to moroccan western sahara[32]. 4.2. phytochemical composition researchers have reported that c. procera has various bioactive compounds in the aerial parts of the plant, including 3-o-rutinoside, kaempferol, isorhamnetin, and flavonoid glycoside 5-hydroxy-3,7-dimethoxyflavone-40-ob-glucopyranoside. the latex of c. procera is particularly rich in bioactive compounds such as cardenolides, proteolytic enzymes, alkaloids, and carbohydrates[33]. c. procera contains cardioactive glycosides, calactin, calotropain, calotropagenin, proceroside, syriogenin, calotoxin, uscharin, uzarigenin, uscharidin, voruscharin, tannins, flavonoids, sterols, and triterpenes[34]. other compounds found include benzoylisolineolon and benzoyllineolone[35], procesterol, a steroidal hydroxy ketone[36], flavonol glycosides[37], organic carbonate with stigmasterol and sitosterol[38]. 4.3. anticancer activity ibrahim et al. conducted a study to evaluate the n-buoh fraction of the root bark of c. procera against three cancer cell lines. seven novel oxypregnane oligoglycosides, calotroposides h-n (1–7; figure 3), were isolated, and their structures were determined using spectroscopic data. the in vitro growth inhibitory activity of these compounds was evaluated against a549 nsclc (deutsche sammlung von mikroorganismen und zellkulturen, dsmz; code acc107), u373 gbm (european collection of cell culture, pmmb 2023, 6, 1; a0000328 9 of 48 ecacc; code 89081403), and pc-3 (dsmz; code acc465) cell lines using the mtt colorimetric assay. the study showed that compounds 4 and 6 were the most active against these cancer cell lines. a significant difference in growth inhibitory activity was observed among the three cell lines tested[39]. 1 2 3 4 pmmb 2023, 6, 1; a0000328 10 of 48 5 6 7 figure 3. structures of the isolated compounds 1–7. 4.4. toxicological properties in a subacute toxicity study, the administration of c. procera extract in animal models did not result in mortality or significant behavioral changes over 28 days. however, a slight decrease in body weight and morphological changes in some organs were observed in treated groups. these changes could result from fat accumulation or decreased appetite rather than toxicity from the extract. phytochemical compounds present in c. procera, such as cardenolides and alkaloids, have been known to have toxic effects. their accumulation over time may affect thrombopoiesis and adversely affect the spleen and liver function, as reported in previous studies[40,41,42]. 4.5. traditional use in morocco, the powder of dried leaves from c. procera is traditionally used as a vermifuge in low doses. however, it has a strong purgative effect when consumed in larger quantities, such as a small amount of latex mixed with semolina porridge. it is typically only used by individuals of solid constitution. the emetic and cathartic properties of latex, known to nomadic communities, are also utilized in treating acute intoxications. the latex and decoction of the bark are also commonly used in veterinary medicine to treat leprosy and scabies[43]. pmmb 2023, 6, 1; a0000328 11 of 48 5. cannabis sativa (c. sativa) 5.1. geographical location c. sativa, commonly known as cannabis, is a flowering plant species belonging to the cannabaceae family, native to central asia. it is widely distributed in egypt and western asia and has been introduced in europe, south america, and north america. in morocco, c. sativa is commonly cultivated in mountainous regions such as the rif, on forest soils rich in humus, and in areas with access to water points. cultivation of c. sativa in morocco requires significant irrigation and is commonly found in regions such as alhucema, tetouan, chaouen, taounate, larache, sefrou, fez, meknes, essaouira and ait ourirn[44]. 5.2. phytochemical composition cannabis (cannabis sativa, or hemp) has been used for multiple purposes (medicinal, recreational, seed oil, industrial fiber, etc.) for thousands of years. its psychoactive and physiologically active constituents, known as cannabinoids, are found in the flowers (and to a cannabis sativa, commonly known as hemp, has a long history of use for various purposes, including medicinal, recreational, and industrial applications. the plant's active compounds, known as cannabinoids, are primarily found in the flowers and, to a lesser extent, in the leaves, stems, and seeds. the most well-known cannabinoid is delta-9-tetrahydrocannabinol (δ9-thc), responsible for the plant's psychoactive effects. other non-psychoactive cannabinoids, such as cannabidiol (cbd), cannabichromene (cbc), and cannabigerol (cbg) have also been found to have medicinal properties. over 545 compounds have been identified in the cannabis plant, with research suggesting potential medical applications for treating various diseases. the first compound isolated from the plant was cannabinol in 1942, followed by cannabidiol in 1963, and delta-9-tetrahydrocannabinol in 1964 by raphael mechoulam and gaoni [45,46,47,48]. 5.3. anticancer activity cannabis (cannabis sativa, or hemp) has been used for various purposes, including medicinal, recreational, seed oil, and industrial fiber, for thousands of years. its active compounds, known as cannabinoids, are primarily found in the plant's flowers and, to a lesser extent, in the leaves, stems, and seeds. δ9-tetrahydrocannabinol (thc) is the main psychoactive cannabinoid, while cannabidiol (cbd), cannabichromene (cbc), and cannabigerol (cbg) are non-psychoactive compounds with medicinal properties. the pmmb 2023, 6, 1; a0000328 12 of 48 cannabis plant also contains other natural compounds, with 545 constituents reported to date. endocannabinoid receptors, cb1 and cb2, are expressed in various tissues and breast cancer cells[39]. mcf-7 (michigan cancer foundation-7) breast cancer cells are highly metastatic estrogen and progesterone receptor-positive cells that express the cb1 receptor, similar to other breast cancer cells and tissues. cannabis-based medicines, as modulators of corticospinal excitability, may provide new therapy options for managing cancer pain, not only for those who do not respond to standard therapies, but also for patients who prefer to try cannabis as a first-line treatment[49,50,51]. the potential of cannabis sativa (c. sativa) extracts as anti-cancer agents were evaluated in a study using mcf-7 breast cancer cells. the cells were exposed to dichloromethane (dcm) extracts of c. sativa samples for 24 and 48 hours. the results indicated that at 24 hours, the exposure to the dcm extracts showed no significant cytotoxicity in the mcf-7 cells. however, all eight extracts from the c. sativa samples showed ic50 values of 65 to 100 μg/ml at 48 hours of exposure. this suggests that these extracts may have moderate cytotoxicity as per the criteria set by the national cancer institute (nci). further study is needed to determine the potential of c. sativa extracts as anti-cancer agents and understand the mechanisms of action via regulation of cb1 receptors and vegf expression in mcf-7 cells[52]. 5.4. toxicological properties the primary psychoactive component of cannabis, tetrahydrocannabinol (thc), interacts with the body's endocannabinoid system, composed of cellular receptors, endogenous ligands, and various intracellular messengers that are involved in the synthesis, degradation, control, and regulation of this system. the endocannabinoid system plays a significant role in modulating the function of the nervous system. according to the scientific literature, the maximum daily dose of thc for humans is around 1mg of thc from 24.5g of cannabis[53]. cannabis (cannabis sativa), commonly known as marijuana, contains various psychoactive and physiologically active compounds called cannabinoids. the primary psychoactive component of cannabis, tetrahydrocannabinol (thc), interacts with the body's endocannabinoid system, which regulates various physiological processes. however, accidental ingestion of cannabis, particularly by children, can lead to severe psychomotor disturbances, such as ataxia, drowsiness, and coma[54]. additionally, cannabis sold in the illegal market may contain potentially toxic pmmb 2023, 6, 1; a0000328 13 of 48 contaminants, such as fecal bacteria, yeast, pesticide residues, and heavy metals, which can lead to respiratory and neurological diseases when inhaled. inhalation of bacteria and yeast can also induce asthma attacks[55,56]. furthermore, some sellers may cut cannabis with other products to increase profit and appeal, potentially leading to dangerous or misleading results[57]. 5.5. traditional use in morocco, c. sativa (indian hemp) has been traditionally used as a sedative for severe pain. in the past, it was also commonly used by surgeons as a sedative and anesthetic before surgical procedures, often in combination with other plants such as hemp and mandrake[50]. 6. chenopodium ambrosioides (c. ambrosioides) 6.1. geographical location dysphania ambrosioides, also known as wormwood, jesuit tea, or mexican tea, is a short-lived annual or perennial herb native to central and south america and mexico. in addition to its native range, it is cultivated in temperate and warm subtropical regions of europe and the united states, such as missouri, new england, and the eastern united states. this plant is also widely distributed in africa, particularly morocco, nigeria, senegal, ghana, and cameroon[58,59]. 6.2. phytochemical composition the essential oil of dysphania ambrosioides, also known as wormwood, was analyzed by adebayo et al. and found to be primarily composed of monoterpene hydrocarbons (76.8%), with the most abundant compounds being α-terpinene (53.4%) and p-cymene (21.1%). additionally, trace amounts of other compounds were identified, including limonene (1.4%), γ-terpinene (0.8%), and carvacrol (1.1%). the presence of carvacrol, an anthelmintic compound, is noteworthy in the context of the traditional use of wormwood for treating parasitic infections. the major phytochemical compounds in dysphania ambrosioides, known as wormwood, are flavonoids and phenolic acids. flavonoids, specifically quercetin and kaempferol derivatives, constitute the majority of the compounds present in the plant, with quercetin 3-o-rutinoside and kaempferol dirhamnoside-o-pentoside being the most abundant. additionally, phenolic acids, such as trans-p-coumaric acid, are present in the pmmb 2023, 6, 1; a0000328 14 of 48 plant. the plant also contains various fatty acids, with polyunsaturated fatty acids, alphalinolenic and linoleic acids, being the most prevalent. these compounds have been previously shown to have potential health benefits, particularly in preventing cancerous diseases[59]. 6.3. anticancer activity the cytotoxic potential of c. ambrosioides was evaluated by testing its essential oil, ethanolic extract, and fractions prepared by liquid/liquid fractionation with dichloromethane, ethyl acetate, and butanol in tumor cell lines (k562 (myeloid leukemia), nalm6 b15 (acute b lymphocytic leukemia), and raji (burkitt's lymphoma)). the results showed that the essential oil displayed significant cytotoxicity against raji cells (ic50 = 1.0 g/ml), and the dichloromethane fraction and ethanol extract demonstrated cytotoxicity against k562 cells (ic50 = 34.0 g/ml and 47.0 g/ml respectively) [60]. the activity of the essential oil is thought to be attributed to the high concentration of ascaridol, as the other primary compound, p-cymene, has been shown to have low cytotoxic activity. the study concluded that ascaridol is responsible for the cytotoxicity observed in the analyzed cells, and that the essential oil and fractions of c. ambrosioides possess cytotoxic potential[61]. 6.4. toxicological properties chronic toxicity studies in albino rats have shown that high doses of c. ambrosioides, ranging from 12.31 to 31.89 g/kg, administered over 42 days, can lead to pathological symptoms such as metaplastic alterations in the mucosal surface of the stomach, congestion of the lungs, and necrosis of the kidney tubules[62]. an overdose of c. ambrosioides oil has resulted in fatalities in humans and rats[63]. the toxic effects are attributed to ascaridol, a compound isolated from the c. ambrosioides oil, which has various biological activities but also exhibits toxic or genotoxic effects[63]. 6.5. traditional use the seeds of dysphania ambrosioides, also known as c. ambrosioides, are traditionally used as a vermifuge in the marrakech region of morocco. however, the whole plant is more commonly used as an infusion or fresh juice to treat gastrointestinal ailments, typhoid, and dysentery in both adults and children and promote lactation. in the sale region pmmb 2023, 6, 1; a0000328 15 of 48 of morocco, the fresh plant of d. ambrosioides is applied topically to treat oral abscesses, ulcerations, and purulent wounds[64]. 7. conium maculatum (c. maculatum) 7.1. geographical location c. maculatum, a member of the apiaceae family, is widely recognized as one of the most toxic plant species. it is native to europe, asia, north africa, north america, australia, and new zealand[65]. additionally, it is present in nearly all south american countries[66]. despite its toxicity, it is not considered a significant problem in animal production in brazil[67]. however, it is well known in argentina as a toxic plant, with reports of acute and teratogenic toxicity[68]. this species is widespread in the country but is particularly prevalent in the pampa region, which is also an area of high animal production. in morocco, it is commonly found in humid areas near buildings[69]. 7.2. phytochemical composition chemical analysis of cicuta maculatum (hemlock) has revealed that all plant tissues contain alkaloids, with the fruits having the highest concentration at up to 1% (w/w). the amount and ratio of alkaloids present can vary depending on the variety of the plant, the ecological conditions, and the stage of phenological development. eight known piperidine alkaloids are present in hemlock, including coniine, n-methylconiine, conhydrin, pseudoconhydrine, and gamma-coniceine. coniine is considered to be eight times more toxic than γ-coniceine . in addition to alkaloids, c. maculatum contains flavonoids, coumarins, polyacetylenes, vitamins, essential oils, and non-volatile oils[70]. chemical analysis of cicuta maculatum (hemlock) has revealed that all plant tissues contain alkaloids, with the fruits having the highest concentration at up to 1% (w/w). the amount and ratio of alkaloids present can vary depending on the variety of the plant, the ecological conditions, and the stage of phenological development[70]. eight known piperidine alkaloids are present in hemlock, including coniine, nmethylconiine, conhydrin, pseudo-conhydrine, and gamma-coniceine. coniine is considered to be eight times more toxic than γ-coniceine (figure 4)[71]. in addition to alkaloids, c. maculatum also contains flavonoids, coumarins, polyacetylenes, vitamins, essential oils, and non-volatile oils[72]. pmmb 2023, 6, 1; a0000328 16 of 48 figure 4. some piperidine alkaloids present in conium maculatum [73]. 7.3. anticancer activity in vitro studies have demonstrated the anticancer potential of c. maculatum extracts against cancer cells. conium, a piperidine alkaloid found in the plant, has been shown to interact with dna and impede the proliferation process and cell cycle. treatment with conium resulted in decreased viability and colony formation of hela cells, as well as an accumulation of reactive oxygen species (ros) and depolarization of the mitochondrial membrane. additionally, conium treatment led to morphological changes in hela cells, fragmentation, and modulation of proteins related to cell proliferation and apoptosis. the mechanism of apoptosis is believed to be related to the upregulation and downregulation of specific proteins. these findings suggest that c. maculatum extract may have the potential as an anticancer agent[74]. conium upregulated reactive oxygen species (ros) activity by stopping cell division early in conium, demonstrating its growth inhibitory effect on cancer cells. incidentally, proliferation is mainly promoted by the protein pakt/akt[75]. nh ch3 n ch3 coniine conicei ne n ch3 ch3 nh ch3 oh n-methylconiine conhydrine nh ch3 oh nh ch3 o pseudoconhydrine conhydrinone pmmb 2023, 6, 1; a0000328 17 of 48 7.4. toxicological properties hemlock (c. maculatum) is a highly toxic plant due to piperidine alkaloids in all plant parts, including leaves, flowers, fruits, seeds, and roots[76]. human poisoning has been reported following ingesting the plant's leaves, roots, or seeds. symptoms of c. maculatum poisoning in humans include rapid loss of lower limb strength (muscle weakness), ataxia, staggering, and tremors. as the effects intensify, there is a loss of control of the upper limbs, total paralysis of legs and arms, loss of ability to chew and loss of sensation, fixed pupils, slow and weak pulse (later becoming fast), rapid breathing, abundant salivation, frequent urination, nausea, convulsions, and a drop in body temperature. ultimately, death occurs due to paralysis of respiration and asphyxia, with the intellect remaining clear until death. acute renal failure appears to be a symptom specific to human intoxication, compared to animal intoxication[76]. the signs of acute c. maculatum intoxication are similar across various animal species. cattle, sheep, and swine exhibit symptoms such as muscle weakness, incoordination, tremors, mydriasis, pressing on the metacarpophalangeal joints, excessive salivation, cyanotic membranes, and cold limbs. the onset of these symptoms is followed by an initial stimulation of the central nervous system, followed by depression, and rapid and shallow breathing, which subsequently transitions to slow and labored breathing, dilated pupils, frequent urination and defecation, coma, and ultimately death due to respiratory paralysis[77]. 7.5. traditional use the medicinal uses of cicuta maculatum, commonly known as water hemlock, are rare today. however, it was traditionally used in low doses to treat neuralgia, sciatica, and rheumatism. the plant is primarily used as an abortifacient as vaginal tampons in the regions of casablanca and rabat in morocco. it is also known for its toxic properties. in the rabat region, fumigations made from the root of water hemlock were used to treat venomous stings and bites[78]. 8. lawsonia inermis (l. inermis) 8.1. geographical location lawsonia inermis, commonly known as henna or mehendi, is a small tree that is primarily cultivated for its leaves. however, the stem bark, roots, flowers, and seeds of the plant have also been utilized in traditional medicine, and it is commonly found in tropical pmmb 2023, 6, 1; a0000328 18 of 48 and subtropical regions. the plant has a long history of use in traditional herbal medicine, particularly in ayurvedic medicine in india, as described in ancient texts. the plant is native to north africa and south-west asia[79]. it is worth noting that the plant is native to the regions of north africa and south-west asia[80]. 8.2. phytochemical composition 2-hydroxy-1, 4-naphthoquinone (hnq; lawsone) is the primary natural pigment in henna leaves, comprising 1.0 to 1.4% of the leaf's composition. other related compounds present in the leaves include 1,4 dihydroxynaphthalene, 1,4-naphthoquinone, 1,2dihydroxyglucoyloxynaphthalene, and 2-hydroxy-1,4 diglucosyloxynaphthalene, in addition to flavonoids (such as luteolin, apigenin, and their glycosides), coumarins (such as esculetin, fraxetin, and scopoletin) and steroids (such as β sitosterol)[81]. additionally, lawsonia inermis leaves have been found to contain a variety of other compounds, including tannin, gallic acid, glucose, mannitol, fat, resin, and mucilage[82]. the plant's bark contains various compounds, including naphthoquinone, isoplumbagin, triterpenoids, hennadiol, and aliphatics (such as 3-methylnonacosan-1-ol). steam distillation of the flowers has yielded an essential oil with a concentration of 0.02%, rich in ionones, with β-ionones predominating[74]. the essential oil primarily comprises aliphatic esters, with relatively few terpenoids present. the major components of the oil include ethyl hexadecanoate (24%), (e)-methyl cinnamate (11.4%), and methyl linoleate (4.1%). other terpenoids present in significant quantities include isocaryophyllene (8.1%), (e)-β-ionone (5.8%), nerylacetone (4.4%), and α-terpineol (3.9%). the leaf oil compositionfound to differ significantly from that of the plant as a whole[82] and the flowers precisely[83]. the β-ionone compound was quantified at 48.6% and 2.5% concentrations in the yellow and red flower essential oils, respectively. however, it was found to be completely absent in the essential oil derived from the entire plant. despite this, the violet odor compound (la β-ionone) was found to occur at a relatively low concentration (5.8%) in the leaf oil. additionally, linalool, which constituted a significant portion of the yellow and red flower oils (19.8% and 12.7%), was detected only in limited amounts (0.7%)[82]. 8.3. anticancer activity the plant lawsonia inermis has a long history of usage as a traditional medicine for cancer treatment. additionally, it has been discovered to have potent anticancer pmmb 2023, 6, 1; a0000328 19 of 48 properties[84]. the compound lawsone found in l.inermis has been demonstrated to possess cytotoxic properties[85]. a study utilizing a crude dichloromethane extract of l. inermis leaves found it to exhibit significant cytotoxic activity against the human breast cancer cell line mcf-7 and the human liver cancer cell line hepg2[86]. an ethanolic extract of l. inermis also increased the life span of mice bearing dalton's lymphoma as cited tumors[87]. qian lian et al. isolated new compounds of bicoumarin, biflavonoid, and biquinone, as well as twelve other known compounds from 4 to 15 (as shown in figure 5), from the dichloromethane (dcm) extract of l. inermis flowers through a series of chromatographic techniques including silica gel, sephadex lh-20, rp-18, and semipreparative hplc. the inhibitory effects of these compounds on the mcf-7, hela, hct-116, and ht29 cancer cell lines were determined using the mtt (tetrazolium salt) assay. compounds 3, 4, and 5 (as shown in figure 5), which can be structurally classified as 1,4naphthoquinones, exhibited significant inhibitory activity. in particular, compounds 3 and 5 showed more potent inhibitory activities than the chemotherapy drug 5-fu. qian lian et al. isolated new compounds of bicoumarin, biflavonoid, and biquinone, as well as twelve other known compounds from 4 to 15 (figure 5), from the dichloromethane (dcm) extract of l. inermis flowers through a series of chromatographic techniques including silica gel, sephadex lh-20, rp-18, and semipreparative hplc[88]. the inhibitory effects of these compounds on the mcf-7, hela, hct-116, and ht29 cancer cell lines were determined using the mtt (tetrazolium salt) assay[88]. compounds 3, 4, and 5 (as shown in figure 5), which can be structurally classified as 1,4naphthoquinones, exhibited significant inhibitory activity. in particular, compounds 3 and 5 showed more potent inhibitory activities than the chemotherapy drug 5-fu[89]. 1 2 pmmb 2023, 6, 1; a0000328 20 of 48 3 4 5 6 r1=oh r2=h 7 r1=oh r2=oh 8 r1=oh r2=oh 9 10 11 pmmb 2023, 6, 1; a0000328 21 of 48 figure 5. structures of compounds 1–15. 8.4. toxicological properties henna is a substance with little or no toxicity, mainly when applied to the skin. given its widespread cosmetic use worldwide over the centuries, and the relatively few allergic reactions reported in the literature, henna is generally considered to be a weak sensitizing agent. studies have shown that an aqueous extract of l. inermis root is slightly toxic when administered orally, causing delayed toxicity symptoms such as paralysis, total body collapse, weakness, sluggishness, and loss of appetite without resulting in death. lawsone, which is considered to be the active component of henna, has been identified as 12 r= (ch2)15ch3 13 14 15 pmmb 2023, 6, 1; a0000328 22 of 48 responsible for its haemotoxicity[89]. when administered to rats, lawsone has been shown to cause decreased hematocrit, reduced hemoglobin level, and an increased spleen weight/liver weight ratio. based on these observations, it is suggested that lawsone is responsible for the observed physiological changes and has been shown to induce oxidative stress related to hemolytic anemia when administered to rats[90]. 8.5. traditional use henna is used in traditional moroccan medicine as a component in infusions to treat conditions such as kidney stones, diarrhea, and ulcers. additionally, it is commonly used in poultices to treat skin conditions such as eczema, mycosis, boils, abscesses, and chapped skin. it is also considered to have astringent, antiseptic, and wound-healing properties and is used to treat umbilical wounds of newborns. henna also treats sprains, dislocations, stretched ligaments, and fractures[91]. 9. nerium oleander (n. oleander) 9.1. geographical location n. oleander, also known as defla in the maghreb and the arab world, is a shrub commonly found in the mediterranean region, particularly along waterways in morocco. it is native or naturalized in a wide geographical area, including regions from mauritania, portugal eastward, through the mediterranean region and the sahara (occurring only sporadically), to the arabian peninsula, the east coast of the united states, south asia, and as far south as yunnan in china[92]. 9.2. phytochemical composition the most abundant components found in the flower essential oil of nerium oleander were neriine (22.56%), followed by digitoxigenin (11.25%), amorphane (8.11%), 1,8cineole (6.58%), α-pinene (5.54%), calarene (5.12%), limonene (5.01%), β-phellandrene (4.84%), terpinene-4-ol (3.98%), sabinene (3.22%), isoledene (2.94%), 3-carene (2.56%), humulene (2.29%), β-pinene (2.01%) and cymen-8-ol (1.67%). the well-known effects of n. oleander are primarily attributed to two glycosides, neriine, and oleandrin alkaloid, which have a cardio-stimulant action[93]. other glycosides such as gentiobiosyloleandrin, gentiobiosylnerigoside and gentiobiosylbeaumontoside extracted from the leaves, have also been found to have diuretic effects and to be effective in treating dermatitis and bruises[94]. pmmb 2023, 6, 1; a0000328 23 of 48 furthermore, the sap of nerium oleander is known to be rich in minerals[95], and αtocopherol. the plant also contains weakly active cardenolides (such as uzarigenin heterosides), inactive cardenolides (such as adynergenin heteroside and digitalose), triterpenoids, resin, tannins, glucose, kerosene, ursolic acid, and vitamin c. the seeds contain glucosides, including oleandrin, odorosides, and adigoside. the plant's bark also includes glucosides, including rosaginoside, nerioside and corteneroside, and the roots contain steroids[96]. 9.3. anticancer activity nerium oleander is commonly used in traditional medicine as an anti-cancer agent. the observed anticancer activities of isolated cardenolides and crude extracts support their use in traditional medicine. the monoglycosidic cardenolides are among the most potent anticancer substances found in the plant. a cold water extract of n. oleander leaves has been tested on more than 380 cancer patients since 1988 with promising results[97,98]. the observed cytotoxic effects are believed to be caused by the inhibition of the na+ / k+ atpase enzyme bound to the plasma membrane[99,100]. 9.4. toxicological properties all parts of nerium oleander contain cardiotonic glycosides whose action on the heart is similar to that of digitalis purpurea (foxglove). poisoning can occur after ingesting even small amounts of plant material. however, the toxic dose varies depending on several factors, such as the concentration of glycosides in the plant, the amount ingested, the age, and the individual's health condition. accidental exposures typically do not result in severe poisoning, as children are unlikely to consume large amounts due to the bitter taste of the leaves[101]. 9.5. traditional use in morocco, the roots of nerium oleander are traditionally used in fumigations to treat headaches, common colds, and diseases of the uterus. the plant stems are also used in traditional medicine to create points of fire for treating rheumatism, and articular and joint pains[102]. pmmb 2023, 6, 1; a0000328 24 of 48 10. nigella sativa (n. sativa) 10.1. geographical location nigella sativa l. (commonly known as black cumin or "habbet el-baraka," meaning "seed of divine grace"), belongs to the family ranunculaceae. it is native to the region of asia minor, including countries such as syria, turkey, saudi arabia, pakistan, and india. the plant is cultivated in the mediterranean region for its seeds, as well as for ornamental purposes. additionally, n. sativa l. is also widely cultivated in morocco[103,104,105]. 10.2. phytochemical composition houghton et al. demonstrated a diverse array of natural compounds in nigella sativa, including lipids, terpene derivatives, flavonoids, alkaloids, and saponins. additionally, n. sativa is an essential source of proteins and minerals[106]. n. sativa seeds have been found to contain 36-38% fixed oil, protein, alkaloids, saponin, and 0.4–2.5% essential oil[107]. the fixed oil primarily comprises unsaturated fatty acids, including arachidic and eicosadienoic acid[108]. the essential oil of n. sativa has been analyzed by brits and bucar using gc/ms. several components have been identified, with the main ones being thymoquinone (27.8%– 57.0%), ρ-cymene (7.1%–15.5%), carvacrol (5.8%–11.6%), t-anethole (0.25%–2.3%), 4terpineol (2.0%–6.6%) and longifolia (1.0%–8.0%). thymoquinone is reportedly readily dimerized form dithymoquinone [109,110]. four alkaloids have been identified as constituents of n. sativa seeds, including nigellicin and nigellidine (figure 6)[111,112]. figure 6. chemical structures of some major constituents of n. sativa seeds. pmmb 2023, 6, 1; a0000328 25 of 48 10.3. anticancer activity the anti-cancer properties of nigella sativa have been primarily attributed to its ability to exert potent antiproliferative, pro-apoptotic, antioxidant, antimutagenic, and antimetastatic effects. many of these anti-cancer activities have been linked to the plant's principal active compound, thymoquinone (tq). studies have shown that tq possesses antiproliferative, pro-apoptotic, antioxidant, antimutagenic, anti-angiogenic, and antimetastatic effects against various cancer cell lines[113,114]. tq is believed to mediate these anti-cancer effects by targeting multiple cellular pathways, including p53, nf-kb, pparg, stat3, mapk, and pi3k/akt signaling pathways[115,116]. in addition to thymoquinone (tq), other phytoconstituents of nigella sativa have also been shown to contribute to the plant's anti-cancer potential. for example, α-hederin, a pentacyclic triterpene saponin found in n. sativa seeds, has been demonstrated to possess practical anti-cancer effects in both in vitro and in vivo studies[117,118]. other phytoconstituents of n. sativa, such as thymol, thymohydroquinone, dithymoquinone, nigellimine-n oxide, nigellicin, nigellidin, and carvacrol, have also been shown to possess anti-cancer and cytotoxic properties. however, further research is needed to fully understand the mechanisms of action that mediate the anti-cancer effects of these phytoconstituents, as the molecular processes behind these effects are not yet fully understood[119,120]. 10.4. toxicological properties acute administration of high doses of nigella sativa seed extract (2g/kg or higher) has been found to cause hypoactivity and respiratory difficulties in animals. these high doses have been found to decrease levels of the antioxidant glutathione (gsh) in the liver, kidney, and heart and cause harm to the liver and kidney, as indicated by significant increases in plasma metabolites and enzymes[121]. however, when thymoquinone, one of the active compounds found in n. sativa, was included in the drinking water of mice at concentrations of up to 0.03% for 90 days, no signs of toxicity were observed, except for a significant decrease in plasma glucose concentrations[122]. toxicity studies in animals have revealed that nigella intake can alter blood parameters such as hemoglobin metabolism, white blood cell, and platelet levels. additionally, total black cumin oil has been found to inhibit certain pro-coagulant prostaglandins, potentially increasing the risk of bleeding. it is also reported that black cumin may have abortifacient properties during the first ten days of pregnancy, but this pmmb 2023, 6, 1; a0000328 26 of 48 effect has not been observed beyond this period. however, it is essential to note that these studies were performed on animals, and more research is needed to determine the potential effects of nigella sativa in humans[123]. 10.5. traditional use in morocco, the powder made from freshly ground nigella sativa seeds is commonly used for various medicinal purposes. these include inhalations for colds, flu, migraines, sinusitis, pulmonary conditions, and asthma. the powder is used as an ointment to treat various skin conditions, such as varicose veins, corns, vitiligo, scabs, hemiplegia, facial paralysis, and limb paralysis. the po wder is also applied to the teeth for relief from dental pain. at low doses, nigella sativa seed powder has traditionally been used as a galactagogue, warming agent, anti -nausea, strengthening agent, vermifuge, emmenagogue, antipyretic, and venom. however, it is essential to note that the medicinal properties of nigella sativa have not been extensively studied, and more research is needed to confirm the effectiveness of these traditional uses [124]. 11. marrubium vulgare (m. vulgare) 11.1. geographical location marrubium vulgare l. (white horehound) is a perennial flowering plant native to north africa, europe, and asia. it has been introduced to other regions, such as japan, southern africa, america, australia, and new zealand, and has been reported to be invasive in many of these territories. however, it is essential to note that invasive species can negatively impact native ecosystems and should be carefully monitored[125]. 11.2. phytochemical composition the yield of essential oil obtained from the aerial parts of marrubium vulgare l. through hydrodistillation was found to be 0.34%. the main chemical compounds identified in the oil were γ-eudesmol (11%), germacrene (10%), d-citronellyformate (10%), βcitronellol (8%), geranyl tiglate (7.1%), and geranyl formate (6.02%). other compounds present in smaller percentages include lendene (5.15%), cyclononasiloxane-octadecamethyl (4.3%), 1,8-cineole (3.75%), geraniol (3.70%), neryl acetate (3.41%), γ-cadinene (3.35%), and b-cubebene (3.30%)[126]. diterpenoids were the top class of compounds present in the aerial parts of m. vulgare[127]. flavonoids are an important class of compounds that are widely distributed in different parts of marrubium vulgare l[128]. trace amounts of alkaloids, ursolic acid (a pentacyclic triterpene) and steroids have also been reported in the aerial parts of m. pmmb 2023, 6, 1; a0000328 27 of 48 vulgare[128]. in 2010, a study reported the isolation of a few normal alkanes and four types of branched alkanes from the aerial parts of m. vulgare[129]. 11.3. anticancer activity a study by mehmet evren okur et al. found that the cytotoxic effects of the methanolic extract of marrubium vulgare l. at a dose of 1mg/ml on u87, ln229, and t98g glioblastoma multiforme (gbm) cell lines resulted in the viability of 69.9% and 71% for u87 and ln229 cells, respectively[130]. in addition, another study found that the essential oil of m. vulgare tested in vitro (using the mtt assay) was cytotoxic against a cervical cancer cell line, hela. the essential oil of m. vulgare was found to inhibit the proliferation of hela cells[131]. zied zarai et al. found that the ethanolic extract of marrubium vulgare l. showed cytotoxic effects by reducing the viability of melanoma (b16) and glioma (u251) cells in a dose-dependent manner[132]. additionally, the essential oil of m. vulgare was tested at different concentrations (3.91–3000 μg/ml) and was found to reduce the viability of hela cells significantly. the study reported that 250 μg/ml of the essential oil could destroy 27% of hela cells, and concentrations above 500 μg/ml destroyed all hela cells. at lower doses, the cells tolerated the oil, and the ic50 (the concentration at which 50% of cells are killed) was found to be 0.258 μg/ml[133]. 11.4. toxicological properties marrubium vulgare l. may contain toxic agents such as psoralen, 8methoxypsoralen, and 5-methoxypsoralen that are responsible for adverse reactions. a study by k. el morabite et al. found that using the plant for analgesic purposes could lead to dermal lesions similar to chemical burns, which could induce dermal-epidermal cleavage up to the formation of vesiculo-bubbles[134]. health risks have also been associated with the handling or ingesting of plants containing psoralen and xanthotoxin. studies in animals have shown th at the administration of psoralen, bergapten (5-methoxypsoralen), and xanthotoxin (8methoxypsoralen) in female rats may cause a reduction in ovarian follicular function and ovulation[135]. 11.5. traditional use in morocco, the decoction of marrubium vulgare l. is traditionally used as an pmmb 2023, 6, 1; a0000328 28 of 48 antidiabetic treatment, either alone or in combination with other plants such as fenugreek, white wormwood, white lupine, thyme, and rue. the juice of the fresh plant is also used for this purpose. additionally, the decoction is used as an antityphoid, antidiarrheal, febrifuge, diuretic, emmenagogue, anti-icteric, expectorant, tonic, and stimulant for bedridden patients. the plant is also commonly used in cataplasms applied to the forehead for fever and on abscesses and furuncles to promote healing[134]. 12. myristica fragrans (m. fragrans) 12.1. geographical location myristica fragrans houtt. is an evergreen tree native to the moluccas (or spice islands) in indonesia. it is widely cultivated in tropical regions, including guangdong and yunnan in china, taiwan, indonesia, malaysia, grenada in the caribbean, kerala in india, sri lanka, and south america[136]. 12.2. phytochemical composition the fresh pericarp, or rind, of the ripe fruit of myristica fragrans houtt. contains an acidic, astringent juice with a distinctive flavor. analysis of the composition of the fruit rind has revealed the presence of protein, fat, minerals, phosphorus, iron, and carotene[136]. additionally, the rind contains up to 14% pectin and 27% fiber[137]. phytochemical analysis of m. fragrans has revealed the presence of various compounds, including essential oil, which makes up about 10% of the seed[138]. a neolignan compound called dihydro-diisoeugenol has been isolated from the hexane and chloroform extract of the arils[139]. furthermore, five phenylpropanoids have been reported in the seed core of the plant, and dihydroguaiaretic acid has been isolated from the nutmeg mass[139,140]. the essential oil of myristica fragrans houtt. from india was subjected to gc-ms analysis, which revealed the presence of 49 compounds. the major constituents identified were sabinene (20.2%), terpinen-4-ol (12.1%), safrole (6.1–10.3%), alpha-pinene (9.7%), phellandrene (6.6%), and gamma-terpinene (5.9%)[141]. a study by m. pal et al. reported that terpinen-4-ol (15.0%), sabinene (13.1%), and gamma-terpinene (11.2%) were found to be the major constituents of the essential oil of m. fragrans from brazil[142]. additionally, the main volatile components of m. fragrans collected from the andaman and nicobar islands were sabinene (41.7%), alpha-pinene (9.4%), and alphapinene (7.3%), terpinene-4-ol (5.8%), limonene (3.7%), and myristicin (2.7%). it is pmmb 2023, 6, 1; a0000328 29 of 48 important to note that the composition of the essential oil of myristica fragrans may vary depending on factors such as geographic location, soil, climate conditions, and plant variety[143]. 12.3. anticancer activity studies have shown that nutmeg extracts from myristica fragrans can suppress the growth of human lymphoid leukemia cells, molt 4 b[144]. dihydroguaiaretic acid, a compound found in m. fragrans, has been shown to inhibit the growth of leukemia, colon cancer, and lung cancer cells in vitro[145]. myristicin, a component of the essential oil of m. fragrans, has been identified as a potential cancer chemopreventive agent[146]. additionally, the mass of m. fragrans has been found to protect against bone marrow genotoxicity in male swiss albino mice[147]. the animal model studies indicated the essential oil of m. fragrans may have chemopreventive effects against dimethylbenz(a)anthracene (dmba) papillomagenesis in mouse skin. they can modulate the formation of dna adducts by aflatoxin in vitro[148,149]. 12.4. toxicological properties toxicity studies have shown that consuming m. fragrans can lead to adverse effects such as weak pulse, hypothermia, delirium, dizziness, and nausea[150]. moteki h and colleagues have reported teratogenic effects in rat fetuses exposed to nutmeg. additionally, studies have found the formation of dna adducts in the livers of adult and fetal mice treated with nutmeg, mace, or myristicin extracts, the primary constituent of the spice nutmeg. safrole, a minor component of nutmeg, has also been found to produce dna adducts in the liver of mice[151]. 12.5. traditional use myristica fragrans, or nutmeg, is a perennial evergreen tree native to indonesia's moluccas (or spice islands). it is widely cultivated in tropical regions worldwide for its seeds, which contain essential oil and other phytochemicals. traditional uses of m. fragrans include treatment for digestion and sexual dysfunction, as well as for general weakness and respiratory conditions. in traditional medicine, the powdered seed is often mixed with honey and consumed orally to treat gynecological disorders[152]. pmmb 2023, 6, 1; a0000328 30 of 48 13. peganum harmala (p. harmala) 13.1. geographical location p. harmala l. is a perennial plant belonging to the family zygophyllaceae, native to the middle east, north africa, and southern europe. it is commonly found in steppe regions, highlands, and the sahara. the plant displays linear, alternate, and sessile leaves and white flowers with five oval petals and numerous yellow stamens. the plant is called "l-harmel" in the arab world[153]. 13.2. phytochemical composition the phytochemistry of p. harmala includes the presence of alkaloids, flavonoids, and anthraquinones[143]. the major beta-carboline alkaloids in p. harmala extracts include harmalin, harmine, harmalol, harmol, and tetrahydroharmine. the total alkaloid content of p. harmala ranges from 2-5%, with the highest concentrations found in the seeds and roots and lower levels in stems and leaves. notably, flowers do not contain significant levels of alkaloids. studies have shown that harmine and harmaline are present in dry seeds at 4.3% and 5.6% (w/w) respectively, harmalol at 0.6% and tetrahydroharmine at 0.1% (w/w). the roots contain harmine and harmol with 2.0% and 1.4% (w/w), respectively[154]. phytochemical analysis of p. harmala has revealed the presence of alkaloids, flavonoids, and anthraquinones. the major beta-carboline alkaloids in p. harmala extracts include harmalin, harmine, harmalol, harmol, and tetrahydro harmine. the total alkaloid content of p. harmala ranges from 2–5%, with the highest concentrations found in seeds and roots. additionally, peganin, isopreganine, dipeganin, and deoxypeganin have been identified in p. harmala. the plant also contains quinazoline alkaloids such as vasicine and vasicinone, and a new β-carboline alkaloid, harmalidine, and pegamine, were also isolated from seeds and aerial parts of p. harmala. phytochemical analysis of p. harmala has revealed the presence of alkaloids, flavonoids, and anthraquinones. the major beta-carboline alkaloids in p. harmala extracts include harmalin, harmine, harmalol, harmol, and tetrahydro harmine. the total alkaloid content ranges from 2-5%, with the highest concentrations in seeds and roots[155,156]. additionally, peganin, isopreganine, dipeganin, and deoxypeganin have been identified in p. harmala. the plant also contains quinazoline alkaloids such as vasicine and vasicinone, and a new β-carboline alkaloid, harmalidine and pegamine, were also isolated from seeds and aerial parts of p. harmala[155,157,158]. the alkaloids vasicine and vasicinone, pmmb 2023, 6, 1; a0000328 31 of 48 both members of the quinazoline class, were initially discovered in the flowers and stems of p. harmala. a new β-carboline alkaloid derivative, characterized as l-thioformyl-8-β-dglucopyranoside-bis 2,3-dihydroisopyridinopyrrol, was isolated from the aerial parts of p. harmala. analysis of the aerial parts of p. harmala revealed the presence of four flavonoids, including 7-o-rhamnoside acetin, 7-o-6''-o-glucosyl-2''-o-(3'''acetylrhamnosyl) glucoside, 7-0-(2'''-0-rhamnosyl-2''-o-glucosylglucoside) and 2'''-orhamnosyl-2''-o-glucosylcytisosideglycoflavone[159]. additionally, two anthraquinones were isolated from p. harmala seeds and identified as 3,6-dihydroxy-8-methoxy-2-methyl anthraquinone (peganone1) and 8hydroxy-7-methoxy-2-methyl anthraquinone (peganone2)[160]. figure 7. some alkaloid content of p. harmala. 13.3. anticancer activity p. harmala has been extensively reported in traditional medicine as a treatment for various diseases, including cancer[161]. previous studies have demonstrated the cytotoxic effects of crude seed extracts of p. harmala, including aqueous, hydroalcoholic, and methanolic extracts, suggesting a significant cytotoxic potential[162,163]. the high alkaloid content of p. harmala extracts has prompted research into their potential cytotoxicity[164]. the activities of the aerial parts, fruit, and roots of p. harmala against several cancer cell lines, namely a549, u373, hs683, mcf7, b16f10, and skmel-28, have not been studied previously. the results from a recent study demonstrated that extracts of total n ch3 nh o ch3 n ch3 nh o ch3 harmalin harmine n ch3 nh oh n ch3 nh oh harmol harmalol pmmb 2023, 6, 1; a0000328 32 of 48 alkaloids from fruits, seeds, roots, and aerial parts decreased the viability of all analyzed cancer cell lines in a dose-dependent manner. the total alkaloid extract (tar) significantly reduced cancer cell viability. these results suggest alkaloids are a primary class of compounds in different parts of p. harmala and are likely responsible for the observed cytotoxic effects. the differences in activity between the other parts of the plant are likely due to variations in alkaloid composition, and a synergistic effect of different alkaloids cannot be excluded[159]. the alkaloids extracted from p. harmala samples inhibit the viability of the breast cancer cell line (mcf7) at a concentration (ic50) lower than the ic50 value reported for total alkaloid extracts of seeds obtained from iranian p. harmala (cell line mcf7; ic50 = 25g/ml) [165]. the components of p. harmala l., the beta-carboline alkaloids harmine and harmaline, have opposing effects on the viability of tumor cell lines. harmine has been observed to have no impact on the cell viability of several cell lines such as hela, c33a, sw480, and ccd-18lu. in contrast, harmaline has been found to significantly reduce the cell viability of control and malignant cell lines in a dose-dependent manner[166]. 13.4. toxicological properties all parts of the p. harmala plant are toxic. previous studies have demonstrated adverse side effects such as heightened respiration, heart rate, and clonic muscle spasms in cattle following intravenous injection of harmine and harmaline (9 mg/kg)[167]. p. harmala poisoning has been observed in all domestic animals, with young camels particularly susceptible during dry seasons[168]. animals that have ingested sublethal doses of p. harmala have exhibited signs of digestive and neurological diseases[160]. a study by shapira et al. investigated the effects of a methanolic extract of p. harmala on the reproduction of female rats. the results showed that administering the extract at a dose of 2.5 g/kg/day for 30 days in meal suspension or suspension significantly extended the diestrus phase by ten days, while the estrous stage duration remained constant. furthermore, the methanolic extract reduced the number of live pups and increased the number of resorptions[169]. 13.5. traditional use p. harmala has been traditionally used as an emmenagogue and abortifacient[170]. the plant's seeds (chebba wa l-harmel) are also believed to protect against the evil eye and evil spirits in certain cultures. in marrakech, rabat, salé, casablanca, and tissint, it is also pmmb 2023, 6, 1; a0000328 33 of 48 used to treat conditions such as icterus, colds, hemorrhoids, intestinal pain, heart disease, female sterility, and uterine diseases. the plant seeds (harmel) are commonly used to treat infant toxicosis and childhood diarrhea[171]. 14. taxus baccata (t. baccata) 14.1. geographical location the geographical range of taxus baccata, commonly known as the yew, covers central and southern europe[172], anatolia, the caucasus, and the elburz mountains in asia[173], and north-western africa[174]. madeira and the azores[175]. in the mediterranean region[176]. as for most other euro-saharan species, t. baccata is found primarily in the mountainous area[177]. in morocco, t. baccata represents the euro-siberian geographical element of the flora[178]. the presence of this species in morocco was first recognized by jahandiez and maire (1931) and emberger (1938)[177]. it has been reported to occur between "beni jaled and beni syel"[179], more specifically in the rif, the central part of the middle atlas and the high atlas[180]. 14.2. phytochemical composition the composition of the essential oils extracted from taxus baccata, commonly known as yew, revealed that aliphatic alcohols, terpenes, aliphatic hydrocarbons, and aliphatic aldehydes were the predominant compounds, contributing, on average, to 86.92% of the total oil composition. the terpene fraction was dominated by monoterpenes (14.41%), while sesquiterpenes represented only 2.31% of the essential oil. the most abundant constituents were two aliphatic alcohols, oct-1-en-3-ol and (3z)-hex-3-en-1-ol, and an oxygenated monoterpene, myrtenol, with a total average content of 46.32%[181]. the twigs of t. baccata were also found to contain a mixture of phenols, taxoids, sterols, and fatty compounds in the dichloromethane-methanol extract (1:1). phenolic compounds were the major constituents of the extract. a new lignan, 4'-odemethylsuchilactone, was isolated as a yellow oil[182]. additionally, three other lignans, suchilactone, diol 3, and (-)secaisolariciresinol, were isolated. suchilactone[~-(trans-3,4-methylenedioxybenzylideneflr-(3,4-dimethoxybenzyl)~ -butyrolactone] was isolated for the first time from a taxaceae species[183].this compound was previously reported from two other species, polygalachinensis and haplophyllumpopovii, and as a pyrolytic product of lignan'shelianthoidin[184,185]. pmmb 2023, 6, 1; a0000328 34 of 48 lignandiol 3, a compound not previously isolated from a natural source, was present in the twigs of t. baccata. it was prepared from suchilactone[186]. upon acetylation with acetic anhydride and pyridine, it yielded a diacetate. the physical and spectral properties of the diacetate were found to be similar to prasanthalin, a compound previously isolated from jatropha gossypiifolia. in addition, the twigs of t. baccata were found to contain the biflavonoid constituents sciadopytisin, ginkgetin, and kayaflavone (figure 8)[73]. figure 8. some biflavonoid constituents of twigs. the taxoids present in the twigs of taxus baccata, commonly known as yew, primarily consisted of rearranged 11(15-1) abieto-taxane skeletons, as indicated by their spectral data. two pure compounds, brevifoliol and 13-decinnamoyl-taxchinin b[74], possessing this rearranged skeleton, were isolated, along with a small taxoid, 10deacetylbaccatin iii, that had the taxane structure. the diterpenoid constituents of the twigs were also found to primarily consist of the abieto-taxane backbone 11(15-1), and taxoids of this type of rearranged structure showed tubulin-binding activity, but not cytotoxicity, in vitro[187]. 14.3. anticancer activity the genus taxus has attracted significant attention due to its content of diterpenic alkaloids, particularly taxol (also known as paclitaxel and registered under the tradename taxol®bms[bristol-myerssquibb]). the anticancer properties of taxol were first discovered in extracts of t. baccata and t. brevifolia in 1971. the anticancer activity of taxol is primarily attributed to its side chain, a c2 benzoyl group, and an oxetane ring. the c3 amide-acyl group is also believed to contribute to its activity in the c13 chain. its cytotoxic action is further enhanced by a hydroxyl group at c2[188]. taxol, a diterpenic alkaloid found in the genus taxus, binds to the surface of r 1 h r 3 h 2 ch3 4 ac pmmb 2023, 6, 1; a0000328 35 of 48 microtubules, specifically the tubulin heterodimer subunit, and promotes the polymerization of these structures even in the absence of gtp (guanosine triphosphate)[189,190]. the interaction of taxol with the tubulin of microtubules leads to the promotion of polymerization and thus produces cytotoxicity and stabilization of microtubules. taxol has been used primarily to treat metastatic ovarian carcinoma, metastatic breast cancer, and non-small cell lung cancer, as the number of cancers treated with taxol is increasing[191]. 14.4. toxicological properties a toxicity study of t. baccata leaf fractions and purified stem fractions in albino mice weighing 20–30g showed elevated levels of alkaline phosphatase and transaminase in mice treated with txa-1 (methanolic extract of the leaf) and txb1 (methanolic extract of the stem). these results indicate liver toxicity as an increase in transaminase (glutamate oxaloacetate transaminase (got) and glutamate pyruvate transaminase (gpt)) and alkaline phosphatase (ap) is a common finding in liver disorders[192]. a marked elevation of serum glutamate oxaloacetate transaminase (sgot) is commonly observed in cases of myocardial infarction and elevated serum glutamate pyruvate transaminase (sgpt) in cases of hepatocellular necrosis. this finding could likely be associated with the presence of taxanes and diterpene amides in the fractions used. it can be concluded that the crude drug, t. baccata, mainly the stem, is highly toxic, despite containing some therapeutic compounds. therefore, the taxanes in different parts of t. baccata may represent a new potential tool for toxicological and pharmacological research[193]. 14.5. traditional use t. baccata, commonly known as yew, has been traditionally used as a medicine for treating rheumatism[194] and diabetes treatment[195]. in the middle atlas region of morocco, a decoction made from the leaves is used as an abortifacient[196]. 15. conclusion the current review focuses on the potential therapeutic value of several toxic plants that have demonstrated anticancer activities and are traditionally used in moroccan medicine. despite their toxicity, these 13 plants were found to have a variety of pharmacological and biological activities due to their diverse phytochemical compositions. additionally, the study highlights the traditional uses of these toxic plants to treat various diseases in morocco, which pmmb 2023, 6, 1; a0000328 36 of 48 warrants further investigation in preclinical and clinical trials to explore their potential therapeutic effects. moroccan cuisine often incorporates gruels, herbal drinks, and spicy drinks, which have been found to have numerous health benefits, including chemo-preventive properties and natural inhibitors of certain infections. when used appropriately and in conjunction with a healthy lifestyle, these traditional remedies may help reduce cancer incidence and provide therapeutic benefits for various human pathologies. author contributions: sd, ach, oeg, ma: writing original draft, editing, visualization, conceptualization. sih, yma: researching articles and references. lcm, hb, ad: reviewing and supervision. all authors have read and approved the manuscript. conflicts of interest: the authors declare no conflict of interest. references 1. ser hl, yin wf, chan kg, et al. antioxidant and cytotoxic potentials of streptomyces gilvigriseus musc 26t isolated from mangrove soil in malaysia. prog microbes mol biol 2018; 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a0000267. doi: a10.36877/pmmb.0000267 http://journals.hh-publisher.com/index.php/pmmb review article long covid-19: psychological symptoms in covid-19 and probiotics as an adjunct therapy angel yun-kuan thye1, loh teng-hern tan1,2, jodi woan-fei law1, priyia pusparajah1, vengadesh letchumanan1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; angelthye.yunkuan@monash.edu (ay-kt); jodi.law1@monash.edu (jw-fl); priyia.pusparajah@monash.edu (pp) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) *corresponding author: vengadesh letchumanan, novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; vengadesh.letchumanan1@monash.edu (vl) received: 13 march 2022; received in revised form: 18 april 2022; accepted: 20 april 2022; available online: 22 april 2022 abstract: there is an increase in mental health sequelae following covid-19 infection, with some studies showing a higher prevalence rate of psychiatric sequelae in post-covid19 survivors than in the general population. this review discusses the possible causes, prevalence, and risk factors of covid-19 associated psychological manifestations, namely anxiety, depression, and post-traumatic stress disorder (ptsd). although the exact cause is yet to be determined, it is likely multifactorial involving environmental, biological, and psychological factors due to the pandemic. variation exists for risk factors and prevalence, but the female gender and psychiatric disorder history seem to be consistent risk factors across several studies. while conventional psychotropic medications are the common therapeutic intervention, probiotics could be a potential adjunct treatment to prevent and treat covid-19 and its associated psychological manifestations. their anti-inflammatory effects have been seen directly via reducing plasma concentration of proinflammatory cytokines or indirectly via the suppression within the kynurenine pathway and restoration of gut permeability. additionally, short-chain fatty acids (scfas) are crucial gut microbial metabolites with essential roles, including signaling along the microbiota-gut-brain (mgb) axis, maintaining blood-brain barrier’s (bbb) integrity, neuronal functions, neurotransmitters, and neurotrophic factors modulation. keywords: mental health; covid-19; anxiety; depression; probiotics 1. introduction the world has been suffering and fighting against the deadly covid-19 pandemic since the end of 2019 [1-3]. the severe acute respiratory syndrome coronavirus 2 (sars-cov2) from the family coronaviridae, has claimed many lives and still spreading stronger with pmmb 2022, 5, 1; a0000267 2 of 13 the emergence of new variants such as the beta and omicron [4-8]. coronavirus has caused not just the current covid-19 pandemic but also past outbreaks-severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) [9, 10]. when this review went to press, over 400 million confirmed cases and 6 million deaths were reported worldwide [11]. vaccines were developed and approved for emergency use by world health organization (who) to protect against severe symptoms, hospitalization, and death [12-15]. many countries took drastic measures by closing their international borders, implementing lockdown, mass screening and testing, contact tracing, and educating the public on using face masks and rtk test kits [16-20]. recently, many studies have reported the clinical characteristics, pathogenesis, epidemiology, and complications of acute covid-19 [21, 22]. however, the long-term consequences of covid-19 remain unclear [23]. the viral infections’ post-infectious sequelae often involve damage to different organs, particularly the brain [24]. furthermore, recognition of mental health consequences of infection rises as covid-19 cases increase [25-27]. a similar trend is seen with previous sars and mers outbreaks that have been associated with longterm neuropsychiatric consequences [28, 29]. given the phylogenetic similarities between the coronavirus subtypes, we use sars and mers data to predict the psychological implications of covid-19 [30]. this review is based on a recently published paper titled “psychological symptoms in covid-19 patients: insights into pathophysiology and risk factors of long covid-19” [9]. this review provides an understanding of the cause, prevalence, and risk factors of covid-19 associated psychological effects, the psychiatric sequelae of covid19, and probiotics as a possible adjunctive therapy. given the impact of this pandemic, we must understand covid-19’s psychological implications so that appropriate and effective health care plans and psychological rehabilitation can be made available to improve the individual functioning of covid-19 survivors [9]. 2. incidence and risk factors of covid-19 on psychological effects although results associated with the psychological symptoms post-covid-19 from many studies are available and increasing, they are mainly from surveys or self-reported by patients. nonetheless, the results are significant and may provide an insight into some of the possible explanations for covid-19’s psychological symptoms [9]. there are many potential causes of covid-19 associated psychological manifestations. they may be associated with virus-infected individuals who are worried about the stigma [31], the outcome of the illness [25], the psychological reactions after covid19 infection, the medical interventions [10], and traumatic memories of severe disease and amnesias [32]. however, the pandemic consequently also affects uninfected individuals. risk factors affecting individuals regardless of infectivity include social isolation [33], anxiety [34], stress in both health care workers and essential workers [35], unemployment, and financial pmmb 2022, 5, 1; a0000267 3 of 13 difficulties [36]. notably, the covid-19 associated psychological effects are likely multifactorial due to the pandemic’s biological, environmental, and psychological factors. a study showed that>15% of sars and mers survivors experienced long-term neuropsychiatric effects [28]. another study showed 42.5% of sars survivors experienced ≥ 1 active psychiatric illness, 54.5% experienced ptsd, 39% had depression, 36.4% had a pain disorder, 32.5% had a panic disorder, and 15.6% had ocd, a sharp increase from the 3.3.% pre-infection prevalence of any psychiatric diagnoses [37]. seeing the similarities between covid-19, sars, and mers infections, we may be able to speculate on the psychological symptoms following covid-19 conditions [9]. with regards to covid-19, according to studies, the prevalence of post-infection anxiety ranges between 6.5-63% [38], while in a study involving hospitalized and non-hospitalized patients, the prevalence rate of depression ranges between 12-48% [39, 40] and for ptsd, at 1-3 months post covid-19, its prevalence rate ranges from 12.146.9% [38]. in terms of risk factors, there is variation in profile for different psychiatric manifestations linked with covid-19 [9]. some of the anxiety risk factors include illness severity [23, 41], female gender [42], medical comorbidities [43], the stigma of covid-19 infection, history of psychiatric illness [44, 45], perceived discrimination, death of a family member and living with children [41], and poor social support [46]. for depression, some reported risk factors are female gender [23, 47-49], illness severity [23, 41, 50], stigma of covid19 infection, history of psychiatric illness [45], perceived discrimination, living with children, more significant total number of symptoms after discharge [41], and poor social support [46]. in contrast, some risk factors of ptsd include a history of psychiatric illness, total duration of isolation, the stigma of covid-19 infection [45], death of a family member, living with children, illness severity, and perceived discrimination [41]. to sum up, there seem to be similarities and associations between risk factors of anxiety, depression, and ptsd with an individuals’ disease severity, the level of social support, and mental health. one study from china found lower perceived social support, adverse media reports, and trauma exposure to be consistent risk factors for anxiety, depression, and ptsd [51]. the risk factor for social support is in line with an israel study that found feeling socially disconnected predicted the presence of ptsd a month post-hospitalization [52]. besides the similarities observed in terms of an individuals’ mental health, a risk factor that seems consistent for various psychological disorders is the female gender, with few studies showing females have 2.2.-2.5 times greater chance of developing psychiatric morbidity after covid-19 infection [38, 53-56]. a study also found that women were more represented among dead covid-19 patients with the common mental disorder than men [57]. this is somewhat consistent with a sars study showing female survivors have a higher risk of anxiety, depression, and stress levels [58]. besides the female gender, covid-19 studies found females with a psychiatric diagnosis history [42, 59-62] and those with psychological symptoms a month post-discharge suffered more in all psychopathological domains [62]. pmmb 2022, 5, 1; a0000267 4 of 13 nonetheless, although an increased severity of post-covid-19 psychiatric symptoms seems to be associated with individuals having a history of the psychiatric disorder [48], even individuals without any history of mental health morbidity (74%) did report symptoms of depression and anxiety post-covid-19 [54]. this is somewhat in agreement with previous sars and mers studies [28, 63], showing a third of patients reporting ≥1 psychological impairment (anxiety, depression, ptsd) > six months post-discharge [63]. 3. psychiatric sequelae of covid-19 the long-term psychiatric manifestation of covid-19 is unclear. however, their prolonged effects could be speculated by understanding covid-19’s effects on the central nervous system (cns) and looking at evidence from previous sars and mers [10]. some sars survivors continued having persistent mental issues at 1-year follow-up, despite improving their physical conditions [58, 64]. in one study, a quarter of the sars survivors experienced significant ptsd symptoms after 30 months [65]. additionally, some had persistent mental consequences that were clinically significant at up to 4 years of follow-up [37]. notably, some studies have demonstrated the presence of psychiatric manifestations postcovid-19 infection. several prospective studies have shown that symptoms of long covid-19 can persist up to 3 months [66], 5 months [67], 6 months [68, 69], and even up to 12 months [70, 71] post-hospitalization. this review will focus on post-covid-19 anxiety, depression, and ptsd. the rates of anxiety, depression, and ptsd in covid-19 survivors have surged [72]. a chinese cohort study showed a significant number of covid-19 patients six months posthospitalization had anxiety/depression (23%) and sleep abnormalities (26%) [23]. another study also showed that 41.3% of patients in iran and a third of patients in italy experienced anxiety and depression post-discharge [73, 74]. a korean study also identified long-term psychological sequelae, accounting for ≥20% of all sequelae [75]. a prospective cohort study in milan with a sample size of 402 found that at 1-month post-covid-19, 55.7% of participants scored ≥1 psychopathological dimension (depression, anxiety, ptsd, and ocd), 36.8% in two, 20.6% in three, and 10% in four [48]. additionally, a single-center study in spain on covid-19 survivors found that, out of 179 patients, some had anxiety, depression, and ptsd at 29.6%, 26.8%, and 25.1%, respectively, two months post-covid-19 [55]. the prevalence of covid-19 associated psychological manifestations ranges between studies and the various psychological symptoms. for instance, studies are reporting higher rates of ptsd (96.2%) [76], depression (60.2%), and anxiety (55.3%) among hospitalized covid-19 patients that are clinically stable than normal controls [77]. however, another china study found that the prevalence rate of clinically significant anxiety, depression, and ptsd symptoms for covid-19 patients post-hospitalization were 10.4%, 19%, and 12.4%, respectively [50, 78]. the difference in rates could be a result of variations in assessment methods and the instruments used to measure these outcomes, time frames for pmmb 2022, 5, 1; a0000267 5 of 13 follow-ups, and the different samples or differences among countries in the implications of cultural or spiritual beliefs to manage the psychological consequences of coronavirus disease [72, 79, 80]. hence, these findings should not be generalized, and study designs with larger-scale sizes and more comprehensive should be conducted. however, according to a study, the prevalence rate of anxiety and depression is much higher compared to the average general adult population in china [81]. this is in agreement with an ethiopian study that showed anxiety and depression rates were higher (61.8%, 55.7%) than before (32%,5.73%) the covid-19 pandemic [46]. 4. probiotics as an adjunct treatment probiotics belonging to the microbial genera are commonly present in the intestinal tract. it has anti-inflammatory properties, helps maintain gut barrier integrity, and restores intestinal homeostasis and microbial balance [9]. research studying probiotics’ application to prevent and treat covid-19 is available [82]. probiotics have been recommended by the guidance (version 5) established by china’s national health commission and national administration of traditional chinese medicine to treat severe covid-19 infections, maintain the balance of intestinal microecology, and prevent secondary bacterial infection. this suggests that first-line medical staff and the chinese government trust the importance of gut microbiota in covid-19 disease [83-85]. regarding covid-19’s psychiatric sequelae, probiotics could be an adjunctive treatment compared to conventional psychotropic medications [9]. many research and clinical trials have been conducted within the last decade, determining probiotics’ effects on mental health, and their efficacy in improving mental illness has been proven in clinical trials [86]. clinical studies showed that probiotic intervention alleviates anxiety and stress, and improves depressed patients’ mental status [87-89]. a survey by büttiker et al. hypothesized that the homeostatic relationship between host, microbiome, and virome, could be decisive in determining the efficiency of subsequent disease susceptibility, immunological responses, and long-term psychopathological effects impact the cns, for instance, covid-19 [90]. the local and systemic production of chemokines, cytokines, and other inflammatory mediators are induced [91]. coronavirus binds directly to angiotensin-converting enzyme 2 (ace-2) receptors in the respiratory epithelial cells, potentially resulting in a cytokine storm that causes widespread inflammation, multi-organ damage, and immune-mediated encephalopathy exhibits convulsions and delirium [10, 92]. this cytokine storm results in asurge in t-helper (th)-1 cytokines, including tumor necrosis factor (tnf)-α, interleukin (il)-1β, ccl2, cxcl10, il-6, and interferon (ifn)-γ, and th-2 cytokines, including il-10, il-4, and il-1 receptor antagonists in the serum of covid-19 patients [62, 93]. significantly, cytokine dysregulation (notably transforming growth factor-β (tgf-β), tnf-α, il-1β, ifnγ, il-6, and il-10) are associated with psychiatric disorders [48, 94-99] and are increased in covid-19 patients [9]. probiotics possibly reduce inflammation as it has anti-inflammatory pmmb 2022, 5, 1; a0000267 6 of 13 effects that have been shown either via the direct observation of reduced plasma concentration of proinflammatory cytokines or indirectly via the suppression within the kynurenine pathway and restoration of gut permeability, which have been associated with the etiopathology of depression [86]. kynurenine and its metabolites have essential roles in mediating inflammatory effects relevant to anxiety, mood, and psychotic disorder [100]. a recent meta-analysis revealed that probiotic intervention could decrease the expression of indoleamine 2,3-dioxygenase 1 (ido); an important enzyme that metabolizes tryptophan to kynurenine in the immune cells and plasma of patients [101]. it is fair to say probiotics could reduce inflammation-induced cns pathology [9]. furthermore, there is a possible link between probiotics and their metabolitesshortchain fatty acids (scfas)with covid-19 and its psychological manifestations. scfas are a crucial gut microbial metabolite that is important in signaling along the microbiota-gutbrain (mgb) axis. the mgb axis concept is defined as the bidirectional communication between the gut microbiota and brain, has been verified in both animal and numerous preclinical and clinical studies, underscoring the involvement of mgb in maintaining health and contributing to various neuropsychiatric disorders [86]. anxiety and depression are some of the neuropsychiatric disorders that have been associated with the mgb axis caused by gut dysbiosis [102-105]. interestingly, there seem to be gut dysbiosis and a few other similarities when comparing the gut microbiota composition of covid-19 individuals and individuals with neuropsychiatric disorders. the similarities include increased opportunistic pathogens, decreased bacterial richness and diversity, depletion of beneficial anti-inflammatory symbiotic bacteria, and particularly scfas producing bacteria [104]. this is consistent with several studies demonstrating alterations in gut microbiome composition in a few neuropsychiatric disorders [104, 106-112]. scfas have several important roles. they maintain the integrity of the blood-brain barrier (bbb) by enhancing the expression of tight-junction proteins [113]. scfas deficiency can increase gut permeability of the gut-blood barrier (gbb), leading to the translocation of bacterial products, increasing cytokine levels, and impacting the bbb integrity [104]. additionally, scfas influence neuronal functions and contribute to microglial maturation as they modulate neuronal activity directly via receptors expressed on neurons, interact with microglia, and function in brain immunity [104, 114]. they have an essential role in reducing inflammation in the brain by downregulating microglial activation and hence, the secretion of the proinflammatory cytokines [115]. besides that, scfas modulate the level of neurotransmitters and neurotrophic factors [116]. neurotransmitters, for instance, serotonin and gamma-aminobutyric acid (gaba), have significant roles in orchestrating the brain’s normal functioning; imbalances in these neurotransmitters trigger stress, anxiety, depression, and impaired cognition [9]. the findings of scfas depleting are worrying and may significantly impact the brain as their deficiency is associated with chronic brain inflammation related to behavioral and cognitive dysfunctions and many brain pathologies pmmb 2022, 5, 1; a0000267 7 of 13 [104]. with that, probiotics may have the potential to be an adjunct treatment of not just covid-19 but along with its associated psychological manifestations as it restores and maintains intestinal homeostasis and microbial balance. 5. conclusion although covid-19 primary vaccination and booster vaccination have been implemented in many countries [12], the psychological manifestations associated with covid-19 are soaring, and so is its attention gained from the public, clinical, and research sectors. the exact mechanisms driving these physiological manifestations and how long they would last are still unclear. however, the effects are possibly significant, given that some covid-19 survivors still experience these symptoms months after hospital discharge. the cause has yet to be determined, but it seems to be multifactorial. it could be due to the direct action of coronavirus on the brain and cns, the indirect effects via systemic inflammatory responses to the virus, or a result of psychological stressors such as being infected, stigma, and the experience of being in the icu [9]. the female gender and a history of psychiatric disorders seem to be consistent risk factors across several studies. however, huge variation exists. covid-19 associated neuropsychiatric disorders have been connected to brain inflammation, changes in the mgb axis, and gut microbiota alteration [104]. therefore, with this connection and the knowledge regarding probiotics, probiotics could be a potential adjunct therapy for preventing and alleviating not just covid-19 but its associated psychological manifestations [9]. nonetheless, more comprehensive research is needed to understand the link between psychosocial, biological-physiological, and immunological aspects of covid-19 and its subsequent psychological manifestations. author contributions: the literature search, data extraction, and manuscript writing were performed by atyk. the manuscript was critically reviewed, proofread, and edited by jl-wf, lt-ht, pp, and vl. pp and vl conceptualized this writing project. funding: no external funding was provided for this research acknowledgments: the authors would like to acknowledge professor shajahan yasin, head of school, jeffrey cheah school of medicine and health sciences, monash university malaysia. conflicts of interest: the authors declare no conflict of interest. references 1. letchumanan v, ab mutalib n-s, goh b-h, et al. novel coronavirus 2019-ncov: could this virus become a possible global pandemic. prog. microbes mol. biol. 2020; 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(lausanne) 2020; 11: 25. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2022, 5, 1; a0000266. doi: 10.36877/pmmb.a0000266 http://journals.hh-publisher.com/index.php/pmmb review article patient’s own medication use during hospitalization hamimatul hayat abdul nasir1, jagjit singh dhaliwal1, hui poh goh1*, long chiau ming1*, daniel vui teck wee2, khang wen goh3, ganesh sritharan4, majid ali5, yaman walid kassab6 article history 1paprsb institute of health sciences, universiti brunei darussalam, gadong, brunei darussalam; 18b3094@ubd.edu.bn (hhan.); jagjit.dhaliwal@ubd.edu.bn (jsd) 2pharmacy department, suri seri begawan hospital, ministry of health, belait, brunei darussalam; daniel.wee@moh.gov.bn (dvtw) 3faculty of data science and information technology, inti international university, nilai, malaysia; khangwen.goh@newinti.edu.my (kwg) 4school of pharmacy, faculty of health and medical sciences, taylor’s university, subang jaya, malaysia; ganesh_alei@hotmail.com (gs) 5school of life and medical sciences university of hertfordshire (hosted by global academic foundation) new cairo, egypt; majid.ali@hotmail.com (ma) 6college of pharmacy, national university of science and technology, muscat, oman; dryamankassab@yahoo.com (ywk) *corresponding authors: hui poh goh and long chiau ming, paprsb institute of health sciences, universiti brunei darussalam, gadong, brunei darussalam; pohhui.goh@ubd.edu.bn (hpg); long.ming@ubd.edu.bn (lcm) received: 5 march 2022; received in revised form: 8 april 2022; accepted: 18 april 2022; available online: 22 april 2022 abstract: medication wasting has been adding to the cost burden on the healthcare system. sustainable interventions such as using patients’ own medications have been implemented to upgrade the medicine management system. this review aimed to describe the studies related to patients’ own medication in hospital settings to explore its positive impacts. current literature has studied the impact of using patients’ own medicines from costeffectiveness, clinical, and patient safety perspectives. the economic impact was represented by determining the cost saved after implementing the patient’s own medications intervention. on the other hand, the efficacy of patient care was proven by reducing the frequency of medication error and prevalence of drug-drug interactions as the intervention was implemented. patients’ knowledge of their medications was also proven to be improved with their own medicines during hospitalization, improving their adherence to their medication treatment. improving patients' adherence to and knowledge of their prescriptions during hospitalization may also aid in the reduction of hospital re-admissions, which could result in a net economic return. keywords: pharmacy; medicine management; patient safety; medication safety pmmb 2022, 5, 1; a0000266 2 of 9 1. introduction in recent years, many cost-saving medicine waste reduction interventions have been explored to mitigate the burden of escalating costs of prescription medications and the prevalence of unused medications at home[1]. recent well-designed studies have confirmed that using patients’ own medication during hospitalization is economically and ecologically friendly. a patient’s own medication is defined as medications brought into a hospital during admission and obtained through hospital prescriptions or community settings[2]. this intervention could tone down the cost burden on the healthcare system or the patients[2]. hence, the cost impact of a patient’s own medication use in the in-patient setting is worth studying. furthermore, this intervention exerted positive implications in patient care, thus making it significant to be explored more in terms of other beneficial impacts it may have to improve the patient-care system. this mini-review addresses the benefits of using a patient’s own medication during admission and the possible solutions to any drawback it may have to optimize the use of finite resources and encourage the implementation of green alternatives for the existing healthcare systems. 2. the conventional hospitalization routine most hospitals have not practiced the use of patients’ own medication during their admission, and new medicines are supplied instead. upon admission, patients’ old medications may have been de-prescribed either to respond to an adverse effect or to evaluate the consequences of continuing versus stopping their current treatments. the term ‘deprescribe’ refers to detecting the potential risks and terminating patients’ medications if it outweighs the potential benefits[3]. discontinuation of regular prescriptions during admission will lead to a major concern, such as the accumulation of unused medications at home, which results in medication waste and, as a result, monetary losses. according to a uk audit commission report, medications worth £90 million were wasted from the disposal of patients’ unused medications, and this wastage could be reduced if the medicines could be reused[4]. medication waste has become a financial burden for healthcare systems in various parts of the world. in many cases, financial losses due to drug waste might have been prevented if significant policy implications had been implemented, and the savings would have aided other healthcare services. this waste signifies a wasted chance of improving the health outcomes of the patients, as well as a loss of health budget allocations that should be better utilized for other healthcare expenses. medication waste has significant economic and environmental consequences since it pollutes the environment without proper disposal, which can harm humans, animals, and plants. generally, an ideal healthcare system should aim to provide the utmost standard quality of care by optimizing the utilization of existing resources and minimizing wastage, especially finite resources such as medications[1]. the audit commission has recommended that the patient’s own medicines during hospitalization should be considered[4]. in most countries, the use of a patient’s own medications during patient's admission is acknowledged, pmmb 2022, 5, 1; a0000266 3 of 9 yet it has not been fully implemented. the healthcare professionals lack awareness of the value of encouraging the use of patients’ own medication in the hospital settings. 3. advantages and disadvantages of using patient’s own medication in wards patients’ medications are often de-prescribed and replaced with new ones during admission. changing medications may also not be acceptable in some instances, mainly if they contain an inactive substance the consumer is allergic[7]. it was advised that if one version of the medication is functioning correctly, it should not be replaced with other medicines unless necessary to avoid the risk of having adverse drug effects. hence, using the patient’s own medication during admission is a safe choice unless necessary changes of medications are required to suit the patients’ conditions[8, 9]. using patients’ own medication is one of the sustainable approaches implemented in many hospitals to improve the medical management system. this is especially true for multiple doses devices such as an inhaler, nasal sprays, or eye drops. another pertinent point is the physician might replace the existing medication, and the whole newly supplied product by the inpatient ward supply will be squandered. boachie-ansah et. al described a patient’s own medication as the drugs brought into a hospital during admission, in which these drugs are acquired via the hospital’s prescriptions or from community settings[2]. the use of patient’s own medication benefits in terms of convenience as the medicines are readily available, reducing the amount of missing and behind-scheduled doses[10]. more importantly, reducing the frequency of changing medications during admission contributed to a lower treatment cost during hospital admission by reducing the wastage of drug disposal of patients’ medications[2, 4]. however, there are limitations in some hospitals as they have a policy where patient’s own medications use is not encouraged, considering the hygiene and storage of the medicines are unknown[12]. nevertheless, it is not entirely discouraged as patients can bring their own medication with the practitioner's authorization in charge. the medications should be checked for their eligibility[12]. if the patient’s own medication is used, documentation should be made in the patient’s health record. due to that, a criteria checklist has been created to ensure that the patient’s own medication is still within the standard[2]. the included assessments are tabulated in table 1. some of the limitations include the problems of starting this protocol, as more staff is required to monitor and check the quality of the medications brought by patients[13]. this is crucial to perform risk management of this patient’s own medication intervention so that minimum standard can be determined to ensure the smoothness of this intervention in the healthcare system[13]. overall, the advantages of implementing this patient’s own medication interventions outweigh the disadvantages. the limitations can be possibly overcome by the involvement of a pharmacist to inspect and identify the quality of the medications brought by the patients before their admission. otherwise, if the medicines are not qualified for use, they can be appropriately disposed of as a step for proper management of medication waste. in order to pmmb 2022, 5, 1; a0000266 4 of 9 bring the economic and environmental implications of medicine wastage to the public's attention, public awareness initiatives are required. hence, raising awareness for patients to bring their own medicines when hospitalized is crucial so that any unused medications can be disposed of properly or reused if they are still within standards. table 1. criteria checklist for patient’s own medication. labelling clear and clean label that must include the name, strength, quantity of the medications with a clear instruction. storage box the medicines brought must be kept in their original storage box. storage storage of the medicines must be according to the manufacturer's suggestions. expiry date the expiry date must be present. quality of medications the medications must be whole, for tablets, and clean. 4. financial impact of using patient’s own medication the introduction of the patient’s own medication seemed to yield a positive result in general and beneficial, especially in cost-savings. the percentage of patient’s own medicines used during hospitalization is quite significant in most studies. hence, it supports the fact that this practice may contribute to cost savings. thus, the patients and their guardians should be encouraged to bring their medicines[8]. patient’s own medication intervention has shown positive results where cost savings were observed. for instance, a total annual savings of the british pound of 24,213 was made after implementing a patient’s own medication during hospitalization in eleven wards of a public hospital located in southern england[14]. a two-week study was done in one children's hospital in england to further evaluate this intervention. half of the study cohort brought their drugs, and three-quarters used their own drugs during hospitalization [8]. three-quarters of these patients were discharged with at least one or two medications, while half were released with the same medicines before they were hospitalized[8]. it was estimated that about the british pound 5,549 was saved within the two weeks[8]. furthermore, another study conducted at four different wards of an australian hospital concluded that 9.9% of annual savings were estimated using a patient’s own medication alone[4]. on the other hand, a study at another hospital showed that half of the warded patients (n=152) brought their medications, and 38 of them brought their medications only after being asked[2]. the patients’ reasons for bringing in the medications include saving money and the continuity of their treatments[2]. it was also reported that 60 of the patients left their medications at home because they had not pmmb 2022, 5, 1; a0000266 5 of 9 been told to bring their medications[2]. the findings reflected that some patients lack the awareness to get their own medications before admissions. literature has shown that the cost of introducing new treatment regimens does not necessarily benefit the patients but adds to the burden in terms of medication cost. a recent study had reported that 40% of new medications started during patients’ admission were discontinued when they were discharged, and 71% of the patients had one of their medications discontinued before their discharge[15]. it was also reported that 10% of the patients had their medications replaced but still within the same class[15]. unless the changes in the regiment are essential and dominant in improving patients’ health, this may act as a deterrent to unnecessary addition and changes in the treatment, which might contribute as one of the sources of the cost burden to the healthcare systems. it is undeniable that excessive spending on medications leads to the potential cost burden of medication wastages[16]. thus, adding new medications hastily should be avoided to prevent further economic burden in the healthcare sector. patient’s own medication implementation has proven its positive financial impact across studies that evaluated this system[8]. if they are not used during hospitalization, returning the patients’ medication to the pharmacy may also contribute to the cost impact of medication use either positively or negatively. depending on how the returned medications were dealt with, either those have been reused or reused disposed[8]. although the estimated cost of returned medications is not on par with newly dispensed medicines, the intervention still gives significant help to reduce medication wastage and cost[1]. summarized details of the financial impact of patients’ own medication use in hospital settings are outlined in table 2. table 2. summary of the financial impact of patient’s own medication use in the hospital settings. no population type sample size country/city year type of study cost saved reference 1 general medical and surgical wards n/a london, united kingdom 2000 prospective £24,213 annually [14] 2 pediatrics 33 patients london, united kingdom 2009 prospective £2,549 within two weeks of study [8] 3 general medical wards 295 patients ghana 2008 prospective 9.9% of annual hospital drug expenditure [2] 4 surgical wards 40 patients canada 2011 prospective $1601.85 within the three weeks of study (including pharmacists’ labor cost) [17] pmmb 2022, 5, 1; a0000266 6 of 9 5. clinical and safety perspectives complete medicine reconciliation is essential to ensure effective patient care and reduce the prevalence of medication errors such as dosing errors, omissions, duplications, or drug interactions during hospitalization. previous studies have proven that the use of patients’ medication during hospitalization has been recognized for its value in completing medicine reconciliation, leading to a more precise drug profile. bringing patients’ own medicines during their hospitalization provides essential details such as patients’ adherence to their medications by checking the pill count and the latest date of refilling their medications. at the same time, the use of patient’s own medicines also requires monitoring by reviewing those medications during admission and discharge to ensure that unsuitable medications are omitted and not continued to be consumed at home[18]. thus, omissions and duplications of crucial medicines can also be prevented if the patients bring their own medication for use, especially when they are on many medications. the use of patient’s own medication may be utilized to produce a precise patients’ medical history to reduce errors such as the risk of misusing medications. it will also make patients’ discharge process more efficient and reduce patients’ confusion after their discharge by continuing familiar treatments[2, 4, 18]. it was found that treatment changes lead to discontinuance or abandonment of expired prescriptions, hence contributing to more wastage[1]. it was undeniable that starting a new regimen and disposing of the patients’ old medications may also lead to error. as in one of the studies, it was observed that at least one error was detected in 12% of warded patients in their newly discharged prescriptions[19]. some of the errors found were the discontinuation of crucial medications in chronic patients and dosing errors[4]. this highlighted the importance of using a patient’s own medicines during admission before adding or changing drug treatments. a study conducted at the surgical and medical ward of a teaching hospital in the united kingdom concluded that the policy of using patients’ own medication reduced medication administration error[20]. similarly, another study conducted in an emergency department setting revealed that significantless errors were found among the patients who brought their own medications compared to those who left their medications[21]. moura et al. also found a higher chance of having drug-drug interactions in hospitalized patients when more drugs are introduced[22]. therefore, this urged the importance of using patients’ own medicines first before changing their drugs regimen entirely. in addition, beers et. al have suggested that some of the new medications introduced during patients’ admission were not necessarily effective, especially when the medications are within the same drug class[15]. these errors should be prevented as they may increase hospital stay length, costs, and mortality[23]. according to studies, the application of a patient’s own medication had an insignificant effect on medication administration errors; thus, the use of a patient’s own medicines was worth to be applied and explored more to improve the medications management system as a whole[20]. additionally, the use of patients' own medication had a good impact on patients’ knowledge of their medications. more than 80% of the total of 731 patients showed more pmmb 2022, 5, 1; a0000266 7 of 9 understanding of their medication use during discharge associated with using their own medicines in the in-patient settings[24]. this might reduce the risk of re-admission to the hospital and thus give a net economic return to the healthcare systems[24]. indirectly, the use of patient’s medication will help enhance patients’ knowledge and understanding of their regimens[24]. concomitantly, it will impact patients’ adherence to taking their medications, which was further believed to help optimize their treatment regimens. this can be demonstrated by a study within the diabetic population where their mean hba1c showed a signification reduction and improved medication adherence by 87.20% with the implementation of patient’s own medication[25]. improving patients’ compliance and understanding of their medications use during hospitalization might help in reducing hospital re-admissions[24, 26]. in one of the studies, the intervention was implemented in small hospitals; 90% of the pharmacy directors in the 300 small hospitals which were the members of american hospital association encouraged the use of patient’s own medications, but under circumstances that the medications’ quality was still exceptional and must be within the practitioners’ order[27]. all these related studies seemed to have found more advantages in implementing sustainable intervention in their healthcare systems. 6. conclusions introducing patient’s own medication use in in-patient settings is to observe its benefits to the healthcare system and for the patients themselves. many studies have shown an increasing trend of net economic saving and patients’ knowledge of their medications with the implementation of patient’s own medication use during hospitalization. the prevalence of medication errors showed a deflating trend, hence, offering a promising strategy to reduce the risk of preventable mistakes. without a doubt, the accomplishments demonstrated in the studies might help reduce medication wastages and cost burden to the healthcare system. its potential effects should be recognized and be considered as a future permanent intervention in the process of delivering healthcare to patients. improving the other multidisciplinary teams’ knowledge of the effectiveness of the patient’s own medication use during hospitalization might also help develop an awareness of this intervention in cutting down medication wastages. not only limited to applying patients’ own medication use during hospitalization, but the continuance of research should also be done to explore further a more sustainable intervention to reduce the cost burden and, at the same time, helps improve the healthcare system. author contributions: conceptualization, hpg, dvtw; methodology, hhan, hpg, lcm, dvtw; software, hhan, hpg, lcm.; validation, gs, ma, ywk.; formal analysis, hhan, kwg, hpg, lcm, dvtw; resources, hhan, jsd, hpg, lcm, dvtw, gs, ma, ywk.; data curation, hhan, gs, ma, ywk; writing—original draft preparation, hhan, hpg, lcm; writing—review and editing, hhan, kwg, jsd, hpg, lcm, dvtw. funding: no external funding was provided for this research. conflicts of interest: the authors declare no conflict of interest. pmmb 2022, 5, 1; a0000266 8 of 9 references 1. makki m, hassali ma, awaisu a, et al, the prevalence of unused medications in homes. pharmacy (basel) 2019; 7(2). 2. boachie-ansah p, anto bp, and marfo afa. reuse of patients' own drugs in hospitals in ghana; the evidence to support policy. bmc health serv res 2019; 19(1). 3. scott s, clark a, farrow c, et al, deprescribing admission medication at a uk teaching hospital; a report on quantity and nature of activity. int j clin pharm 2018; 40(5): 991-996. 4. james cr, leong ck, martin rc, et al. patient's own drugs and one‐stop dispensing: improving continuity of care and reducing drug expenditure. j pharm pract res 2008; 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12(3): 266-272. 23. sorensen ca, de thurah a, lisby m, et al. cost-consequence analysis of self-administration of medication during hospitalization: a pragmatic randomized controlled trial in a danish hospital setting. ther adv drug saf 2020; 11: 2042098620929921. 24. van herpen-meeuwissen lj, van den bemt bj, derijks hj, et al. economic impact of patient’s own medication use during hospitalisation: a multicentre pre-post implementation study. int j clin pharm 2019; 41(6): 1658-1665. 25. lim pc, chung sj, tan ty, et al. comparing the cost, glycaemic control and medication adherence of utilizing patients’ own medicines (poms) versus usual dispensing among diabetic patients in an outpatient setting. daru j pharm sci 2021; 29(1): 125-132. 26. sokol mc, mcguigan ka, verbrugge rr, et al. impact of medication adherence on hospitalization risk and healthcare cost. medical care 2005; 521-530. 27. norstrom pe, brown cm. use of patients’ own medications in small hospitals. oxford university press, 2002. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2022, 5, 1; a0000278. doi: 10.36877/pmmb.a0000278 http://journals.hh-publisher.com/index.php/pmmb original research article comparative analysis of deep learning algorithm for cancer classification using multi-omics feature selection nur sabrina azmi1, azurah a samah1*, vivekaanan sirgunan1, zuraini ali shah1, hairudin abdul majid1, chan weng howe1, nies hui wen1, nuraina syaza azman1 article history 1faculty of computing, universiti teknologi malaysia, 81310 johor bahru, johor, malaysia; nsabrina36@graduate.utm.my (ns); vivekaanan16@gmail.com (vs); aszuraini@utm.my (zas); hairudin@utm.my (ham); cwenghowe@utm.my (cwh); huiwennies@utm.my (nhw); nsyaza7@graduate.utm.my (nsa) *corresponding author: azurah a samah; faculty of computing, universiti teknologi malaysia, 81310 johor bahru, johor, malaysia; azurah@utm.my (aas) received: 16 may 2022; received in revised form: 27 september 2022; accepted: 4 october 2022; available online: 6 october 2022 abstract: advancement of high-throughput technologies in omics studies had produced large amount of information that enables integrated analysis of complex diseases. complex diseases such as cancer are often caused by a series of interactions that involve multiple biological mechanisms. integration of multi-omics data allows more advanced analysis using features from various aspects of biology. however, analysing cancer multi-omics data on a large scale could be challenging due to the high dimensionality of the data. the recent development of advanced computational algorithms, especially deep learning, had sparked numerous efforts in applying these algorithms in multi-omics studies. this study aims to investigate how deep learning algorithms, namely stacked denoising autoencoder (sdae) and variational autoencoder (vae) can be used in cancer classification using multi-omics data. moreover, this study also investigates the impact of feature selection in multi-omics analysis through the implementation of an embedded feature selection. the multi-omics data used in this study includes genomics, methylomics, transcriptomics and clinical data for a case study of lung squamous cell carcinoma. the classification performance has been compared and discussed in terms of the effectiveness of different models and the impact of feature selection. results showed that vae outperforms sdae with 91.86% accuracy, 22.73% specificity and 0.21% matthews correlation coefficient (mcc). keywords: genomic; cancer classification; multi-omics; deep learning; feature selection; recursive feature elimination; stacked denoising autoencoder; variational autoencoder 1. introduction cancer is a complicated and heterogeneous disease to human health as it has contributed to a tremendous number of deaths worldwide [1]. cancer research has been carried out by past researchers for decades. the classification of cancer based on molecular level research attracted the attention of numerous researchers, as it offered a systematic, precise and objective diagnosis for different types of cancer [1]. clinical practices these days prefer pmmb 2022, 5, 1; a0000278 2 of 10 classifying cancer based on its tissue or cell type origin as well as pathogens [2]. types of genomic data that are regularly consolidated for cancer diagnosis include gene expression, dna methylation, mrna expression, microrna (mirna) expression and also protein expression [3]. integration of these components is expected to improve our understanding of the clinical and biological importance [4]. integrative analysis of multi-omics data is the key to connecting cancer genetics, clinical and epidemiological information to ensure patients receive an effective and proper diagnosis [3]. accordingly, one of the significant goals of cancer multi-omics study is to discover possible cancer subtypes using molecule-level signatures, which would be handy in successful diagnosis and treatments [4]. multi-omics analysis has become increasingly popular in biomedical research as these omics with different unique features of their own are being integrated using effective integration strategies to obtain valuable data for solving biological problems. however, analysing cancer multi-omics data on a large scale could be genuinely challenging as it requires an efficient and robust computational algorithm [4]. this is due to the heterogeneity of distinct omics data in terms of scale, dimension and quality, requiring subtle processing. fortunately, deep learning has grown into a formidable force in handling big data, which now stands as one of the most powerful architectures in overcoming complex problems related to biological data. it has been continuously refreshing the state-of-the-art performance of many machines learning tasks and facilitating the development of numerous disciplines. besides, deep learning allows computational models composed of multiple processing layers to understand data representations with multiple levels of abstraction [5]. deep learning has also clearly demonstrated its power in promoting bioinformatics, including sequence analysis, structure prediction and reconstruction, biomolecular property and function prediction, biomedical image processing and diagnosis, biomolecule interaction prediction and systems biology [6]. since computational methods are being widely exploited in the bioinformatics field lately, stacked denoising autoencoder (sdae) is considered a handy algorithm in terms of gene regulatory targets discovery, disease detection and drug discovery [7]. in addition, variational autoencoder (vae) has shown promising character when applied for single omics-based research carried out for drug discovery and disease diagnosis as well. since the model is unsupervised and entirely data driven, it does not rely on existing omics annotations [8]. that is why these two deep learning algorithms were chosen. this paper aims to compare the performance of two deep learning architectures namely vae and sdae using a multiomics dataset derived from a case study. the performance of these models will be measured in terms of model accuracy and loss rate as integrated multi-omics data will be analysed thoroughly. the rest of this paper is further organised as follows. section ii illustrates the literature review for detailed information in this study while in section iii explains all the steps taken to complete this analysis. then, section iv describes the result and discussion of the model performance and ends with a conclusion included in section v. pmmb 2022, 5, 1; a0000278 3 of 10 2. materials and methods experimental workflow has been done to understand further the processes and steps involved in implementing the algorithms and methodologies involved as a part of this research. figure 1 demonstrates the experimental framework of this study. figure 1. experimental framework 2.1. omics dataset the cancer dataset used in this study was lung squamous cell carcinoma (lusc) obtained from the multi-omics cancer benchmark tcga pre-processed data publicly available at multi-omics cancer benchmark repository [9]. the url for the dataset is http://acgt.cs.tau.ac.il/multi_omic_benchmark/download.html. table 1 shows the summary of lusc data. table 1. summary of lusc data. types of datasets omics field num. of patients num. of features gene expression genomics 553 20531 dna methylation methylomics 388 1046 mirna expression transcriptomics 413 5000 survival data (clinical) 626 164 2.2. data pre-processing data pre-processing is very important for integrative analyses towards multi-omics to reduce unnecessary biases and noises [10]. the retrieved omics dataset has been pre-processed previously. however, in this study, we recheck the presence of missing values in the dataset. the patient survival data is a representation of two different class labels labelled "0" and "1". pmmb 2022, 5, 1; a0000278 4 of 10 patients who are occupied with "primary tumour" samples are marked with "1" while patients having "solid tissue normal" samples are marked "0". class imbalance issues were also monitored throughout the data preparation process, whereby the initial class count showed that only 35 patients had "solid tissue normal" (10.17 %) while the other 309 patients (89.83 %) had "primary tumour" (refer to table 2). to overcome this issue which may lead to unequal representation of classes and misclassifications, we proposed smote as an oversampling technique, whereby minority class gets oversampled by creating synthetic examples instead of oversampling with unfamiliar replacement [11]. the presence of class labels in the form of survival data encourages supervised cancer classification and aids the process of predicting omics features that contributes the most to analyses of patients with lusc. table 2. details of classes in survival data (clinical). data type class survival data (clinical) primary tumour solid tissue normal number of samples 309 35 the pre-processed omics data which were splitted into a ratio of 75% was used for training while the remainder of the 25% was used for testing. additionally, data normalisation was also carried out in this phase using min-max normalisation method. the major goal of normalisation process was to scale every feature into the range of 0 to 1. it becomes imperative to carefully use these pre-processing steps as they have a huge influence on the integrative analysis [12]. 2.3. multi-omics integration analysis of multi-omics has become increasingly popular among professionals from the biomedical sector [13]. in conjunction with that, integrating multiple layers of information has been hugely challenging for researchers due to data heterogeneity [14]. to conduct the data integration of multi-omics, the integration strategy used in this study was early integration. this strategy was implemented after data has been pre-processed. then, the preprocessed data were concatenated and the resulting data was formed into a single large matrix before having fed into model training. the early integration is common due to its simplicity, ease of implementation and mainly its ability to reveal the effects of interactions between the different layers by combining variables from each omics [15]. table 3 summarises the number of samples and features after data integration. table 3. summary of the number of samples and features after data integration. types of datasets num. of sample num. of features multi-omics 344 18663 pmmb 2022, 5, 1; a0000278 5 of 10 pandas, a python library has been utilised to concatenate all the three omics relatively to each other. this was done to ensure that a complete multi-omics dataset comprising of patients with all three omics was produced. "patient id" has been used as the index or target data whereby only patients whose data was presented at all 4 data types, including patient survival (clinical) data, were eligible for further analysis. 2.4. feature selection the multi-omics dataset consisted of a large volume of data that could hike the computational cost rate. therefore, we decided to undergo feature selection which has successfully reduced the dimensionality and complexity of the multi-omics dataset. in general, feature selection is a technique majorly used for dimensionality reduction, which is necessary for several fields such as machine learning, pattern recognition, statistics and even data mining [16]. feature selection performs subset selection based on feature relevance and consistency [16]. in this study, we choose the svm-rfe method which is a recursive feature reduction technique that utilises svm weights as a criterion for ranking [17]. svm-rfe’s key concept is to remove features that have the lowest squares of weight in each of the iteration. svmrfe technique searches for a subset of features by going around all the features available to be iterated and finally removing a certain amount of features, leaving the desired number of data available. for this study, a non-linear svm-based rfe has been implemented to eliminate the least relevant features in predicting target variables. input: training data {𝑥𝑖 , 𝑦𝑖 } 𝑖=1 𝑁 output: ranked feature list 𝑅 initialize: 𝑆 = {1, 2, … , 𝐷}𝑖 𝑅= ∅ while 𝑆 is not empty, do: 1. restrict the features of 𝑥𝑗 to the remaining 𝑆 2. get weight vectors by training svm 3. compute the ranking criteria 𝑐𝑘 = 𝑤𝑘 2, 𝑘 = 1, … , |𝑆| 4. find features with lowest value of 𝑐𝑘, called feature 𝑝 5. add feature 𝑝 into 𝑅 (𝑅 = {𝑝} 𝑅) 6. remove feature 𝑝 from s (𝑆 = 𝑆\𝑝) algorithm: feature selection based on svm-rfe pmmb 2022, 5, 1; a0000278 6 of 10 the procedure that has been carried out in our case study was highly similar to the recursive process of the feature removal method in general: 1. train classifier to find the weight vector (𝑤). 2. calculate criteria of ranking for all features. 3. dispose features with the lowest rating criterion value. features used in the iteration had to be removed with backward feature elimination. the ranking score is given according to the components of svm’s weight vector 𝑤: 𝑤 = ∑ 𝑎𝑘 𝑦𝑘 𝑥𝑘𝑘 (1) in our case, multi-omics dataset was aggregated into two portions termed "features" and "targets". this step is taken to ensure svm-rfe based feature selection is made without interfering with independent variables such as class labels during the biological data ranking process. table 4 below shows the number of the multi-omics feature before and after the implementation of the feature selection technique. table 4. implementation of svm-rfe technique for feature selection. before svm-rfe after svm-rfe total features 18663 12000 2.5. stacked denoising autoencoder (sdae) and variational autoencoder (vae) implementation the significance of this study is in the development of two deep learning approaches that are capable classify cancer using multi-omics data. the models used in this study were sdae and vae. the models were designed to analyse the contribution of integrated multiomics data in identifying cancer patients, easing early diagnosis of lusc. sdae model was designed with an early stopping with a patience rate of 50 and the loss rate monitoring was set to ensure that the sdae model development stops training when the monitored metric has stopped improving. the architecture of dae was implemented with 50% gaussian noise with a batch size of 4 and lasso (l1) kernel regulariser. the input dimension of dae was 12,000 features and the output dimension was 500, along with the rectified linear unit (relu) activation function and adaptive moment estimation (adam) optimiser. later, sdae with 7 layers of dimensions of 12,000, 10,000, 8,000, 6,000, 4,000, 2,000 and 500, were built with similar parameters as dae. the performance of this model was observed and analysed at the end of the experimental works. next, an input of 12,000 features has been fed into the input layer, with the learning rate set to 0.0005 for the vae model. the vae model was built with the compression of the input layer into a mean and log variance vector of the size latent dimension set into 100. each pmmb 2022, 5, 1; a0000278 7 of 10 layer was initialised with glorot uniform weights and each step includes dense connections, batch normalisation and relu activation funnelled separately. both encoder and decoder layers were implemented with ridge regression (l2) to add a squared magnitude of coefficient as a penalty term to the loss function. each vector of latent dimension length was connected to the omics input tensor. the hidden layer took two keras layers as input to the custom sampling function layer with a latent dimension output. meanwhile, the decoding layer contained a single layer and sigmoid activation function. adam optimiser and binary cross entropy loss rates were observed as the model stores trained layer weights and reconstructed multi-omics features. lastly, the performance of this model to classify cancer was also analysed and compared to sdae. 2.6. classifier analysis the model accuracy for sdae and vae models in cancer classification was calculated through training and testing/validation phases. the percentage of the correctly assigned multi-omics features into its proper classes, namely benign or malignant tumours was measured and the result obtained was well assessed. both models' training and testing phases require different sets or portions of data to avoid overfitting. integrated multi-omics data was split into training and testing data before being fed into the model. the efficiency of both deep learning architectures was evaluated by accuracy, sensitivity, precision, f1score, model loss, specificity and mcc. 3. results to identify the best performing deep learning model in integrated multi-omics data, both existing sdae and developed vae models were compared and analysed in detail to obtain a firm justification, supporting our research objectives. both models were accessed using a similarly structured model known as artificial neural network (ann). table 5 shows the comparative analysis of sdae and vae models based on several evaluation metrices as described below. table 5. performance measurement of both autoencoder models. deep learning architecture accuracy (%) sensitivity (%) precision (%) f1-score (%) model loss (%) specificity (%) mcc (%) sdae 43.97 % 59.48% 28.86 % 37.17% 0.69 % 29.31% -0.05% vae 91.86 % 98.48 % 93.18 % 95.19 % 0.25 % 22.73% 0.21% pmmb 2022, 5, 1; a0000278 8 of 10 figure 2. sdae model loss figure 3. vae model loss 4. discussion the above table 5 is made up of analysis done on two models (sdae and vae) implementing similar models (ann), data preparation and hyper-parameters including optimisers, loss functions, dropouts batch size as well as epochs. integrated multi-omics data (12,000 features) were fed into both models accordingly to identify how these models interpret and reconstruct these features accurately, leading to an optimal reconstruction rate. vae model has outperformed sdae model in terms of an overall performance rate. vae model has achieved 91.86% accuracy, while sdae only managed to produce 43.97%. meanwhile, sdae produce 59.48% in sensitivity while vae produce 98.48%. the sensitivity indicates the proportion of actual positive cases (cancerous patient) which is correctly predicted as positive. this means vae performed well in classifying the majority class label the lusc patients. moreover, the sdae precision was 28.86% while the vae model precision was 93.18%. precision defines the number of positive cases (cancerous patient) over the total number of misclassified and correctly classified positive cases (cancerous patient). in this case, the sdae model has performed poorly in classifying cancerous patients. the f1-score which depended on the weighted average of sensitivity and precision scored 37.17% for the sdae model while for the vae model, the score was 95.19%. this shows that vae has a better classification and feature reconstruction rate compared to sdae. figure 2 and 3 shows the loss rate for both models in training and testing. the vae model managed to produce a lesser loss rate (0.25%) compared to sdae (0.69%). however, the specificity value for sdae was higher (29.31) than vae (22.73%). in addition, the mcc value for vae was 0.21% because it was closer to 1 than the sdae mcc value, which produces a negative value of -0.05%. in the mcc score, when the proposed model lies at +1, it shows a good model. this indicates that the vae model extracts more information from multi-omics features in comparison to the sdae model. confusion matrix-based elements can be considered additional supporting factors for our claim showing that vae was better than sdae in handling complex multi-omics data. pmmb 2022, 5, 1; a0000278 9 of 10 5. conclusions in conclusion, the performance of sdae and vae models were compared to extract meaningful features from integrated multi-omics data that enable the classification of lusc cells among patients. as a result, vae has outperformed sdae in producing excellent accuracy and minimal model loss rate. it was useful to use the weights of the vae model to extract genes that were also useful for cancer prediction and possess the potential as biomarkers or therapeutic targets. one major drawback of these deep learning approaches is the requirement for large data sets, which are limited for certain cancer tissue. with that, the performance of both models will improve and reveal more meaningful patterns when more omics data becomes available. together, deep learning models are highly scalable to large input data. therefore, future studies should analyse different cancer types to identify cancerspecific biomarkers. moreover, there is also a high potential to identify cross-cancer biomarkers through the analysis of aggregated heterogeneous cancer data. author contributions: conceptualization, methodology, aas, ns, vs, ham, nsa; literature search nsa, ns, aas, vs, zas; experiment, result analysis and validation, vs, ham, ns, nsa, nhw; writing—original draft preparation, vs, aas, nsa, ns; writing—review and editing, aas, nsa, ns, cwh; proofread, ham, aas, cwh, zas. funding: this work was funded by the malaysian ministry of higher education through the fundamental research grant scheme (grant number: frgs/1/2018/ict02/utm/02/11). acknowledgments: the authors would like to express gratitude to the malaysian ministry of higher education for the financial sponsorship of this study through the fundamental research grant scheme (grant number: frgs/1/2018/ict02/utm/02/11). the study is also supported by the faculty of computing, universiti teknologi malaysia. conflicts of interest: the authors declare no conflict of interest. references 1. duan r, gao l, gao y, hu y, xu h, huang m et al. evaluation and comparison of multi-omics data integration methods for cancer subtyping. plos computational biology. 2021;17(8):e1009224. 2. sompairac n, nazarov p, czerwinska u, cantini l, biton a, molkenov a et al. independent component analysis for unraveling the complexity of cancer omics datasets. international journal of molecular sciences. 2019;20(18):4414. 3. manzoni c, kia d, vandrovcova j, hardy j, wood n, lewis p et al. genome, transcriptome and proteome: the rise of omics data and their integration in biomedical sciences. briefings in bioinformatics. 2016;19(2):286-302. 4. wu d, wang d, zhang m, gu j. fast dimension reduction and integrative clustering of multi-omics data using low-rank approximation: application to cancer molecular classification. bmc genomics. 2015;16(1). 5. lecun y, bengio y, hinton g. deep learning. nature. 2015;521(7553):436-444. 6. li y, huang c, ding l, li z, pan y, gao x. deep learning in bioinformatics: introduction, application, and perspective in the big data era. methods. 2019;166:4-21. 7. xu a, chen j, peng h, han g, cai h. simultaneous interrogation of cancer omics to identify subtypes with significant clinical differences. frontiers in genetics. 2019;10. pmmb 2022, 5, 1; a0000278 10 of 10 8. pomraning k, kim y, nicora c, chu r, bredeweg e, purvine s et al. multi-omics analysis reveals regulators of the response to nitrogen limitation in yarrowia lipolytica. bmc genomics. 2016;17(1). 9. multi-omic cancer benchmark [internet]. acgt.cs.tau.ac.il. 2022 [cited 26 september 2022]. available from: http://acgt.cs.tau.ac.il/multi_omic_benchmark/download.html 10. wang d, gu j. integrative clustering methods of multi-omics data for molecule-based cancer classifications. quantitative biology. 2016;4(1):58-67. 11. chawla n, bowyer k, hall l, kegelmeyer w. smote: synthetic minority over-sampling technique. journal of artificial intelligence research. 2002;16:321-357. 12. luo j, wu m, gopukumar d, zhao y. big data application in biomedical research and health care: a literature review. biomedical informatics insights. 2016;8:bii.s31559. 13. vasaikar s, straub p, wang j, zhang b. linkedomics: analyzing multi-omics data within and across 32 cancer types. nucleic acids research. 2017;46(d1):d956-d963. 14. zhang w, li f, nie l. integrating multiple ‘omics’ analysis for microbial biology: application and methodologies. microbiology. 2010;156(2):287-301. 15. hasanin t, khoshgoftaar t, leevy j, seliya n. investigating random undersampling and feature selection on bioinformatics big data. 2019 ieee fifth international conference on big data computing service and applications (bigdataservice). 2019;346-356. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology review article 1 a review on mangrove actinobacterial diversity: the roles of streptomyces and novel species discovery jodi woan-fei law1,2, priyia pusparajah1, nurul-syakima ab mutalib3, sunny hei wong4, bey-hing goh5*, learn-han lee1* 1novel bacteria and drug discovery research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, selangor darul ehsan, malaysia 2institute of biomedical and pharmaceutical sciences, guangdong university of technology, guangzhou 510006, pr china 3ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia 4li ka shing institute of health sciences, department of medicine and therapeutics, the chinese university of hong kong, shatin, hong kong 5biofunctional molecule exploratory research group (bmex), school of pharmacy, monash university malaysia, selangor darul ehsan, malaysia abstract : in the class actinobacteria, the renowned genus streptomyces comprised of a group of uniquely complex bacteria that capable of synthesizing a great variety of bioactive metabolites. streptomycetes are noted to possess several special qualities such as multicellular life cycle and large linearized chromosomes. the significant contribution of streptomyces in microbial drug discovery as witnessed through the discovery of many important antibiotic drugs has undeniably encourage the exploration of these bacteria from different environments, especially the mangrove environments. this review emphasizes on the genus streptomyces and reports on the diversity of actinobacterial population from mangroves at different regions of the world as well as discovery of mangrove-derived novel streptomyces species. overall, the research on diversity of actinobacteria in the mangrove environments remains limited. a total of 19 novel streptomyces spp. isolated from mangroves between the year 2009 early 2019, notably from china, india, malaysia, and thailand. hence, it will be worthwhile to encourage the study of these bacteria from mangroves of different locations. keywords: streptomyces; actinobacteria; mangrove; environment; forest received: 06th january 2019 accepted: 30th april 2019 published online: 22nd may 2019 *correspondence to: learn-han lee, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. lee.learn.han@monash.edu; leelearnhan@yahoo.com; bey-hing goh, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. goh.bey.hing@ monash.edu citation: law jwf, pusparajah p, ab mutalib ns, et al. a review on mangrove actinobacterial diversity: the roles of streptomyces and novel species discovery. prog microbes mol biol 2019; 2(1): a0000024. introduction the phylum actinobacteria is among the earliest lineages within the prokaryotes which appears to exist since 2.7 billion years ago[1]. being one of the major lineages in the domain bacteria, this phylum consists of many genera which contributed to its vast diversity with reference to the morphological and physiological variations as well as metabolic abilities[2-4]. in the recent road map of phylum actinobacteria revised by ludwig et al. (2012)[5], this phylum currently comprises 6 classes, namely “actinobacteria”, “nitriliruptoria”, “acidimicrobiia”, “coriobacteriia”, “thermoleophilia”, and “rubrobacteria”. the largest class “actinobacteria” consists of 15 orders, including order streptomycetales that contains only the family streptomycetaceae, where the type genus streptomyces within this family is undeniably one of the most extensively studied genera in the phylum actinobacteria[5-7]. the class actinobacteria can be categorized into two groups: (1) the streptomyces which represents the dominant genus, and (2) the non-streptomyces genera (e.g. sinomonas spp., mumia spp., microbacterium spp. etc.) which also known as rare actinobacteria[8-13]. the genus streptomyces was first introduced by professor dr. selman abraham waksman and professor dr. arthur trautwein henrici in the year 1943[14]. the name streptomyces means “twisted fungus” in greek and it is given due to its morphology which appears to be simicopyright 2019 by law jwf et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) 2 lar to that of filamentous fungi; streptomyces is derived from the combination of actinomyces which means “ray fungus” in greek and streptothrix which means “twisted hair” in greek[14,15]. this genus is made up of a group of aerobic, gram-positive, non-motile, and multicellular filamentous microorganisms that are commonly found in soil, often recognized as persistent soil saprophytes[16-19]. besides, most of the members of streptomyces produce a characteristic earthy odour due to the production of geosmin[7,20]. this group of bacteria has been greatly explored for their pharmaceutically important natural antibiotics or other bioactive compounds, and industrially useful enzymes[1,17,21]. as a result of the continuous efforts in isolation and screening of streptomyces for their biologically active secondary metabolites, the genus streptomyces presently contains 848 species and 38 subspecies that are validly identified (http://www.bacterio.net/streptomyces. html, accessed on 15th january 2019)[22]. unique features of streptomyces streptomyces bacteria are uniquely complex microorganisms, primarily well-known as prolific producers of secondary metabolites with bioactivities such as antimicrobial, anticancer, antioxidant, and immunosuppressive activities[19,20,23,24]. production of secondary metabolites is not essential for bacterial growth, but it is crucial to facilitate the defence mechanisms of the hosts as to ensure survival during unfavourable environmental conditions[6]. it is remarkable that streptomycetes can generate secondary metabolites with various interesting bioactivities and extraordinary chemical diversity, although the roles play by these molecules in the survival of the host are still not fully understood other than the antibiotics which benefit the host by thwarting the growth of competing microbes in unfavourable environment[18,25]. actually, there are several notable features of streptomyces that could account for their complex secondary metabolism. one of the unique features of streptomycetes is their multicellular life cycle involving complex morphological changes. upon spore germination, they grow by tip extension and the development of a network of branched filaments known as the substrate mycelium (vegetative phase) is initiated. as they age, aerial multinucleated mycelium is formed which subsequently experience synchronous cell division leading to the generation of monoploid compartments where each will differentiate into resistant spores (reproductive sporulation phase) (figure 1)[18,26,27]. according to the observation on agar plate media, the substrate mycelium of streptomyces grows into and across the surface of agar plate through extension and branching, whilst the aerial mycelium erected on top of the substrate mycelium that appears above the surface of agar plate with granular, powdery, floccose or velvety appearance[26,28]. apparently, the morphological differentiation event in streptomyces is related to the sensing of essential nutrients depletion[18,27]. meanwhile, streptomyces starts to produce secondary metabolites when encountering nutritional deficiencies in a stressful environment and this process is referred to physiological differentiation[29]. therefore, there is a close association between morphological differentiation and physiological differentiation in the life cycle of streptomyces. during the shifting phase from vegetative to aerial growth and sporulation, metabolic development is activated and thus many interesting secondary metabolites are produced to ensure the survival of streptomyces in stressful environments[27,29,30]. furthermore, many of the streptomyces isolates can produce diverse pigments or pigmented secondary metabolites that responsible for the colour of substrate and aerial mycelia of streptomyces colonies on agar plate media[26,30]. another distinctive feature of streptomyces is that members of this genus have linearized chromosomes, commonly with a large size of over 8 mbp and a high g+c content of approximately 67-78 mol %[32-34]. in the year 2002, the first complete genome sequence of streptomyces was reported a model actinomycete, streptomyces coelicolor a3(2) with a genome size of 8,667,507 bp, thus, became the largest completely sequenced bacterial genome at that moment[35]. in the following year, the complete genome sequence of an industrial strain streptomyces avermitilis was reported to consist of 9,025,608 bp[36]. since then, many streptomyces strains have been subjected to whole genome sequencing to at least the draft stage and these genome sequences are publicly available[37]. these evidences show that the genome size of streptomyces is at least almost two times larger as compared with other well-studied bacteria such as bacillus subtilis with genome size of 4,214,810 bp, and escherichia coli k-12 with genome size of 4,639,221 bp[20,38,39]. the large genome size correlates with the ability of streptomyces to produce large amount of diversified secondary metaboa review on mangrove actinobacterial... figure 1. schematic representation of the life cycle of streptomyces (adapted from barka et al. (2016)[31]). 3 lites. for instance, bentley et al. (2002)[35] reported that the genome of streptomyces coelicolor a3(2) was found to contain over 20 gene clusters related to/involved in the biosynthesis of secondary metabolites. besides, ikeda et al. (2003)[36] noted the presence of over 30 biosynthetic gene clusters streptomyces avermitilis associated to various secondary metabolites. therefore, these special features of streptomyces have contributed to the complexity of the organisms, causing them to be capable of producing a vast array of interesting or novel bioactive secondary metabolite. roles of streptomyces in microbial drug discovery the search for antibacterial agents from streptomyces had in fact began since the early 1940s, before the reclassification of actinobacteria and the introduction of genus streptomyces. pioneered by professor dr. selman a. waksman, who ventured into the discovery of antagonistic soil-inhabiting microbes towards pathogenic bacteria with his team of scientists[40]. the outcome of their investigation led to the isolation of first antibiotic designated as actinomycin, a specific bacteriostatic and bactericidal agent, produced by streptomyces antibioticus (formerly known as actinomyces antibioticus)[20,41,42]. a few years later, waksman and his team achieved a breakthrough in streptomyces research, which was the discovery of streptomycin as a cure for many diseases[40,43]. streptomycin was the first discovered broad-spectrum antibiotic for effective treatment of tuberculosis caused by mycobacterium tuberculosis – a deadly pathogen. streptomycin was derived from streptomyces griseus, the first streptomyces to be utilized for industrial production of this antituberculosis drug[1,25,40,43,44]. from this event, professor selman a. waksman was awarded with the nobel prize in physiology or medicine in 1952 for his successful discovery of streptomycin[6]. eventually, scientists intensified the search for antibiotics from streptomyces. consequently, streptomyces has become the producers of over 75% of the commercially valuable antibiotics applied in human and veterinary medicines[45]. a variety of antibiotics belonging to different classes can be sourced from streptomyces, for instances: clavulanic acid (class: β-lactam) from streptomyces clavuligerus[46], neomycin (class: aminoglycoside) from streptomyces fradiae[47], vancomycin (class: glycopeptide) from streptomyces orientalis[48], and tetracycline (class: tetracycline) from streptomyces aureofaciens[49]. after a period of more than 50 years since the amazing achievement of professor selman a. waksman, streptomycetes continue to surprise the scientific community by their ability in synthesizing functionally and structurally varied bioactive compounds. these bacteria have relentlessly showcasing their significance in the drug discovery and development. recently, professor satoshi omura and professor william cecil campbell were bestowed with the nobel prize of physiology or medicine in 2015 for the successful discovery of avermectin (which later chemically modified to ivermectin) from streptomyces avermitilis. avermectin is a strong anthelmintic agent effective against many nematodes, arachnids, and insects. the semisynthetic derivative ivermectin is an effective treatment for onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) in human[50-52]. to date, researchers are still actively searching for bioactive streptomyces strains around the world due to their enormous potential in producing effective drug candidates and many efforts have been put to further explore the potential of these organisms in producing other clinically important compounds (examples: anticancer, antioxidant etc.) in addition to antibiotic agents[53]. occurrence of streptomyces from different habitats terrestrial and aquatic environments member of streptomyces are ubiquitous in soil and aquatic sediments, existing either in a metabolically active state or relatively inactive state in the form of spores. streptomyces is among the significant microbial populations in most terrestrial soils, thus, the soil is the most extensively studied habitat as a rich source of streptomycetes[54]. additionally, streptomycetes have a substantial role in soil ecology through their involvement in biodegradation. these organisms are capable of degrading recalcitrant polymers present in soil and plant litter, such as lignocelluloses, chitin, and keratin[54,55]. soil-derived streptomycetes have been the producers of pharmaceutically important bioactive compounds and valuable enzymes. therefore, streptomyces of terrestrial soil origin has been greatly exploited in many drug screening programs for the past decades. moreover, it is common to find streptomyces in marine environments where they were frequently assumed to be derived from spores that wash-in from adjacent terrestrial habitats[54]. nevertheless, several studies have proven the existence of indigenous marine streptomyces, although their distributions and ecological roles remain elusive[56-59]. there are also evidences for the presence of streptomyces in other aquatic environments such as freshwater lakes and rivers[54,60,61]. living organisms furthermore, some streptomycetes exist as symbionts instead of free-living soil bacteria. they create a symbiotic relationship with other organisms such as plants and invertebrates, whereby these interactions can either be beneficial or parasitic in some cases[62]. streptomycetes that associated with plants could lead a pathogenic or endophytic lifestyle. while the presence of phytopathogenic streptomyces species are scarce, but one of the most notable phytopathogenic streptomycetes is streptomyces scabies. streptomyces scabies is the primary causative agent of common scab disease in potato and other crops including beet and radish[63]. as for endophytic streptomyces species, they could live as plant commensals whilst occasionally provide certain beneficial properties to their hosts such as protection from phytopathogens or plant growth promotion. also, studies have demonstrated that endophytic streptomycetes could be attractive biocontrol agents due to their ability in generating antibacterial or antifungal agent that can eradicate phytopathogens [19,62,64]. there are several streptomyces species have been found to form protective mutualistic symbioses with insects such as european beewolf, southern pine beetles, and attine ants, in which basically the streptomyces offers law jwf et al. 4 protection to its host or host’s resources against pathogens through the production of antibiotics, in return, the host feeds the streptomyces[62,65-67]. apart from forming symbioses with insects, many reports have revealed the associations between streptomyces species and marine sponges as well as cone snails though the symbiotic relationships between these organisms remain inconclusive as further studies are required to address these queries[62,68,69]. other unique environments interestingly, streptomyces bacteria happen to be widely distributed in extreme and underexplored environments such as deserts[70], caves[71], hot springs[72], mangroves[33,73], mountain plantations[55], arctic and antarctic regions[74,75]. these environments are often characterized by certain fluctuations in environmental conditions, extreme high/low temperature, acidic/alkaline ph, limitation of nutrients, or high radiation[1]. existence of streptomyces in these unusual environments have demolished the traditional paradigm pertaining the restricted predominance of streptomyces in terrestrial soil and aquatic environments. it is relatively unsurprising that streptomycetes can occur in these environments because they can exist for an extended period as resting arthrospores; these spores are resistant to water and high temperatures, also, they will germinate when sufficient nutrients are available[16,54]. these bacteria also possess physiological and metabolic flexibility that could trigger the production of secondary metabolites to facilitate their survival under extreme conditions[1,74]. exploration of streptomyces from underexplored mangrove recent drug discovery research seeks to discover novel and useful bioactive compounds by emphasizing on the screening of streptomyces species from uncommon and underexplored areas. there is increasing evidence that bioprospecting of streptomyces from dynamic environment like the mangroves are producing positive outcomes[6]. similarly, mangroves have been regarded as one of the rich resources for isolating streptomyces bacteria, which also includes novel streptomyces species[76]. there has been increasing number of mangrove-derived streptomyces strains exhibiting potent bioactivities such as antimicrobial, anticancer, and antioxidant[20,77-81]. it is possible that mangrove streptomycetes have unique metabolic capacity on the synthesis of bioactive secondary metabolites. therefore, it is worthwhile to pursue new groups of streptomyces from less investigated ecological systems such as the mangroves in hopes to unearth novel and/or useful bioactive compounds from these organisms. the mangrove environment the mangroves stand out as an exceptional type of forest distributed along the intertidal zone between the terrestrial and the sea of tropical and subtropical regions around the world[82]. mangroves are described as assemblage of trees and shrubs adapted to thrive in dynamic environmental settings of extreme tides, high salinity, high temperature, and high sedimentation[82,83]. despite those harsh environmental conditions, these “rainforests of the seas” are very productive and biologically significant ecosystems, for which they provide a superb nursery habitat for a wide range of flora and fauna[84]. of course, mangrove forests offer invaluable benefits towards human society besides sustaining biodiversity. these wetland forests provide ecosystem goods that are vital to sustain human well-being, which cover food source (e.g. fish, shrimps), boat building materials, furniture, firewood, and traditional medicine[85]. mangrove forests also provide protection by buffering salinity changes, stabilizing shorelines through reducing coastal erosion, and minimizing severe impact of natural disasters like floods, tidal waves, hurricanes, and tsunamis[82,86,87]. the latest update on global distribution of mangroves was reported in the 2010 world atlas of mangroves, with the inputs from several organizations including the international tropical timber organization (itto), international society for mangrove ecosystems (isme), food and agriculture organization (fao), united nations environment programme (unep), and others. it was estimated that the mangrove area was a total of 152,361 km2 found in 123 countries and territories, based on satellite imagery captured in year 1999 – 2003[88,89]. the distribution of mangroves in each region is summarized in the chart illustrated in figure 2. the largest proportion of mangroves is found in southeast asia, where indonesia, malaysia, and myanmar are the top three mangrove-rich countries within this region[82,89]. bacterial diversity of mangrove mangroves have a highly diverse microbial community comprising numerous different species of fungi and bacteria. in the mangrove sediments, bacteria are the primary decomposers of organic residues and the main contributors in the carbon cycle. many of them are associated with ecologically important roles such as nitrogen fixation, phosphate solubilising, sulphate reducing, enzyme producing, photosynthetic anoxygenic, and methanogenic[90,91]. based on previous research findings, the bacterial assemblages which dominated the mangrove ecosystems include phyla proteobacteria, actinobacteria, firmicutes, bacteroidetes, chloroflexi, acidobacteria, cyanobacteria, nitrospirae, fusobacteria, and planctomycetes[92-96]. given the fact that bacteria control the nutrient availability in the environment, so the abundance of these bacteria present in mangroves could result in an increased amount of nutrient levels in the sediments, thereby controlling vegetation patterns, stimulating growth of many organisms, and promoting species diversity[91,97,98]. above all, the periodic tidal flushing and constant fluctuation in salinity could induce changes in metabolic pathways in microorganisms for their adaptation to the mangrove environment. henceforth, these factors could encourage the advent of “new” species that have the potential to produce uncommon metabolites which may be compelling to the pharmaceutical and biotechnology industry[45,94,99]. diversity of actinobacteria in mangrove actinobacteria are important soil inhabitants and the exploration of actinobacteria in mangroves has been gaining attention from researchers around the world due a review on mangrove actinobacterial... 5 to their incredible contribution to drug discovery. in the recent years, there are several reports regarding the diversity of actinobacteria in mangroves, mainly from the east asia, south asia, and southeast asia regions. the actinobacteria population in mangrove forests of china of east asia region was investigated by hong et al. (2009)[100]. in the report, more than 2,000 actinomycetes were isolated from soil and plant samples obtained from eight mangrove sites. furthermore, many of these actinomycetes exhibited biological activities such as growth inhibition of human colon tumor 116 cells, antimicrobial activity against candida albicans and staphylococcus aureus, inhibition of protein tyrosine phosphatase 1b (ptp1b), caspase 3 and aurora kinase a. it was found that the bioactive strains were belonged to 13 genera and most of them were from the genus streptomyces (table 1). the study also revealed that most of the bioactive actinomycetes were originated from soil samples. on the other hand, a study conducted by wei et al. (2010)[101] demonstrated that 77 out of 118 actinobacterial strains isolated from china mangrove plant samples exhibited antibiotic property and these bioactive strains were from the genus streptomyces, micromonospora, nocardia, nocardiopsis, saccharothrix, and lentzea. in the south asia region, one of the largest areas of mangroves is situated in india. a few researchers have reported the actinobacterial diversity in india’s mangroves. for instance, the distribution of actinobacteria in mangrove forests of sundarbans in india was examined by mitra et al. (2008)[102] and the actinomycetes isolated from soil samples were found to belonged to 9 genera with potential antimicrobial activity. in addition, rajkumar et al. (2012)[76] had unveiled the isolation of 116 actinomycetes belonging to 7 genera from sediment samples collected from five sites of bhitarkanika mangroves (table 1). these studies have showed that streptomyces emerged as the most prevalent genus detected in the mangroves of india[76, 102]. as for the southeast asia region, lee et al. (2014)[45] discovered a high level of diversity of actinobacteria in mangrove forest located at peninsular malaysia. a total of 87 actinobacterial isolates from mangrove soil samples were identified to belong to 11 genera and 48 of these isolates exerted antibacterial activity to at least 1 of the 12 pathogens tested, which were bacillus subtilis, bacillus cereus, enterococcus faecalis, staphylococcus epidermidis, methicillin-resistant staphylococcus aureus, klebsiella pneumonia, klebsiella oxytoca, salmonella typhi, pseudomonas aeruginosa, acinetobacter calcoaceticus, yersinia pseudotuberculosis, and aeromonas hydrophila. additionally, diversity of actinobacteria in mangrove soil of indonesia was reported by retnowati et al. (2017)[103], for which they had revealed mangrove actinomycetes with antibacterial activity belonging to the genus amycolatopsis, nocardiopsis, streptomyces, and saccharomonospora. the studies on the actinobacterial population in mangroves of different locations are summarized and listed in table 1. among the many genera of class actinobacteria, there is no doubt that the genus streptomyces is the most biologically active microorganisms. as a result, the exploration of streptomyces in mangrove environments has been the centre of attraction, which subsequently lead to the discovery of novel species. table 2 describes the discoveries of novel streptomyces species isolated from several mangrove areas. meanwhile, there are some studies focusing on the diversity of only the rare actinobacteria. the rare actinobacteria are recognized as non-streptomyces strains of actinomycetes and the frequency of isolation of these actinomycetes through conventional methods is often lower as compared to that of streptomyces[108,109]. one of the examples include the study conducted by naikpatil et al. (2011)[110] who investigated the rare actinobacteria from mangrove sediments of karwar, india. a total of 53 rare actinomycetes were isolated in the study and identified as actinoplanes, actinomadura, microbispora, and micromonospora. another research led by ismet et al. (2013)[111] described the diversity of rare actinomycetes in mangrove rhizosphere soil samples collected from several mangrove sites in bangladesh. rare actinomycetes belonging to actinomadura, actinoplanes, catellatospora, longispora, micromonospora, microbispora, nocardia, nocardiopsis, nonomuraea, rhodococcus/gordonia, saccharomonospora, streptosporangium, and virgisporangium were isolated from the soil samples. figure 2. the global distribution of mangrove (information obtained from itto (2012))[89]. law jwf et al. 6 a review on mangrove actinobacterial... region author country mangrove location source diversity of actinobacteria east asia hong et al. (2009)[100] china i. hainan province: danzhou, haikou, sanya and wenchang ii. guangdong province: shenzen and zhanjiang iii. fujian province: xiameng iv. guangxi province: beihai soil and plant samples actinoplanes, actinomadura, arthrobacter, isoptericola, microbacterium, microbispora, micrococcus, micromonospora, nocardia, nonomuraea, rhodococcus, streptomyces, and verrucosispora. wei et al. (2010)[101] china guangxi province: shankou mangrove nature reserve plant samples lentzea, micromonospora, nocardia, nocardiopsis, saccharothrix, and streptomyces. liao et al. (2010)[104] china guangxi province: beihai soil samples streptomyces and nocardiopsis. jiang et al. (2018)[105] china guangxi zhuang autonomous region: beilun estuary national nature reserve plant samples actinoplanes, agrococcus, amnibacterium, brachybacterium, brevibacterium, citricoccus, curtobacterium, dermacoccus, glutamicibacter, gordonia, isoptericola, janibacter, kineococcus, kocuria, kytococcus, leucobacter, marmoricola, microbacterium, micrococcus, micromonospora, mycobacterium, nocardia, nocardioides, nocardiopsis, pseudokineococcus, sanguibacter, streptomyces, and verrucosispora. south asia mitra et al. (2008)[102] india indian sundarbans: i. bally jetty ii. pakhiralaya 1 iii. pakhiralaya 2 iv. jamespore v. dattar forest vi. bally forest soil samples actinomadura, actinoplanes, nocardia, nocardiopsis, micromonospora, saccharopolyspora, streptomyces, streptosporangium, and streptoverticillium. rajkumar et al. (2012)[76] india sites of bhitherkanika: i. kola creek ii. bhiterkkanika iii. baguludia iv. kalibhanchidia v. thanidia sediment samples actinomadura, actinomyces, actinopolyspora, micromonospora, nocardiopsis, saccharopolyspora, and streptomyces. karthikeyan et al. (2013) [106] india tamil nadu: ennoor soil samples actinokineospora, actinopolyspora, amycolata, glycomyces, microbispora, microtetraspora, micropolyspora, nocardia, nocardiopsis, promicromonospora, saccharothrix, saccharopolyspora, streptomyces, streptoverticillium, spirillospora, and thermomonospora. southeast asia lee et al. (2014)[45] malaysia pahang: tanjung lumpur soil samples gordonia, leifsonia, microbacterium, micromonospora, mycobacterium, nocardia, nocardioides, sinomonas, streptacidiphilus, streptomyces, and terrabacter. malek et al. (2015)[107] malaysia pahang: tanjung lumpur soil samples actinophytocola, gordonia, micromonospora, mycobacterium, pseudonocardia, rhodococcus, and streptomyces. retnowati et al. (2017)[103] indonesia gorontalo province: torosiaje soil samples amycolatopsis, nocardiopsis, streptomyces, and saccharomonospora. table 1. actinobacterial diversity present in mangroves at east asia, south asia, and southeast asia regions. 7 no. strain name and designation mangrove sampling site source reference 1. streptomyces avicenniae mccc 1a01535t national mangrove reserve in fujian province, china rhizosphere of mangrove plant avicennia mariana xiao et al. (2009)[112] 2. streptomyces xiamenensis mccc 1a01550t national mangrove reserve in fujian province, china sediment xu et al. (2009)[113] 3. streptomyces sundarbansensis ms1/7t sundarbans mangrove forest, india sediment arumugam et al. (2011)[114] 4. streptomyces shenzhenensis 172115t shenzhen, china sediment hu et al. (2011)[115] 5. streptomyces sanyensis 219820t sanya, hainan province, china composite sediment sui et al. (2011)[116] 6. streptomyces qinglanensis 172205t qinglan harbour, wenchang, hainan, china composite soil hu et al. (2012)[117] 7. streptomyces pluripotens musc 135t tanjung lumpur mangrove forest in pahang, peninsular malaysia soil lee et al. (2014)[33] 8. streptomyces ferrugineus hv38t thailand soil ruan et al. (2015)[118] 9. streptomyces mangrovisoli musc 149t tanjung lumpur mangrove forest in pahang, peninsular malaysia soil ser et al. (2015)[119] 10. streptomyces gilvigriseus musc 26t tanjung lumpur mangrove forest in pahang, peninsular malaysia soil ser et al. (2015)[120] 11. streptomyces mangrovi ha11110t dongzhaigang national nature reserve, hainan, china soil wang et al. (2015)[121] 12. streptomyces malaysiense musc 136t tanjung lumpur mangrove forest in pahang, peninsular malaysia soil ser et al. (2016)[78] 13. streptomyces antioxidans musc 164t tanjung lumpur mangrove forest in pahang, peninsular malaysia soil ser et al. (2016)[122] 14. streptomyces humi musc 119t tanjung lumpur mangrove forest in pahang, peninsular malaysia soil zainal et al. (2016)[123] 15. streptomyces euryhalinus ms 3/20t lothian island of sundarbans mangrove forest, india sediment biswas et al. (2017)[124] 16. streptomyces colonosanans musc 93jt mangrove forest in kuching, sarawak, malaysia soil law et al. (2017)[125] 17. streptomyces nigra 452t zhangzhou, fujian province, china rhizosphere soil of mangrove plant avicennia marina chen et al. (2018)[126] 18. streptomyces caeni ha15955t sanya, china mud huang et al. (2018)[127] 19 streptomyces monashensis musc 1jt mangrove forest in kuching, sarawak, malaysia soil law et al. (2019)[128] table 2. mangrove-derived novel streptomyces species published from year 2009 to early 2019. conclusion research on the diversity of microbial community in the class actinobacteria originating from different environments and countries has been thoroughly conducted due to their ecological importance and astonishing biotechnological potential. nevertheless, there is still limited knowledge pertaining the diversity of actinobacteria in the mangrove environments given that there are over 150, 000 km2 of mangroves distributed at many different parts of the world. it will be valuable to explore the diversity of actinobacteria from mangrove given that they have been producers of numerous useful bioactive compounds and enzymes, especially from streptomyces. the discovery novel streptomyces in addition to bioprospecting of streptomyces population from underexplored mangrove environments could therefore increase the chances of uncovering new sources of natural products for various applications. authors contributions the literature review and manuscript writing were performed by jw-fl, pp, n-sam, shw, b-hg, and l-hl. l-hl founded the research project. law jwf et al. 8 conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. acknowledgments this work was supported by pvc award grant (project no. pvc-ecr-2016), external industry grant (biotek abadi – vote no. gba-808138 and gba-808813) awarded to l-hl. reference 1. shivlata l and tulasi s. thermophilic and alkaliphilic actinobacteria: biology and potential applications. front microbiol 2015; 6: 1014. 2. gao b and gupta rs. phylogenetic framework and molecular signatures for the main clades of the phylum actinobacteria. microbiol mol biol rev 2012; 76(1): 66-112. 3. chandra g and chater kf. developmental biology of streptomyces from the perspective of 100 actinobacterial genome 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law1, kei-xian tan1, sunny hei wong2, nurul-syakima ab mutalib3*, learn-han lee1,4,5* 1novel bacteria and drug discovery research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, selangor darul ehsan, malaysia 2 li ka shing institute of health sciences, department of medicine and therapeutics, the chinese university of hong kong, shatin, hong kong 3ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia 4biofunctional molecule exploratory research group, biomedicine research advancement centre, school of pharmacy, monash university malaysia, selangor darul ehsan, malaysia 5center of health outcomes research and therapeutic safety (cohorts), school of pharmaceutical sciences, university of phayao, phayao, thailand abstract : the 16s ribosomal rna gene is the gold standard for taxonomic identification and phylogenetic study of most of the bacteria. however, the resolution of 16s rrna gene is found to be insufficient to distinguish closely related species within the genus streptomyces due to large size of streptomyces member. thus, it is essential to find alternative phylogenetic gene markers with higher discriminatory power in addition to 16s rrna gene for the phylogenetic analysis of streptomyces species in order to effectively indicate the evolutionary relationships among streptomyces members at intra and interspecific level. this article aims to discuss the resolution power of ribosomal genes (16s rrna and 23s rrna gene), of protein-coding genes (gyrb and trpb gene) and between ribosomal and protein-coding genes in order to identify gene that could provide better resolution for phylogenetic studies of streptomyces. keywords: streptomyces; actinobacteria; taxonomic; review; method received: 8th october 2018 accepted: 15th november 2018 published online: 14th december 2018 *correspondence to: learn-han lee, novel bacteria and drug discovery research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia; lee.learn.han@ monash.edu. nurul-syakima ab mutalib, ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia; syakima@ppukm.ukm.edu.my citation: law jwf, tan kx, wong sh, et al. taxonomic and characterization methods of streptomyces: a review. progmicrobes mol biol 2018; 1(1): a0000009. introduction members of actinobacteria consist of different metabolic and physiological characteristics while expressing different morphological characteristics such as coccoid and fragmenting hyphalor branched mycelium forms[1]. actinobacteria possess various lifestyles, for instances, as soil inhabitants (example: streptomyces), gastrointestinal commensals (example: bifidobacterium) and pathogens (example: mycobacterium and corynebacterium)[2]. streptomyces is the largest genus located within the family streptomycetaceae, belonging to the phylum actinobacteria [2]. streptomyces bacteria are biologically active at which they can produce secondary metabolites that have various biological effects[3]. currently, there are over 10 000 bioactive compounds have been derived from streptomyces [3-5]. prior to the late 1950s, it was believed that members of streptomyces are eukaryotes instead of prokaryotes due to their distinctive life cycle which is similar to the life cycle of multicellular eukaryotes [6]. as a matter of fact, streptomyces consists of multicellular, gram positive, filamentous aerobic, and mycelial bacteria that live mainly in soil as saprophytes[7]. these bacteria have special features such as complex life cycle involving the development of substrate mycelium with extensive branches containing ll-diaminopimelic acid as main diamino acid and carries aerial hyphae that are able to differentiate into spores or arthrospores[8,9]. spores allow extended survival of streptomyces species in an inactive form in the soil because they are known to be resistant to both water and nutrients deficiencies as well as high temperature in unfavourable environments[7]. another important feature of streptomyces includes copyright 2018 by law jwf, et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) 2 the generation of diverse pigments that form the colour of aerial and substrate mycelia[10]. interestingly, some streptomyces bacteria exist as marine symbionts, plant root symbionts in rhizophere, growing on gamma-irradiated surfaces or thermal springs[11]. meanwhile, some streptomyces bacteria are human, animal or plant pathogens such as streptomyces scabies that stimulates potato scab[12]. the genus streptomyces within the order actinomycetales has spectacular diversity in morphology, genomic size, genomic g+c content, and amount of coding sequences. streptomyces bacteria are noted by their large and linearized chromosomes with approximately 8.5-12 mb in size and high gc content of about 67-78%[3,9,13]. the large genome of streptomyces could account for their ability to produce many secondary metabolites[14]. several studies have reported the discovery of over 20 biosynthetic gene clusters in the genome of streptomyces which could be associated with the production of secondary metabolites[4]. for examples, the genome of streptomyces coelicolor a3(2) was found to contain more than 20 gene clusters related to numerous metabolites[15]; genome of streptomyces avermitilis was found to contain more than 30 gene clusters related to numerous metabolites[16]. importance of streptomyces being an important soil microbial population, streptomyces has been extensively screened and isolated over the years due to its production of natural biologically active secondary metabolites that are medically and commercially important[4]. it has been an ongoing effort for the screening of novel streptomyces since the discovery of streptomycin and actinomycin from streptomyces griseus and streptomyces antibioticus respectively[17,18]. there are over 75 % of useful antibiotics that are commercially available for the use in both human and veterinary medicines have been produced by various streptomyces species[19]. many antibiotics belonging to different classes have been derived from streptomyces. for instances: chloramphenicol (chloramphenicol) from streptomyces venezuelae, neomycin (aminoglycoside) from streptomyces fradiae, clavulanic acid (β-lactam) from streptomyces clavuligerus, vancomycin (glycopeptide) from streptomyces orientalis, tetracycline (tetracycline) from streptomyces aureofaciens, and nystatin (polyenepolyol macrolide) from streptomyces noursei[20-22]. other than the capacity of producing antibiotics, streptomyces bacteria are producers of many other bioactive compounds with high functional and structural diversity such as anti-parasitic, anticancer/antitumour, antifungal, biocatalysts, biopesticides, immunosuppressive, and herbicides biological control agents[23-26]. they are also producers of enzymes which are important in environmental, food biotechnology and other industries[27]. streptomyces produces wood or fungal cell wall hydrolytic enzymes which include hemicellulases, chitinases, glucanases and cellulases to decompose a variety of plant-based polysaccharides, insoluble polymers like chitin and cellulose[28]. they are also involved in biodegradation and bioremediation at which they can degrade lignin and lignin-related aromatic compounds[28,29]. it is important to continue searching for novel streptomyces from different habitats in hope to find novel secondary metabolites such as new antibiotics to combat against pathogens which have gained resistance towards existing antibiotics via chromosomal mutation, vertical or horizontal gene transfer[30]. it is also essential to search for effective antimicrobial compounds to fight against infectious disease agents[31]. studies indicated that researchers have only discovered a fraction of antibiotics generated by streptomyces species. hence, it is still possible to isolate novel antibiotics from terrestrial streptomyces despite of being screened continually over the past half century[21]. nevertheless, exploring novel streptomyces from other areas could lead to the findings of other new/interesting compounds[32]. many studies have shown the successful isolation of novel streptomyces species from different environments including marine sediments[33], lakes[34], caves[35], mangroves[36], deserts[37], peat swamps[38], and plants[39]. researchers also found that many novel streptomyces species possess important bioactivities such as antioxidant, anticancer, and antifungal[40,41]. bioactive metabolites from streptomyces secondary metabolites are different from primary metabolites as they are not essential for growth, reproduction and other metabolisms in the cell[42]. they played an important role in the survival and defense mechanism for their producers to compete in a stressful environment which has a high amount of other metabolically active bacteria[32]. hence, prolific production of secondary metabolites is an advantage trait of streptomyces. streptomyces produces secondary metabolites when encountering nutritional stresses from the surrounding environment and the production of secondary metabolites is referred as physiological differentiation[43]. there is a close association between morphological differentiation and physiological differentiation in the life cycle of streptomyces[44]. it is apparent that morphological differentiation is related with the sensing of nutrient deficiencies and environmental stresses[8]. therefore, the generation of various secondary metabolites such as antibiotics or antifungals are initiated during the morphological transition of streptomyces from vegetative to aerial growth stage[44]. in most cases, production of antibiotics is depending on the spatial and temporal control of morphogenesis, metabolism, gene expression and metabolites flux[45]. in fact, bioactive secondary metabolites are produced by metabolic pathways that can be linked to primary metabolism; both secondary and primary metabolite pathways share intermediates, also, the intermediate or end products of primary metabolism are usually the precursors used to synthesize secondary metabolites[31,46]. moreover, the composition of the culture medium will also affect the capacity of streptomyces species in producing bioactive compounds[4,42]. bioactive secondary metabolites are synthesized extrataxonomic and characterization methods... 3 cellularly, which can be isolated and purified with highest purity from fermentation broth by using a combination of different extraction and purification methods. often, the methods utilized for extraction and purification of secondary metabolites are dependent on their applications. these methods include simple processes such as solvent extraction, chemical precipitation, and processes utilizing advance instruments such as ion-exchange chromatography, high performance liquid chromatography (hplc) and many more [4,31]. sometimes, these secondary metabolites may be obtained in the form of crude extracts which can be further subjected to chemical profiling using liquid chromatography-mass spectrometry (lc-ms) or gas chromatography-mass spectrometry (gc-ms) and thus allowing the prediction of compounds present in the extracts prior to purification [47,48]. taxonomic and characterization methods of streptomyces due to large-scale isolation and screening over the years, there were nearly 3000 species of streptomyces named by year 1970 with most of them identified only on patent literature in the past[49]. most organisms were given a new name solely based on small variations in cultural and morphological characteristics or for the sake of meeting publications or patent requirements. the issues arisen from this situation include several confusions in the criteria for streptomyces speciation, risks of misclassificaiton as well as concerns on the competency of the individual who describing the new organism as reported by trejo (1970)[50]. fortunately, amendments on their taxonomy have been carried out to address these issues [49,50]. to date, streptomyces genus consists of 844 species and 38 subspecies that are validly described (www.bacterio.net). taxonomic characterization of streptomyces is undeniably more complicated and challenging as a result of the large amount of described species in the genus streptomyces which is relatively greater as compared to that of other microbial genera[51]. the techniques used for characterization of streptomyces have improved over time; from classical morphological classifications such as spore chain morphology, colour of substrate and aerial mycelia to numerical taxonomic analyses that include phenotypic characterization based on standardized sets and, now, applying molecular and phylogenetic analyses[51,52]. international streptomyces project (isp), a traditional approach international streptomyces project (isp) is one of the approaches for the classification of streptomyces strains since 1964 with the objective of producing reliable characterization of existing type strains of both streptomyces and streptoverticillium species. isp was amended and enacted by the subcommittee on taxonomy of actinomycetes of the committee on taxonomy of the american society of microbiology and the subcommittee on taxonomy of actinomycetes of the international committee on bacteriological nomenclature[51,53]. isp works by sending type and neotype strains of species under these genera to a minimum of three experts in different nations to be investigated under strict standardized experimental and media settings for the identification of morphological characteristics, pigmentation, and carbon utilization profiles of the strains[53,54]. the protocols and outcomes of isp studies were previously published and the resulting identified type strains were deposited in a few service collections which are internationally recognized[53,55,56]. although isp has always been a standard method for the classification and characterization of streptomyces, however, it is not the best approach since it is heavily depending on limited number of selected features or standard phenotypic criteria that are highly based on morphological and pigmentation characteristics[51]. hence, other methods have been established to further assist in the classification and characterization of bacteria. molecular characterization of streptomyces, a modern approach identification and characterization methods have evolved to molecular and phylogenetic characterizations with analysis of gene sequences which targets predominantly linear 16s rrna gene sequences due to the advent of polymerase chain reaction (pcr), dna-dna hybridization (ddh) and dna sequencing approaches[57]. molecular revolution in the last 30 years allowed not only further insights into molecular cell biology but also brought huge improvement to the study of evolution, conservation and ecology. in other words, genetic techniques enable taxonomic identification of bacteria which can hardly be delineated based on morphological characteristics alone, accompanied with higher efficiency in terms of rapid and high-throughput identification[58]. molecular characterization polymerase chain reaction (pcr) pcr is invented in 1980s by the american biochemist, kary mullis, as a revolutionary method[59]. the invention of pcr plays a crucial role in improving the knowledge of dna evolution and phylogenetic relationships among streptomyces species. an adequate amount of a particular gene or dna fragment is needed to enable the detection and identification of gene sequence. to achieve this practical amount of dna, the application of pcr is able to specifically and rapidly amplify any dna stretch within size limits which is flanked by synthetic oligonucleotide known as primers to produce up to millions or billions of copies of a particular nucleotide sequence in approximately two hours[60]. these primers with approximately 20 base pairs in length will determine the 5’-3’ end of the target region of the double-stranded dna which is the target of amplification. primers are used to initiate dna synthesis while heat-stable dna polymerase such as taq polymerase is used to produce a new dna strand enzymatically from single-stranded dna template by adding nucleotides. pcr amplification is operated in a thermal cycler that law jwf et al. 4 consists of repeated cycles of heating and cooling reaction at a series of specific temperature to melt and replicate the target dna sequence[60,61]. there are three major steps in a pcr amplification process: denaturation, annealing, and extension[61,62]. the pcr reaction begins when high temperature (e.g. 90-97 ºc) is used to melt and separate the double-stranded dna molecule for one to several minutes to form single-stranded dna. next, primers are used to anneal to the dna template strands at a lower temperature of 50-65ºc. basically, the annealing temperature is approximately 3-5 ºc lower than the lowest melting temperature of primers pair used. dna polymerase then extents and synthesizes new complementary dna strand at the end of the annealed primers at approximately 72 ºc for 2 to 5 minutes. consequently, the original dna is duplicated at which each of the new dna molecules are made up of one new and one old dna strand. these two new dna strands can then be used as dna template to produce more copies in coming cycle. this cycle of denaturing, annealing, extending and synthesizing is repeated up to 30 or 40 times to produce up to billions of identical copies of the original dna sequence. pcr is widely used in laboratories for many applications including sequencing, genetic engineering and cloning[61]. the application of pcr is certainly useful for the rapid identification of an organism at genus or species level, depending on the primers used. many studies have been utilizing pcr for the identification of the genus streptomyces, especially via the detection of 16s rrna gene[63-65]. molecular characterization box-pcr box-pcr is a repetitive element sequence-based pcr (rep-pcr) fingerprinting technique which possesses high discriminatory power and can be used for typing purpose[66]. it is based on the box dispersed-repeat motif, initially found in streptococcus pneumonia but appears to be common in most of the bacterial species. this 154 base pair(bp)-box elements comprising 3 subunits (boxa 59bp, boxb 45bp, and boxc 50bp) represent the naturally occurring, highly conserved and repetitive sequence elements present in multiple copies which are found in most gram-negative and some gram-positive bacterial genomes[66,67]. box-pcr can simultaneously survey many dna regions scattered in the genome of bacteria because the box repetitive sequences are interspersed throughout the bacterial genome[68]. the primer used in box-pcr (box a1r primer) targets and amplifies selectively the regions located between box elements, thereby resulting in dna amplification products of different sizes which serve as dna fingerprints of the bacteria. these amplified fragments will then be resolved using gel electrophoresis to produce box-pcr genomic fingerprinting profile for differentiation at species, subspecies, and strain level[67,68]. box-pcr is inexpensive, efficient and easy to perform, concurrently offering equivalent or superior discriminatory power as compared to other methods such as restriction fragment length polymorphism (rflp) and amplified fragment length polymorphism (aflp)[68]. molecular characterization dna-dna hybridization (ddh) ddh is a dna-based molecular technique used to indicate genetic distance or genetic similarity between species, particularly between those closely related species, through genome-wide comparisons. in the 1960s, ddh has been employed by bacterial taxonomists and it is well known as the gold standard for species delineation and for taxonomic evaluation of strain [69-71]. dna-dna reassociation method is often conducted in conjunction with other tests such as morphological, physiological and phylogenetic tests [70]. ddh is a process where two dna molecules from different biological sources or organisms are mixed and heated to form single-stranded dna before cooling step. the dna of one of these sources is label while another is unlabeled. these single-stranded dna are then allowed to reanneal to reform the hybrid double helices or dna-dna hybrids based on contingent sequence homology of the two organisms. besides that, values of ddh are usually expressed in percentage to quantitatively estimate the genome-wide similarity of the dna sequence. for instance, two strains that are from same species will have a high value of hybridization which is typically more than 70% while two strains of different species but same genus would have around 30-60% of hybridization value. if the unlabeled dna is from the same organism as the labelled dna then the hybridization value would be 100% [72]. ddh analysis is one of the assays included in polyphasic taxonomy which is performed in taxonomic studies involving the delineation of taxa at all levels[73]. the application of ddh can be seen in many recent studies pertaining the discovery of novel streptomyces, for instances, discovery of streptomyces mangrovisoli sp. nov.[74], streptomyces humi sp. nov.[75], and streptomyces gilvigriseus sp. nov.[76]. henceforth, emphasizing the importance of ddh as a criterion for the delineation of bacterial species despite being time-consuming and labor-intensive[69]. molecular characterization – next generation sequencing (ngs) the rapid evolution of new molecular technologies leading to the development of dna sequencing technologies has revolutionized biological and biomedical related research. these studies assist scientists in many applications including molecular cloning, search of pathogenic genes, comparative and evolutionary studies[77]. in this context, the sequencing of bacterial genome has becoming more common and being routinely applied in many microbial studies. during the early 1990s, dna sequencing is based on the sanger’s method, also identified as dideoxy chain termination sequencing chemistries. however, the sanger’s “first generation sequencing” method has eventually reached a plateau in terms of technical development, along with several limitations faced by this technique when comes to whole genome sequencing such as time consuming and resource intensive[78-80]. in 2005, the introduction of “next generation sequenctaxonomic and characterization methods... 5 ing” (ngs) technologies has offer more advantages than sanger’s method in the aspects of producing massive volume of data at rapid high-throughput and cost-effective manners[77,78]. besides, the latest ngs technologies operate differently than the sanger’s method as they employ various massively parallel sequencing instruments that are commercially available, for examples, 454 (roche), solexa (illumina), and solid (applied biosystems) platforms[81]. all of these instruments have their respective work flows, but they generally share some similar features: (1) simplified initial preparatory steps, (2) library preparation by dna fragmentation and amplification, and (3) sequencing reaction is conducted followed by automated detection using imaging systems[78,81,82]. with the availability of numerous massively parallel sequencing platforms and the dramatic decrease in the costs for sequencing, it is anticipated that ngs will be routinely used for the whole genome sequencing of bacterial genome, hence, enables the extraction of biologically or clinically useful insights from the genome sequence[32,81]. target genes for streptomyces characterization ribosomal rna genes the taxonomic classification of streptomyces species based on morphological, biochemical, nutritional and physiological characterizations are challenging and often problematic [51,83,84]. therefore, molecular approaches including phylogenetic analysis can be an improvement in evolutionary and diversity studies. the rrna gene sequences which code for the ribosomal subunits such as 16s, 23s and 5s rrna are the target of phylogenetic analysis due to the highly conserved rrna structure in all cells throughout evolution[51]. majority of the rrna folding have important functions regardless of the divergence in primary sequence. precise spatial relationships are needed to produce functional ribosomes, resulting in some regions of rrna genes which are linked with other components in ribosome are conserved[85,86]. the rrna gene sequences are universally distributed across distantly related strains, and therefore these sequences can be aligned precisely to make measurement of true differences between them easier [87,88]. however, these rrna genes also consist of hypervariable regions such as gamma region that diverged over evolutionary time which can be used for species discrimination. ribosome is defined as large ribonucleoprotein which synthesizes protein and highly conserved among the three life kingdoms. for prokaryotes, ribosome is made up of two subunits which are the small 30s subunit and large 50s subunit. the small 30s subunit consists of a 16s rrna and 20 proteins while large 50s subunit consists of a 5s rrna, a 23s rrna and over 30 proteins. thus, the conserved regions in rrna genes are used as tools to study distant phylogenetic relationships while regions between the conserved regions with higher mutation rates are used to discriminate closely related bacteria. it is also important to note that within the highly constrained rrna genes that are essential for survival of organisms, horizontal gene transfer events are usually implausible to happen[86]. ribosomal rna genes 16s rrna gene in general, the 16s rrna gene is the gold standard in the identification of taxonomic and phylogenetic relationships among different bacteria[83]. the 16s rrna gene is an essential constituent found in all bacteria, containing highly conserved regions and variable regions with approximately 1600 nucleotides long (bp)[88]. the 16s rrna gene can clearly distinguish the three main kingdoms which include archaea, bacteria and eukarya. the hypervariable regions of 16s rrna gene in a single genome provides sufficient sequence variation for phylogenetic discrimination[88]. generally, 16s rrna gene is still sensitive enough for other genera but unfortunately seems to be less sensitive for streptomyces. previously, many studies attempted to utilize sequences from variable regions of 16s rrna gene to form taxonomic structure within the streptomyces genus. however, the variation was too low to distinguish between closely related streptomyces species/ strains as they might exhibit highly similar or identical 16s rrna gene sequences, with the addition of overspeciation issue within the genus[89,90]. alternately, streptomyces strains with highly similar or identical 16s rrna gene sequences appear to be of different species[49]. furthermore, there are multiple copies of 16s rrna gene or heterogeneous rrna operons within a single genome have been found in some bacteria[87]. this might suggest that there is a potential for the occurrence of horizontal gene transfer events in the rrna genes[87,90]. hence, phylogenetic construction based on 16s rrna gene sequences could not assure well-resolved phylogenetic trees that represent relationship between bacterial species particularly within genus streptomyces[9,12,51]. the 16s rrna gene sequences provide low resolution as phylogenetic marker for streptomyces species. furthermore, insignificant phenotypic differences, limitations of dna fingerprinting and dna-dna hybridization appear to ineffectively elaborate taxonomic grouping of streptomyces species within the genus streptomyces[91]. this suggests that it is challenging for streptomyces taxonomic classification, thus, methods with higher resolution are crucial to evaluate taxonomic grouping among closely related streptomyces species. ribosomal rna genes 23s rrna gene the 23s rrna gene is formerly less preferred to be used for phylogenetic study as compared to 16s rrna gene due to costing consideration[9]. however, the use of 23s rrna gene in phylogenetic analysis is eventually being highlighted because of several factors including reduced sequencing costs due to the advancement of technology such as next generation dna sequencing[92]. the 23s rrna gene offers similar advantages as 16s rrna gene with additional properties owing to its longer sequence length of approximately 3000 bp such as better resolution due to greater sequence variation, unique insertions and/or deletions, and more characteristic sequence regions[9,86,92,93]. recent study conducted by chaves et al. (2018)[94] revealed that the application of 23s rrna gene can be utilized as an alternative to 16s rrna gene for phylogenetic study of streptomyces species which also appeared to be more discriminative than the 16s law jwf et al. 6 rrna gene. in addition, previous study showed that broadrange primers for 23s rrna genes with a similar universality degree to that of 16s rrna genes can be generated based on the conserved regions of 23s rrna genes[92]. protein-coding genes the use of protein-coding genes as phylogenetic markers has become more common as they could determine genome relatedness with higher accuracy and might replace ddh for species taxonomy in the future[70,83,95,96]. currently, multilocus sequence analysis (mlsa) technique that involves the use of a combination of a few proteincoding genes (also referred as housekeeping genes) has been widely applied to deduce bacterial phylogeny and aims to buffer phylogeny distortions caused by recombination[97]. this approach could be an alternative to ddh technique since it is reproducible with comparable resolution to ddh[89]. studies have reported that there was a high correlation determined between ddh and mlsa at which mlsa evolutionary distance of 0.007 based on fivegene schemes corresponds to ddh value of 70% species cut-off point for species delineation of the genus streptomyces[91,97,98]. protein-coding genes are proven to improve the resolution and robustness at streptomyces species level and can be used as an acceptable approach for species differentiation. especially the gyrase b (gyrb) and tryptophan b (trpb) housekeeping genes can provide greater discriminatory power of closely related strains due to their higher percentages of variable regions and higher molecular evolution rates as compared to 16s rrna gene[98]. the higher evolutionary rate could be explained by the synonymous substitutions predominantly at the third position of codons in these protein coding genes that occur without affecting the amino acid sequences[9]. numerous streptomyces phylogenetic analyses were conducted using protein-coding genes due to their higher resolution power, for examples, taxonomic studies of streptomyces griseus[83,98] and streptomyces hygroscopicus [91], and diversity studies on the family streptomycetaceae[9,51]. protein-coding genes dna gyrase subunit b, topoisomerase type ii (gyrb) the gyrb gene is one of the structural genes of dna topoisomerases; the dna topoisomerases are responsible for dna replication, recombination, transcription and repair, also controlling supercoiling level[99]. the gyrb gene encodes for the subunit b protein of bacterial dna gyrase (dna topoisomerase type ii) that is responsible for dna replication and involves in direct dna repair mechanism[96,100]. the specific functions of dna gyrase include supercoiling relaxed closed circular double-stranded dna negatively with atp hydrolysis, whilst relaxing supercoiled dna molecule in the absence of atp hydrolysis[100,101]. the gyrb gene is found universally distributed among different species of bacteria. as compared to ribosomal 16s rrna gene, gyrb gene has faster molecular evolution rate and also rarely undergo horizontal gene transfer[96,101]. many studies have proven that the consistency between the results of ddh and gyrb-based phylogenetic analysis[70,96,101], whereby approximately 98.5 % gyrb gene sequence similarity could corresponds to dna-dna relatedness threshold value of 70 % for species delineation[70,102]. protein-coding genes tryptophan synthase subunit b (trpb) the trpb gene encodes tryptophan synthase subunit b which is responsible for amino acid biosynthesis. it catalyzes the last two steps of tryptophan biosynthesis by causing indole and serine to be condensed irreversibly to produce tryptophan in the pyridoxal phosphate-dependent reaction[100,103]. protein-coding genes such as trpb gene is often used for phylogenetic analysis because it is selectively neutral and is not affected by selection pressure for amino acid changes. this implied that trpb gene locus is independent as it underwent evolution[83]. the trpb gene has been used individually or in combination with other protein-coding genes for the taxonomic characterization of streptomyces in previous studies and had successfully resolved the phylogenetic relationships between closely related subspecies and species of actinobacteria due to the high genetic variation within these genes as compared to ribosomal genes[9,51,83,89,90,104]. tools and databases to fetch genes basic local alignment search tool (blast) blast stands for ‘basic local alignment search tool’ and it is a sequence similarity search program, accessible as an independent tool or via a web interface. it works by comparing the query sequences either nucleotide or protein sequences to an appropriate nucleotide or protein sequence database. this is to determine type sequences that resemble the query sequence above a particular threshold level. evolutionary relationships between sequences can then be indicated based on the sequence similarity found between query and type strains[105]. ezbiocloud database ezbiocloud is the latest improved version of open access database developed by chunlab inc., prospers the former versions namely eztaxon and eztaxon-e (https:// www.ezbiocloud.net/). it is an integrated database that contains comprehensive hierarchical taxonomy of both bacteria and archaea domains from phylum to species level, represented by quality controlled 16s rrna gene and genome sequences[106]. in the initial version of database, eztaxon, contains 16s rrna gene sequences of all validly described prokaryotic type strains which serves as a web-based tool for the analysis of 16s rrna gene sequences including the determination of pairwise nucleotide similarity values of the gene sequences[107]. eztaxon was later evolved to the next generation database known as the eztaxon-e. extaxon-e database was enhanced by including species with non-validly published names, uncultured phylotypes, and the incorporation of a new function involves the estimation of degree of completeness in sequencing. besides, sequences of all species and phylotypes within this database have undergone comprehensive phylogenetic analysis based on 16s rrna gene sequences to produce a complete hierarchical taxonomic system[108]. despite the identification and comparison of 16s rrna genes being a reliable method taxonomic and characterization methods... 7 to determine the identity of a strain of interest, however, it comes with a limitation where it does not guarantee that two strains with nearly identical 16s rrna gene sequences belong to the same species, alternatively they could be of different species[109]. hence, analyzing bacteria genome sequences has become a method to overcome this issue. the latter ezbiocloud united database contains information on both 16s rrna gene and genome sequences as well as several bioinformatics tools such orthoani. furthermore, all genomes in this database were quality filtered where the taxonomic identification at genus, species or subspecies levels was performed according to the algorithm comprising a combination of gene-based search and orthoani[106]. currently, ezbiocloud holds up to 81,151 taxa and 99,418 qualified genomes (available at https://www.ezbiocloud. net/dashboard, accessed on august 2018). national center for biotechnology information (ncbi) genbank database ncbi is a national resource for molecular biology information established in year 1988, which involves in the development of computational biology and software for genome analysis[110]. the ncbi is the host of the genbank a comprehensive public online database that contains dna sequences, supporting bibliographic and biological annotation (http://www.ncbi.nlm.nih.gov/genbank/)[111]. newly discovered nucleotide sequences such as the non-coding dna region, a particular gene region or coding region of a dna sequence, and whole genome shotgun as well as other sequence data will be submitted to this database often by individual laboratories/authors and bulk submissions from high-throughput sequencing projects. direct submissions to genbank can be done via a web-based form named as bankit or stand-alone submission program known as sequin. genbank is established as part of the international nucleotide sequence database collaboration, thus, it contains data from other databases such as the european molecular biology laboratory (embl), and dna databank of japan (ddbj). consequently, genbank holds dna nucleotide sequences for more than 300,000 validly described species[111,112]. phylogenetic trees reconstruction phylogenetic is the study of evolutionary relationship while phylogenetic analysis is the estimation of these evolutionary relationships. phylogenetic indicates changes caused by evolution and understand relationships between ancestor and its descendants to estimate divergent time and the evolution in a family[113,114]. phylogenetic trees can be built by choosing an appropriate phylogenetic information marker (e.g. a particular dna gene sequence, rna) of prokaryotes or eukaryotes to determine the degree of relatedness between species, family, genus or order and their hypothetical common ancestors[1,51,115]. phylogenetic trees consist of nodes and branches, the node is the taxonomic unit or evolutionary event while branch is link between two adjacent nodes. the length of a branch represents the divergence. branching presents an estimated pedigree of evolutionary relationships among various organisms[115]. clade is a monophyletic taxon with a set of descendants which are originated from a single most common ancestor, at which the members of a clade are more closely related to one another than members of other clades. members of the same clade possess a common evolutionary history and share unique features which could not be found in distant ancestors[1,114]. there are two types of phylogenetic trees which are: rooted tree that consists of a common ancestor and unrooted tree which has no common ancestor. an outgroup is the least related operational taxanomic unit to the group of taxa being studied. it is used to root a tree and as a reference to determine evolutionary distance[115]. bootstrapping analysis bootstrapping is one of the statistical methods to determine the reliability of tree branch arrangement/topology, in other words, to estimate the confidence intervals on phylogenies. the phylogenetic tree built is not the actual representation of the evolutionary relationships, instead, it is an estimation of these relationships. hence, bootstrapping analysis is requested to calculate the reliability of that estimate. columns in a multiple aligned sequences are resampled during bootstrapping process to produce many new alignments sets and replace the original dataset. the bootstrapping process is continued for at least 100 times while phylogenetic trees are produced from all these alignments sets. the final outcomes determine the number of time a particular branch point is generated out of the total number of the built phylogenetic trees. the branching point will be more valid when the number of occurring time is larger. bootstrap values are statistically significant if they are ranged between 90 to 100 %. phylogenetic trees are produced multiple times and the trees produced are not always identical during every production. for instance, a node with a particular cluster descending from it might produce bootstrap value of 90 % when a thousand of trees are built randomly, thereby inferring that node with that identical cluster descending from it appears for nine hundred times out of the one thousand times the tree were rebuilt[115-117]. bioedit sequence alignment editor software bioedit is a free of charge biological sequence alignment editor and sequence analysis software program available online for windows. bioedit is commonly used in many molecular biology studies, it provides auto-integration with clustal w multiple alignments, manipulation, trimming and editing of aligned sequences. the sequences of type strains are aligned with query sequences to determine differences between them. besides consisting options for sequence analysis, it also provides links to external analysis programs such as blast[118,119]. clustal w alignments alignment process is the crucial key for the construction of phylogenetic trees at which poor alignment would result in incorrect phylogenetic tree[120]. an alignment is considered as good if it consists of no gaps. regions which do not align between sequences should be deleted before building of phylogenetic trees. clustal w method is a computational multiple alignment methods that law jwf et al. 8 align sequences based on an explicitly phylogenetic criterion or known as a ‘guide tree’. the generation of guide tree is based on the initial pairwise sequence alignments while its guide tree is formatted as a phylip tree file. clustal w tool aligns all the sequences according to the matching identical or similar regions found between these sequences. the ‘w’ stands for ‘weighted’ which refers to the different weights that are applied to parameters and sequences in different regions of the alignment. during the pairwise alignments, distance matrix is calculated among each sequence. distance is defined as amount of identical matches divided by the length of sequences regardless of gaps[121]. mega software molecular evolutionary genetics analysis (mega) software provides various tools for analyzing dna and protein sequences according to evolutionary perspective. mega is widely utilized for assembling sequence alignments, constructing phylogenetic trees, mining online databases, testing selection, estimating molecular evolution rate and divergence times to study molecular evolutionary histories of genes, genomes and species[122,123]. several versions of mega have been developed, where the initial version mega 1 had made available since early 1990s[124]. mega version 1 has been further evolved up till mega version 6, in which the progress involved multiple upgrades for the use in microsoft windows including improved computational algorithms and statistical methods[122,123]. mega 7 was later introduced as the latest 64-bit version with greater computational power that capable of handling memoryintensive analysis of large datasets[125]. recently, mega x has been developed as cross-platform version that is able to run natively on linux and microsoft windows with additional upgrade includes the application of multiple computing cores for various molecular evolutionary analyses[123]. tree-building methods tree building methods can be divided into two different methods: (1) distance-based, and (2) character-based. character-based methods generate trees that optimize the distribution of actual data for each character. examples of character-based methods are maximum likelihood and maximum parsimony algorithms. distance methods calculate pairwise distances based on certain measure, followed by elimination of the actual character data while only utilize the fixed distances to obtain trees. hence, pairwise distances are not fixed because they are identified by the tree topology. neighbour-joining is one of the commonly used algorithm in distance-based methods[114,126]. character-based method this method works by using character data in every step of phylogenetic analysis at which aligned sequences such as dna sequences are used directly during tree analysis. it also enables the reliability of the position of each base in an alignment to be assessed based on the positions of all other bases. character-based methods examine every alignment column individually and determine the tree which best accommodates all of the information[114,126]. character-based method maximum likelihood (ml) algorithm ml algorithm builds a tree based on mathematical models the probability models to deduce the evolutionary distances. it determines the evolutionary model and tree which consist of the highest likelihood of generating the observed data. it searches the most likely tree from all the trees of the given dataset with the help of a tree model for nucleotide substitutions. ml is calculated for each base position in the multiple alignment by determining the likelihood that a certain pattern of variation at a particular site would be generated by a particular substitution reaction when a particular tree and the overall observed base frequencies are provided. likelihood is the total of the probability of each particular variation pattern produced by a particular substitution process. a tree that best accounts for the high number of variations of the dataset given based on likelihood calculations will be generated[1,114,127]. character-based method maximum parsimony (mp) algorithm the mp algorithm builds shortest phylogenetic tree that minimizes the amount of evolutionary change or consists of the fewest amount of evolutionary changes such as nucleotide substitution to explain the evolutionary differences found among different taxa. the mp algorithmtree contain of the least parallel changes as compared to nj and ml algorithms. ancestral relationship can be determined as the mp algorithm uses all the evolutionary information in every nucleotide bases[1,114,127,128]. distance-based method this method build tree according to the evolutionary distance or total of dissimilarity between two aligned sequences. a true tree will be generated based on this method if all the event of genetic divergence in the sequence were recorded accurately. the sequence data will first be converted into pairwise distance in order to build tree using the matrix of pairwise distances between gene sequences in an alignment regardless of the character data. moreover, the tree is generated according to the distance values and clustering algorithm[128]. distance-based method neighbour joining (nj) algorithm nj algorithm is a commonly used distance-based method for the reconstruction of phylogenetic trees[113]. it calculates the amount of differences between two sequences and the actual tree is then generated based on the matrix of distance values[113,127]. the tree construction begins when the most similar sequences or pairs of closest neighbours are joined. then, a node (common ancestor) is successively inserted between them before next closest neighbours are chosen and added with node. the process is repeated until a tree with a combination of nodes that gives the smallest total branch lengths is produced. nj algorithm works by trying to locate all neighbours in correct positions before correcting the length of branches[129]. it is frequently used by users due taxonomic and characterization methods... 9 to its fast computation speed and high accuracy which is equivalent or better than other computationally intensive algorithms[114]. substitution model substitution model is used to determine the probability or rate and how one nucleotide replaces or substitutes another. they calculate evolutionary distance between sequences based on the observed differences found between sequences. this evolutionary model works based on the measurement of normalized distance which is the mean degree of change per length of aligned dna sequences. thus, calculations of distance between sequences are improved. selection of substitution model is important as it influences the alignment and tree building and thus the right substitution model could result in most accurate phylogenetic relationship[1,114]. substitution model kimura-2 parameter kimura-2 parameter is used to calculate evolutionary distances and similarity values in nj tree-building algorithm. it is an evolutionary model which states that transitions which is a change between pyrimidines or between purines (c to t or a to g) occur more frequently as compared to transversions which is a changed between purines to pyrimidines or vice versa (a to t or c to g) when the substitution rate is unequal. hence, the rate of transitional nucleotide substitutions is greater than rate of transversional nucleotide substitutions[130,131]. substitution model tamura-nei this is used to discriminate the rates of two types of transitional substitutions, at which transitional changes between a↔g (purine ↔purine) may occur at different rate as compared to that between c↔t (pyrimidine ↔ pyrimidine) while transversions happen at same rate and this rate can be different from both transitional substitutions rates. therefore, two different types of transitional substitutions rates and transversional substitutions rate are estimated individually with the consideration of base composition bias which refers to unequal frequency of all four nucleotides[132,133]. general discussion the identification and taxonomic characterization of streptomyces species could be relatively challenging mainly because of the large amount of species present in this genus. several techniques are often required to be conducted simultaneously to produce a more reliable result for classification of bacterial species. fortunately, the rapid progress in molecular techniques has greatly improve the efficiency of bacterial species identification and characterization. traditional phenotypic approach may not be sufficient for describing or differentiating taxa as certain phenotypes changes could be caused by horizontal gene transfer event[57,73]. the incorporation of both molecular genotyping and phenotyping methods could assist in determining the correlations between both traits and thus lead to a better understanding of the streptomyces taxonomy[57]. the application of pcr, ddh, and ngs have govern modern taxonomic studies and these techniques are important to generate genotypic data for classification and allocation of taxa on phylogenetic tree[73]. ribosomal rna genes and protein-coding genes are commonly used molecular markers for phylogenetic studies of streptomyces (figure 1). the rrna genes are typically considered as best phylogenetic markers as they are universally distributed across living organisms and they made up of a combination of highly conserved, variable, and hypervariable regions that evolve at varying rates, thereby facilitate the differentiation among distinct bacterial groups[73,134,135]. the rrna genes have slower evolutionary rate as compared to the protein coding genes, hence, rrna genes are important phylogenetic markers for analyzing the phylogenies of distantly related species[134]. among the rrna genes, 16s rrna gene acts as a very powerful discriminatory tool in resolving phylogenetic relationships and this gene has been the most sequence phylogenetic marker[135]. however, it is arguable whether the gene sequence of 16s rrna gene should be the gold standard for streptomyces phylogeny. many studies have suggested that 16s rrna gene sequence alone is not sufficient enough to discriminate between closely related species, notably species within streptomyces genus due to its low resolution at species level[51,98]. this issue is because of many species in the genus streptomyces share identical or highly similar 16s rrna gene sequence in addition to highly similar phenotypes[9,51]. henceforth, the implication of other molecular targets with higher resolution in addition to the 16s rrna gene is recommended for the phylogenetic analysis within the genus streptomyces to observe their actual relationships. as an alternative, the 23s rrna gene is employed in taxonomic classification of streptomyces species. the 23s rrna gene offers better resolution than 16s rrna gene as it has more variable regions. in the early days, it appears that 23s rrna gene has somewhat lost favor to 16s rrna gene as phylogenetic marker in taxonomic classification which might due to difficulties in developing broad-range sequencing primers and sequencing of larger genes. though the use of 23s rrna gene has now eventually increased with time and advances in sequencing technology[86]. nevertheless, it is important to acknowledge that intragenomic or intraspecific diversity of rrna genes is a common phenomenon in streptomyces that could lead to false identification regarding phylogenetic relatedness and ancestry when there is only a single gene sequence used[86,95,136]. besides, rrna genes may not be able to provide high resolution when it comes to distinguishing streptomyces species as the functional constraints on both 16s and 23s rrna genes for them to be highly conserved to maintain the precise spatial relationships for producing functional ribosomes. conserved regions are suitable for investigating organisms with distant relationships, whilst variable regions with faster evolutionary rate are suitable for differentiating organisms that are closely related[86,91]. ribosomal genes were demonstrated to be insufficient to discriminate between law jwf et al. 10 closely related streptomyces species by a number of studies despite of being a powerful tool in elucidating interand intrageneric evolutionary relationships[83,91,137]. protein-coding genes (housekeeping genes) such as gyrb and trpb genes have exhibit potential for taxonomic classification of streptomyces at species level and have been shown to be effective discriminatory tools for phylogenetic analysis. also, the utilization of multiple protein-coding genes in mlsa has been conducted in several biosystematics studies of streptomyces species[51,83,91]. proteincoding genes produce higher resolution in discriminating streptomyces at species level than the rrna genes because of the presence of more variable regions in the gene sequences[9,91]. additionally, protein-coding genes such as gyrb gene tend to exhibit higher number of base substitutions as compared to rrna genes. ochman et al. (1987)[138] reported that 16s rrna gene had average base substitution rate of 1 % in 50 million years whereas the average base substitution rate of gyrb gene at the synonymous sites was found to be about 0.7 % to 0.8 % in one million years. as a result, the higher percentage of base substitution rate accounts for higher rate of evolution of protein-coding genes than rrna genes and thus improve their resolution as phylogenetic markers that capable of determining the relationships between closely related streptomyces species with the generation of stable phylogenetic trees[9,91]. the advent of ngs technology has increase the availability of whole genome sequences of many prokaryotes, along with the rapid progression of bioinformatic tools which are important for analyzing these sequences. the inclusion of whole genome sequencing data could further enrich the prokaryotic systematics by overcoming the limitations of current approaches (e.g. ddh, 16s rrna gene sequences etc.)[139]. there is an increasing evidence that phylogenetic and taxogenomic approach offers robust classification as it is capable of resolving complex taxa below family level (intrageneric and intrafamily)[12,139-141]. therefore, providing reliable information on the relationship and differentiation of prokaryotes at species and genera level. the taxonomic classification of prokaryotes utilizing genomic data is likely becoming the norm in near future. figure 1. framework of ribosomal genes and protein-coding genes as molecular markers for phylogenetic analysis based on different algorithms. conclusion streptomyces bacteria have been a focus in systematic research due to the various beneficial properties they can offer towards mankind. however, there are taxonomic chaos and overspeciation problem occurred within the genus streptomyces. thus, it is important to search for solutions that could ease the taxonomic classification this complex taxon. several important molecular makers including ribosomal genes and protein-coding genes where each has their own advantages and limitations in determining the phylogenetic relationships of streptomyces species. the availability of various molecular techniques, bioinformatic tools, databases, sequence editing and phylogenetic tree reconstruction software have greatly assist in taxonomic studies of streptomyces. advances of ngs and associated bioinformatic tools will revolutionize taxonomic practice through the integration of genomic approach. it is anticipated that genome-based phylogenetic analysis will further improve the identification and taxonomic classification of members in the genus streptomyces. authors contributions the literature review and manuscript writing were performed by jw-fl, k-xt, shw, n-sam, and l-hl. l-hl founded the research project. conflict of interest the authors declare that the research was conducted in taxonomic and characterization methods... 11 the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. acknowledgments this work was supported by the mosti escience fund (02-02-10-sf0215) and external industry grants from biotek abadi sdn bhd [vote no. gba-808138 and gba808813] awarded to l-hl. reference 1. reddy bn. basics for the construction of phylogenetic trees. web central biology 2011; 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vengadesh letchumanan1*, wen-si tan2, wai-fong yin3, kok-gan chan3,4 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2illumina singapore pte ltd, woodlands industrial park e1, singapore 3division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia 4international genome centre, jiangsu university, zhenjiang 212013, pr china abstract: the members of vibrionaceae family are gram-negative bacterium are ubiquitous in marine and estuarine environments. this diverse group of bacteria include many pathogenic strains that potentially cause infection to human and aquaculture animals. vibrio cholerae, vibrio parahaemolyticus and vibrio vulnificus are among the few recognized as a major, worldwide cause gastroenteritis, particularly in countries where seafood consumption is high. the control of these vibrios has been a hurdle due to the rising numbers of antibiotic resistant strains in the environments. we report the genome sequence of vibrio sp. oull4 isolated from shellfish. the availability of this genome sequence will facilitate the study of its antimicrobial traits, as well as add our knowledge of vibrio sp. diversity and evolution. keywords: vibrionaceae; infection; gastroenteritis; antibiotic; genome received: 27th february 2020 accepted: 24th march 2020 published online: 6th april 2020 citation: letchumanan v, tan w-s, yin w-f et al. genome sequence of vibrio sp. oull4 isolated from shellfish. prog microbe mol biol 2020; 3(1): a0000066. https://doi.org/10.3687/pmmb.a0000066 introduction seafood production has double over the years to meet the rising consumer demand for seafood. this involuntary action has exposed aquatic animals to bacterial infections[1,2]. this situation gets complicated and worsen by the emergence of resistant vibrio sp. strains, which hampers medical care. vibrio sp. is a gram-negative halophilic bacteria that belongs to the vibrionaceae family[3–8]. they naturally inhabit the aquatic surroundings and associated with aquatic animals for example crustaceans, molluscs and fish[9–13]. the world health organization (who) has acknowledged antibiotic resistance as a public health hazard that affects millions of people worldwide[14]. due to excessive use of antibiotics in the aquaculture sector, the incidence of resistance accelerated, mostly among foodborne pathogens such as vibrio sp.[15–23], listeria sp.[24–26], and salmonella sp.[27–32]. the resistant foodborne pathogens poses a threat and challenge to drug discovery programmes worldwide[33,34]. therefore, it is important to continuously monitor and manage the resistant vibrio sp. in seafood and environments. vibrio sp. oull4 strain was isolated from shellfish originated from a supermarket in selangor, malaysia. the strain presented a large yellow colony on selective media—thiosulphate citrate bile salt sucrose (tcbs) agar. the antibiotic susceptibility test was performed to determine to resistance phenotype of vibrio sp. oull4 strain. the strain was resistant to 11/14 antibiotics tested, namely the ampicillin, ampicillin/sulbactam, 3rd generation cephalosporin (cefotaxime, ceftazidime), aminoglycoside (amikacin, gentamicin, kanamycin), suphamethox/trimethoprim, oxytetracycline, tetracycline, and chloramphenicol. this is a worrying scenario as the antibiotic resistant profile exhibited by the strain is among the recommended antibiotics agents used in treatment if vibrio sp. infection[35–37]. the vibrio sp. oull4 strain was selected for genome sequencing to further explore and understand the antibiotic resistant traits. data description the genomic dna of vibrio sp. oull4 was extracted using masterpuretm dna purification kit (epicentre, il lumina inc., madison, wi, usa) prior to rnase (qiagen, copyright @ 2020 by letchumanan v and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: vengadesh letchumanan, jeffrey cheah school of medicine and health sciences, monash university malaysia; vengadesh.letchumanan1@monash.edu. 2 usa) treatment[38,39]. the dna quality was quantified using nanodrop spectrophotometer (thermo scientific, waltham, ma, usa) and a qubit version 2.0 fluorometer (life technologies, carlsbad, ca, usa). illumina sequencing library of genomic dna was prepared using nexteratm dna sample preparation kit (illumina, san diego, ca, usa) and library quality was validated by a bioanalyzer 2100 high sensitivity dna kit (agilent technologies, palo alto, ca) prior to sequencing. the genome of oull4 strain was sequenced on miseq platform with miseq reagent kit 2 (2 x 250bp; illumina inc, san diego, ca, usa)[40]. the trimmed sequences were de novo assembled with clc genomic workbench version 5.1 (clc bio, denmark). contigs with at least 200bp and 30-fold coverage were selected for gene prediction and annotation. the bacteria identity was also checked by local blast against ncbi prokaryotic 16s rrna database. prodigal (version 2.6.1) was utilized to predict the bacteria gene coding sequence (cds) from the draft genome[41]. gene annotation was performed by local blast of translated predicted cds against ncbi-nr database and on rapid annotation using subsystem technology (rast) server[42]. presence of rrna and trna genes were detected using rnammer and trnascan se version 1.21[43,44]a total of 59 contigs were generated with n50 size of 201,133bp. the assembled genome size of vibrio sp. oull4 contains 4,146,642 bp, with an average genome coverage of 54-fold with a g + c content of 45.4% (table 1). the whole genome project was deposited at ddbj/embl/genbank under accession mqvk00000000. the version described in this paper is the first version mqvj00000000. it is composed of 59 contigs and there were 3,743 protein coding genes (out of a total of 3,898 predicted gene) (table 1). table 1. general features of vibrio sp. oull4 draft genome. attribute value genome size (bp) 4,146,642 g + c content % 45.4 dna scaffold 59 total genes 3,898 protein coding genes 3,743 rna genes (5s, 16s, 24s) 5, 3, 1 pseudo genes 55 the analysis obtained from rast server revealed 493 subsystems (figure 1). the annotated genome has 63 genes responsible for resistance to antibiotic and toxic compounds including 25 genes for multidrug resistance efflux pumps, one gene for beta-lactamase, and two genes for tetracycline resistance. the presences of these genes in the genome is closely related to the phenotypic resistance exhibited by the strain toward ampicillin, cefotaxime, oxytetracycline, and tetracycline. genome sequence of vibrio... figure 1. subsystem category distribution of vibrio sp. oull4 (based on rast annotation server). vibrio sp. oull4 is a multidrug resistant strain—resistant to 11/14 antibiotics tested. the resistant phenotype and genes of genome illustrates how extensive antibiotics have been used in aquaculture sector. some of the resistance phenotype seen in this strain possibly due to the misuse of permitted antibiotics in asian aquaculture industry namely tetracycline, quinolone, oxytetracycline, sulphonamide, and trimethoprim[45]. soon, our dependency to antibiotics will eventually deprive the efficacy of clinical antibiotics. we will need to resort to non-antibiotic approach such as bacteriophage application or natural plant antimicrobials to manage vibrio infections in the aquaculture[46–50]. we also could adapt quorum sensing method to understand the various signalling molecules of vibrio sp. these information are useful in the management of virulence traits[51]. in summary, the application of antibiotics in aquaculture should be reviewed and monitored in order to ensure the 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cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; angelthye.yunkuan@monash.edu (ay-kt) *corresponding author: jodi woan-fei law, novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; jodi.law1@monash.edu (jw-fl) received: 30 september 2022; received in revised form: 29 october 2022; accepted: 30 october 2022; available online: 31 october 2022 abstract: almost three years have passed since the start of the covid-19 pandemic. the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has constantly been mutating, producing variants with evolutionary advantages. a total of 5 variants of concern (vocs) have emerged since the start of the covid-19 pandemic, including alpha (b.1.1.7), beta (b.1.351), delta (b.1.617.2), gamma (p.1), and omicron (b.1.1.529). however, as of october 2022, only the omicron variant remains a voc. as compared to the previous variants, although omicron has the most extensive mutations but it appears to have lower severity and risk of hospitalization. symptoms of omicron infection seem to also differ from previous variants. omicron is highly transmissible and infectious and seems to have immune evasion capability. this is worrying as even after covid-19 vaccination has been implemented globally, there are findings that covid-19 vaccines may not be able to provide complete protection against omicron. this review aims to provide insight into the characteristics of omicron, including its symptoms, severity, risk of hospitalization, transmissibility and infectivity, immune evasion, and impact on the effectiveness of covid-19 vaccines. keywords: covid-19; sars-cov-2; omicron; severity; transmissibility 1. introduction the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) caused a severe pneumonia outbreak in china in late december 2019 [1] . then in early march 2020, it was declared a global pandemic known as covid-19 [2,3] . since the emergence of sars-cov-2 from wuhan, hubei province of china, in december 2019 [2,4] , this viral strain has spread like wildfire across the globe. it has undergone multiple mutations resulting in the emergence of variants of concern (vocs), which are variants with evolutionary advantages [5] . this is alarming and led to global havoc as many lives were lost upon infection, and neither was pmmb 2022, 5, 1; a0000280 2 of 10 there a cure nor had the global vaccination program been initiated [5] . during the peak of the pandemic, mounting research was being conducted towards developing vaccines effective against sars-cov-2. there was indeed a rapid development of covid-19 vaccines to put a halt to the spread and severity of covid-19 infection. some vaccines that were developed include coronavac (sinovac biotech), bbibp-corv (beijing institute of biological products/sinopharm), comirnaty (bnt162b2) (pfizer), mrna-1273 (moderna), chadox1 ncov-19/covishield (astrazeneca/university of oxford), nvx-cov2373 (novavax), covaxin (bbv152b)(bharat biotech international limited), ad5-nco (cansino biologics), sputnik v (gamaleya research institute), ad26.cov2.s (janssen pharmaceutical companies) [6] . as soon as some covid-19 vaccines were approved by the world health organization (who) under the emergency use listing, many countries implemented a national vaccination program, encouraging people to be vaccinated [7,8] . however, factors such as waning immunity, reduction in the protective effect of covid-19 vaccines against voc, or some risk groups having inadequate protection from the primary vaccination led to the development of covid-19 booster vaccine that targets improved and prolonged protection against covid-19 [8] . up until october 2022, there have been a total of 5 variants of concern (vocs), namely b.1.1.7 (alpha), b.1.351 (beta), b.1.617.2 (delta), p.1 (gamma) [5,9,10] , and b.1.1.529 (omicron) [11–13] . however, as of october 2022, the only circulating variant listed as a voc by the world health organization (who) is omicron [14] . when this review went to press, 629,740,541 confirmed infections and 6,587,818 deaths were recorded worldwide (as of 29 th october 2022) [15] . this review aims to provide some background information on omicron, and discuss its characteristics in terms of symptoms, severity and hospitalization, transmissibility and infectivity, immune evasion and the impact on vaccine effectiveness. 2. background of omicron b.1.1.529, or omicron, is the fifth voc and the only mutant of sars-cov-2 that remains a voc as of october 2022 [14] . the first sequenced omicron case was reported on the 11 th of november 2021 from botswana, and a couple of days later, another sequenced case from hong kong in a traveler from south africa [16] . although the first omicron case in south africa was in a covid-19 patient diagnosed on the 9 th of november 2021 [16] , on the 24th of november, only the omicron variant was reported to the who in south africa [17] . later on the 26 th of november 2021, only b.1.1.529 was designated as a voc, named omicron by the who [18] . three theories have been proposed with regard to the origin of omicron. this includes the possibility of omicron being circulated and evolving from a hidden population; omicron might have evolved from a cat-and-mouse game between virus and host in some immunocompromised patients; and omicron may have originated from animal reservoirs and transmitted back to humans. the possibility of these theories with pmmb 2022, 5, 1; a0000280 3 of 10 current evidence has been analyzed by du et al. [19] . this agrees with sun et al.'s findings, which revealed omicron has five mouse-adapted mutation sites, suggesting this variant might have evolved in a mouse host [20] . the omicron variant has five lineages: ba.1, ba.2, ba.3, ba.4, and ba.5 [14] . it possesses numerous mutations, a significant concern implicating the covid-19 pandemic. compared to the original wild-type strain, omicron contains more than fifty mutations [20] . furthermore, omicron’s spike protein has 26–35 amino acids that differ from the original sars-cov-2 and the delta variant [21] . omicron has some deletions and more than thirty mutations in the spike protein, with a few overlapping with those in alpha, beta, gamma, or delta vocs [16,22] . the list of spike mutations present on previous and current vocs will be listed in table 1 [23] . surprisingly, compared to omicron, the previous vocs only have nine to twelve mutations on their spike protein region. this finding is in agreement with another study [22] . additionally, fifteen out of the thirty-plus mutations happen on the receptor-binding domain (rbd). this has significant implications as the rbd is the binding site to the host angiotensin-converting enzyme 2 (ace-2) receptors which allows sars-cov-2 to enter the body, but also the key target of neutralizing antibodies produced by immune response and therapeutic antibodies [20,22] . table 1. spike mutations of sars-cov-2 variants: alpha, beta, gamma, delta, and omicron. sars-cov -2 variants of concern spike mutations reference total mutations alpha 69–70hv and 144y deletions, n501y, d614g, a570d, p681h, t716i, s982a, d1118h,*e484k, *s494p, *k1191n (*found only in some sequences) [5,24] 12 beta l242–244 deletions, a701v, d215g, d80a, d614g, e484k, k417n, n501y, r246i, l18f [5] 10 gamma k417t, e484k, n501y, l18f, t20n, p26s, d138y, r190s, d614g, h655y, v1176f, t1027i [5,25] 12 delta 156–157 deletions, d614g, d950n, l452r, t19r, t478k, p681r, r158g, *g142d (*found only in some) [5,9] 9 omicron a67v, δ69-70, t95i, g142d/δ143-145, δ211/l212i, ins214epe, g339d, s371l, s373p, s375f, k417n, n440k, g446s, s477n, t478k, e484a, q493r, g496s, q498r, n501y, y505h, t547k, d614g, h655y, n679k, p681h, n764k, d796y, n856k, q954h, n969 k, l981f [23] 32 pmmb 2022, 5, 1; a0000280 4 of 10 3. characteristics of omicron 3.1. symptoms, severity, and risk of hospitalization of omicron besides the extent of mutations, other critical differences between the omicron variant and previous vocs are the symptoms, severity, and risk of hospitalization after infection. reports suggest omicron infection is less severe than previous variants [26,27] . in a south korean study that involved only 40 omicron-infected patients, some were asymptomatic (47.5%), while some had mild symptoms (52.5%) that resolved within a few days. the initial presenting symptoms include sore throat (25%), fever (20%), headache (15%), cough (12.5%), sputum (12.5%), runny nose (10%), myalgia (5%), fatigue/weakness (2.5%), and loss of taste or smell (2.5%) [27] . additionally, a united kingdom (uk)-based study involving symptomatic covid-19-positive patients who had received at least two doses of the covid-19 vaccine found that sore throat was more common during omicron prevalence. at the same time, loss of smell was less common, and there was a 25% lower hospital admission rate than delta [28] . on a side note, findings of a lower hospital admission rate were consistent with the south african private health insurer discovery health in johannesburg, which found a 29% lower risk of hospital admission among omicron-infected individuals compared to those infected with delta [29] . a study from france and denmark also found a lower hospitalization rate (2% vs. 1.2%) in omicron-infected patients [26,30] . furthermore, both france study and the south korean study found that the loss of taste (9%; 2.5%) and smell (8.3%; 2.5%) were less common in omicron-infected patients, which were somewhat consistent with the uk study that found loss of smell to be less common [26–28] . although the france study found symptomatic cases presents mostly with mild symptoms of asthenia/fatigue, cough, fever, headache, myalgia, sore throat or runny nose but the symptomatic cases makes up 89%, which differs from the south korean study with only 52.5% [26,27] . the omicron symptoms mentioned in the uk study suggest less respiratory tract involvement in omicron infections. moreover, their findings indicate a shorter period of illness and potential infectiousness [28] . this might be due to omicron having a lower replication competence in the human lungs, which is compatible with the lower disease severity as presented by hui et al. [31] . 3.2. transmissibility and infectivity of omicron omicron is found to be highly transmissible. for instance, in south africa, the mean number of 280 covid-19 cases per day in the week before the detection of omicron rose to 800 cases per day in the following week. furthermore, there is a higher early doubling time in the fourth wave (omicron) compared to previous waves in the gauteng province of south pmmb 2022, 5, 1; a0000280 5 of 10 africa [16] . based on the increase of omicron cases and sequencing data in gauteng, omicron is estimated to have the ability to infect three to six times as many individuals as delta over the same period [32] . within months, omicron has become the dominant variant in many countries and has spread rapidly across the globe. in the uk, 1,239 omicron cases were found on the 12th of december 2021, bringing the total cases to 3,137. on the 15 th of december 2021, within days, the confirmed cases increased to a total of 4,671 in britain, the most significant daily increase [21] . in the united states (us), the first omicron case was detected in san francisco, california, and was identified on the 1 st of december 2021. as of 15 th of december 2021, omicron cases have been detected in at least 31 states in the us and washington dc [21] . the enhanced sars-cov-2 transmission [21] , and infectivity [33] are associated with omicron mutations. karim et al. suggest that if the overlapping omicron mutations maintain their known effects, increased transmissibility is expected, mainly due to mutations near the furin cleavage site [16] . this somewhat agrees with thakur et al., that state mutations at h655y, n679k, and p681h in the s1-s2 furin cleavage site of the omicron may be associated with enhanced transmissibility [34] . on the other hand, hui et al. suggest that omicron has an intrinsic capacity for increased transmission due to its quick and increased viral replication efficiency in the human bronchi compared with earlier lineages [31] . with regards to an increased infectivity, lupala et al. found that the binding energies for ace2-rbd structures for omicron is lower than the sars-cov-2 wild type, indicating stronger binding interactions and hence enhanced infectivity [33] . besides that, as a consequence of the extensive mutations in omicron, the surface of the spike protein is highly positively charged. based on the molecular dynamic simulation, nie et al., that compared to the wild type, the omicron spike has a much stronger interaction with polysulfates. this consequently favors omicron binding, leading to cell entry and infection. furthermore, the p681h mutation located next to the -rrarfurin cleavage site indicates an increased infectivity potential [23] . additionally, using an artificial intelligence model (topnetmab), chen et al. demonstrated that omicron might be more than 10 times more contagious than the original sars-cov-2. it is more infectious than other variants, roughly 2.8 times as infectious as delta, primarily due to omicron’s rbd mutations n501y, n440k, and t478k [35] . pmmb 2022, 5, 1; a0000280 6 of 10 3.3. immune evasion and impact on the effectiveness of covid-19 vaccines against omicron omicron seems to have an enhanced immune escape capability. due to omicron’s extensive mutations, there have been serious concerns regarding the neutralization antibodies and reduced efficacy of covid-19 vaccines. in south africa, it is found that individuals who have received any vaccines from pfizer-biontech, oxford-astrazeneca, and johnson and johnson reported breakthrough infections [36] . additionally, 83% of omicron cases in denmark involved fully or booster-vaccinated individuals [30] . furthermore, a study conducted by the south african centre of excellence in epidemiological modelling and analysis through late november 2021 that analyzed 35,670 reinfections among close to 2.8 million positive tests found the proportion of reinfections increased greatly as omicron spreads. this suggests earlier infection only provides half as much protection against the omicron variant compared to the delta variant [37] . this is concerning, as it seems that omicron has a more remarkable immune evasion ability and more extensive mutations on the spike protein than previous vocs. moreover, we know that sars-cov-2 enters the human host via the spike protein, and covid-19 vaccines are targeted at the spike protein. so, the one important question is whether the currently available covid-19 vaccines effective against this new voc. preliminary results from teams in south africa, germany, sweden, and the pfizer-biontech collaboration suggest that protection provided by current covid-19 vaccines will not be lost entirely and booster vaccines should strengthen immunity against omicron [21,38] . a study conducted at the africa health research institute in durban, south africa, involving 12 individuals who received pfizer-biontech vaccines, showed a 40 times less potent serum against omicron than previous sars-cov-2 strains. this finding is consistent with results reported by pfizer-biontech in a press release dated 8 th of december 2021 [38] . hoffmann et al. also found that compared to b.1, the antibodies induced upon bnt/bnt immunization neutralized the omicron spike with 34-fold reduced efficiency, indicating that two doses of bnt may not provide enough protection against omicron infection [39] . a study by chen et al. used structures of 185 known antibody-rbd complexes and detected that the vaccine escape capability of omicron is almost 14 times as high as that of delta. on the other hand, based on an artificial intelligence model, chen et al. also found omicron’s rbd mutations e484a, k417n, and q493r might decrease the efficacy of eli lilly monoclonal antibody cocktail. in contrast, the rbd mutation q498r may moderately reduce the effectiveness of astrazeneca monoclonal antibody cocktail tixagevimab and pmmb 2022, 5, 1; a0000280 7 of 10 cilgavimab [35] . furthermore, according to cao et al., numerous single mutations of omicron can impair neutralizing antibodies of different epitope groups. in particular, neutralizing antibodies in group a-d, the epitopes that overlap with the ace-2 binding motif, are largely escaped by g446s, e484a, q493r, and k417n. interestingly, their study found more than 85% of the tested neutralizing antibodies were escaped by omicron, suggesting this new variant could result in substantial humoral immune evasion and potential antigenic shifting [40] . although omicron is likely to affect vaccine effectiveness but based on a pfizer biontech study, booster vaccines may still be able to provide protection against omicron as results showed individuals who received a booster dose had neutralizing antibody levels against omicron comparable to those against sars-cov-2 variants that were triggered by two vaccine doses [38] . furthermore, a uk study found that patients infected during omicron prevalence receiving the third dose of vaccine presented with higher symptom duration reduction than patients infected during delta prevalence [28] , suggesting a decrease in severity. these findings indicate that a booster dose can still provide some protection against omicron. 4. conclusions omicron is the most mutated variant of the sars-cov-2 vocs to date. despite the rapid development of covid-19 vaccines and the worldwide implementation of the national vaccination program, omicron still spreads across the globe and remains a voc. compared to the delta prevalence, there is a difference in symptom, severity, and hospital admission upon omicron infection. both severity and hospital admission decreased during omicron prevalence, and symptoms tend to affect the respiratory tract less. after an omicron infection, sore throat appears to be one of the most prevalent symptoms, while the loss of taste and smell is significantly less prevalent than during the delta. despite the lower severity and hospital admission, omicron appears more transmissible, infectious, and likely capable of immune evasion. the current covid-19 vaccines seem to have lower effectiveness against omicron, but boosters still provide some protection. furthermore, existing public health prevention measures implemented towards sars-cov-2 strains and their vocs should be continued to protect and prevent one against omicron infection [41, 42] . these measures include wearing a face mask, proper hand hygiene, social distancing, and avoiding crowded places or poorly ventilated areas. in conclusion, combining vaccination and public health measures is crucial in preventing and decreasing the severity of sars-cov-2 infection. pmmb 2022, 5, 1; 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185(3): 447–456. e11. 40. cao y, wang j, jian f, et al. omicron escapes the majority of existing sars-cov-2 neutralizing antibodies. nature 2022; 602(7898): 657–663. pmmb 2022, 5, 1; a0000280 10 of 10 41. hoo he, loh hc, ch’ng ash, et al. positive impacts of the covid-19 pandemic and public health measures on healthcare. prog microbes mol bio 2021; 4(1). 42. rayanakorn a, leong sl, chaiprom p, et al. cost-effectiveness of public health strategies on covid-19 control: a systematic review. prog microbes mol bio 2022; 5(1). author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2022, 5, 1; a0000279. doi: 10.36877/pmmb.a0000279 http://journals.hh-publisher.com/index.php/pmmb original research article incisor malocclusion using cut-out method and convolutional neural network muhamad farhin harun1, azurah a samah1*, muhammad imran ahmad shabuli1, hairudin abdul majid1, haslina hashim1, nor azman ismail1, syiral mastura abdullah2, aspalilah alias3,4,5 article history 1faculty of computing, universiti teknologi malaysia, 81310 johor bahru, johor, malaysia; muhamadfarhin@gmail.com (mfh); imranaini96@gmail.com (mias); hairudin@utm.my (ham); haslinah@utm.my (hh); azman@utm.my (nai) 2faculty of dentistry, universiti sains islam malaysia, jalan pandan utama, 55100 kuala lumpur, malaysia; syiral@usim.edu.my (sma) 3department of basic sciences and oral biology, faculty of dentistry, universiti sains islam malaysia, kuala lumpur, malaysia; aspalilah@usim.edu.my (aa) 4forensic odontology unit, department of imaging & pathology, ku leuven, belgium; aspalilah@usim.edu.my (aa) 5department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia; aspalilah@usim.edu.my (aa) *corresponding author: azurah a samah; faculty of computing, universiti teknologi malaysia, 81310 johor bahru, johor, malaysia; azurah@utm.my (aas) received: 17 may 2022; received in revised form: 25 september 2022; accepted: 4 october 2022; available online: 6 october 2022 abstract: malocclusion is a condition of misaligned teeth or irregular occlusion of the upper and lower jaws. this condition leads to poor performance of vital functions such as chewing. a common procedure in orthodontic treatment for malocclusion is a conventional diagnostic procedure where a dental health professional takes dental x-rays to examine the teeth to diagnose malocclusion. however, the manual orthodontic diagnostic procedure by dental experts to identify malocclusion is time-consuming and vulnerable to expert bias that results in delayed treatment completion time. recently, artificial intelligence technology in image processing has gained attention in orthodontics treatment, accelerating the diagnosis and treatment process. however, several issues concerning the dental images as input of the classification model may affect the accuracy of the classification. in addition, unstructured images with varying sizes and the problem of a machine learning algorithm that does not focus on the region of interest (roi) for incisor features bring challenges in delivering the treatment. this study has developed a malocclusion classification model using the cut-out method and convolutional neural network (cnn). the cut-out method restructures the input images by standardising the sizes and highlighting the incisor sections of the images which assisted the cnn in accurately classifying the malocclusion. from the results, the implementation of the cut-out method generates higher accuracy across all classes of malocclusion compared to the non-implementation of the cut-out method. pmmb 2022, 5, 1; a0000279 2 of 16 keywords: malocclusion; orthodontics; incisor; classification; convolutional neural network; class activation mapping, cut-out method; image processing 1. introduction malocclusion is a common dental problem. it is a dentoskeletal condition that can impact patients' quality of life and social interactions by affecting both function and aesthetics [1]. malocclusion may aggravate or even create health problems such as eating disorders, migraines, speech difficulties and sleep disorders [2]. the genesis of malocclusion is complicated by several genetic, environmental and local factors and it can be caused by a skeletal or dental-related discrepancy [3]. various types and symptoms give difficulties to dental solutions to overcome the problems. the conventional manual classification procedure by the dentist consumes time and requires more evaluation processes. at the same time, most studies have not confirmed the anticipation of which orthodontic treatments that applies modern technology will minimise treatment time [4–8]. there are three main classes of malocclusion, which are class i, class ii and class iii malocclusion [9]. the classes are used to classify the condition of teeth and to give a clear view of the malocclusion problem to the dentist. manual analysis by the dentist and orthodontic experts is currently the most common technique for the majority of orthodontic problems. the manual malocclusion classification procedure is time-consuming due to the need for a thorough manual evaluation by an expert. some dental images are challenging to classify, costly to collect and revolve around various legal problems for patient privacy [10]. the manual classification procedure may consume weeks to months. this lengthy and laborious diagnostic process contributed to a long waiting time for patients waiting for malocclusion treatment. the computational deep learning-based model proposed in this study is able to speed up the classification process with better decision consistency at a reduced cost. this will lead to improved quality of services in terms of reduced cost of treatment and waiting time for patients waiting for malocclusion treatment at various dental clinics. this paper is organised as follows. section ii presents the literature review on categories of malocclusion, convolutional neural network (cnn), previous studies on the classification of malocclusion and class activation mapping (cam). this is followed by section iii on the methodology of the study. briefly, section iii highlights the main activities in conducting the study, including cnn development, dataset, experimental design, performance measure and data analysis. in section iv, the experimental results are presented and discussed. this paper continues in section v on results and discussion. the paper ends with a conclusion that is included in section vi. 2. literature review 2.1. categories of malocclusion as described by world health organization (who), malocclusion is a handicapping dentofacial anomaly, which refers to irregular occlusion or abnormal craniofacial pmmb 2022, 5, 1; a0000279 3 of 16 relationships which can affect aesthetic appearance, function, facial harmony and psychosocial well-being [6]. malocclusion is the third most popular oral pathologic disorder after tooth decay and periodontal disease that requires special treatment. malocclusions such as deep overbite, midline deviation, extreme overjet, anterior cross bite, mal-alignment, and open bite are common in clinics. the angle malocclusion classification based on the relative position of the permanent maxillary first molar is commonly used for assessing malocclusion [11]. the four front teeth in the upper and lower jaws are called incisors. the central incisors are the two incisors on either side of the midline, while the lateral incisors are the teeth next to the central incisors [12]. the condition of the problem based on the british standard institute (bsi) also categorizes the classes into class i, class ii/1, class ii/2 and class iii, as shown in table 1 and figure 1. table 1. incisor classification description [13]. class description class i lower incisors' incisal edge bites on or below upper incisors' cingulum plateau class ii/1 upper incisors are upright or proclined and lower incisors bite behind the upper incisors' cingulum plateau class ii/2 lower incisors bite behind the cingulum plateau and the higher incisors are retroclined class iii maxillary incisors' cingulum plateau is anterior to mandibular incisors' cingulum plateau figure 1. incisor classification. other types of teeth, such as canines, molars and premolars, have their own malocclusion classification. all incisor classification patterns will be the same for three malocclusion classes, and class ii/1 and ii/2 will be combined to form class ii malocclusion. it should be emphasised that the incisor section will decide the outcome of the execution processes, which will be classified into one of three malocclusion classifications based on the image input. pmmb 2022, 5, 1; a0000279 4 of 16 2.2. convolutional neural network (cnn) cnns have recently been used in medical trials and the findings showed that this approach is one of the potential computer-aided methods for medical practitioners [14]. this medical imaging study works to design orthodontic procedures based on cnn all imaging modalities used in orthodontics. as shown in figure 2, the cnn cycle will conclude the cephalometric x-ray implemented together to be evaluated using cnn. along with the growing demands for dental healthcare, a recent advance in the image recognition technology using the cnn model brings drastic improvements in diagnostic imaging and is eagerly awaited in the field of orthodontics as well [15]. figure 2. convolutional neural network (cnn). when there is a paucity of training data for image classification, the learnt model may be fooled by irrelevant local information, which might be considered noise. furthermore, the overall structure of a cnn model, rather than the particular local pixels, has a significant impact on its performance in most classification tasks and if pixels within local structures change in a way that does not impair the overall perspective of a picture, the model should be able to accurately recognise the input image [16]. overfitting is a serious difficulty in training robust cnn models and regularisation is a method to prevent cnn from overfitting [17]. dropout is a regularisation method where square areas of an image is obscured. as visualised in figure 3, the technique is important to help present a method for efficiently mixing a large number of distinct neural network topologies. figure 3. dropout method implementation. pmmb 2022, 5, 1; a0000279 5 of 16 during training, hidden unit activations are set to zero with a pre-set probability, resulting in dropout and when evaluating the network, all activations are maintained. however, the output is scaled according to the dropout probability [18]. this strategy approximates averaging over an exponential number of smaller sub-networks and it performs well as a robust type of bagging that prevents feature detectors from co-adapting within the network [19]. 2.3. previous studies on classification of malocclusion despite a few attempts to develop more understanding of malocclusions, the orthodontic discipline is still mostly confined to edward h. angle's malocclusion classification, which was developed nearly 30 years before cephalometry was introduced [13,20].the studying of the lateral cephalogram image features is a conventional method for malocclusion class detection that is still used today. as time passes, researchers have employed a variety of techniques to improve the malocclusion classification procedure [21]. the complete structure of the teeth can be examined through the cephalogram images as mentioned earlier. for the development of the study, results of classification based on incisor focus are extremely limited. 2.4. class activation mapping (cam) to sort different classes, the cam method has been used to highlight the classspecific areas that the network learned from the data [22]. the cam method can also be combined with fine-grained visualisation techniques to produce high-resolution classdiscriminative representations. this applies to a wide range of cnn model families without any architectural modifications or re-training. however, their effects, are often accompanied by random noises that are unrelated to the image's target item and the weight does not adequately capture the value of each activation map [23]. the class-specific area highlighted using the cam method can assist the understanding of how the cnn operates on classification and what features the cnn had extracted. 3. materials and methods 3.1. materials this retrospective study uses existing data recorded by the faculty of dentistry, universiti sains islam malaysia (usim), which does not require any written informed consent from the patient. the patient id was replaced with a unique id and no personal details were exposed during the execution of this study. this study has been approved by the research and ethics committee of usim. the ethics approval code for this study is usim/jkep/2020-88 3.2. methodology there are four main activities in conducting the study, as shown in figure 4. the four main activities are problem articulation, data collection, experimental design, performance measure and analysis. pmmb 2022, 5, 1; a0000279 6 of 16 figure 4. four phases of research methodology. the problem articulation phase starts with understanding malocclusion, cnn modelling solution and techniques used in the malocclusion classification study. in total, there were 454 images, including 166 incisor images of class i, 254 incisor images of class ii and 125 incisor images of class iii used in this study. the next phase is data collection, where lateral cephalogram images of the entire facial skeleton used in this study were gathered as input to measure the classification performance of cnn for analysis. there were several tasks in the experimental design phase, which were data preprocessing (table 2), development of the cnn model, hyperparameter tuning, implementation of the cut-out method and cam (refer to figure 6). each image input is fixed at a size of 128x128 pixels. the objective of this phase is to design the cnn model to identify patterns by analysing the structure of each image in each class. the pattern of the incisor is identified as a significant feature based on the single input image. to produce good roi, the cut-out method is implemented to improve the performance of cnn. thus, the size of the image data was adjusted to enhance the malocclusion classification. then, cam highlights the extracted features from cnn in terms of visualisation. table 2. data augmentation parameters. parameter value/type zoom range 0.5 shear range 0.2 height shift range 0.2 width shift range 0.2 fill mode nearest lastly, the performance measure and analysis were conducted. a confusion matrix which consisted of four elements, as shown in figure 5 was generated to aid in the analysis of the results. there are four elements in the confusion matrix which are true positive (tp), false positive (fp), true negative (tn) and false negative (fn). pmmb 2022, 5, 1; a0000279 7 of 16 figure 5. confusion matrix. the accuracy, precision, recall and f1 score are measures derived from the confusion matrix. accuracy is a measure of how many correct predictions the proposed model made for the complete test dataset. precision is a measure of the correctness of a positive prediction. on the other hand, recall is a measure to determine how many true positives get predicted out of all positives in the dataset. finally, the f1 score is a statistic that takes into account both precision and recall. equation 1 to 4 shows the formulation of performance measures used in this study. 𝑎𝑐𝑐𝑢𝑟𝑎𝑐𝑦 = 𝑇𝑃+𝑇𝑁 𝑇𝑃+𝑇𝑁+𝐹𝑃+𝐹𝑁 (1) 𝑝𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛 = 𝑇𝑃 𝑇𝑃+𝐹𝑃 (2) 𝑟𝑒𝑐𝑎𝑙𝑙 = 𝑇𝑃 𝑇𝑃+𝐹𝑁 (3) 𝐹1 − 𝑆𝑐𝑜𝑟𝑒 = 2 × 𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛×𝑅𝑒𝑐𝑎𝑙𝑙 𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛+𝑅𝑒𝑐𝑎𝑙𝑙 = 2𝑇𝑃 2𝑇𝑃+𝐹𝑃+𝐹𝑁 (4) 4. experimental results 4.1. experimental setup the flow of the experimental setup for this study is as shown in figure 6. the layers were implemented in a sequence to establish a series of evaluations toward input during training. the cnn layers of the study are as specified in table 3. figure 6. the flow of experimental setups. pmmb 2022, 5, 1; a0000279 8 of 16 table 3. cnn layers architecture. layer specification conv2d input size = 128×128×3 3×3 convolution, 16 filters conv2d 3×3 convolution, 16 filters batch normalization activation relu maxpooling2d 2×2 max-pooling conv2d 3×3 convolution, 32 filters conv2d 3×3 convolution, 32 filters batch normalization activation relu maxpooling2d 2×2 max-pooling conv2d 3×3 convolution, 64 filters conv2d 3×3 convolution, 64 filters batch normalization activation relu maxpooling2d 2×2 max-pooling conv2d 3×3 convolution, 128 filters conv2d 3×3 convolution, 128 filters batch normalization activation relu maxpooling2d 2×2 max-pooling flatten dropout 50% dropout activation relu dense 3 filters softmax softmax 4.2. hyperparameters the hyperparameter settings for the cnn model were configured to deliver better outcomes and maximise the capabilities of the training process. the parameters are as shown in table 4. pmmb 2022, 5, 1; a0000279 9 of 16 table 4. hyperparameters and training options for cnn. hyperparameters and training options assigned values training optimisation function adam initial learn rate 1e-07 maximum epoch 200 training:validation:test ratio 7:2:1 learning rate 0.001 number of iterations 200 4.3. cut-out method the input data was in the form of single images. most of the images were unstructured images where the size of images varied from one image to another. additionally, the incisor section for each image was relatively small and confined. the cut-out method was used to restructure the image such that it focuses on the incisor section and provided a clear view for model development. the size of the image input was initially different for all images included in the study. therefore, size adjustments were required. the adjustments involve changing the pixel size of an image manually for horizontal and vertical scaling using a resize function attained from the opencv library to scale the image and create a cut-out effect. the newly restructured image which includes the incisor part is displayed and scaled as shown in figure 7. figure 7. cut-out technique from a selected input image. the cut-out method was further validated during the post-processing phase using cam heatmap which is further discussed in the next section (b. cam heatmap validation and analysis). 5. results and discussion in this section, a confusion matrix for classification, accuracy and loss from the model is presented. additionally, a heatmap from cam is included and the validation of the cut-out method is discussed to give additional perspective to the evaluation. there are others malocclusion classification studies using cnn on different malocclusion datasets. as our cut-out method is a very niche improvement over the private dataset, it was very difficult to pmmb 2022, 5, 1; a0000279 10 of 16 conduct a one-to-one comparison with a published dataset. instead, we measured the effectiveness of our experimental framework with other malocclusion studies. 5.1. confusion matrix the performance measures of incisor classification were analysed using the confusion matrix. the result of the analysis is shown in figure 8. figure 8. confusion matrix of classification processes. for the true and false values, nine-sectioned blocks were set up to represent each class. details of the performance matrices at a 75% accuracy and 0.64 loss rates are shown in table 5. table 5. accuracy, precision, recall and f1-score. precision recall f1-value support class i 0.67 0.27 0.38 15 class ii 0.75 0.88 0.81 17 class iii 0.76 0.96 0.85 23 the recall value of 0.96 for class iii was the highest among the other classes. a high recall value indicates low misclassification of the predicted class. features for class i and class ii were quite similar to each other, which was why their recall values were lower than class iii. the recall value for class i was the lowest amongst the classes presented, which indicates that the model cannot correctly identify the features for class i. from the results obtained, it was seen that many of the class i samples were misclassified as either class ii or class iii. class iii achieved the highest f1value based on its corresponding precision and recall values. the performance of incisor classification for malocclusion was highly satisfied for class iii. in contrast, class i achieved the lowest f1 value as both of its corresponding precision and recall values were low. pmmb 2022, 5, 1; a0000279 11 of 16 5.2. cam heatmap validation and analysis the cam heatmap was used to validate the cnn results. the validation was done using single image classification. in particular, cam heatmap was used to detect the focal area of the incisor part for a single image. a sample of a single image was chosen to demonstrate the validation process. figure 9 and figure 10 show the heatmap output of the same image. in figure 9, the heatmap output was generated without implementing the cutout method. on the other hand, figure 10 shows a heatmap output that was generated after a cut-out method has been implemented. figure 9. heatmap output without cut-out method. figure 10. heatmap output with cut-out method. both figure 9 and 10 revealed significant differences between each other. as shown in figure 9, without the cut-out method, the focus section was spread to regions that were irrelevant to the study's observation. furthermore, the focus was not on a precise section, which has led to unfavourable outcomes. on the other hand, with the implementation of the cut-out method, figure 10 shows that the focus was specific on the incisor areas. thus, the results were more accurate and convincing, with the implementation of the cut-out method. it was also evident that the implementation of cut-out method and cam validation has greatly assisted the highlighting and classifying of the incisor areas. further validation analysis of pmmb 2022, 5, 1; a0000279 12 of 16 the cut-out method using the heatmap was done on samples of each class. the results of the validation analysis are shown in table 6. as shown in table 6, a sample from each class was validated with and without the cut-out method. for all classes, the implementation of the cut-out method resulted in the highest accuracy. for instance, a sample of class ii with id 317 implemented a cut-out method yielded the highest accuracy of 0.827 of being classified as class ii. similarly, the sample from class i (sample id of 46) and class iii (sample id of 456) yielded the highest accuracy of 0.700 and 0.598, respectively of being classified as class i and iii. table 6. heatmap result analysis. sample confident level class 1 class 2 class 3 sample id: 317 [class 2] without cut-out 0.330 0.332 0.336 with cut-out 0.050 0.827 0.121 sample id: 46 [class 1] without cut-out 0.335 0.330 0.333 with cut-out 0.700 0.172 0.127 sample id: 456 without cut-out 0.331 0.333 0.334 [class 3] with cut-out 0.148 0.253 0.598 both validation on a single sample and the three samples that represented each class demonstrated that the implementation of the cut-out method and cnn model have assisted in the identification and marking of specific incisor areas for specific samples, which further assisted in the classification of malocclusion. satisfying results with the implementation of the computational deep learning-based model were obtained not only in this study but also in other malocclusion studies conducted by other researchers. table 7 summarised two malocclusion studies that have implemented deep learning methods for malocclusion classification, which are closely similar to our study. results obtained by the research listed in table 7 are more promising than ours and remarks in column four of table 7 stated the justification of the higher results that were obtained in comparison to our study. these remarks have open room for further improvement to our study, to attain better results in the future. both malocclusion studies listed in table 7 used different data types. thus, different data pre-processing was implemented. this brings the importance of cnn modelling in better understanding and focusing on the features. in our study, the focus was more on the cut-out method to highlight the incisor feature. although we have carried out several data augmentations, the focus should be on improving the image quality as it could help highlight the feature much better. both studies listed in table 7 used customised cnn deep learning models to work better with specific samples. the results of our study may be further improved by integrating pmmb 2022, 5, 1; a0000279 13 of 16 the cut-out method with a customised cnn model. as the current study is a stepping stone toward understanding more about the behaviour of cnn when classifying the incisor dataset, enhancement of the image quality and customisation of the cnn deep learning model are improvements that can be included in our future work. the proposed improvements will be deeply analysed as they could open more research opportunities in the future. table 7. malocclusion studies. malocclusion study dataset type overall result remarks molar-incisorhypomineralisation (mih) [24]. intraoral photographs. (normal image) the result has an overall accuracy of 95.5%. 1. the samples used are clear and high quality. 2. the feature in the samples is clearly defined. 3. deep cnn architecture was used which improved the efficiency at the cost of longer training time. incisors, canines, premolars, and molars [25]. grey-level image the best result obtained of accuracy is 87%. 1. rigorous augmentation on a limited sample of the dataset. 2. customised pooling architecture. 6. conclusions in this study, the main objective related to the development of an improved classification model based on incisor data using the cut-out method and cnn modelling to facilitate the classification of malocclusion has been achieved with satisfying results. input images that have been restructured with a focus on the incisor section using the cut-out method produced higher accuracy and lower loss in classifying malocclusion as compared to images that have not been implemented with the cut-out method. despite this achievement, few works can be carried out in the future to expand the research further and improve existing results. one of improvements is to automate the cut-out method which currently was implemented manually in this study. the implementation of the cut-out method manually may cause inconsistent sizes of incisor region in some of the samples/images. an automated and adaptive cut-out method is therefore suggested to generate better consistency in the sizes of the incisor region for every sample, which may produce more accurate focal region detection. author contributions: conceptualization, methodology, literature search, mias, mfh, aa; validation, nai, mias, aa; data collection, mfh, sma, aa; writing—original draft preparation, mias, ham; reviewed, editing, proofread, aas, hh. funding: this work was funded by the malaysian ministry of higher education under fundamental research grant scheme (grant number: frgs/1/2021/ict02/utm/02/5). acknowledgments: the authors would like to express gratitude to the malaysian ministry of higher education for the financial sponsorship of this study through the fundamental research grant scheme pmmb 2022, 5, 1; a0000279 14 of 16 (frgs/1/2021/ict02/utm/02/5). the study is also supported by the faculty of computing, universiti teknologi malaysia and faculty of dentistry, universiti sains islam malaysia. conflicts of interest: the authors declare no conflict of interest. references 1. koskela a, neittaanmäki a, rönnberg k, palotie a, ripatti s, palotie t. the relation of severe malocclusion to patients’ mental and behavioral disorders, growth, and speech problems. eur j orthod. 2021;43(2):159-164. doi:10.1093/ejo/cjaa028 2. leavy km, cisneros gj, leblanc em. malocclusion and its relationship to speech sound production: redefining the effect of malocclusal traits on sound production. am j orthod dentofac orthop. 2016;150(1):116-123. doi:10.1016/j.ajodo.2015.12.015 3. al-qurashi, h., al-farea, m., alshamrani, h., almasoud, n. n., & nazir ma. orthodontic treatment needs and association between malocclusion and oral hygiene behaviors. pakistan oral dent j. 2018;38(1):62-66. accessed september 20, 2022. https://podj.com.pk/index.php/podj/article/view/137 4. jung mh. factors influencing treatment efficiency: a prospective cohort study. angle orthod. 2021;91(1):1-8. doi:10.2319/050220-379.1 5. mavreas d, athanasiou ae. factors affecting the duration of orthodontic treatment: a systematic review. eur j orthod. 2008;30(4):386-395. doi:10.1093/ejo/cjn018 6. zou j, meng m, law cs, rao y, zhou x. common dental diseases in children and malocclusion. int j oral sci. 2018;10(1). doi:10.1038/s41368-018-0012-3 7. pinto as, alves ls, maltz m, susin c, zenkner jea. does the duration of fixed orthodontic treatment affect caries activity among adolescents and young adults? caries res. 2018;52(6):463-467. doi:10.1159/000488209 8. moresca r. orthodontic treatment time: can it be shortened? dental press j orthod. 2018;23(6):90-105. doi:10.1590/2177-6709.23.6.090-105.sar 9. hengsheng l, jun y. management of malocclusion in children and adolescents. decisions in dentistry. published 2020. accessed september 20, 2022. https://decisionsindentistry.com/article/management-ofmalocclusion-in-children-and-adolescents/ 10. guibas jt, virdi ts, li ps. synthetic medical images from dual generative adversarial networks. 31st conf neural inf process syst (nips 2017). 2017;(31):1-9. http://arxiv.org/abs/1709.01872 11. alkhadra t. characteristic of malocclusion among saudi special need group children. j contemp dent pract. 2017;18(10):959-963. doi:10.5005/jp-journals-10024-2156 12. grawish me, grawish lm, grawish hm. permanent maxillary and mandibular incisors. in: dental pmmb 2022, 5, 1; 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a0000279 16 of 16 022-04552-4 25. li z, wang sh, fan rr, cao g, zhang yd, guo t. teeth category classification via seven-layer deep convolutional neural network with max pooling and global average pooling. int j imaging syst technol. 2019;29(4):577-583. doi:10.1002/ima.22337 author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2022, 5, 1; a0000272. doi: 10.36877/pmmb.a0000272 http://journals.hh-publisher.com/index.php/pmmb review article gut microbiome in obsessive compulsive disorder: potential of probiotics as an adjuvant therapy grace yong-en kong1, vengadesh letchumanan1, loh teng-hern tan1,2, jodi woanfei law1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, subang jaya, selangor, 47500, malaysia; emrysgrace@gmail.com (gy-ek), vengadesh.letchumanan1@moansh.edu (vl) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) *corresponding author: jodi woan-fei law, novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, subang jaya, selangor, 47500, malaysia; jodi.law1@monash.edu (jw-fl) received: 13 august 2022; received in revised form: 03 november 2022; accepted: 18 november 2022; available online: 30 november 2022 abstract: the gut-brain axis concept has become an exciting area of research in psychiatry. gastrointestinal inflammation and gut microbiome dysbiosis have been associated with mental health disorders. obsessive-compulsive disorder (ocd) is a debilitating and complex mental illness that cannot be completely curable, stemming from many causes and risk factors. generally, there is limited research on ocd and its association with the gut microbiome compared to other psychiatric conditions such as depression and anxiety. this review aims to provide insights into the association of gut microbiome and gastrointestinal inflammation with ocd. besides, the role of probiotics as a potential therapy will be discussed in this review. the studies compiled in this review demonstrated variations in the gut microbial composition, often with lower microbial diversity in ocd patients compared to the controls. the gut microbiome is also involved in regulating the immune system. alteration in certain groups of gut bacteria could give rise to inflammation and manifestations of gastrointestinal symptoms in ocd patients. as an approach to restoring the balance of the gut microbiome, probiotics serve as an effective solution. in vivo animal studies showed that probiotics can potentially improve ocd symptoms. nevertheless, clinical trials are required to determine the efficacy of probiotics as an adjuvant therapy to alleviate ocd symptoms. keywords: ocd; mental health; obsession; compulsion; gut microbiota pmmb 2022, 5, 1; a0000272 2 of 10 1. introduction obsessive-compulsive disorder (ocd) is a debilitating chronic neuropsychiatric disorder with a 2.3% lifetime prevalence and an average age of onset at 19.5 years old [1]. it is characterized by persistent intrusive thoughts (obsessions), often paired with repetitive behaviors (compulsions) that can alleviate anxiety [2, 3]. the etiology of ocd is still unclear. it may involve various factors such as genetic vulnerability, neurobiological dysfunction, and environmental influences. recent research has also found that glutamate and inflammation could be involved in ocd [4]. the current ocd treatment options focus on serotonin imbalance and cognitive behavioral therapy [3]. however, up to 40% of patients do not respond to selective serotonin reuptake inhibitors (ssris). those who respond tend to require higher dosages of ssris than those used in depression [4]. hence, there is an increased risk of dose-dependent adverse effects such as gastrointestinal symptoms and sexual dysfunction. meanwhile, only one-third of treatment-resistant ocd may respond to augmentation with antidopaminergic agents [4]. there is a need for research to understand the pathophysiological mechanisms of ocd further to discover new approaches for treatment-resistant ocd and augment existing therapies. recent advances in sequencing technologies have enabled research exploring the human microbiome. the gut microbiome may have a role in modulating inflammation and has been implicated in the pathogenesis of psychiatric conditions such as depression and anxiety [5]. few studies have hypothesized that the gut-brain axis may be implicated in ocd because anxiety is a significant component of the disorder [6, 7]. many ocd risk factors are known to disrupt the microbiome, such as stress and pregnancy [6, 8]. animal studies have also shown anxiety and ocd-like behavior altered by microbial treatments such as probiotics and germ-free environments [9-11]. this review discusses the current literature on the association of gut microbiome and gut inflammation with ocd. additionally, this review offers information on the roles of probiotics as a potential adjuvant therapy for ocd. literature search was performed using keywords: “obsessive compulsive disorder”, “ocd”, “gastrointestinal microbiome”, “gut microbiome”, “inflammation”, and “probiotics” on three databases, including ovid medline, proquest, and scopus. there are limited studies available in this area of research; hence, studies of pans (paediatric acute-onset neuropsychiatric syndrome) and pandas (paediatric autoimmune neuropsychiatric disorder associated with streptococcal infections) will not be excluded from this review. considering the clinical overlap of pans/pandas with ocd phenotypes, these findings may be useful for comparison and discussion [12]. children with pans are characterized by an acute onset of tics, ocd, or various other psychiatric disorders. meanwhile, pandas is a subset of pans associated with infection by group a streptococci [13]. treatment for pandas includes antibiotics, immunomodulatory medications, ssris, and cognitive-behavioral therapy [13]. thirteen english-language articles, including clinical trials and animal studies, were selected for qualitative analysis based on their relevance to this review. pmmb 2022, 5, 1; a0000272 3 of 10 2. the association of gut microbiome and ocd the human gut microbiome consists of numerous bacteria species essential in metabolizing indigestible compounds, producing vitamins, developing gut-associated lymphoid tissue (galt), and preventing pathogenic colonization [8, 14]. the gut microbiome profile is primarily stable in adults but can be affected by factors such as stress, infections, inflammation, diet, antibiotics, and probiotics [5]. researchers discovered the importance of the gut microbiome in health and its implications for various diseases, such as cardiometabolic diseases, neuropsychological diseases, autoimmune diseases, cancers, and many more [15-20]. one of the most studied areas is the gut-brain axis (gut-microbiome-brain axis) [21]. the gut-brain axis describes the bidirectional relationship between the gut microbiome and the central nervous system. stress and emotions can affect gut function, while the gut can modulate cognition and emotions [22]. researchers have attempted to explore the connection between gut microbiome composition and the development of mental illnesses like ocd. turna et al. [12] investigated the stool microbiome and inflammatory markers of ocd patients, and the confounding variables were taken into consideration, such as depression, antibiotics, probiotics, and ssri intake. the 21 cases of ocd patients recruited in the study were not depressed and not on medications, matched to a control of the same age and sex. the ocd patients had lower species richness and evenness than the control group and a significantly lower relative abundance of 3 genera: oscillospira, odoribacter, and anaerostipes [12]. odoribacter is from the phylum bacteroidetes, while oscillospira and anaerostipes are from the phylum firmicutes and class clostridia . these three genera of bacteria can produce butyrate, a shortchain fatty acid (scfa) that provides energy and maintains the integrity of the gut epithelium, preventing “leaky gut” that would increase intestinal permeability to various compounds that can cause inflammation [23]. therefore, the lower abundance of these bacteria could indirectly indicate the lower production of butyrate, which could confer health benefits to the host. a study by domènech et al. [24] also showed that ocd patients tended to lower αdiversity compared to the control group, which indicated lower species richness and evenness of the gut microbiome in ocd patients. the relative abundance of rikenellaceae, especially the genus alistipes, was increased in ocd patients. this family has a positive association with intestinal inflammation in mice [25]. meanwhile, the relative abundance of the family prevotellaceae was reduced compared to the control [24]. several genera in this family, especially prevotella, were reported to be reduced in children with other disorders, such as autism. prevotella has been discovered to help prevent pathogen colonization as a commensal in the gut microbiome [26]. compared to controls, ocd patients had a reduction in the abundance of 2 genera within the lachnospiraceae family, which were agathobacter and coprococcus [24]. coprococcus can be associated with dopac (a dopamine metabolite) synthesis, which may contribute to ocd via the dopaminergic pathway [24]. pmmb 2022, 5, 1; a0000272 4 of 10 in addition, domènech et al. [24] was the first study to investigate the oropharyngeal microbiome of ocd patients. the oropharyngeal samples also exhibited lower bacterial diversity than the stool samples. however, streptococcus bacteria, a potential biomarker of ocd associated with pandas, was not detected as the top 15 most abundant species in both ocd and control samples. the ratio of fusobacteria to actinobacteria was significantly lower in the ocd group as compared to the control group due to ocd patients having an increase of species within the actinobacteria and coriobacteria classes (particularly the genera actinomyces and atopobium). in contrast, the control group had a higher percentage of fusobacteria population than the ocd group. the study suggested that the fusobacteria to actinobacteria ratio was correlated with the presence of the coprococcus genus, which may be implicated in the pathogenesis of ocd. furthermore, nikolova et al. [27] recently conducted a meta-analysis based on the studies by turna et al.[12] and domènech et al.[24], and they found that ocd patients had 15 taxa of gut microbes that were significantly different in abundancies compared to controls. however, compared to other disorders like depression and anxiety, there were insufficient studies of ocd for more in-depth comparisons, such as analyzing the specific taxa involved and checking for overlap with other disorders. meanwhile, quagliariello et al. [28] investigated the gut microbiome of 30 patients with pans/pandas. they discovered an increase in anti-streptolysin o titer (asot) correlated with increased levels of odoribacter and reduced levels of dehalobacterium, corynebacterium, gemella, and lactobacillus. the younger age group of children at 4-8 years old had an increased abundance of bacteroidetes, especially bacteroides, odoribacter, and oscillospira, and a reduction in firmicutes . in general, the gut microbiota of the younger pan patients lacked several pathways involved in modulating inflammation and neurological function. in the older group of pan children above 9 years of age, there was a greater abundance of peptostreptococcaceae and erysipelotrichaceae with reduced rikenellaceae and barnesiellacea [28]. besides, it is important to note that the different findings in the two age groups might also be due to the gut microbiome changes throughout different stages of life [13]. animal studies have also provided evidence of the changes in the gut microbiome of mice or rats with ocd behaviors. for instance, jung et al. [29] conducted an experiment by inducing compulsive checking and locomotor sensitization (ocd-like behaviors) in 15 rats by injecting the dopamine agonist quinpirole. the rats injected with quinpirole were found to have changes in several bacterial communities compared to the control group of 16 rats injected with saline. after statistical analysis of fecal samples, 25 gut bacteria clusters were found to be likely altered due to long-term injection of quinpirole. there were 22 clusters from the firmicutes phylum, with the highest prevalence in the lachnospiraceae family, followed by the ruminococcaceae family. this increase in ruminococcaceae is consistent with another study that investigated the gut microbiome of rats injected with an indirect dopamine agonist [30]. the remaining 3 clusters consisted of deferribacteres, proteobacteria, and tenericutes phyla [29]. pmmb 2022, 5, 1; a0000272 5 of 10 nevertheless, jung et al. [29] could not confirm the relationship between changes in the gut microbiome and the onset of obsessive-compulsive behaviors. the changes in the gut microbiome could be due to chronic drug injections or other confounding factors like stress. rather than directly stimulating behavioral change via the gut-brain axis, jung et al. proposed that behavior change only reflects the supportive role of the gut microbiome. this alternative infrastructure support model suggested that gut bacteria adapt to different circumstances to fulfill their roles in processing nutrition and immune regulation [29]. with this model, a change in the gut microbiome reflects the presence of adapting to support different physiological demands. the firmicutes phylum consists of gram-positive bacteria that produce butyrate, a short-chain fatty acid (scfa), as their primary end-product that can be used for energy, fatty acid oxidation, and lipolysis [31]. in this study, the changes in these microbes reflected a shift towards free fatty acids (ffa) utilization, which may be an adaptation of the gut bacteria to support energy use requirements because ffa is a denser energy source. the obsessivecompulsive behaviors induced by quinpirole, such as enhanced locomotor sensitization, and actions of compulsive checking, can increase the energy requirements of the rats [29, 31]. another study by scheepers et al. [32] examined deer mice with large nest building (lnb) behavior, which is considered a compulsive-like behavior in mice. the gut microbiome of 11 deer mice expressing lnb was compared to 11 normal nest-building (nnb) deer mice. the study found that both groups have significantly different overall gut microbiome compositions. in nnb deer mice, there was an increased prevalence of prevotella and anaeroplasma compared to lnb mice. prevotella and anaeroplasma have anti-inflammatory properties that may be protective against developing nnb behavior [32]. meanwhile, lnb mice have an increased prevalence of desulfovermiculus, aestuariispira, peptococcus, and holdemanella compared to nnb mice. desulfovermiculus and peptococcus are hydrogen sulfide-releasing bacteria linked to inflammation and injury to the gut mucosa, suggesting that ocd-like behavior development could be driven through the gut-brain axis [32]. overall, α-diversity in the gut microbiome was lower in ocd [24, 28] and pans/pandas patients [12], as compared to controls, indicating the presence of fewer bacterial species with a more unbalanced distribution. firmicutes was one of the phyla being affected in the gut microbiome of the ocd population. a younger group of pans/pandas showed increased oscillospira [28]. besides, ruminococcaceae and lachnospiraceae also increased in ocd-like rats, which were attributed to increased metabolic demands [29]. however, these were inconsistent with the findings in other clinical trials showing a reduction within the families lachnospiraceae [24], oscillospira [12], and anaerostipes [12]. the odoribacter family was found to be reduced in ocd patients [12], but increased in younger pans/pandas patients [28]. these organisms could produce butyrate to prevent intestinal permeability and inflammation. the discrepancy in the findings of gut microbiome composition levels in pans/pandas as compared to adult ocd patients may reflect changes in the microbiome throughout the lifetime or as a compensatory response to an abnormal childhood level of the microbe. pmmb 2022, 5, 1; a0000272 6 of 10 the rikenellaceae family, especially the alistipes genus, was increased in ocd patients, and these bacteria were associated with gut inflammation [24]. meanwhile, the family prevotellaceae was reduced in ocd patients [24]. these findings were consistent with the animal study, in which higher levels of prevotella and anaeroplasma in normal rats than in ocd-like rats, with these bacteria having anti-inflammatory properties [32]. other organisms shown to be altered in the ocd population include desulfovermiculus, aestuariispira, peptococcus, and holdemanella, which are increased in mice with ocd-like behaviors [32]. a raised anti-streptolysin o titer indicating streptococcal infection was correlated with reduced levels of dehalobacterium, corynebacterium, gemella, and lactobacillus in pans/pandas patients. thus, streptococcal infection could alter the gut microbiome composition by favoring certain gut bacteria that promote a pro-inflammatory state [28]. 3. relationship between gastrointestinal diseases and ocd obsessive-compulsive, anxiety and depression symptoms can arise due to a need to control unpredictable gastrointestinal symptoms that affect the quality of life [33]. young adults with chronic gastrointestinal diseases such as coeliac disease, inflammatory bowel disease (ibd), and irritable bowel syndrome (ibs) were significantly more likely to have obsessive-compulsive, depression, and anxiety symptoms with more mentally unhealthy days than their healthy controls [34-36]. another study on adult ibd patients found that those with active disease have more obsessive-compulsive, depression, anxiety, and psychosis symptoms than those in remission [33]. stress and psychiatric disorders can alter the gut microbiome. a dysfunctional gut microbiome can lead to inflammation and manifestations of gastrointestinal symptoms. a cross-sectional study on men in the korean army found that obsessive-compulsive and somatization behaviors are independent predictive factors for functional gastrointestinal disorders (fgids) [37]. in this study, men with fgid were also found to have more psychopathological symptoms than controls, assessed by a modified symptom checklist-90r (scl-90-r) [37]. serving in the army is a stressful environment that may precipitate psychiatric symptoms and alter the gut microbiome. fgid includes conditions such as irritable bowel syndrome (ibs), functional heartburn, or functional diarrhea, of which ibs is the most common fgid. ibs is hypothesized to involve the gut-brain axis, with ibs patients having higher rates of anxiety and depression [36]. ocd patients are also found to have a higher prevalence of ibs, with 47.6% meeting the rome iii criteria for ibs, compared to 4.5% of controls [36]. the outcome is consistent with an earlier study that showed 35.1% of ocd patients meeting the rome i criteria for ibs compared to that 2.5% of controls [38]. compared to controls, ocd patients were also found to have more severe gastrointestinal symptoms such as reflux, indigestion, bowel dysfunction, and abdominal symptoms [36]. pmmb 2022, 5, 1; a0000272 7 of 10 4. potential therapeutic roles of probiotics in ocd given that a dysfunctional gut microbiome could serve as a contributing factor to psychiatric disorders, probiotics may be an alternative remedy to alleviate these conditions. probiotics have been shown to reduce depression-like symptoms, improve gut function and regulate corticosteroid release in animal studies [11, 39]. currently, the use of probiotics as a therapeutic regime for ocd remains unclear. evidence on the effect of probiotics on ocd patients is limited. a secondary analysis performed on a study regarding the impact of the probiotic lactobacillus helveticus r0052 and bifidobacterium longum r0175 (pf) revealed obsessive-compulsive scores in 25 healthy subjects improved after 30 days of pf intake [40]. a case report on a 16-year-old boy with autism spectrum disease, ocd, and self-injurious behavior (sib) showed a significant reduction of ocd symptoms (as rated by his parents) and a decrease in sib episodes [41]. he was given saccharomyces boulardii at 6 capsules daily for 3 months, then began to wean the dose by 4 capsules per week [41]. in 2014, researchers in the united states produced ocd-like behaviors in house mice with ru 24969, a serotonin receptor agonist [10]. pretreatment with 2-weeks and 4-weeks of lactobacillus rhamnosus gg resulted in mice with lesser ocd-like behavior than controls. pretreatment with 2-weeks of the probiotic was found to be comparable to 4-weeks fluoxetine pretreatment in preventing the induction of ocd behaviors. in 2020, researchers in iran induced ocd-like behaviors in 4 groups of 6 rats with a chronic injection of quinpirole (a dopamine agonist) to compare with 1 group of rats injected with normal saline [11]. for 4 weeks, the first group of rats was given the probiotic lactobacillus casei shirota (l. casei) as an intervention, the second group was given fluoxetine, the third group was given fluoxetine, and the probiotic, while the fourth group was given no treatment as the control group [11]. l. casei has been reported to improve autism spectrum disorder symptoms and improve mood by having an anti-inflammatory effect [11, 42]. rats treated with l. casei, fluoxetine, or both showed improvement in ocd symptoms in terms of their exploratory behavior. the expression of 3 genes involved in the serotonergic pathway of ocd pathogenesis (brain-derived neurotrophic factor [bdnf], 5-hydroxytryptamine receptor type2a [htr2a], and solute carrier family-6-member-4 [slc6a4]) were measured using pcr analysis. quinpirole led to decreased expression of bdnf, increased htr2a, and no difference in slc6a4 compared to controls. in contrast, the treatment with fluoxetine or probiotics resulted in the upregulation of bdnf and downregulation of htr2a [11]. l. casei shirota appeared to modulate these gene expressions and alleviate ocd symptoms. 4. conclusion adult ocd patients, children with pans/pandas, and mice/rats with ocd-like behaviors demonstrated changes in the gut microbiome composition. the gut bacteria that have been identified to be altered prominently include the firmicutes phylum (ruminococcaceae, lachnospiraceae, oscillospira, anaerostipes), genus odoribacter, rikenellaceae family, and prevotellaceae family. other organisms shown to be altered in ocd include desulfovermiculus, aestuariispira, peptococcus, holdemanella, pmmb 2022, 5, 1; a0000272 8 of 10 dehalobacterium, corynebacterium, gemella, and lactobacillus. the supplementation of probiotics to modify the gut microbiome composition and reduce ocd symptoms are successful in two animal studies involving probiotics lactobacillus rhamnosus gg and lactobacillus casei shirota (l. casei). there is a need to raise awareness of ocd and encourage research in this area. it is crucial to study and discover the specific gut microbes that might trigger the development of ocd or as a biomarker of the disease to select the appropriate probiotic as adjuvant therapy. to date, the studies investigating the gut microbiome composition of the ocd population are still limited, and their relationship remains inconclusive as the gut microbiota changes can be either part of the aetiology of ocd or a consequence of the condition. there is room for improvement in future studies pertaining to the gut microbiome and ocd. for instance, a germ-free mouse model experiment can be explored to determine if changes in the gut microbiome could lead to the development of ocd. meanwhile, future studies should consider confounders that can affect the microbiome, such as medication usage, comorbidities like depression or anxiety, stress, or diet. it may also prove useful to analyze the severity of ocd symptoms with regards to the microbiome profile or if there are any differences in treatment-responsive compared to treatment-resistant patients. author contributions: the literature searches, data collection, and manuscript writing were performed by gy-ek. the manuscript was critically reviewed, proofread, and edited by lt-hl, and vl. the project was conceptualized and supervised by jw-fl. funding: no external funding was provided for this research. acknowledgments: this work was inspired by the jeffrey cheah school of medicine and health sciences “med5101 scholarly intensive placement (sip)”, monash university malaysia. conflicts of interest: the authors declare no conflict of interest. references 1. ruscio am, stein dj, chiu wt, et al. the epidemiology of obsessive-compulsive disorder in the national comorbidity survey replication. mol psychiatry 2010; 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17(6): 3841. 42. santocchi e, guiducci l, prosperi m, et al. effects of probiotic supplementation on gastrointestinal, sensory and core symptoms in autism spectrum disorders: a randomized controlled trial. front psychiatry 2020; 11. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2021, 4, 1; a0000233. doi: 10.36877/pmmb.a0000233 http://journals.hh-publisher.com/index.php/pmmb original research article incidence, antibiotic susceptibility and characterization of vibrio parahaemolyticus isolated from seafood in selangor, malaysia vurmila venggadasamy1, loh teng-hern tan2, jodi woan-fei law1, hooi-leng ser1, vengadesh letchumanan1*, priyia pusparajah1* article history 1 jeffrey cheah school of medicine and health sciences, monash university, bandar sunway, subang jaya, selangor, 47500, malaysia; vven17@student.monash.edu (vv); jodi.law1@monash.edu (jw-fl); ser.hooileng@monash.edu (h-ls) 2 clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) *corresponding author: priyia pusparajah, priyia.pusparajah@monash.edu (pp); vengadesh letchumanan, vengadesh.letchumanan1@monash.edu (vl) received: 29 july 2021; received in revised form: 25 august 2021; accepted: 27 august 2021; available online: 07 september 2021 abstract: vibrio parahaemolyticus is one of the major foodborne pathogens owing to its cause of infectious diseases such as gastroenteritis. these diseases are often associated with the consumption of contaminated seafood. this study aims to investigate the presence of v. parahaemolyticus, their virulence, antibiotic profiles, and plasmid profiles from 77 different kinds of shellfish samples collected from wet markets and supermarkets in selangor, malaysia. high densities of vibrio species ( > 5 log cfu/g) were found in 14/16 groups of shellfish. among 77 presumptive v. parahaemolyticus isolates, 43 (55.8%) were positive for the toxr gene, confirming the identity of the isolates at the species level. however, none of the v. parahaemolyticus isolates harboured the virulence tdh and trh genes. the antibiotic susceptibility of the v. parahaemolyticus isolates revealed that most of them were resistant to ampicillin (95.3%), ampicillin-sulbactam (81.4%), cefotaxime (37.2%) and imipenem (23.3%). the plasmid profiles of the v. parahaemolyticus isolates showed that 41.9% (18/43) possess at least one plasmid. our results indicate the v. parahaemolyticus isolates are continuously exposed to various antibiotics in the environments, thus consuming the seafood carries a potential health risk to consumers. the antibiotic resistance conferred by the species necessitates an immediate plan to approach the usage of antibiotics differently. keywords: vibrio parahaemolyticus; shellfish; prevalence; virulence; antibiotic resistance 1. introduction seafood is a well-known source of omega-3 long-chain polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid, protein, carnitine, vitamin a, b, d, pmmb 2021, 4, 1; a0000233 2 of 34 and e, and minerals (i.e., magnesium, selenium, iodine, calcium, phosphorus, iron, and zinc)[1–3]. these active compounds are shown to reduce the risk of preterm deliveries, triglyceride levels in type 2 diabetes mellitus, sarcopenia in the elderly and prostate cancerrelated mortalities[3–6]. the global consumption of fish has been increasing steadily at an annual rate of 3.1% from 1961 to 2017. this is attributed to the steady increase in the production of these aquatic animals through aquaculture[7]. in 2018, the aquaculture industry produced 114.5 million tonnes of seafood consumed, in which southeast asia contributed to 17% of the total world production[7, 8]. the aquaculture industry of malaysia is a significant contributor to the overall production of seafood in southeast asia[9]. the increasing production of aquaculture products locally is attributed to the rise in seafood demand internationally and domestically[10]. food and agriculture organization (fao) of the united nations reported that the domestic fish intake in malaysia is 55.9 kg per capita, thrice as much as the global average of fish consumption[9]. this finding demonstrates the importance of seafood as a source of animal protein in malaysia. two of the most widely exported aquaculture products in malaysia are cockles and shrimps[9]. the microbial spoilage of seafood can occur when contaminated water, sediments and sewage runoffs enter water bodies[1,11–13]. in addition, once seafood is harvested, cross-contamination can also take place at any point during rearing, handling, preparing, processing, transporting, and storing[1,11,12]. shellfish are prone to contamination by a plethora of organisms, resulting in shellfish-borne outbreaks[14]. some of the most common foodborne pathogens include vibrio species[15–19], salmonella species[20–25], escherichia coli, listeria monocytogenes[26–30], plesiomonas shigelloides, hepatitis a virus, and calicivirus[14,31]. vibrio parahaemolyticus causes infectious diarrhoea associated with the consumption of raw or contaminated seafood[32–36]. the incubation period of the disease caused by this pathogen varies between literature. however, symptoms can occur as early as four hours post-infection[37,38]. v. parahaemolyticus causes gastroenteritis, which presents with watery diarrhoea, nausea, vomiting, abdominal pain, fever and chills[38]. the disease is self-limiting and lasts for approximately three days in healthy individuals[32–34]. the immunocompromised individuals may experience severe inflammatory diarrhoea, which could lead to septicaemia and death[34,39]. pathogenic v. parahaemolyticus possess tdh and trh genes that encode for thermostable direct haemolysin (tdh) and thermostable direct haemolysin-related (trh), respectively, and is the primary virulence factors produced by v. parahaemolyticus[40,41]. these haemolysins are responsible for the enterotoxic, cytotoxic and haemolytic actions of the enteropathogen[33,34,42–46]. environmental isolates of v. parahaemolyticus that do not possess tdh and trh genes produce other virulence factors to exert pathogenicity. studies have shown that non-tdh and non-trh producing v. parahaemolyticus can still maintain their pmmb 2021, 4, 1; a0000233 3 of 34 enterotoxicity and cytotoxicity activities through other mechanisms, which includes but is not limited to thermolabile haemolysin (tlh), type iii secretion systems (t3sss) and type vi secretion system (t6sss)[47–51]. the widespread usage of antibiotics has exerted selection pressure on bacteria, leading to resistance against these therapeutic agents. antibiotics are excessively used in the healthcare, agriculture, and aquaculture industries[52–54]. this has resulted in the vibrio species developing multi-drug resistance against common antibiotics[55–57]. generally, bacteria may possess intrinsic resistance genes in the chromosomes or acquire resistance genes via plasmids[55,58]. mobile genetic elements such as plasmids are transferred to other bacteria via horizontal gene transfer or vertical gene transfer. the ubiquitous existence of vibrio species in aquatic animals poses a risk to humans as multi-drug resistant vibrio species can be transferred directly to humans via seafood consumption[1,59]. the risk is higher in malaysia due to the growing aquaculture industry and high consumption of seafood locally[9,10]. to date, v. parahaemolyticus isolated from seafood have shown resistance to β– lactams (penicillin and ampicillin), third generation cephalosporins (cefotaxime and ceftazidime), second-generation cephalosporins (cefuroxime), first-generation cephalosporins (cephalothin), bacitracin, amikacin, and vancomycin[60–62]. frequent surveillance of the microbial status of seafood is crucial to monitor the prevalence of v. parahaemolyticus. the various virulence mechanisms exhibited by the pathogen contributes to the pathogenicity of bacteria, resulting in foodborne infections in human hosts. besides that, the overuse and misuse of antibiotics have raised concerns regarding the multi-drug resistant strains of v. parahaemolyticus. therefore, the focus of this study is to determine and characterise the prevalence, virulence, antibiotic resistance profile and plasmid profiles of v. parahaemolyticus isolated from seafood. 2. materials and methods 2.1 sampling this study included two types of bivalve mollusc (i.e., short-necked clam and blood clam) and crustacean (i.e., tiger prawn and indian white shrimp). a total of 77 seafood samples were collected from two wet markets and two supermarkets in selangor, malaysia. upon collection, the samples were kept in separate sterile sealed bags and transported to the laboratory for analysis[63]. pmmb 2021, 4, 1; a0000233 4 of 34 2.2 enumeration of presumptive vibrio species the enumeration of vibrio species from seafood samples was conducted based on the bacterial analytical manual of fda and fao/who risk assessment tool for vibrio parahaemolyticus and vibrio vulnificus[63,64]. 10 g of the sample were weighed and placed in a sterile stomacher beg (bagmixer® 400w, interscience, france), and added with 90 ml of apw with 2% w/v nacl, ph 8.5. the samples are stomached for 90 s. this produces the first 10-1 dilution[65]. the subsequent tenfold dilutions (i.e., 1:10, 1:100, 1:1000, 1:10,000) were prepared by serial dilutions[66]. hicrome™ vibrio (himedia™, india) agar was used in the identification of vibrio species in food samples[67]. the spread plate technique was employed for the enumeration of vibrio species[68]. the hicrome™ vibrio (himedia™, india) agar plates were inoculated with 100 µl of each dilution of the homogenate in triplicate. the agar plates were then incubated at 37°c for 18 hours. after that, the total number of vibrio colonies were identified and enumerated. v. parahaemolyticus colonies are round, bluish-green, opaque and flat, whereas v. cholerae colonies are round, purple, opaque and flat on the hicrome™ vibrio (himedia™, india) agar plates[69]. 2.3 isolation of presumptive vibrio parahaemolyticus the isolation of vibrio species from seafood samples was conducted based on the fda bacterial analytical manual and fao/who risk assessment tool[63,64]. the homogenate in the filter bags was incubated at 37°c for 18 hours to revitalise stressed bacterial cells. after that, 50 µl of the homogenate was streaked onto the selective hicrome™ vibrio (himedia™, india) agar plates. the inoculated agar plates were then incubated at 37°c for 18 hours. subsequently, presumptive v. parahaemolyticus colonies (based on the colony morphology) were picked and purified on tryptic soy agar (tsa) (himedia™, india) supplemented with 2% w/v sodium chloride[70,71]. the agar plates were then incubated at 37°c for 18 hours. vibrios generally form large, cream coloured colonies on tsa plates[66]. the purified single colonies were then kept in semisolid nutrient agar until further analysis. 2.4 genomic dna extraction the genomic dna of presumptive v. parahaemolyticus was extracted by the direct boiled cell lysate method, as described in previous studies[61,72]. the v. parahaemolyticus colonies in the semisolid nutrient agars were revived in tryptic soy broth (tsb) (himedia, pmmb 2021, 4, 1; a0000233 5 of 34 india) with nacl (2% w/v) and incubated in a shaker incubator at 200 rpm and 37°c for 18 hours. one and a half millilitre of the overnight bacterial culture was transferred into microcentrifuge tubes and centrifuged for 5 minutes at 10,000 rpm. centrifuging the bacterial suspension allows the concentration of the bacterial population in the suspension[72]. the supernatant was discarded and the pellets were resuspended in 1 ml of sterile ultrapure water, vortexed and heated at 95°c for 7 minutes in a water bath. the application of heat causes the release of bacterial dna strands from the cells[72]. the cell lysate was then cooled in ice for 5 minutes before centrifuging for 1 minute at 10,000 rpm. lastly, the supernatant with the nucleic acid was pipetted into new 1.5 ml microcentrifuge tubes. the dna samples were stored at -20°c until further analysis. 2.5 identification of vibrio parahaemolyticus using toxr-pcr a singleplex pcr assay targeting the toxr gene was employed in the molecular identification of the v. parahaemolyticus isolates (n = 77) at the species level (table 6)[73]. the pcr assay was performed in a final volume of 20 µl: 2 µl of dna template, 10 µl of 2x taq plus pcr smart mix 1 (solgenttm, korea), 6 µl of sterile ultrapure water, 1 µl of primer toxr-f and toxr-r each. this process was completed using the supercycler: thermalcycler (kyratec, australia) with the following thermal conditions: pre-denaturation of dna at 95°c for 4 minutes, 35 cycles of denaturation at 94°c for 1 minute, annealing at 68°c for 1 minute and extension at 72°c for 30 seconds and final elongation at 72°c for 5 minutes[74]. the pcr product was separated by electrophoresis in 1.5% agarose gel and visualised under a gel documentation system (chemidoctm xrs, bio-rad, usa). vibrio parahaemolyticus vp20 was employed as a positive control. 2.6 detection of virulence genes, tdh and trh the toxr-positive isolates were subjected to a duplex pcr assay targeting the virulence genes, tdh and trh (table 1)[43]. the pcr assay was performed in a final volume of 20 µl: 2 µl of dna template, 10 µl of 2x taq plus pcr smart mix 1 (solgenttm, korea), 4 µl of sterile ultrapure water, 1 µl of primer tdh-f, tdh-r, trh-f and trh-r each. this process was completed using the supercycler: thermalcycler (kyratec, australia) with the following thermal conditions: pre-denaturation of dna at 94°c for 3 minutes, 30 cycles of denaturation at 94°c for 1 minute, annealing at 58°c for 1 minute, extension at 72°c for 1 minute and final elongation at 72°c for 5 minutes[74]. the pcr product was separated by electrophoresis in 1.5% agarose gel and visualised under a gel documentation system pmmb 2021, 4, 1; a0000233 6 of 34 (chemidoctm xrs, bio-rad, usa). vibrio parahaemolyticus vp20 was employed as a positive control. table 1. primers used in the identification and virulence gene detection primer primer sequence (5ʹ → 3ʹ) amplicon size (bp) reference toxr-f toxr-r gtcttctgacgcaatcgttg atacgagtggttgctgtcatg 368 [75] tdh-f tdh-r gtaaaggtctctgacttttggac tggaatagaaccttcatcttcacc 269 [76] trh-f trh-r ttggcttcgatattttcagtatct cataacaaacatatgcccatttccg 500 [76] 2.7 antibiotic susceptibility test the v. parahaemolyticus isolates were examined for susceptibilities against 14 antibiotics (table 2). these antibiotics were selected based on their importance in the clinical setting and aquaculture industry. quinolones (nalidixic acid and levofloxacin) and thirdgeneration cephalosporin (cefotaxime and ceftazidime) are indicated in severe vibriosis, whereas tetracycline is recommended in cholera[77–80]. the ampicillin-sulbactam covers bacterial infections that are resistant to ampicillin[81]. carbapenem (imipenem) and aminoglycoside (amikacin, gentamicin and kanamycin) are broad-spectrum antibiotics used in infections caused by gram-negative and gram-positive bacteria[82–85]. the v. parahaemolyticus isolates were also tested against trimethoprim-sulfamethoxazole and chloramphenicol, which are used in pneumocystis jirovecii pneumonia and eye infections resulting from methicillin-resistant staphylococcus aureus (mrsa), respectively[86,87]. on the other hand, oxytetracycline is an important drug in the aquaculture industry[88]. the isolates were subjected to the antibiotic susceptibility test by the kirby disc diffusion method[89]. this method has been employed in several studies investigating the antibiotic sensitivity in v. parahaemolyticus isolated from food samples[90,91]. all the isolates were revived in tryptic soy broth (tsb) (himedia, india) supplemented with nacl (2% w/v) and shaken at 37°c for 18 hours at 200 rpm. after the incubation, 100 µl of the suspension was swabbed uniformly onto the surface of the mueller-hinton (himedia, india) agar plate with a sterile cotton swab. the agar plates were then incubated at 37°c for 18 hours. the diameter of the inhibition zones was measured and interpreted according to the pmmb 2021, 4, 1; a0000233 7 of 34 guidelines provided by clinical and laboratory standards institute m45[92]. the mar index was calculated using the formula below, which was initially developed by krumperman [93]. 𝑀𝑢𝑙𝑡𝑖𝑝𝑙𝑒 𝑎𝑛𝑡𝑖𝑏𝑖𝑜𝑡𝑖𝑐 𝑟𝑒𝑠𝑖𝑠𝑡𝑎𝑛𝑐𝑒 (𝑀𝐴𝑅) = 𝑎 𝑏 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑛𝑡𝑖𝑏𝑖𝑜𝑡𝑖𝑐𝑠 𝑡ℎ𝑎𝑡 𝑡ℎ𝑒 𝑖𝑠𝑜𝑙𝑎𝑡𝑒𝑠 𝑎𝑟𝑒 𝑟𝑒𝑠𝑖𝑠𝑡𝑎𝑛𝑡 𝑡𝑜 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑛𝑡𝑖𝑏𝑖𝑜𝑡𝑖𝑐𝑠 𝑡ℎ𝑒 𝑖𝑠𝑜𝑙𝑎𝑡𝑒𝑠 𝑎𝑟𝑒 𝑒𝑥𝑝𝑜𝑠𝑒𝑑 𝑡𝑜 table 2. the list of antibiotics tested in this study[94–96] class of antibiotics antibiotics concentration (μg) penicillin ampicillin 10 β-lactamase inhibitor ampicillin-sulbactam 30 third-generation cephalosporin cefotaxime 30 ceftazidime 30 carbapenem imipenem 10 aminoglycoside amikacin 30 gentamicin 30 kanamycin 30 dihydrofolate reductase inhibitor trimethoprimsulfamethoxazole 25 tetracycline tetracycline 30 oxytetracycline 30 quinolone nalidixic acid 30 levofloxacin 5 anti-50s antimicrobial chloramphenicol 30 2.8 plasmid profiling the bacterial isolates were revived in tryptic soy broth (tsb) (himedia, india) supplemented with nacl (2% w/v) and shaken at 37°c in a shaker incubator (200 rpm) for 18 hours. 1.5 ml of the suspension were transferred into a new microcentrifuge tube, and this was used for the plasmid extraction using the gf-1 plasmid dna extraction kit (vivantis technologies, malaysia). the plasmid dna was separated by gel electrophoresis in 1.0% agarose gel and visualised under uv light with a gel documentation system (chemidoctm xrs, bio-rad, usa). pmmb 2021, 4, 1; a0000233 8 of 34 2.9 statistical analysis the analysis of the data was conducted using the statistical analysis software, ibm® spss® statistics version 26. a one-way anova test was performed to determine if the difference between the mean vibrio count in the four types of shellfish samples were statistically significant. besides that, a chi-squared test was used to determine whether the differences in the prevalence of vibrio parahaemolyticus in seafood from wet markets and supermarkets were statistically different. another chi-squared test was employed to study the relationship between different types of establishments and the number of v. parahaemolyticus isolates with a mar index of more than 0.2. a difference was considered statistically significant when p < 0.05. 3. results 3.1 microbial load of total vibrio species a total of 77 shellfish comprising of short-necked clam (paratapes undulatus) (n = 19), blood clam (tegillarca granosa) (n = 20), tiger prawn (penaeus monodon) (n = 19) and indian white shrimp (penaeus indicus) (n = 19) were sampled from two wet markets and two supermarkets in selangor, malaysia. the mean vibrio count is calculated according to the sample type from the respective sampling sites (table 3). out of 16 groups of shellfish samples, 14 groups were contaminated with more than 5 log cfu/g of vibrio species, which is the minimum bacterial load required to cause symptoms of vibriosis in human hosts[61]. the only group of samples with a mean vibrio count lower than the infectivity limit are tiger prawns from wet market 1 and indian white shrimps from supermarket 2. overall, the mean density of vibrio species in all the shellfish samples ranges from 4.66 ± 0.40 to 8.95 ± 0.00 log cfu/g. the highest count is seen in short-necked clams from wet market 2, followed by blood clams from wet market 1 (6.06 ± 0.01 log cfu/g) and short-necked clams from wet market 1 (5.98 ± 0.04). the lowest vibrio count is detected in tiger prawns from wet market 1. the highest mean vibrio count in each seafood sample type from all the markets, 6.52 log cfu/g, is seen in short-necked clam samples, followed by 5.77 log cfu/g in blood clams, 5.50 log cfu/g in indian white shrimps and 5.34 log cfu/g in tiger prawns. one-way anova test performed showed a significant difference between the mean of vibrio count in the four types of shellfish (p = 0.005). mean vibrio count in indian white shrimps is significantly lower than short-necked clams (-1.03, 95% ci = -1.95 -0.10, p = 0.022). besides that, tiger prawns also have mean vibrio count that is significantly lower than shortnecked clams (-1.19, 95% ci = -2.11 -0.27, p = 0.005). however, the mean vibrio count was not statistical significant between the blood clam and short-necked clam samples (0.76, 95% ci = -1.68 0.17, p = 0.005). pmmb 2021, 4, 1; a0000233 9 of 34 table 3. mean total vibrio species count listed according to the type of seafood samples and sampling sites. sample mean of total vibrio count (log cfu/g) mean ± standard deviation wet market 1 wet market 2 supermarket 1 supermarket 2 paratapes undulatus (short-necked clam) 5.98 ± 0.04 8.95 ± 0.00 5.71 ± 0.17 5.46 ± 0.08 tegillarca granosa (blood clam) 6.06 ± 0.01 5.75 ± 0.02 5.42 ± 0.03 5.84 ± 0.03 penaeus monodon (tiger prawn) 4.66 ± 0.40 5.79 ± 0.05 5.21 ± 0.08 5.67 ± 0.05 penaeus indicus (indian white shrimp) 5.41 ± 0.11 5.74 ± 0.05 4.89 ± 0.21 5.95 ± 0.04 3.2 molecular identification of vibrio parahaemolyticus the pcr assay revealed positive amplification of the toxr gene with 368 bp amplicon band in 55.8% (43/77) of the presumptive vibrio parahaemolyticus isolates (table 4). the incidence of vibrio parahaemolyticus in the shellfish samples was highest in samples collected from supermarket 1 at 70.0% (14/20), followed by wet market 1 at 61.0% (11/18). supermarket 1 presented the lowest prevalence of v. parahaemolyticus at 35% (7/20). among the samples, v. parahaemolyticus is most prevalent in tiger prawn 78.9% (15/19), followed by indian white shrimp 68.4% (13/19), blood clam 45.0% (9/20) and short-necked clam 31.6% (6/19) (table 2). the results of a chi-squared test performed showed no significant association between the number of toxr-positive v. parahaemolyticus isolates in shellfish from wet markets and supermarkets (51.2% vs. 48.8%, p = 0.539). none of the 43 v. parahaemolyticus isolates harboured the thermostable direct haemolysin (tdh) and thermostable direct haemolysin-related (trh) virulence genes. pmmb 2021, 4, 1; a0000233 10 of 34 table 4. prevalence of vibrio parahaemolyticus in the shellfish samples sites wet market 1 wet market 2 supermarket 1 supermarket 2 overall type of shellfish total no. of isolates toxrpositive (%) total no. of isolates toxrpositive (%) total no. of isolates toxrpositive (%) total no. of isolates toxrpositive (%) total no. of isolates toxrpositive (%) paratapes undulatus (shortnecked clam) 5 1 (20.0) 4 1 (25.0) 5 3 (60.0) 5 1 (20.0) 19 6 (31.6) tegillarca granosa (blood clam) 5 4 (80.0) 5 2 (40.0) 5 0 (0.0) 5 3 (60.0) 20 9 (45.0) penaeus monodon (tiger prawn) 3 3 (100) 6 4 (67.0) 5 3 (60.0) 5 5 (100) 19 15 (78.9) penaeus indicus (indian white shrimp) 5 3 (60.0) 4 4 (100) 5 1 (20.0) 5 5 (100) 19 13 (68.4) total 18 11 (61.0) 19 11 (58.0) 20 7 (35.0) 20 14 (70.0) 77 43 (55.8) 3.3 antibiotic susceptibility test and multiple antibiotic resistance (mar) indices the antibiotic resistance profiles of all 43 v. parahaemolyticus isolates are summarised in table 5. a vast majority of the bacterial isolates were resistant to ampicillin (95.3%). similarly, resistance towards the combination antibiotic, ampicillin-sulbactam was also high (81.4%). the antibiotic resistance profile of the v. parahaemolyticus isolates towards third-generation cephalosporins showed mixed results. there was nearly an equal distribution between isolates that were resistant (37.2%) to cefotaxime and sensitive (34.5%) towards the said drug. in contrast, most isolates were susceptible to ceftazidime (55.8%), with a high proportion of the isolates showing intermediate resistance towards this antibiotic (37.2%). although most of the v. parahaemolyticus isolates (76.7%) were susceptible to the carbapenem antibiotic, imipenem, 23.3% (10/43) were resistant to this antibiotic. this pmmb 2021, 4, 1; a0000233 11 of 34 finding is worrying because carbapenems are broad-spectrum antibiotics commonly used as the last line of drug in gram-negative and gram-positive bacterial infections[82,83]. table 5. antibiotic resistance profile of the vibrio parahaemolyticus isolated from the shellfish. antibiotics resistant n (%) intermediate n (%) sensitive n (%) ampicillin 10µg 41 (95.3) 0 (0.0) 2 (4.7) ampicillin-sulbactam 30µg 35 (81.4) 5 (11.6) 3 (7.0) cefotaxime 30µg 16 (37.2) 12 (27.9) 15 (34.9) ceftazidime 30µg 3 (7.0) 16 (37.2) 24 (55.8) imipenem 10µg 10 (23.3) 0 (0.0) 33 (76.7) amikacin 30µg 1 (2.3) 3 (7.0) 39 (90.7) gentamicin 30µg 2 (4.7) 1 (2.3) 40 (93.0) kanamycin 30µg 4 (9.3) 9 (20.9) 30 (69.8) trimethoprim-sulfamethoxazole 25µg 0 (0.0) 0 (0.0) 43 (100.0) tetracycline 30µg 8 (18.6) 3 (7.0) 32 (74.4) oxytetracycline 30µg 7 (16.3) 1 (2.3) 35 (81.4) nalidixic acid 30µg 1 (2.3) 1 (2.3) 41 (95.3) levofloxacin 5µg 1 (2.3) 0 (0.0) 42 (97.7) chlorampenicol 30µg 0 (0.0) 0 (0.0) 43 (100.0) in contrast, the v. parahaemolyticus isolates were highly susceptible to quinolones (levofloxacin, 97.7%; nalidixic acid, 95.3%), aminoglycosides (gentamicin, 93%; amikacin, 90.7%; kanamycin, 69.8%) and tetracyclines (oxytetracycline, 81.4%; tetracycline, 74.4%). unsurprisingly, all the isolates were sensitive towards trimethoprim-sulfamethoxazole and chloramphenicol. the isolates exhibited mar indices ranging from 0.07 to 0.36. the highest mar index (0.36) was seen in 5/43 (7.0%) bacterial isolates. these isolates were resistant to penicillin, beta-lactamase inhibitors, third-generation cephalosporins, carbapenems and, either quinolones or tetracyclines. the highest frequency of the mar index was 0.21, followed by 0.29. 37.2% (16/43) of the v. parahaemolyticus isolates exhibited a mar index of 0.21, whereas 25.9% (11/43) had a mar index of 0.29. among all 43 v. parahaemolyticus isolates, only one (2.3%) did not exhibit mar as it was sensitive to all the antibiotics included in the study. interestingly, all 10 v. parahaemolyticus isolates that were resistant to pmmb 2021, 4, 1; a0000233 12 of 34 imipenem demonstrated mar indices ranging from 0.21 to 0.36, which is the highest mar index reported in this study. bacterial isolate with a mar index of more than 0.2 connotes the origin of the isolate from a high-risk source of contamination. this means that a particular strain of vibrio parahaemolyticus was exposed to an increased number of antimicrobials[97,98]. in the present study, 30/43 v. parahaemolyticus isolates had a mar index of more than 0.2. cumulatively, this was expressed by 69.8% of the bacterial isolates which were resistant to 3 to 5 different antibiotics tested. of the 30 v. parahaemolyticus isolates with a mar index exceeding 0.2, an equal number of bacterial strains were isolated from wet market and supermarket samples. a chi-squared test was performed to study the relationship between different types of establishments and the risk of contamination. the analysis shows that there was no significant difference in the risk of contamination among different sources of shellfish (50% vs. 50%; p = 0.054). 3.4 plasmid profiles of vibrio parahaemolyticus all 43 toxr-positive v. parahaemolyticus isolates were subjected to plasmid profiling and the profiles are illustrated in table 6. among all the v. parahaemolyticus isolates, 18 (41.9%) isolates harboured at least one plasmid, whereas the majority did not possess any plasmids (25/43; 58.1%). interestingly, all the isolates without plasmids (excluding vv34) were resistant to at least 1 antibiotic with mar indices ranging from 0.07 to 0.29 (table 7). cumulatively, the number of plasmids in the v. parahaemolyticus isolates ranges from one to four, whereby 20.9% (9/43), 14.0% (6/43), and 4.7% (2/23) of the isolates had one, two and three plasmids, respectively. only one (2.3%) strain of v. parahaemolyticus harboured four plasmid dna bands with molecular weights of 1.5 kb, 5.2 kb, 7 kb and above 10 kb. this v. parahaemolyticus isolate, vv40, was resistant to four antibiotics which are ampicillin, ampicillin-sulbactam, cefotaxime and imipenem. in contrast, the vv34 strain, which was susceptible to all the antibiotics tested, did not harbour any plasmids. the molecular weight of the plasmids that reside in the v. parahaemolyticus isolates ranges from 1 kb to more than 10 kb. the plasmid dna bands in each strain of bacteria were categorised according to the number of plasmids and plasmid sizes. with that, 14 different plasmid patterns were encountered among the v. parahaemolyticus isolates. the most common plasmid pattern seen is 1.1, which refers to the possession of one plasmid with a size of more than 10 kb. this pattern has been identified in 4/43 (9.3%) v. parahaemolyticus isolates. pmmb 2021, 4, 1; a0000233 13 of 34 besides that, vv09, vv30 and vv37, which were resistant to five out of 14 antibiotics tested, had relatively bigger plasmids. vv37 has only one plasmid that is more than 10 kb but is resistant to five different antibiotics, namely, ampicillin, ampicillinsulbactam, cefotaxime, imipenem, and tetracycline. vv30 has plasmids with the molecular weight of more than 10 kb and 10 kb, whereas vv09 has 10 kband 2kb-sized plasmids. strikingly, 9 out of 10 (90%) vibrio parahaemolyticus isolates that were resistant to imipenem had one to four plasmids, sizes ranging from 1.5 kb to more than 10 kb. vv38 is the only bacterial strain that was resistant to imipenem but did not harbour any plasmids. table 6. plasmid profiles of the vibrio parahaemolyticus isolates from shellfish samples number of plasmids plasmid pattern plasmid size (kb) number of isolates (%) vibrio parahaemolyticus isolates 0 25 (58.1) vv02, vv10, vv11, vv12, vv21, vv22, vv23, vv25, vv26, vv27, vv34, vv38, vv39, vv41, vv42, vv43, vv52, vv56, vv57, vv58, vv65, vv68, vv69, vv70, vv75 1 1.1 >10 4 (9.3) vv37, vv45, vv48, vv49 1.2 10 2 (4.7) vv29, vv67 1.3 7 1 (2.3) vv19 1.4 5.2 1 (2.3) vv64 1.5 1.5 1 (2.3) vv71 2 2.1 >10, 10 1 (2.3) vv30 2.2 10, 4 1 (2.3) vv14 2.3 10, 2 1 (2.3) vv09 2.4 7, 6.2 1 (2.3) vv07 2.5 7, 4 1 (2.3) vv24 2.6 6.2, 5.2 1 (2.3) vv01 3 3.1 10, 6.2, 2.5 1 (2.3) vv08 3.2 10, 5.2, 2 1 (2.3) vv35 4 4.1 >10, 7, 5.2, 1.5 1 (2.3) vv40 pmmb 2021, 4, 1; a0000233 14 of 34 table 7. plasmid profiles and antibiograms of vibrio parahaemolyticus isolates. number of plasmids plasmid pattern plasmid size (kb) mar index antibiogram isolates 0 0.0 vv34 0.07 amp vv22, vv39 0.14 amp/sam vv02, vv26, vv52, vv57, vv65 0.14 amp/ot vv43 0.21 amp/sam/te vv12, vv23 0.21 amp/caz/k vv10 0.21 amp/sam/k vv11 0.21 amp/ctx/k vv21 0.21 amp/ak/k vv25 0.21 amp/ipm/ot vv38 0.21 amp/sam/ot vv41 0.21 amp/sam/te vv68 0.21 sam/ctx/te vv69 0.21 amp/sam/ctx vv75 0.29 amp/sam/ctx/ot vv27, vv42, vv70 0.29 amp/sam/te/ot vv56 0.29 amp/sam/ctx/ipm vv58 1 1.1 > 10 0.21 amp/sam/cn vv49 0.29 amp/sam/ipm/ot vv45 0.29 amp/sam/ctx/ipm vv48 0.36 amp/sam/ctx/ipm/te vv37 1.2 10 0.14 amp/sam vv67 1.2 10 0.29 amp/sam/ctx/cn vv29 1.3 7 0.14 amp/sam vv19 1.4 5.2 0.21 amp/sam/ipm vv64 1.5 1.5 0.21 amp/sam/ctx vv71 pmmb 2021, 4, 1; a0000233 15 of 34 number of plasmids plasmid pattern plasmid size (kb) mar index antibiogram isolates 2 2.1 > 10, 10 0.36 amp/sam/caz/ipm/te vv30 2.2 10, 4 0.14 amp/sam vv14 2.3 10, 2 0.36 amp/sam/caz/ipm/na vv09 2.4 7, 6.2 0.21 amp/sam/ctx vv07 2.5 7, 4 0.14 amp/sam vv24 2.6 6.2, 5.2 0.29 amp/sam/ctx/ipm vv01 3 3.1 10, 6.2, 2.5 0.21 amp/sam/ctx vv08 3.2 10, 5.2, 2 0.29 amp/sam/ctx/te vv35 4 4.1 > 10, 7, 5.2, 1.5 0.29 amp/sam/ctx/ipm vv40 amp (ampicillin), sam (ampicillin-sulbactam), caz (ceftazidime), ctx (cefotaxime), ipm (imipenem), ak (amikacin), cn (gentamicin), k (kanamycin), te (tetracycline), ot (oxytetracycline), na (nalidixic acid), lev (levofloxacin), sxt (trimethoprim-sulfamethoxazole), c (chloramphenicol) 4. discussion the vibrio species are commonly found in marine environments, living in the water, sediments, plankton, aquatic animals, and flora[1,38,49,99]. therefore, it is essential to continuously monitor the density of these pathogens to ensure the safety of seafood. the microbial load of vibrio species in the shellfish sampled in this study is reported in table 3. in this study, vibrio species were recovered from all the seafood sampled from all four sampling locations. the mean total count of vibrio species ranged from 4.66 ± 0.40 to 8.95 ± 0.00 log cfu/g. as of now, the microbiological limits of v. parahaemolyticus count in seafood is not regulated in malaysia[88]. however, letchumanan and colleagues suggested that a minimum of 5 log cfu/g of vibrio count is required to cause symptoms of vibriosis in human hosts[61]. in the present study, 14/16 groups of shellfish samples listed in table 3 were contaminated with more than 5 log cfu/g of vibrio species. considering this, groups of shellfish that harboured a mean vibrio count of more than 5 log cfu/g can potentially become a health hazard to humans. the highest mean total vibrio count detected in this study was higher than the microbial load of vibrio species reported by other studies conducted on seafood. in the study done by letchumanan et al.[61] in malaysia, the mud crabs (scylla serrate), flower crabs (portunus pelagicus), carpet clams (paphia textile) and hard shell clams (meretrix meretrix) pmmb 2021, 4, 1; a0000233 16 of 34 contained a microbial load of 2.45 to 6.63 log cfu/g. another study done by the same author reported the microbial load of vibrio species ranging from 4.36 log cfu/ml to 6.34 log cfu/ml in the prawns sampled from malaysia[74]. it is also noteworthy that the findings of this study are higher than the results obtained by lamon et al.[100] in italy. the mean vibrio count in the mediterranean mussel (mytilus galloprovincialis) and grooved carpet shell (ruditapes decussatus) sampled in the study were well below 5 log cfu/g before the purification process[100]. the upward trend in the mean concentration of vibrio species calls for the attention of the public health body to monitor the levels of this enteropathogen closely in seafood, especially in shellfish. high levels of vibrio species have been detected in many types of seafood samples from this region[61,74,88]. this can be attributed to the ubiquitous existence of these organisms in estuaries and coastal regions compounded by the hot climate in malaysia, which promotes the growth of vibrio species[1,38,49,88,99]. nevertheless, this study has shown that the mean count of vibrio species was significantly different among the four species of shellfish (p < 0.05). the mean vibrio count in short-necked clams was significantly higher than in both species of shrimps analysed in this study. to our best knowledge, the present study is the first to statistically analyse the difference in the microbial load of vibrio species among different types of shellfish. despite the increased risk of contamination associated with all types of shellfish, the higher density of vibrio species in short-necked clams can be linked to the filter-feeding habits of these molluscs. bivalves such as the short-necked clams feed by filtering water through their gills, allowing bacteria and other contaminants to mobilise and concentrate in the digestive tract[13,32,101,102]. however, the findings cannot be extrapolated to all types of bivalve molluscs. this is because the microbial load of vibrio species in the blood clams sampled in this study were not significantly higher than both species of crustaceans, disallowing a general correlation to be formed. nonetheless, the findings of this study indicate that all shellfish are potential vehicles of transmission of the vibrio species. the high risk of contamination directly translates into a high risk of gastroenteritis related to the consumption of crustaceans and bivalve molluscs. to avert the incidence of vibriosis, centers for disease control and prevention (cdc) recommends boiling or steaming shellfish for an appropriate duration of time[78]. as evidenced by liu et al.[103], the direct exposure of shellfish to high temperatures has been shown to reduce the concentrations of the pathogen to undetectable levels (< 3 mpn/g), consequently ensuring safe consumption of seafood. the toxr gene is commonly selected as the target gene in the detection of vibrio parahaemolyticus in seafood[10,60,74,88,104]. this is because the toxr-pcr assay is highly pmmb 2021, 4, 1; a0000233 17 of 34 accurate attributable to the heterogeneity in the nucleotide sequence of toxr gene among the species in the vibrio genus[105,106]. a total of 43/77 (55.8%) presumptive v. parahaemolyticus isolates from the shellfish samples were positive for the toxr gene, confirming the presence of the pathogen. tiger prawn was detected at the highest incidence rate (78.9%), followed by indian white shrimp (68.4%), blood clam (45.0%) and short-necked clam (31.6%). the high prevalence of v. parahaemolyticus in these shellfish samples can be attributed to the temperature of seawater in tropical countries like malaysia, which provides a conducive environment for v. parahaemolyticus to survive and grow in seafood[1,10,104,107]. interestingly, the highest prevalence of v. parahaemolyticus was found in tiger prawns, which contained the lowest mean vibrio count among all the shellfish samples. conversely, the short-necked clams with the lowest incidence of the pathogen possessed the highest microbial load of vibrio species. these findings are in line with previous studies[98,108], which supports the use of pcr amplification over the direct plating method in identifying v. parahaemolyticus as the molecular detection technique has shown to produce more accurate results than the latter. there is a considerable amount of literature concerning v. parahaemolyticus in seafood from asia. despite the subtle variation in the methodologies employed, ultimately, most studies have consistently used the toxror tlh-pcr amplification technique, which allows accurate comparisons of findings to be made[90,109–113]. the overall prevalence of v. parahaemolyticus in the shellfish sampled in this study is far lower compared to most studies done in other tropical and subtropical countries. in malaysia, narayanan et al.[90] reported that 85.71% (120/140) of blood clam (anadara granosa), shrimp (penaeus species), surf clam (paphia undulata) and squid (loligo species) were positive for v. parahaemolyticus. besides that, narayanan et al.[90] found that 96.8% (31/32) of the shellfish from kerala, india, were contaminated with the pathogen. in bangladesh, 69.44% of the shrimps harvested from farms contained v. parahaemolyticus, as outlined by siddique et al.[110]. in east asia, recent studies showed a varying incidence of the pathogen in shellfish. the occurrence of v. parahaemolyticus was reported as 20.0% and 14.0% in short-necked clams (paphia variegata) and white shrimps (penaeus vannamei) from china, which is lower than our findings[111]. in contrast, oysters (crassostrea gigas) sampled in kang et al.[113] showed a higher prevalence (85.5%) of the bacteria in their study. in the present study, we found that the occurrence of v. parahaemolyticus was slightly higher in wet market samples as compared to the supermarket samples (51.2% vs. 48.8%). to our knowledge, this is the first study to show that the prevalence of v. parahaemolyticus was not significantly associated with the type of establishments the shellfish are obtained from (p > 0.05). unlike the present study, tan et al.[88] demonstrated a significantly lower pmmb 2021, 4, 1; a0000233 18 of 34 prevalence of v. parahaemolyticus in short mackerels (rastrelliger bachysoma) from hypermarkets compared to mini market and wet markets samples (83.3% vs. 89.1% vs. 95.2%; p < 0.05). in contrast, the study by lee et al.[98] reported a lower prevalence of the pathogen in wet market fish samples compared to fishes from the supermarket (47% vs. 53%). the mixed evidence on the contamination rate of shellfish in different establishments supports the idea outlined by previous studies[61,114]. these studies suggested that crosscontamination can take place at any point during handling, preparing, processing, transporting, and storing of shellfish, irrespective of the sampling site[61,114]. if the cold chain is broken, multiplication of v. parahaemolyticus cells can occur as a result of the exposure of shellfish to room temperatures[115]. therefore, temperature control plays a vital role in the preservation of shellfish. liu et al.[103] and vasudevan et al.[116] have highlighted the importance of freezing in maintaining the freshness of shellfish up to the point of human consumption. ice crystals that form during frozen storage disrupt bacterial cell structures, consequently terminating viable v. parahaemolyticus cells in shellfish[103]. historically, the two well-defined haemolysins encoded by the tdh and trh genes are more frequently found in v. parahaemolyticus isolated from clinical samples than environmental and food samples[98,110,117]. therefore, these putative genes provide the most discriminatory ability to detect pathogenic strains of v. parahaemolyticus[40,41]. generally, the occurrence of pathogenic strains of v. parahaemolyticus depends on the isolation rate of the bacteria in seafood samples. as stated in the previous section, the prevalence of foodborne pathogens varies according to different geographical sites and species of seafood. therefore, direct comparisons of the incidence of tdhand trh-positive v. parahaemolyticus among individual studies should be interpreted cautiously. although a high prevalence of v. parahaemolyticus was detected in the shellfish samples, none of the v. parahaemolyticus strains was positive for tdh and trh genes. this shows that the expression of tdh and trh does not exist among the v. parahaemolyticus strains isolated from the shellfish sampled from the wet markets and supermarkets in selangor. the findings of our study are in agreement with several studies undertaken around this region. a study by narayanan et al.[90] demonstrated that all 140 samples of the bivalve molluscs and crustaceans obtained from selangor did not present with either tdh or trh genes. similar results were also found in the study conducted by kang et al.[113], whereby none of the oysters (crassostrea gigas) was contaminated with tdhor trh-positive strains of vibrio parahaemolyticus. pmmb 2021, 4, 1; a0000233 19 of 34 previous studies performed in asia have demonstrated a low occurrence of the tdh and trh in the v. parahaemolyticus isolates from seafood. in lee et al.[98], 4 out of 165 (2.4%) toxr-positive v. parahaemolyticus isolates from fish samples were positive for trh gene, whereas none of the isolates expressed the tdh. the oysters (crassostrea gigas) sampled in south korea were contaminated with 9.1% of trh-positive vibrio parahaemolyticus. contrastingly, none of the isolates was positive for tdh[118]. hu et al.[111] demonstrated that the 2.6% (2/77) and 1.3% (1/77) of v. parahaemolyticus isolated from shellfish samples from china were positive for tdh and trh gene, respectively. despite the low prevalence of pathogenic strains of the bacteria reported in most studies, a minority of the studies conducted have illustrated a higher incidence of pathogenic v. parahaemolyticus strains in seafood. for instance, 21.95% of shrimp and 18.75% of fish samples from china were contaminated with trh-positive vibrio parahaemolyticus, whereas the prevalence of tdh was 7.32% and 15.63% in shrimps and fish, respectively[119]. as outlined by raghunath[41], the vast difference in the occurrence of tdhand trh-positive strains demonstrated by individual studies is attributable to the different sampling sites, source of shellfish and detection method of the bacteria. there is still insufficient data to conclude that all the v. parahaemolyticus strains in this present study are non-pathogenic, based entirely on the absence of tdh and trh genes. this is because several other mechanisms are involved in the mediation of virulence in this pathogen, such as the tlh, t3ss, t6ss, biofilm, siderophore and protease production[15,120– 123]. besides that, the demonstration of β-haemolytic action, which is highly specific to tdh, was recently seen in tdh and trh-negative strains of vibrio parahaemolyticus[90,124]. this raises questions on the involvement of mechanisms apart from the tdh, the kanagawa phenomenon (kp) action of the bacteria[43,125,126]. despite the absence of tdhand trh-positive v. parahaemolyticus strains in the present study, continuous monitoring of the shellfish sold in this region is important to ensure the food safety of seafood products. however, it is imperative that future work targets other virulence factors to determine the pathogenicity of the v. parahaemolyticus strains in seafood. the antibiotic susceptibility test was performed for all 43 v. parahaemolyticus isolates from the shellfish samples. the results show that nearly all of the bacterial isolates (95.3%) were resistant to ampicillin. however, resistance to β-lactams such as ampicillin is not uncommon in v. parahaemolyticus. due to the misuse and overuse of ampicillin in aquaculture, high incidences of ampicillin-resistant v. parahaemolyticus have been reported extensively in literature worldwide[55,61,90,98,118,119]. the increasing trend in the minimum inhibition concentration (mic) of ampicillin from 64 µg/ml in 2011 to 128 µg/ml in 2013, as outlined by al-othrubi et al.[60], have highlighted the worsening case of resistance against pmmb 2021, 4, 1; a0000233 20 of 34 this drug. although ampicillin is not used in the management of vibriosis, these findings are of great concern as it impedes the role of ampicillin in the empirical management of bacterial infections[55]. besides that, most of the v. parahaemolyticus isolates were also resistant to ampicillin-sulbactam (81.4%). alarmingly, this value is much higher than those reported by other studies in malaysia[74,88,90]. although the sample source and detection methods could have contributed to the differences, the resistance to β-lactam/β-lactamase inhibitor drug in this study should still be highlighted, considering the significance of the drug in treating infections caused by β-lactamase-containing s. aureus, h. influenzae and e. coli[81,127]. a notable resistance pattern was observed with cefotaxime (37.2%) in the present study. on the contrary, most of the v. parahaemolyticus isolates were found to be sensitive to ceftazidime (55.8%). similar resistance patterns to third-generation cephalosporins were also demonstrated by other studies, including the study done by narayanan et al.[90]. in lee et al.[98], more than half (52%) of the v. parahaemolyticus isolates were resistant to cefotaxime, whereas 48% of these isolates exhibited resistance to ceftazidime. meanwhile, kang et al.[118] reported that most isolates (63.6%) from oysters (crassostrea gigas) were sensitive to cefotaxime. this study also demonstrated that none of the v. parahaemolyticus strains was resistant to the aforementioned drug. the discrepancies in the literature regarding the resistance of v. parahaemolyticus to third-generation cephalosporins reflects on the antibiotic-prescribing practices in the local setting. however, the resistance to β-lactams like this group of antibiotics is a significant public health threat. the recent discoveries of extended-spectrum beta-lactamases (esbl) producing strains of v. parahaemolyticus in korea and india raise concerns about the spread of these strains locally[128,129]. carbapenems such as imipenem are proven effective in providing complete coverage in human infections resulting from esbl-producing organisms[130,131]. carbapenems are also frequently used as the drug of last resort in infections caused by gram-positive and gramnegative bacteria[82,83]. the over-reliance on carbapenems have therefore resulted in the increasing resistance to these group of drugs. this is evidenced by the 23.3% resistance rate to imipenem in the present study. these results tie well with similar studies done in this region. lee et al.[98] have reported that 12.0% of the v. parahaemolyticus isolated from marine fish and freshwater fish were resistant to imipenem. similarly, a low incidence (2%) of imipenem resistance was detected in v. parahaemolyticus isolated from banana prawns and red prawns from malaysia[74]. letchumanan et al.[61] showed that 0.5% out of 200 isolates from crustaceans and bivalve molluscs were resistant to the same drug. contrary to these findings, narayanan et al.[90] and siddique et al.[110] found that all the v. parahaemolyticus isolates from the seafood samples were susceptible to carbapenems such as imipenem and pmmb 2021, 4, 1; a0000233 21 of 34 meropenem. the low resistance rates show that the global resistance to this group of antibiotics is still at the initial stage. hence, immediate action is needed to preclude the further spread of carbapenem resistance among v. parahaemolyticus in seafood. in the present study, high susceptibility rates to quinolones (levofloxacin, 97.7%; nalidixic acid, 95.3%) were seen. these findings are in line with other literature that has reported similar susceptibility patterns to quinolones[90,119]. the consistently high sensitivity of v. parahaemolyticus to quinolones reinforces the usage of these antimicrobials in the management of vibriosis, thus reducing mortality rates caused by the disease[77,78]. the isolates in this study were highly susceptible to trimethoprim-sulfamethoxazole (100%), chloramphenicol (100%), aminoglycosides (gentamicin, 93%; amikacin, 90.7%; kanamycin, 69.8%) and tetracyclines (oxytetracycline, 81.4%; tetracycline, 74.4%). similarly, a study reported high susceptibility of v. parahaemolyticus isolates to chloramphenicol (91.04%), tetracycline (83.58%), and aminoglycosides such as gentamicin (74.63%) and amikacin (65.67%)[88]. several other studies have also found high sensitivity rates to these groups of antibiotics in v. parahaemolyticus from food sources[90,132]. in this study, the mar index of the v. parahaemolyticus isolates ranges from 0.00 to 0.36. three isolates exhibited resistance to five antibiotics, yielding the highest mar index value seen in this study. although the mar index provides a good measure of the severity of antibiotic resistance in the samples, comparisons of mar indices between studies are impossible to make due to the variation in the types of antibiotics tested and the total number of antibiotics used in individual studies. for example, narayanan et al.[90] demonstrated that the mar indices of v. parahaemolyticus isolates ranged from 0.00 to 0.71. the isolate with the highest mar index was resistant to 17 out of 24 antibiotics. however, in siddique et al.[110], the mar index ranged from 0.07 to 0.27, and the highest mar index was demonstrated by v. parahaemolyticus isolates that were resistant to four out of 15 antibiotics. bacterial isolates with a mar index of more than 0.2 reflects the origin of the strain from contaminated sources such as aquaculture and agriculture farms[97,98]. the excessive usage of antibiotics in these sectors exerts selection pressure on the microflora in the water and soil, resulting in the growth of multi-drug resistant organisms[53,59,133]. alternatively, isolates that have mar indices less than 0.2, are thought to have originated from a low-risk source with lesser exposure to antibiotics[98]. in this study, 69.8% of the v. parahaemolyticus isolates had mar indices of more than 0.2. a chi-squared test showed that the number of bacterial isolates with a mar index of more than 0.2, was not significantly different between wet market and supermarket samples (p = 0.054). this suggests that the shellfish from wet pmmb 2021, 4, 1; a0000233 22 of 34 markets and supermarkets had similar levels of exposure to antibiotics. seafood samples from both locations are at equally high risk of transmitting multidrug resistant strains. the advent and widespread usage of antibiotics for over 80 years have exerted selection pressure on bacteria such as v. parahaemolyticus. this has resulted in resistance against these therapeutic agents, as demonstrated in the present study. antibiotics like ampicillin are excessively used in the agriculture industry and healthcare sectors[52]. antimicrobials are used to prevent, treat and control diseases and promote the growth of marine products in the aquaculture industry[52,53]. in the healthcare setting, antibiotics are often used to treat self-limiting infections and indefinite diagnoses[52,134]. ultimately, these anthropogenic activities involving the widespread usage of antibiotics have resulted in v. parahaemolyticus developing multi-drug resistance against common antibiotics[55]. the development of multi-drug resistance in v. parahaemolyticus is a public health and therapeutic concern. this is because these resistance genes can spread to human hosts through the consumption of contaminated seafood. resistance genes can also be transmitted to human hosts via the lateral gene transfer of mobile genetic elements with resistance genes, from v. parahaemolyticus to other human pathogens[1,59]. plasmids are one of the mobile genetic elements that contain important genes for bacterial survival, such as genetic elements encoding antibiotic resistance[135,136]. the selection pressure exerted by the frequent exposure to antibiotics promotes the transmission of plasmids with resistance genes among bacterial cells[53,59,133]. therefore, profiling plasmids in bacteria like v. parahaemolyticus provides a better understanding of the mediation of antibiotic resistance in these bacteria. in this study, 18 out of 43 (41.9%) v. parahaemolyticus isolates possess one to four plasmid dna bands, ranging from 1 kb to more than 10 kb in size. these findings are in line with other studies that have detected plasmid dna in v. parahaemolyticus isolated from seafood[61,98,137]. however, the majority of the bacterial isolates in the present study did not harbour any plasmid. based on the plasmid profiles of the isolates mentioned, 25/43 v. parahaemolyticus isolates (58.1%) were devoid of any plasmids. interestingly, all except one of the bacterial strains (vv34) without plasmid were resistant to at least one antibiotic tested in this study. it can be postulated that the antibiotic resistance in these v. parahaemolyticus isolates was intrinsically mediated via chromosomes. since v. parahaemolyticus exists ubiquitously in aquatic environments, they are constantly exposed to residues of antibiotics used in aquaculture, farming, and healthcare sectors. antibiotics used in these economic sectors are often released into wastewater treatment plants[138]. the residual antibiotics in wastewater, compounded by antibiotics used in aquaculture, promote the growth of resistant pmmb 2021, 4, 1; a0000233 23 of 34 v. parahaemolyticus via the mutation of genes that control the activity of antibiotics in the cell[53,59,133,139]. once a resistant mutant emerges, the antibiotic terminates strains sensitive to the antibiotic. ultimately, this allows the resistant v. parahaemolyticus to thrive in the marine environment[139]. the findings of our study are in strong agreement with previous studies which have reported chromosomal mediation of antibiotic resistance in their respective studies[61,98,140]. the plasmid profiles of the v. parahaemolyticus isolates from the shellfish samples showed that the isolates that were resistant to the highest number of antibiotics tested (5/14) had relatively larger plasmids. therefore, we cannot rule out the possibility of a relationship between plasmid sizes and the number of antibiotic resistance genes possessed by a bacterium. it is worth noting that in most cases, larger plasmids in gram-negative bacteria are conjugative plasmids. these plasmid dna bands possess a higher number of dna base pairs which codes for the conjugation function of the bacteria[141]. another striking pattern encountered in this study is the possession of at least one plasmid in most of vibrio parahaemolyticus isolates (9/10; 90%) that were resistant to imipenem. this suggests the possibility of the imipenem resistance being acquired through laterally transferable plasmids that encode for carbapenemase genes[142,143]. however, existing studies have only described the chromosomal mediation of carbapenem resistance in v. parahaemolyticus[98,144]. therefore, further analysis through plasmid curing assay is required to determine the role of plasmids in the antibiotic resistance phenotype of the v. parahaemolyticus isolates in this study[145]. there are several potential non-antibiotic methods that been effective against v. parahaemolyticus and dealing with antibiotic-resistant strains. recently, bacteriophages or phage therapy has regained renewed interest in controlling vibriosis and multidrug-resistant bacteria[80,146,147]. these phages are host specific, induces bacteriolysis and immediate counter action, readily isolated, cost effective, and generate less adverse effect compared to antibiotics[148–150]. the use of phage therapy in the aquaculture sector will eventually reduce the dependency for antibiotics and will allow the bacteria strains to lose their resistance traits[151–153]. besides, recent literatures have been evidence in bioprospecting for natural products derived from plants[154,155], microbial origins[156–158] or animals with potential antimicrobial properties to fight against multidrug vibrio strains[159–161]. streptomyces sp., a soil derived bacteria has exhibited valuable properties to be biocontrol agent of vibrio[150,162–165] and as probiotic in aquculture or animal husbandry[166–170]. these natural bacteria are safer and does not couse any resistance traits similar to antibiotics. hence, the application of phage therapy pmmb 2021, 4, 1; a0000233 24 of 34 and streptomyces sp. probiotics should be introduced in aquaculture sectors as a tool agaisnt bacterial infections and reduce the dependency towards antibiotics. 5. conclusion in summary, high densities of vibrio species ( > 5 log cfu/g) were found in 14 out of 16 groups of shellfish, increasing the risk of foodborne infection in human hosts. while most of the seafood analysed (55.8%) were contaminated with v. parahaemolyticus, none of the isolates harboured the genes encoding for tdh and trh. this shows that all the strains of the bacteria are non-pathogenic. however, the complexity in the virulence mechanism of this bacteria calls for future studies to explore other factors facilitating the pathogenicity of v. parahaemolyticus. besides that, the bacterial isolates exhibited resistance to different antibiotics. high resistance rates were seen towards common antibiotics such as ampicillin, ampicillin-sulbactam, cefotaxime and imipenem. the seriousness of antibiotic resistance is also reflected in the high proportion (69.8%) of v. parahaemolyticus isolates exhibiting a mar index of more than 0.2. this is a threat to public health because the transmission of antibiotic resistance genes can occur via the lateral transfer of plasmids among bacterial cells. since 41.9% of the v. parahaemolyticus isolates were shown to have plasmid dnas, it is imperative to take immediate action to curb multi-drug resistance in v. parahaemolyticus. the relevant agencies should closely monitor and control the use of antibiotics in aquaculture, aqriculture and animal husbandry. author contributions: vv performed the research work, data analysis and manuscript writing. lt-ht, jwfl, h-ls, vl, and pp provided conceptualisation, technical support, and proofreading. pp set up this research project. all authors have read and agreed to the published version of the manuscript. funding: this bachelor of medical science research project was funded by jeffrey cheah school of medicine and health sciences. acknowledgments: authors would like to thank professor dr. shajahan yasin, professor, and head of school, jeffrey cheah school of medicine and health sciences, monash university malaysia for his endless support. conflicts of interest: the authors declare no conflict of interest. references 1. elbashir s, parveen s, schwarz j, et al. seafood pathogens and information on antimicrobial resistance: a review. food microbiol 2018; 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vengadesh.letchumanan1@monash.edu (vl) received: 30 october 2022; received in revised form: 29 november 2022; accepted: 02 december 2022; available online: 04 december 2022 abstract: the novel coronavirus, sars-cov-2, part of the coronaviridae family, was discovered in december 2019 in china. world health organization named it covid-19 and by march 2020, it had spread worldwide, causing a global pandemic. despite continuous efforts to prevent the spread of the virus, the virus seemed always to be a step ahead of humankind as it rapidly evolved to produce new variants. these variants have higher transmissibility than the original virus and were responsible for new waves of infections. the first variant was alpha, followed by the discovery of beta, gamma, delta, and omicron, being the latest variant of concern. although vaccines were distributed worldwide to reduce the severity of the disease when infected with sars-cov-2, the emergence of new variants raised doubts about the efficacy of the available vaccines. studies showed a decrease in neutralizing antibodies during infection with xbb and bq subvariants compared to infections caused by other variants. fortunately, early evidence shows that the mrna vaccines are still effective against the current circulating omicron sublineages of the coronavirus. nevertheless, continuous genomic surveillance of the coronavirus is still important to detect any new variants of concern to assess their potential to threaten public health. the efficacy of vaccines and treatment options available against the latest circulating variant of sars-cov-2 should also be periodically evaluated. this ensures that the treatment and vaccines used are safe and effective against the virus to protect the public from another health crisis. keywords: covid-19; coronavirus; sars-cov-2; xbb; bq.1 pmmb 2022, 5, 1; a0000282 2 of 11 1. introduction in december 2019, a cluster of pneumonia cases in china prompted the investigation into the causative agent of the outbreak [1]. these patients presented with fever, fatigue, dry cough, and shortness of breath and thus were given the initial diagnosis of viral pneumonia. however, further studies conducted via whole genome sequencing revealed the identity of the causative agent to be a novel coronavirus [2]. this novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), spread rampantly across the globe. the virus is spread via respiratory droplets and can stay in the air for up to three hours [3, 4]. studies also showed that this novel virus was associated with the manifestation of mental health issues such as anxiety, depression, and post-traumatic stress disorder during and postinfection [5, 6]. the highly contagious nature of sars-cov-2 led to the declaration of the covid-19 pandemic by the world health organization (who) in march 2020. the virus spread globally [7-16], garnering over 633 million confirmed cases and claiming more than 6.5 million lives worldwide as of november 2022 [17]. as the pandemic began to unfold, world leaders quickly implemented various strategies to control the spread of the virus, such as travel bans, social distancing protocols, lockdowns for areas with outbreaks, contact tracing, and mandatory masking [18, 19]. mass testing for the coronavirus was also done to detect the virus during the early stages of disease development to prevent outbreaks. nevertheless, the world was waiting on sars-cov-2 vaccines to be developed to more efficiently control the disease and reduce mortality rates due to the coronavirus. in december 2020, the world rejoiced when pfizer introduced the first covid-19 vaccine, comirnaty, and the vaccine was promptly deployed to various nations worldwide [20]. following pfizer, the covid-19 vaccines from moderna, astrazeneca, johnson & johnson, sinopharm, sinovac, bharat biotech, novavax, and nuvaxovid have also been approved for emergency use listing (eul) by who for use as of 12 january 2022 (table 1) [21]. these vaccines have also been deployed as to meet the demands for vaccines worldwide. with the administration of covid-19 vaccines, infection rates, the severity of disease, and mortality rates of the coronavirus have reduced. the vaccines proved to be effective in controlling the virus, and people worldwide were hopeful that the pandemic would end. although there is currently no cure for covid-19, other treatment options have been explored to treat the viral infection. in general, covid-19-positive patients are given symptomatic treatment such as ibuprofen or acetaminophen for patients with fever. in patients with difficulty breathing or low oxygen saturation, oxygen therapy will be given to prevent hypoxia. in severe cases, the patient may be on a ventilator to ensure adequate oxygen supply. in addition, the us food and drug administration (fda) has approved remdesivir, an antiviral drug, to treat covid-19 [22-24]. remdesivir effectively prevented the replication of the viral genome of sars-cov-2 [21]. the fda also approved monoclonal antibodies (mab) for the treatment and prophylaxis of covid-19. among the approved mabs are antisars-cov-2-mabs, bamlanivimab, casirivimab and imdevimab [25]. the administration of monoclonal antibodies stimulates the immune system of the host to recognize and respond pmmb 2022, 5, 1; a0000282 3 of 11 more effectively to the coronavirus, reducing the reproductivity of the virus and minimizing the harm caused by the virus. table 1. vaccines approved for emergency use listing (eul) by who. manufacturer vaccine pfizer/biontech comirnaty (bnt162b2) astrazeneca/oxford covishield (chadox1ncov-19) johnson & johnson/janssen jcovden (ad26.cov2.s) moderna spikevax (mrna-1273) sinopharm verocell sinovac coronavac bharat biotech covaxin (bbv152) novavax covovax, nuxavoid (nvx-cov2373) however, sars-cov-2 began mutating rapidly, producing new variants of the coronavirus [26, 27]. the emergence of variants has led to sudden outbreaks in places that previously had covid-19 cases under control [28]. some of the variants have also been reported to have immune escape properties [29], which could potentially decrease the effectiveness of the covid-19 vaccines. they are also likely to cause reinfections in those previously infected, raising major concern of new waves of infections and increased disease severity. the emergence of variants of concern (voc) has also led to the administration of covid-19 booster vaccines aimed at improving and prolonging protective immunity against the disease [30]. who has reported the vocs are alpha, beta, gamma, delta, and omicron variants [31]. this data has been consistently updated to track the latest variant causing the most disease worldwide. by staying updated on the latest variant of the coronavirus, which caused the sudden increase in covid-19 infections, necessary precautions and management strategies can be implemented to control the outbreaks and reduce the negative effects of the disease on public health. 2. variants of sars-cov-2 sars-cov-2 belongs to a coronaviruses (covs) group in the coronaviridae family, and the virus is classified as a β-cov which mainly infects mammals [32]. coronaviruses are pleomorphic enveloped viruses with a single-stranded rna genome [33]. further analysis of the novel coronavirus showed that about 82% of the sequence identified sars-cov-2 with pmmb 2022, 5, 1; a0000282 4 of 11 the well-known sars-cov and middle east respiratory syndrome coronavirus (merscov). sars-cov-2 also shared over 90% sequence identity with sars-cov and merscov for essential enzymes and structural proteins [32, 34]. spike proteins on the surface of the sars-cov-2 virus enable the virus to enter the host cell by interacting with the host cell receptor angiotensin-converting enzyme 2 (ace2). upon entry into the host cell, the viral rna is released and triggers the replication of viral rna to synthesize structural and nonstructural proteins for further propagation of the virus in the host [35]. in 2020, before any vaccines were successfully developed to manage covid-19 outbreaks, sars-cov-2 had already mutated and produced a voc, the alpha variant (b.1.1.7), in the united kingdom in september. the alpha variant is the product of 23 mutations from the original strain and has been found to have higher transmissibility than its predecessors. although the alpha variant has not been found to cause more severe illness than the other sars-cov-2 strains, the higher transmission rate enabled the virus to spread from the united kingdom to 52 other countries within four months of its discovery [36, 37]. subsequently, in october 2020, the beta variant, b.1.351, a new variant, was detected in nelson mandela bay, south africa [38]. the beta variant contained a mutation in the receptor-binding domain of the spike protein (n501y), which increases transmission and two additional mutations (k417n and e484k), which increased antibody resistance [39]. the increased transmissibility proved detrimental as the beta variant displaced other variants in south africa [38]. the rapid increase in covid-19 cases bangladesh was mainly attributed to this variant [40]. furthermore, this strain was neutralized less effectively by antibodies produced after vaccination or natural infection with other strains [41, 42]. two months after the beta variant was detected, another voc was reported in brazil, the gamma (p.1) variant, which contributed to a second wave of infection in manaus. the virus had once again evolved and 17 amino acid changes were made, including 10 in the spike protein [26]. following the report in manaus, it was reported in salvador, northeast brazil, that an increased proportion of younger adults without comorbidities had the severe disease during the second covid19 wave due to the gamma variant [43]. this strain continues to spread to more than 36 countries worldwide [26]. meanwhile, in india, the delta (b.1.617.2) variant was identified in maharashtra in december 2020. this voc rapidly spread throughout india, displacing the pre-existing lineages, including the alpha variant [44, 45]. this lineage of the coronavirus had l452r spike receptor-binding motif (rbm) substitution which increased infectivity and reduced its susceptibility to neutralizing antibodies [46]. in addition, the delta variant was more efficient at replication than the alpha variant in the human airway epithelial systems. the spike protein on b.1.617.2 mediates highly efficient syncytium formation, which lowered the virus's sensitivity towards inhibition by neutralizing antibodies as compared to the original sarscov-2 [44]. moreover, researchers in bahrain compared the recovery patterns infected with the alpha variant to those infected with the delta variant. the results showed that patients with the delta variant recovered slower than those with the alpha variant. their hospital pmmb 2022, 5, 1; a0000282 5 of 11 stays were more extended with higher shedding of the virus regardless of their vaccination status [47]. when vocs of the novel coronavirus began to emerge and were still rapidly evolving in 2020, the distribution of vaccines against covid-19 had just started, raising concerns about the efficacy of the vaccines as they were developed based on the original sars-cov2. one study in france found that two doses of mrna vaccines (pfizer or moderna) were still effective against the original sars-cov-2 and its alpha, beta, and gamma variants [48]. however, a report in india showed that covishield, the viral vector vaccine by astrazeneca, was less effective against the delta variant than the other variants. breakthrough infections were reported when patients who received the covishield vaccine tested positive for covid19 caused by the delta variant [44]. nevertheless, vaccines were continuously rolled out to reach the high demands of nations worldwide to control the spread of the virus. the goal was to achieve herd immunity as soon as possible to protect populations at high risk of contracting the virus. 3. latest variant of concern: omicron xbb, bq.1 the continuous evolution of the covid-19 virus led to the highly mutated omicron (b.1.1.529) variant that is widely known today [28, 49]. with each evolution, the covid-19 virus carries more mutations than its previous variant, which is evident in the omicron variant [50]. researchers from italy published the first 3d omicron image indicating that the variant has several mutations, mainly on the spike protein [51]. first isolated in south africa in november 2021, the omicron variant quickly spread to other continents, becoming the dominant variant globally by february 2022 [52] (figure 1). the widespread virus once again triggered global concern, and travel bans were implemented [51]. despite efforts to prevent the virus from spreading, the omicron variant managed to increase the daily cases in the united states to over a million by december 2021. omicron, being very efficient spreaders of covid-19, began mutating to produce subvariants. although a plethora of subvariants have been produced, not all of them are voc. who introduced “omicron subvariants under monitoring” in its variant tracking system to highlight, prioritize and monitor the voc, potentially impacting public health. in october 2022, the latest omicron subvariants under monitoring are xbb and bq.1. xbb is a recombinant of omicron ba2.10.1 and ba.2.75, which were previously identified and characterized in 2021 [28]. first isolated in singapore, xbb has a global prevalence of 1.3% as of the first week of october 2022 and has been reported in 35 countries, such as japan, india, malaysia, and australia [53-55]. this subvariant raises concern as there is early evidence from singapore and india demonstrating that the virus has a higher reinfection risk. reinfections occurred in individuals previously infected with pre-omicron variants of sarscov-2. it has also begun to cause new waves of infections leading to spikes in the daily number of confirmed cases in many countries. as for the bq.1 subvariant, they carry significant spike mutations in key antigenic sites and have been detected in 65 countries. bq.1 has shown a significant growth advantage and a higher reinfection risk than other pmmb 2022, 5, 1; a0000282 6 of 11 omicron subvariants. the mutations producing bq.1 could have produced an immune escape advantage, thus allowing it to displace other circulating lineages, and increasing the risk of reinfections [56]. at the time of writing, there is no data on omicron xbb and bq.1 subvariants that show increased severity of disease by these subvariants, however, authorities remain vigilant in monitoring and assessing future available data to determine whether these subvariants pose a threat to the public. figure 1. illustration of covid-19 variant of concern (voc) transmission, symptoms and vaccines. the rapid emergence of omicron subvariants raises further concern concerning the efficacy of the current treatment options and vaccines available to fight against the virus. one study found that the effectiveness of antivirals was not diminished in treating omicron subvariants. still, the efficacy of mabs has been greatly reduced due to the immune escape of the subvariants [57]. therefore, an alternative immunotherapy for covid-19-positive patients is covid-19 convalescent plasma (ccp). ccp is a polyclonal preparation consisting of antibodies with many specificities and represents all isotypes, making it more challenging to defeat a single variant [57]. it has been reported that if administered as early as within nine days of the onset of symptoms, ccp can reduce the risk of disease progression leading to hospitalization in unvaccinated individuals [58]. however, albeit effective, ccp can potentially cause severe adverse reactions in the recipient [59]. for instance, bronchospasms, transfusion-related acute lung injury, and circulatory overload have been reported in patients with comorbidities such as cardiorespiratory disorders and renal impairment. the administration of donor plasma to the recipient may elicit severe allergic reactions such as serum sickness and anaphylaxis, which have been associated with bronchospasms [60]. pmmb 2022, 5, 1; a0000282 7 of 11 a study found that the omicron ba.5 bivalent vaccine by pfizer or moderna booster dose had better neutralization against the newly emerged omicron sublineages than the parental mrna vaccines. moreover, the study also found that individuals previously infected with sars-cov-2 developed a higher and broader neutralization against the omicron sublineages after receiving the ba.5 bivalent booster vaccine. however, they reported the subvariants bq.1.1 and xbb.1 demonstrated the greatest evasion against vaccine-elicited neutralization [61]. a separate study found that in vaccinated individuals infected with bq.1.1, there was a drop by a factor of seven in neutralizing antibody titers, indicating that the subvariant can escape from neutralization more effectively than its predecessors [62]. any future development of omicron-based vaccines will be challenging as the virus rapidly evolves and produces diverse subvariants circulating and co-existing in our environment. 4. conclusion covid-19 is the first ever documented coronavirus pandemic in history which has caused over 6.5 million deaths worldwide in under three years since its discovery. the populations at a higher risk of getting infected are the elderly, individuals with pre-existing medical conditions, and the immunocompromised [63]. in addition, many recovered individuals have also reported post-covid conditions, which can last for weeks or even months after infection [64, 65]. symptoms of long covid include fatigue, cough, chest tightness, breathlessness, palpitations, myalgia, difficulty focusing, and brain fog [66, 67]. healthcare systems across the globe were taking a toll due to sudden outbreaks or surges in covid-19-positive cases. declines and disruptions in health services not related to covid19 were also reported. the sudden increase in covid-19 cases caused by the emerging variants places a considerable burden on the healthcare systems, significantly reducing their capacity to treat other diseases or ailments [68]. without a doubt, covid-19 has dramatically disrupted the normality of our lives. thus, consistent monitoring and tracking of the latest variant of sars-cov-2, which is causing detrimental changes in covid-19 epidemiology in terms of increased transmissibility, increased virulence, worsening disease progression, and reducing the effectiveness of vaccines, is crucial in the fight against covid-19. continuous genomic surveillance of the virus is essential to identify new variants that could threaten public safety and take the necessary measures to prevent another public health crisis. author contributions: the literature search, data extraction, and manuscript writing were performed by kyl. the manuscript was critically reviewed, proofread, and edited by vl. kyl and vl conceptualized this review writing project. funding: no external funding was provided for this research. conflicts of interest: the authors declare no conflict of interest. references 1. zhou p, yang x-l, wang x-g, et al. a pneumonia outbreak associated with a new coronavirus of probable bat origin. nature 2020; 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a0000271. doi: a10.36877/pmmb.0000271 http://journals.hh-publisher.com/index.php/pmmb review article role of garlic in chronic diseases: focusing on gut microbiota modulation wei quan lim1, jia ying cheam1, jodi woan-fei law1, vengadesh letchumanan1, learn-han lee1*, loh teng-hern tan1,2* article history 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia. wlim0012@student.monash.edu (w-ql); jche0331@student.monash.edu (j-yc); jodi.law1@monash.edu (jw-fl) vengadesh.letchumanan1@moansh.edu (vl) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia *corresponding author: learn-han lee, novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia; lee.learn.han@monash.edu (l-hl); loh teng hern tan, clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) received: 18 july 2022; received in revised form: 13 october 2022; accepted: 25 october 2022; available online: 1 november 2022 abstract: garlic is a herb and has been used as a spice with a long history in different types of cuisine. garlic and its components are believed to be able to bring benefits to the health of an individual. the gut microbiota is closely related to an individual's health, and garlic is shown to have an effect on the gut microbiota as well. hence, this literature review aims to portray the uses of garlic and its bioactive constituents on human health, particularly looking at how it modulates the gut microbiota and subsequently affects an individual's health directly or indirectly. these studies have shown the ameliorative effects of garlic and its bioactive constituents on various chronic diseases, including hypertension, diabetes mellitus, hyperlipidaemia and liver diseases. keywords: garlic; allicin; alliin; gut microbiome; metabolic diseases 1. introduction chronic diseases, such as hypertension, diabetes mellitus, obesity, hyperlipidaemia, and complications, including myocardial infarction and cerebrovascular accidents, are the major health concerns surrounding people all the time. it takes time, effort, and money to manage the diseases from diagnosis to treatment. they appear as some form of burden to the people, and most of the time, uncontrolled diseases will lead to complications that may pmmb 2022, 5, 1; a0000271 2 of 12 severely affect the well-being of humans. awareness regarding the diseases has been raised, and people started taking steps to prevent the diseases and their complications. initiatives can be made in the simplest form, including lifestyle modifications (diet modification and exercise) and taking medications. regarding preventative methods, garlic has been used traditionally in improving health. garlic as a spice has not only been used to improve the taste of food, but it also has the potential to regulate an individual's health. recently, it has been believed that garlic helps to alter the gut microbiota in good ways and further improves health. various chronic illnesses can be prevented by supplementing garlic into our diet. garlic and its constituent allicin, diallyl disulphide, can act as anti-inflammatory and antioxidant agents to help cope with diseases. gut microbiota plays a vital role in maintaining human health. the gut microbiome consists of different types of bacteria. a balanced gut microbiome system helps maintain good health by forming a good immune system and defence against pathogens[1–7]. the components in the gut microbiota are largely affected by many factors, including inheritance, diet, and drugs[8–11]. as inheritance is a non-modifiable factor, diet and drugs are those we can take into account and modify accordingly to help improve our health[12–14]. the gut microbiota is in mutualism with the host, and they provide benefits to each other. the gut microbes feed on the food we consume, and on the other hand, they help to degrade and convert those complex substances to enable the gut to absorb the nutrients. garlic has been part and parcel of our diet, and taking garlic can help to modulate the gut microbiota in a good way so that the beneficial microorganisms or the probiotics can be retained while the pathogenic species can be eliminated. consumption of garlic in the long term will help to improve health indirectly. this review aims to investigate recent studies to determine the benefits of taking garlic in daily life and the role of garlic and its constituents in modulating human health via gut microbiome modulation. besides that, this review also provides an overview of how garlic prevents chronic diseases and explores the potential use of garlic as a therapeutic agent for diseases. 2. an overview of garlic and its constituents and their effect on gut microbiota garlic has been used widely in different cuisines. it can be consumed raw or crushed, depending on the consumers’ preferences. garlic is thought to be bringing a lot of benefits, especially towards the health of an individual. a person with a healthy gut microbiota will have a healthy immune system. garlic and its constituents have been shown to modulate the gut microbiota composition by selectively stimulating beneficial microorganisms' growth and suppressing pathogenic species' growth. gut microbiota is correlated to an individual's health because it affects the absorption and elimination of the food we eat. this is particularly more observed with patients with metabolic diseases as those chronic diseases are highly pmmb 2022, 5, 1; a0000271 3 of 12 dependent on diet and lifestyles[15]. in the literature review, the aim is to investigate the effect of garlic on gut microbiota, in turn, its impact on an individual's health. 3. composition of garlic garlic (allium sativum l.) is mainly comprised of water, followed by carbohydrates, protein, and organosulfur compounds. fructans are the main component of carbohydrates, and they can provoke the growth of probiotic bacteria in the gut and subsequently improve the immune system[1]. organosulfur compounds (osc) are available in most plants, but garlic's amount is higher than the other plants. the primary osc from garlic are γ-glutamyls-allyl-l-cysteines (g-sac). g-sac is a metabolite that will later be converted to alliin and is thought to be responsible for antimicrobial activity in the gut[1]. alliinase will be activated during the crushing process of garlic, and it converts alliin into allicin. allicin has a variety of actions on the gut, including antimicrobial, anticancer, antioxidant, and immunemodulating activities. allicin will be absorbed in the intestines through the villi, and it can bypass the stomach without being affected by the low ph of the gastric acid. allicin is then metabolised into diallyl disulphide in the bloodstream and transported to various organs. allicin and its metabolites can be interconverted by the liver cells. this keeps allicin readily available as it can only be transported in the form of diallyl disulphide. the metabolites formed from allicin include diallyl disulphide, s-allylmercaptocysteine, s-allyl cysteine, and so on, and they are those responsible for bringing benefits to the health[16]. apart from that, garlic also contains a variety of minerals and vitamins, including potassium, zinc, iron, sodium, and water-soluble vitamins[17]. 4. the effect of garlic and its constituents on gut microbiota gut microbiota consists of the bacteria grown in the intestines and have a mutualistic relationship with the human host. bacteroidetes and firmicutes are the significant part of the gut microbiota, and other bacteria reside in the human gut[18]. numerous studies have investigated the relationship between garlic and its effect on gut microbiota and health[1,19– 22]. through these studies, garlic has been shown to be able to modulate gut microbiota[1,19,21]. one of the studies by chen et al.[23] has demonstrated that garlic supplementation in the diet increases lachnospiraceae and decreases prevotella[1]. lachnospiraceae is the normal flora found in the gut, and it plays a role in producing short-chain fatty acid (scfa). scfas are the primary source of nutrients for colonic epithelial cells, and they exert an antiinflammatory effect in the gut[24]. another study by filocamo et al.[19] suggested that using garlic powder can inhibit the growth of microorganisms in the gut. however, lactobacilli (lactic acid bacteria) have shown resistance towards garlic powder, and these bacterial species can grow and replicate despite consuming garlic regularly. lactobacilli are the probiotics that have an essential role in forming the barrier in the gastrointestinal system[25]. apart from that, they act as anti-inflammatory agents and help to prevent cancer and inflammation in the intestines[26–30]. studies also showed that the components present in garlic have bactericidal effects on some bacteria species, including escherichia coli, salmonella typhimurium and neisseria gonorrhoeae[20,31,32]. those bacteria are the pathogen pmmb 2022, 5, 1; a0000271 4 of 12 that predisposes the host to infections[33,34]. a study by maeda et al.[35] investigated the effect of taking aged garlic extracts on the gut microbiota profile. after seven weeks of consuming aged garlic extracts, there is an increase in the lactobacillus and a decrease in several bacteria species, including clostridium cluster xviii and prevotella. the use of garlic has been shown to be beneficial to the host. garlic supplementation in the diet can expand the diversity and richness of the gut microbiome and, at the same time, help lower the pathogenic species in the gut[36]. the ratio of firmicutes to bacteroidetes is closely related to obesity, and obese patients tend to have a higher firmicutes/bacteroidetes ratio compared to a healthy individual[21]. the firmicutes/bacteroidetes ratio will increase with ageing and implementing a high-fat diet[23]. allicin, the main osc present in crushed garlic, has an antibacterial effect on the gut. consumption of garlic has been shown to reduce the ratio, and this indirectly deals with gut dysbiosis caused by a prolonged high-fat diet[23]. a study from zhang et al.[22] demonstrates that alliin (an active component in garlic) consumption can modify the gut microbiota, especially allobaculum sp. the role of allobaculum spp. in the gut is to utilise glucose[37], which helps to weight loss for obese individuals[38]. another study finds that alliin can induce the modulation of components in the gut microbiota. the study shows that alliin supplementation results in a decrease in the abundance of lachnospiraceae and an increase in the abundance of ruminococcaceae[39]. those are the common normal flora found in the gut, and they assist the host in degrading the complex materials from plants, such as cellulose and hemicellulose, and further aid in the absorption of the substrate to yield energy for the hosts[40]. those substrates cannot be digested and broken down by the host as there is no enzyme specifically targeting those substrates, and the host must rely on the gut microorganisms. besides, the study suggested that alliin consumption helps to improve lipid and glucose profiles in the long term[39]. this result is consistent with the increase in ruminococcaceae as it has been proved that ruminococcaceae has a protective function against long-term weight gain in the host[41]. besides alliin, fructan is also the main component found in garlic. supplementation of garlic fructan (gf) can promote the growth of bifidobacterium in the gut[42]. bifidobacterium is the beneficial normal flora in the gut, and it is linked to many health benefits, including preventing colon cancer and reducing the symptoms of inflammatory bowel disease[43]. bifidobacterium probiotic has been used to treat diarrhoea and necrotising enterocolitis in preterm neonates[43,44]. another study by zhang et al.[45] focuses on the effect of inulin-type fructan on the gut microbiota. after treatment with inulin, a rise in the number of short-chain fatty acid-producing bacteria and lactobacillus is evidenced[45]. apart from that, a drop in desulfovibrio is also present in the study. desulfovibrio bacteria is considered a pathogen in the human host, and it is often associated with ulcerative colitis and intraabdominal infections[46,47]. inulin-type fructan can affect the gut microbiota. it is noted that those receiving inulin-type fructan have softer stools, which actually improves the hosts' constipation issues[48]. pmmb 2022, 5, 1; a0000271 5 of 12 organosulfur compounds in garlic are the hydrogen sulphide (h2s) donors in the body[49]. study shows that h2s can prevent the dysbiosis of gut microbiota caused by nonsteroidal anti-inflammatory drugs (nsaids) and alcohol[50]. this helps balance the system after the host is exposed to external factors like alcohol and drugs. besides that, h2s has been shown to prevent inflammation and foster the restoration of the damaged tissues in the gut [50]. propyl‐propane thiosulfonate (ptso) from garlic also exerts anti-inflammatory effects and helps restore the epithelial tissues' barrier function in the intestines[51]. this suggests that garlic may help with inflammatory bowel diseases in the future. 5. the effect of garlic on chronic diseases many studies have shown that garlic can directly or indirectly improve the health of an individual, which may link to the modulation of gut microbiota (figure 1). supplementation of garlic in diet can affect health in a good way. the human gut microbiota helps to degrade complex substances, produce short-chain fatty acids, and maintain homeostasis in the intestines. when there is microbial dysbiosis, problems start in the host, such as metabolic syndromes, allergies, and neurological and gastrointestinal diseases[52–55]. figure 1. summary of garlic's gut microbiome modulation effect in improving overall health and metabolic syndromes. 5.1 hyperlipidaemia practising a diet high in fat content in the long term affects gut microbiota composition. the high-fat diet increases the number of pathogenic bacteria such as proteobacteria, desulfovibrio, and actinobacteria in the gut. these pathogenic bacteria tend to be pro-inflammatory and affect energy uptake, leading to obesity[56]. in previous studies, garlic has been shown beneficial in lowering cholesterol levels and ameliorating the lipid profile. one shows that consuming garlic can improve dyslipidaemia caused by a high-fat diet[23]. another study found that intake of aged garlic extract may help decrease fat uptake pmmb 2022, 5, 1; a0000271 6 of 12 from the intestines and thus suppressing the deposition of triglycerides in the liver[35]. black garlic melanoidins (mld) can target obesity issues as they effectively slow down the weight gain process caused by the high-fat diet. besides, mld can also inhibit lipid accumulation in the liver and assist the lipid metabolism process, improving the lipid profile among patients with dyslipidaemia[57]. a high-fat diet usually leads to a change in the gut microbiota, and a study reveals that implementing low molecular and high molecular melanoidins can reverse the changes and eventually relieve the disrupted rhythm of gut microorganisms[58]. allicin from garlic helps the browning of white adipose tissue and further prevents the gaining of weight caused by a high-fat diet[59]. allicin can stimulate lipolysis in the body as well as contribute to thermogenesis, and these are ways to burn fat and relieve obesity issues[59]. inulin, as a substrate found in garlic, is also able to promote weight loss in patients with obesity. after 3 months of an inulin-rich diet, the participants experienced a drop in their bmi and had better blood pressure and sugar profile[60]. 5.2 liver diseases a high-fat diet causes metabolic syndrome such as obesity, eventually leading to liver diseases. dysbiosis of gut microbiota can lead to liver inflammation and fibrosis. this occurs through the change in the yielding of energy from the diet, an increase in intestinal permeability that leads to the presence of bacterial products in the bloodstream, the formation of ethanol and changes in the metabolism of choline, bile acid, and lipid[61]. choline deficiency predisposes an individual to non-alcoholic fatty liver disease by increasing lipid deposition in the liver[62]. not only does garlic target dyslipidaemia, but garlic also helps with liver inflammation. garlic supplementation in the diet can relieve inflammation in the liver by reducing alanine transaminase (alt) and aspartate transaminase (ast) and normalising the lipid profile. this suggests that garlic may potentially treat alcoholic liver fibrosis[63]. diallyl disulphide was used to address the issue of liver inflammation, and it was found that diallyl disulphide can suppress inflammation, lipid metabolism, and peroxidation[64]. nonalcoholic steatohepatitis (nash) can be targeted using garlic as a therapeutic agent. another study has used garlic essential oil and diallyl disulphide, and the result shows that they can prevent non-alcoholic fatty liver disease (nafld) by suppressing inflammation and lipid accumulation[65]. a study from shunming et al.[66] revealed that high amounts of raw garlic could help lower the prevalence of nafld among men but not women. this can be due to allicin's bioactivity, which exhibits anti-inflammatory and antioxidant effects that stop the progression of nafld [66]. on the other hand, a study by yang et al.[67] shows that diallyl disulphide (dads) can modulate the gut microbiota by lowering the number of bacteroides and raising the number of firmicutes. these have been seen in patients with obesity and those on a high-fat diet. dads alters the expression of genes responsible for lipid metabolism, and a low dose of dads, along with a high-fat diet, increases fat deposition in the liver[67]. the results from this study are different from the other studies where garlic has consistently shown to have the ability to target obesity and liver diseases. more studies need to be done to explore garlic's effect on patients' lipid profiles. pmmb 2022, 5, 1; a0000271 7 of 12 5.3 hypertension gut microbiota plays a vital role in regulating blood pressure as well. they modulate the uses of the energy, metabolism of catecholamines in the gut, and transportation of ions through the gastrointestinal system[68]. production of short-chain fatty acids switches on receptors such as g-protein coupled receptors and olfactory receptors, which work to balance blood pressure[68]. a rise in firmicutes to bacteroidetes ratio puts an individual at a higher risk of developing hypertension, and garlic can help to lower the ratio. on the other hand, lactobacilli helps to inhibit angiotensin-converting enzymes and subsequently lowers blood pressure[68]. this works similarly to the drugs ace inhibitors used for hypertensive patients. garlic can lower blood pressure among hypertensive patients. kyolic aged garlic supplementation prevents the stiffening of arteries caused by ageing via lessening the pulse wave velocity and thinning the blood to lower the risk of thrombosis[69]. this overall improves the cardiovascular health of an individual. allicin found in garlic is able to inhibit the formation of trimethylamine-n-oxide (tmao)[70]. the gut microbiota forms tmao through the metabolism of carnitine, and it acts to promote atherosclerosis[70]. hence, we can deduce that consumption of garlic in the long term will decrease the risk of atherosclerosis. furthermore, garlic-derived organic polysulfides have been shown to exhibit vasoactivity. a study showed that several garlic-derived organic polysulfides acted as an h2s donors and boosted endogenous h2s production in blood and tissues. h2s can promote vasorelaxation of the vessels and indirectly help promote the health of the cardiovascular system[71]. a more recent animal study by hsu et al.[72] revealed that garlic oil supplementation protected adult male offspring from hypertension induced by a perinatal high-fat diet given to the mother during pregnancy and lactation. interestingly, garlic oil was shown to mediate the hypertension preventive effect with modulations of gut microbiota composition, microbiota-derived metabolite scfas, h2s-generating pathway, and no bioavailability. genus lactobacillus was found to be increased in abundance, while tunricibacter and staphylococcus were decreased in the garlic oil supplementation group[72]. 5.4 diabetes mellitus patients with diabetes mellitus tend to show some changes in the gut microbiota to some extent. there is an increase in the pathogens and a decrease in the butyrate-producing bacteria in the intestines[73]. diabetic patients have a higher number of lactobacillus species in the gut, and those species have a positive correlation with fasting glucose and hba1c levels[74]. garlic has a beneficial effect on the blood glucose profile of diabetic patients as well. aged garlic extract used in the study has been demonstrated to improve insulin insensitivity[35]. another study focused on the effect of alliin and found that the use of alliin increased the sensitivity to insulin and augmented glucose homeostasis in the body[39]. this will prevent fluctuation of glucose levels after meals and help diabetic patients better control pmmb 2022, 5, 1; a0000271 8 of 12 their serum glucose levels. a study by zhang et al.[75] also found that allicin from garlic maintained glucose homeostasis, suggesting that it could be used to treat metabolic disorders. 6. conclusion consumption of garlic has been demonstrated in many studies that it can modulate the gut microbiota by enhancing probiotics' growth and suppressing pathogenic microorganisms' growth. studies showed that garlic has the potential to become a complementary alternative medicine to a lot of diseases. thus, garlic is a great functional food containing bioactive constituents with immense potential to be developed as an antiinflammatory, lipid-lowering, glucose-lowering, and antihypertensive agent. numerous studies have been conducted, and garlic has been portrayed as a beneficial agent for health conditions. it can modulate gene expression in the gut and the gut microbiota. apart from that, garlic can be used to improve blood pressure, blood glucose profile, and lipid profile, and most importantly, garlic is a natural organic substance. it can effectively reduce the number of medications needed by a patient by substituting them with natural substances. however, most studies were done on the animal model. future studies should be conducted on a large scale in humans to understand better the underlying mechanisms of garlic in conferring better health outcomes. author contributions: w-ql performed the literature search, critical data analysis, and manuscript writing. j-yc, jw-fl, vl, l-hl, and lt-ht provided critical editing and proofreading. lt-th and l-hl conceptualized this review writing project. acknowledgments: this work was inspired by the jeffrey cheah school of medicine and health sciences “med5101 scholarly intensive placement (sip)”. conflicts of interest: the authors declare no conflict of interest. references 1. shreiner ab, kao jy, and young vb, the gut microbiome in health and in disease. curr opin gastroenterol 2015; 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65(11): 2001116. 73. grigorescu i and dumitrascu dl, implication of gut microbiota in diabetes mellitus and obesity. acta endocrinol (copenh) 2016; 12(2): 206–214. 74. blandino g, inturri r, lazzara f, et al., impact of gut microbiota on diabetes mellitus. diabetes metab 2016; 42(5): 303–315. 75. zhang c, he x, sheng y, et al., allicin-induced host-gut microbe interactions improves energy homeostasis. faseb j 2020; 34(8): 10682–10698. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. pmmb 2022, 5, 1; a0000269. doi: 10.36877/pmmb.a0000269 http://journals.hh-publisher.com/index.php/pmmb original research article differential gene expression analysis of papillary thyroid carcinoma reveals important genes for lymph node metastasis wan fahmi wan mohamad nazarie1, azliana mohamad yusof 2, francis yew fu tieng1, nur fadhlina mohamad pakarulrazy1, rohaizak muhammad3, shahrun niza abdullah suhaimi3, nani harlina md latar3, sazuita saidin1, isa mohamed rose4, learn-han lee5, nurul-syakima ab mutalib1, 5, 6* article history 1ukm medical molecular biology institute, universiti kebangsaan malaysia, bandar tun razak, 56000 cheras, kuala lumpur, malaysia; wanfahmi5785@gmail.com (wfwmn); francistieng@yahoo.com.my (fyft); nurfadhlinamohamadpakarulrazy@gmail.com (nfmp); sazuita@ukm.edu.my (ss) 2cytogenetics and molecular diagnostics laboratory, pantai premier pathology sdn. bhd., 55100 kuala lumpur, kuala lumpur, malaysia; azlianayusof@gmail.com (amy) 3department of surgery, faculty of medicine, universiti kebangsaan malaysia, bandar tun razak, 56000 cheras, kuala lumpur, malaysia; rohaizak@ppukm.ukm.edu.my (rm); shahrunniza@ppukm.ukm.edu.my (snas), naniharlinalatar@ukm.edu.my (nhml) 4department of pathology, faculty of medicine, universiti kebangsaan malaysia, bandar tun razak, 56000 cheras, kuala lumpur, malaysia; isa@ppukm.ukm.edu.my (imr) 5novel bacteria and drug discovery research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 subang jaya, selangor, malaysia; lee.learn.han@monash.edu (l-hl) 6faculty of health sciences, universiti kebangsaan malaysia, 50300 kuala lumpur, kuala lumpur, malaysia *corresponding author: nurul syakima ab mutalib, ukm medical molecular biology institute, universiti kebangsaan malaysia, bandar tun razak, 56000 cheras, kuala lumpur, malaysia; syakima@ppukm.ukm.edu.my (n-sam) received: 05 june 2022; received in revised form: 11 july 2022; accepted: 13 july 2022; available online: 17 july 2022 abstract: background: papillary thyroid carcinoma (ptc) is the most prevalent thyroid cancer. we explored the differential gene expression (deg) of the transcriptional regulation in thyroid cancer with the main aim of determining the genes involved in lymph node metastasis (lnm) in ptc. methods: we employed a bioinformatics pipeline for rna-seq analysis in ptc with and without lnm at the gene expression level. we performed read mapping, read quantitation, and deg analysis using star, cufflinks, and cuffdiff, respectively. subsequently, functional annotation and pathway enrichment were carried out using funrich (functional enrichment analysis tool). results: expression profiling revealed changes in the ptc with lnm (33 genes at p-value < 0.05 and log2 fold change |1.0|) mailto:wanfahmi5785@gmail.com mailto:francistieng@yahoo.com.my mailto:sazuita@ukm.edu.my mailto:azlianayusof@gmail.com mailto:rohaizak@ppukm.ukm.edu.my mailto:shahrunniza@ppukm.ukm.edu.my mailto:isa@ppukm.ukm.edu.my pmmb 2022, 5, 1; a0000269 2 of 19 compared to the adjacent normal thyroid, whereas 69 genes showed differential expressions in the ptc with lymph node negative (lnn) versus adjacent normal thyroid. we identified 31 significant degs in ptc lnm versus ptc lnn; and 44 significant degs between adjacent normal thyroid tissues from ptc lnm and ptc lnn. the biological processes of genes expressed at higher levels in ptc lnm compared to ptc lnn were involved in cell communication, energy pathway, and metabolism, whereas ion transport, energy pathways, and regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolisms, were downregulated. conclusion: these findings provide further evidence for the role of cellular transport regulatory processes in metastatic disease. keywords: papillary thyroid carcinoma; lymph node metastasis; lymph node negative; differentially expressed genes 1. introduction the number of new cases of thyroid cancer including papillary thyroid carcinoma (ptc) is steadily increasing over the past decade especially in asian countries compared to european countries[1]. thyroid cancer has a high survival rate because of early detection through screening and advances in medical treatment[2,3]. there are four types of thyroid cancer namely follicular (ftc), medullary (mtc), anaplastic (atc) and papillary (ptc), and the latter is the most common, contributing 80% to 85% of all thyroid cancers[4,5]. the survival rate for ptc is high (98%) in europe and north america due to improved diagnosis, management, and treatment of the disease[6]. yet, a subset of ptc patients displayed aggressive features associated with poor prognosis, and one of the features is the presence of lymph node metastasis (lnm)[7]. several studies have shown the different molecular landscapes of lnm in various types of cancer, including oral, pancreatic, colorectal, and breast cancer[8]. however, the gene expression profile in lnm of ptc has not been extensively done, with only a handful of published findings[9]. understanding the molecular and cellular mechanisms of lnm in ptc is required to improve prognostication. in this study, we investigated the gene expression profiles of ptc with and without lnm and conducted a comprehensive analysis of gene expression of thyroid tissue samples via rna-seq. the differentially expressed genes (degs) identified in ptc with and without lnm might be the potential biomarkers and provide the basis for further exploration of the mechanisms of metastatic disease in ptc. 2. materials and methods 2.1. ethics statement and sample collection this study was conducted according to the universiti kebangsaan malaysia research ethics committee (ukmrec) (reference: ukm 1.5.3.5/244/umbi-2015-002). ten freshpmmb 2022, 5, 1; a0000269 3 of 19 frozen tumour ptc tissues specimens from the cancer biobank were subjected to cryosectioning and stained with haematoxylin and eosin (h&e). the pathologist reviewed the percentage of tumour cells and normal cells in the slides. tumour tissues containing more than 80% of cancer cells and normal tissues with less than 20% necrosis were subjected to rna extraction. 2.2. total rna isolation according to the manufacturer’s protocol, total rna was isolated from the samples using allprep dna/rna/mirna isolation kit (qiagen, germany). the total rna quality and quantity were carried out using an nd-1000 spectrophotometer (thermo scientific, usa) and qubit™ fluorometer (invitrogen, usa). before library preparation, the rna quality was measured using an eukaryote total rna nano chip on bioanalyzer 2100 (agilent technologies, usa). 2.3. library preparation and rna sequencing libraries were constructed using the ion total rna-seq v2 kit (life technologies, usa) according to the manufacturer’s protocol. briefly, 2 µg of total rna was subjected to ribosomal removal using ribominus eukaryote kit (life technologies, usa). ercc rna spike-in control mixes (thermo scientific, usa) was added into rrna depleted-rna before rna fragmentation using rnase iii. the fragmented rna was quantified using qubit™ fluorometer (invitrogen, usa) and agilent rna 6000 nano kit (agilent technologies, usa), followed by hybridization and ligation to adaptors, reverse-transcribed, purified, size-selected, and amplified. the final library concentration was assessed using agilent high sensitivity dna chip (agilent technologies, usa) and normalized from 100 pm to 130 pm for clonal amplification. clonal amplification was performed on ion chef system using ion pi ic 200 kit followed by sequencing using pi bc v2 chip on ion proton system (all from life technologies, usa). 2.4. read mapping and gene quantification raw reads (fastq files) were processed using afterqc[10] to clean the data and obtain high-quality reads by removing reads that contain adapter or poly-n and low quality reads. gc content of the clean data was then calculated. all downstream analyses were based on clean data with high quality. sequencing reads were mapped with star (v2.0.9[11,12] to the human genome sequence assembly (grch38.89). the mapped reads of each sample were assembled using cufflinks[13]. 2.5. differential gene expression differential gene expression (deg) analysis was performed using cuffdiff[13]. four comparisons were performed: ptc lnm (lymph node metastasis) versus ptc lnn (lymph node negative), ptc lnm versus normal thyroid, ptc lnn versus normal thyroid, and normal thyroid from ptc lnm patients versus normal thyroid from ptc lnn. genes with pmmb 2022, 5, 1; a0000269 4 of 19 a p-value of less than 0.05 and showing a log2 fold change |1| were considered as significantly differentially expressed. 2.6. gene ontology and kegg pathway enrichment analysis next, we performed a gene ontology (go) analysis of these degs. functional enrichment analysis focusing on biological processes was performed on datasets by using funrich software[14]. settings were set at a default parameter, and fold enrichment with unadjusted p-values were computed using a modification of the fisher's exact test. we used kegg, a database resource for understanding high-level functions and utilities of the biological system, to test the statistical enrichment of differential expression genes in kegg pathways[15]. 3. results 3.1. data characterization of sequencing and mapping our rna-seq yielded 22.2 to 80.3 million reads. using star aligner, the proportion of reads that were successfully mapped to the ensembl reference genes ranged from 77% to 85%, indicating that the data produced are of high quality. gene expression levels were estimated using cufflinks plotted in figure 1 with pearson correlation coefficients of the estimated abundances between the two tissues. it showed that the gene quantification with cufflinks was consistent across different comparisons. figure 1. differential gene expression analysis of ptc and normal thyroid tissue. (a) density plots showing the expression level distribution for all genes in the two tissues. fpkm = fragments per kb of transcript per million fragments mapped. (1b) the scatter plot of global expression for each comparison between samples where the pearson correlation coefficient (pcc) is shown. each dot represents one gene that has detectable expression in either tissue. pmmb 2022, 5, 1; a0000269 5 of 19 3.2. differential gene expression we identified 33, 69, 31, and 44 significant degs between the ptc lnm versus normal, ptc lnn versus normal, ptc lnm versus ptc lnn, and normal thyroid from ptc lnm versus normal thyroid from ptc lnn, respectively (table 1). the complete lists of the degs were provided in supplementary table s1. upand down-regulated genes comparing ptc lnm versus ptc lnn were shown in figure 2. in the group comparison of ptc lnm versus normal thyroid, up-regulated transport-related genes in the ptc lnm included gabre and nup160, while down-regulated genes were ryr3 and hba2. in the ptc lnn versus normal thyroid, the upregulated genes included lcn2, ryr3, kcnq3, gria3, slc13a5, and slc34a2 but downregulated transport-related genes were not found. while in the group comparison of ptc lnm versus ptc lnn, there were two up-regulated transport-related genes, slc13a5 and sybu, and three down-regulated transport-related genes, which were grid1, scn8a, and trpm2. in the normal thyroid from both ptc lnm and ptc lnn, 17 up-regulated and 27 down-regulated with only two transport-related genes were being differentially expressed. the down-regulated transport-related genes were ryr3 and tmem199. the overlap of the degs among the four different groups was shown in a venn diagram in figure 3. table 1. significant differently expressed genes (p-value < 0.05 and log2 fold change |1|) in ptc lnm versus ptc lnn, normal thyroid (ptc lnm) versus normal thyroid (ptc lnn), ptc lnm versus normal, and ptc lnn versus normal group. group ptc lnm versus normal thyroid ptc lnn versus normal thyroid ptc lnm versus ptc lnn normal thyroid (ptc lnm) versus normal thyroid (ptc lnn) up-regulated gene 14 50 14 17 down-regulated gene 19 19 17 27 total 33 69 31 44 lnm: lymph node metastasis; lnn: lymph node negative; ptc: papillary thyroid cancer pmmb 2022, 5, 1; a0000269 6 of 19 figure 2. expression level of five upand down-regulated in ptc lnm versus ptc lnn. (2a) boxplots illustrated fpkm expression value in ptc lnm versus ptc lnn (slc13a5 and sybu) and (2b) boxplots demonstrated fpkm expression value in ptc lnm versus ptc lnn (grid1, scn8a, and trpm2). figure 3. venn diagram representing differentially expressed transport-related genes overlapping between the samples. the gene list from each group comparison showed the frequency of transport-related genes. pmmb 2022, 5, 1; a0000269 7 of 19 3.3. enriched functions of differentially expressed gene the functional analysis showed that the primary biological processes altered in ptc lnm versus normal thyroid were protein metabolism, metabolism, energy pathways, cell growth and/or maintenance, and transport. these processes are frequently involved in cancer. to improve our understanding of the function of the degs, we performed an enrichment analysis of gene ontology (go) for the up-and down-regulated genes, as illustrated in figure 4. first, we performed enrichment tests to detect the functional categories for significantly upand down-regulated genes detected by cuffdiff in the ptc lnm, ptc lnn, and adjacent normal tissue using the funrich software. the go categories that were greatly enriched in the regulated genes from ptc lnm versus normal thyroid, ptc lnn versus normal thyroid and ptc lnm versus ptc lnn were selected. the upand down-regulated genes in all comparisons in ptc were categorized into 177 functional categories. we identified 14 up and 19 down-regulated genes in ptc lnm versus normal thyroid and 50 up-regulated and 19 down-regulated genes in ptc lnn versus normal thyroid. for instance, the biological processes for up-regulated genes, which include “protein targeting”, “inflammatory response”, “regulation of cell growth”, “cell differentiation” and “transport” are essential to cancer progression[16,17]. whilst the upregulated genes in ptc lnn versus normal included “cell growth and maintenance”, “energy pathway”, “pyrimidine salvage”, “transport”, “ion transport” and “protein metabolism”. on the contrary, genes that were downregulated in ptc lnn versus normal thyroid included those involved in “cell communication”, “cell differentiation”, “cell proliferation”, “steroid hormone receptor signaling pathway”, and “vesicle-mediated transport” and “apoptosis”. interestingly, the biological process of “transport” appeared in most of the group comparisons suggesting that transport genes could potentially govern the metastasis cancer progression in thyroid cancer. detailed analysis of functional annotation was performed using funrich to analyze which kegg pathways were enriched with ptc-specific down-regulated genes. the pathways enriched with degs are listed in table 2. rho gtpases activate cit interaction pathway was affected in two out of four pairwise comparisons (ptc lnm versus normal thyroid and ptc lnm versus ptc lnn, and other gene regulation alterations in the extracellular matrix (ecm) organization pathway were involved in cancer tissue. furthermore, the chrebp activates metabolic gene expression pathway was enriched in the degs identified from the ptc tissue. pmmb 2022, 5, 1; a0000269 8 of 19 figure 4. gene enrichment biological processes across the different groups. pmmb 2022, 5, 1; a0000269 9 of 19 table 2. kegg pathway of enriched differentially expressed genes. comparison pathway id pathway name p-value fdr ptc lnm versus normal thyroid 1270056 acyl chain remodeling of dag and tag 1.272e4 2.480e2 1269514 rho gtpases activate cit 4.759e4 4.640e2 ptc lnn versus normal thyroid 1270114 chrebp activates metabolic gene expression 6.05e04 2.62e01 1270254 non-integrin membrane-ecm interactions 9.19e04 3.99e01 1470923 interleukin-4 and 13 signaling 1.29e03 5.59e01 1270244 extracellular matrix organization 1.44e03 6.26e01 ptc lnm versus ptc lnn 1269514 rho gtpases activate cit 5.60e04 5.93e02 1457800 met activates ptk2 signaling 7.79e04 8.25e02 1108786 mucin core 1 and core 2 oglycosylation 1.22e03 1.30e01 1270253 laminin interactions 1.22e03 1.30e01 1383022 fgfr2 alternative splicing 1.53e03 1.63e01 ptc lnm (normal) versus ptc lnn (normal) 852705 micrornas in cancer 2.38e05 7.58e03 142173 serine biosynthesis (phosphorylated route) 2.98e03 9.48e01 cit: citron rho-interacting serine/threonine kinase; chrebp: carbohydrate response element binding protein; dag: diacylglycerol; ecm: extracellular matrix; lnm: lymph node metastasis; lnn: lymph node negative; met: mnng hos transforming gene; ptc: papillary thyroid cancer; ptk2: protein tyrosine kinase; tag: triacylglycerol pmmb 2022, 5, 1; a0000269 10 of 19 go analysis was further refined to identify which degs were involved in transport, which was an important component of metastasis in ptc and illustrated in table 3. for in silico validation, the expression signatures of selected thyroid-specific genes (grid1, kcnq3, and slc34a) were retrieved from the rna-seq data of primary tumors in the cancer genome atlas (tcga) portal (figure 5). table 3. transport-related genes expression in rna-seq data of thyroid cancer. group ptc lnm versus normal thyroid ptc lnn versus normal thyroid ptc lnm versus ptc lnn normal thyroid (ptc lnm) versus normal thyroid (ptc lnn) upregulated gene gabre, nup160 lcn2, ryr3, slc34a2, kcnq3, slc13a5, gria3 slc13a5, sybu downregulated gene ryr3, hba2 grid1, scn8a, trpm2 ryr3, tmem199 gabre: gamma-aminobutyric acid type a receptor subunit epsilon; gria3: glutamate ionotropic receptor ampa type subunit 3; grid1: glutamate receptor delta-1 subunit; hba2: haemoglobin alpha 2; kcnq3: potassium voltage-gated channel subfamily q member 3; lcn2: lipocalin 2; lnm: lymph node metastasis; lnn: lymph node negative; nup160: nucleoporin 160; ptc: papillary thyroid cancer; ryr3: ryanodine receptor 3; scn8a: sodium voltage-gated channel alpha subunit 8; slc13a5: solute carrier family 13 member 5; slc34a2: solute carrier family 34 member 2; sybu: syntabulin; tmem199: transmembrane protein 199; trpm2: transient receptor potential cation channel, subfamily m, member 2 pmmb 2022, 5, 1; a0000269 11 of 19 figure 5. gene expression overview of tcga data; higher in thyroid cancer. (5a) grid1 (5b) slc34a2 and (5c) kcnq3. pmmb 2022, 5, 1; a0000269 12 of 19 4. discussion our study aimed to identify genes responsible for lymph node metastasis in ptc. we profiled the whole transcriptome of ptc lnm, ptc lnn, and normal thyroid tissues using ribosomal-reduced rna-seq. research on thyroid cancer progression has been well-studied using microarray and rna-seq. we identified several transporter-related genes associated with ptc lnm, ptc lnn, and adjacent normal thyroid. ion transport process happens during the multi-stage cancer progression, from a normal cell to cancer cell and finally to metastasis. our results suggested an important role of transporter genes during lymph node metastasis in thyroid cancer. we identified 177 degs within the various comparison groups. however, a kegg pathway enrichment analysis showed that the regulated genes were significantly enriched in the rho gtpases activated cit pathway in two group comparisons: ptc lnm versus ptc lnn and ptc lnm versus normal thyroid. this pathway is closely related to cancer progression[18,19]. for instance, aberrant activity of rho small g-proteins, particularly rac1 and their regulators, is a hallmark of cancer and contributes to the tumorigenic and metastatic phenotypes of cancer cells[20]. two up-regulated transporter genes (gabre and nup160) and two down-regulated genes (ryr3 and hba2) were identified in ptc lnm versus normal thyroid. the gabre gene encodes for the gamma-aminobutyric acid type a receptor epsilon subunit, which is a multisubunit chloride channel that facilitates the synaptic transmission in the central nervous system (cns). the mir‐224/452 cluster is co‐expressed with its host gene gabre located in the intron of gabre gene coding for gamma-aminobutyric acid (gaba) receptor[21], and its expression is directly activated by transcription factor e2f1 through transactivation of the gabre gene[22]. this gene is up-regulated in medulloblastomas, lung cancer, bladder cancer, prostate cancer, and colorectal cancer[23–26]. many studies reveal that mirnas are involved in carcinogenesis, and mirna expression has been found in many types of cancer[27–29]. in contrast, the gabre∼mir-452∼mir-224 locus has been reported to be downregulated and hypermethylated in prostate cancer, suggesting its potential as a new epigenetic candidate biomarker for prostate cancer diagnosis and prognosis[24,30]. it has also been upregulated in hepatocellular carcinoma[31]. mir-224 is also up-regulated in ptc, mtc, ftc and atc[32]. many studies on different types of cancer suggest that mir-224 promotes tumorigenesis in colorectal cancer[33,34], non-small cell lung cancer[35], medullary thyroid carcinoma[36] and hepatocellular carcinoma[37]. in contrast, some studies show that it suppresses tumorigenesis in prostate cancer[24,30], and breast cancer[38,39]. gabrb2, which is part of the gaba receptors gene family, plays a crucial role in the lymph node metastasis of ptc[40]. its function is closely related to the nervous system, but its role in cancer remains uncertain[41]. we identified two down-regulated genes in ptc lnm versus normal thyroid, namely, nup160 and ryr3. the nup160 gene encodes the nuclear pore complex protein nup160, which mediates molecule transport across the nuclear envelope[42] and is thought to be pmmb 2022, 5, 1; a0000269 13 of 19 involved in cancer progression[43]. up-regulation of this gene may indicate its role in lymph node metastasis. one study reported a novel frequent fusion gene, nup160-slc43a3, in angiosarcoma patients and deduced that this fusion protein might cause rapid tumour progression[44]. this novel fusion could also provide a possible therapeutic target for many human malignancies[45,46]. our findings suggest an oncogenic role of gabre and nup160 in thyroid carcinogenesis. the ryr3 gene encodes a ryanodine receptor, which releases calcium from intracellular storage for use in many cellular processes[47,48]. it is imperative for the growth, morphology, and migration of breast cancer cells, and studies have shown that it is commonly expressed in breast cancer[49,50]. functional ryanodine receptor is also involved in the apoptosis of in vitro human prostate cancer lncap cells[51]. this gene's gene ontology (go) analysis included calcium ion binding and calmodulin binding. additionally, this gene is also involved in transport, suggesting that it might be involved in the process of metastasis in ptc. in comparing tumour samples of ptc lnm and ptc lnn, we identified two upregulated transport-related genes (slc13a5 and sybu) and three down-regulated transportrelated genes (grid1, scn8a, and trpm2). the slc13a5 (solute carrier family 13 members 5) gene encodes for proteins that transport citrate from the cytoplasm into cells and is mostly expressed in liver cancer[52] via regulation of metabolic processes[53]. it is a sodiumcoupled transporter that facilitates the cellular uptake of citrate and plays vital role in synthesizing fatty acids and cholesterol[52]. syntabulin-kinesin-1 family member 5b (sybu), a subset of the kinesin motor-adaptor complex is important for axonal transport and is involved in neuronal development [54]. it encodes syntabulin, a microtubule-related protein that facilitates the transport of vesicles to neuronal processes[55]. functional analysis of sybu revealed its involvement in various binding function such as protein binding, microtubule binding, syntaxin-1 binding, and kinesin binding[56]. it has been reported to be over-expressed in pancreatic adenocarcinoma, breast cancer and bladder cancer[57]. thus far, no study has been conducted on this gene involvement in thyroid cancer. in our study, three glutamate receptor subunits: grid1 (ionotropic glutamate receptor), scn8a (sodium voltage-gated channel) and trpm2 (transient receptor potential cation channel) were significantly up-regulated in ptc lnm tissues. ion transport is believed to be involved in cancer progression, starting with transforming a normal cell into a cancer cell and until the metastasis stage[58]. moreover, this derangement in the ion transport mechanism is one of the hallmarks of cancer and could be a key event in tumour metastasis [59]. the study by stepulak et al. also suggested glutamate as a potential growth factor in tumorigenesis [60]. glutamate receptor subunits: nr1-nr3b, glur1-glur7, ka1, ka2, and mglur1-mglur8, have been shown to be differentially expressed in multiple cancer cell lines (te671, sk-nas, ftc 238, sk-lu-1, moggccm, rpmi 8226, a549, ht 29, jurkat e6.1, t47d, ls180, u87-mg, and u343)[61,62] and tumours including thyroid cancer, hepatocellular carcinoma pmmb 2022, 5, 1; a0000269 14 of 19 (grik1), colorectal cancer (mglur4)[63], melanoma (mglur1)[64], and serous ovarian adenocarcinoma (gria2)[65]. the grid1 gene showed increased expression in colorectal cancer and breast cancer[66], and down-regulated expression in non-small cell lung cancer of node-positive cases[67]. thereby, grid1 might play an essential role in the progression of ptc. it could also be used as a prognostic biomarker and a therapeutic target[68]. another gene up-regulated in ptc lnm is scn8a which encodes a protein of the ion pore region of the voltage-gated sodium channel (vgsc) and is crucial for the rapid membrane depolarization[69]. this gene is over-expressed in cervical cancer and prostate cancer[70]. additionally, the expression of vgsc is associated with cancer cell migration, invasion, and metastasis[71]. inhibition of vgsc subunits has been suggested as a suitable treatment strategy to decrease the metastatic spread of prostate cancer[70]. a detailed review of vgsc by wang et al. indicated that upregulation of scn8a gene might increase cancer cell growth and promote metastasis. they also suggested that inhibiting sodium channels in both immune and cancer cells might reduce metastases[37]. based on our results, the trpm2 gene, which encodes for the non-selective calciumpermeable cation channel, was up-regulated in ptc lnm. our study showed concordance with other studies where trpm2 was over-expressed in many cancers, including but not limited to bladder cancer[72], gastric cancer[73(p2)], lung cancer, and breast cancer[74]. in 2013, liu and the coauthors reported activation of trpm2 gene by irradiation via parp1 activation. they contributed to the irreversible loss of salivary gland function, suggesting that the disordered expression of trpm2 might be associated with the occurrence of head squamous cell carcinoma (hscc)[75(p2)]. in addition, a group of researchers from china proposed that trpm2 was essential in the regulation of migration and survival of scc cancer cells. at the same time, another study described that trp channels were clinically valuable for cancer diagnosis and prognosis[76]. our study identified a prominent association between grid1, scn8a, and trpm2 with lnm in ptc. these findings suggest that the upregulation of grid1 and scn8a is related to ptc metastasis. nevertheless, further studies are required to explore the potential of grid1 and scn8a as molecular biomarkers of ptc. three thyroid-specific gene expression signatures (grid1, kcnq3, and slc34a) were up-regulated in thyroid cancer based on the rna-seq data of primary tumours in the cancer genome atlas (tcga) portal, and this observation was concordant with our results. in our study, the kegg pathway and go enrichment analysis showed many transport pathways involved in lymph node metastasis. future studies could focus on these transporter genes and its related pathways in cancer, such as the glucose and hexose pathways, which had been discussed previously by adekola et al.[77]. the functional analyses of genes differentially enriched in ptc lnm and ptc lnn further supported the hypothesis that lymph node metastasis was associated with specific essential genes and pathways. the results from our study showed the similarities found in terms of the role of crucial transport pathways, especially from the perspective of metastasis in ptc. we screened the degs linked to the transport-related pathways to ptc by using the pmmb 2022, 5, 1; a0000269 15 of 19 tcga database to confirm our findings. among the 17 transport-related degs, only four genes (gabre, hba2, kcnq3, and slc34a2) showed similar expressions to our conclusions. another 13 genes required further validation to confirm the results. 5. conclusion in conclusion, we have identified key mrna signatures of ptc lnm and ptc lnn. the degs might be promising biomarkers of ptc lnm and could provide a basis for further exploration of the tumorigenesis ptc. these genes may play critical roles in metastasis and survival in ptc patients. these results offer new insights on metastasis in ptc and eventually lead to better diagnosis and possibly new therapeutic targets for thyroid cancer. author contributions: n-sam conceived the idea, supervised the project, and applied for the grant that funded the work. amy and ss performed laboratory experiments. wfwmn performed the bioinformatics analyses and generated the tables and figures. rm, snas and nhml are the thyroid surgeons involved in specimen collection, and imr is the pathologist who confirms the diagnosis. wfwmn, n-sam, fyft, nfmp and l-hl wrote and edited the manuscript. funding: this study was funded by the fundamental research grant scheme (frgs) from the ministry of higher education malaysia (frgs/1/2014/skk01/ukm/03/1). conflicts of interest: the authors declare no conflict of interest. references 1. vaccarella s, franceschi s, bray f, et al. worldwide thyroid-cancer epidemic? the increasing impact of overdiagnosis. n engl j med 2016; 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a0000274. doi: 10.36877/pmmb.a0000274 http://journals.hh-publisher.com/index.php/pmmb original research article development of a semiconductor sequencing-based panel for screening individuals with lynch syndrome ryia-illani mohd yunos1, nurul-syakima ab mutalib1, janice khor sheau sean2, sazuita saidin1, mohd ridhwan abd razak1, isa md. rose3, ismail sagap4, luqman mazlan4, rahman jamal1* article history 1ukm medical molecular biology institute (umbi), universiti kebangsaan malaysia, kuala lumpur, malaysia; ryia.yunos@ppukm.ukm.edu.my (rimy); syakima@ppukm.ukm.edu.my (ns-am); sazuita@ukm.edu.my (ss); ridhwanrazak@ppukm.ukm.edu.my (mrar) 2life sciences solutions, thermo fisher scientific inc.; janice.khor@thermofisher.com (jk) 3department of pathology, faculty of medicine, universiti kebangsaan malaysia, kuala lumpur, malaysia; isa@ppukm.ukm.edu.my (ir) 4department of surgery, faculty of medicine, universiti kebangsaan malaysia, kuala lumpur, malaysia; ismailsagap@ppukm.ukm.edu.my (is); luqman@ppukm.ukm.edu.my (lm) *corresponding author: rahman jamal; ukm medical molecular biology institute (umbi), universiti kebangsaan malaysia, kuala lumpur, malaysia; rahmanj@ppukm.ukm.edu.my (rj) received: 21 june 2022; received in revised form: 23 july 2022; accepted: 28 july 2022; available online: 31 july 2022 abstract: lynch syndrome is a genetic disorder associated with mutations in mismatch repair (mmr) genes that are linked to the development of colorectal cancer. individuals with this condition have a lifetime risk of developing cancer at around 20% to 65%. due to the autosomal dominant inheritance pattern, close biological relatives are also at high risk. early detection of crc may lead to better health outcomes and considerable savings in treatment costs. therefore, our objective is to develop a rapid screening method for ls. we designed an ion ampliseq™ custom panel, which includes four mmr genes associated with ls (mlh1, mlh2, msh6, and psm2), a downstream gene (epcam), and a gene that indicates sporadic crc (braf) for sequencing on the ion torrent pgm™ sequencer. sequencing was performed on 16 dna samples derived from various stages of crc. the sensitivity and specificity of the identified mutations were determined by sequencing the serially diluted dna from two human cancer cell lines, hct 116 and ln-18. an average of 92 % of reads were mapped to the target region with 98 % uniformity. there was no amplicons dropout across all samples, and 58 variants were chosen for validation in 19 samples using massarray and sanger sequencing. we achieved 87% specificity, 97% accuracy, and 100% sensitivity for detecting variants at an allele frequency of more than 13% using this ls gene panel. with the development of this method, ls crc can be detected at an early stage using this rapid and sensitive approach. keywords: lynch syndrome, colorectal cancer, mmr genes, next generation sequencing, gene panel mailto:third_author@domain.com pmmb 2022, 5, 1; a0000274 2 of 17 1. introduction colorectal cancer (crc) is the third most common cancer in men (10.9% of the total cases) and the second in women (9.5% of the total cases) [1]. crc is a major global health burden and causes significant morbidity and mortality. almost 55% of crc cases occur in developed regions [1]. in malaysia, crc is the commonest cancer among males and the second most common cancer among females [2]. this cancer is widely preventable through the various interventions that the community can adopt, such as adopting a healthy lifestyle and regular medical screening [3, 4]. in general, there are three classifications of crc based on the increasing hereditary risk of cancer [5, 6]. the most common type is sporadic crc, which accounts for about 70% of all cases. the other two are familial and hereditary crcs [5, 6]. familial crc (20%) refers to patients with at least one biological family diagnosed with crc, but there is no specific germline mutation or obvious pattern of inheritance [6]. hereditary crc (10%), also known as lynch syndrome (ls), is caused by the inheritance of germline mutations in highly penetrant cancer susceptibility genes. ls is an autosomal dominant disease, in which if one of the parents has the disease, there is a 50% chance that the offspring will inherit it. this syndrome is marked by an increased risk of developing crc and other cancers, such as the endometrium, ovary, stomach, hepatobiliary tract, urinary tract, brain, and skin [7, 8]. besides, the risk of developing a second primary crc is high (approximately 16% within ten years), and the risk of cancer in a first or second-degree family member is around 45% for men and 35% for women by the age of 70 [9]. while the last group is the least common, this group is crucial in understanding the molecular mechanisms of carcinogenesis. it has important implications for the screening and follow-up of patients and their families [6]. mutations in dna mismatch repair (mmr) genes; namely, mutl homologue 1 (mlh1), muts homologues 2 and 6 (msh2 and msh6), and postmeiotic segregation increased 2 (pms2), are the main causes of lynch syndrome[10]. mutations in the mmr genes have prevented the repair of base mismatches, minor insertions, and deletions that might result in cancers, which is the reason for the loss of mmr function in a cell. [10]. global data show that mlh1 accounts for 39% of entries in the international society for gastrointestinal hereditary tumours (insight) database (www.insight-group.org/mutations/), msh2 for 34%, msh6 for 20%, and pms2 for 8% [11]. identification of mmr gene mutation confirms the clinical diagnosis of ls and therefore identifies the individuals who should undergo routine surveillance for associated cancers. another gene, epcam, which was not categorised in the mmr group but is located adjacent to one of the mmr genes (msh2), was also identified as the cause of this disease. mutation of the 3' end of epcam can affect msh2 gene expression, whereby epcam 3' deletion can lead to inactivation of the msh2 gene [12, 13]. therefore, epcam should also be included in the screening process for ls. besides, a marker such as the braf v600e mutation may help to distinguish sporadic from hereditary or familial tumours [14, 15]. the braf gene, which belongs to the raf-ras gene family, encodes a cytoplasmic serine/threonine kinase, a key component of the mitogenactivated protein kinase signalling pathway. in 15% of sporadic crcs, somatic mutations in pmmb 2022, 5, 1; a0000274 3 of 17 the braf gene, primarily at codon 600, are found. because braf gene mutations are mostly linked to sporadic crc, testing of this gene will essentially rule out the diagnosis of ls [15]. experts in pharmacoeconomics agree that crc is linked to increased economic burden [16, 17]. the annual incidence is forecasted to increase by ~80 % from the current 1.2 million estimated cases over the next two decades. a majority of the increase is expected to be contributed by less developed regions [16]. the long-term cost of managing crc was estimated to go up to $50,175 per patient in 2008 [16]. hence, the efforts to control crc are becoming critical [18, 19]. the current standard for diagnosis of ls includes mutation detection using polymerase chain reaction (pcr) followed by sanger sequencing. using this technique, mutations in the six genes mentioned earlier must be screened one at a time. one diagnostic laboratory charges around us$1 000 per gene. testing six genes will easily cost the patient more than us$6,000 for such tests, excluding any pre-screening test (including msi analysis, ihc, and braf test for v600e), before the genetic screening (http://fightlynch.org/physicians-info/genetic-testing/). hence, this approach is expensive, low throughput, and time-consuming, primarily because at least five or six genes may have to be analysed, and their mutational spectra are extensive [19]. developing a benchtop next generation sequencing (ngs) instrument that allows the analysis of multiple genes simultaneously is a superior method to sanger sequencing. the higher throughput capabilities of this technology remarkably reduced the cost of mutation screening and shortened the turnaround time. several diagnostics service centres abroad, such as invitae and the university of chicago (genetic services laboratory) in the usa, have already been offering this screening service but at approximately us$1500 to us$3000 per patient. however, if we develop this test locally in malaysia, the cost will be around us$628. to the best of our knowledge, our country has no diagnostic service provider yet that offers the mutation screening of these six genes using ngs technology. therefore, we aim to develop a relatively affordable, fast, specific, and sensitive screening for ls using the ngs approach. 2. materials and methods 2.1 tumour specimens and cell lines sixteen specimens of crc with known mutations were selected from the umbippukm biobank. these samples have been analysed for variant detection via whole exome sequencing [20] and denaturing high-performance liquid chromatography (dhplc) in our previous studies [21]. the average age of patients was approximately 66 years old (range 44 – 75 years). with regards to cancer stage, 13% (n=2) of the patients were of dukes' a, 38% (n=6) were dukes' b, 43% (n=7) were dukes' c, and the remaining 6% (n=1) were dukes' d. hct 116 is a cell line with a heterozygous mutation of mlh1: p.s252x, c.755c>a while the ln-18 cell line is negative for this mutation. this mutation is a verified cosmic variant. the dna extraction was performed using the qiaamp dna mini kit (qiagen, germany) according to the manufacturer's protocol. the quality and quantity of the extracted dna were assessed using the qubit fluorometer (invitrogen, usa), nanodrop 2000 spectrophotometer (thermo fisher scientific, us), and agarose gel electrophoresis. pmmb 2022, 5, 1; a0000274 4 of 17 2.2 ion ampliseq custom panel design ion ampliseq custom panels (life technologies, us) covering the entire coding region of mlh1, msh2, msh6, pms2, braf, and 3' untranslated region (utr) of epcam was designed using the ion ampliseq designer v4 (https://ampliseq.com). the designed gene panel contains a pool of 115 primers for multiplex amplification of genomic regions of interest. 2.3 ion ampliseq library preparation libraries were constructed using ion ampliseq™ library kit 2.0 and ion ampliseq™ custom panel (both from life technologies, us) using ten ng of genomic dna according to the protocol suggested by the manufacturer. genomic dna was amplified by pcr for each primer pool (two primer pools for this panel), followed by partial digestion of primer sequences with fupa reagent. the amplicons were then ligated to the adapters with the addition of a barcode from the ion xpress™ barcode adapters 1-16 before being subjected to amplification and purification. amplified libraries were assessed on the agilent® bioanalyzer® instrument using the high sensitivity dna kit (both from agilent technologies, us). libraries were diluted to 60 pm, and eight libraries were combined in one pool before proceeding with template preparation. 2.4 template preparation and sequencing enriched template-positive ion sphere™ particles were prepared for sequencing on ion chef™ system (life technologies, us). combined libraries (eight libraries) were loaded onto an ion 318™ bc chip and subsequently sequenced on ion torrent pgm (it-pgm) sequencer (life technologies, us). ion pgm™ hiq™ sequencing kit (life technologies, us) was used for sequencing up to ~400 bp library inserts to improve the indel sequencing accuracy of targeted resequencing panels. 2.5 sensitivity analysis the it-pgm platform's mutation detection sensitivity was tested by sequencing serially diluted dna from a human colorectal adenocarcinoma cell line, hct 116. the dna from hct 116 was diluted in the dna obtained from a malignant glioma cell line, ln-18 (crl2610; atcc, us) in the ratio of 1:3, 1:9, and 1:19, resulting in 25%, 10%, and 5% dilution. 2.6 validation of mutations by the agena massarray system and sanger sequencing fifty-eight variants identified across 19 samples (including three from cell lines) were validated via massarray (agena bioscience, ca). the four wells containing 58 assays for variant screening were developed in our laboratory using the massarray® assay design suite to achieve universal thermal cycling conditions for all assays. dna was amplified and subjected to single-base primer extension using the iplex gold kit (agena bioscience, ca) and analysed using the maldi-tof (mass-assisted laser desorption/ionisations-time of flight) mass spectrometry (agena bioscience, ca). our four-well multiplex primer extension assay was designed to assess the mutational status of 58 variants identified in mlh1, msh2, msh6, pms2, epcam, and braf by it-pgm. the target regions were amplified using 20 ng of dna per well, followed by dephosphorylation of the unincorporated nucleotides by treatment with alkaline phosphatase. the amplified product was used as the template for a pmmb 2022, 5, 1; a0000274 5 of 17 locus-specific single base extension with mass-modified dideoxynucleotides. primers for pcr amplification and single base extension were designed using the massarray® assay design suite and were synthesised by integrated dna technologies, idt (coralville, ia). maldi-tof analysed the mass of products of a single base extension for single nucleotide polymorphism detection using typer analyzer software (agena bioscience, ca). variants that were not successfully validated by massarray (agena bioscience, ca) were subjected to sanger sequencing for further validation. pcr was performed using genomic dna as a template and primer pairs flanking the variant sites. the pcr products were purified using the qiaquick pcr purification kit (qiagen, germany) according to the manufacturer's instructions. the cycle sequencing was performed using the big dye terminator v3.1 (life technologies, us). the cycle sequencing products were then purified using ethanol precipitation, and sequencing was carried out using the abi 3130xl genetic analyser capillary electrophoresis (life technologies, us). the results were analysed using the basic local alignment system tool (blast) [22] and sequence scanner (applied biosystem, us). 2.7 data analysis we analysed the sequencing data using three pipelines to perform the alignment and variant calling. first, the sequencing data were processed using ion torrent suite™ software v4.4.2.1 running on the torrent server. the pipeline included signal processing, base calling, adapter trimming, pcr duplicate removal, alignment to the human genome 19 reference (hg19), quality control of mapping quality, coverage analysis, and variant calling. coverage analysis and variant calling were performed using torrent variant caller plugin software v4.4.2.1 in the torrent server. the variant caller parameter setting was germline pgm low stringency. we obtained the fastq files from torrent suite software and subjected them to quality assessment and pre-processing using fastqc software [23]. fastqc: a quality control tool for high throughput sequence data. available online at http://www.bioinformatics.babraham.ac.uk/projects/fastqc]. it is important to ensure only high-quality bases were used for the variant analysis. we performed alignment and variant calling from here using another two additional pipelines. firstly, the fastq files were mapped to the human reference genome using bowtie2 v2.2.6 [24], and variant calling was performed using samtools/bcftools (mpileup) v0.1.19 [25]. secondly, the alignment was performed using burrows-wheeler aligner (bwa) v0.7.12 [26], and indel realignment, base recalibration and variant calling were performed using gatk-picard pipeline 3.4-46 [27]. the variants were further annotated using annovar [28] against alternative allele frequency data in 1000 genomes project (1000g2014oct_all) (the 1000 genomes project consortium, 2015) [29], dbsnp version 138 (snp138) [30], clinvar (clinvar_20150629) [31] and cosmic version 70 (cosmic70) [32]. 2.8 determination of specificity, sensitivity, and accuracy of ion torrent pgm gene panel assay we applied a method by parikh et al. (2008) [33] to determine sensitivity (probability of being tested positive when disease present), specificity (probability of being tested negative pmmb 2022, 5, 1; a0000274 6 of 17 when disease absent), and accuracy (measured by sensitivity and specificity). true positive, true negative, false positive, and false negative variants were analysed across 19 samples by sanger sequencing and massarray. below is the equation used to determine the specificity, sensitivity, and accuracy: 3. results 3.1 the ngs panel performance the six genes (mlh1, msh2, msh6, pms2, epcam, and braf) were sequenced in the 19 samples (including three samples from cell lines), generating a mean of 664 260 reads per sample. on average, 92% of these reads mapped to the targeted regions, with uniformity across all targets being 98%. the percentage of reads mapped to the target was a bit low due to the paralogous gene to pms, which is pms2cl. nevertheless, other genes were wellcovered. no amplicons dropout was observed across all samples. however, to determine whether coverage was substantially lower for any particular region, we calculated the proportion of amplicons covered less than 40x. at about 500x sequencing coverage, 2.6% of the amplicon was covered less than 40x. at about 4000x sequencing coverage, the percentage of amplicons covered less than 40x decreased to 1.7%. no amplicons were covered at less than 40x when sequencing coverage reached 9000x. the amplicons covered less than 40x covered mlh1 and msh6 genes. hence, variants identified in these amplicons will require careful interpretation. on the other hand, an evenly distributed mean depth of coverage 4000-5000x for all six genes across 19 samples was achieved. 3.2 comparison of three different data analysis pipelines the data were analysed using three different pipelines for comparison. from the analysis, we found that an average of 99.9% of the reads were mapped to the genome. tmap gave the highest percentage of mapped reads to the target region, 92%. in terms of the number of called variants, gatk gave the highest number of variants compared to the other two pipelines (table 1). specificity = sensitivity = number of true positives number of true positives + number of false negatives pmmb 2022, 5, 1; a0000274 7 of 17 table 1. comparison of mapped reads and variants called between three different pipelines. mapping and alignment bwa bowtie2 tmap percentage of mapped reads to the reference genome 99.9 99.9 99.7 percentage of mapped reads to target region 86.9 86.8 92 variant calling bwa-gatk bowtie2-samtools tmap-tvc snps 105 68 93 indels 209 14 26 3.3 sequencing on ion torrent pgm and variants detection upon the completion of data analysis using three different pipelines, 58 variants were found to be overlapped and chosen for validation in 19 samples (resulting in a total of 381 variants) (figure 1). this analysis classified six variants as pathogenic according to clinvar [31] (assessed on 13th may 2016), as shown in table 2. the ion torrent pgm identified two missense mutations in mlh1 and msh2 genes in patients c360t and c76t, respectively. both are pathogenic variants as classified by clinvar. a single base-pair substitution in the msh2 gene (c to t) with a variant frequency of 52% was identified in patient c76t and validated by the massarray (figure 2a). braf v600e mutation was also clearly identified by the ion torrent pgm in patient c41t. this mutation is a single base pair substitution in the braf gene resulting in a substitution of valine with a glutamic acid residue, and the massarray confirmed this. the reliability of the mutation call was evident by the high total sequencing coverage of 11,223x (figure 2b). patient c36t harbour at least one pathogenic missense mutation in msh6 with a variant frequency of 50%. we compared one of the pathogenic variants identified in patient c337t with the in-house exome data. interestingly, the mutation we detected using the lynch panel was somatic in our previous exome study [20]. the variant was covered at about 30% in the exome and targeted panel. this was also confirmed by massarray (figure 3). pmmb 2022, 5, 1; a0000274 8 of 17 figure 1. overlapped variants were identified from three different data analysis pipelines. table 2. list of identified pathogenic variants. sample id gene protein change dna change variants frequency coverag e variants coverage dbsnp id clinvar c41t braf p.v600e c.1799t> a 30% 11223 3348 rs113488022 pathogenic c360t mlh1 p.c77y c.230g>a 68% 3522 2381 rs63750437 pathogenic hct116_ln-18-5% hct116_ln-1825% mlh1 p.s252x c.755c>a 13% 30% 4793 5645 632 1670 rs63750198 pathogenic c76t msh2 p.q215x c.643c>t 52% 3348 1748 rs63751274 pathogenic c337t msh2 p.r389x c.1165c>t 30% 7513 2235 rs587779075 pathogenic c36t msh6 p.y214y c.642c>t 50% 4000 1992 rs1800937 pathogenic pmmb 2022, 5, 1; a0000274 9 of 17 figure 2(a). a pathogenic variant was identified in the msh2 gene of patient c76t. figure 2(b). a pathogenic variant was identified in the braf gene of patient c41t. pmmb 2022, 5, 1; a0000274 10 of 17 figure 3. a pathogenic variant in the msh2 gene of patient c337t, comparing whole exome sequencing (matched normal and tumour sample) and lynch panel. 3.4 sensitivity analysis of ion torrent pgm gene panel assay serially diluted dna from two human cancer cell lines: hct 116 and ln-18, were sequenced and used to determine the sensitivity of the ion torrent pgm technology for variant identification. dna from hct 116 was diluted into dna from ln-18, resulting in 25%, 10%, and 5% dilution, respectively. from the sequencing, we managed to identify the mutation with a variant frequency of about 25 to 30% and 10-13% for 25% dilution and 10% dilution, respectively, using three different pipelines (table 3). we have validated the variant detected in our sensitivity sample using sanger sequencing. however, due to the limit of detection by sanger sequencing, we could not identify as low as 5% of allele frequency (figure 4). table 3. variant frequency of mlh1, c.755c>a in serially diluted dna hct 116 and ln-18. samples details bowtie2-samtools bwa-gatk tmap-tvc hct116_ln-18 25% coverage frequency (ref) frequency (alt) 5,603 70.79% 29.21% 2757 74.95% 25.05% 1,670 70% 30% hct116_ln-18 10% coverage frequency (ref) frequency (alt) not detected 2,403 89.01% 10.99% 632 87% 13% pmmb 2022, 5, 1; a0000274 11 of 17 figure 4. validation of mlh1, c.755c>a using sanger sequencing in (a) serially diluted dna from hct 116 to ln-18 at 25% and (b) serially diluted dna from hct 116 to ln-18 at 5%. 3.5 determination of specificity, sensitivity, and accuracy of ion torrent pgm gene panel assay the develop panel's specificity, sensitivity, and accuracy were determined by comparing the number of variants identified by the ion torrent pgm with variants detected by massarray or sanger sequencing. our ls panel achieved 87% specificity, 100% sensitivity, and 97% accuracy. 4. discussion lynch syndrome (ls) is inherited in an autosomal dominant manner, which means that a parent with ls has a 50% (1 in 2) chance of passing the condition on to their children. it also will not skip a generation, meaning the grandchildren will not be affected if the children do not inherit ls [34]. it is important to note that people who inherit ls have a significantly increased risk of developing cancer, not the disease itself. not all people who inherit mutations in these mentioned genes will develop cancer. however, screening of ls may help in the early detection of crc and save the treatment cost. it also helps in managing the psychological impact and emotions of the individuals. non-carriers may avoid unnecessary surveillance programs and experience relief from worries. the carriers can minimise their risk by applying a healthy lifestyle and getting annually screened via colonoscopy. therefore, developing a rapid and sensitive method to screen ls is strategic, especially in countries aiming to reduce the incidence of crc. according to the american college of medical genetics and genomics (acmg) guideline [35], the standard procedure for evaluating tumour tissue for msi is immunohistochemistry of four mmr proteins. however, msi status alone is insufficient to diagnose ls as 10-15% of sporadic crc exhibit msi. methylation test on mlh1 and somatic braf pathogenic variants may help identify those tumours more likely to be pmmb 2022, 5, 1; a0000274 12 of 17 sporadic than hereditary [36]. the molecular genetic testing of the mmr genes can be performed to identify a germline pathogenic variant when findings from these two tests are consistent with ls. we ventured into the development of rapid tests using ngs based panel to screen individuals with ls. as braf gene mutation is predominantly associated with sporadic crc, we've included the braf gene in our panel to rule out the diagnosis of ls. it requires a small amount of dna and provides simultaneous detection of variants in six genes with high accuracy and sensitivity. upon analysis using three different pipelines, we shortlisted 58 highly confident variants to be validated via sanger sequencing and massarray. six of these 58 variants were pathogenic based on clinvar in five patients. we observed that the tmap-tvc pipeline outperformed the other two pipelines, bowtie2-samtools and bwagatk, regarding alignment to the genome and mapping to the target. it is known that ion semiconductor sequencing platforms, such as ion torrent pgm, suffer from inaccuracy in detecting variants in the homopolymer regions [37]. these homopolymer errors often lead to inaccurate local alignment results, and false-negative detection and careful interpretation of variants located in this region are critical. however, using open-source software such as gatk and samtools, we showed that the false negatives could be minimised using appropriate bioinformatics analysis [37]. in this study, we aim to determine the developed panel's sensitivity, specificity, and accuracy rather than the pipeline used. to overcome the issue of false-negative detection in homopolymer regions, we performed sequencing on the ion pgm™ system using ion pgm™ sequencing 400 kit (thermo fisher scientific) and the ion pgm™ hi-q™ sequencing kit (thermo fisher scientific). hiq chemistry was claimed to reduce mapping errors to 49 errors for 10kbp mapped compared to 89 errors using the former kit. additionally, reduction of insertion and deletion (indel) errors, including homopolymer errors, by 80% across 400 base pair read lengths on the ion pgm™ system, resulting in higher data quality [38]. this was also supported by a study in the forensic genetic laboratories by churchill et al. in 2016. this suggests that reliable and accurate data can be generated, and the identification of variants in the homopolymeric region can be improved using ion torrent hi-q™ sequencing chemistry [39]. the lifetime risk of crc in ls has been estimated in various ways, and it appears to depend on gender and the mutated mmr gene [40]. most studies estimate the lifetime risks of crc for mlh1 and msh2 gene mutation carriers to be between 30 and 74% of crc. patients with msh6 mutations, on the other hand, have a decreased lifetime risk of colorectal cancer, ranging from 10% to 22%, compared to 15% to 20% in those with pms2 mutations [41]. in ls patients, the average age of crc diagnosis is 44 to 61 years, compared to 69 years in sporadic cases of crc [42]. crc is becoming more common in young people, with one out of every ten new cases affecting those under 50 years old [43]. we observed a pathogenic heterozygous variant in the msh2 gene of patient c76t, substitution from c to t. since we have documented the presence of a mutation in this 44year-old man who was diagnosed with dukes' d crc, testing for at-risk individuals in the family is possible. genetic counselling is recommended for this patient and for other family members at risk for carrying this mutation. extensive investigation of mmr gene mutation status in the family may be required to rule out ls if they meet the amsterdam criteria or bethesda guidelines. pmmb 2022, 5, 1; a0000274 13 of 17 on top of mutations identified in mmr genes, we observed a braf v600e mutation in 81 years old women with colorectal cancer dukes' b (moderately differentiated adenocarcinoma). it is known that mutations in the four mmr genes (mlh1, msh2, msh6, and pms2) account for only half of the ls cases identified by pedigree criteria [44]. most sporadic colorectal tumours show no mismatch repair defects [45]. on the other hand, 12–15% of crc with defective mmr and msi-h phenotype is primarily due to hypermethylation of the mlh1 gene promoter, not to the germline. in more detailed analyses, braf mutations were not detected in those cases with a germline mutation in either mlh1 or msh2 mutations [45]. domingo and colleagues 2004 reported that 40% of sporadic msi-h tumours harbour braf v600e, but none was found in 111 tested ls tumours [36]. thus, we conclude that this 81-year-old woman probably has a sporadic form of crc. we also compared the data obtained from the ngs panel with the whole exome sequencing (wes) data of the same patients we analysed. whole exome sequencing of the tumour and matched normal tissue of patient c337t was previously performed. we identified one pathogenic variant, msh2: p.r389x, c.1165c>t, in the tumour tissue via wes and targeted sequencing using our developed panel. however, this variant was not observed in the matched normal tissue of the c337t patient. thus, we suggest that this mutation is most probably a somatic mutation. our finding is also supported by a study from haraldsdottir et al. in 2014, suggesting that deficiency can arise from somatic mutations. in the study, they observed some patients with mmr deficiency during screening for ls, and 70% of these patients acquired somatic mutations rather than germline mutations in mmr genes [46]. thus, it is recommended that patients with crc and mmr deficiency not explained by germline mutations might undergo analysis for somatic mutations in mmr genes to guide future surveillance guidelines. however, we would like to highlight some of the limitations and shortcomings of the present study. one main concern is the limited number of dukes' a crc patients, which restricts the generality of this applied method for an early stage of crc with ls. samples from tumour tissues and cell lines were insufficient to address its accuracy. results of detected variants from adjacent normal tissues should be employed to confirm its efficacy for early detection of ls. 5. conclusions in summary, multiplex pcr followed by ngs is helpful for screening individuals with ls by detecting germline mutations in the mmr genes. including braf gene mutation screening in our panel will assist in differentiating sporadic crc from ls. we achieved 87% specificity, 97% accuracy, and 100% sensitivity for detecting variants at more than 13% allele frequency. overall, this ampliseq-based panel was specific and sensitive enough for mutation analysis of mmr genes and can be incorporated into daily clinical practice. however, further investigation is warranted on detecting indels in mmr genes and pms2 (due to highly homology to pms2l). author contributions: rimy, nsam, and jkss conceived and designed the experiments; nsam and jkss designed the gene panel; rimy performed the next generation sequencing and validation using massarray; ss and mrar performed sanger sequencing; rimy and kjs analysed the data; is and lm provide the tissue samples, imr is the pathologist who assessed the tumour percentage of the samples, rimy prepared the manuscript, figures, and tables, nsam and rj provide critical insights of the scientific contents. all authors read and approved the final manuscript. funding: this work was funded by universiti kebangsaan malaysia dana pembangunan penyelidikan grant 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1316. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology review article 1 the rising of “modern actinobacteria” era jodi woan-fei law1, vengadesh letchumanan1, loh teng-hern tan1, hooi-leng ser1, bey-hing goh2, learn-han lee1* 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. 2biofunctional molecule exploratory research group (bmex), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. abstract: the term “modern actinobacteria” (mod-actino) was coined by a malaysian scientist dr. lee learn-han, who has great expertise and experience in the field of actinobacteria research. mod-actino is defined as a group of actinobacteria capable of producing compounds that can be explored for modern applications such as development of new drugs and cosmeceutics. mod-actino members consist of already identified or novel actinobacteria isolated from special environments: mangrove, desert, lake, hot spring, cave, mountain, arctic and antarctic regions. these actinobacteria are valuable sources for various industries which can contribute directly/indirectly towards the improvement in many aspects of our lives. keywords: modern; bioactive; actinobacteria; environment; bioprospecting received: 16th february 2020 accepted: 23rd march 2020 published online: 29th march 2020 citation: law jw-f, letchumanan v, tan lt-h et al. the rising of “modern actinobacteria” era. prog microbe mol biol 2020; 3(1): a0000064. https://doi.org/10.3687/pmmb.a0000064 introduction the actinobacteria has a long evolutionary history for it has existed on earth around 2.7 billion years ago, anteceding the great oxidation event that occurred 2.3 billion years ago[1, 2]. in the bacteria kingdom, ancient actinobacteria is one of the major phyla associated with the early colonization of land and they play important roles in assisting earth’s ecosystems function[2]. as one of the most primitive lineages among prokaryotes, actinobacteria have extraordinary diversity of morphology and function[3,4]. this phylum consists of free-living gram-positive bacteria with a variety of morphological features including coccus, rod, and complex fragmenting hyphal that develops into branched mycelium[3,5]. these bacteria can be found predominantly in terrestrial soil and marine ecosystems[6]. actinobacteria have significant functions, for instances, they are important agents of global carbon and nitrogen cycles; agents of bioremediation; probiotics in humans and animals; pathogens of humans, animals and plants; producers of enzymes and clinically important metabolites[1,3,7]. following the pioneering research led by professor waksman, the ’52 nobel laureate who revealed streptomycin antibiotic from streptomyces griseus, actinobacteria have since become the “star” in the scientific community[8,9]. essentially, the investigation of novel actinobacteria (genera or species) and bioprospecting of active isolates have intensified around the world, often through random large-scale sampling of environment, selective isolation and subsequently bioactivity screening of isolates[6]. this resulted in the discovery and screening of over thousands of species of actinobacteria. historically, the actinobacteria were documented as a controversial kind of microorganisms due to their diverse and unique appearances, for which, several of them resemble the appearance of fungi[10]. the taxonomy of phylum actinobacteria has been revised over time and the recent roadmap has been proposed with 6 major classes in the phylum, namely: actinobacteria, acidimicrobiia, coriobacteriia, nitriliruptoria, rubrobacteria, and thermoleophilia. class actinobacteria is the largest among others as it consists of 15 orders: actinomycetales, actinopolysporales, bifidobacteriales, catenulispocopyright 2020 by law jw-f and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: learn-han lee, novel bacteria and drug discovery research group (nbdd), microbiome and biore-source research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. lee. learn.han@monash.edu 2 rales, corynebacteriales, glycomycetales, jiangellales, kineosporiales, micrococcales, micromonosporales, propionibacteriales, pseudonocardiales, streptomycetales, streptosporangiales, and frankiales[11,12]. the genus streptomyces (order: streptomycetales, family: streptomycetaceae) is the most famous actinobacteria as they have been greatly studied due to their tremendous bioactive potentials[7]. the era of modern actinobacteria (modactino) actinobacteria have been distinguished for their prolific production of antibiotics. from the 1950s to 1970s, approximately 60% of new antibiotics were predominantly isolated from streptomycetes[13]. eventually, researchers have further exposed the presence of actinobacteria in special and extreme environments with the increasing efforts to discover new metabolites from various microbial sources. this essentially leads to a significant paradigm shift in the exploration of actinobacteria, such instances include the isolation of actinobacteria from underexplored unique habitats and the investigation of their secondary metabolites with different activities other than antimicrobials (e.g. antioxidant, anticancer)[14]. furthermore, the non-streptomyces genera (e.g. sinomonas, microbacterium, nocardia) which referred as the “rare actinobacteria” have shown growing importance as valuable sources in discovery of novel bioactive secondary metabolites[15]. malaysia research star award winner, dr. lee learn han — who has great expertise and experience in the field of actinobacteria research, coined the term “modern actinobacteria” (mod-actino) to define actinobacteria with modern applications (figure 1). in this context, the term refers to actinobacteria that synthesize natural products with new interesting bioactivities in recent years, for examples, drug leads with anti-viral (hiv), anti-protozoa (malaria), antioxidant, and neuroprotection properties as well as compounds utilized for cosmetic formulation. in addition, this term covers actinobacteria which produce approved drugs and have been subjected to drug repurposing effort. mod-actino also inclusive of known or novel actinobacteria that have been discovered from special environments. the rising of... figure 1. the ideas of “modern actinobacteria” (mod-actino) proposed by dr. lee learn han. by the end of 20th century, actinobacterial natural products have been found to exert extensive biological activities comprising antibacterial (against antibiotic resistant strains), antifungal, antiparasitic, immunosuppressant, antioxidant, and anticancer agents[8,16–22], numerous actinobacterial bioactive compounds are well-known for the treatment of plant, animal, and human diseases. for instances, kasugamycin is a marketed antifungal antibiotic produced from streptomyces kasugaensis which used for the control of rice blast caused by phytopathogenic fungus magnaporthe oryzae[23,24]. moreover, several chemotherapeutic drugs such as bleomycin (from streptomyces verticillus) and doxorubicin (from streptomyces peucetius) that have been introduced into clinical use are of actinobacterial origin[25–27]. another remarkable drug discovery event from genus streptomyces is achieved by professor william c. campbell and professor satoshi omura through the isolation of a new “miracle” drug avermectin from streptomyces avermitilis (renamed as streptomyces avermectinius)[28]. avermectin was later being refined into the safest and most potent derivative known as ivermectin. ivermectin is an antiparasitic drug effective against helminths, arachnids and insects. it was marketed in 1981 for veterinary use around the world and subsequently approved for human use in 1987. ivermectin is administered for treatment of onchocerciasis and lymphatic filariasis in many parts of the world. this “miracle” drug has revolutionized the treatment of these devastating parasitic dis3 law jw-f et al. eases, thereby improving the health of millions of individuals. resultantly, the 2015 nobel prize in physiology or medicine was awarded (with one half jointly) to professor william c. campbell and professor satoshi omura[28,29]. research on actinobacteria is still ongoing as they never cease to amaze us with their vast potential of bioactive secondary metabolite production. studies conducted nowadays, towards the 21st century, have gradually revealed the immense ability of actinobacteria in producing compounds with new captivating bioactivities far more than expected. this is witnessed through findings of compounds with in vitro anti-human immunodeficiency virus (hiv) activity produced by actinobacteria[30–32]. one of the earliest research studies on this was reported by chokekijchai et al. (1995)[33], for which a new antihiv polypeptide was obtained from a streptomyces sp. isolated from soil sample collected in japan. besides, a recent study conducted by ding et al. (2010)[34] had successfully isolated a novel pentacyclic indolosesquiterpene — xiamycin produced by mangrove-derived streptomyces sp. gt2002/1503 which is active against hiv. apart from anti-hiv activity, a number of actinobacteria were documented to produce compounds (e.g. borrelidin, metacycloprodigiosin, bafilomycin a1) with promising activity against human malaria parasite (plasmodium falciparum) [35–37]. furthermore, studies also reported the production of neuroprotective substances by actinobacteria that may be potential medicines for brain ischemia and other neurodegenerative diseases such as multiple sclerosis, parkinson’s diseases, and alzheimer’s disease[38,39]. as an example, hayakawa et al. (2013)[40] revealed a new neuroprotective compound isolated from streptomyces sp. rai20 indanostatin, which is also the first reported 1,3-indanone from bacteria. the compound was found to partially protect c6 glioma cells (derived from rat neural tumors induced by n-nitrosomethylurea) against glutamate toxicity which could be useful as treatment for cerebral ischemic disorders. likewise, the possibility of incorporating actinobacterial bioactive metabolites in modern skin care cosmetics has further uplift the value of mod-actino. the human skin is the largest organ of our integumentary system which could face esthetic issues such as freckles, acne, and aging. dahal et al. (2016)[41] proposed the addition of actinobacterial derived resources into cosmetics products for beneficial effects which could enhance the appearance of human skin such as anti-acne, anti-aging, skin whitening, and antioxidant effects. in the study, 12 strains of actinobacteria belonging to the genera streptomyces, actinokineospora, and calidifontibacter exhibited antibacterial activity against skin pathogens staphylococcus epidermidis and propionibacterium acnes. the crude supernatant of these actinobacteria also demonstrated promising tyrosinase inhibition, elastase inhibition, and antioxidant activities. another research conducted by tan et al. (2019)[42] had reported the isolation of a mangrove streptomyces sp. mum273b which possessed antioxidant and uvb protective properties. hence, actinobacterial derived resources can be added to cosmetics applications to improve skin conditions by providing skin whitening effects, acne vulgaris treatment, anti-aging effects, antioxidant effects, and anti-uv properties. interestingly, there is an increasing number of studies that support the concept of using actinobacteria as probiotics in animal feed especially for aquaculture[43]. probiotics in aquaculture are expected to confer health benefits to the host such as growth enhancement, improvement in nutrient digestion and immune response, also, to assist in prevention of bacterial infection through production of inhibitory compounds[43,44]. a few number of studies have suggested the utilization of actinobacteria as potential probiotic strains against shrimp and fish pathogenic vibrio spp.[45–49]. meanwhile, the members of streptomyces and bacillus are also compelling probiotic strains as they have been shown to be capable of promoting growth and increasing resistance against bacterial infections in fishes and shrimps[50–52]. most studies recommended the genus streptomyces as the most potent actinobacteria probiotic for aquaculture mainly due to their ability to produce a multitude of extracellular enzymes and antibiotics, and to form heatand desiccation-resistant spores[44,50]. therefore, these mod-actino will be a great asset to the biopharmaceutical, agriculture, aquaculture, and cosmetic industries. aside from the exploration of actinobacteria-derived compounds for development of novel drugs, research also emphasizes on the investigation of drug repurposing. drug repurposing (drug repositioning/reprofiling/retasking) is defined as an approach to search for new applications of approved or investigational drugs that are beyond the scope of the original medical indication[53]. previously approved actinobacteria-derived drugs such as rapamycin (sirolimus; produced by streptomyces hygroscopicus) was initially known as an antifungal agent[54]. rapamycin was approved as an immunosuppressant for the prevention of allograft rejection in 1999 due to its strong suppression of interleukin-2 (il-2)-stimulated t cell proloferation[55]. it is a macrolide and an allosteric inhibitor of mammalian target of rapamycin (mtor)[55,56]. the mtor is a serine/threonine protein kinase and it is often upregulated in different types of cancers. as a result, researchers are determined to examine its anticancer potentials. rapamycin has been verified to be a potent immunosuppressant and a promising anticancer/antitumor agent that can be used as a single agent or in drug combination[57–59]. thus, this demonstrated one of the criteria of mod-actino where the actinobacterial compounds exhibited different bioactivities from their originally identified bioactivity. presence of mod-actino in special environments actinobacteria are sporulating organisms that possessed astonishing capability to generate extraordinary properties[60–62]. this is often associated with their complex morphological changes in their multicellular life cycle and their large genome size as observed particularly in streptomycetes[3,11,63]. the complexity of these organisms has enabled them to thrive in extreme and special environments[15] such as the arctic and antarctic regions[64,65], mountain plantations[66], glaciers[67], caves[68], deserts[69], hot springs[70], and mangroves[71–75]. these environments are special in terms of physical parameters (e.g. unusually high/low temperature, 4 radiation, pressure) or chemical conditions (e.g. acidic/ alkaline ph, high salinity, low levels of nutrients and moisture)[76,77]. the actinobacteria evolved by developing unique defense mechanism that enables them to survive under hostile and extreme conditions. consequently, actinobacteria from special and extreme environments may be thermotolerant, acidtolerant, alkalitolerant, psychrotolerant, halotolerant, haloalkalitolerant or xerophilous[76]. in addition, several novel genera/species have been discovered from these special environments. for instances, mumia flava gen. nov., sp. nov. (family nocardioidaceae)[78], barrientosiimonas humi gen. nov., sp. nov. (family dermacoccaceae)[79], and monashia flava gen. nov., sp. nov. (family intrasporangiaceae)[80] were each novel species of a new genus isolated from mangroves in malaysia; actinocrinis puniceicyclus gen. nov., sp. nov. (family actinospicaceae) [81] isolated from acidic spring; and desertiactinospora gelatinilytica gen. nov., sp. nov. (family streptosporangiaceae) isolated from desert[82]. besides, other novel species of rare actinobacteria were also identified such as microbacterium mangrovi sp. nov.[83] and sinomonas humi sp. nov.[84] from mangroves; rhodococcus kroppenstedtii sp. nov.[85] and micromonospora acroterricola sp. nov.[86] from desert; and nonomuraea monospora sp. nov.[87] from cave soil. in fact, recent studies also uncovered many novel bioactive actinobacteria which originated from these unique niches. there are multiple novel streptomyces strains recovered from mangrove environments with useful bioactivities, for examples, streptomyces colonosanans sp. nov. (antioxidant and anticancer)[88], streptomyces monashensis (antioxidant and anticancer)[27,89], streptomyces mangrovisoli sp. nov. (antioxidant)[90], streptomyces pluripotens sp. nov. (antibacterial)[91], and streptomyces malaysiense sp. nov. (antioxidant and anticancer)[92]. many compounds produced by mod-actino exhibit important properties which can be developed into new drugs/drug leads with higher efficacy in the near future. harnessing the potentials of mod-actino and conclusions with the growing importance of actinobacteria in various fields, the advancement in molecular biology especially in this post-genomic era can assist us to reach a higher level of understanding of these organisms by studying their genome. the availability of next generation sequencing (ngs) technologies and the -omics methods (metagenomics, metaproteomics) have greatly assisted in overcoming the issue on detection of unculturable bacteria as well as contributed to the research on actinobacteria biosynthetic gene clusters and their secondary metabolites production[93]. lately, there is an increase in the number of new genome sequences of actinobacteria which have been made available to the public. majority of them were resulted from projects aimed to understand the connection of secondary metabolites productions or to evaluate new actinobacterial natural products to their biosynthetic pathways via genome mining[94]. in particular, the bioactive actinobacteria strains have been subjected to whole genome sequencing to further appreciate their biological importance in bioactive metabolites or enzyme production[95–104]. it is anticipated that the accessibility to large sets of actinobacterial genome sequences will provide us a more thorough understanding of actinobacteria phylogeny and facilitate in the identification of medically useful new natural products[105]. members of mod-actino are valuable sources for various industries which can contribute directly/indirectly towards the improvement in many aspects of our lives. mod-actino will be the “key” microorganisms to further improve human health and wellbeing in the modern society. authors contributions the research and manuscript writing were performed by jw-fl, vl and l-hl. lt-ht, h-ls and b-hg provided vital guidance of the research and proof of the writing. the research project was founded by jw-fl and l-hl. conflict of interest the authors declare that there is no conflict of interest in this work. reference 1. lewin, gr, carlos, c, chevrette, mg, et al. evolution and ecology of actinobacteria and their bioenergy applications. annu rev microbiol 2016; 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71(3): 495–548. the rising of... progress in microbes and molecular biology artikel review 1 mikrorna – molekul versatil dalam kanser tiroid azliana mohamad yusof1, nurul syakima ab mutalib2* 1cytogenetics and molecular pathology diagnostics laboratory, pantai premier pathology sdn bhd, kuala lumpur 2ukm medical molecular biology institute, ukm medical center, universiti kebangsaan malaysia, cheras, kuala lumpur abstrak : mikrorna adalah molekul bebenang tunggal rna kecil, dengan kepanjangan di antara 18 hingga 25 nukleotida. molekul pengawalatur ini mampu menyasarkan lebih dari satu gen, dan dianggarkan mengawal 30% daripada keseluruhan gen manusia. mikrorna tidak diendahkan oleh saintis beberapa dekad sebelum ini kerana ia tidak mengekod sebarang protein lantas dianggap jujukan sampah (junk sequence), namun sejak penemuan lin-4 yang mengawalselia perkembangan larva caenorhabditis elegans, semakin banyak kajian bertumpu ke arah molekul ini. sebagai molekul pengawalatur (regulatory molecule), mikrorna adalah calon kajian yang sesuai untuk penyelidikan kanser tiroid di mana landskap mutasi genomnya secara relatif lebih senyap berbanding kanser lain. hanya sebilangan kecil gen bermutasi dikenalpasti dalam kanser tiroid dan mekanisme molekular patogenesis kanser ini masih kurang jelas. artikel ini bertujuan untuk mendedahkan pembaca terhadap mikrorna dan faedahnya dalam kajian kanser tiroid, serta perkembangan terkini kajian tentang mikrorna dalam kanser ini. penekanan terhadap peranan mikrorna dalam kanser tiroid, dengan memfokus kepada versatiliti molekul ini dalam aplikasi diagnosis, prognosis, serta sebagai biopenanda dalam mengenalpasti pesakit yang tidak avid terhadap radioiodin akan turut dibincangkan. kata kunci: mikrorna; kanser; tiroid; biopenanda diserahkan: 18th may 2019 diterima: 15th june 2019 diterbitkan: 05th july 2019 *pengarang untuk surat-menyurat: nurul syakima ab mutalib, ukm medical molecular biology institute, ukm medical center, universiti kebangsaan malaysia, cheras, kuala lumpur; nurulsyakima@gmail.com citation: mohamad yusof et al, ab mutalib ns. mikrorna – molekul versatil dalam kanser tiroid. prog microbes mol biol 2019; 2(s1): a0000025. mikrorna (mirna) mirna merupakan molekul bebenang tunggal rna kecil bersaiz di antara 18 hingga 25 nukleotida. ia memainkan peranan penting dalam mengawal tahap ekspresi gen dan protein berdasarkan jujukan komplimentari separa atau penuh dengan molekul sasaran rna pengutus (messenger rna; mrna) (li et al., 2010). mirna memodulasi ekspresi gen dengan menindas kestabilan mrna dan/ atau merencat translasi kepada protein (landgraf et al., 2007). beberapa mirna dikategorikan sebagai onkogen atau penindas tumor (esquela-kerscher dan slack, 2006). setiap mirna mempunyai potensi untuk meregulasi lebih daripada satu mrna sasaran dan dianggarkan 30% daripada gen dalam badan manusia dikawal oleh mirna (esquela-kerscher dan slack, 2006). penemuan mikrorna berdekad-dekad yang lalu kewujudan mirna tidak diendahkan dan kepentingannya diabaikan kerana ketika itu saintis hanya memfokus pada gen yang mengekod protein. namun apabila mirna pertama iaitu lin-4 ditemui oleh lee dan rakan-rakan dalam caenorhabditis elegans (c. elegans) (lee et al., 1993), pengetahuan mengenai mirna telah berkembang secara pesat. pada tahun tersebut, victor ambros, rosalind lee dan rhonda feinbaum mendapati bahawa lin-4, iaitu gen yang mengawal masa perkembangan larva c. elegans tidak mengekod sebarang protein tetapi sebaliknya menghasilkan sepasang rna kecil dengan saiz kira-kira 22 dan 61 nukleotida, di mana rna kecil yang lebih panjang itu diramalkan untuk membentuk struktur gelung stem (stem loop) yang merupakan pelopor kepada rna kecil yang bersaiz 22 nukleotida (lee et al., 1993). makmal ambros dan ruvkun menyedari bahawa lin-4 rna ini mempunyai jujukan yang komplemen dengan beberapa lokasi dalam kawasan tidak diterjemah pada 3’ (3’ untranslated region; 3’ utr) gen lin-4 yang dihipotesiskan sebagai pengantara penindasan protein lin-14 (lee et al., 1993; wightman et al., 1993, 1991). wightman dan rakan-rakan kemudiannya membuktikan kepentingan lin-4 dalam regulasi lin-14, di mana lin-4 menyebabkan penurunan ekspresi protein lin-14 tanpa perubahan mrnanya yang nyata (wightman et al., 1993). rna lin-4 yang lebih pendek (22 nukleotida) itu kemudiannya dikenali sebagai pengasas sekumpulan rna kecil yang terlibat dalam pengawalaturan gen dan dinamakan sebagai mirna. tujuh tahun selepas penemuan lin-4, mirna copyright 2019 by mohamad yusof et al. and hh publisher. this work under licensed under the creative commons attributionnoncommercial 4.0 international lisence (cc-by-nc 4.0) 2 kedua iaitu let-7 telah dijumpai oleh reinhart et al. pada tahun 2000 (reinhart et al., 2000). let-7 merupakan mirna yang mengawal transisi l4 kepada tahap dewasa dalam perkembangan larva (reinhart et al., 2000). dalam badan manusia, let-7 telah ditemui pada peringkat ekspresi yang berbeza dalam kebanyakan tisu termasuklah jantung, otak, paru-paru, buah pinggang, kolon, usus kecil dan timus (pasquinelli et al., 2000). penemuan let-7 dalam pelbagai spesis mencetuskan revolusi dalam kajian mirna. sehingga kini, beribu-ribu mirna telah diklasifikasikan dalam pelbagai jenis spesis termasuklah manusia. repositori jujukan mirna secara atas talian seperti pangkalan data mirbase telah diwujudkan dan sebanyak 24,521 lokus mirna daripada 206 spesis di mana ia menghasilkan 38,589 mirna matang telah direkodkan (kozomara et al., 2019; kozomara dan griffiths-jones, 2011). sehingga kini, sebanyak 1,917 prekursor dan 2,654 mirna matang telah diperihalkan dalam genom manusia (kozomara et al., 2019). biogenesis dan mekanisme mikrorna hampir kesemua gen mirna ditranskripsi oleh polimerase rna ii (pol ii) dalam nukleus dengan mirna primer (pri-mirna) bertutup (capped), disambat (splice) dan dipoliadenilat (polyadenylated) (lee et al., 2004). kira-kira 30% mirna diproses daripada intron gen mengekod protein, manakala mirna yang lain diekspres daripada lokus gen mirna (lin dan gregory, 2015). primirna boleh menghasilkan sama ada mirna tunggal atau mengandungi dua atau lebih mirna daripada transkrip primer yang sama. pri-mirna ini dibelah oleh mikropemproses yang terdiri daripada drosha dan dgcr8 (digeorge syndrome critical region 8) (gregory et al., 2004). selepas itu, prekursor mirna (pre-mirna) dieksport keluar daripada nukleus ke dalam sitoplasma melalui interaksi exportin-5 dan ran-gtp. dalam sitoplasma, pre-mirna diproses oleh dicer1, enzim rnase iii yang memotong hujung 5’ dan 3’ pre-mirna (park et al., 2011). dicer1 bersamasama transactivation-responsive rna-binding protein (trbp) melekat pada rna bebenang ganda dua (bernstein et al., 2001). trbp menghubungkan dicer1 dengan protein argonaute (ago1, ago2, ago3 atau ago4) untuk terlibat dalam himpunan kompleks penyenyapan mirna-teraruh (mirna-induced silencing complex; mirisc) (chendrimada et al., 2005). satu bebenang mirna matang (bebenang panduan) terikat pada protein argonaute dan dikekalkan dalam mirisc untuk memandu komplek dan famili protein gw182 kepada sasaran komplemen mrna bagi penyenyapan gen pasca-transkripsi (lin dan gregory, 2015). mirna matang mengesan jujukan komplemen pada kawasan 3’ utr mrna sasaran melalui jujukan benih (seed sequence) pada posisi 2 hingga 7 dalam jujukan mirna. perbandingan antara mirna dengan rna bukan pengekod panjang (long noncoding rna; lncrna) rna bukan pengekod (noncoding rna; ncrna) merupakan molekul rna fungsian yang tidak ditranslasikan kepada protein. ia dibahagikan mengikut saiz (ncrna pendek dan ncrna panjang), fungsi (rna penyelenggara dan rna pengawalaturan) dan arah transkripsi (sense/ antisense, dwiarah, intronik dan intergenik), mempunyai julat molekul yang luas dengan pelbagai fungsi dan sifat seperti mrna, trna, mirna dan lncrna (kunej et al., 2014). ncrna pendek mempunyai saiz kurang daripada 200 nukleotida manakala lncrna mempunyai saiz lebih daripada 200 nukleotida. semenjak beberapa tahun yang lalu, banyak kajian lebih menumpukan kepada ncrna pendek seperti mirna, pirna dan sirna (kunej et al., 2014). ncrna yang paling banyak dikaji adalah mirna. berbanding pengetahuan tentang mirna yang lebih meluas, pemahaman mengenai lncrna adalah masih terhad namun ianya semakin berkembang. pelbagai mekanisma pengawalaturan transkripsi ekspresi gen oleh lncrna telah dilaporkan. melalui kajian-kajian ini, lncrna dilihat membantu memodifikasi epigenetik dna dengan merekrut komplek ubah suai kromatin kepada lokus yang spesifik (gupta et al., 2010). lncrna boleh diklasifikasikan kepada beberapa kelas iaitu ncrna intergenik, ncrna intronik, lncrna utr, transkrip antisense, transkrip pseudogen, ncrna seperti-penggalak (enhancer-like ncrna), ncrna mitokondria, repeatassociated ncrna dan ncrna satelit (gullerova, 2015). kebanyakan fungsi lncrna berkaitan dengan kapasiti mereka untuk mengikat pada rna, dna dan protein (varilh et al., 2015). famili lncrna yang pertama ditemui adalah xist (bersaiz ~17 kilobes) (clemson et al., 1996). lncrna berpotensi digunakan sebagai indikator aktiviti transkripsi lokus atau gen (derrien et al., 2011). sama seperti mirna, lncrna juga memainkan peranan sebagai pengaktif atau penindas ekspresi protein (varilh et al., 2015). mikrorna dan penyakit manusia kajian terdahulu melaporkan bahawa mirna adalah penting dalam pelbagai proses biologi dan deregulasi mirna boleh menyebabkan pelbagai jenis penyakit dalam manusia (li dan kowdley, 2012). oleh itu, pemahaman tentang bagaimana mirna mengawalatur proses sel normal dan sesuatu penyakit adalah penting (giza et al., 2014). mutasi, deregulasi atau ketidakfungsian biogenesis mirna dan sasarannya mendorong kepada kerintangan tapak jalan biokimia dan fisiologi yang terlibat dalam perkembangan dan evolusi penyakit dalam manusia. dengan menggunakan ramalan in silico, beberapa hubungan antara mirna dengan penyakit telah dikenalpasti (giza et al., 2014). perbezaan ekspresi mirna di antara tisu normal dan tumor telah dikaji dalam pelbagai jenis kanser termasuklah kanser payudara, paru-paru, tiroid papilari, pankreas, serviks, kolon, glioblastoma dan limfoma (giza et al., 2014). tambahan pula, perubahan ekspresi mirna juga dilaporkan dalam penyakit bukan kanser seperti skizofrenia, penyakit neurodegeneratif seperti alzheimer dan parkinson, penyakit berkaitan dengan imun serta penyakit jantung (li dan kowdley, 2012). deregulasi mirna dalam kanser telah dilaporkan buat kali pertama pada 2002, apabila dua kluster mirna iaitu mir-15 dan mir-16 telah dikenalpasti pada 13q14.3, kawasan delesi yang lazimnya berlaku dalam leukemia limfosit kronik (chronic lymphocytic leukemia; cll) (calin et al., 2002). sejak itu, kajian mengenai permikrorna – molekul versatil... 3 anan mirna dalam kemampuan untuk menyerang tisu persekitaran dan metastasis banyak dilaporkan (hanahan dan weinberg, 2011). antara mirna yang sering dilaporkan dalam pelbagai jenis kanser adalah mir-17-92, mir21, mir-221/222, let-7, mir-15/16, mir-200 dan mir-34 (hayes et al., 2014). terdapat penemuan awal yang mengatakan bahawa beberapa virus mampu mengawalatur sel perumah mirna bagi mengawal kemandirian mereka dan virus juga boleh mengekod mirna mereka sendiri, seperti virus polioma, adenovirus dan virus herpes (gregory dan damania, 2009). pada masa manuskrip ini ditulis, sebanyak ~ 86,162 penerbitan saintifik mengenai mirna telah boleh dicapai melalui rangkaian pubmed. mikrorna dalam kanser tiroid inisiasi dan progresi kanser tiroid berlaku melalui akumulasi berturutan pelbagai perubahan genetik dan epigenetik termasuklah pengaktifan dan penyahaktifan mutasi somatik, deregulasi mrna dan/atau mirna serta metilasi gen (nikiforov dan nikiforova, 2011). berbanding kanser lain, kanser tiroid menunjukkan predisposisi genetik yang kuat seperti yang dibuktikan dalam kajian populasi kes-kawalan, di mana risiko untuk mendapat kanser ini meningkat sekurang-kurangnya lapan kali ganda di kalangan ahli keluarga yang rapat berbanding populasi umum (risch, 2001). namun, tiada predisposisi gen pengekod-protein dilaporkan (swierniak et al., 2013). maka, adalah dihipotesiskan bahawa mekanisme karsinogenesis tiroid mungkin melibatkan interaksi beberapa gen penetrasi rendah (low penetrance gene) dan gen pengawalatur seperti mirna (swierniak et al., 2013). kajian mengenai penentuan dan perbandingan profil ekspresi mirna dalam kesemua jenis tumor tiroid, korelasi pola ekspresi mirna dengan mutasi onkogen yang spesifik dan kelebihan diagnosis pengesanan spesifik mirna dalam penilaian prabedah nodul tiroid telah dilakukan (nikiforova et al., 2008). sebanyak tujuh mirna (mir-146b, mir-155, mir-187, mir-197, mir-221, mir-222 dan mir-224) mempunyai peningkatan ekspresi yang signifikan dalam tumor tiroid. tambahan pula, jenis histopatologi tumor tiroid yang berbeza mempunyai profil mirna yang berlainan, dimana ia mencerminkan mutasi bersifat onkogen yang spesifik (nikiforova et al., 2008). melalui kajian ini, set mirna yang terhad mempunyai aplikasi diagnostik dengan kadar ketepatan yang tinggi bagi mengesan kanser tiroid dalam sampel pembedahan dan aspirasi jarum halus (fine-needles aspiration; fna) prabedah (nikiforova et al., 2008). meskipun pendekatan kumpulan kawalan normal dan pelantar yang digunakan adalah berbeza, namun terdapat beberapa mirna dengan corak ekspresi yang konsisten sekurang-kurangnya daripada tiga kajian berbeza. antara mirna yang sering dilaporkan dalam kajian kanser tiroid papilari ialah mir-221, mir-222 dan mir146b. mir-221 yang berekspresi tinggi sering dikaitkan dengan ciri-ciri klinikopatologi agresif dan mutasi braf dalam kanser tiroid papilari (zhou et al., 2012). melalui kajian oleh diao et al. (2016), mir-221 menindas ekspresi timp3 dengan mengikat pada 3’utr timp3. sebaliknya, ekspresi timp3 adalah tinggi dengan kehadiran perencat mir-221 (diao et al., 2017). mir-146a dan mir-146b tergolong dalam famili mir-146. banyak kajian melaporkan bahawa mir-146 diekspres dengan tinggi dalam kanser tiroid papilari berbanding tisu tiroid normal (cancer genome atlas research network, 2014; shi et al., 2018). melalui asai fungsian menggunakan sel selanjar kanser tiroid manusia bcpap, mir-146b menyebabkan migrasi dan proliferasi sel kanser tiroid papilari dengan merendahkan regulasi ekspresi irak1. tambahan pula, ekspresi mrna irak1 adalah rendah secara signifikan dalam sampel tisu kanser tiroid papilari berbanding spesimen normal bersebelahan dan hal ini menunjukkan bahawa irak1 mempunyai korelasi songsang yang kuat dengan mir-146b dalam kanser tiroid papilari (chou et al., 2016). mikrorna sebagai onkogen dan penindas tumor perubahan ekspresi mirna dalam kanser dapat menyokong hipotesis di mana mirna memainkan peranan yang penting dalam sesuatu jenis kanser. mirna mempunyai profil ekspresi yang berlainan dalam kanser berbanding dengan tisu normal dan profil ini berbeza di antara pelbagai jenis kanser (lu et al., 2005) mirna mempunyai dua peranan dalam kanser bergantung kepada status ekspresinya. mirna yang diekspresi dengan tinggi dalam tumor dikenali sebagai ‘onkomir’ yang lazimnya merangsang perkembangan kanser dengan merencat gen penindas tumor dan/atau gen yang mengawal proses pembezaan sel atau apoptosis (esquela-kerscher dan slack, 2006). selain itu, terdapat sesetengah mirna diekspresi dengan rendah dalam sel kanser. mirna ini dikenali sebagai mirna penindas tumor dan berfungsi menghalang perkembangan kanser dengan merencat onkogen dan/atau gen yang mengawal proses pembezaan sel atau apoptosis (esquela-kerscher dan slack, 2006). mir-21 yang terletak pada kromosom 17 merupakan antara mirna yang lazim dilaporkan sebagai onkomir dalam pelbagai jenis kanser. ekspresi mir-21 yang tinggi lazimnya dikaitkan dengan prognosis lemah di kalangan pesakit kanser. mir-21 diekspresi dengan rendah secara signifikan dalam pesakit kanser tiroid papilari berulang berbanding kanser tiroid papilari tanpa tumor berulang (sondermann et al., 2015). ekspresi menurun mir-21 dikaitkan dengan kanser prostat (ren et al., 2014) tetapi mir-21 diekspresi dengan tinggi dalam kolon (oue et al., 2014), paru-paru (m. yang et al., 2013) dan payudara (wang et al., 2015). di antara sasaran mir-21 dalam kanser payudara adalah icam-1 (terao et al., 2011). dengan menggunakan kaedah transfeksi dan pengklonan, mir-21 merencat ekspresi icam-1, menunjukkan bahawa ia merupakan sasaran terus (direct target). perencatan icam-1 secara aruhan-atra yang bergantung kepada mir-21 ini adalah konsisten dengan aktiviti pro-motiliti protein dalam sel kanser payudara (strell et al., 2010). tambahan pula, beberapa sasaran mir-21 dapat dilihat dalam pelbagai jenis kanser seperti pten dalam kanser kolorektal (xiong et al., 2013) dan paru-paru (zhang et al., 2010) serta pdcd4 dalam kanser peparu (yang et al., 2015) dan renal (yuan et al., 2017) deregulasi let-7 telah banyak dilaporkan dalam mengawalatur proliferasi dan pembezaan sel semasa mohamad yusof et al. 4 perkembangan pelbagai spesis (boyerinas et al., 2010). konsisten dengan penemuan ini, ekspresi let-7 adalah rendah dalam banyak jenis kanser apabila dibandingkan dengan tisu normal dan semasa perkembangan tumor. ekspresi rendah let-7 dilaporkan berkaitrapat dengan jangka hayat yang pendek di kalangan pesakit kanser paru-paru (takamizawa et al., 2004). beberapa kajian juga melaporkan bahawa aktiviti penindas tumor let-7 melingkungi pelbagai jenis kanser termasuklah gastrik (ishiguro et al., 2014), kolon (mizuno et al., 2018) dan ovari (biamonte et al., 2019). mikrorna dalam apoptosis apoptosis merupakan proses biologi kematian sel yang aktif dan kompleks di mana ia terlibat dalam penyingkiran sel yang tidak dikehendaki semasa proses perkembangan, infeksi dan homeostasis tisu (adlakha dan saini, 2016). ia terjadi melalui dua tapak jalan pengisyaratan iaitu tapak jalan bersifat apoptosis intrinsik dan ekstrinsik di mana ia dicetuskan oleh molekul boleh larut yang mengikat kepada reseptor membran plasma atau pelbagai rangsangan mitokondrion (mukhopadhyay et al., 2014). antara mirna yang terlibat dalam apoptosis adalah mir-181b. dengan menggunakan sel selanjar kanser tiroid papilari tpc1, mir-181b diekspresi dengan rendah hingga menyebabkan perencatan tumbesaran sel dan menggalakkan apoptosis dengan mensasarkan cyld (li et al., 2014). gen kanser terbahagi kepada dua kumpulan kerana sel kanser selalunya dikelaskan melalui kematian sel yang rendah dan proliferasi sel yang meningkat. kumpulan pertama iaitu onkogen meningkatkan proliferasi dan mengurangkan apoptosis. manakala kumpulan kedua iaitu penindas tumor menjalankan fungsi yang berlawanan (peng dan croce, 2016). deregulasi apoptosis merupakan satu langkah signifikan dalam kanser (hanahan dan weinberg, 2011). tidak seperti sel-sel normal, sel-sel kanser adalah di bawah tekanan yang berterusan, seperti tekanan onkogenik, ketidakstabilan genomik dan hipoksia sel (fernald dan kurokawa, 2013). sebagai tindak balas kepada tekanan ini, tapak jalan intrinsik apoptosis biasanya diaktifkan. namun, sel-sel kanser mampu mengelakkan tindak balas selular ini dengan mematikan laluan apoptosis (fernald dan kurokawa, 2013). apoptosis turut dikawalselia oleh mirna dalam perkembangan kanser tiroid seperti yang dibuktikan oleh carvalheira et al. (2015). melalui asai lusiferase yang menggunakan sel selanjar kanser tiroid wro dan tpc1, mir-106b mensasarkan c1orf24 dengan mengikat pada kawasan 3’utr. ini menyumbang kepada pengurangan ekspresi c1orf24 yang seterusnya meningkatkan apoptosis serta merencat migrasi sel dalam kanser tiroid (carvalheira et al., 2015). dalam satu kajian lain, ma et al. melaporkan bahawa peningkatan ekspresi mir-34a menggalakkan tumbesaran sel dan merencat apoptosis dengan mensasarkan gas1 melalui tapak jalan pi3k/akt/bad (ma et al., 2013). mikrorna dalam diagnosis dan prognosis pelbagai kajian dan analisis terhadap tahap ekspresi mirna dalam tumorigenesis melaporkan bahawa mirna memainkan peranan yang besar sebagai penanda prognostik dan/atau diagnostik. ini adalah penting bagi membezakan pelbagai jenis tumor serta meramal perubahan klinikal (kavitha et al., 2014). perkembangan pesat teknik yang canggih seperti mikroatur, penjujukan rna kecil, tindakbalas polimerase berantai kuantitatif (quantitative polymerase chain reaction; qpcr) dan teknologi antisense digunakan untuk memberi impak yang signifikan dalam onkologi klinikal pada masa hadapan (rufino‐ palomares et al., 2013). sejak mirna menjadi faktor utama dalam mengenalpasti identiti sel, mirna juga boleh menjadi molekul yang berguna dalam diagnosis kanser. menurut liu et al. (2014), profil mirna yang terekspres secara unik dan signifikan melalui analisis mikroatur mirna dan pemprofilan mirna berasaskan prob, mampu membezakan tisu kanser daripada tisu bukan kanser (liu et al., 2014). dengan menggunakan teknik qpcr mirna, pelbagai mirna diekpresi dengan tinggi dan rendah dalam kanser tiroid papilari, kanser tiroid folikular, dan kanser tiroid atipikal. mir-125b dilaporkan diekspresi dengan tinggi dalam kanser tiroid papilari apabila dibandingkan dengan tisu normal tetapi ia diekspresi dengan rendah dalam atc (pallante et al., 2006). melalui kajian oleh vriens et al. yang menggunakan pendekatan teknik qpcr, empat mirna (mir100, mir-125b, mir-138 dan mir-768-3p) mampu membezakan sampel tisu benigna dan malignan tiroid. mirna ini menunjukkan potensinya sebagai penanda mirna dalam kanser tiroid (vriens et al., 2012). tambahan pula, profil ekspresi mirna juga mengandungi maklumat yang penting berkaitan dengan prognosis pesakit kanser. sebagai contoh, ekspresi mir-21 dan mir-155 yang tinggi boleh meramal kanser berulang dan kemandirian yang tidak menggalakkan di kalangan pesakit kanser paru-paru bukan sel kecil (yang et al., 2013) dengan mengawalatur socs1, socs6, dan pten (xue et al., 2016). mikrorna dalam metastasis kadar kematian pesakit kanser menjangkau 90% disebabkan oleh metastasis (budczies et al., 2014). metastasis merupakan proses kompleks yang memerlukan sel kanser untuk terpisah daripada tumor primer dan memasuki tisu yang berdekatan melalui membran dasar (basement membrane), memasuki saluran darah (intravasatan), hidup dalam peredaran darah, keluar daripada sistem peredaran darah ke bahagian metastasis (pengektravasatan) dan masuk serta membiak pada persekitaran yang baru seterusnya membentuk tumor metastasis (kolonisasi) (chan dan wang, 2015). peralihan epitelium kepada mesenkima (epithelial-mesenchymal transition; emt) merupakan langkah penting dalam deretan metastasis, dikategorikan dengan kehilangan pelekatan sel melalui penindasan e-cadherin dan pengaktifan gen yang berkait rapat dengan invasi dan motiliti (nisticò et al., 2012). melalui kajian in vitro dan in vivo, mirna dilaporkan mempunyai peranan dalam mengawal proses metastasis. mir-10b merupakan mirna pertama yang dilaporkan mempunyai peranan dalam menggalakkan kanser metastasis (ma et al., 2007). ma dan rakan-rakan menunjukkan bahawa mir-10b menindas sintesis promikrorna – molekul versatil... 5 tein hoxd10, lantas meninggikan ekspresi rhoc dalam kanser payudara serta meningkatkan invasi dan migrasi sel kanser. selain itu, ekspresi mir-141 adalah rendah dalam tisu kanser tiroid dan ia berkait rapat dengan metastasis nodus limfa melalui perencatan substrat reseptor insulin (irs2) (dong et al., 2016). selain itu, mir-146b-3p, mir-146b-5p dan mir-222 dilaporkan sebagai biopenanda yang berpotensi untuk mengenalpasti sentral metastasis nodus limfa dalam kanser tiroid papilari (han et al., 2016). wang et al juga melaporkan bahawa perubahan ekspresi yang tinggi pada mir-2861 dan mir-451 dalam kanser tiroid papilari dengan metastasis nodus limfa merupakan penanda mirna unik yang berkaitrapat dengan prognosis dan perkembangan kanser ini (wang et al., 2013). mikrorna dalam tindakbalas terhadap terapi radioiodin kanser tiroid dirawat dengan beberapa kaedah seperti pembedahan, kemoterapi dan/atau radioterapi. pembedahan diikuti oleh terapi radioiodin (i-131) dianggap rawatan yang optimal untuk tumor berisiko tinggi. namun, progresi kanser tiroid sering juga dikaitkan dengan perbedaan sel kanser dan menyumbang kepada penurunan tindakbalas terhadap terapi radioiodin dalam sebilangan kecil pesakit. pesakit yang tidak avid kepada i-131 (non-rai avid patient) selalunya mempunyai prognosis yang buruk (sciuto et al., 2009; xing et al., 2013). beberapa mirna termasuklah mir-339-5p dan mir-195 mempunyai peranan dalam mengawalatur gen yang spesifik dalam tiroid, terutamanya simporter sodium-iodin (sodium-iodine symporter; nis), iaitu protein utama yang terlibat dalam tindakbalas terhadap rawatan i-131 (lakshmanan et al., 2015). seperti yang dinyatakan sebelum ini, mir-146b adalah antara mirna yang paling kerap dideregulasi dalam kanser tiroid. ekspresi mir-146b tinggi yang selalunya ditemui dalam kanser tiroid juga mungkin turut menyumbang kepada kerintangan terhadap i-131. mir-146b mampu merencat translasi nis mrna dan menyebabkan penurunan paras protein, kemudiannya menyebabkan pengurangan pengambilan i-131 oleh sel kanser melalui nis (riescoeizaguirre et al., 2015). penemuan ini disokong oleh satu kajian fungsian lain di mana perencatan ekspresi mir-146b meningkatkan sensitiviti sel kanser terhadap i-131 (li et al., 2015). kehilangan keupayaan untuk menumpukan i-131 adalah salah satu penyebab utama penyakit radioiodin-refraktori dalam kanser tiroid papilari. untuk mengenal pasti perbezaan ekspresi mirna dalam pesakit kanser tiroid papilari dengan metastasis paru-paru yang i-131 avid dan i-131 tidak avid, qiu dan rakan-rakan mengkaji paras mirna dalam serum pesakit. berbanding kumpulan pesakit yang i-131 avid, mir-106a adalah antara mirna yang mempunyai ekspresi tinggi yang signifikan dalam pesakit kanser tiroid papilari dengan metastasis paru-paru yang i-131 tidak avid. analisis bioinformatik seterusnya menunjukkan bahawa mir-106a adalah mirna teras yang mengawal selia 193 gen dalam rangkaian yang terlibat dalam keavidan terhadap i-131 (qiu et al., 2015). pada tahun seterusnya, kumpulan penyelidik tersebut menyiasat lebih lanjut mekanisme mir-106a dalam tindak balas terapi radioiodin. analisis fungsian mereka menunjukkan bahawa mirna-106a menyasarkan retinoic acid receptor beta (rarb),mengawal ekspresi nis dan reseptor tsh serta merubah fungsi penyerapan i-131 dalam sel kanser tiroid (shen et al., 2016). kajian tersebut membuka peluang kepada aplikasi perencatan mir-106a dalam pesakit kanser tiroid papilari i-131 tidak avid. rumusan punca kenapa kanser tiroid adalah antara kanser yang lazim berlaku di malaysia, terutamanya di golongan wanita adalah masih kurang jelas. kegagalan kajiankajian sebelum ini untuk mengenalpasti gen utama yang menyebabkan kanser tiroid mungkin berpunca dari interaksi di antara dua atau lebih gen yang terlibat. oleh itu, kajian mengenai molekul pengawalatur ekspresi gen seperti mirna, adalah lebih relevan dalam mengkaji dan memahami patogenesis kanser tiroid. tidak dinafikan lagi, mirna adalah molekul yang versatil dalam kanser tiroid. peranan molekul kecil ini adalah sangat meluas, bermula dari diagnosis hinggalah kepada mempengaruhi tindakbalas terhadap rawatan. walaupun kanser tiroid mempunyai prognosis yang baik berbanding kanser lain, masih ada sebilangan pesakit berisiko tinggi yang mempunyai prognosis buruk dan cenderung untuk mendapat kanser berulang. kajian mendalam tentang fungsi dan peranan mirna mampu menyumbang kearah kaedah pengesanan dan rawatan yang lebih baik untuk pesakit kanser tiroid. rujukan adlakha, y.k., saini, n., 2016. 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wang, o.-c., 2012. overexpression of mir-221 is associated with aggressive clinicopathologic characteristics and the braf mutation in papillary thyroid carcinomas. med. oncol. northwood lond. engl. 29, 3360–3366. https://doi.org/10.1007/s12032-012-0315-8 geran penulis merakamkan sekalung penghargaan kepada kementerian pendidikan tinggi atas penganugerahan skim geran penyelidikan fundamental (frgs/1/2014/ skk01/ukm/03/1) untuk penyelidikan tentang mikrorna dalam kanser tiroid papilari. law jwf et al. pmmb 2022, 5, 1; a0000277. doi: a10.36877/pmmb.a0000277 http://journals.hh-publisher.com/index.php/pmmb review article exploring the safety and effects of covid-19 vaccination in patients with autoimmune disease kirttna solai vairavan1, loh teng-hern tan1,2, jodi woan-fei law1, priyia pusparajah1, vengadesh letchumanan1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia. kvai0001@student.monash.edu (ksv) loh.teng.hern@monash.edu (lt-ht); jodi.law1@monash.edu (jw-fl); priyia.pusparajah@monash.edu (pp) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia *corresponding author: vengadesh letchumanan, novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia; vengadesh.letchumanan1@monash.edu (vl) received: 18 september 2022; received in revised form: 3 november 2022; accepted: 10 november 2022; available online: 16 november 2022 abstract: the covid-19 pandemic has quickly become the most significant public health phenomenon, effectively eclipsing the h1n1 and ebola crises that came before it. it can spread rapidly and has caused the death and disability of many worldwide. vaccines are our most effective line of defense against the rapidly spreading and mutating virion. still, there is significant vaccine hesitancy among those with autoimmune conditions who fear the vaccine may cause them more harm than good. this scoping review explores the safety, outcomes, and effects of covid-19 vaccines in autoimmune patients. online databases; pubmed, ovid medline, and scopus were used to search published literature evaluating the effectiveness and side effects of covid-19 vaccines in patients with autoimmune conditions. the search results were limited to 4 distinct autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and myasthenia gravis). thirty-seven studies were retrieved and assessed on the safety, effects, and outcomes of covid-19 vaccination in patients with the chosen autoimmune conditions. overall, the risk of flares and the development of severe side effects after vaccination was low. most autoimmune patients showed a good antibody response to vaccination, especially after the second dose. this review provides a favorable impact of vaccination in patients with autoimmune conditions. keywords: covid-19; vaccines; autoimmune patients; rheumatoid arthritis; systemic lupus erythematosus; psoriasis; myasthenia gravis pmmb 2022, 5, 1; a0000277 2 of 21 1. introduction the covid-19 outbreak, which emerged in china late in 2019, has since quickly spread worldwide to become the most infamous health crisis of our time [1-8]. world health organization (who) declared covid-19 a pandemic caused by severe-acute-respiratorysyndrome-coronavirus-2 (sars-cov-2) [9-11]. covid-19 has caused high morbidity and mortality, socioeconomic burden, and pressure on healthcare systems. all these factors can only be reduced by achieving herd immunity against covid-19 thru vaccination. the virus attacks the host respiratory system and causes flu-like symptoms upon exposure. the disease manifests with a mild respiratory infection, cough, headache, and fatigue at the early stage [12]. still, as the disease progresses, it may lead to acute respiratory failure with severe complications such as multiorgan failure [13-15]. there is a greater risk of infection among the elderly, patients with autoimmune diseases, and immunocompromised patients [15, 16]. given the severity of illness in autoimmune patients, clinicians and healthcare professionals have been advocating the importance of being vaccinated. pharmaceutical companies raced against time to develop covid-19 vaccines. the most common covid-19 vaccine platforms that are currently in use include inactivated virus vaccines (sinovac, covaxin, sinopharm), mrna (pfizer-biontech and moderna), and adenovirus vector (astrazeneca, cansino, sputnik v) [17-19]. these vaccines have produced robust humoral responses and presented good outcomes in most vaccinated populations [20]. vaccinations help to protect the host by creating an antibody response without having the person experience severe illness or post-covid-19 conditions. existing literature suggests that autoimmune patients are at high risk of contracting severe covid-19 and emphasizes the need for vaccination [21, 22]. however, hesitancy has increased in patients with autoimmune diseases since there is limited data on the safety of covid-19 vaccines in autoimmune patients. studies comparing the consequences of different vaccine types between patients and healthy controls are also unavailable [21]. in addition, the disease onset and vaccination can trigger flares. flares are cumbersome to the patient and would thus discourage them from taking the vaccine; the simple possibility is enough to make them wary of the vaccine roll-out. on top of that, many patients with autoimmune conditions are also on immunosuppressants to keep their conditions at bay. this immunosuppressed state may impair their ability to mount an immune response and even increase their risk of vaccine side effects. localized pain on the injection site, fever, body aches, and headaches are among the typical covid-19 vaccine side effects that people have reported worldwide [23]. esquivelvalerio and colleagues studied the impact of six different covid-19 vaccines on patients with autoimmune rheumatic diseases. they found that localized pain, fatigue, headache, and muscle ache are the most prevalent adverse effects in this group of patients [24]. it is also reported hepatitis b, and influenza vaccines trigger the flares of autoimmune diseases by molecular mimicry inducing autoimmunity [25-27]. this review explores the safety, outcomes, and effects of covid-19 vaccines in autoimmune patients. pmmb 2022, 5, 1; a0000277 3 of 21 2. methodology three databases (pubmed, ovid medline, and scopus) were searched using search keywords ‘rheumatoid arthritis, systemic lupus erythematosus, psoriasis, myasthenia gravis, and covid-19 vaccines’ to retrieve studies evaluating the effectiveness and side effects of covid-19 vaccines in patients with autoimmune conditions. this review search was focus to four distinct autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and myasthenia gravis). the included studies were published in english between january 2020 and january 2022. conference proceedings, editorials, dissertations, literature reviews, commentaries, and conference abstracts were excluded. articles were then screened using covidence based on the inclusion and exclusion criteria (table 1). table 1. the inclusion and exclusion criteria. criteria specific criteria inclusion • studies evaluate the effectiveness and side effects of covid-19 vaccines in patients with autoimmune diseases. • population: patients with rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and myasthenia gravis. exclusion • studies related other than covid-19 vaccines and autoimmune diseases. • conference proceedings, commentaries, editorials, dissertations, literature reviews, and conference abstracts. 3. results 3.1. search results three electronic databases were chosen to conduct the search process with search terms. the search retrieved 523 studies from pubmed, ovid medline, and scopus. a total of 204 duplicates were removed from the resulting studies, and 319 articles were kept for the title and abstract screening. based on the inclusion and exclusion criteria, 59 full-text articles met the eligibility criteria, and 260 articles were excluded. in the end, 37 articles were chosen and included in this review. the scoping review process is shown in figure 1. pmmb 2022, 5, 1; a0000277 4 of 21 figure 1. the prisma flowchart 3.2. characteristics of included studies of the 37 articles reviewed, the findings were divided into 15 studies on rheumatoid arthritis, 11 studies about psoriasis, seven on systemic lupus erythematosus (sle), and four on myasthenia gravis. 3.2.1. rheumatoid arthritis of the 15 rheumatoid arthritis studies, ten evaluated the administration of mrna vaccines; two discussed mrna vaccines and viral vector vaccines; one looked at mrna vaccines and inactivated vaccines; the remaining two explored inactivated either alone or with viral vector vaccines (table 2). all the study participants were adults (>18 years old). the number of participants varied from 5493 patients in one of the cohort studies to 1 patient in the case report. spinelli et al. [28] and geisen et al. [29] found that the vaccine side effects in those with rheumatoid arthritis were similar to those without autoimmune conditions. local injection site pain was the most common complaint by the patients. furer and colleagues studied the flare of herpes zoster following bnt162b2 mrna covid-19 vaccination in patients with rheumatic diseases. the study found six patients with aiird (autoimmune inflammatory rheumatic diseases) developed mild herpes zoster after the first dose of the vaccine and were given oral antiviral therapy with a resolution of herpes zoster symptoms. five patients completed the second covid-19 vaccine dose without other pmmb 2022, 5, 1; a0000277 5 of 21 adverse effects. they concluded that further studies are required to assess the safety of the mrna-based covid-19 vaccines in patients with aiird and the reactivation of zoster [30]. table 2. summary of studies related to rheumatoid arthritis (ra). study vaccine given population therapies used response cherian et al. [31] chadox1 ncov-19 (viral vector vaccine) or bbv152 (inactivated vaccine) 724 patients with rheumatoid and musculoskeletal disease (rmd). 523 had autoimmune rheumatic disease (aird) and 201 had non-aird in aird group, steroids (n=97) csdmards (n=468) biodmards (n=27) 436 (60.22%) participants had at least one adverse effect (ae). four patients reported a flare of arthritis that resolved in 5 days. geisen et al. [29] biontech/pfizer or moderna (mrna vaccine) 42 healthy controls and 26 patients with chronic inflammatory disease (cid) in cid group, biodmards (n=20) csdmards (n=8) steroids (n=7) sars-cov-2 antibodies and neutralising activity detected in all participants. no severe adverse effects were observed. ferri et al. [32] bnt162b2 and mrna-1273 (mrna vaccines) 478 unselected asd patients (mean age 59 ± 15 years), namely 101 ra, 38 sle, 265 ssc, and a miscellanea of 74 systemic vasculitis. the control group included 502 healthy individuals. in asd group, glucocorticoids, mycophenolatemofetil, rituximab increased prevalence of non-response to vaccines was observed in patients with asd-related interstitial lung disease and those treated with glucocorticoids, mycophenolatemofetil, or rituximab. furer et al. [30] bnt162b2 (mrna vaccination) 491 patients with rheumatoid disease and 99 healthy controls not available prevalence of herpes zoster was 1.2% in patients with the rheumatic disease compared with none in controls. pmmb 2022, 5, 1; a0000277 6 of 21 watad et al. [33] chadox1 ncov-19 (viral vector vaccine), bnt162b2 (mrna vaccine), or mrna-1273 (mrna vaccine) collection of cases (n = 27, 17 flares, and 10 new-onset imid) in patients with imid in 28 days following covid vaccination mixed group including biologics, steroids, and dmards flares were temporally associated with vaccination, but no way to determine causation. iancovici et al. [34] bnt162b2 (mrna vaccine) 12 rheumatoid arthritis patients who were treated with janus kinase inhibitors. 26 healthy controls. janus kinase inhibitors reduced levels of anti-spike antibodies in patients taking janus kinase inhibitors. b cell responsiveness to sars-cov-2 in patients with rheumatoid arthritis was low. simander et al. [35] mrna vaccine 53 patients with ra, 46 patients with spondyloarthropathy and 169 healthy participants dmards (disease-modifying anti-rheumatic drug) seroconversion rates after 1st immunisation was 54% in patients with inflammatory arthritis compared with 98% in control group. seroconversion was 100% in both groups after second dose. seroconversion was reduced in individuals receiving dmard therapy. spinelli et al. [28] mrna vaccine 126 patients with rmd and 85 controls. 70 patients were taking immunosuppressants and/or biologics. 30 patients were taking hydroxychloroquine. 5 patients had confirmed arthritis flares. most common side effect was injection site pain. pmmb 2022, 5, 1; a0000277 7 of 21 li et al. [36] coronavac, bnt162b2 5493 patients with rheumatoid arthritis immunosuppressants, corticosteroids, nsaids, cs/bdmards no significant link between rheumatoid arthritis flares and vaccination. lukaszuk et al. [37] bnt162b2 vaccine (mrna vaccine) 50-year-old female with rheumatoid arthritis methotrexate antibody levels kept increasing but at a lower rate than in patients not receiving immunomodulatory therapies. madelon et al. [38] mrna vaccine 37 patients with rheumatoid arthritis (ra) or multiple sclerosis (ms), 22 healthy controls rituximab in patients with ra and ocrelizumab in patients with ms patients on anti-cd20 treatment can mount potent t-cell responses to mrna covid-19 vaccines. picchiantidiamanti et al. [39] bnt162b2 vaccine (mrna vaccine) 167 controls and 35 rheumatoid arthritis patients ctla-4-inhibitors, il-6 inhibitors sufficient immune responses induced by the covid-19 mrna vaccine were present in majority of ra patients who underwent temporary suspension of immunosuppressive treatment during vaccination. schreiber et al. [40] bnt162b2 and mrna-1273 (mrna vaccines 243 patients with rheumatoid arthritis, spondyloarthropathy or psoriatic arthritis cs/bdmards seventy-two patients (32%) had an insufficient igg response. the median igg level in patients treated with cs/bdmard combination therapy was significantly lower compared to patients without any dmard treatment. pmmb 2022, 5, 1; a0000277 8 of 21 schumacher et al. [41] bnt162b2 (mrna vaccine), mrna1273 (mrna vaccine), astrazeneca/oxford (viral vector vaccine), ad26.cov2.s (viral vector vaccine) 102 patients with idiopathic rheumatic disease on treatment with rituximab rituximab (rtx) • prednisone (n=81) • methotrexate (n=23) 65 patients (64%) showed a negative antibody level after vaccination. seyahi et al. [42] coronavac (inactivated vaccine) 82 hospital workers with rmd and 300 controls. rituximab (rtx), dmards among rmd patients, those using immunosuppressive drugs were significantly less likely to have detectable antibodies than those of treatment. 3.2.2. psoriasis of the 11 psoriasis studies, ten focused on mrna vaccines, and one focused on inactivated vaccines (table 3). one of the above studies included results from patients taking viral vector vaccines, but no studies focused on that subset of vaccines. participants were all adults above 18, and the study population ranged from 1 to 436. five studies focused on the immune response following vaccination, while the remaining 6 explored the risk of exacerbation following anti-covid-19 vaccination. no studies looked specifically into the side effects of vaccination in psoriatic patients. generally, the risk of psoriatic flare following vaccination seems very low, but it can happen. usually, it is not severe and easily managed. the outcomes of vaccination are promising; most patients can mount a detectable immune response even if it is not as significant as that seen in the normal population. methotrexate has shown itself to reduce the immune response produced by the body. table 3. summary of studies related to psoriasis. study vaccine given population therapies used response haberman et al. [43] bnt162b2 (mrna vaccination) patients with imid (n=51). 24 subjects had psoriasis, 22 had rheumatoid arthritis in imid group, methotrexate anticytokine biologics those patients with imid on background methotrexate (n=45) achieve an adequate. pmmb 2022, 5, 1; a0000277 9 of 21 and 5 had other imids. healthy subjects as controls (n=26). a second independent a validation cohort of controls (n=182) and patients with imid (n=31) tnf inhibitors in controls, nil response in only 62.2% of cases. patients with imid on methotrexate do not demonstrate increased cd8+ t-cell activation after vaccination. huang et al. [44] moderna mrna-1273, astrazenecaoxford azd1222 32 unimmunized controls and 51 vaccinated psoriasis patients 49 patients with psoriasis were on biologics. fifteen episodes of exacerbation in the vaccinated group, which is higher than two episodes in the control group. skroza et al. [45] mrna vaccine 436 patients with moderate-severe psoriasis, both male and female, in treatment with biologics biologics. none of the vaccinated patients in concomitant therapy with anti-psoriatic immunomodulating agents developed adverse events (adr). musumeci et al. [46] pfizer mrna bnt162b2 (mrna vaccine) and moderna m1273 (mrna vaccine) 50 patients with stable plaque psoriasis 24 patients were treated with antitnf, 14 with antiil 17, 7 with antiil 12-23 (ustekinumab), and 5 with anti-il 23 (guselkumab). none of the patients experienced any side effects or a psoriatic flare. only one patient treated with infliximab reported an exacerbation of psoriasis after the vaccine. venerito et al. [47] bnt162b2 vaccine (mrna vaccine) 40 patients with psoriasis tnf inhibitors continuing tnfi throughout the vaccination did not hamper immunogenicity. wei et al. [48] moderna mrna-1273 (mrna vaccine) 8 patients biologics 7 patients experienced an acute exacerbation of psoriasis and 1 had newonset psoriasis. pmmb 2022, 5, 1; a0000277 10 of 21 lopez et al. [49] pfizer (mrna vaccine) 58-year-old man with a history of psoriasis 4 days post-second dose of vaccine not available acute exacerbation of psoriasis. mahil et al. [50] bnt162b2 (mrna vaccine) 82 participants after second vaccination. methotrexate (n=14), tnfinhibitors (n=19), il-7 inhibitors (n=14), il-23 inhibitors (n=20) all participants had detectable spike-specific antibodies following the second dose, and all groups demonstrated similar neutralising antibody titres against wild-type, alpha, and delta variants. by contrast, a lower proportion of participants on methotrexate had detectable t-cell responses following the second vaccine dose, compared with controls. mahil et al. [51] bnt162b2 (mrna vaccine) 121 participants were enrolled, 84 with psoriasis receiving immunosuppressive treatment and 17 controls. methotrexate (n=17), tnfinhibitors (n=27), il-7 inhibitors (n=15), il-23 inhibitors (n=25) seroconversion rates were lower in patients receiving immunosuppressants than in controls, with the lowest rate in that receiving methotrexate. onsun et al. [52] coronavac (inactivated vaccine) a 72-year-old male psoriasis patient 4 days post-vaccination not available generalised pustular psoriasis flare. pavlostsky et al. [53] bnt162b2 (mrna vaccine) 51 psoriasis patients on treatment with systemic immune modifiers systemic immune modifiers, especially biologics forty-nine patients had a positive antibody response. 3.3.3. systemic lupus erythematosus (sle) of the 7 sle studies, 3 were case reports, 2 were cohort studies, and the remaining two were case series and case-control studies (table 4). six studies focused on mrna vaccines alone, and only 1 study focused on mrna vaccines and viral vector vaccines. there is a distinct lack of studies looking into the effect of viral vector vaccines and inactivated pmmb 2022, 5, 1; a0000277 11 of 21 vaccines in patients with sle. the results of the studies looking at vaccination outcomes in patients with sle were similar to those found in rheumatoid arthritis and psoriasis. patients with sle could produce an immune response to the vaccine, but it was weaker than that seen in healthy controls. assawaksakul et al. [54] added on the potency of the booster in stimulating a stronger immune response and suggested that the viral vector vaccine may not be as effective as the mrna vaccine in patients with sle. regarding disease exacerbations, multiple studies found that the vaccination triggers sle flares but is not severe enough to warrant hospitalization or emergency care. they have commonly identified side effects of the vaccine, including headache, fatigue, muscle pain, and local injection site pain, similar to that seen in non-autoimmune patients. table 4. summary of studies related to systemic lupus erythematosus (sle). study vaccine given population therapies used response assawasaksakul et al. [54] pfizer/biontech (mrna vaccine) or chadox1 (viral vector vaccine) booster 8 healthcare workers in thailand with known sle who had previously completed the coronavac series (2 doses of inactivated vaccine) mycophenolate mofetil azathioprine calcineurin inhibitor 6 patients had a strong immune response to the booster. 1 patient was heavily immunosuppressed, and the other had taken the viral vector vaccine rather than the mrna vaccine. during the study period of 2 months, no sle flares were recorded. most common side effects reported were injection site pain, fatigue and fever. bartels et al. [55] bnt162b2 (mrna vaccine) 285 subjects with either sle or ra. 128 patients with sle and 154 patients with ra. biologics 5 patients had severe adverse events (1.8%). no patients were hospitalized or died. fatigue, headache, muscle pain and joint pain were the most reported side effects. pmmb 2022, 5, 1; a0000277 12 of 21 hidaka et al. [56] bnt162b2 (mrna vaccine) 53-year-old japanese woman 2 weeks post-second dose of vaccine not available. new onset evans syndrome. izmirly et al. [57] bnt162b2 (mrna vaccine) or mrna-1273 (mrna vaccine) 90 sle patients and 20 healthy controls. hydroxychloroquine prednisone immunosuppressants (ex. azathioprine, mycophenolate mofetil, methotrexate, etc.) sle patients produced significantly lower antibodies compared to healthy controls. the use of any immunosuppressant or prednisone was associated with decreased vaccine response. post-vaccination flares occurred in 11.4% of patients, 1.3% were severe. joseph et al. [58] moderna (mrna vaccine) 54-year-old woman 4 days post-second dose of vaccine mycophenolate mofetil subacute cutaneous lupus erythematosus flare. zavala-flores et al. [59] bnt162b2 (mrna vaccine) 100 patients with sle hydroxychloroquine azathioprine 27 patients experienced sle flares. the predominant type of flare was arthritis, followed by dermal manifestations. pain at the inoculation site was the most common side effect. all side effects were mild. nune et al. [60] bnt162b2 (mrna vaccine) 24-year-old previously well male 2 weeks post-second dose of vaccine nil new onset of sle pmmb 2022, 5, 1; a0000277 13 of 21 3.3.4. myasthenia gravis of the 4 myasthenia gravis studies, 1 was a single center case series, while the remaining 3 were single case reports (table 5). the case series was conducted for one month and looked at 22 participants registered on an mg database. all 3 case reports looked at the elderly population (age>60 years old). the participants of the case series ranged from 25 to 73 years old. three studies looked at the worsening/onset of myasthenia gravis symptoms [6163], whereas one looked at the immune response elicited by vaccination [64]. tagliaferri et al. [62] and chavez et al. [63] reported a myasthenia gravis crisis in a patient following a second vaccination dose. these two articles suggest that covid-19 vaccination may be a potential trigger for myasthenia gravis, especially the second dose. plymate and colleagues showed that immune response was minimal in a myasthenia gravis patient after receiving two doses of the mrna vaccine, but she then demonstrated an excellent response to the third dose given 85 days later [64]. this suggests that patients with myasthenia gravis may require booster shots to mount an adequate immune response. ruan et al. [61] reported a retrospective case series demonstrating the safety of inactivated vaccines in patients with myasthenia gravis, defined as mild/absent worsening of symptoms. table 5. summary of studies related to myasthenia gravis. study vaccine given population therapies used response ruan et al. [61] coronavac vaccine (inactivated vaccine) 22 patients with mg receiving the covid-19 vaccine. 10 have ocular mg and 12 have generalized mg. azathioprine (n=12) mycophenolate mofetil (n=3) steroid and azathioprine (n=3) none (n=4) 20 patients did not have any worsening of mg symptoms. 2 patients reported mild mg symptoms worsening. tagliaferri et al. [62] moderna covid-19 vaccine (mrna vaccine) a 77-year-old man with a fiveyear history of mg 1 week postsecond dose of vaccine prednisone and pyridostigmine myasthenia gravis crisis plymate et al. [64] bnt162b2 covid-19 vaccine (mrna vaccine) a 74-year-old woman diagnosed with generalized mg 44 months 1440 mg mycophenolate sodium and prednisone 11 mg daily. 2 doses of mrna vaccination failed to elicit detectable circulating vaccinespecific igg or ifn-γ t cell responses. pmmb 2022, 5, 1; a0000277 14 of 21 before the initial sars-cov-2 vaccination chavez et al. [63] bnt162b2 covid-19 vaccine (mrna vaccine) 82-year-old man, two days postsecond dose of vaccine nil. new late-onset myasthenia gravis. 4. discussion there is a distinct lack of studies focusing on viral vectors and inactivated vaccines in these four different autoimmune disease patients. each vaccine has a different mechanism of action and, therefore, will have other interactions and effects on the human body, even if they aim to accomplish the same outcome. as such, it isn't easy to conclude the outcomes, safety, and side effects of the vaccines (figure 2). figure 2. the outcome, side-effect and safety of covid-19 vaccine on autoimmune disease patients. 4.1. outcomes overall, most autoimmune patients can mount a significant immune response to the vaccine even if the potency is less than that of healthy participants. several studies also seem to show a reduced response after the first dose but a much better response after the following doses. for example, a study by simander et al. [35], wherein the seroconversion rate after the pmmb 2022, 5, 1; a0000277 15 of 21 first dose was only 54% amongst participants with rheumatoid arthritis as opposed to 98% in the control group, but the seroconversion rates in both groups were 100% after the second dose. a case series by assawaksakul et al. that looks at 8 sle patients found a much stronger immune response in patients following a booster dose of the vaccine [54]. the case report by plymate et al. depicted an older woman who received two doses of the pfizer/biontech vaccine but mounted a negligible immune response following vaccination. she then took a third booster dose 85 days later and showed significant improvement [64]. these studies indicate that patients with autoimmune conditions may benefit more from multiple doses of vaccination than the general population. as mentioned above, taking rituximab and dmards reduces the immune response in patients with rheumatoid arthritis. schreiber et al. [40] identified a 32% non-response rate in patients taking dmards to manage their arthritis. picchianti-diamanti et al. [39] found that suspending the medication during vaccination allowed all patients in their study to develop a strong and sufficient immune response to the vaccine. patients are taking their medication for a reason. while we want them to get the most out of their vaccination, any decision to stop or modify treatment should be discussed and decided with the advice and guidance of a clinician lest they end up with chronic pain or severe flares. rituximab is a form of b-cell-depleting therapy. the study by madelon et al. found that patients taking rituximab could still produce good immune responses [38]. still, the study by schumacher et al. found that 64% of patients taking rituximab were vaccine nonresponders. schumacher’s study had a larger population of 102 participants and found a correlation between the antibody response elicited and the interval since rituximab administration [41]. interval dosing may allow clinicians to optimize vaccine outcomes without stopping the medication that keeps the patient’s arthritis in remission. however, there is still a small risk of non-response, which should be considered. since a booster dose seems to offer some benefit in improving outcomes in patients with sle and myasthenia gravis, it can be considered to optimize vaccine efficacy in patients with rheumatoid arthritis as well, barring any contraindications. prednisone, janus kinase inhibitors, and methotrexate hamper the immune response produced by the vaccine, but they don’t seem to cause non-response. as such, it is still beneficial for patients taking these medications to take the anti-covid-19 vaccine. venerito et al. find that tnf inhibitors do not impair the vaccine's efficacy [47]. a review by wack et al. finds that tnf inhibitors may still cause some impairment compared to healthy controls, and therefore the immune response may still be less robust [65]. interval dosing or suspension of the medication may play a part in all autoimmune patients taking immunosuppressants. still, the decision should be on a case-by-case basis and with the advice and guidance of a clinician. pmmb 2022, 5, 1; a0000277 16 of 21 4.2. safety myasthenia gravis is an autoimmune neuromuscular junction disorder caused by antibodies to the acetylcholine receptor [66]. it causes fatigable weakness, most evident after repeated muscle use or with the day's progression. exacerbations of the condition are usually caused by infection, especially since the advent of this pandemic. since infection with sarscov-2 can cause a worsening, it warrants the suggestion that vaccination against sarscov-2 could also trigger an exacerbation. studies suggest vaccination is unlikely to trigger an exacerbation, with only 2 participants reporting slight worsening symptoms that quickly resolved within a few days [61-63]. more studies with bigger sample sizes must be conducted before establishing a solid link between symptom flares and vaccination. there is also a lack of studies looking into different types of vaccines and their potential link to myasthenia gravis exacerbations. spinelli et al. studied 126 participants, and five rheumatoid arthritis patients had arthritic flare after vaccination [28]. thankfully, the flares were self-limiting and resolved within a few days with some symptomatic management. the study by izmirly et al. looked at the risk of sle flares and found the risk to be about 11.4%, only 1.3% were severe, and none were so severe that they required hospital care [57]. huang and tsai studied 32 unimmunized psoriatic patients and 51 immunized patients. they reported that the immunized group patients presented exacerbations compared to the non-immunized group [44]. the mean psoriatic arthritis severity index (pasi) score also increased from 3.1 to 8 following vaccination. as such, there is undoubtedly some correlation between the vaccine and autoimmune conditions worsening or fluctuating across the board. flares can be triggered by various factors, from infections to stress to poor medication adherence. hence, it is difficult to determine whether a flare has resulted from vaccination, disease progression, or perhaps even a combination. overall, we can see that the risk of disease flares post-covid-19 vaccination is significant. still, the risk of a flare severe enough to require hospitalization or emergency care remains rare and almost exclusive to the odd case report. most of the flares are mild and self-limiting. given the increased risk of morbidity and mortality in patients with comorbidities, it may be better for autoimmune patients to be vaccinated rather than remain unvaccinated. the final decision should be discussed and well-thought-out, considering the patient’s risk factors and disease severity. for patients with mild autoimmune conditions in remission, getting vaccinated is likely the lesser of two evils. 4.3. side effects herpes zoster virus activation was identified as a potential side effect of vaccination in patients with rheumatoid arthritis by furer et al. [30]. this study documents six patients who developed their first episode of herpes zoster shortly after vaccination. the prevalence amounted to roughly 1.2% in patients with autoimmune rheumatic conditions as opposed to none in the healthy controls. herpes zoster viral activation has been reported in connection pmmb 2022, 5, 1; a0000277 17 of 21 to other vaccines, such as trivalent influenza, hepatitis a, and rabies vaccines. potential mechanisms that might explain the link between mrna-covid-19 vaccination and herpes zoster reactivation are related to the stimulation of innate immunity through toll-like receptors (tlrs) by mrna-based vaccines. tlr signaling has been implicated during the reactivation of herpesviruses, a process essential for these viruses to maintain themselves in the host. tlrs are much more highly expressed in patients with rheumatic disease compared to the general population. this might be why the prevalence of herpes zoster activation following vaccination is much higher in this group compared to the controls. hidaka et al. document a case of evans syndrome following covid-19 vaccination [56]. evans syndrome, a rare condition characterized by the coexistence of autoimmune hemolytic anemia (aha) and idiopathic thrombocytic purpura (itp), is associated with sle. interestingly, a case of evans syndrome following the influenza vaccine has also been reported. looking at this, we can conclude that an overactive immune response following vaccination is the most likely trigger causing these sle patients to progress to evans syndrome. it is rare, and there has only been one case so far, but it is worth keeping in mind. geisen et al. discussed side effects in vaccinated patients with chronic autoimmune conditions, but the side effects identified are quite similar to those seen in the general population; headache, malaise, body aches, injection site pain, fever, etc. [29]. a few other studies in this review have come to a similar conclusion. as mentioned, there is a lack of studies looking into specific side effects elicited by vaccination in patients with myasthenia gravis and other autoimmune conditions. it would be essential to establish if there are any condition-specific side effects that clinicians should investigate when counseling these patients on vaccination. 5. conclusions with covid-19 vaccines becoming widely available and the pandemic once again proving itself to be more resilient than the pandemics that came before it, clinicians need to be prepared to discuss the risks and benefits of vaccination with their autoimmune patients. based on the information available, we can deduce that the benefits of vaccination in this population group still outweigh the risks and severe complications are rare if present. neither an autoimmune condition nor any concurrent treatment is a contraindication for the vaccination, but immunosuppressants have shown to reduce the vaccine's efficacy. this can be countered by interval dosing or temporary suspension of the medication, but these are not solutions that would work for everyone. a third vaccination dose to boost immune response appears safe and effective. the decision should be made after viewing each patient as a whole and considering their needs and wants, the severity of their condition, and any limitations they may have. the decision to be vaccinated should not be rushed, especially if patients are hesitant. patients should be monitored for any undesirable side effects and be able to return to the hospital if they face any severe adverse events. it is also essential that more studies should be carried on to understand better the safety and efficacy of different vaccines in various autoimmune conditions. pmmb 2022, 5, 1; 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a0000175. doi: 10.36877/pmmb.a0000175 http://journals.hh-publisher.com/index.php/pmmb review the chemistry of gut microbiome in health and diseases agnes wei yin lau 1 , loh teng-hern tan 1 , nurul-syakima ab mutalib 1,2 , sunny hei wong 3 , vengadesh letchumanan 1* , learn-han lee 1* article history 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia. weiyin.agnes.0816@gmail.com (aw-y.l); loh.teng.hern@monash.edu (ltht); vengadesh.letchumanan1@monash.edu (vl); lee.learn.han@monash.edu (l-h.l) 2ukm medical molecular biology institute (umbi), ukm medical centre, universiti kebangsaan malaysia, kuala lumpur, malaysia; syakima@ppukm.ukm.edu.my (n.-s.a.m) 3li ka shing institute of health sciences, department of medicine and therapeutics, the chinese university of hong kong, shatin, hong kong, china; wonghei@cuhk.edu.hk (s.h.w) *corresponding author: vengadesh letchumanan, vengadesh.letchumanan1@monash.edu; learn-han lee, lee.learn.han@monash.edu received: 30 november 2020; received in revised form: 31 january 2021; accepted: 2 february 2021; available online: 8 february 2021 abstract: there are trillions of microbes residing in our body, with their collective genomes much more than human genomes. they have been living in a close relationship with us and play a role in various biological functions. the human microbes begin to build up in utero, accumulate, and fluctuate until a set point is achieved around three years of age. the gut microbiome is altered by several factors, which include age, diet, and antibiotic use. after the exposure, the microbe may shift back to retain its balance, but some factors may leave a permanent footprint on the gut flora. this may be significant as our review has shown the relationships between microbes and diseases. when the homeostasis of gut-microbes is disrupted, multiple mechanisms have been shown to contribute to diseases' development. the balance between protective and pathogenic microbes must be kept in check to prevent disease onset. with a better understanding of this relationship, we will investigate the potential methods to modify the gut flora as the background of developing therapeutic options. there are already some therapeutic options such as prebiotics, probiotics, and fecal transplantation, but their clinical use is limited and restricted. therefore, there is still a need to investigate the characteristic microbiome association with gut-dysbiosis-related diseases, which may help manage the disease and develop diagnostic and monitoring tools. this review aims to discuss our gut microbiome and its association with human health and diseases. keywords: microbes, gut microbiome, homeostasis, gut-dysbiosis, diseases. mailto:syakima@ppukm.ukm.edu.my mailto:wonghei@cuhk.edu.hk pmmb 2020, 4, 1; a0000175 2 of 40 1. introduction it has been a chaotic start to the new year 2021, with a rising number of covid-19 cases worldwide [1-4]. people are still suffering from this disease, exhibiting asymptomatic to severe symptoms, and losing their loved ones to the disease [5-7]. the covid-19 pandemic has brought in many changes to our daily and professional life. people around the globe adopted the “new norm” – together, we fight against this virus. these days homes are our new offices – where everything is held virtually, and our social life has all been altered. the work from home concept has brought new challenges to our professional, personal, and family life. in whatever circumstances, we should always devote our time to a healthy lifestyle and fitness. foremost, we should understand our body before we step into changing our life. a range of microorganisms colonizes our body, comprising bacteria, viruses, eukaryotic, and archaea, collectively forming the microbiome community [8]. it is a vast active population consisting of nearly 100 trillion microbes and weighing as heavy as our human brain [9]. every organ has distinct microbial communities that vary in composition and function, with its aggregate function and metabolic capacity [8]. they live in a symbiotic relationship with the host and responsible for various roles, including protecting against pathogens, digestion of nutrients, and metabolism of drugs and substances [10]. these microbiotas are incredibly dynamic. they continuously fluctuate around a set point and vary from site to site [11, 12]. also, the composition of the microbiome is determined by multiple environmental and local factors. for instance, the organ site's ph and substrate determine microbes suitable for surviving and living in the community [13, 14]. among the different microbiomes on different human body sites, the gastrointestinal tract has the most affluent microbes’ community. so, what makes a good gut microbiome community and how they play in health and diseases. there are hundreds to thousands of distinct bacterial species in our gut, some can be beneficial or pathogenic. the bacteroidetes and firmicutes phyla dominates the main pool of community in the gut [13]. our healthy gut microbiome produces short-chain fatty acids (scfa), which is crucial in regulating the gut's functional and structural integrity [15, 16]. the gut community plays a vital role in developing gastrointestinal mucosal immunity, an essential part of our immune defense system [17-19]. any disruption of this balanced ecosystem has been influenced and implicated by the gut microbiome. these include cancer, autoimmune disorders, inflammatory bowel disorders, diabetes, psoriasis, eczema, asthma, and autistic spectrum disorder (asd) [20-27]. the evidence is mounting on gut microbiome association in health and diseases. our biologists are exploring and studying the gut ecosystem, in order to prevent the development of diseases. there is an growing interest in potential novel treatments, preventive and diagnostic methods for the diseases using homeostasis of the gut microbiome as the stepping stone [28, 29]. this review aims to discuss human health, particularly examining the association between the gut microbiome and health and diseases (figure 1). pmmb 2020, 4, 1; a0000175 3 of 40 figure 1. the association of gut microbiome with health and disease. probiotics, prebiotics, dietary changes, and fecal transplantation are potential or current therapeutic options for gut-dysbiosis associated diseases. 2. origins of our gut microbiome we have always thought humans are born with sterile guts, not until recent contradicting evidence supporting in-utero transmission of microbes [30]. the advancement of genetic sequencing has given us the privilege to investigate the microorganisms in maternal tissues better than before [31]. studies of the meconium microbiome have enabled us to understand that gut colonization occurs before birth [32]. this is evident by the similarity in meconium microbiota among newborns even if delivered via different methods (vaginal delivery and cesarean delivery) [33]. also, the endometrium and placenta are not wholly sterile [34]. they may act as possible sources for gut colonization to occur in the fetus. however, it is still unclear how and when exactly colonization of the fetus’ gut occurs. meanwhile, ingestion of amniotic fluid has been suggested as one of the possible routes [35]. when the infant is being delivered, the process allows colonization of the microbiome to occur. thus, arises the controversy of whether the mode of delivery can affect human microbiota. newborns delivered via cesarean section instead of vaginal delivery have been observed to lack the composition of maternal vaginal and fecal bacteria (e.g., lactobacillus and prevotella) [36]. however, recent studies have shown that by two months of age, the delivery mode subsides effects. other confounders may cause the discrepancy of gut flora between infants delivered vaginally or via cesarean section [37, 38]. for instance, maternal comorbidities are implicated in cesarean delivery and lower exclusive breastfeeding rates in this group of mothers [39]. interesting to know that breastfed infants have a lower diversity of gut flora before six months of age than their non-exclusively breastfed counterparts. pmmb 2020, 4, 1; a0000175 4 of 40 however, exclusively breastfed seems to be a protective factor against bowel diseases [40]. thus, the author postulates that a less diverse gut microbiome may be required for the first few months to develop a healthy gut [40]. perhaps, more studies are needed to investigate the significance of differences in gut microbiome between these two groups of infants. infants will gradually build up their unique microbiome via exposure to many factors [8]. biological and environmental factors can all leave footprints on the composition of the human microbiome [41]. when infants start a solid diet around six months of age, the initial diversity gut microbiome begins flourishing with different microbes. this diet contributes to forming a complex and diversifying gut microbiome, which achieves its stability at around three years of age [31, 42]. besides, a study has shown that our diet can affect our gut microbiome [43, 44]. for instance, a high fiber diet has increased bacteroidales and firmicutes [45] population, potentially commensal to a healthy gut. however, as part of aging, dysbiosis of the gut could occur [46]. aging is a physiological process associated with diminished gut microbiome diversity and slowly weakened gut functional integrity [46]. today, our dependency on antibiotics has played a vital role in forming our gut diversity and community. dethlefsen and relman's study revealed an alteration of the gut community after using ciprofloxacin for 3 to 4 days [47]. however, despite the gut microbiome's tendency to shift back to its initial composition, the recovery was observed to be incomplete [47]. thus, the effect of repeated or long-term use of antibiotics on gut flora remains unknown. further research is required to understand the role of antibiotics in gut dysbiosis completely. 3. gut microbes and diseases in the early days, people believe in galenism, which explains diseases resulting from an imbalance in bodily fluids or humor [48]. not until the 16th century, with the development of a microscope, the model by parcelsians which regards tiny inorganic particles as the culprit of diseases are justified [48]. however, this exciting concept submerged for decades until louis pasteur and robert koch successfully demonstrated germ theory's validity. they proved that microorganisms could be the causative agents of diseases [49]. scientists have observed the symbiotic relationship between the host-microbe system in fecal and oral microbiota [8]. in addition, environmental microbes have been seen to live in an involved community [50]. human microbes are a community of microorganisms that interact with one another and the host so delicately that its disruption may lead to various health diseases and mental health disorders. there are blooming studies on the association between human microbes and many diseases. these studies have demonstrated gut dysbiosis's relationship in human health [20, 51-54]. 3.1. gastrointestinal diseases 3.1.1. inflammatory bowel diseases pmmb 2020, 4, 1; a0000175 5 of 40 inflammatory bowel disease (ibd) is the chronic inflammation of the gastrointestinal tract, consisting of ulcerative colitis and crohn’s disease. the pro-inflammatory state in ibd can be attributed to the dysregulated host’s inflammatory response in genetically susceptible individuals towards the gut microbiome [55]. the environmental factors are then responsible for the disease's onset and recurrences [56, 57]. when comparing the gut microbiota of individuals with ibd and healthy counterparts, there is a significant discrepancy between them [58-60]. it is observed that individuals with ibd have a higher population of pro-inflammatory microbes (proteobacteria, escherichia spp., fusobacterium spp.) and a lower population of protective microbes (bacteroidetes, firmicutes, butyrate-producing spp.) [40, 61, 62]. by reducing butyrate-producing species responsible for short-chain fatty acids (scfa) production, it is not surprising that individuals with ibd have a lower concentration of these molecules [63]. the significance of lacking these molecules and reduced population of protective microbes [64] is that they have been shown to play an anti-inflammatory role in immune responses. the deficit in these microbes can lead to a pro-inflammatory state, parallel with the disease state in individuals with ibd. when discussing the differences between the gut microbiome of ibd individuals and healthy counterparts, it is unsure whether it is a cause of the disease or its result. however, recent studies have been able to show that genetic factors such as nucleotide oligomerization domain (nod 2), caspase recruitment domain-containing protein 9 (card 9), and autophagy-related 16-like 1 (atg16l1) [65-67] may be attributed to the differences. the expression of these genes is responsible for the anti-inflammatory and antimicrobial effect in the gastrointestinal tract. besides, they also help to regulate the homeostasis of the gut microbiome by determining its composition. studies revealed that nod-2 deficient mice have a higher gut commensal population and a higher vulnerability to be colonized by pathogenic microbes [68, 69]. on the other hand, atg16l1 mice have been observed to have a higher bacteroides species population with an anti-inflammatory role [70]. thus, altering this gene expression can disrupt the gut's colonization and its inflammatory response and defense against pathogens. when extrapolating this to the human host, it explains the vulnerability of genetically susceptible individuals to disruption of gut homeostasis, leading to inflammatory bowel disease [71]. the terminal ileum is the most affected segment of the gut in crohn’s disease and the colon segment in ulcerative colitis. this corresponds to the gastrointestinal tract sites in individuals with ibd with a higher microbes concentration than healthy individuals [72]. the parallel distribution of microbes and affected gut segments in ibd illustrates the effect of gut dysbiosis in the disease activity of ibd. moreover, mice that have received fecal microbiome from mice with colitis have been observed to develop colitis [73]. besides that, improvement of ibd symptoms by using antibiotics, probiotics, and fecal transplantation, suggest that pmmb 2020, 4, 1; a0000175 6 of 40 dysbiosis of gut flora contributes to the pathogenesis of the disease [74]. the disease activity of ibd is correlated to the biodiversity of the gut microbiome [75]. they have half the gut microbiome's diversity compared to healthy individuals [76]. interestingly, swidsinski et al. have identified the characteristic spatial composition of fecal flora in crohn’s and ulcerative colitis [77]. the difference in abundance of mucosaassociated or fecal faecalibacterium prausnitzii can be potentially used as a biomarker to differentiate between the two diseases [78]. it is observed that crohn’s disease has depleted f. prausnitzii with normal leukocyte count. in contrast, ulcerative colitis has a higher abundance of f. prausnitzii with increased leukocytes in the fecal mucus transition zone [77]. however, compared to healthy individuals, individuals with ibd have less diversity in subtypes of f. prausnitzii, and there is a shift in the phylotype distribution of f. prausnitzii species [79]. some f. prausnitzii subtypes are disease-specific, allowing discrimination between individuals with ibd and healthy counterparts [79]. perhaps we may consider analyzing punched fecal cylinders obtained from individuals with ibd to diagnose and monitor the disease, given the characteristic composition and spatial distribution of gut microbes in individuals with ibd [77, 78]. 3.1.2. gastrointestinal malignancies a) colorectal cancer colorectal cancer is one of the most common forms of cancer that affects both genders [80]. it is known that chemoresistance is a hurdle in colon cancer therapies. hence, there is an interest to study the anti-colon cancer potential of novel streptomycetes as an effective treatment for colon cancer [81-85]. recently, studies revealed the role of colon microbes in the carcinogenesis of colorectal cancer [69]. these studies' evidence is supported by the positive correlation between microbes' concentration and cancer cells' distribution. cancers have been linked with our immune and genetic dysregulation. the gut microbes are known to involve themselves in cancer development by altering the gut into pro-inflammatory and procarcinogenic states. some of the microbes can act as ‘bacteria drivers’ which damage the gut epithelial dna. this can lead to hyperproliferative epithelial cells, the cells' ability to evade apoptosis, and so on, which are the cancer hallmarks [86, 87]. besides, the ‘bacteria drivers’ can disrupt the gut integrity, favoring the proliferation of pathogenic ‘passenger microbes’, which plays a synergistic effect with the ‘bacteria driver’ [86, 87]. wu et al. have shown specific gut commensal mechanisms in activating the host’s immune system, creating a favorable proinflammatory state for cancer development [88]. this may be postulated as the disruption of gut homeostasis, allowing the overgrowth of pathogenic commensal, leading to the development of colorectal cancer. pmmb 2020, 4, 1; a0000175 7 of 40 fusobacterium nucleatum is a causative effect in the development of colorectal cancer [89, 90]. this microbe has been observed to have a negative relationship with the number of cd3+ t cells in individuals with crc [89, 91]. they can bind via the bacteria-derived protein fap2 on exclusively human t cell immunoglobulin and itim domain (tigit) molecules thus inhibiting natural killer cells in removing cancer cells [89]. this evades the tumour suppression process, thus contributing to the development of tumors [89]. also, f. nucleatum spp. can recruit cd11b+ to promote a pro-inflammatory and oncogenic environment, which further supports its virulence in causing crc [92, 93]. interesting to note that, with the use of metronidazole, mice study has shown that the population of f. nucleatum is reduced and the retarded tumor growth and proliferation of cancer cell. perhaps further investigation down this path can allow for the potential use of antimicrobial in managing colorectal cancer [90]. there is also a discrepancy between the gut microbiome in individuals with colorectal cancer and healthy individuals. individuals with colorectal cancer have higher population of streptococcus bovis [94], bacteroides fragilis [95, 96], clostridium septicum [97], fusobacterium spp. [92], enterococcus faecalis [98] and escherichia coli [99]. studies have observed that these microbes are associated with carcinogenesis via different virulence factors [96, 100, 101]. they can alter host metabolism, immune system, and production of genotoxins, which can align the gut environment towards pro-carcinogenic state [102]. arthur et al. have shown the mechanisms of virulence factors and metabolites of certain microbes in the progression of colorectal cancer [103, 104]. this further supports that the gut microbes do play a vital role in developing colorectal cancer, and more research is needed to identify the exact microbiota associated with the disease. in addition, recent studies have shown that microbes can form biofilms that can hold them together forming a shield against external agents [105]. they proliferate within the biofilms and damaging the e-cadherin in the colon[106]. this increases intestinal permeability, activates the local inflammatory response, and induces gut dysbiosis [106]. this has been seen in mice studies in which biofilms formation has been an imperative factor in promoting the oncogenic potential of microbiome in crc development [107, 108]. meta-analysis studies have shown an increase in protein, choline, and mucin [109, 110]. moreover, it is associated with the enhanced fermentation process, gluconeogenesis, and secondary bile acids production with reduced carbohydrate metabolism [109, 110]. this portrays a shift from the microbiome that utilizes carbohydrate metabolism in healthy gut to use amino acids and fats. furthermore, individuals with crc have a higher concentration of amino acids in the feces and gastrointestinal epithelium, suggesting the association between meat-rich high-fat diets with colorectal cancer [109, 110]. this is plausible as diet is known to be one of the factors that can modify the gut microbiome [92, 93], which influences our health. pmmb 2020, 4, 1; a0000175 8 of 40 b) gastric cancer with the emergence of louis pasteur and robert koch's germ theory, there is a bloom in research on the microbiome's role in diseases. in the late 19th century, the relationship between gastric microbes and gastric cancer is proposed despite the fact that the gastric organism's predominant organism is yet to be identified [111]. this was later denied by heinemann and ecker, who observed that the microbes suggested by the previous study were lactobacillus. reduction in gastric acidity has allowed the proliferation of this species, and it is not causative of gastric cancer [112]. it is not until the late 19th century, with the discovery of helicobacter pylori, researchers begin to relate the association between gut microbiome with gastric cancer [113]. the h. pylori affect approximately 50% to 60% of the world’s population [114, 115]. it is known to cause gastric cancer in 1-3% of those with h. pylori infection [116] and contributes to around three-quarters of all gastric cancer cases [117]. they are multiple strains of h. pylori, and each possesses different virulence factors that can cause genetic mutation, thus interfering with gastric epithelial cellular division, function, and immune responses [118, 119]. one of the proposed mechanisms is that h. pylori can produce reactive oxygen species that can lead to dna mutations, aberrant methylation, and oxidative damage, leading to loss of functioning tumor suppressor genes [120]. another virulence factor worth noting is the caga protein. caga is associated with a higher risk of premalignant and malignant gastric lesions [121]. the h. pylori utilize its adhesion molecules for prolonged colonization of gastric epithelium and injecting its oncogenic proteins, including caga, into the gastric epithelial cells. this oncogenic protein then leads to alteration of the morphology of epithelial cells and disrupts the epithelial intercellular junction. in addition, these molecules trigger metaplasia and dysplasia of the epithelial lining which leads to the development of gastric cancer [122124]. the h. pylori possess vaca toxin, which can disrupt the mitochondrial membranes, creating vacuoles in the cytoplasm, causing apoptosis of the gastric epithelium. this is aligned with the mechanism of h. pylori-related atrophic gastritis, which subsequently undergoes metaplasia and dysplasia of the epithelium, leading to the development of gastric cancer [120]. moreover, these virulence factors promote inflammation and create an environment favorable for tumorigenesis [118, 125]. the disrupted gut epithelial cells will begin to increase uncontrollably, leading to gastric cancer. saenz et al. have proposed that gastric epithelium can respond to injury by cellular plasticity and reprogramming. however, the mechanism of dedifferentiation and differentiation is vulnerable to the accumulation of genetic mutations, which can further predispose the host to gastric cancer [126]. this is supported by the stem cell-like cells observed in the transition of epithelial to mesenchymal cells in the process of transitioning to gastric malignancy [127]. pmmb 2020, 4, 1; a0000175 9 of 40 besides h. pylori, other microbes may also contribute to gastric cancer development with or without the aid of h. pylori infection. when observing individuals with h. pylori infection's gut microbiome, they have a higher proportion of actinobacteria, firmicutes, and bacteroidetes. [128]. this discrepancy is significant because bacteroidetes has been shown to promote gastric cancer in genetically predisposed germ-free mice to a similar extent as genetically predisposed specific pathogen-free mice [129]. this indicates that microbes can play a part in carcinogenesis. gen genetically predisposed specific pathogen-free mice to appear to have more severe and vigorous gastric cancer progression than genetically predisposed germ-free mice [130].this illustrates that h. pylori can alter the gut microbiome by disrupting commensal, which confer protection and promotes pathogens' growth, enhancing its carcinogenic effect. it also shows that pathogenic microbes can alter the gut microbiome into a pro-carcinogenic environment that can favour more severe disease development. fortunately, with the availability of treatment option for h. pylori infection, we can eradication this pathogen before it causes detrimental harm to our body. 3.2. infectious diseases 3.2.1. clostridium difficile infection clostridium difficile infection beautifully demonstrates gut dysbiosis as the pathogenesis of the disease. the infection is due to pathological overgrowth of an organism following antibiotic use [131]. c. difficile is part of the gut flora, and its population is regulated by the homeostasis of the gut microbiome [131]. antibiotics are used to disrupt this equilibrium, reduce microbe diversity, and cause a significant shift in composition [131, 132]. gu et al. have shown that antibiotic use is associated with a diminished number of putative butyrateproducing anaerobes and increased endotoxin-producing opportunistic pathogens [131]. this may explain the opportunistic overgrowth of c. difficile in patients with recent antibiotic use. the c. difficile infection is caused by the toxin released by the microbes following germination of the c. difficile spores [133, 134] and bile acids are critical for the germination of these spores. the liver produces bile acids that are secreted into the gastrointestinal tract. some of these acids tracked down the colon, which is subsequently bio-transformed by the gut flora into secondary bile acids [135, 136]. the biotransformation happens via the catalyst activity of 7α‐dehydroxylation and some other enzymes [134, 135]. this balance may be disrupted by the use of antibiotics, which results in the accumulation of primary bile acids (cholic acid, chenodeoxycholic acid) and reduction in secondary bile acids (deoxycholic acid, lithocholic acid), thus favoring the germination of c. difficile spores [137]. kang et al. have reported that some of the commensal possessing bile acid 7α‐ dehydroxylating property, for instance, clostridium scindens and clostridium sordellii can inhibit the in vivo growth of c. difficile [135]. they secrete tryptophan derived endogenous antibiotics to outcompete other microbes in the complex gut ecosystem [135]. interestingly, the secondary bile acids produced by the 7α‐dehydroxylating bacteria themself can further pmmb 2020, 4, 1; a0000175 10 of 40 enhance the endogenous antimicrobial effect [135]. thus, any disruption in this regulating mechanism of the gut will reduce the protective microbes’ population, which favors the growth of c. difficile. 3.2.2. human immunodeficiency virus infection human immunodeficiency virus (hiv) is an on-going global public health issue. with the development of highly active anti-retroviral therapy (haart), the incidence of acquired immunodeficiency syndrome (aids) has significantly reduced. mortality of hiv has dropped tremendously from 80% to 50% within ten years since its discovery [138]. however, haart treatment does not seem to revert the chronic inflammatory status in individuals with hiv [139]. this pro-inflammatory state in hiv individuals has been shown to drive the disease progression [140, 141], on top of its associated high morbidity and mortality [142, 143] researchers investigated the chronic inflammatory state's etiology in individuals with hiv, including individuals treated with haart with undetectable viral load. they noticed a massive reduction of cd4 t cells in the gut, especially those responsible for enhancing gut barrier integrity [144, 145]. vujkovic-cvijin et al. took a step further to show that altering the gut immune system in hiv individuals can be linked to disruption of the gut microbiome with its associated local inflammation [146]. it is still inconclusive on the findings regarding the shift of gut microbiome in individuals with hiv. there is a consensus on reducing gut microbiome diversity, but the gut microbiome's specific signature in individuals with hiv is yet to be identified [147-149]. some common findings include the increase in prevotella spp. and decrease in a decrease in bacteroides spp. these microbes are proposed to have different gut immunology roles, and the alteration in their abundance can affect gut diversity. for instance, prevotella spp. can create an inflammatory response by activating myeloid dendritic cells, colonic t cells, expression of cd40, and some other inflammatory mediators [147]. meanwhile, the bacteroides spp. is responsible for protecting gut health by expression polysaccharide-a (psa). this molecule inhibits the activation of th17, which is a subset of cd4 + t cells [150, 151]. the species also stimulates the invariant natural killer t in the gut-associated lymphoid tissue [152-154]. together with the th17 cells, they are responsible for controlling the gut microbiome's homeostasis and regulating the microbiome's translocation. reduction in th17 cells indicates the loss of security of the gut barrier, in which the tight intercellular junctions are disrupted, thus allowing gut microbes to enter the systemic circulation. this creates a persistent inflammatory state in individuals with hiv [155, 156], even under haart treatment [151, 157]. when isolating the fecal microbiome from individuals with hiv individuals, it is observed to have higher levels of activated t cells and monocytes, which is positively correlated with the host's viral load [158]. even in individuals receiving antiretroviral therapy (art), there is still activation of adaptive immunity despite a lesser extent than individuals not receiving hrt [158]. neff et al. also suggested that tumor necrosis factor α and toll-like pmmb 2020, 4, 1; a0000175 11 of 40 receptor-2 are the immune mediators that activate the inflammatory response in individuals with hiv. besides the ‘leaky’ gut hypothesis, the gut microbiome's pro-inflammatory properties in hiv individuals can lead to a chronic inflammatory state [158]. there are still gaps to be filled in terms of the gut microbes' involvement in the progression of hiv disease so that adjuvant therapies that may restore gut homeostasis can be developed [159], thus reducing hivassociated morbidity and mortality. 3.3. metabolic disorders 3.3.1. obesity microbes have many potential roles in our gut, ranging from digestion, regulation of absorption to metabolism of drugs and substances. they can also synthesize molecules such as short-chain fatty acids, affecting our immune and metabolic systems [160]. unsurprisingly obesity is part of metabolic disorders [161]. the disruption of gut homeostasis can affect the balance energy uptake from diet and its expenditure [162]. this can be explained by the role of microbes in nutrient handling. they can break down indigestible diet and convert them into short-chain fatty acids (scfa), for instance, acetate, butyrate, and propionate. these substances are then involved in different functions and activities in various organs. butyrate, acetate, and propionate are required to produce glucose and lipids in the liver [163]. they stimulate the enteroendocrine l cells to release glucagon-like neuropeptided-1 and release local factor peptide yy, regulating lipid digestion and lipid metabolism on top of deposition of fatty acids in the liver. meanwhile, butyrate also acts as a significant energy source for the colon's epithelial cells [163]. the concentration of these end products is dependent on the gut flora of the host as different gut microbiome are associated with varying properties of metabolism. turnbaugh et al. have shown that genetically obese mice have higher acetate and butyrate levels in the gastrointestinal tract and lower energy content in their feces than genetically lean mice [164]. this illustrates that the gut community in genetically obese mice are better in energy extraction than their lean counterparts. on the other hand, some of these scfa end products can have a beneficial effect on health. for instance, butyrate and propionate have been seen to enhance satiety [148, 149]. furthermore, butyrate enhances mitochondrial function and oxidation of fats, increasing the energy expenditure [101]. thus, it is reasonable to suggest that different gut microbiome compositions can have other metabolic effects that can affect the hosts' metabolic profile. it is also observed that obese host has reduced gut microbes biodiversity [164] and higher firmicutes to bacteroidetes ratio versus healthy individuals [165, 166]. the firmicutes are associated with the production of scfa and utilized in lipogenesis [167]. interestingly, this discrepancy in firmicutes to bacteroidetes ratio can be reversed via weight loss demonstrated in mice studies [168] and in individuals who have undergone bariatric surgery [169, 170]. this characteristic of gut microbiota and its reversibility illustrate that specific microbe are more favorable to a specific metabolic phenotype in nutrient handling and metabolism. meanwhile, pmmb 2020, 4, 1; a0000175 12 of 40 bacteroidetes are responsible for the breakdown of branched-chain amino acids (bcaas) which are strongly associated with obesity [171]. thus, reduction of the species in obese individuals can explain the higher circulating levels of bcaas [171], which results in hyperphagia [172]. however, other study results on the composition of the obesity-related gut microbiome are contradicting. to date, the data is still inconclusive on the exact composition of the obesity-related gut microbiome, other than the common findings on reduced diversity of gut flora in obese individuals [165]. even so, animal studies on gut microbes and obesity are still quite promising. when fecal microbes from obese or lean twins were transplanted to germ-free mice, they showed similar metabolic phenotypes and adiposity as their host-source [173]. when microbes from lean cotwins were transplanted to obese mice fed with a regular diet, they successfully prevented the obese mice from further gaining adipose tissue source [173]. this illustrates the possibility of transmitting the lean and obese phenotype by transplantation of gut microbes and the influence of different gut microbiome ecosystems on individuals' metabolic profile. furthermore, microbes play a significant role in energy harvesting, which can impact the host’s metabolic profile. it is observed that transplanting gut microbes from conventionally raised mice to germ-free mice can cause significant gain in body fat despite reduced food consumption [174]. this can be explained by microorganisms' ability to ferment indigestible dietary carbohydrates, which can then be absorbed and converted into lipids [174]. these lipids are then further promoted to be stored as adipose tissues [174, 175]. this can be extrapolated on obese individuals such that they may possess characteristic gut microbiota that has enhanced efficacy in extracting energy from the diet, thus contributing to their excess storage. 3.3.2. type 2 diabetes type 2 diabetes (t2dm) is a chronic disease characterized by increased blood sugar level, relative insulin deficit, and insulin resistance [26, 176]. it is also associated with other metabolic abnormalities such as incretin deficiency, raised glucagon level, and increased lipids breakdown [177, 178]. t2dm, as with other chronic diseases, has been attributed to gut dysbiosis. this has been illustrated by backhead et al. in which different gut microbiota has demonstrated different glucose metabolism profile, even when the mice are of similar genotype and controlled for their diet pattern [174]. this shows that a diverse gut microbiome with different glucose metabolism capabilities and lower glucose metabolism can predispose the host to t2dm. it is also long known that t2dm is associated with low-grade systemic inflammation, which leads to insulin resistance of the host [179]. this pro-inflammatory state can be led back to the host’s microbiome which can play a role in metabolism and immunology. as in other chronic diseases, reducing gut microbial diversity has been observed in individuals with t2dm [180]. this allows the overgrowth of pathogenic microbes to promote local inflammation by activating innate immunity [180]. the inflammatory mediators from the pmmb 2020, 4, 1; a0000175 13 of 40 immune response or microbes’ toxins disrupt the intestinal barrier, allowing gut content, including microbes, to enter the systemic circulation [180]. the immune system is then activated by the bacteria and toxin in the blood, which creates a low-grade systemic inflammation that is pro-insulin resistant [180]. overall, gurung et al. have reported a consistent increase in fusobacterium, ruminococcus, and blautia genera, decreasing bacteroides, bifidobacterium akkermansia, faecalibacterium, and roseburia genera in individuals with t2dm [181]. among these protective microbes, bifidobacterium genus is the strongest protective factor against t2dm [182, 183]. animal studies have successfully demonstrated the reduction in blood glucose level when introduced to bifidobacterium spp. [184, 185]. also, losing these protective gut microbes can contribute to the chronic inflammatory state in t2dm individuals. these protective microbes can inhibit pro-inflammatory mediators. for instances, tnf-α, il-8,il-1β, cd36, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, and c-reactive protein [186, 187]. other butyrate-producing microbes also produce metabolites that suppress nf-kb [188]. without them, it is not surprising that the inflammatory pathway will be dominant, which causes chronic inflammation in the host. interesting to note that individuals with t2dm have a lower population of roseburia spp. [189]. the roseburia spp. has been inversely associated with the plasma glucose level in individuals with t2dm [190]. this shows that different microbes have different metabolic properties, which may either confer towards or against the disease activity of t2dm. as discussed, some gut commensal, especially clostridium spp. is involved in converting primary into secondary bile acids [191]. these bile acids can then affect glucose metabolism by activating membrane-bound, g-protein-coupled receptor tgr 5 and nuclear farnesoid x receptor (fxr). these receptors can be activated to inhibit the enzymes involved in gluconeogenesis [192]. also, tgr 5 membrane receptor on the enteroendocrine l cells has been suggested to increase the secretion of glucagon-like peptide1 (glp-1). glp-1 is an incretin hormone that enhances the insulin effect and increases satiety [193]. on the other hand, fxr is shown to improve glucose tolerance and increase insulin sensitivity [191]. thus, gut dysbiosis that interferes with bile acids' biotransformation can have a ripple effect on glucose metabolism and glucose level regulation, thus contributing to t2dm. in addition, it is known that the gut microbiome can metabolize nutrients into scfas [194]. most of these end products are then absorbed and utilized by the host for various functions [194]. the scfas include butyrate and propionate, beneficial to the host's energy balance and metabolism [194]. for example, propionate stimulates glp-1 and peptide yy secretion, which reduces energy intake [195]. sanna et al. have shown that the gut microbiome that produces butyrate can enhance beta cells' function. in contrast, those with reduced propionate absorption can confer an increased risk of t2dm [194]. in short, various mechanisms can implicate the development of t2dm, and maintenance of the homeostasis of gut flora is the key to prevention. pmmb 2020, 4, 1; a0000175 14 of 40 3.4. pulmonary health and asthma the lower airways were once thought to be sterile until recent years; studies have demonstrated microbes in the lungs, even in healthy individuals [196, 197]. lungs microbes are detectable right after birth [198], and their composition is enriched by the upper airway [199]and oropharyngeal microbes [200]. the gut-lung axis concept was also suggested in parallel to the increased attention given to the correlation between human microbes and diseases. this has been demonstrated by schuijt et al., in which gut microbes depleted mice have a more disseminated disease, complications, and higher mortality when infected with streptococcus pneumonia [201]. when fecal microbes were transplanted into pneumococcal infected mice with depleted gut microbes, the lungs' inflammatory response was reduced [201]. this shows that gut microbiota can enhance host defense against lung infection. asthma is an atopy related respiratory disease characterized by chronic inflammation of the smaller airways [202]. when comparing the microbiome of asthmatic to healthy individuals, it is found that asthmatic patients have a higher population and diversity of microbes [197, 203]. the colonization of an infant’s oropharyngeal by moraxella, haemophilus, and streptococcus before one year of age has been shown to predict the risk of developing asthma later on [204]. while moving to the comparison of the gut microbiome, a study showed a reduction in the population of genera faecalibacterium, lachnospira, veillonella and rothia in infants with increased risk of developing asthma in the first hundred days of life [205]. meanwhile, another study showed an increase in clostridium neonatale and lachnospira in the infant's gut flora, which is predictive of asthma risk at the pre-school age [206]. recently, there is increased research on the gut-lung axis concept, a bi-directional relationship between the gut and the lungs [207]. they are proposed to be involved in regulating a ‘common mucosal immunological system’ [208]. there is increasing evidence that one mucosal compartment's inflammation can impact the distal mucosal site [208]. this is in line with the dysbiosis of both lungs and gut microbiome observed in individuals with asthma. in addition, childhood use of broad-spectrum antibiotics has also been associated with increased asthma development later [209, 210]. this further illustrates that disruption of the homeostasis of the gut microbiome can affect the lungs' immunity. however, the exact mechanism is still unknown, and more studies are needed to understand this mucosal immunological system and the bi-directional relationship between compartments [208]. if it is indeed evident, the concept can be beneficial in explaining various diseases, including asthma, associated with airway mucosa inflammation. besides that, the composition of the lung microbiome is related to its treatment responsiveness. corticosteroid-resistant patients are populated by proteobacteria (haemophilus, neisseria), which produces endotoxin triggering chronic inflammation [211]. in contrast, fusobacterium and bradyrhizobium, which have lower endotoxicity, were found pmmb 2020, 4, 1; a0000175 15 of 40 in treatment sensitive patients [211]. moreover, the severity can also influence the gut flora's alignment with the gut-lung axis concept. bisgaard et al. demonstrated that the gut microbiome could be modified by viruses [212]. this allows the overgrowth of specific pathogens that are associated with severe asthma. there are evidence implying a high fiber diet can reduce asthmatic symptoms by modifying the gut microbiome's composition [213]. it is shown that gut microbes can ferment soluble high fiber diets to produce short-chain fatty acids with an immunomodulatory effect [213]. the molecules can counteract the proinflammatory state in asthmatic individuals' airways, modifying the disease activity. 3.5. mental health the gut-brain axis concept can be traced back to the early 20th century, where the gut's functional integrity and microbes were attributed as part of the pathophysiology of mental health disorders [214]. researchers back then have believed that autointoxication can affect one’s mental health [214]. they suggested that toxic gut content can have a profound impact on the brain [214]. in recent years, there are increasing studies supporting this bi-directional relationship between the brain and the gut [215, 216]. the gut microbes produce metabolites and molecules that can modulate the immune system, which interacts with the brain; vice versa, the brain can alter the composition of gut microbiome via neural signaling [217]. homeostasis of the gut microbiome has been shown to protect intestinal and blood-brain barrier integrity. disruption of this balance allows pathogenic microbes to overgrow and induce local inflammation. they then increase the gut permeability, allowing translocation of gut content into the systemic circulation, which triggers a systemic immune response [218]. a pro-inflammatory state is shown to alter the integrity of the blood-brain barrier [219]. this allows immunomodulatory substances and pathogenic microbes to reach the brain [219]. on the other hand, some gut microbes produce metabolites that indirectly modify the brain function via the neural signaling pathway [220]. in short, this illustrates the imbalance in the gut ecosystem can affect brain function, which predisposes individuals to the development of mental health disorders. various studies have supported this view where a pro-inflammatory state has been observed in individuals with mental health disorders. for instances, raised inflammatory mediators, inflammatory markers, and antibodies specific to gut commensal is observed in individuals with major depressive disorders [221, 222], higher activated level of c1q in individuals with schizophrenia, and increased intestinal permeability [223]and inflammatory state in individuals with autistic spectrum disorders [224]. besides that, some gut microbes are known to secrete substances that can act as neuromodulators [225]. for example, lactobacilli spp. produces gamma-aminobutyric acid; bacillus, escherichia and saccharomyces spp. produce noradrenaline; streptococcus, enterococcus, and candida spp. produce serotonin (5-ht), and bacillus produces dopamine [226]. the balance of these neuronal signaling messengers is crucial in the functioning of pmmb 2020, 4, 1; a0000175 16 of 40 cognition and behavior of individuals. matsumo et al. exhibited that dopamine concentrations were two fold higher in germ-free (gf) mice than in ex-germ-free mice [227]. meanwhile, another study showed that gf mice have a higher level of 5-ht and their metabolite in the hippocampus. this can occur without alteration in the expression of genes responsible for their production [228]. a ‘healthy gut flora’ is essential in the balance of neurotransmitter levels of the brain. the alteration in neurotransmitters has been attributed as the mechanism behind various mental diseases. it can be extrapolated that the homeostasis of the gut ecosystem is important in preserving healthy mental states. 3.6. autism spectrum disorder dsm-5 defines autism spectrum disorder (asd) as a neurodevelopmental disorder characterized by impaired communication and interactions with repetitive and/or restricted behaviors [229]. the disease is considerably associated with various comorbidities, including epilepsy, bowel disorders, and sleep disorders [230]. the exact cause of autism spectrum disorder is still unknown despite strong genetic evidence [231, 232]and environmental factors. genetic factors have been estimated to contribute to half of the asd cases [231, 232], with the remaining contributed by ecological and other risk factors. considering the gut-brain axis concept, studies have explored the possible association between the gut microbiome and asd. asd children were found to have more bowel disorders, including abdominal pain, constipation, diarrhea, and gastric reflux [233, 234]. it will be worth finding a causal relationship between gut dysbiosis, which contributes to bowel disorders, and autistic behavior in asd individuals. exciting to note that not until recently, the effect of microbiota transfer therapy has been shown to improve the behavioral symptoms of asd for at least 8 weeks [235]. this further supports the direction of investigating the link between the gut microbiome and asd as it shows the possibility of reversing autistic behavior with alteration of gut flora. it is found that children with asd have a deficit in gut integrity [236] and a discrepancy in the gut microbiome's n composition instead of healthy individuals. studies over the years have noted the differences in gut microbiota between healthy versus asd individuals, with some common findings including clostridium spp., prevotella spp., bifidobacterium spp., candida spp., and firmicutes spp., to be greater in population and lacking anaerobic bacteria in individuals with asd [237-241]. the significance of gut flora composition is that there were events where children treated with antibiotics developed chronic diarrhea, followed by regressive symptoms [237, 240, 242, 243]. these clinical progressions may be due to the altered gut microbiome's antibiotics' consequences [244]. clostridium spp., especially c. bolteae, c. perfringens , c. difficile [237] and c. histolyticum [239], is abundant gut flora of asd children. it is suggested that they play a role in developing autistic behavior via neurotoxin ρ-cresol production [243, 245], which is evident pmmb 2020, 4, 1; a0000175 17 of 40 by the raised serum level of p-cresol chemically similar metabolites in asd children [245]. the toxin produced by the gut microbes can be transported by the vagus nerve directly into the brain [243]. the toxin then affects the cleaving of synaptic vesicle membrane protein, affecting the release of neurotransmitters [243]. this lowered neurotransmitter level causes behavioral changes, which are observed to be in parallel with the characteristic behavior of asd children [243]. in addition, a higher concentration of clostridium spp. is associated with more severe autistic symptoms [246]. the notion is further supported by the transient improvement of asd symptoms in individuals with regressive autism treated with vancomycin, which is an antibiotic with activity against clostridium spp. [247]. this suggest that clostridium spp. may be a potential cause of regressive autism, but subsequent controlled studies have yet to replicate the results. besides that, candida spp. has also been recently found to be associated with asd [246]. gut dysbiosis in individuals with asd has allowed this commensal's overgrowth, producing propionic acids and ammonia [248]. these metabolites have been shown to react and result in beta-alanine production, chemically like inhibitory neurotransmitter gamma-aminobutyric acid (gaba) found in the brain. assuming if the beta-alanine can cross the blood-brain barrier [249], they can occupy the gaba receptors [250] in human brain tissues, thus stimulating the compensatory production of gaba [251]. this excess in gaba is in keeping with the increased gaba concentration observed in children with asd [251]. further studies can be done to determine whether the raised level of beta-alanine observed in individuals with asd does function similarly as suggested and subsequently triggering the remaining chain of reaction, leading to the development of asd [252]. other organisms are being studied for their association with asd, but no significant results have been found, or the mechanisms of them causing asd is yet to be explained [253]. as discussed, gut dysbiosis is a possible mechanism that leads to the development of asd. factors that may alter the gut flora include antibiotics [253], diets, variation in genetic and medical heterogeneity, and comorbidities. pinpointing the exact cause of gut dysbiosis, which subsequently leads to asd impossible [254]. finally, increased intestinal permeability in asd children allows the escape of gut content into the systemic circulation, thus leading to a systemic pro-inflammatory state. this corresponds with the chronically raised cytokine levels observed in individuals with asd, and some of these inflammatory mediators (il-1β, il-6, il-8, and il-12p40) are linked explicitly to low social interaction and communication [255, 256]. in short, further studies are needed to identify the exact organisms and their mechanisms in the development of asd and recognize factors that may alter the homeostasis and integrity of the gut, leading to the development of asd. pmmb 2020, 4, 1; a0000175 18 of 40 3.7. alzheimer’s disease alzheimer’s disease (ad) is a neurodegenerative disease characterized by progressive cognitive function loss [257]. the most widely accepted mechanism that leads to ad is the deposition of amyloid plaques and neurofibrillary tangles [257]. however, the exact cause of this deposition is still unknown and is believed, to date, to be multifactorial [258]. with the emergence of the gut-brain axis, ad has also been attributed to gut dysbiosis [257, 259]. disease progression of ad has been hypothesized to be due to chronic inflammation following deposition of amyloid plaques [260]. however, recent studies have shown that these plaques possess antimicrobial activities, suggesting that neuroinflammation may cause the deposition of these plaques instead of vice versa [258]. moreover, a study suggests that amyloid plaques and neuroinflammation deposition is a vicious cycle [261]. thus, there is now a chronic inflammatory state in the brain tissue of individuals with ad. more studies are needed to fill the gap of causal effect relationships between amyloid plaques and neuroinflammation. when considering the possibility of the gut microbiome affecting neuronal functions, animal studies have shown a significant reduction of amyloid plaques in germ-free mice [262]. nonetheless, when gut microbiota is reintroduced, the mice demonstrated an increase in cerebral amyloid plaques pathology [262]. this is further supported by several studies that showed that gut infection is associated with ad development [258]. for example, individuals infected by borrelia burgdorferi and h. pylori have been observed to have raised serum aβ40 and aβ42, which is a biomarker of ad [263]. these pathogens may also contribute to ad's development by inducing an inflammatory response in the brain [264]. ad individuals have an abundance of pro-inflammatory microbiome and reduced anti-inflammatory gut microbes [265]. some of these pathogenic microbes can secrete pro-inflammatory neurotoxins, which can affect the neurofunction [266]. another pathology observed in ad is the reduction of bdnf proteins in the hippocampus [258]. it is showed that probiotics have been able to reverse this in the mice model, suggesting that specific gut microbiome are protective and needed for the brain's normal functioning [267]. recent breakthroughs in discovering the superior temporal lobe and hippocampus lysates of individuals with ad are saturated with lipopolysaccharide [266]. this could be due to the gram-negative gut microbes, which can stimulate local inflammation, disrupting the gut barrier, allowing the translocation of gut microbes, and endotoxins into the systemic circulation [268]. when these molecules such as lipopolysaccharides reach the brain, it can induce local inflammation or affect the nerve functions directly or indirectly [269]. on the other hand, some gut microbes can produce neuroprotective molecules such as short-chain fatty acids, and losing these protective microbes can alter brain tissue's normal function [258]. for instance, li et al. have shown that clostridium butyricum species can enhance the intestinal and blood brain barrier structural integrity and improve neurodegeneration symptoms [270]. pmmb 2020, 4, 1; a0000175 19 of 40 is it also known that our gut microbiome changes physiologically as we age [258]. there is an increase in proteobacteria and reduction of probiotics (bifidobacteria) and scfa producing microbes [271]. the significant decrease in functioning microbiome, especially those with scfa is their deficit associated with raised inflammatory mediators [272]. this aging-related pro-inflammatory state is known as inflamm-aging. it is the common pathology for a wide range of age-related diseases, including degeneration of cognitive functions in ad [273] and increased ad prevalence with age. 4. emerging treatments probiotics probiotics are live microorganisms that have numerous benefits for our health when consumed [274]. the story of probiotics begins about a century ago, where the process of fermentation was initially used as a means of food preservation [275]. with people starting to notice the health benefits of consuming fermented food, this food was adapted for various uses. for example, treatment of diseases and consumption for energy and strength [275]. elie metchnikoff, an immunologist, suggested that probiotics found in fermented food can improve health and increase lives [274]. this, later, created a ripple effect, encouraging more scientists to venture into the discovery of probiotics. they believe that not all bacteria are pathogenic, and some of them can replace the pathogenic flora, creating a healthy gut microbiome. with the blooming studies on the gut health axis, therapies to create a ‘healthy’ gut flora are always searching. with the known effect of probiotics, it is not surprising that probiotics are proposed as one of the therapeutic options. probiotics are suggested to confer its effect by preventing pathogenic microbes' overgrowth, enhancing the gut's structural and functional integrity, and modulating the gut immune system [18]. this effect is conferred by the probiotics' cell-surface architecture, such as their surface proteins and capsule. this can be done by various mechanisms that include enhancing natural killer cells, immune phagocytes, or upregulation of antibodies [276, 277]. to date, there are numerous studies on various health diseases and probiotics use, with some showing promising results. for instance, probiotics use has been shown to reduce the duration of diarrhea in acute gastroenteritis and c. difficile infection [278, 279], inducing more prolonged remission in ibd [280, 281]and modulating the immune response in patients with allergy [282, 283]. some probiotics strains can increase antiinflammatory mediators' concentration, such as tumor necrosis factors, which can abate diseases [276, 284]. traditionally, the most widely used yeast strain is the saccharomyces cerevisiae, whereas the most common bacterial probiotics are the bifidobacterium and lactobacillus species [285]. for instance, l. plantarum, l. rhamnosus, b. bifidum, and b. breve [286]. there is a variation of the functions between different probiotics strains, even if there are of the same species [285]. for example, strains that initially fail to prevent necrotizing enterocolitis pmmb 2020, 4, 1; a0000175 20 of 40 in infants [287], will successfully prevent sepsis when replaced with another formulation [288]. thus, it will be imperative to evaluate the functions and efficacy of each of these strains of probiotics for disease treatment [285]. another issue with probiotics is worth noting because its overall effectiveness is also influenced by the host factors, including the baseline gut microbiome [28]. maldonado et al. have shown that the efficacy of probiotic bifidobacterium longum subsp. longum ah1206 is the highest if the host has low abundance of b. longum and carbohydrate metabolizing genes [289] however, disappointing results contradict the positive outcomes with probiotics use [290292]. also, there were reported events of adverse effects associated with probiotics consumption [293]. the significant side effects are particularly concerned in individuals with an immunodeficiency. they are at risk of sepsis, bacteremia, or endocarditis with probiotics consumption [285]. this makes the current recommendation of probiotics as a therapeutic option impossible. the studies cannot conclude as there is always discrepancy on the type of probiotics, criteria of the selected population, and the outcomes [293]. thus, we cannot know which particular strains are beneficial and which populations will benefit the most from probiotics consumption [293]. besides that, there are also insufficient studies to identify people at risk of consuming probiotics [293]. this issue needs to be addressed in view that probiotics may be a potential therapeutic option in the future. furthermore, there is still difficulty explaining probiotics' outcomes on human health via mechanisms found in laboratory studies [290]. this makes it difficult to extrapolate the effect of probiotics on human diseases [290]. it is known that alteration of gut microbes can affect the gut's functional and structural integrity, which can affect other host’s organ systems. with this strong relationship between gut dysbiosis and various health diseases, it is still worth continuing the search for the effect of probiotics on human health. the probiotic use has been recommended in some clinical guidelines for children in antibiotic-associated diarrhea, acute infectious diarrhea, necrotizing enterocolitis, infantile colic, ulcerative colitis, and irritable bowel disease [294, 295]. however, there is still no clinical recommendations for the clinical use of probiotics in adults [28]. current research has been exploring different human commensals other than the traditional genera bifidobacterium and lactobacillus in view that different strains have distinctive effects on the host [28]. this research also needs to be gradually moved on to human trials after confirming its safety profile and efficacy on animal experiments [296]. it is also essential to understand that the outcomes depend on the formulations, participants, doses, and clinical endpoint [28]. perhaps, with more standardized trials done, we will be able to come out with a more definite conclusion on the clinical use of probiotics [290]. prebiotics and dietary fibers fiber is plant-derived food that cannot be digested by the human gastrointestinal tract [297]. instead, they need to be fermented by gut microbes to extract their nutrients [298]. they are various kind of fibers which can be derived from different plants [299, 300]. each of them pmmb 2020, 4, 1; a0000175 21 of 40 possesses specific properties and has other effects on the host when consumed [300]. if the fibers can modify the gut microbiome in such a way that confers health benefits, they can be considered prebiotics [301]. this modification includes altering the microbiome's composition, regulating the activities and metabolites produced by the microbes beneficial to the host [302]. a diet low in fibers has been shown to shift the gut microbiome drastically. human trials have demonstrated that a switch from a high fiber plant-based diet to a fiber-deprived meatbased diet can cause significant changes within as short as 24 hours [303]. also, fibers deprived of fiber is associated with reduced biodiversity of the gut microbiome [304]. this is supported by the depleted diversity of gut microbiota observed in mice fed with fiber depleted diet [305]. however, this deprivation is reversible by re-introducing a high fiber diet [306]. this reversibility of gut flora biodiversity may shine a light on various diseases that have been associated with the low diversity of gut microbes. this suggests that a diet high in fibers can help maintain the gut microbiome's homeostasis, preventing pathogenic microbes from causing various physical and mental diseases. the fermentation end products are mainly short-chain fatty acids (scfa), which confers invaluable benefits to human health [303]. for instance, lactobacilli spp. produces metabolites, including scfa, via fermentation of fibers with anti-carcinogenic effect [307]. they have been shown to prevent colorectal cancer by reducing the ph of colorectal cancer [307]. on the other hand, lacking of scfa has been observed in individuals with ibd due to their depletion of scfa producing bacteria [308]. depriving scfa means the host has lost its ability to inhibit pro-inflammatory state activation, as in individuals with ibd. this is supported by the reduction of inflammatory markers via the consumption of prebiotics [309]. moreover, specific prebiotics such as psyllium, beta-glucans, and pectins are classified as high glycemic index food [299]. they possess unique properties of slowing glucose absorption, which can benefit patients with metabolic disorders [299, 310]. interestingly, despite the unknown mechanism, prebiotics has been shown to suppress type 2 t helper responses, which can have an immunomodulatory effect on individuals with atopy diseases [311, 312]. arslanoglu et al. have shown that infants fed with prebioticsupplemented hypoallergenic formula had a lower risk of allergies in pre-school age [313]. moreover, animal studies have shown that prebiotics that can promote the growth of protective gut microbes, in which intestinal permeability is reduced, the concentration of endotoxin is decreased, thus reducing the prevalence of metabolic disorders [314] when extrapolating prebiotic use into clinical use, the evidence is still insufficient compared to probiotics [28]. there are currently no clinical guidelines recommending prebiotic use in neither the pediatric nor adult population [28]. as with probiotics, studies have shown that prebiotics' effectiveness depends on the baseline gut flora, which can be influenced by multiple factors such as age, diet, and lifestyle [315, 316]. besides that, formulations, participants, doses, and clinical endpoints must be considered when planning pmmb 2020, 4, 1; a0000175 22 of 40 future research. all these factors can influence prebiotic use's effectiveness compared with probiotics [28]. when discussing therapies' safety profile, prebiotics seems to have minimal lifethreatening side effects compared to probiotics [317]. they are mostly harmless and just indigestible fibers. thus, the most probable side effects are the osmotic functions of the prebiotics' fermented product [317]. for example, prebiotic consumption can cause osmotic diarrhea, flatulence, and abdominal cramps [317]. interestingly, the safety profile is dosedependent, and the therapeutic dose is usually low. this implicates that if consumed appropriately, the side effects of prebiotics are usually mild [318]. a further advantage of prebiotics is that it’s convenient in production, storage, and transportation compared to probiotics and fecal transplantation [317]. it does not need to be preserved via a cold chain makes the whole process of making prebiotics available to the patients much easier [317]. overall, a diet high in fibers and prebiotics has multiple benefits for our health and can play a disease-modifying role in various diseases. with more human trials done, perhaps it can be a potential intervention and recommended therapeutic option for many conditions. fecal microbiota transplantation fecal microbiota transplantation (fmt) transfers gut microbes from a donor to a recipient, either by infusion into the colon by colonoscopy or delivered via upper gastrointestinal tract such as capsule ingestion, by gastroenteric tube or endoscope [303]. this procedure was reported as early as 1958 by eiseman et al., where few patients with pseudomembranous colitis recovered after receiving fmt [319]. fischer et al., also showed the beneficial effect of fmt on individuals with severe and severe-complicated clostridium difficile infection [320]. they have shown promising results with a significant cure rate in all the participants [320]. the increasing studies on the association between gut microbes and health diseases have created ripples to modify the gut microbiota as a therapeutic option for gut dysbiosis related health diseases [268, 321]. with the success of fmt in the treatment of clostridium difficile infection, fmt seems to be a plausible method to help modify other diseases related to disruption of the gut microbiome. currently, fmt has been adopted by the u.s guidelines second-line treatment for recurrent clostridium difficile infection [322]. at the same time, the european proposed fmt guidelines after the first episode of severe and refractory c. difficile infection [323]. it is also showing promising experimental results in gut dysbiosis related diseases such as ibd [324, 325], metabolic syndrome [326], and autism spectrum disorder[235] but all of them are still in the experimental phase [23]. the donor stools for fmt can be obtained from either universal donors via stool banks or patient-directed donors [327]. patient-directed donors have been less frequent as it is more time-consuming to collect, screen, and process, leading to treatment delay [328]. also, the donor chosen may feel vulnerable for their confidential information [329]. thus, the most preferred option to date is the universal donor fmt. stools are collected from pmmb 2020, 4, 1; a0000175 23 of 40 young adults who have passed the screening test [330]. the advantage of universal donors is that recipients can receive microbiome from multiple donors who may provide a greater diversity of microbes instead of receiving only the gut flora from a single donor. this is further supported by an rct which showed that multidoor recipients showed improvement in the disease progression of ulcerative colitis [331]. with the increase in popularity and evidence on the efficacy of fmt, stool banks such as openbiome have emerged [332]. they are usually strict criteria to screen donors, standardized protocols for stool handling, processing, and delivering. however, there is a concern that some infections may go undetectable, which may risk infecting the recipients [332]. to note that a few years back, the fda has been alerting health practitioners and patients on the potentially severe and life-threatening infections due to fmt. however, in these cases, pathogens have been identified from the fecal transplantation product [333]. thus, a regulated protocol on fmt should be detailed enough to maximize reproducible outcomes and minimize adverse events to patients [334]. other than the risk of infection, typical side effects with fmt are loose stools and bloating, which usually resolve spontaneously within a day [335]. also, the long-term impact and risks of fmt are still unknown. longer-term follow-ups must fill this gap. lastly, it is still unknown what constitutes a ‘healthy’ gut microbiome, which can be the ‘ideal’ fecal microbiota for transplantation. we are assuming healthy individuals have ‘healthy’ microbiota. more research on the genomes of microbes required for good health is crucial for understanding the mechanism behind fmt [336]. 5. conclusion from this review, we know a strong association between the gut microbiome and various diseases, regardless of whether it is a physical or mental disorder. the structural and functional integrity of the gastrointestinal tract is imperative in the maintenance of good health. disruption of gut homeostasis has been demonstrated to contribute to the development of diseases. meanwhile, the microbes can be categorized into protective and pathogenic subgroups. different species have their mechanism in disrupting or protecting the gut flora. if the gut's homeostasis is lost, local inflammation will lead to gut microbes' translocation, leading to a pro-inflammatory state observed in various diseases. some toxins or beneficial scfa molecules can also influence other systems such as metabolic and neurofunction by several pathways. moreover, we have now known that we may reverse or alter gut-dysbiosis-related diseases by modifying the gut microbiome [337]. we have reviewed that probiotics, prebiotics, and fecal transplantation are potential or current therapeutic options for gut-dysbiosis associated diseases. despite supporting data, more standardized studies are still needed to obtain a definitive outcome for their safety and beneficial use in humans. if it is proven to be effective, studies can even move on to investigate their use in disease prevention, especially with prebiotics that has minimal side effects in human studies. in conclusion, there is a strong pmmb 2020, 4, 1; a0000175 24 of 40 association between the human microbiome and diseases. this is a potential field further to investigate the characteristic microbiome for every gut-dysbiosis related disease. with that, it may be possible to modify illnesses that have never been possible before, such as autistic spectrum disorder, which will be life changing. author contributions: aw-yl performed the literature search, critical data analysis as well as manuscript writing. lt-ht, n-sam, shw, l-hl and vl provided review, editing, and proofreading for this manuscript. l-hl and vl conceptualize this review writing project. funding: the seed funding funded this work from microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, (vote number: mbrs/jcsmhs/02/2020) awarded to vl. acknowledgments: professor dr. shajahan yasin, professor, and head of school, jeffrey cheah school of 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review and meta-analysis. am j gastroenterol 2013; 108(4): 500-508. 330. osman m, stoltzner z, o'brien k, et al. donor efficacy in fecal microbiota transplantation for recurrent clostridium difficile: evidence from a 1,999-patient cohort. in open forum infectious diseases. 2016. oxford university press. 331. paramsothy s, kamm ma, kaakoush no, et al., multidonor intensive faecal microbiota transplantation for active ulcerative colitis: a randomised placebo-controlled trial. lancet 2017; 389(10075): 1218-1228. 332. kim ko and gluck m, fecal microbiota transplantation: an update on clinical practice. clin endosc 2019; 52(2): 137. 333. smith m, kassam z, edelstein c, et al., openbiome remains open to serve the medical community. nat biotechnol 2014; 32(9): 867-867. 334. petrof eo, gloor gb, vanner sj, et al., stool substitute transplant therapy for the eradication of clostridium difficile infection:‘repoopulating’the gut. microbiome 2013; 1(1): 3. 335. kelly bj and tebas p, clinical practice and infrastructure review of fecal microbiota transplantation for clostridium difficile infection. chest 2018; 153(1): 266-277. 336. yang y, tian j, and yang b, targeting gut microbiome: a novel and potential therapy for autism. life sci 2018; 194: 111-119. 337. lee l-h, ser h-l, letchumanan v, et al., the role of gut microbiome in traditional chinese medicine syndromes: focusing on the spleen deficiency syndrome. gut 2020; 69(suppl 2): a19. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. 1. introduction 2. origins of our gut microbiome 3. gut microbes and diseases 3.1. gastrointestinal diseases 3.1.1. inflammatory bowel diseases 3.1.2. gastrointestinal malignancies a) colorectal cancer b) gastric cancer 3.2. infectious diseases 3.2.1. clostridium difficile infection 3.2.2. human immunodeficiency virus infection 3.3. metabolic disorders 3.3.1. obesity 3.3.2. type 2 diabetes 3.4. pulmonary health and asthma 3.5. mental health 3.6. autism spectrum disorder 3.7. alzheimer’s disease 4. emerging treatments 5. conclusion references progress in microbes and molecular biology review article 1 novel coronavirus 2019-ncov: could this virus become a possible global pandemic vengadesh letchumanan1a*,nurul-syakima ab mutalib2a, bey-hing goh3, learn-han lee1* 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, bandar sunway, selangor darul ehsan, malaysia. 2ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia 3biofunctional molecule exploratory research group (bmex), school of pharmacy, monash university malaysia, 47500, bandar sunway, selangor darul ehsan, malaysia. athe authors contributed equally to the writing of this review abstract: in december 2019, the emerging of a new coronavirus-induced pneumonia created a distress among people in china and the international community. this virus was identified as novel coronavirus 2019-ncov. at the time this review went to press, the virus has spread to across 27 countries, infecting 17,488 people and caused 362 deaths. the transmission of 2019-ncov to individuals of different countries is predominantly through close contact with an infected person. based on the available data, there is a rising trend of infected and death cases. at this point, there is no specific drug or vaccine to treat infected patients besides supportive care. nevertheless, researchers and doctors around the world have reported the success of using existing antiviral drugs to treat patients. keywords: novel coronavirus, 2019-ncov, supportive care, global pandemic, treatment received: 3rd february 2020 accepted: 5th february 2020 published online: 6th february 2020 citation: letchumanan v, ab mutalib n-s, goh b-h, et al. novel coronavirus 2019-ncov: could this virus become a possible global pandemic. prog microbes mol biol 2020; 3(1): a0000068. https://doi.org/10.36877/pmmb.a0000068 introduction a new year and a new decade kicked off with a global health threat and challenge, the novel coronavirus 2019ncov. news emerged in early december 2019 of pneumonia outbreak by an unknown aetiology, where it is believed to be contracted at wuhan’s huanan seafood wholesale market[1]. on 31st december 2019, the world health organization (who)’s chinese office was notified of pneumonia cluster cases, which led to the closing of the market on 1st january 2020[1]. the chinese authorities and who identified it as a new type of coronavirus, better known as 2019-ncov[2]. the 2019-ncov virus belongs to same coronavirus family which includes the severe acute respiratory syndrome (sars) — the virus that caused an eventual 8,096 cases and 744 deaths in 2002-2003[3]. the virus became an instant hot news and raised the fear of the international community. the health organizations along with scientists and clinicians worked around the clock to uncover the information of 2019-ncov. within days of detecting the initial cluster of cases, the complete genome sequence of 2019-ncov with a sequence length of 29,903bp was deposited on genbank and global initiative on sharing all influenza data (gisaid)[4,5]. the genome sequence information is beneficial in the development of primers for surveillance tests and diagnostic kits[3]. pharmaceutical companies and researchers around the world have been working on different approaches to produce an effective drug or vaccine for 2019-ncov[6]. these efforts accelerated after over 30 genome sequence of 2019-ncov have been publicly deposited in genbank (table 1)[7]. table 1. genome sequence of 2019-ncov deposited in genbank. genbank accession gene region locality mt008023 m italy, rome mt008022 m italy, rome mt007544 complete australia mn997409 complete usa mn996531 complete wuhan mn996530 complete wuhan copyright 2019 by letchumanan v et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) *correspondence: learn-han lee, jeffrey cheah school of medicine and health sciences, monash university malaysia, selangor, malaysia; lee.learn.han@monash.edu; leelearnhan@yahoo. com. vengadesh letchumanan, jeffrey cheah school of medicine and health sciences, monash university malaysia, selangor, malaysia. vengadesh.letchumanan1@monash.edu 2 mn996529 complete wuhan mn996528 complete wuhan mn996527 complete wuhan mn994468 complete usa mn994467 complete usa mn988713 complete usa mn988669 complete china mn988668 complete china mn985325 complete usa mn975268 s china mn975267 s china mn975266 s china mn975265 rdrp china mn975264 rdrp china mn975263 rdrp china mn975262 complete china mn970004 rdrp thailand mn970003 rdrp thailand mn938390 s shenzhen rdrp:rna-dependent rnapolymerase; s: surface epidemiology 2019-ncov reservoir researchers compared the genome sequence of 2019ncov with a viral database and suggest it belongs to betacoronavirus originated from bats. however, the specific wildlife host as virus reservoir is yet to be confirmed since various live animals was sold at the market[8,9]. a comprehensive genome sequence analysis was carried out by researchers from beijing, china to determine the possible animal reservoir. their evolutionary analysis suggested that snakes are the most likely wildlife animal as virus reservoir for 2019-ncov[10]. conversely, scientists affiliated with uk-based universities disputed the findings by chinese researchers, and affirmed that 2019ncov is most closely related to bats[11]. zhou and colleagues from china also reported this virus is potentially bat origin[12]. pushing aside on the debate whether bats or snakes are the real culprit of 2019-ncov, this zoonotic virus has spread to over twenty-seven countries worldwide. the spreading wuhan, the epicenter has been left deserted amid the deadly coronavirus-induced pneumonia outbreak. in aim to contain the virus spread, the local authorities ordered a city-wide lockdown in wuhan, the sprawling capital of central china’s hubei province on 23 january 2020[1,13]. although, early reports revealed limited human to human spread, but the recent drastic increase of infected cases proven the latter assumption was wrong. it is indeed a human-to-human transmission and the virus is spreading in a fast pace to other chinese cities, asian countries and other parts of the world[14,15] (table 2). this fast-spreading virus has claimed 362 lives and infected 17,488 people in 27 countries at the time this editorial went to press (figure 1). china tops all the countries with over 17,000 infected patients and 361 deaths (table 3), this figure increases daily with an estimated transmission rate/basic reproductive number (ro) 3.0-4.0 (3–4 newly infected cases from 1 case)[16]. on 2nd february 2020, a death of a 44-year-old chinese man from wuhan in philippines marked the first death that occurred outside of china. the patient showed initial signs of improvement, however his condition deteriorated within the last 24 hours and he succumbed[17]. the concern raises as transmission of 2019-ncov to other countries besides china is mainly by people who have travelled from the epicenter of the outbreak or had close contact with an infected person. for instance, in malaysia, eight confirmed cases are patients of china nationality, and they have been under isolation hospitalization until fully recover from the infection. of note, based on the existing epidemiological data, the incubation period of this virus is 14 days. 2019-ncov: could this... figure 1. illustration of novel coronavirus 2019-ncov transmission across the global. as of 3 february 2020 , this virus has spread to over 27 countries and alarming a fear to the international community. 3 table 2. the worldwide spread of coronavirus 2019-ncov. countries confirmed cases death cured china 17,302 361 512 hong kong 15 taiwan 10 japan 20 1 thailand 19 7 singapore 18 korea 15 australia 12 2 germany 10 malaysia 8 us 11 vietnam 8 1 france 6 united arab emirates 5 canada 4 india 3 italy 2 philippines 2 1 russia 2 uk 2 cambodia 1 finland 1 nepal 1 spain 1 sri lanka 1 1 sweden 1 macau 8 source: national health commission of the people’s republic of china and worldometer on wuhan coronavirus outbreak https://www.worldometers.info/coronavirus/. table 3. the spread of coronavirus 2019-ncov in china. province confirmed cases death cured hubei 11177 350 295 zhejiang 724 36 guangdong 725 14 henan 566 2 15 hunan 521 17 anhui 408 8 jiangxi 391 18 chongqing 312 2 8 jiangsu 271 7 szechuan 254 1 13 shandong 259 7 beijing 212 1 12 shanghai 203 1 10 fujian 179 shaanxi 128 guangxi 127 7 yunnan 114 5 hebei 113 1 3 heilongjiang 118 2 2 liaoning 74 1 hainan 70 1 4 shanxi 66 2 tianjin 56 1 ganxu 51 3 guizhou 46 2 ningxia 31 inner mongolia 34 1 jilin 31 1 xinjiang 24 hong kong 15 qinghai 13 taiwan 10 macau 8 tibet 1 source: national health commission of the people’s republic of china, as of 3rd february 2020 letchumanan v et al. 4 management measures lock-down and medical facilities the occurrence of this virus has placed the chinese government in a strong crisis management and preparedness for a pandemic. the government imposed a complete lockdown of wuhan and about a dozen of other cities, in an attempt to prevent the virus from spreading further within china. the effectiveness of this implementation has been disputed by many experts globally. professor chandy john, a former president of america society of tropical medicine and hygiene mentioned that a number of potential issues may arise including basic human rights concern, inadequate healthcare facilities or medication for the sick people, and people who aren’t sick — unable to leave and may fall ill after exposure to the virus. however, he still agrees that prevention of travel is one the effective way to contain the virus and reduce the transmission[18]. likewise, a bioethicist at new york university also expressed an agreement for wuhan quarantine and added that it is a prudent action implemented by the government[18]. since the majority of infected cases are in wuhan, the chinese government has invested huge capital in building two new hospitals: huoshenshan hospital and leishenshan hospital that would be operational in early february 2020[19,20]. in addition, the chinese government has approved the sale of two virus detection kits and a sequencing system by genomic company bgi group, to aid and enhance the identification of this novel virus. according to bgi sources, their test kit is able to identify the novel 2019-ncov within three hours, while the other kit can assist to differentiate and the diagnose infections. one hundred thousand test kits have been dispatched to the country’s worst hit regions[21]. global effort and protection measures in other parts of the nation and several countries has taken precaution measures to control the spread of coronavirus. health screening in all entry and exit points (airports, port terminals, train stations) have been beef-up in effort to contain the virus[22]. passengers who present any clinical symptoms of fever, flu and cough, are placed in an isolation and quarantine for further evaluation and treatments. many airlines have also drawn-up preventive measures by not providing hot meals, blankets and newspapers in the plane, in aim to contain the virus and reduce the personal contact among passengers[23]. the healthcare organization has been promoting awareness and preventive measures that should be followed by the members of the public. people should adopt good hygiene practice by washing their hands with water and soap, or use hand sanitizer, always wear a mask, avoid crowded public locations, and avoid close contact with any infected person. a person should directly report to their respective healthcare centers if they are presenting any symptoms and other risk factors of 2019-ncov infection including had a travel history or close contact with a confirmed patient. the novel coronavirus 2019-ncov has created an impact to the public healthcare and to our community. in a recent interview, dr huang chaolin, the vice director of wuhan jinyintan hospital, revealed that there might be multiple places where the virus was first originated and transmitted to humans[24]. it is because the first few patients who was admitted at the hospital had no direct exposure to wuhan’s huanan seafood wholesale market, which was considered the primary source of the epidemic. judging from the whole situation, there could be multisources, nonetheless the authorities have no clue about other sources. the chinese center for disease control and prevention have collected environmental samples from wuhan’s huanan seafood wholesale market, and over thirty samples were found to contain the nucleic acid of the virus. if the virus is from multi-sources, experts warned the situation would become worsen in the coming days if efforts taken to contain the virus are ineffective[24]. treatment options currently, there is no specific drug or vaccine available to treat this virus infection besides supportive care. a patient will present symptoms of fever, fatigue, dry cough, shortness of breath, runny nose and sore throat [25]. patients with underlying comorbidities for example diabetes, hypertension, chronic lung disease, asthma, cardiovascular diseases, and immunocompromised are prone for severe complication of 2019-ncov infection [25,26]. hence, the older group and children are advice to take extra precaution measures to avoid been contract with 2019-ncov. in hospitals, doctors are doing their best to treat and save the lives of their patients, without the effective drug or vaccine. doctors from thailand successfully treated a 71-year-old patient with a combination of antivirals that used to treat flu (oseltamivir) and hiv (lopinavir and ritonavir) [27]. where else, in the united states (us), doctors treated a 2019-ncov positive patient with experimental gilead sciences drug remdesivir. remdesivir was given to the 35-year-old patient and his symptoms improved without any side effects [28]. although the combinations of antiviral treatments have shown positive effect in treating 2019-cov patients, further tests should be done to confirm the effectiveness of these antivirals. hopefully, this novel coronavirus does not exhibit any resistance phenotype towards flu antivirals e.g. oseltamivir-resistance, which was observed previously in influenza a h7n9 virus [29]. conclusion the rapid rising of infected cases and deaths alarm experts, who fear the virus 2019-ncov would likely become a pandemic across the globe. the lethally of this new virus is yet to be known, but now findings suggest that the virus may be spreading from person to person via the digestive system [30,31]. there is an increasing trend of infected cases in china and around the worldwide countries. nevertheless, the china government, who and respective healthcare bodies are doing their best to understand and manage the coronavirus outbreak. on 30th january 2020, who declared the novel 2019-ncov coronavirus outbreak as a public health emergency of international concern (pheic), and this brings an impact to china and international community 2019-ncov: could this... 5 [32,33]. the pheic will see the mobilization of international response to work to together to contain the virus threat. hopefully, this announcement would help with controlling the spread of the virus. in view of the present virus outbreak, the community should adapt to the new lifestyle changes. social distancing must be a part of their daily life. avoid being proximity with people, mass gatherings, and functions. in certain countries, their public has the tendency to greet others by hugging or a handshake. without realizing, they probably might be spreading or contracting the virus by their actions. now, we may want to stop this culture for the time being. another aspect is self-quarantine if one exhibit any symptoms of infection or had a close contact with a confirmed patient. this measure is proven to be an effective tool to contain the spread of any airborne virus. the nation may feel stressed and unpleasant if a lock-down is imposed in their country or asked to wear mask. looking back at history, lock-down and wearing mask was seen to be an effective step implemented by dr wu lien-teh in effort to control the spread of pneumonic plague virus in 1910. dr wu lien-teh, a malaysian born doctor gained fame by ending the pneumonic plague in china and encouraging people at that time to wear gauze-and-cotton masks, controlled the people’s movement, instructed to hospitalize all infected patients, carried out disinfection in open areas, mass burials of the deaths, and prohibited close contact [34]. his endless effort managed to contain the plague within seven months (march 1911). in summary, the novel coronavirus 2019-ncov outbreak is a reminder to the international community of an emerging virus infection that need constant surveillance, rapid diagnosis, and robust research to understand the biology features of this novel virus. there is a possibility this novel coronavirus would become a global pandemic which the severity and people’s susceptibility is yet to be forecasted by the experts. if it becomes a pandemic, the developing countries may not have enough resources to cope this deadly virus. in addition, countries may experience severe setbacks as the world economic may paralyze and will take months for the recovery. hence, the world should get prepared with adequate preventive measures to contain the spread of this deadly virus. the international community should have the preparedness for a possible pandemic outbreak and adhere to all the precaution measures. the healthcare sector should develop effective countermeasures and ensure all medical resources like masks. ppes, and ventilators are readily available in all hospitals. well, it is rather safer to be over prepared than being under prepared on facing this novel coronavirus 2019-ncov. conflict of interest the authors declare that there is no conflict of interest in this work. author contributions vl and n-sam performed the literature search, critical data analysis and writing of this review. technical support and proofreading was contributed by b-hg and l-hl. this review writing was founded by l-hl. acknowledgement this work was supported by pvc award grant (project no. 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(accessed on 31 january 2020). 33. the coronavirus outbreak is now a public health emergency of international concern here’s what that means. available online: https:// time.com/5774747/coronavirus-who-public-health-emergency/. (accessed on 30 january 2020). 34. lee, kh., wong, dtk., ho, tm., et al. dr wu lien-teh: modernising post-1911 china’s public health service. sing medic j 2014; 55(2): 99-102 pmmb 2022, 5, 1; a0000275. doi: 10.36877/pmmb.a0000275 http://journals.hh-publisher.com/index.php/pmmb review article malaysia’s breakthrough in modern actinobacteria (mod-actino) drug discovery research angel yun-kuan thye1, vengadesh letchumanan1, loh teng-hern tan1,2, jodi woan-fei law1*, learn-han lee1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, 47500, malaysia; angel.thye1@monash.edu (ay-kt); vengadesh.letchumanan1@monash.edu (vl) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia; loh.teng.hern@monash.edu (lt-ht) *corresponding author: learn-han lee and jodi woan-fei law; novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, subang jaya 47500, malaysia; lee.learn.han@monash.edu (l-hl); jodi.law1@monash.edu (jwfl) received: 15 july 2022; received in revised form: 28 august 2022; accepted: 3 september 2022; available online: 12 september 2022 abstract: actinobacteria are well-known producers of metabolites with medicinal value. the application of actinobacterial compounds has been expanded to other fields, including agriculture, aquaculture, and cosmeceutical. with this, the term “modern actinobacteria” (mod-actino) was first coined by a malaysian researcher, associate professor dr. lee learn han, to define actinobacteria with modern applications. the present review aims to highlight the mod-actino research achievements in malaysia. the malaysian modactino strains are capable of exerting a wide range of bioactivities such as antimicrobial/anti-mrsa, anticancer, antioxidant, antifungal, and antimalarial. research on mod-actino is highly encouraged to harness the benefits of actinobacteria and unravel important metabolites for various applications. keywords: modern actinobacteria; actinomycetes; streptomycetes; drug discovery; anticancer; antioxidant; antimicrobial 1. introduction the global spread of new emerging diseases and existing diseases have been a public health burden. researchers constantly explore earth’s natural resources to discover novel disease prevention and treatment drugs. the need for new drugs has led to the unraveling of bioactive products deriving from microorganisms found on earth [1]. interestingly, a study predicted that earth houses up to one trillion microbial species and that there are as many as 99.999% microbial taxa yet to be discovered [2]. within the bacteria domain, the phylum actinomycetota (synonym actinobacteria) represents one of the largest taxonomic units pmmb 2022, 5, 1; a0000275 2 of 21 among 18 major lineages [3, 4], comprising 6 classes: actinomycetes (synonym actinobacteria or actinomycetia), acidimicrobiia, nitriliruptoria, coriobacteriia, rubrobacteria, and thermoleophilia [5]. generally, actinobacteria is the largest class, and it can be characterized into two groups, namely streptomyces (dominant genus) and non-streptomyces, also known as “rare actinobacteria” [6, 7]. streptomyces and rare actinobacteria are important sources of novel bioactive secondary metabolites. associate professor dr. lee learn han coined “modern actinobacteria” (mod-actino) to define actinobacteria with modern applications. briefly, this term applies to: (1) actinobacteria that synthesize natural products with new bioactivities; (2) actinobacteria that produced approved drugs and have been subjected to drug repurposing efforts; and (3) known or novel actinobacteria found from the unique environment [8]. this review aims to unravel the discovery of mod-actino from the diverse ecosystems in malaysia. this review will also offer insights into the vast bioactive properties of malaysia’s mod-actino, such as anti-mrsa/antimicrobial, anticancer, antioxidant, antifungal, and antimalarial. 2. actinobacteria reservoir actinobacteria are the most prolific source of secondary bioactive metabolites, with diverse structural complexity [9, 10]. they demonstrate beneficial applications for humankind in agricultural, medical, and industrial fields [9][3]. notably, around 50% of actinobacteria originate from the genus streptomyces, and approximately 75% of commercially valuable antibiotics have been derived from streptomyces [11]. many bioactive compounds have been isolated from actinobacteria in recent years, particularly from the genus streptomyces. streptomyces-derived compounds produce bioactivities such as antimicrobial, anticancer, antioxidant, antifungal, and antimalarial [12-16]. given the significant contribution by actinobacteria, current research focuses on exploring new metabolites from these microorganisms. researchers are beginning to tap into underexplored locations or extreme environments to isolate actinobacteria and investigate their secondary metabolites. actinobacteria are abundant in nature, and they are crucial soil inhabitants. the isolation source of actinobacteria was initially from terrestrial areas, which then expanded to freshwater and marine environments [1]. to date, actinobacteria are widely distributed across various underexplored areas and extreme environments, including mangroves [17], caves [18], mountain plantations [19], deserts [20], hot springs [21], and even in the arctic and antarctic regions [22, 23]. in malaysia, the most notable discoveries include the identification of numerous novel and bioactive streptomyces species isolated from mangrove soil. these novel streptomyces species also harbored valuable bioactive molecules. asia has one of the most extensive mangrove coverages, with a total global mangrove area of 42%. as a matter of fact, 3.7% of the global mangrove coverage is located in malaysia [24]. the mangrove ecosystem is one of the most productive environments. it is an underexplored source of actinobacteria with a huge potential to harvest biologically active compounds [7, 25]. a study investigating actinobacteria from tanjung lumpur mangrove forest (city of kuantan, pahang state, pmmb 2022, 5, 1; a0000275 3 of 21 malaysia) unraveled that there was indeed a high level of diversity within the phylum actinobacteria. out of the 87 isolates, 59.8% (n = 52) belonged to the genus streptomyces. in fact, they also discovered several rare actinobacteria, namely sinomonas, streptasidiphilus, leifsonia, and terrabacter, which were genera not commonly found in mangrove environments. additionally, most actinobacteria isolates were found to possess detectable biosynthetic genes polyketide synthetase (pks) and non-ribosomal polyketide synthetase (nrps), which indicated the ability to produce bioactive secondary metabolites [11]. therefore, mangrove is a good source for bioactive actinobacteria discovery. 3. mod-actino novel species discoveries in malaysia 3.1. novel streptomyces in malaysia many novel streptomyces species have been isolated and identified from malaysia’s mangrove forest. several of the novel streptomyces species that were discovered in tanjung lumpur (pahang) include streptomyces pluripotens (musc 135t, musc 137t) [14, 26-28] streptomyces malaysiense (musc 136t) [29-31], streptomyces gilvigriseus (musc 26t) [32, 33], streptomyces mangrovisoli (musc 149t) [34, 35], and streptomyces antioxidans musc 164t [17, 36]. there are also some novel streptomyces species that were discovered in kuching (sarawak), such as streptomyces monashensis (musc 1jt) [37-39], and streptomyces colonosanans (musc 93jt) [39, 40]. 3.2. novel rare-actinobacteria in malaysia several novel rare actinobacteria strains have been discovered in malaysia, including monashia flava (musc 78t) [41], microbacterium mangrovi (musc 115t) [42], sinomonas humi (musc 117t) [43], and mumia flava (musc 201t) [44]. these novel rareactinobacteria strains were isolated from the mangrove soil of tanjung lumpur, kuantan, in the state of pahang. 4. mod-actino in malaysia with important bioactivities 4.1. anti-methicillin-resistant staphylococcus aureus (mrsa) activity streptomyces is the largest antibiotic-producing genus that can produce antibiotics of different classes, including macrolides (tylosin from streptomyces fradiae), β-lactams (cephamycin and clavulanic acid from streptomyces clavuligerus), aminoglycosides (streptomycin from streptomyces griseus), tetracycline (tetracycline from streptomyces aureofaciens), and glycopeptide (vancomycin from streptomyces orientalis) [15, 45-50]. however, the improper use of antibiotics has led to an issue with multidrug-resistant pathogens, such as, carbapenem-resistant enterobacteriaceae (cre), vancomycinresistant enterococcus (vre), and methicillin-resistant staphylococcus aureus (mrsa). mrsa is non-susceptible to conventional antibiotic therapy, which results in life-threatening infections, posing a significant challenge to the health care system [51-53]. increased antibiotic resistance is associated with biofilm formationa vital pathogen virulent factor. biofilm is a slimy layer comprised of a cluster of bacterial cells encased pmmb 2022, 5, 1; a0000275 4 of 21 within a hydrated matrix of polysaccharides and proteins, which can attach to animate or inanimate surfaces [51, 54]. biofilm acts by protecting bacteria from the host immune system and antibiotics, causing the bacterial cell encapsulated within the biofilm to be more resistant to conventional antibiotics than their planktonic counterpart [51, 55]. biomedical deviceassociated infections, which are often persistent, recurrent, and hard to treat, are primarily due to biofilms formed by staphylococcus aureus [51]. hence, it is crucial to discover new compounds targeting the biofilm to halt the spread of antibiotic-resistant pathogens. a few potent anti-mrsa compounds produced by streptomyces spp., such as polyketomycin, marinopyrrole, and griseusin a, showed promising results as future clinical drugs [9, 56]. the ability of streptomyces spp. to offer sources for new antibiotics against mrsa has been highlighted through actinobacteria studies in malaysia. for example, the novel bacteria sister strains musc 135t and musc 137t, with the name streptomyces pluripotens, were isolated from tanjung lumpur mangrove soil. interestingly, these two strains have been confirmed to be the same species, but only s. pluripotens musc 135t strain exhibited antimicrobial activity. the study showed that s. pluripotens musc 135t exhibited broad-spectrum bacteriocin activity against pathogens, including mrsa strain atcc baa44 (inhibition zone of 10.5mm), aeromonas hydrophila atcc 7966t (4mm), and salmonella typhi atcc 19430t (4mm) [27, 44]. s. pluripotens musc 135t demonstrated the strongest antibacterial activity against mrsa; thus, it can be a valuable source of the antimrsa agent. furthermore, s. pluripotens musc 135t exhibited antioxidant properties and produced metabolites with anti-colon cancer properties [27, 57]. additionally, the analysis of the draft genome of musc 135t (7,480,269 bp) revealed 72 biosynthetic gene clusters contributing to secondary metabolite production. in particular, 5 clusters demonstrated 85% or more similarities with known gene clusters: albaflavenone (100% similarities), ectoine (100%), desferrioxamine or deferoxamine (100%), antimycin (93%), and informatipeptine (85%) [27]. the streptomyces sp. musc 125 (genome size of 7,656,461bp), also isolated from tanjung lumpur mangrove soil, had shown promising anti-mrsa and anti-biofilm compounds. this strain displayed a high 16s rrna sequence similarity with s. pluripotens musc 135t (99.93%), streptomyces cinereospinus (99.24%), and streptomyces mexicanus (99.17%) [58][59]. streptomyces sp. musc 125 methanolic extract was demonstrated to be effective in inhibiting biofilm formation by mrsa at 1/8 × mic (equivalent to 1.5625 mg/ml), the lowest concentration tested [58]. although the methanolic extract of streptomyces sp. musc 125 showed anti-mrsa activity, the mic values of methanolic extract streptomyces sp. musc 125 were relatively high compared to other recent studies [58, 60, 61]. besides, streptomyces sp. musc 125 possessed the biosynthetic gene clusters encoding for polyketide synthase type 1 (pks i) and pks ii. pks i is the primary enzyme for the biosynthesis of macrocyclic polyketides, whereas pks ii plays a role in aromatic polyketides synthesis. in fact, there are drugs derived from streptomyces that are macrocyclic polyketides (erythromycin) or aromatic polyketides (doxorubicin, tetracyclin) [58, 62]. hence, streptomyces sp. musc 125 is a promising producer of cryptic polyketides that can be valuable therapeutic agents [58]. pmmb 2022, 5, 1; a0000275 5 of 21 streptomyces sp. suk 25 isolated from medicinal plants in malay peninsular had demonstrated to be a potential anti-mrsa agent. results showed that streptomyces sp. suk 25 crude extracts exerted antimicrobial activity against mrsa atcc 49476, mrsa atcc 43300, mrsa atcc 33591, and the inhibition zones were 30mm, 20mm, and 21mm respectively. streptomyces sp. suk 25 showed more potent inhibition activity than the vancomycin control. streptomyces sp. suk 25 exhibited strong inhibitory activity with mic value of 1.95µg/ml, at aeration of 140rpm, but not at higher speeds [63]. based on the above findings, it is evident that streptomycetes possess anti-mrsa activity. particularly, s. pluripotens musc 135t had demonstrated to be a strong broadspectrum bacteriocin against several pathogens and exerted significant anti-mrsa activity, which strengthens its potential to be a good source of antibiotics targeting mrsa. 4.2. anticancer and antioxidant properties anticancer and antioxidant are interrelated in the event of cancer due to the association between cancer initiation and progression with oxidative stress, characterized by increased free radicals [36, 64]. free radicals can cause modifications or cause damage to critical cellular macromolecules, for instance, dna, lipid, and protein. these effects consequently compromise the functioning of dna repair system, resulting in an increased mutation rate and an increased risk of cancer [34, 40]. antioxidants exert their free radicals scavenging ability to prevent harmful effects of excessive free radicals during oxidative stress [40]. several common anticancer drugs have been previously derived from streptomyces[65]. these drugs are pentostatin, bleomycin, mitomycin c, aclarubicin, and doxorubicin [40]. moreover, the antioxidants that have been sourced from streptomyces spp. include antiostatins a1to a4 and b2 to b5 [66], benthocyanins a, b, c [67, 68], carazostatin [69], and carbazoquinocins a to f [70]. in malaysia, numerous studies have revealed the anticancer and antioxidant properties of novel streptomyces and rare actinobacteria species, especially those isolated from malaysian mangroves. the malaysian mod-actino with anticancer and antioxidant properties that will be discuss in detail are streptomyces colonosanans (musc 93jt) [39, 40], streptomyces monashensis (musc 1jt) [37-39], streptomyces antioxidans musc 164t [17, 36], streptomyces mangrovisoli (musc 149t) [34, 35], streptomyces malaysiense (musc 136t) [29-31], streptomyces pluripotens (musc 137t) [34], monashia flava (musc 78t) [41], microbacterium mangrovi (musc 115t) [42], and sinomonas humi (musc 117t) [43]. musc 93jt and musc 1jt are novel streptomyces strains isolated from the mangrove soil in kuching, sarawak [71]. musc 93jt was known by its colon-healing properties, hence, the name streptomyces colonosanans; while musc 1jt was known by its antioxidative activity and its given name is streptomyces monashensis [38-40, 72]. s. pmmb 2022, 5, 1; a0000275 6 of 21 colonosanans musc 93jt extract had demonstrated anticancer activity against human colon cancer cell lines (hct-116, ht-29, caco-2, and sw480) without significant cytotoxic effect against human normal colon cells (table 1). the musc 93jt extract also exhibited potent antioxidant activity. for instance, the extract (2mg/ml) exhibited 11.80 ± 3.75% of 2,2′azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (abts) radicals scavenging activity, 50.06 ± 1.95% of metal chelating activity, and 83.32% ± 2.62% superoxide dismutase(sod)like activity. additionally, this strain can produce chemo-preventive related metabolites via gas chromatography-mass spectrometry (gc-ms) analysis [40]. for example, compound 1,2benzenedicarboxylic acid, mono(2-ethylhexyl) ester was detected in musc 93jt extract, and this compound had been reported to have potential antibacterial, antifungal, and cytotoxic activities [40, 73, 74]. most of the chemical compounds identified via gc-ms (table 1) are recognized for their anticancer and antioxidant activities. thus, these compounds might contribute to the antioxidant and cytotoxic properties exerted by musc 93jt. furthermore, antibiotics & secondary metabolite analysis shell (antismash) analysis detected 23 biosynthetic gene clusters in musc 93jt. four of the biosynthetic gene clusters exhibited more than 70% similarities to known gene clusters, and 1 cluster was linked to ectoine production (75% gene similarities)a protective molecule [75]. s. monashensis musc 1jt has a genome size of 10,254,857 bp. s. monashensis musc 1jt extract demonstrated significant antioxidative activity, as it exhibited metal chelating activity of 75.50 ± 1.44%, and exerted up to 83.80 ± 4.80% sod-like activity. these results suggest s. monashensis musc 1jt can produce antioxidant(s), targeting oxidative stress. in terms of its cytotoxic activity, s. monashensis musc 1jt extract exhibited significant cytotoxic effects against the colon cancer cell lines hct-116 and sw480. however, it demonstrated the highest cytotoxic activity against sw480 with the lowest cell viability of 81.7% ± 4.0% when tested at the highest concentration of 400µg/ml [38, 39] (table 1). a total of 14 compounds were detected via gc-ms (table 1). similar to s. colonosanans musc 93jt, compounds pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-; pyrrolo[1,2a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl); pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)-; and phenol, 2,4-bis(1,1-dimethylethyl)were detected in s. monashensis musc1jt [38, 39]. whole genome analysis of s. monashensis musc 1jt revealed the presence of biosynthetic gene clusters, where more than half of them were hypothesized as protein kinase synthases and non-ribosomal protein kinase synthetases, potentially producing compounds such as micromonolactams (100% known gene cluster similarities) and indogoidine (100%) [37]. other studies have reported indigoidine exerted antioxidant and antibacterial activities [76, 77]. additionally, a gene cluster linked to the biosynthesis of desferrioxamine b (83%) was also detected [37]. therefore, the detection of pmmb 2022, 5, 1; a0000275 7 of 21 these compounds in s. monashensis musc 1jt via gc-ms and genome analysis has increased its value as a good microbial source for drug discovery. s. antioxidans musc 164t (genome size: 9,118,065 bp) isolated from the mangrove soil at pahang exhibited antioxidative activities [36][17]. using 2,2-diphenyl-1-picrylhydrazyl (dpph) assay, the antioxidant activity at 2mg/ml of methanolic crude extract was 18.31 ± 2.03%. the extract also reduced abts radical (up to 30.38 ± 2.27), chelated ferrous ion (up to 43.66 ± 0.98%), and exerted 53.09-79.84% for the sod assay. furthermore, whole genome and bioinformatic analyses of s. antioxidans musc164t revealed the presence of biosynthetic gene clusters associated to siderophores production such as desferrioxamine b, reaffirming its antioxidative potential [17]. s. mangrovisoli musc 149t is another mangrove-derived novel streptomyces that showed antioxidative potential as it exhibited significant free-radical scavenging up to 36.5 ± 3.0% at the highest concentration of 2mg/ml. notably, the compound pyrrolo[1,2a]pyrazine-1,4-dione, hexahydrowas detected in the methanolic crude extract of s. mangrovisoli musc 149t. pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydrohas been reported to demonstrate a strong antioxidant activity by other studies, and thus, further strengthens the hypothesis that this could be one of the compounds contributed to musc 149t antioxidative potential [34, 78, 79]. the antioxidant potential of s. mangrovisoli musc 149t is further highlighted with the detection of siderophores biosynthetic gene clusters – desferrioxamine b [35]. s. malaysiense musc 136t (genome size: 7,963,326 bp) demonstrated promising antioxidant activity and cytotoxic potential against cancer cells [30][29]. it exhibited up to 68.27 ± 3.67% sod-like activity. s. malaysiense musc 136t extract was also found to possessed the highest cytotoxic activity against human colon cancer hct-116 cells with the lowest cell viability of 48.8 ± 4.1% (at 400 μg/ml concentration) (table 1). this occurrence was postulated to be mediated via the p53-dependent apoptosis pathways, as it was reported that there was an increase in p53 protein expression and a decrease in intracellular glutathione levels in hct-116 cells treated with the extract. this phenomenon was accompanied by morphological changes related to cell death, such as the presence of shrunken cells. additionally, s. malaysiense musc 136t extract demonstrated the ability to produce chemopreventive related metabolites through the compounds identified via gc-ms [30]. (table 1) the antismash analysis based on the whole genome sequence of s. malaysiense musc 136t detected 36 biosynthetic gene clusters, whereby 7 clusters demonstrated more than 80% similarities to known gene clusters associated to ectoine, terpene, thiopeptide, lantipeptide, pmmb 2022, 5, 1; a0000275 8 of 21 and desferrioxamine. the production of desferrioxamine by s. malaysiense musc 136t was hypothesized to contribute to the strain’s cytotoxicity against colon cancer cell lines [29] s. pluripotens musc 137t, the sister strain of musc 135t, has antioxidative and cytotoxic potentials. the antioxidant activity of s. pluripotens musc 137t tested via dpph free radical scavenging assay was 35.03 ± 3.74% at 2mg/ml of extract. the cytotoxicity of s. pluripotens musc 137t extract was tested against various human cancer cell lines, including colon cancer cells: hct-116, caco-2, sw480, and ht-29; breast cancer cell: mcf-7, lung cancer cell: a549; prostate cancer cell: du145; and cervical cancer cell: ca ski. among the listed cancer cells, breast cancer cell mcf-7 was the most susceptible with the lowest ic50 of 61.33 ± 17.10 μg/ml, followed by hct-116 (83.72 ± 7.17 μg/ml), a549 (147.20 ± 19.23 μg/ml). the chemical profile of s. pluripotens musc 137t was determined by gc-ms analysis, as shown in table 1 [28]. on the other hand, several rare actinobacteria strains also have shown anticancer potentials [80]. monashia flava musc 78t [41], microbacterium mangrovi musc 115t [42, 81], and sinomonas humi musc 117t [43] are novel rare actinobacteria strains that were isolated from the mangrove soil of tanjung lumpur, kuantan, in the state of pahang. the anticancer properties of these rare actinobacteria strains have been examined against two human cancer cell lines: colon cancer ht-29 cell line and cervical carcinoma ca ski cell line. the methanolic crude extracts of monashia flava musc 78t and microbacterium mangrovi musc 115t demonstrated significant cytotoxicity against ca ski cells with a dosedependent response. on the contrary, the extracts of monashia flava musc 78t and microbacterium mangrovi musc 115t showed a lower cytotoxic activity against ht-29 cells. for sinomonas humi musc 117t, the extract exhibited significant cytotoxicity towards ht-29 cells only [80]. based on gc-ms analysis, the number of compounds detected in the crude extracts of monashia flava musc 78t, microbacterium mangrovi musc 115t, and sinomonas humi musc 117t were 20, 6, and 10 respectively [80] (table 1). the bioactivities exhibited by these rare actinobacteria could be due to the production of phenolic and pyrazine compounds known for their antioxidant and anticancer/antitumor activities [80]. overall, the malaysian mod-actino strains have good antioxidant and anticancer properties. they are also capable of producing pyrrolopyrazine and phenolic compounds which are known for their antioxidant activity [34, 36, 78, 79, 82, 83]. in particular, pyrrolo[1,2a]pyrazine-1,4-dione, hexahydrowere detected in many of the strains mentioned including s. colonosanans musc 93jt [40], s. monashensis musc 1jt [38, 39], microbacterium mangrovi musc 115t [80], and sinomonas humi musc 117t [43]. furthermore, s. monashensis musc 1jt [37], s. antioxidans musc 164t [17], s. mangrovisoli musc 149t [35], s. pluripotens musc 136t [29], and s. pluripotens musc 137 [28] were capable of producing desferrioxamine. desferrioxamine or deferoxamine is an iron chelator that remove excess iron and could inhibit the growth of tumor cells via intracellular iron pool depletion [84, 85]. besides, these streptomyces strains appeared to exhibit higher cytotoxicity towards human colon cancer cells, particularly hct-116 and sw480. many other actinobacteria with antioxidative and/or anticancer activities have also been discovered from malaysian pmmb 2022, 5, 1; a0000275 9 of 21 environments, including streptomyces sp. musc 125 [58, 59], streptomyces sp. mum 212 [86], streptomyces sp. mum 256[73, 87], streptomyces sp. mum 265 [88], streptomyces sp. mum 292 [89], streptomyces sp. musc 14 [90, 91], streptomyces sp. musc 125 [58], streptomyces sp. musc 5 [92], and streptomyces sp. musc 11 [93]. thus, the findings highlight that these mod-actino strains are valuable sources for drug discovery. table 1. the cytotoxic activity of malaysian novel actinobacteria strains (mod-actino) against cancer cells and the chemical compounds detected via gas-chromatography mass spectrometry (gc-ms). strain source cell lines highest cytotoxicity against cancer cell line range of activities against the most sensitive cell line compounds detected via gaschromatography mass spectrometry (gc-ms) streptomyces colonosanans musc 93jt [40] mangrove soil, kuching, sarawak colon cancer cell lines: hct-116, ht-29, caco-2, and sw480 sw480 lowest cell viability of 63.6% ± 3.0% after being treated with the highest concentration of crude extract (400µg/ml) • 2(5h)-furanone • 1-nonanol • phenol, 2,4-bis (1,1-dimethylethyl) • benzoic acid, 4-ethoxy-, ethyl ester • pentanoic acid, 2,2,4-trimethyl-3carboxyisopropyl, isobutyl ester • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl) • 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester pmmb 2022, 5, 1; a0000275 10 of 21 streptomyces monashensis musc 1jt [38, 39] mangrove soil, kuching, sarawak colon cancer cell lines: hct-116 and sw480 sw480 lowest cell viability of 81.7% ± 4.0% after being treated with the highest concentration of crude extract (400µg/ml) • pyrazine, 2,5-dimethyl • pyrazine, trimethyl • 2-pyrrolidone • 2-piperidinone • indolizine • pyrazine, 3,5-dimethyl-2-propyl • phenol, 2,4-bis(1,1-dimethylethyl) • benzoic acid, 4-ethoxy-, ethyl ester • (3r,8as)-3-methyl-1,2,3,4,6,7,8,8aoctahydropyrrolo[1,2-a]pyrazine1,4-dione • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro • phenol, 3,5-dimethoxy • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) • 9h-pyrido[3,4-b]indole • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl) streptomyces malaysiense musc 136t [30] mangrove soil, tanjung lumpur, kuantan, pahang colon cancer cell lines: hct-116 and ht-29 lung cancer cell line: a549 cervical cancer cell line: ca ski hct-116 lowest cell viability of 48.8 ± 4.1% after being treated with the highest concentration of crude extract (400 μg/ml) • isomeric dihydro-methyl-furanone • 1-pentadecene • phenol, 2,5-bis (1,1-dimethylethyl) • (3r,8as)-3-methyl-1,2,3,4,6,7,8,8aoctahydropyrrolo[1,2-a]pyrazine1,4-dione • 1,4-diaza-2,5dioxobicyclo[4.3.0]nonane • tetradecanoic acid, 12-methyl-, methyl ester • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) • pentadecanoic acid, 14-methyl-, methyl ester • deferoxamine streptomyces pluripotens musc 137t [28] mangrove soil, tanjung lumpur, kuantan, pahang colon cancer cell lines: hct-116, caco-2, sw480, and ht-29 breast cancer cell line: mcf-7 mcf-7 lowest ic50 of 61.33 ± 17.10 μg/ml • 2,2-dimethoxybutane • benzeneacetamide • phenol, 2,5-bis(1,1-dimethylethyl) • (3r,8as)-3-methyl-1,2,3,4,6,7,8,8aoctahydropyrrolo[1,2-a]pyrazine1,4-dione • 2,5-cyclohexadiene-1,4-dione pmmb 2022, 5, 1; a0000275 11 of 21 lung cancer cell line: a549 cervical cancer cell line: ca ski prostate cancer cell line: du145 • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro • 1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane • deferoxamine • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-phenylmethyl) monashia flava musc 78t [80] mangrove soil, tanjung lumpur, kuantan, pahang colon cancer cell line: ht-29 cervical carcinoma cell line: ca ski ca ski cell viability of 62% (estimated based on the graph in the study) after being treated with the highest concentration of crude extract (200 μg/ml) • 2-methylpyrazine • pyrrole, 2-methyl • pyrazine, 2,5-dimethyl • 2,3,4-trithiapentane • pyrazine, 2-ethyl-6-methyl • pyrazine, 2-ethyl-5-methyl • pyrazine, trimethyl • pyrazine, 3-ethyl-2,5-dimethyl • 4h-pyran-4-one, 3-hydroxy-2methyl • 1h-indole • 2,4-di-tert-butyl phenol • 1h-pyrrole, 2-phenyl • 1-naphthalenamine, n-ethyl • 3,4-dimethyl-2-phenyl-1h-pyrrole • (3r,8as)-3-methyl-1,2,3,4,6,7,8,8aoctahydropyrrolo[1,2-a]pyrazine1,4-dione • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro • methyl 13-methyltetradecanoate • hexadecanoic acid, methyl ester • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) • 3-benzyl-1,4-diaza-2,5dioxobicyclo[4.3.0]nonane microbacterium mangrovi musc 115t [80] mangrove soil, tanjung lumpur, kuantan, pahang colon cancer cell line: ht-29 cervical carcinoma cell line: ca ski ca ski cell viability of 54% (estimated based on the graph in the study) after being treated with the highest concentration • methyllaurate • 2,4-di-tert-butyl phenol • (3r,8as)-3-methyl-1,2,3,4,6,7,8,8aoctahydropyrrolo[1,2-a]pyrazine1,4-dione pmmb 2022, 5, 1; a0000275 12 of 21 of crude extract (200 μg/ml) • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro • 1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane • 3-benzyl-1,4-diaza-2,5dioxobicyclo[4.3.0]nonane sinomonas humi musc 117t [80] mangrove soil, tanjung lumpur, kuantan, pahang colon cancer cell line: ht-29 cervical carcinoma cell line: ca ski ht-29 cell viability of 80% (estimated based on the graph in the study) after being treated with the highest concentration of crude extract (200 μg/ml) • butanoic acid, 3-methyl • butanoic acid, 2-methyl • 2,4-di-tert-butyl phenol • (3r,8as)-3-methyl-1,2,3,4,6,7,8,8aoctahydropyrrolo[1,2-a]pyrazine1,4-dione • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro • methyl n-pentadecanoate • 1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane • 5,10-diethoxy-2,3,7,8-tetrahydro1h,6h-dipyrrolo[1,2-a:1′,2′d]pyrazine • methyl 14-methylhexadecanoate • pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl) 4.3. neuroprotective effect the mod-actino strains isolated from malaysia are capable of exerting a neuroprotective effect. in addition to the antioxidative effect, s. antioxidans musc 164t extract exhibited potent neuroprotective activity on neuronal sh-sy5y cells against hydrogen peroxide (h2o2). hence, it resulted in the highest cell viability of 80.62 ± 2.75% when pre-treated at 400µg/ml [17]. furthermore, microbacterium mangrovi musc 115t extract was reported to exhibit a similar neuroprotective effect against oxidative stressinduced cytotoxicity on neuronal sh-sy5y cells. the extract protected neuronal sh-sy5y cells against h2o2 challenged at a low concentration of 6.25 µg/ml, and the maximum efficacy was at 12.5µg/ml. microbacterium mangrovi musc 115t extract also demonstrated neuroprotective activity on dementia-induced cytotoxicity as the neuronal cells were protected from streptozotocin (stz)-induced neuronal damage at extract concentrations ranging from 6.25 − 2.5µg/ml. as for monashia flava musc 78t, the extract produced a neuroprotective effect against hypoxia-induced cytotoxicity. it protected neuronal sh-sy5y cells from cobalt (ii) chloride (cocl2) insult at a concentration of 6.25 − 50µg/ml [80]. pmmb 2022, 5, 1; a0000275 13 of 21 4.4. antifungal activity plants can be vulnerable to diseases, and conventional treatment using chemical fungicides may provide an absolute cure, but it may also cause environmental pollution. hence, there is a need for an alternative solution. an increasing amount of research focuses on using antagonist microbes as biological control agents for an environmentally friendly approach. many studies have shown that actinobacteria are capable of suppressing plant diseases [94]. herein, this review will discuss the potential of malaysian mod-actino strains as biological control agents against phytopathogenic fungi in plant diseases. the targeted plants include rice, oil palm, banana plantlet, and chili. rice is one of the staple foods, particularly in asia, whereby the annual consumption of rice reaches >110kg per capita. oryza sativa is one of the rice species that humans consume [95]. however, phytopathogenic infection in rice may lead to crop yield losses, and some fungi produce compounds that are potentially toxic upon consumption. therefore, to prevent such occurrence, there is a need to discover effective biocontrol agents against these phytopathogens in rice [12]. the two main pathogens of rice, fusarium oxysporum and pyricularia oryzae (p.oryzae) are causative agents of root rot and blast in rice, respectively [96]. a study by awla et al. found that streptomyces isolate upmrs4, from rice fields soils of tanjung karang, in the state of selangor, produces bioactive antifungal compounds and exhibited good antifungal activity against p. oryzae. ethyl acetate extract exhibited the highest inhibition of 98.33% towards mycelial growth of p.oryzae compared to the control at a 100µg/ml concentration and had an effective inhibitory concentration (eic) of 1.562µg/ml. besides, from the ethyl acetate crude extract, gc-ms detected 22 volatile compounds for which some of these compounds might account for direct inhibition of bacterial and fungal pathogens. compounds detected include pyrrolo[1,2-a] pyrazine-1,4dione, hexahydro-3-(2-methylpropyl), pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3(phenylmethyl), and ergotamine. additionally, liquid chromatography-mass spectrophotometry (lc-ms) detected 35 different compounds, which include fungicidal compounds amicomacin, fungichromin, n-acetyl-d, l-phenylalanine, and rapamycin. thus, streptomyces sp. isolate upmrs4 can be a promising candidate as biocontrol agent against p.oryzae [97]. in regards to oil palm, two studies reported on using actinobacteria isolates to inhibit ganoderma boninense, the main causal agent of basal stem rot (bsr) disease in oil palm (elaeis guineensis jacq.) [94, 98]. rhizosphere soil samples from 15 healthy oil palms were collected from four oil palm plantations in peninsular malaysia with a severe history of g. boninense infection. findings of the in vitro study showed that approximately 13.5% of isolates exhibited percentage inhibition of radial growth (pirg) of more than 80% towards g. boninense. also, 21 of these isolates exerted an antagonistic effect, causing the abnormal growth of g. boninense. the culture filtrates of four of these 21 isolates (aga 043, aga 048, aga 347, and aga 506) inhibited and showed potential production of metabolites against g. boninense [94]. the researchers then continued analyzing these 4 isolates and found all they belonged to the genus streptomyces. the isolates aga347 and aga506 showed 99% similarity with s. hygroscopicus subsp. hygroscopicus and s. ahygroscopicus, pmmb 2022, 5, 1; a0000275 14 of 21 respectively. under glasshouse conditions, the study reported that powder formulation of aga347 was the most effective in reducing bsr in seedlings by 73.1%. on the other hand, formulations using the known antifungal producer streptomyces noursei, aga043, aga048, and aga506 reduced bsr by 47.4%, 30.1%, 54.8% and 44.1%, respectively [98]. hence, streptomyces spp. has biocontrol potential against g. boninense in oil palm. getha et al. also investigated the antagonistic effect of streptomyces sp. against fusarium oxysporum f.sp. cubense (foc), a pathogen that causes the wilt disease of bananas. the selected streptomyces strain g10 was isolated from port dickson, in the state of seremban. this strain g10 was assigned to the clade streptomyces violaceusniger, and it exhibited a strong antagonistic activity against different pathogenic races of the fusarium wilt pathogen. strain g10 exhibited in vitro antibiosis as shown by inhibition zones, and the inhibited fungal colonies showed lysis of hyphal ends. furthermore, the in vitro antagonistic effects against foc were found to be due to the production of antifungal metabolite by strain g10 [99]. results of in vivo experiment revealed that planting hole and roots of four -weekold tissue culture derived ‘novaria’ banana plantlets treated with strain g10 suspension at 108 cfu/ml demonstrated a significant decrease in wilt severity in plantlets inoculated with 104 spores/ml foc race 4. the plantlets treated with strain g10 showed a 47% and 53% reduction in the final disease severity for leaf symptoms and rhizome discoloration, respectively, compared to the untreated plantlets. however, strain g10 may provide better control at lower foc inoculation concentrations. to sum up, streptomyces violaceusniger strain g10 could interfere with the banana wilt disease cycle and may be a valuable biological control agent for the fusarium wilt disease of bananas [100]. another study discovered that streptomyces isolated from rhizosphere soil of chili plants collected from a chili farm in ulu chuchoh and sungai burung, in the state of selangor, exhibited a range of in vitro inhibitory activity against three different dominant species of colletotrichum including c. acutatum, c. gloeosporioides, and c. capsici. streptomyces strain p42 was selected as a biological control agent under greenhouse conditions as it demonstrated the highest inhibitory activity against all three fungi species and had high chitinase activity. strain p42 was identified as belonging to the streptomyces rochei clade and it could protect chili plants from anthracnose disease under greenhouse conditions. therefore, it is a promising biocontrol agent of anthracnose disease in chili plants [101]. thus far, streptomyces isolates derived from different malaysian environments are capable of exhibiting antifungal properties and thereby suggesting their use as potential biocontrol agents for various plants such as rice, oil palm, banana plantlet, and chili. 4.5. antimalarial activity malaria is a human parasitic disease that affects many parts of the world. according to the world health organization (who), an estimated 241 million malaria cases were recorded globally in 2020, in 85 malaria-endemic countries. the increase in drug resistance in the greater mekong subregion is worrisome as the parasite plasmodium falciparum had pmmb 2022, 5, 1; a0000275 15 of 21 developed partial resistance to artemisinin, which is the core compound of the best antimalarial drugs. it is fortunate for malaysia that there were no cases of non-zoonotic malaria for three consecutive years [102]. additionally, chloroquine is one of the antimalarial drugs used worldwide in the 20th century. currently there is an emergence of parasites with chloroquine resistance after decades of utilizing chloroquine as a treatment regime. plasmodium falciparum is the most malignant of the four human malaria parasite species and has shown foci of chloroquine resistance in southeast asia since the late 1950s [103]. hence, finding alternative bioactive compounds capable of combating these resistant parasites is crucial. a few studies in malaysia also demonstrated the capability of mod-actino in producing valuable compounds with antimalarial activities, for instance, streptomyces sp. h11809 [104, 105] and streptomyces sp. suk 10 [16] a study conducted by dahari et al. isolated streptomyces sp. h11809 from a soil sample in imbak valley and likas, in the state of sabah, east malaysia. the acetone crude extract of h11809 showed potent in vitro inhibition on the growth of plasmodium falciparum 3d7 (pf 3d7) with ic50 value of 0.57 ± 0.09 µg/ml. dibutyl phthalate (dbp) produced by streptomyces sp. h11809 showed active anti-plasmodial activity against pf 3d7 (ic50 4.87 ± 1.26 µg/ml equivalent to 17.50 µm). hence, the study suggested that dbp is the bioactive compound exerting antimalarial activity via glycogen synthase kinase 3β (gsk3β) inhibition [104]. another bioactive compound known as nocardamine (desferrioxamine e) was also discovered from streptomyces sp. h11809 chloroform extract. nocardamine exhibited moderate antimalarial activity against pf 3d7, with ic50 of 1.5 μm, which was more potent than dbp [105]. besides that, zin et al. discovered an endophytic streptomyces sp. suk 10 from the bark of shorea ovalis tree in malaysia that produces a bioactive compound known as gacidin w, a potential low-toxicity antimalarial agent. mice model experiment of gancidin w against plasmodium berghei pzz1/100 showed an inhibition rate of almost 80% of the parasite at the concentration of 6.25 and 3.125 μg/kg body weight on the last day of the test. furthermore, 50% (n-3) of mice treated with gancidin w at a concentration of 3.125 μg/kg body weight survived until 291.13 ± 0.5 days after inoculation of infection, which is roughly the life span of normal mice (12-18 months). hence, this suggests that gancidin w is one of the metabolites contributing to the in vivo antimalarial activity, as demonstrated in the animal model [16]. 5. conclusions nature is a treasure chest for novel and biologically active actinobacteria discovery. this review presents mounting evidence of mod-actino discovery from malaysia. modactino from malaysia can produce medically valuable bioactive metabolites, including anti-mrsa/antimicrobial, anticancer, antioxidant, antifungal, and antimalaria metabolites. these metabolites could be further isolated and developed into useful drugs to help improve human health and well-being. nature is undoubtedly a rich source for novel streptomyces discovery. it is anticipated that more novel strains await to be explored from unique pmmb 2022, 5, 1; a0000275 16 of 21 environments, evidenced by their discoveries from mangrove environments. in summary, the mod-actino strains from malaysia are valuable resources worth further investigation. author contributions: ay-kt performed the literature search, critical data analysis, and manuscript writing. lt-ht performed proofreading and editing. vl, jw-fl, and l-hl provided 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the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology editorial note 1 pmmb covid-19 bulletin: spain (18th april 2020) hooi-leng ser1*, vengadesh letchumanan1, jodi woan-fei law1, loh teng-hern tan1, nurul-syakima ab mutalib2, learnhan lee1* 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia abstract: the growing threat of pandemics around the world has contributed to rising uncertainty during various periods of age throughout history, with notable instances such as the bubonic plague in the sixth century caused by the pathogen yersinia pestis, and more recently middle east respiratory syndrome (mers) caused by the coronavirus mers-cov in 2012. fast forward to the last month of 2019, the who china country office was informed of pneumonia cases with unknown etiology, which later on determined as a new type of coronavirus known as sars-cov-2. zooming into european countries, spain is no stranger to coronavirus-related pandemics, given that this country was the first to publicly report the 1918 flu pandemic caused by the h1n1 coronavirus. unfortunately enough, spain is once again not spared from the current ordeal, recording the highest confirmed cases of covid-19 (diseased caused by sars-cov-2) in europe, with cumulative cases of 190,839 and 20,002 deaths. the spanish government has taken counter-measures for the management of covid-19, consisting of the announcement of “state-of-emergency” and subsequent extension to control movements, setting up research grants to expedite drug and vaccine development in addition to introduction of the use of artificial intelligence as an official channel to provide advice and enquires about covid-19. these are all part of a collective effort worldwide in alleviating dwindling healthcare resources in hopes of starving the virus of hosts by introducing travel restrictions and movement control as well as giving more time for a cure to be engineered. through the advancement in technologies, improved trade, heightened human mobility and as well as the spurning of social media, we are able to closely monitor the latest progress of this pandemic; working together to remove the gloomy clouds before us will eventually lead to a solution of this pandemic. keywords: covid-19; novel coronavirus; spain; pandemic; pmmb received: 18th april 2020 accepted: 19th april 2020 published online: 22nd april 2020 citation: ser h-l, letchumanan v, law jw-f, et al. pmmb covid-19 bulletin: spain (18th april 2020). prog microbes mol biol 2020; 3(1): a0000074. https://doi.org/10.3687/pmmb.a0000074 main text as a member of the european union since january 1986, the population in spain was reported to reach nearly 47 million with healthy life years (at birth) of 68 years for both male and female in 2018 based on report by world bank and eurostat[1,2]. looking at the total of death (n = 418,516) in 2016, 80.5 % of them occurred in those above age of 70 years or older, while only 3.5 % of these cases were related to communicable diseases (n = 14,847; 95% uncertainty interval = 13,208–16,482)[3]. as a matter of fact, the health reporting system in spain has evolved much since the infamous health crisis during the world war i, known as the “spanish flu” or “la grippe” and later on 1918 (h1n1) influenza pandemic[4–8]. the estimated worldwide mortality caused by this pandemic ranged from 20 to 100 million deaths. even though spain was the first country to publicly report this pandemic in the past, the true source of this pandemic remains a mystery till this date[6,7]. fast forward to more than 100 years later, another copyright @ 2020 by ser h-l and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: hooi-leng ser, novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; hooileng_ser@y7mail.com; learn-han lee, novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; 2 pandemic is befalling humanity again; on 11th march 2020, the director-general of world health organization (who) dr tedros adhanom ghebreyesus announced the disease covid-19 to be the latest pandemic, caused by the novel coronavirus, sars-cov-2 as the number of cases increased drastically — as high as 13fold compared to china where first few cases had been reported in december 2019[9–12]. taking a view at the european countries, spain was not spared from this novel coronavirus and the first case was reported on 31st january involving a german tourist in the canary islands, who has been in contact with people who had travelled to china (figure 1)[10,11,13]. it was at this juncture the health authorities begun to set an official test protocol to have those experiencing breathing difficulties, fever and with travel history to china’s hubei province over the past 15 days screened. these criteria were later broadened to include those with obvious symptoms and a history of travel to “hot-spots” such as northern italy. within two months of the first case, the spanish government has taken a lot of counter-measures in trying to reduce the widespread, including school shutdowns and later on flight control along with gatherings (of more than 1,000 people) at closed venues in hardest-hit hit areas. by the second week of march, the european countries have reported more than 5,000 cases, with spain ranking second in terms of most badly affected countries[11,13,14]. on the 14th march, the spanish government used a royal decree (463/2020) to declare a “state-of-emergency”, starting on 15th march which only allowed people to drive alone to perform basic needs activities such as procuring food or medication, attending health centres and financial institutions, returning to one’s primary residence and caring for vulnerable people[15,16]. another exception was those who are working in essential services. however, despite all of these measures, the number of confirmed cases in spain continued to rise, leading to the subsequent announcement of extension on the “state-of-emergency” till midnight of 12th april and non-essential workers were ordered to remain indoors[17]. along with these major announcements, the spanish health ministry has also been constantly updating the general public regarding information on the disease along with advice on personal hygiene and preventive measures. the incorporation of artificial intelligence in management of the pandemic in spain has also been deemed to be an important step to provide official response and inspire confidence in the public[18]. the conversational assistant known as hispabotcovid19 is a “chatbot service” set up by the government, allowing individuals to obtain immediate response about covid-19 via whatsapp app, regardless of time and place. covid-19: spain... in a televised address to the nation, the spanish prime minister pedro sanchez pointed out that the “lockdown” has begun to show positive results noted by the “the start of the decrease in the epidemic”[19,20]. shortly after the approval of a royal decree to extend the state of emergency until midnight of 26th april by the extraordinary council of ministers, the government published “a comprehensive guide on good practices in work centres to prevent the spread of covid-19, coinciding with the return of their work force, on monday (13th april) or tuesday (14th april), encompassing all those workers in non-essential activities that cannot work from home”[21,22]. besides promoting the “essential distancing” of 2 metres, the guideline recommends “travelling to work by means that do not involve more than two people being in the same place” and encourages individuals to take the appropriate hygiene measures including avoiding more than one person using a row of seats for those travelling in a car or in a vehicle for hire and for those commute via public transport are recommended to use a non-medical face mask. in the same announcement, the government has also emphasized that an individual who has symptoms or have been in close contact with people affected by the virus are recommended not to go to the work centre until he/she is confirmed not to be at risk or a risk to others, and also encouraged individuals to contact the covid-19 hotline (available in each region) and refer to the published list of 10 actions (https://www.mscbs.gob.es/ figure 1. graph of confirmed cases and important events including actions taken by the spanish government for the management of covid-19 (data on number of confirmed cases retrieved from coronavirus resource center, john hopkins university of medicine). 3 ser h-l et al. profesionales/saludpublica/ccayes/alertasactual/ncovchina/documentos/20200325_decalogo_como_actuar_ covid19.pdf) in the event of displaying symptoms. apart from ensuring supply of materials such as personal protective equipment, detection kits and ventilators to regional health authorities, the government has currently appointed several logistical points around the country to distribute a total of 10 million face masks at main public transport hubs “to those people who need to travel to work on those means of transport where it is more complex to respect the safety distance”[23–25]. having that said, the ministry of health still recommends working from home as the top priority, while the state law enforcement officers will be performing corresponding checks at suburban and underground stations and at transport hubs to make sure that only authorized traveller are commuting under the royal decree on the state of emergency. the spanish minister of health salvador illa has also pointed out in early april that the government would not make the use of face masks compulsory, unless the public can gain access to them easily and the ministry is still studying the measure before rolling it out[26,27]. the covid-19 pandemic is indiscriminate, posing as an “unseen threat” to every nation in the world. as of 18th april, the total number of confirmed cases in spain is recorded at 190,839 and death cases were reported to be 20,002 according to the data reported by coronavirus resource center, john hopkins university of medicine[13, 14]. the spanish government has recently expressed their support to the work of who to fight this crisis. as the public research organization of the government, the carlos iii health institute has approved another four new research projects to tackle sars-cov-2 and is now supporting 15 research projects under the covid-19 fund with a total allocation of 5 million euros[28]. for instance, one of the newly financed project carried out by university of seville, institute of biomedicine of seville and group of technical specialists in the deactivation of explosive artefacts of the national police is looking at building optical visualisation system to detect residues of virus, by using cameras that cater for different light spectrum. on the other hand, the institute for biomedical research and innovation of cadiz is exploring the potential use of nanosensors as diagnostic tool to identify immunoglobulin antibodies (igg) which are produced by the host upon exposure to the virus. another two latest research projects are more focused at therapeutic interventions — (a) carrying evaluation on approved medication(s) which could be effective against covid-19 via supercomputing, and (b) looking into possible modification of a spanishproduced vaccine, known as mtbvac (which is being developed against tuberculosis) to be used in the battle against covid-19. it is particularly exciting for the vaccine development project carried out the university of zaragoza as the invention is already in in its final preclinical phase. in the event that the vaccine is capable of generating non-specific immunity against sars-cov-2, this could mean a vital breakthrough that permits next phase of clinical trial in humans. tackling a pandemic is surely not an easy task. based on the protocols established by the who, every nation can be a “noah”, setting up surveillance system for diseases and notifying the who in a timely manner to ensure adequate preparedness plans[29–31]. with leaders over the world indicating that “failure is not as option”, it is indeed dire times now as the spread of the virus cripples cities around and crashes the economies worldwide[32,33]. it may not be saving astronauts or rescuing animals as in the movies but instead focusing on real life which is redefining the earth’s inhabitant way of life as we write. with every nation joining hands together in this battle against the “unseen enemy”, it will just be a matter of time for the scientists and clinicians to discover the panacea — be it vaccine or drug to tackle this life-threatening coronavirus. conflict of interest the authors declare that there is no conflict of interest in this work. reference 1. the world bank group. population, total — spain | data [internet]. 2019. available at: https://data.worldbank.org/indicator/sp.pop. totl?locations=es [accessed 17 april 2020]. 2. eurostat. healthy life years statistics [internet]. 2020. available at: https:// 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available at: https://www. lamoncloa.gob.es/lang/en/gobierno/news/paginas/2020/20200408covidassistance.aspx 19. la moncloa. government to ask lower house to extend state of emergency to 25 april. 2020. available at: https://www.lamoncloa.gob.es/lang/ en/presidente/news/paginas/2020/202004040emergency-extend.aspx 4 20. jones j. spain to extend state of emergency to april 26 as rise in infections slows. the star (world) [newspaper on the internet]. available at: https://www.thestar.com.my/news/world/2020/04/04/spain039sdaily-coronavirus-death-toll-falls-for-second-day-in-row [accessed 17 april 2020]. 21. extraordinary council of ministers, la moncloa. council of ministers approved new extension of state of emergency. 2020. available at: https://www.lamoncloa.gob.es/lang/en/gobierno/councilministers/ paginas/2020/20200410council-extr.aspx 22. la moncloa. government launches a guide on good practices at work centres in response to covid-19. 2020. available at: https://www.lamoncloa.gob.es/lang/en/gobierno/news/ paginas/2020/20200411good-practices.aspx 23. la moncloa. government has distributed 6.7 million face masks since 10 march. 2020. available at: https://www.lamoncloa.gob.es/lang/en/ gobierno/news/paginas/2020/20200326face-masks.aspx 24. la moncloa. government distributes 64 million units of material to regional health authorities to combat covid-19. 2020. available at: https://www.lamoncloa.gob.es/lang/en/gobierno/news/ paginas/2020/20200408covid19-material.aspx 25. la moncloa. government distributes 10 million face masks to be handed out as of monday around spain to workers at main transport hubs. 2020. available at: https://www.lamoncloa.gob.es/lang/en/gobierno/news/paginas/2020/20200412face-masks.aspx 26. sappal p. spain promises masks for everyone if it makes face covering compulsory to combat covid-19. euro weekly news (world) [newspaper on the internet]. available at: https://www.euroweeklynews.com/2020/04/05/spain-promises-masks-for-everyone-if-itmakes-face-covering-compulsory-to-combat-covid-19/ [accessed 15 april 2020]. 27. sappal p. obligatory use of face masks in spain to combat coronavirus is delayed. the olive press (national) [newspaper on the internet]. available at: https://www.theolivepress.es/spain-news/2020/04/07/ obligatory-use-of-face-masks-in-spain-to-aid-combat-coronavirus-isdelayedd/ [accessed 15 april 2020]. 28. la moncloa. covid-19 fund to finance new projects for ‘visual’ detection of virus, diagnostic nanotechnology, super-computing to discover pharmaceuticals and development of potential vaccine. 2020. available at: https://www.lamoncloa.gob.es/lang/en/gobierno/news/ paginas/2020/20200415covid19-fund.aspx 29. world health organization. department of communicable disease surveillance and response: who recommended surveillance standards. who/cds/csr/isr/99.2–126 p./http://www. who. int/emc; 2007. 30. world health organization. protocol for the assessment of national communicable disease surveillance and response systems: guidelines for assessment teams. geneva: world health organization; 2001. 31. swaan c, van den broek a, kretzschmar m, et al. timeliness of notification systems for infectious diseases: a systematic literature review. plos one. 2018;13(6). 32. babubal v. ‘failure not an option for frontliners’. new straits times (nation) [newspaper on the internet]. available at: https://www.nst.com.my/ news/nation/2020/03/577958/failure-not-option-frontliners [accessed 17 april 2020]. 33. rahim r. china’s cgtn names dr noor hisham one of world’s top doctors. the star (nation) [newspaper on the internet]. available at: https://www.thestar.com.my/news/nation/2020/04/15/china039s-cgtnnames-dr-noor-hisham-one-of-world039s-top-doctors [accessed 17 april 2020]. covid-19: spain... progress in microbes and molecular biology editorial note 1 pmmb covid-19 bulletin: united kingdom (22nd april 2020) loh teng-hern tan1*, vengadesh letchumanan1, hooi-leng ser1, jodi woan-fei law1, nurul-syakima ab mutalib2, learn-han lee1* 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 2ukm medical molecular biology institute (umbi), ukm medical centre, university kebangsaan malaysia, kuala lumpur, malaysia abstract: covid-19 has greatly impacted the world and posed an enormous public health threat. the united kingdom is hit harder by the covid-19 crisis than any other european countries, besides italy, spain and france. the uk government has come under heavy criticism for its response to covid-19, with lack of preparedness, shortages of personal protective equipment and covid-19 testing. despite the lockdown is in place to slow the spread of covid-19, uk death toll continues to surge. as of 21st april 2020, more than 120,000 confirmed covid-19 cases and 16,000 deaths had been recorded in uk. keywords: novel coronavirus; sar-cov-2; covid-19; united kingdom; pmmb. received: 23rd april 2020 accepted: 24th april 2020 published online: 26th april 2020 citation: tan lth, letchumanan v, ser hl, et al. pmmb covid-19 bulletin: united kingdom (22nd april 2020). prog microbes mol biol 2020; 3(1): a0000078. https://doi.org/10.3687/pmmb.a0000078 main text on the late december 2019, the novel coronavirus sarcov-2, identified as the cause of an outbreak of severe pneumonia in china, has quickly spread to all corners of the world. by 21st april, the number of reported confirmed cases of covid-19 has exceeded 2.3 million globally[1,2]. covid-19 has greatly impacted the world and posed an enormous public health threat which has not been seen in a respiratory virus ever since the 1918 h1n1 influenza pandemic[3]. after the assessment of the alarming levels of spread and severity by who, covid-19 is characterized as a pandemic with more than 118,000 cases in 114 countries, and 4291 deaths recorded on 11th march 2020. sar-cov-2 becomes the first coronavirus which causes a pandemic. on 13th march, europe became the epicentre of the pandemic[4] with more reported cases and deaths than the rest of the world combined, excluding china, reported by dr. tedros adhanom ghebreyesus (director-general of who). since the beginning of the outbreak at the end of january, the world has been alarmed by the clear message from china and the scientific modelling study[5] that the covid-19 was on the trajectory to become a global epidemic. however, within the month of february, the absence of isolation, quarantine and control tracing had further exacerbated the situation in united kingdom (uk) leading to the upsurge of covid-19 confirmed cases and number of critically ill patients overwhelm the healthcare system of the country. the sars-cov-2 virus does not discriminate between social classes, nationalities, ethnicities or ideologies, as shown by the rapid domino effect of infections in uk government officials, even the prime minister, boris johnson succumbed to covid-19 and was admitted to intensive care on 6th april 2020[6]. as of 21st april 2020, more than 120,000 confirmed covid-19 cases and 16,000 deaths had been recorded in uk[1]. the first case of covid-19 in uk was confirmed on 31st january 2020 (figure 1), consisted of two members of a family of chinese nationals staying in a hotel in york. they were identified in the community and copyright @ 2020 by tan lth and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: loh teng-hern tan, novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; loh.teng.hern@monash.edu. learnhan lee, novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia; lee.learn.han@monash.edu. 2 transferred to the regional infectious disease unit at hull university teaching hospitals directly from their hotel[7]. the index case (a), who is a 50-year-old healthy female, entered uk on 23rd january 2020 without any symptoms. however, she developed symptoms of fever, sore throat, dry cough and malaise on the third day of arrival. meanwhile, a previously healthy 23-year-old male (case b) returned to uk from hubei province on 6th january 2020, who had a close contact with case a on 28th january 2020, developed myalgia and dry cough. promptly, case b sought advice from the national health service (nhs) and followed by admission to hospital due to being possibly at risk of covid-19[8]. although the first cases in uk was identified without any clear case definitions, the decision to test was performed purely because of high clinical suspicion and information about the distribution of infection. however, this show that case definitions should evolve rapidly with any newly emerging infection[7] pmmb covid-19 bulletin... figure 1. illustrations of confirmed covid-19 cases and deaths and the brief response plan taken by the uk government. the stacked bar graph shows the daily confirmed cases and deaths since the first case reported in uk while the line graph shows the cumulative confirmed cases. a heat map shows the distribution of confirmed cases across uk as of 21st april 2020. as the covid-19 pandemic progresses, the global community started implementing a broad range of measures depending on their own capacity in the respect of healthcare system, financial status, sociopolitical networks and self-sufficiency[9]. countries like singapore and south korea have achieved relative control over the virus because of widespread testing, contact tracing and social distancing measures. the who-china joint mission on coronavirus disease also successfully demonstrated that immediate and decisive public health responses are critical to hinder or delay hundreds of thousands of cases in china[10]. meanwhile, the uk government faces mounting criticism and rigorous scrutiny of its actions in preparations for and response to covid-19. for instance, “when this is all over, the nhs england board should resign in their entirety,” quoted by the editor-in-chief richard horton of the lancet, expressing his disappointment over the inefficient response taken by the uk government amid the covid-19 pandemic[11]. he also commented that the failure of contain-delay-mitigate-research strategy, which was launched on 3rd march 2020 by the uk government as the preparedness plan for tackling covid-19, was attributed to the relatively lower testing capacity in uk. richard horton also expressed his concern over the new plan of uk government shifting to suppress-shield-treat-palliate may be too late to deal with the consequence of this pandemic with the lack of preparedness, putting the lives of front-line staff and patients at stake[11]. the uk government launched action plans for tackling covid-19 which includes four phases: contain, delay, research and mitigate[12]. the initial containment measures aimed to prevent covid-19 from spreading by implementing approaches include the early detection, isolation and care of the infected patients and coupled with contact tracing and screening. these approaches have seen to be effective in country like china which implemented one of the so-called ‘draconian’ and controversial containment approaches by almost forcing the entire population to stay home. although many argued that these extreme measures are unlikely to be replicated in europe, which curtail human rights and cripple economies, italy government eventually had resorted to impose the national lockdown measures on 9th march 2020 as a consequence of the overstraining of the country’s healthcare system[13]. case finding, contact tracing and testing and strict quarantine are the imperative measures in public health to control infectious diseases[14]. however, contact tracing started in the uk but stopped early in the epidemic, due to the questioning on its effectiveness. the reasons of 3 tan lth et al. on hospitals, redeployment of clinical staff, recalling of retired doctors, newly graduated medical students and many clinicians may also be asked to practise outside their defined areas of expertise in responding to the unprecedented hospital demand[20]. in this challenging time, the situation in the healthcare setting is further compounded by the apparent increased risk of infection among healthcare workers, the understaffed service because of illness or self-isolation as well as shortages of personal protective equipment (ppe) in the past month. despite the government has repeatedly assured the healthcare workers that millions of units of ppe have been delivered to the frontline[21], a survey by british medical association (bma) showing that more than half of doctors working in high risk environment with either shortages or no supply of adequate face masks and no access to eye protection[22]. in addition to unpreparedness of the government and shortages of personal protective equipment, uk is also falling short on testing. testing has regarded as one of the most effective strategies to curb with the rapid spreading covid-19, thereby it offers the authorities the opportunity to isolate the infected patients and stem the spread of covid-19. ‘test, test, test’ is the mantra of the who and other countries in response to the pandemic[13]. meanwhile, tests for healthcare workers are only becoming available in uk. south korea has been among the most aggressive when it comes to testing, in contrary to the early stages in uk where the government officials had even talked up a theory of allowing the disease to spread while protect the vulnerable in the society. as of 20th march 2020, there were only around 957 tests per million in uk as compared to countries like south korea (6182 tests per million), italy (3019 tests per million) and australia (4294 test per million) [23]. on 2nd april 2020, reports showed that only 2,000 nhs frontline workers out of about half a million have been tested for coronavirus. on the same day, the health secretary, matt hancock, pledged that there will be 100,000 tests for coronavirus available daily by end of the month of april across uk [24]. at the time of writing, various countries are wondering when and how to ease coronavirus lockdowns as curves flatten and covid-19 cases start to fall in some european countries, including denmark, germany, switzerland and austria[25]. the who has also warned that lifting the lockdowns should be done slowly and only when additional capacity in the healthcare system is in place to isolate cases and contact-tracing[26]. meanwhile, the uk will probably have to maintain some level of social distancing for another 18 months or until a vaccine for the novel coronavirus is available, neil ferguson said, an epidemiologist and government adviser from imperial college, who has helped shape the government’s response to the pandemic. furthermore, the health secretary also commented on 16th april that it was still too early to lift the lockdown[27]. conflict of interest the authors declare that there is no conflict of interest in this work. discontinuation of control tracing, which is against the who recommendation, have not been explained by the uk government. it seems to be linked to the shift from “contain” to “delay” by the uk government’s action plan on 12th march 2020, when control tracing was substituted instead of coupling with other control measures[14]. despite facing the criticisms on the slow decision made to move to delay from contain phase, the delay phase aims to slow the spread and push the peak of cases towards the spring and summer months to reduce pressure on the already overwhelmed nhs, in order to give time the researchers to comprehend the virus and potentially reduce the transmission given there is a seasonal element – as happens with influenza[7]. a modelling study by the researchers at imperial college london has forced the uk government to shift their response plans for covid-19, showing that the uk’s heath service would soon be overwhelmed with severe cases of covid-19 and resulted more than 500,000 deaths if no alternatives are taken by the government[15]. almost immediately, the prime minister boris johnson announced new stringent restrictions on the movement of populations on 16th march 2020[16]. ‘urging everyone to practice social distancing and avoiding contact with others, households quarantine for 14 days if develops symptoms of covid-19 and isolation of the high risk group (pregnant women and aged over 70) for 12 weeks’, represented a critical factor in jolting the uk government into changing its policy which had previously only told people with symptoms to isolate at home for a week and suggested people over 70 may have to self-isolate[16]. moreover, the study also suggests that population-wide social distancing applied to the population has the largest impact to reduce onward transmission. in addition, the combination of population-wide social distancing with other measures, such as self-isolation of cases and closure of school and university, which require the minimum policy has the potential to suppress transmission below the threshold of r=1 and effectively reduce the case incidence[15]. on 23rd march 2020, the prime minister officially announced to place uk in a state of lockdown, whereby all nonessential businesses are to close with immediate effect and residents are only allowed to go out to shop for essential items. the uk government’s decision to impose this restrictions came under the increasing pressure to curb the spread of the virus and the joint efforts by the public health specialist, epidemiologists, scientist and doctors urging the government with the evidence on the best strategy to flatten the peak of covid-19 outbreaks and to widen covid-19 testing[17]. initially, there were even reports on whether it is worthwhile to impose restriction, while also reports on “zero prospect” of lockdown of the city london[18]. nevertheless, this enforced social distancing is absolutely crucial to save lives, to protect vulnerable in society and to ensure the healthcare system can cope and care for patients. according to another modelling study, a lockdown scenario of 8 days (17th march 2020) earlier than on the 24th march 2020 would have reduced the deaths from over 81,000 to below 19,000 by the end of a 12 week lockdown[19]. apart from the lockdown intended to ease the pressure 4 reference 1. world health organization. coronavirus disease 2019 (covid-19). situation report－92, 2020 [22 april 2020]. available from: https://www.who.int/docs/default-source/coronaviruse/situationreports/20200421-sitrep-92-covid-19.pdf?sfvrsn=38e6b06d_4 2. vengadesh l, ab mutalib ns, goh bh, et al. novel coronavirus 2019-ncov: could this virus become a possible global pandemic. prog microbes mol biol 2020; 3(1): a0000068. 3. lee vs, chong wl, sukumaran sd, et al. computational screening and identifying binding interaction of anti-viral and anti-malarial drugs: toward the potential cure for sars-cov-2. prog drug discov biomed sci 2020; 3(1). 4. ser hl, vengadesh l, law jwf, et al. pmmb covid-19 bulletin: spain (22 april 2020). prog microbes mol biol 2020; 3(1): a0000074. 5. wu jt, leung k, and leung gm. nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study. the lancet 2020; 395(10225): 689–697. 6. cowper a. covid-19: testing times for the government—but not for nhs staff. bmj 2020; 369: m1433. 7. moss p, barlow g, easom n, et al. lessons for managing highconsequence infections from first covid-19 cases in the uk. the lancet 2020; 395(10227): e46. 8. lillie pj, samson a, li a, et al. novel coronavirus disease (covid-19): the first two patients in the uk with person to person transmission. j infect 2020; 80(5): 578–606. 9. world health organization. covid-19 strategy update 14 april 2020,2020 [20 april 2020]. available from: https://www.who.int/ publications-detail/covid-19-strategy-update-13-april-2020. 10. world health organization. report of the who-china joint mission on coronavirus disease 2019 (covid-19) 16-24 february 2020, 2020 [20 april 2020]. available from: https://www.who. int/docs/default-source/coronaviruse/who-china-joint-mission-oncovid-19-final-report.pdf 11. horton r. offline: covid-19 and the nhs&-”a national scandal”. the lancet 2020; 395(10229): 1022. 12. the lancet respiratory m. covid-19: delay, mitigate, and communicate. the lancet resp med 2020; 8(4): 321. 13. cohen j and kupferschmidt k. countries test tactics in ‘war’ against covid-19. sci 2020; 367(6484): 1287–1288. 14. pollock am, roderick p, cheng k, et al. covid-19: why is the uk government ignoring who’s advice? bmj 2020; 368: m1284. 15. ferguson n, laydon d, nedjati gilani g, et al. report 9: impact of non-pharmaceutical interventions (npis) to reduce covid19 mortality and healthcare demand. imperial college covid-19 response team 2020. 16. mahase e. covid-19: uk starts social distancing after new model points to 260 000 potential deaths. bmj 2020; 368: m1089. 17. iacobucci g. covid-19: uk lockdown is “crucial” to saving lives, say doctors and scientists. bmj 2020; 368: m1204. 18. cowper a. andy cowper: covid-19—government communications need to build and sustain trust. the bmj opinion 2020 [21 april 2020]. available from: https://blogs.bmj.com/bmj/2020/03/20/andy-cowpercovid-19-government-communications-need-to-build-and-sustaintrust/. 19. dropkin g. covid-19 uk lockdown forecasts and r0. medrxiv 2020. available from: https://doi.org/10.1101/2020.04.07.20052340 20. willan j, king aj, jeffery k, et al. challenges for nhs hospitals during covid-19 epidemic. bmj 2020; 368: m1117. 21. department of health and social care. coronavirus (covid-19): personal protective equipment (ppe) plan, 2020 [21 april 2020]; available from: https://www.gov.uk/government/publications/ coronavirus-covid-19-personal-protective-equipment-ppe-plan. 22. british medical association. bma survey finds doctors’ lives still at risk despite government pledges on ppe,2020 [21 april 2020]; available from: https://www.bma.org.uk/news-and-opinion/bmasurvey-finds-doctors-lives-still-at-risk-despite-government-pledgeson-ppe. 23. feilding c and anand k. coronavirus testing: which countries are leading? 2020 17th april 2020]; available from: https:// g r a p h i c s . r e u t e r s . c o m / h e a lt h c o r o n av i r u s g l o b a l testing/0100b5lc45h/. 24. department of health and social care. health secretary sets out plan to carry out 100 000 coronavirus tests a day, 2020 [21 april 2020]; available from: https://www.gov.uk/government/news/healthsecretary-sets-out-plan-to-carry-out-100000-coronavirus-tests-a-day. 25. brandon s. these european countries are starting to lift their coronavirus lockdowns. world economic forum, 2020. [20 april 2020]; available from: https://www.weforum.org/agenda/2020/04/ these-european-countries-are-starting-to-end-their-lockdowns/. 26. gilbert m, dewatripont m, muraille e, et al. preparing for a responsible lockdown exit strategy. nat med 2020. 27. day m. covid-19: european officials warn that exiting lockdown will be “very long” and will require cooperation. bmj 2020; 369: m1549. pmmb covid-19 bulletin... progress in microbes and molecular biology genome report 1 whole genome sequence of streptomyces colonosanans strain musc 93jt isolated from mangrove forest in malaysia hooi-leng ser1†, jodi woan-fei law1†, wen-si tan2, wai-fong yin3, kok-gan chan3,4* 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. 2illumina singapore pte ltd, woodlands industrial park e1, singapore. 3division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia. 4vice chancellor office, jiangsu university, zhenjiang 212013, pr china. †these authors contributed equally to the work. abstract: under the family actinobacteria, streptomycetes are ubiquitous in nature, producing a wide spectrum of bioactive compounds including antibacterial, antioxidant, anticancer and immunomodulatory properties. during a screening programme in malaysia, streptomyces colonosanans musc 93jt was isolated as a novel streptomyces sp. from the mangrove soil in sarawak. the strain exhibited potent antioxidant activities and cytotoxic activity against several human cancer cell lines. due to these data, the strain was subjected to whole genome sequencing to uncover its genomic potential and further improve the understanding of the strain. the genome of musc 93jt consists of 7,015,076 bp (g + c content of 69.90%), carrying a total of 5,859 protein coding genes. analysis using a bioinformatics tool, antismash predicted a total of four biosynthetic gene clusters which displayed similarity of more than 70% to known gene clusters and one of which was associated with the production of a natural protectant, ectoine. displaying selective toxicity that kills only cancer cells, ectoine has showed its potential to be developed as therapeutic agents for humans. altogether, the current project clearly highlights the importance of under-explored environment like mangrove in natural product discovery. the availability of whole genome sequence musc 93jt warrants subsequent in-depth investigation and optimization for the production of bioactive compounds which can be exploited for the health and wellbeing of mankind. keywords: streptomyces; anti-cancer; mangrove; genome; musc 93jt; actinobacteria received: 18th february 2020 accepted: 20th march 2020 published online: 25th march 2020 citation: ser h-l, law jw-f, tan w-s, et al. whole genome sequence of streptomyces colonosanans strain musc 93jt isolated from mangrove forest in malaysia. prog microbes mol biol 2020; 3(1): a0000061. https://doi.org/10.3687/pmmb/ a0000061 short introduction streptomycetes are filamentous bacteria that can be found in various ecosystems and most well-known for their ability to produce secondary metabolites which can be exploited for the benefits of mankind[1–7]. for instance, the isolation of streptomycin from streptomyces griseus described by professor waksman and his team was a major breakthrough back in the 1950s, being the first effective treatment against the causative agent of the great white plague, mycobacterium tuberculosis[8,9]. even though more than 60 years have passed, drug discovery studies investigating bioactive potential of streptomyces sp. from various habitat did not regress, but more efforts are now being poured into the investigation of their genomic potential[10–19]. streptomyces colonosanans musc 93jt was recovered from mangrove forest soil located at sarawak, malaysia during a screening programme for bioactive streptomycetes[10,20]. forming light yellow aerial and vivid yellow substrate mycelium on isp 2 agar which is a typical trait of streptomycetes, musc 93jt was designated as novel species of genus streptomyces which is closely related to streptomyces malachitofuscus nbrc 13059t (99.2% sequence similarity), strepcopyright © 2020 by ser h-l and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: kok-gan chan, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia; kokgan@um.edu.my 2 tomyces misionensis nbrc 13063t (99.1%), and streptomyces phaeoluteichromatogenes nrrl 5799t (99.1%) based on phylogenetic analysis using their 16s rrna genes. nonetheless, fermentative extracts of musc 93jt displayed potent antioxidant activity and anticancer activity against several human colon cancer cell lines without significant cytotoxic effect against human normal colon cells. the type strain for musc 93jt is available at two culture collection centres with accession of (= dsm 102042t = mccc 1k02298t). based on the biosystematics study using a polyphasic approach, the strain was selected for whole genome sequencing to explore its genomic potential, particularly the production of bioactive compounds that are responsible for its anticancer and antioxidant activities[10,21,22]. data description genomic dna of musc 93jt was obtained using masterpure™ dna purification kit (epicentre, illumina inc., madison, wi, usa) and subjected to rnase (qiagen, usa) treatment[23–25]. following that, dna quality check was conducted with nanodrop spectrophotometer (thermo scientific, waltham, ma, usa) and a qubit version 2.0 fluorometer (life technologies, carlsbad, ca, usa). construction of dna library was done using nextera™ dna sample preparation kit (nextera, usa) and the library quality was checked by bioanalyzer 2100 high sensitivity dna kit (agilent technologies, palo alto, ca). paired-end sequencing was performed on miseq platform with miseq reagent kit 2 (2 × 250 bp; illumina inc., madison, wi, usa)[26,27]. after trimming, the paired-end reads were de novo assembled on clc genomics workbench version 7 (clc bio, denmark), which resulted in 166 contigs and an n50 contig size of approximately 99,963 bp. the genome size of musc 93jt comprised 7,015,076 bp, with an average coverage of 53.0-fold and g + c content of 69.90 %. the genome sequence of musc 93jt has been deposited at ddbj/ embl/genbank under accession of mlyp00000000. table 1. general genomic features of streptomyces colonasanans musc 93jt. streptomyces colonasanans musc 93jt genome size (bp) 7,015,076 contigs 166 contigs n50 (bp) 99,963 g + c content % 69.90 genome coverage 53.0x protein coding genes 5,859 trna 66 rrna (5s, 16s, 23s) 3, 1, 1 the assembled genome was annotated using rapid annotation using subsystem technology (rast)[28]. gene prediction was performed using prodigal version 2.6, while ribosomal rna (rrna) and transfer rna (trna) were predicted using rnammer and trnascan se version 1.21, respectively[29–31]. the analysis from rast revealed 5,859 protein-coding genes, along with a total of 71 rna genes (figure 1). based on rast system, most of the protein-coding genes were shown to be involved in amino acids and derivatives metabolism (9.18%), followed by carbohydrates metabolism (6.21%) and protein metabolism subsystems (4.91%). further analysis on antibiotics & secondary metabolite analysis shell (antismash) detected presence of 23 biosynthetic gene clusters in musc 93jt genome using “strict” detection settings (version 5.1.1)[32,33]. among the four biosynthetic gene clusters which displayed similarity of more than 70% to known gene clusters, one cluster was associated with the production of ectoine (75 % gene similarities). ectoine is commonly expressed by bacteria to survive in harsh environments, protecting these microorganisms against extreme osmotic stress[34–38]. as a compatible solute, ectoine has been shown to be safe as it does not interfere with the host’s metabolism while offering some beneficial effects including antioxidant and protection against ionizing radiation[39–42]. apart from that, a recent study by sheikhpour et al. (2019) showed that ectoine induced apoptosis in lung cancer cells without affecting normal cells. as a natural protectant, ectoine seems to be a promising protective agent to be developed for human use, particularly against chronic inflammatory diseases and cancer[43,44]. on top of that, there has been many studies reported ectoine-based spray or lozenges showed superior efficacy in treating acute pharyngitis and/or laryngitis, proposing its potential use as adjuvant treatment for anti-inflammatory or anti-infective drugs[45,46]. the detection of this biosynthesis gene cluster within the genome of musc 93jt reflects the bioactive potential of mangrove-derived actinobacteria (including rare actinomycetes and streptomycetes and further highlighting the possible development of this strain as “mini-factories” for the production of protective molecule like ectoine[47–49]. with the emerging role of probiotics in regulating human diseases caused by gut dysbiosis (i.e. imbalance in gut microbial population), ectoine as a osmoprotectant could potentially increase the viability of probiotics in food and prolong its shelf life[50–60]. with the availability of the whole genome sequence of musc 93jt, these data would greatly accelerate the medium optimization process and allow genomic manipulations to maximize the production of bioactive compounds including ectoine. conflict of interest the authors declare that there is no conflict of interest in this work. acknowledgement this work was supported by the university of malaya for high impact research grant (um-mohe hir nature microbiome grant no. h-50001-a000027 and no. a000001-5001) and ppp grant (pg090-2015b) awarded to k-gc. musc 93t genome... 3 reference 1. bérdy j. thoughts and facts about antibiotics: where we are now and where we are heading. j antibiotics 2012; 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7(1): 44. 59. omara am, sharaf ae, atef a, et al. optimizing ectoine biosynthesis using response surface methodology and osmoprotectant applications. biotechnol lett 2020: 1–5. 60. lee lh, chan kg, stach j, et al. the search for biological active agent(s) from actinobacteria. front microbiol 2018; 9: 824. musc 93t genome... pmmb 2022, 5, 1; a0000268. doi: a10.36877/pmmb.0000268 http://journals.hh-publisher.com/index.php/pmmb review article cost-effectiveness of public health strategies on covid-19 control: a systematic review ajaree rayanakorn1,2*, siew lian leong2, pattaranai chaiprom1,3, shaun wen huey lee2* article history 1faculty of public health, chiang mai university, chiang mai 50200 thailand; pattaranai_cha@cmu.ac.th (pc) 2school of pharmacy, monash university malaysia, jalan lagoon selatan, 47500, bandar sunway, selangor, malaysia; leong.siewlian@monash.edu (sll) 3faculty of science, lampang rajabhat university, lampang 52100 thailand; pattaranai@pru.ac.th (pc) *corresponding authors: ajaree rayanakorn; faculty of public health, chiang mai university, chiang mai 50200 thailand; ajaree.rayanakorn@cmu.ac.th; ajaree.rayanakorn1@monash.edu (ar); shaun wen huey lee; school of pharmacy, monash university malaysia, jalan lagoon selatan, 47500, bandar sunway, selangor, malaysia; shaun.lee@monash.edu (swhl) received: 08 june 2022; received in revised form: 12 july 2022; accepted: 15 july 2022; available online: 17 july 2022 abstract: the covid-19 pandemic has been one of the greatest public health challenges imposing significant economic and societal costs. a wide range of public health interventions (phis) have been implemented to control the virus, with many aggressive measures that led to economic downturn and social calamity. however, evidence concerning their impacts in terms of costs and benefits of the best buy strategy is limited. this systematic review aimed to provide a critical summary of full economic evaluations (ees) to inform decisions concerning their adoptions. a systemic search in 7 relevant databases and other sources were conducted. out of 11,584 and 11 records identified from databases and other sources, a total 31 full ees focusing on phis were included. majority of studies included were in good quality and from the us and upper-middle, and high-income countries whereas only 6 studies were from low and middleincome countries. suppression/containment was the most deployed strategy (n=19), followed by screening/detection (n = 8), and protection (n = 4). aggressive elimination strategy usually results in more lives or qalys saved compared to mitigation strategies but at a very high cost. the trade-off between aggressive and loose suppressions depends on several factors including timing of implementation, duration, epidemiological characteristics of the virus, and the healthcare capacity. tight and timely adoption of effective intervention at the early stage of pandemic is key in shrinking the number of cases. using a combination approach is generally more cost-effective compared to a single intervention. personal protective measure is highly cost-effective in protecting healthcare workers in a high prevalence scenario and when it is adopted together with social distancing strategy. future studies to address the flaws of current 2 of 26 pmmb 2022, 5, 1; a0000268 evidence are warranted. this review provides important insights regarding adoption of phis and their cost-effectiveness which would be useful to inform policy decisions in response to covid19 and future pandemics. keywords: systematic review; covid-19; sars-cov-2; public health intervention; costeffectiveness 1. introduction the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which was first reported in wuhan, china in december 2019[1], and declared a global pandemic on march 11, 2020[2] has spread to every corner of the world and become one of the greatest public health challenges globally with over 503,000,000 confirmed cases and 6,218,000 deaths worldwide as of april 15h, 2022[3]. recent evidence has shown exhibition of neurological, gastrointestinal, and long-term complications among post covid-19 survivors[4-6]. the possible mechanism of which is the use of the angiotensin-converting enzyme 2 (ace-2) receptors which are expressed in the central nervous system and various organs for sars-cov-2 entry to the host cell[5], and the inflammatory responses caused by the virus[6]. various public health strategies to slow the infection have been implemented[7]. despite the success in virus containment, many of extreme non-specific measures have also inadvertently resulted in substantial economic and social costs[8, 9], of which those with lower socioeconomic status or scarce resource settings are disproportionally impacted[10-12]. as such, policymakers need to design a low-risk yet cost-effective method for managing this pandemic. to date, there have been several epidemiological models of these interventions published. however, most previous reviews mainly focused on clinical outcomes without considering the costs and the effectiveness of interventions[13]. the evidence concerning costeffectiveness is limited with conflicting findings. this systematic review was conducted to update the results from two previous systematic reviews on the cost-effectiveness and feasibility of such public health measures[14, 15] with a significant distinction in including only full economic evaluations focusing on public health interventions (phis) for the covid-19 pandemic. the findings would be essential to inform decisions for practical approaches to slowing down the infection and minimizing the economic impact. 2. materials and methods the study reporting was done according to the preferred reporting items for systematic reviews and meta-analyses (prisma) statement[16, 17]. the study protocol was registered in prospero under protocol number crd42021243848. 3 of 26 pmmb 2022, 5, 1; a0000268 2.1. search strategy we performed a systematic search in relevant databases including cinahl, the cochrane library, econlit, embase, medrivx, pubmed, and web of science using medical subject headings (mesh) terms “((covid* or covid-19 or covid19 or (sars-cov2)) and (economic or cost-effectiveness analysis or cost-benefit or cost-utility or costeffectiveness)” without language restriction from 2019 up until 19 march 2021 (online supplementary appendix 1). the search results from all databases were merged and duplicates were removed. additional references were sought by scanning the reference lists. the primary outcome was incremental cost-effectiveness ratio (icer) per any health outcome unit gain or averted. an incremental cost-effectiveness ratio (icer) is a summary of measures representing the economic value of an intervention, compared with an alternative, calculated by dividing the difference in total costs by the difference in the total health outcome measure[18]. the ratio presents additional cost incurred per one more health unit gained which is useful to inform policy decisions. articles were included if they contained a full economic evaluation (ee) on phis in response to covid-19 control and prevention; included the comparison of phis with do nothing or other alternative strategies; and had one of outcome of interests. systematic review, meta-analysis, review, and editorial letters were excluded. 2.2. study selection and data extraction pc performed searching and initial screening. the screened articles were rechecked against the inclusion and exclusion criteria which were independently reviewed by ar and sll. data extraction was performed by ar and sll and checked for accuracy by swhl. any disagreement was discussed until consensus was reached. in case, there was more than one publication of the same study identified, the most recent publication would be referred to. information including study setting, study design, intervention, comparisons, perspective, an incremental cost-effectiveness ratio (icer)/net monetary benefit (nmb), willingness to pay (wtp) threshold, time horizon, and main findings were extracted. the original currency of costs was converted into 2020 international dollars (i$) according to methods recommended by turner et al.[19]. briefly, local currencies were inflated to 2020 value using the local consumer price indices (cpis) and later converted to international dollars using the purchasing power parity (ppp) using the average exchange rate for the year 2020[20]. 2.3. risk of bias assessment the quality of studies included was assessed according to the drummond 10-point checklist guidelines, the standard tool for appraisal the methodological quality of cost4 of 26 pmmb 2022, 5, 1; a0000268 effectiveness analysis[21, 22]. each item in the checklist has a potential score of 1. the aggregate results reflect the overall study quality ranging from poor quality (score of 1 to 3), average quality (score of 4 to 7), and good quality (score of 8 to 10)[23]. ar and sll independently appraised the quality of the included economic evaluations, and all ratings were checked for accuracy by swhl. 3. results 3.1. study selection we identified 11,584 records from searches in seven relevant databases. after duplicates were removed, there were 10,183 articles. after title and abstract screening, 10,135 irrelevant articles that did not meet the inclusion criteria were excluded. of 48 articles reviewed for fulltext eligibility, twenty-five were excluded, leaving 23 studies included in the review. additionally, eleven articles were identified from other sources (7 articles from websites, 4 articles from citation searching) and reviewed in full text, of which three studies were excluded. therefore, 31 studies were finally included (23 from databases and 8 from other sources) in the systematic review[24-54] (figure 1). the details of 28 studies excluded after full-text evaluation were presented in the online supplementary appendix 3. figure 1. flow diagram of search strategy and study selection. 5 of 26 pmmb 2022, 5, 1; a0000268 3.2. study characteristics all included studies were published between 2020 and 2021. thirteen studies were from the us[29, 31, 36-38, 40, 41, 43, 47-49, 51, 52], four studies were from china[28, 34, 44, 45], three were from india [32, 42, 50], and two studies each were from germany [33, 46] and the united kingdom[53, 54] while one study each was from australia[24], south africa[26], israel[27], ghana[30], morocco[35]. one study evaluated data from sweden and denmark[25] and one study was an ee that investigated the cost-effectiveness of personal protective equipment (ppe) in 139 low and middle-income countries (lmics)[39]. a summary of study characteristics is presented in table 1. 3.3. study design and time horizon thirteen studies conducted cost-effectiveness analysis using life-years saved[25, 26, 33], number of human protected[28], positive patients detected[46], infection or cases[30, 35, 38-40, 44, 51], and years of life lost averted[47] as health outcome measures. eight studies performed a costutility analysis in which six studies reported outcomes in terms of quality-adjusted life years (qalys)[29, 34, 36, 37, 53, 54] while one each reported the outcome in terms of health-adjusted life years (halys)[24] and disability-adjusted life-year (dalys) saved[45]. seven studies focused only on cost-benefit analysis [31, 32, 41-43, 48, 49]. three study employed both cost-utility and costeffectiveness analysis[27, 50, 52] using the number of death[27, 50], infection averted and qalys[52] as outcome measures. three studies used a stochastic agent-based model (abm)[24, 28, 47], a computational model for simulation that capture the behavior of individuals in the model (agents) and their interactions with other agents and the environment[55]. fourteen studies applied the susceptibleexposed-infected-recovered (seir) or sir model, a compartment disease model computing how sars-cov2 infects the population[27, 32, 34, 36-38, 43-45, 49, 51-54]. a decision-tree analytic model was implied in four studies[33, 39, 40, 42]. the others used different approaches including a dynamic covid-19 microsimulation model[26], a decision-analytic model deploying a monte carlo simulation[29], multi-region discrete-time mathematical modeling[35], the institute for health metrics and evaluation (ihme) model[31], and the penn wharton model[41]. the time horizon ranged from 30 days to 30 years. nine studies projected the outcomes ranging from 30 to 150 days[29, 31, 34, 38, 44, 45, 47, 52, 54] while twelve studies used the time span from 6 months to 1 years[24-27, 33, 36, 37, 39, 42, 46, 50, 53]. the time duration of 30 years was assumed in a cba[43]. the other studies did not report the duration. the discount rate was not applied in most studies due to a short timeframe while discounting rates ranging from 3% to 5% were reported in 8 studies[24, 31, 32, 36, 37, 41, 43, 45]. 6 of 26 pmmb 2022, 5, 1; a0000268 3.4. willingness-to-pay thresholds different willingness to pay (wtp) thresholds were used ranging from $3,250 to $200,000 depending on country-specific context and health outcomes. studies from the us generally employed thresholds spanning from $8,500 to $200,000 per qaly, yll or infection averted [29, 36-38, 40, 41, 47, 52] except a cba in which the threshold of $10 million per value of statistical life (vsl) was used[43]. thresholds adopted by chinese studies were lower, at $15,000 to $47,000 per qaly or dalys saved[28, 34, 45], while one study applied a cost-effectiveness threshold of three times of gdp per capita[28]. similarly, an ee from india applied a costeffectiveness threshold of 1 to 3 gdp per capita[50] based on the who guideline for wtp threshold[56]. two studies from the uk used wtp thresholds of £20,000-£30,000 per qaly ($28,589-$42,884), as recommended by the national institute for clinical excellence (nice)[53, 54]. the threshold was not stated in 11 studies[30-32, 35, 39, 42, 44, 46, 48, 49, 51]. 3.5. perspectives twelve studies used a societal perspective[25, 28, 29, 31, 33, 37, 39, 42, 46-48, 52] and six studies used a health system perspective[26, 27, 34, 36, 50, 51] in their analyses. three studies used both the health system and societal perspective[24, 45, 53], of which one study adopted a partial societal perspective using gross domestic product (gdp) cost in addition to healthcare expenditure[24]. ten studies did not specify the perspectives used in their analyses[30, 32, 35, 38, 40, 41, 43, 44, 49, 54]. 7 of 26 pmmb 2022, 5, 1; a0000268 table 1. summary of study characteristics. no author, year country study design population interventions comparison perspective unit of icer wtp threshold time horizon discount rate screening/detection 1 atkeson et al. 2021[48] usa cba general population strategy 1. 10-day screening testing strategy 2. 5-day screening testing strategy 3. 3-day screening testing no additional screening test societal nr nr nr nr 2 baggett et al. 2020[51] usa cea, extended seird (ceacov) model adults residing in homeless shelters • symptom screening, pcr, and hospital • symptom screening, pcr, and acs • universal pcr testing and hospital • universal pcr and acs • universal pcr and temporary housing • hybrid hospital • hybrid acs no intervention: only basic infection control practices are implemented in shelters healthcare sector cost/case prevented nr 4 months na 3 du et al. 2021[47] usa cea (a stochastic individualbased chainbinomial model) general population strategy 1: daily antigen test plus 1week isolation strategy 2: daily antigen test plus 2week isolation strategy 3: antigen test every 7 days plus 1-week isolation. strategy 4: antigen test every 7 days plus 2-week isolation. strategy 5: antigen test every 14 days plus 1-week isolation. strategy 6: antigen test every 14 days plus 2-week isolation. strategy 7: antigen test every 28 days plus 1-week isolation. strategy 8: antigen test every 28 days plus 2-week isolation. symptom-based testing and isolation (status-quo strategy) societal cost/yll $100 000 per yll averted 150 days na 4 jiang et al. 2020[34] china cua (salird model) suspected people with covid-19 and covid19 patients being discharged three reverse transcription-pcr (rtpcr) tests two reverse transcription-pcr (rt-pcr) tests health system cost/qaly 64,644 rmb ($15,444.37) 43 days (23 january 6 march 2020) na 5 losina et al. 2020[52] usa. cua, extended seird (ceacov) model university students, faculty and community members 4 npis include social distancing, maskwearing policies, isolation, and laboratory testing in various combinations no intervention modified societal cost/infection prevented; cost/qaly $150,000 per qaly 105 days na 8 of 26 pmmb 2022, 5, 1; a0000268 no author, year country study design population interventions comparison perspective unit of icer wtp threshold time horizon discount rate 6 neilan et al. 2020 [36]† usa cua, extended seird (ceacov) model general population strategy 1. pcr-severe-only strategy 3 (symptomatic + asymptomatic-once): symptomatic and one-time pcr for the entire population strategy 4: symptomatic + monthly testing strategy 2 (symptomatic): hospitalised and pcr covid-19-consistent symptoms with selfisolation healthcare system cost/qaly $100,000 per qaly 180 days (may 1 nov 1, 2020) 3% 7 paltiel et al. 2020[38] usa cea (seir model) university students aged < 30 years old 1. weekly screening 2. screening every 3 days 3. screening every 2 days 4. daily symptom-based screening nr cost/ infection averted $8,500 per infection averted 80 days na 8 zafari et al. 2020[29] usa cua, decisionanalytic model deploying a monte carlo simulation (the columbia covid-19 model) columbia university students and staff cdc guidelines +additional screening and preventive measures include: 1. symptom-checking mobile application 2. standardizing mask 3. thermal imaging camera 4. one-time testing for sars-cov2 on entry 5. weekly testing for sars-cov2 6. upgrades to ventilation systems or installation of far-ultraviolet c lighting systems cdc guidelines (social distancing, protective measures, and maintaining a healthy environment) alone societal cost/qaly $200,000 per qaly 91 days na suppression/containment 9 asamoa et al. 2020[30] ghana cea (a deterministic model) general population strategy 1, u1 only (the effective testing and quarantine when borders are opened. strategy 2, u2 only (intensifying the usage of nose masks and face shields through education.) strategy 3, u3 only (cleaning of surfaces with home-based detergents.) strategy 5, u5 only (fumigating commercial areas such as markets. strategy 6, combines the use of control ui, i = 1,..,5 strategy 4, u4 only (safety measures adopted by asymptomatic and symptomatic individuals such as practicing proper cough etiquette) nr cost/ infection averted nr nr nr 10 blakely et al. 2021[24] australia (victoria) cua, an agent-based model (abm) general population the four policy options: 1. aggressive elimination strategy 2. moderate elimination strategy 3. tight suppression strategy 4. loose suppression strategy business-as usual or no covid-19 health system and partial societal cost/haly $15,000 per haly 12 months 3% 9 of 26 pmmb 2022, 5, 1; a0000268 no author, year country study design population interventions comparison perspective unit of icer wtp threshold time horizon discount rate 11 broughele t al. 2021[31, 57]† usa cba, model from the institute for health metrics and evaluation (ihme) general population suppression policies enforced by the u.s states. (government suppression scenario) only targeted “mitigation” was practiced including case isolation, household quarantine, and social distancing among elderly and high-risk populations societal nr note: net mortality benefit (nmb) was reported nr 42-65 days 5% 12 dutta et al. 2020[32] india cba (susceptibleinfectedrecovered (sir) model) general population national lockdown without lockdown nr nr note: reported in net benefits nr nr 4% 13 gandjour 2020[33] germany cea (decision tree) general population successful lockdown: icu capacity exceeded by 50%, 100%, 200%, and 300% no intervention societal cost/lys gained €101,493 ($136,133) per life years gained 1 year na 14 khajji et al. 2020[35] morocco cea (a multi-region discrete-time mathematical modeling) general population strategy 1: protecting susceptible individuals from contacting the infected individuals in the same region 1 strategy 2: protecting and preventing susceptible individuals from contacting the infected individuals in the same region or in other regions strategy 3: protecting susceptible individuals, preventing their contact with the infected individuals, encouraging the exposed individuals to join quarantine centers strategy 4: protecting susceptible individuals, preventing their contact with the infected individuals, encouraging the exposed individuals to join quarantine centers and the disposal of the infected animals strategy 3: protect susceptible individuals, prevent their contact with infected individuals, and encourage the exposed individuals to join quarantine centers. nr cost/case averted nr nr nr 15 miles et. al. 2021[54] united kingdom cua, extended seird (imperial college covid-19 response team model) general population lockdown do nothing nr cost/qaly; cost/lys £30,000/qaly ($42,884); £20,000/ lys ($28,589) 3 months na 16 mol and karnon 2020[25] sweden and denmark cea general population strict lockdown strategy (denmark) flexible social distancing strategy (sweden) societal cost/lys $100,000 per life-year saved 6 months na 10 of 26 pmmb 2022, 5, 1; a0000268 no author, year country study design population interventions comparison perspective unit of icer wtp threshold time horizon discount rate 17 padula et al. 2020[37] usa cua (markov model using seir structure) general population 1. social distancing 2. treatment 3. vaccination do nothing societal cost/qaly $50,000 per qaly 365 days 3% 18 reddy et al. 2021[26] south africa (kwazulu -natal) cea (markov) general population public health intervention strategies below: 1. ht+ct 2. ht+ct+ic 3. ht+ct+ic+ms 4. ht+ct+ic+qc 5. ht+ct+ic+ms+qc healthcare testing (ht) health sector cost/lys $3250 per lifeyear saved 360 days na 19 scherbina 2020[49] usa cba (sir model) general population suppression policy extended by 6, 10, 12, 15, 18-week suppression extended by 2-week nr nr note: reported in nmb nr nr nr 20 schonberger et al. 2020[41] usa cba (the penn wharton model) general population limited reopening with social distancing full reopening and reduced social distancing nr cost/qaly $125,000 per qaly nr 3% 21 sharma and mishra 2020[42] india cba (decision tree) general population national lockdown no lockdown societal cost/case averted nr 1 year na 22 shlomai et al. 2020[27] israel cea and cua (seird model) general population non-selective nationwide lockdown focused isolation of individuals at high exposure risk health sector* cost/death averted; cost/qaly $15,243$17,366 per qaly 200 days na 23 thunstrom et al 2020[43] usa cba (sir model) general population social distancing policy no social distancing policy nr cost/ live saved (vsl) $10 million/live saved (vsl) 30 years 3% 24 wang et al. 2020[28] china cea, the stochastic agent-based model (abm) general population single strategies: 1. personal protection 2. isolation-and-quarantine 3. gathering restriction 4. community containment combination of public health measures: 1. personal protection (mask wearing and hand washing) and isolation-andquarantine program (program a) 2. gathering restriction and isolationand-quarantine, program (program b) 3. personal protection and community containment (program c) 4. personal protection, isolation-andquarantine, and gathering restriction (program d) no intervention societal cost/human protected icer < 3 times of per capita gdp ($47,155.59) nr na 25 xu et al. 2020[44] china cea (a spatialtemporal general population 1. epidemiological control including identification of infected cases, tracing their close contact tracing 2. local social interaction control no restrictions nr cost/ infection averted nr 30 days na 11 of 26 pmmb 2022, 5, 1; a0000268 no author, year country study design population interventions comparison perspective unit of icer wtp threshold time horizon discount rate explicit seir model) 3. inter-city travel restriction 26 zala et. al. 2020[53] united kingdom cua, extended seird (imperial college covid-19 response team model) general population 1. mitigation policy: individual case isolation, home quarantine, social distancing advice for people aged > 70 years old 2. suppression 1: mitigation+social distancing+school closure, triggered "on" when there are 100 icu cases/week, and "off" when weekly cases halve to 50 cases 3. suppression 2: suppression 1 triggered "on" when there are 400 icu cases/week, and "off" when weekly cases halve to 200 cases unmitigated (do nothing) nr note: societal perspective could be assumed based on costs included. cost/qaly nice wtp: £20,00030,000 ($28,58942,884) more general estimates of the social value: £10,00070,000 ($42,883100,061) 1 year na 27 zhao et al. 2021[45] china cua (sier model) general population strategy b: 1 week delay movement restriction strategy c: 2 weeks delay movement restriction strategy d: 4 weeks delay movement restriction strategy a: rapid implementation of movement restriction healthcare and societal cost/daly 70,892 rmb per disabilityadjusted lifeyear saved ($16,937) 100 days 3% protection 28 bagepallye t al. 2021[50] india cea, and cua (a decision tree and markov model) general population 1. surgical mask 2. n-95 respirator (fit and non-fit tested) 3. hand hygiene 4. surgical mask + hand hygiene do nothing health system cost/case prevented; cost/qaly 146,709.29 inr ($6671.77) 1 year na 29 ebigbo et al. 2021[46] germany cea patients presenting for endoscopy strategy 2: no routine pre-endoscopy virus test; additional use of ffp-2 and water-resistant gowns for all procedures strategy 3: decentralized point of care antigen test; use of surgical masks, goggles, gloves and apron for all procedures strategy 4: decentralized point of care antigen test; additional use of ffp-2 and water-resistant gowns for all procedures irrespective of test result. strategy 5: centralized laboratorybased rapid pcr test; use of surgical masks, goggles, gloves and apron for all procedures strategy 6: centralized laboratorybased rapid pcr test; additional use of ffp-2 and water-resistant gowns for all procedures irrespective of test result. strategy 7: centralized laboratorybased standard pcr test; use of surgical strategy 1. no routine pre-endoscopy virus test; use of surgical masks, goggles, gloves and apron for all procedures nr note: societal perspective could be assumed based on costs included cost/ number of patients who tested positive nr 1 year na 12 of 26 pmmb 2022, 5, 1; a0000268 no author, year country study design population interventions comparison perspective unit of icer wtp threshold time horizon discount rate masks, goggles, gloves and apron for all procedures strategy 8: centralized laboratorybased standard pcr test; additional use of ffp-2 and water-resistant gowns for all procedures irrespective of test result. 30 risko et. al. 2020[39] lmics cea (decision tree) hcws full personal protective equipment (ppe) supply per the who best practice guidelines to maintain a low rate of hcw infection inadequate ppe with absence of one or more ppe elements societal cost/hcw life saved; cost/hcw case averted nr 30 weeks na 31 savitsky, et al. 2020[40] usa cea (decision tree) hcws on labor and delivery universal screening universal ppe nr cost/hcw case averted $25000 per hcw case averted nr nr acs, alternative care site; cdc, the centers for disease control and prevention; ct, contact tracing; ht, healthcare testing; ic, isolation center; ms, mass symptom screening; qc, quarantine centres; cba, cost-benefit analysis; cea, cost-effectiveness analysis; cua, cost-utility analysis; daly, disability adjusted life years; hcw, healthcare worker, icer, incremental cost-effectiveness ratio; lmics, low-middle income countries; na, not applied; npis, nonpharmaceutical interventions; nr, not reported; qaly, quality adjusted life years; rmb, the renminbi or chinese yuan (¥); seir, susceptible-exposed-infected-recovered; salird, susceptible-asymptomatic-presymptomatic-symptomatic-recovered-deceased; usa, the united states of america; vsl, value of statistical life; wtp, willingness to pay; yll, years of life lost; yls, years of life saved; *not reported by the authors but interpreted from methodology and key findings; †the most recent publication was cited. pmmb 2022, 5, 1; a0000268 13 of 26 3.6. public health interventions and cost-effectiveness results 3.6.1. screening strategies of the 31 studies, eight explored cost-effectiveness of screening strategies[29, 34, 36, 38, 44, 47, 48, 51, 52]. key findings are presented in the online supplementary appendix 4. three studies examined the cost-effectiveness of antigen testing[38, 47, 48]. atkeson et al.[48] compared the costs and benefits of a 10-day, 5-day, and 3-day nationwide covid-19 screening testing regime coupled with self-isolation among those testing positive vs. no federal testing program. the net economic benefits were found to be greater with early introduction and projected to avert between 28,000 to 91,000 deaths with an increased in gdp ranging between $8 to $46 billion[48]. du et al.[47] assessed eight antigen testing strategies with different testing frequencies in combination with 1 or 2-week isolation vs. symptom-based testing and isolation. they found that weekly testing following with 2-week isolation upon a positive test result was preferred at the reproduction number (r0 or rt) of 2.2 (icer = $31,267 per yll averted, median nmb = $2,378 billion) while monthly testing followed by 1-week isolation was the most cost-effective under low transmission scenarios at r0 of 1.2 (icer = $52,500 per yll averted, median nmb $257 billion)[47]. paltiel et. al.[38] reported screening every 2 days with a 70% sensitivity rapid test as the optimal strategy vs. symptom-based screening under a university setting to keep rt below 2.5 (icer = $5,700 per infection averted). another two ees of covid-19 mitigation strategies in college settings implied the added value of nonpharmaceutical interventions (npis) in combination with laboratory testing (lt)[29, 52]. losina et. al.[52] examined the cost-effectiveness of 24 covid-19 mitigation strategies in various combinations compared with no intervention and found that extensive social distancing and mandatory mask-wearing policies were very cost-effective (icer = $224/infection prevented, $25,485/qaly). adding routine laboratory testing would further decrease infections at a relatively higher cost (icer = $482/infection prevented, $121,643/qaly) while keeping campuses closed may result in more infections due to the lack of structured programs to enforce mask-wearing and social distancing. zafari et. al.[29] compared additional control measures to the centers for disease control and prevention (cdc) guidelines and found that the effectiveness of interventions varied greatly according to the covid-19 prevalence rate. at a low prevalence rate of 0.1%, a symptom checking application was cost-saving relative to cdc guidelines alone whereas using a symptom checking application and 2-ply mask-wearing was cost-saving at a prevalence rate of 2.0%. however, the latter approach would result in university closure after 18 days due to many covid-19 cases. three ees compared the use of rt-pcr strategies in identifying potential covid-19 cases. the chinese study[34] comparing 3 rt-pcr vs. 2 rt-pcr strategy concluded that the 3test strategy was cost-saving compared to the 2-test strategy resulting in 850 qalys gain and a net monetary benefit of cn¥ 104 million ($4.86 million). neilan et al.[36] compared different pcr testing approaches and demonstrated cost-saving of pcr testing with any covid-19-consistent symptoms compared to the hospitalized strategy (pcr testing only severely symptomatic pmmb 2022, 5, 1; a0000268 14 of 26 individuals) while symptomatic + asymptomatic monthly approach resulted in the most favorable outcomes in reducing infections and deaths but would be cost-effective only in the surging scenario at re of 2.0 (icer = $33,000/qaly). an american study[51] assessing 7 different combinations of symptom screening, pcr, hospital-based covid-19 care, alternative care sites (acs), and temporary housing strategies relative to no intervention among adults residing in homeless shelters showed that daily symptom screening paired with acs strategy and universal pcr every 2 weeks (hybrid acs) was the optimal strategy compared with no intervention in all scenarios (icer = $-2,549.02/prevented case). 3.6.2. suppression/containment nation-wide lockdown/aggressive suppression policies a total of 19 studies reported the cost-effectiveness of suppression or containment strategies with conflicting findings. ten studies compared a strict lockdown strategy or suppression policy to a flexible social distancing policy or no intervention as a reference strategy[24, 25, 27, 31-33, 42, 49, 53, 54]. among these, 4 studies reported the substantial costs of the lockdown which far exceeded its benefits and did not justify its value[25, 27, 32, 54]. in contrast, the other studies found lockdown to be cost-effective/saving[24, 31, 33, 42, 49, 53] depending on the context. two studies[25, 27] showed that a strict lockdown measure could potentially save more lives or life-year saved but was not cost-effective compared to a more focused approach (icers = $45,104,156/death averted or $4.5m/qaly; icer = $137,285/lys) while a cba conducted in india[32] reported the negative net benefits of lockdown vs. no lockdown under all scenarios varying from -9,125 to -23,232 rs. billion ($-415 to $-1,052 billion). on the contrary, another cba from the same country[42] concluded that lockdown was a cost-saving intervention compared to no lockdown in saving 2.74 rs. trillion ($1.25 trillion) equating to an annual gdp of 1.86%. two cuas from the uk using the nice guideline as the wtp threshold (£30,000 or $42,884 per qaly) reported inconsistent findings[53, 54], of which one study[54] demonstrated significant fewer benefits of 3-month lockdown compared to its costs accounting for the net extra economic costs of £59 billion ($84 billion) relative to the easing restrictions even on the most conservative assumption. in contrast, another study[53] showed the icers of suppression policies vs. an unmitigated (do nothing) to be below the threshold. in line, a cua conducted in australia compared 4 covid-19 strategies; aggressive and moderate elimination, tight suppression and loose suppression to a no covid-19 scenario, which favored elimination (moderate and aggressive) strategies over a 1-year pandemic[24]. broughel et. al.[31] and gandjour[33] focused on national lockdown/suppression policies compared with mitigation strategies or no intervention also demonstrated the positive net benefits of suppression policies on economic impact and flattening the curve. pmmb 2022, 5, 1; a0000268 15 of 26 timing and duration of policy implementation zhao et. al.[45] examined the cost-effectiveness of early movement restriction policies (mrps) relative to delayed mrps by 1, 2, and 4-week in china and identified the rapid mrp as the “dominant” strategy vs. all other approaches. consistently, another chinese study[44] comparing three anti-epidemic policies for covid-19 emphasized the high cost-effectiveness of comprehensive epidemiological control measures at the early stage. at the same time, in-city, inter-city travel restrictions had minimal impact with high costs. scherbina[49] assessed the costs and benefits of suppression policy extension by 6, 10, 12, 15, and 18-week compared with lifting the lockdown after 2-week. the optimal duration of lockdown suggested ranges between 10 and 19 weeks depending on its effectiveness in reducing the number of new cases. 3.6.3. social distancing policy the benefits of the social distancing policy were noted in three studies from the u.s.[37, 41, 43]. padula et. al.[37] compared three interventions: social distancing, covid-19 treatment, and vaccination vs. do nothing, of which all the three options dominated the do-nothing approach. thunstrom et. al.[43] estimated the net benefits of social distancing policy relative to the uncontrolled scenario to be $5.16 trillion. schonberger et. al.[41] considered cost-benefit of a full reopening with reduced social distancing, and a return to shelter-in-place (sip) relative to continued limited reopening with social distancing. a limited reopening with partial mitigation dominated a full reopening and sip policies in terms of qalys gained and gdp costs under the condition that an effective treatment/vaccine could be deployed within 11.1 months 3.6.4. a wide range of interventions four ceas[26, 28, 30, 35] compared a range of strategies using a stepwise approach across the general population regarding the number of infections prevented or life-year saved, and costs. wang et. al.[28] compared single strategies of either personal protection (mask-wearing and hand washing), isolation-and-quarantine, gathering restriction, community containment or a combination of public health measures with no intervention in two scenarios (1 imported case, and 4 imported cases). among all strategies, the joint strategy of personal protection and isolationand-quarantine was optimal among all strategies. however, all interventions except personal protection and gathering restriction were cost-effective if cases were low. reddy et. al.[26] evaluated various phis vs. healthcare testing only among those presenting at healthcare centers (ht) in south africa at different epidemic growths (re 1.2, 1.5, and 2.6). at base case (re 1.5), strategies involving healthcare testing, contact tracing, isolation center, mass symptom screening, and quarantine center (ht+ct+ic+ms+qc) was the most cost-effective (icer $340/yls), followed by ht+ct+ic+ms (icer = $590/yls) whereas ht+ct+ic+qc was the optimal strategy at a low prevalence scenario (re 1.1-1.2). the cost-effectiveness was sensitive to epidemic growth that all combinations of control measures were outpaced and ineffective at re 2.6. pmmb 2022, 5, 1; a0000268 16 of 26 another study from morocco[35] compared 4 different preventive strategies by progressively adding more restrictions in a stepwise manner ranging from implementing awareness campaigns to prevent population movement, encourage contacts to join quarantine centers, and dispose of the infected animals. among all strategies considered, awareness/security campaigns to avoid exposure to the infected population and encourage the exposed individual to join quarantine centers was the most preferred strategy. according to a study from ghana[30], safety measures including social distancing, hand washing, and cough etiquette were the most cost-effective strategy and dominated the other 5 interventions considered while a combination of all strategies was dominated (less effective and more costly). 3.6.5. protection four economic evaluations focused on personal protective equipment (ppe) as protective measures[39, 40, 46, 50], of which two ceas found that that ppe was highly cost-effective in maintaining a low rate of infection among hcws[39, 40]. risko et. al.[39] demonstrated costeffectiveness of full ppe supply defined as supply availability allowing full adherence to the world health organization (who) best practice guidelines[58] compared with an “inadequate” scenario in 139 lmics with a mean icers of $59, and $4,309 per hcw case averted and life saved respectively, and the societal return of 7.93%. savitsky et. al.[40] compared ppe with universal screening to prevent covid-19 transmission among hcw and reported that universal screening was generally the preferred option. in contrast, universal ppe was cost-effective under the high prevalence scenario (29.5 to 34.3%) and cost-saving for planned cesarean delivery. a german cea[46] examined the cost-effectiveness of eight different combinations of preendoscopy virus testing and protection strategies including additional use of ff-2 masks, waterresistant gowns, poc antigen test, pcr vs. routine protective measures with no pre-endoscopic virus testing among asymptomatic patients presenting at endoscopy unit. the study suggested that the icer values were lowest when a strategy of poc antigen testing without the use of high-risk ppe was implemented whereas at higher prevalence rates of 1% and 5%, the lowest icers were achieved with rapid poc antigen testing paired with high-risk ppe use. in contrast, bagepally et. al.[50] conducted cea and cua in india comparing the use of 5 different protective measures (surgical mask, n-95 respirator fit tested and non-fit tested, hand-hygiene, surgical mask+hand hygiene) vs. do-nothing approach and found that none of the interventions were cost-effective while hand hygiene appeared to be less expensive compared to other interventions. 3.7. sensitivity analysis most studies performed one-way sensitivity analysis, three of which[28, 40, 51] also conducted 2-way sensitivity analyses. probabilistic sensitivity analyses (psas) were carried out in 10 studies[26, 27, 29, 36, 37, 39, 40, 45, 46, 50]. nineteen studies[24, 28, 29, 32, 33, 38-40, 43, 44, 46-49, 51-54, 57] used scenario analyses in which only two[24, 47], and one studies[43] provided cost-effectiveness acceptability pmmb 2022, 5, 1; a0000268 17 of 26 curves (ceacs) and break-even analysis respectively. sensitivity analyses used were not reported in 3 studies[35, 41, 42] (table 2). table 2. sensitivity analyses. no author, year country 1-way 2-way 3-way psa ceac scenario 1 atkeson, a. et al 2021[48] usa • • 2 baggett, tp. et. al. 2020[51] usa • • • 3 du, z. et al. 2021[47] usa • • 4 jiang, y. et al. 2020[34] china • 5 losina, e. et al. 2020[52] usa • • 6 neilan, am. et al. 2020[36] usa • • 7 paltiel, ad. et al. 2020[38] usa • 8 zafari, z. et al. 2020[29] usa • • • 9 asamoah, jkk. et al. 2020[30] ghana • 10 blakely, t. et al. 2021[24] australia • • • 11 broughel, j. et al. 2021[31, 57] united states • 12 dutta, m. et al. 2020[32] india • 13 gandjour, a. 2020[33] germany • • 14 khajji, b. et al. 2020[35]* morocco 15 miles, dk. et al. 2021[54] uk • 16 mol, b and karnon, j 2020[25] sweden and denmark • 17 padula, wv. et al. 2020[37] usa • • 18 reddy, kp. et al. 2021[26] south africa • • 19 scherbina, a. 2020[49] usa • 20 schonberger, rb. et al.2020[41]* usa 21 sharma, n. et al. 2020[42]* india 22 shlomai, a. et al. 2020[27] israel • • 23 thunstrom, l. et al 2020[43] usa • • 24 wang, q. et al. 2020[28] china • • • pmmb 2022, 5, 1; a0000268 18 of 26 no author, year country 1-way 2-way 3-way psa ceac scenario 25 xu, l. et al. 2020[44] china • • 26 zala, d. et al 2020[53] uk • • 27 zhao, j. et al. 2021[45] china • • 28 bagepally, bs. et al. 2021[50] india • • 29 ebigbo, a. et al. 2021[46] germany • • 30 risko, n. et al. 2020[39] lmics • • 31 savitsky, lm. et al. 2020[40] usa • • • • note: ceac, cost-effectiveness acceptability curve; lmics, lowand middle-income countries; psa, probability sensitivity analysis; *not reported 3.8. risk of bias assessment the risk of bias was assessed in all studies (table 3). according to the drummond 10-point checklist, 21 of economic evaluations[24-29, 34, 36-40, 43, 45, 47, 49-53, 57] included were rated 8 to 10 indicating the methodology and analyses used were of high quality. the others were rated a score of 4 to 7 and classified as average quality. a summary of the rating and the checklist detail is provided in the online supplementary appendix 5. table 3. summary of rating using the 10-item drummond’s checklist. no author, year q1 q2 q3 q4 q5 q6 q7 q8 q9 q10 screening/detection 1 atkeson et al 2021[48] 2 baggett et al. 2020[51] 3 du et al. 2021[47] 4 jiang et al. 2020[34] 5 losina et al. 2020[52] 6 neilan et al. 2020[36] 7 paltiel et al.2020[38] 8 zafari et al. 2020[29] suppression/containment ? ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + — — — — — — — — — — pmmb 2022, 5, 1; a0000268 19 of 26 no author, year q1 q2 q3 q4 q5 q6 q7 q8 q9 q10 9 asamoah et al. 2020[30] 10 blakely et al. 2021[24] 11 broughel et al. 2021[31, 57]† 12 dutta et al. 2020[32] 13 gandjour 2020[33] 14 khajji et al. 2020[35] 15 miles et al. 2021[54] 16 mol and karnon 2020[25] 17 padula et al. 2020[37] 18 reddy et al. 2021[26] 19 scherbina et. al. 2020[49] 20 schonberger et al. 2020[41] 21 sharma and mishra 2020[42] 22 shlomai et al. 2020[27] 23 thunstrom et al 2020[43] 24 wang et al.2020[28] 25 xu et al. 2020[44] 26 zala et. al. 2020[53] 27 zhao et al 2021[45] protection 28 bagepally et al. 2021[50] 29 ebigbo et al. 2021[46] 30 risko et. al. 2020[39] 31 savitsky et al. 2020[40] note: plus signs represent yes (low risk of bias); minus signs, no (high risk of bias); question marks, unclear (unclear risk of bias). — — — ? ? ? ? ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + — — — — — — — — — — — — — — — — — — — — — — + + + + + — + — + + + + + + + + + — + + + — + — ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + — — — — + + — + — + + — — — ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + — — — — — pmmb 2022, 5, 1; a0000268 20 of 26 4. discussion this systematic review summarizes the cost-effectiveness of full economic evaluations on public health interventions in response to covid-19. we identified 31 full ees focusing on phis regarding covid-19 prevention, of which majority are in good quality. the number of included studies is slightly more than the 2 previous reviews[14, 15] , possibly due to the extensive inclusion of several databases and other sources including citation searching. the findings concerning costeffectiveness results, assessment of methodological quality, strengths and limitations would provide invaluable insights for policymakers regarding optimal strategies under different contexts and health economists concerning existing evidence gaps to be addressed in future ees. suppression/containment strategies were most frequently examined among the included studies (n =19), followed by screening/detection (n = 8), and protection (n = 4). using a combination strategy is generally more cost-effective than a single intervention approach. however, certain conditions including the number of infected cases, timing, effectiveness, and adherence to intervention should be considered. wang et. al.[28] reported a combination of phis involving personal protection and isolation-and-quarantine as the optimal strategy compared to isolation-and-quarantine alone in both 1 and 4 imported cases scenarios. in contrast, neither personal protection nor gathering restriction was cost-effective when there was more than 4 imported cases were imported. the decline in the effectiveness of isolation and quarantine was more pronounced as decreasing quarantine probability and increasing delay time especially in sporadic outbreak scenario. in a south african study[26], joint strategies involving healthcare testing, contact tracing, isolation center, mass symptom screening, and quarantine center dominated healthcare testing alone across the general population at re 1.5. however, the efficacy of isolation and quarantine markedly decreases at high epidemic growth (re 2.6). a high prevalence with many cases can lead to a higher probability of contacting with the infected population among susceptible individuals resulting in exacerbation of the infection. a nationwide lockdown usually results in more lys and qalys compared to mitigation strategies or loose suppression but with tremendous costs[25, 27, 49]. the effectiveness of this extreme measure has been controversial with concerns regarding basic human rights and limited healthcare facilities[59]. the primary purpose of lockdown is to flatten the pandemic curve, allowing time for preparing the healthcare infrastructure before replacing it with less restrictive measures to delay the growth of new cases. the trade-off between aggressive and relaxing control measures depends on several factors, including the health system capacity, virus mutation, vaccine effectiveness, timing, and duration. the optimal time of lockdown before its incremental benefits fall below the incremental costs ranges between 10 to 19 weeks[49] which largely relies on the policy’s effectiveness in reducing the number of new cases. contact tracing and isolation would be most effective when there is a relatively small number of cases. as the number of infections increases, susceptible individuals are more likely to be exposed to the virus resulting in a rapidly rising of infected population in the community. these epidemiological measures would be insufficient to control the transmission and can lead to an overwhelming health system. therefore, pmmb 2022, 5, 1; a0000268 21 of 26 the timely and effective suppression strategy at the early stage of transmission at sufficient duration would be critical for a significant reduction in the number of new cases and a reversal of the epidemic whereas alternating suppression strategy would be economically inefficient[49]. comprehensive testing and identification of the infection have a significant impact on reverse the pandemic trend. more frequent testing prevents more infections but at a relatively high cost[52]. expanding screening strategies provides greater benefits in reducing infections as re increases whereas icers significantly rise as re decreases but reduce as costs decline. at a high prevalence rate with rapid disease transmission, periodic universal screening combined with 1-2 week isolation subsequently to a positive test result is potentially to be cost-effective[36, 38, 47] while at a low prevalence rate, restricting testing to those with covid-19 symptoms combining with self-isolation and other protective measures is likely to be economically preferred strategy[29, 36, 51]. specificity, the turnaround time, and the test cost are also important factors in determining costeffectiveness. high specificity is matter far more than sensitivity in controlling the outbreak which can result in overwhelming number of false positives and quarantine center capacity[38]. lowering the test cost would enable more frequency and expansion of testing capacity for cases detection with a lower icer value. a rapid turnaround time would facilitate timely isolation of infected individuals while a longer turnaround time over a day period imposes a higher number of new cases and costs[51]. laboratory screening and contact tracing are tedious and exhaustive work. this epidemiological approach requires extensive manpower and heavily relies on the health system capacity. in limited-resource settings, particularly lmics with underfunding of the health system, elimination strategies may need to be deployed for sufficient transmission control. the utilization of acs is helpful in covid-19 management among the homeless and socioeconomic disadvantaged population. this approach would avoid the fixed costs and avert hospitalizations to preserve beds for those exhibiting severe conditions. the hybrid acs strategy among those with pending test results or mild to moderate symptoms is associated with substantially reduced infections at a lower cost than hospital care management in achieving similar clinical outcomes[51]. personal protective measures, including ppe, ffp-2, n-95 respirator, surgical masks are potentially cost-effective in high prevalence situations especially when combined with rapid antigen testing among hcws[39, 40], and patients undergoing specific conditions procedures[40]. in contrast, applying such preventive measure broadly across the general population is unlikely to be cost-effective particularly in low-resource settings[50]. the usage of surgical/2-ply mask in combination with other phis including symptom screening, extensive social distancing was suggested to be cost-effective in college settings[29, 52]. social distancing reduces contact hours between infected and susceptible individuals while masks decreases infectivity among cases[52]. therefore, a protection strategy should be enforced among high-risk groups e.g., hcws or implemented alongside with social distancing strategy to optimize the benefits. pmmb 2022, 5, 1; a0000268 22 of 26 high cost-effectiveness interventions exist at the beginning of the endemic. this involves tight and timely comprehensive epidemiological measures to contain the virus whereas a travel ban or lockdown which impose substantial economic and societal costs may have minimal impact at this stage. therefore, in a low prevalence scenario, stringency, and enforcement of effective phis in minimizing exposed individuals is more important than implementing aggressive policies that paralyze the whole society. no intervention or mediocre stringency of social distancing is generally the worst choice and not an acceptable option unless there is sufficient evidence to justify or achievement of herd immunity, which is unlikely to be the case under the current situation. as the transmission progresses with the increasing number of infected cases, aggressive policies may be necessary to reverse the spread in reaching the turning point and clearance of cases, of which strong enforcement is essential as the stricter the control, the lower number of cases[44, 60]. due to a large stochastic variation in sars-cov-2 infection, there is no universal optimal strategy. the success of phis depends on epidemiological characteristics of the disease (re), policy enforcement, and stringency, vaccine, and treatment effectiveness, and the health system capacity. the fact that major sources of infections and mortality are concentrated in elderlies and vulnerable populations reinforces the need for more focused phis targeting on high-risk individuals. despite the widely available of covid-19 vaccines and treatment[61], evidence has shown that vaccination alone is not sufficient to control the virus. with emerging variant of concerns which spread more rapidly with higher transmissibility[62] and disproportionate covid19 vaccination uptake and acceptance, effective phis are still essential to curtail the spread of the infection. this systematic review has notable limitations. first, the studies included in the review were widely different in terms of study design, setting, perspective, time horizon, population, wtp thresholds, and type of interventions. therefore, direct comparison of the findings would be limited. the fact that majority of included ees are from the us and upper-middle, and highincome countries with only 6 studies from lmics (india, ghana, morocco, and 139 lmics) may impede transferability of findings especially in low-resource settings. however, most studies were of good quality which could be attributable to our stringent criteria in explicitly including only full ee focusing on phis. none of the included studies accounted for the impacts from long covid19 and irreversible medical conditions. the costs of lockdown including disruption in education, maternal and child health programs, increased domestic violence, and the benefits including the environment rebound and decreased road traffic accidents/injuries were not captured in the analyses. therefore, the estimations might have been underestimated. in addition, the results are largely based on estimations from the first wave of the covid-19 pandemic which may not reflect the current pandemic situation concerning evolving variants of concerns, and the immunity developed against the virus through past infection and vaccination, wanning of vaccine efficacy and the extent of booster vaccines administration and protection[63]. with the constantly growing covid-19 literature, an updated review could be carried out to capture new evidence. finally, pmmb 2022, 5, 1; a0000268 23 of 26 most included studies assumed a homogenous population and employed static models which can lead to overestimation of sars-cov2 prevalence[36, 38]. therefore, future ees considering on these limitations from a societal perspective is warranted. 5. conclusion tight and timely implementation of phis is essential to flatten the curve of the pandemic. the cost-effectiveness of epidemiological control measures depends on the stringency, enforcement, timing, and adherence to interventions. a combination of strategies focusing on specific targets is likely to be more cost-effective than non-selective widescale measures or a single strategy. the epidemiological characteristics of the virus, the health system capacity, and local contexts should be considered in adopting phis. more ees particularly in lmics to address existing evidence gaps should be mandated. author contributions: ar and swhl conceptualized and designed the study. pc conducted searching and initial screening under supervision of ar. ar and sll independently performed data extraction and quality assessment which were checked for accuracy by swhl. ar drafted the manuscript, created the tables and figures with support from swhl. swhl and ar edited the manuscript. the manuscript was reviewed and approved by all authors. funding: this work was supported by cmu junior research fellowship program (jrcmu2564_062). acknowledgments: the authors thank puttarin kulchaitanaroaj of department of pharmacy practice and science, college of pharmacy, university of iowa for kind suggestions concerning costs inflation and conversion during the data extraction process. conflicts of interest: the authors declare no conflict of interest. references 1. zhou p, yang x-l, wang x-g, et al. a pneumonia outbreak associated with a new coronavirus of probable bat origin. nature 2020; 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3(1). 60. chen s, zhang z, yang j, et al. fangcang shelter hospitals: a novel concept for responding to public health emergencies. lancet. 2020; 395(10232): 1305-1314. 61. loo k-y, letchumanan v, ser h-l, et al. covid-19: insights into potential vaccines. microorganisms. 2021; 9(3): 605. 62. shiehzadegan s, alaghemand n, fox m, et al. analysis of the delta variant b.1.617.2 covid-19. clin pract 2021; 11: 778-784. 63. thye a, tan l-h, law j, et al. covid-19 booster vaccinesadministration in different countries. prog microb mol biol 2021; 4(1). author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted. progress in microbes and molecular biology review article 1 effect of chronic kidney diseases-associated pruritus on patients’ sleep quality, well-being and its management inayat ur rehman1,2*, tahir mehmood khan3,4 1department of pharmacy, green campus, abdul wali khan university mardan, pakistan 2novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 3institute of pharmaceutical sciences (ips), university of veterinary & animal sciences (uvas), pakistan 4biofunctional molecule exploratory research group (bmex), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia abstract: chronic kidney diseases-associated pruritus (ckd-ap) affects the patients’ mental and physical health, potentially resulting in fatigue, depression, and directly affecting quality of sleep. hemodialysis patients were reported to experiencing moderate to extreme ckd-ap, thus exhibited higher possibilities of remaining awake at night while sleeping in the day. therefore, ckd-ap is attributed toward nocturnal awakenings and difficulty falling asleep. this condition (ckd-ap) significantly impacts the quality of life (qol), triggering sleep disturbance, mood changes, and uncontrollable scratching. ckd-ap patients have a compromised qol that is generally linked to limited personal freedom and control due to lengthy treatment time. overall, the loss of freedom has wider implications, such as altering marital, family, and social relationships. thus, this writing highlights the vital effect of chronic kidney diseases-associated pruritus on patients’ sleep quality, social and mental well-being and providing comprehensive management and treatment options to improve patients’ quality of life. keywords: chronic kidney diseases-associated pruritus; malaysia; pakistan; sleep quality; well-being; management received: 27th february 2020 accepted: 24th march 2020 published online: 6th april 2020 citation: rehman iu and khan tm. effect of chronic kidney diseases-associated pruritus on patients’ sleep quality, wellbeing and its management. prog microbes mol biol 2020; 3(1): a0000067. https://doi.org/10.3687/pmmb.a0000067 introduction chronic kidney diseases-associated pruritus (ckd-ap) influences the patients’ mental and physical capacity, resulting in fatigue, depression, and quality of sleep[1–6]. hemodialysis patients, experiencing moderate to extreme ckd-ap, exhibit higher chances of being awake at night while sleeping in the day. hence, ckd-ap is attributed toward nocturnal awakenings and difficulty to sleep[2,3,7,8]. pruritus is an undesirable disorder that stimulates itching and could negatively affect sleep quality and affecting the quality of life[9]. ckd-ap significantly influences patients’ quality of life, causing sleep disturbance, mood changes, and uncontrollable scratching[10]. ckd-ap could cause serious problems such as discomfort, anxiety, depression, sleep disorders, and an overall negative effect on one’s physical and mental health. about 42% of chronic kidney disease patients on dialysis experienced ckd-ap with intensity from moderate to severe, and also correlated with other health-related complications such as poor sleeping quality and poor quality of life[1]. sleep disorders account for chronic fatigue which is connected with disturbed day and night rhythm, causing a negative impact on physical and mental ability[11]. furthermore, ckd-ap is associated to higher risk of mortality in dialysis patients[10]. chronic inflammatory skin diseases such as pruritus, psoriasis, and atopic eczema have a considerable impact on ckd patients qol, including psychological health, physical well-being, family relationships and social development[12]. among all the psychological complications, depression is common and has a serious impact on the quality of life of ckd patient and their caregivers. copyright @ 2020 by rehman iu and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: inayat ur rehman, department of pharmacy, green campus, abdul wali khan university mardan, pakistan; inayat.rehman@monash. edu. 2 it has a negative impact on social, economic, and psychological well-being[13]. ckd-ap has a substantial effect on patients qol as it may cause serious discomfort, depression, anxiety[11] depression is more frequently seen in ckd patients mainly between the 3rd to 9th years of treatment, with mostly female patients being affected. depression is exhibited mainly in the form of sadness, anxiety, depressed mood, poor self-esteem, pessimism about the future, decreased libido, sleeps disorders, and reduced appetite[14]. quality of life of patients having ckd is adversely affected by rising intensity of ckd-ap; and is correlated with higher mortality risk[15]. ckd-ap patients have a compromised qol that is mostly linked to limited personal freedom and control due to lengthy treatment time. generally, the loss of freedom has wider implications, altering marital, family, and social relationships[16]. ckd-ap has a negative impact on the social well-being of female patients on hemodialysis as compared to male patients[17]. a strong association exist between qol score and ckd-ap intensity; therefore treating ckdap may improve the qol of ckd patients[15]. ckd-ap should be frequently assessed and effectively managed to reduce associated morbidity, mortality and to improve the overall quality of life. guidelines for the management of ckd-ap ckd-ap is graded as one of the most common dermatological complications among patients on hemodialysis. due to the refractory nature of ckd-ap and its unknown pathophysiology, there is no definitive cure for its management. even though a wide range of therapeutic agents has been utilized for its management, however no therapy has been established for proper management of ckd-ap. based on the proposed hypothesis for the pathogenesis of ckd-ap, different treatment options shall be discussed in this writing. alteration in hemodialysis techniques studies have suggested the role of optimum dialysis rates along with the use of dialyzer membranes can play a role in ckd-ap. the increasing dose of dialysis may improve ckd-ap[18–20]. shaldon (1993)[21] suggested that short dialysis sessions and underdialysis could lead to patient malnutrition and death. indeed, dialysis time of fewer than three hours and 30 minutes has been associated with a doubled rate of patient mortality as compared to patients dialyzed for four hours and being dialyzed thrice weekly. likewise, among dialysis patients, clearance was strongly correlated with an increased duration time of dialysis[22]. high-flux hemodialysis is one the most frequent blood purification method used worldwide, but in developing countries, low-flux dialysis is the main method of extracorporeal blood purification therapy due to poor economic conditions. this method is not effective in removing the middle-molecule uremic toxins that contributes toward ckd-ap[23]. ko et al. (2013)[24] also supports the notion that use of low-flux dialyzer has significant association with the aggravation of ckd-ap. as the high flux dialyzers efficiently remove averagesized molecules[25]. the occurrence of ckd-ap can be reduced by use of high flux hemodialysis, as it significantly contributes toward better improvement in patients’ ckd-ap intensity[26,27]. chen et al. (2009)[28] reported the use of high permeability hemodialysis (ultrafiltrate coefficient, 40 ml/h/mm hg) in having significant improvement in ckd-ap with high-permeability as compared to conventional hemodialysis (ultrafiltrate coefficient, 5.5 ml/h/mm hg). the use of hemodiafiltration with hemoperfusion is also effective in relieving ckd-ap[29]. the intensity of ckd-ap is also reduced by the use of biocompatible dialysis membrane (polymethylmethacrylate [pmma])[30,31]. local pharmacological therapies (topical treatments) emollients and topical analgesic agents emollients such as high-water-content emollient[32,33], glycerol and paraffin[34] are the favored topical treatment of ckd-ap if xerosis (dry skin) is present. aqueous gels with higher water content (containing 80g of water and 20g of aloe vera extract, squalane, naturally-derived vitamin e, silk powder with no artificial and synthetic substances) can help to relief discomfort of ckd-ap[33]. topical analgesic agents are also useful in the treatment of ckd-ap such as pramoxine hcl 1% lotion is reported to be useful in relief of ckd-ap[35]. multiple studies showed that topical capsaicin 0.025% cream were effective for localized ckd-ap[36–38]. suzuki et al. (2015)[39] stated that capsaicin act by desensitization of nociceptive nerve endings depletion of substance p causing blocking of the conductor of pruritus. tacrolimus ointment the effects of tacrolimus ointment in relieving ckd-ap is uncertain. with some studies indicating that it is effective in relieving ckd-ap[40,41], nevertheless, in a randomized control trial, tacrolimus 0.1% ointment showed no effect of among patients on hemodialysis over control group[42]. topical cromolyn sodium the use of topical cromolyn sodium 4% was reported as more effective in decreasing ckd-ap as compared to placebo[43]. gamma linolenic acid (gla) enriched cream chen et al. (2006)[44] reported that gamma linolenic acid enriched cream contributes significantly improvement in ckd-ap severity. sarna and eurax lotions both sarna lotion (0.5% of each camphor, menthol, and phenol) and eurax lotion (10% crotamiton) has been reported to be effective in improving ckd-ap[45]. systemic therapies although local pharmacological therapies are effective for the management and treatment of localized ckd-ap, yet for the management of generalized ckd-ap, systemic effect of chronic kidney... 3 rehman iu et al. therapies are used and shall be discussed here: oral histamines antihistamines are a widely used to relieve itch. they are classified into 2 categories: “histamine receptor antagonists such as hydroxyzine, diphenhydramine, loratadine, or cetirizine and medications that prevent the release of histamine like the mast cell stabilizers cromolyn sodium and ketotifen”[46]. researchers reported that the used of histamine receptor antagonist for the management of pruritus and antipruritic activity have been generally unsuccessful[47–49]. furthermore oral antihistamnes cannot be recommended as first line option for treatment of pruritus due to dangerous side effect[46]. while mast cell stabilizers are reported to be effective in the management of pruritus. it is stated that ckd-ap severity is reduced by using ketotifen therapy[50,51], cromolyn sodium[52,53], zinc sulfate[54,55] and nicotinamide[56]. gabapentin and pregabalin the use of neuroleptic agents such as gabapentin and pregabalin to manage ckd-ap has increased. but patients should be monitored closely for potential side effects from these agents. many studies reported favorable effects of gabapentin in treating ckd-ap. studies showed that intervention with gabapentin 100mg[51,57]; gabapentin 300mg[58–60] and gabapentin 400mg[61] could significantly improve ckd-ap intensity. kobrin (2017)[62] stated that gabapentin 100mg is the preferred initiating dose after each dialysis session, and the dose may be gradually increased to 350mg daily. however, a dose greater than 350mg daily are not recommended in dialysis patients. pregabalin could be used in patients who are unable to tolerate gabapentin[63]. pregabalin 25mg daily is the preferred initiating dose and can be gradually increased to 75mg daily. dose greater than 75mg daily are not recommended in dialysis patients[64]. while pregabalin 50mg[65] and pregabalin 75mg[66] were reported to improve ckdap intensity significantly. opioid imbalance treatment the overstimulation of central mu-opioid receptors or antagonism of kappa-opioid receptors is a contributing factor in ckd-ap. therapies treating the opioid imbalance are employed to improve ckd-ap among patients. studies showed that mu-opioid receptor antagonists such as naltrexone 50mg were effective in the relief of pruritus[67]. but a study by pauli-magnus et al. (2000)[68] indicated no effect of naltrexone in the relief of pruritus [68]. kappa opioid receptor agonists indicated good result in ckd-ap patients on hemodialysis, and the widely used kappa opioid receptor agonist is nalfurafine[69]. nalfurafine 2.5µg[70] and nalfurafine 5µg[69,70] displayed effectiveness in management of ckd-ap. furthermore nalbuphine hydrochloride 60 mg and 120mg extended-release tablet (mu-opioid receptor antagonist and kappa opioid receptor agonist) were reported as effective in the management of ckd-ap[71,72]. other systemic treatments thalidomide 100mg[73], montelukast 10mg[74], cholestyramine 5gm[75], sertraline (selective serotonin reuptake inhibitor)[76–78] were reported as effective in the management and reduction of ckd-ap. phototherapy studies were conducted and indicated potential effects of phototherapy on ckd-ap. the narrowband ultraviolet b phototherapy[79,80] was reported to be effective in the management of ckd-ap. however, study by ko et al. (2011) [81] indicated that narrowband ultraviolet b phototherapy showed no significant improvement in ckd-ap. nevertheless the potential carcinogenic effect of ultraviolet radiation requires serious consideration[82]. while hsu et al. (2009)[83] reported that thermal therapy with far-infrared rays could effectively improving ckd-ap intensity. alternative treatment alternative therapies such as acupressure, acupuncture and homeopathic verum medication were used for treatment and management of ckd-ap. acupressure therapy at li-l11 point[84] and auricular acupressure[85] were stated to be effective in the management of ckd-ap. acupuncture therapy which block spinal cord release of opioid-like substances, if applied at quchi (li11) acupoint is an easy, safe and effective ways in relieving ckd-ap[86]. a systematic review on acupuncture for treatment of ckd-ap in end-stage renal disease patients reported the beneficial effect of acupuncture intervention but also reported the high risk of bias[87]. assessment of sleep quality and quality of life several validated and self-designed questionnaires were used to assess the sleep quality and quality of life among patients having ckd-ap undergoing dialysis. validated questionnaires for sleep assessment the pittsburgh sleep quality index (psqi) pittsburgh sleep quality index (psqi) is one of the most commonly used questionnaires for assessment of sleep quality among ckd-ap patients[88–91]. it assess the self-rated sleep quality over the past one month. this questionnaire consists of “19 items and seven domains: subjective sleep quality, sleep duration, sleep latency, sleep disturbances, habitual sleep efficiency, use of sleep medication, and daytime dysfunction”, and responses were rated on a 4-point likert scale[92,93]. the overall score was calculated by totalling the scores of the seven domains (range: 0 to 21)[94]. psqi score of 5 and ≥ 5 were classified as bad sleepers and psqi < 5 classified were as good sleepers[94]. epworth sleepiness scale the epworth sleepiness scale (ess) is a simple and inexpensive measure for evaluation of daytime sleepiness, it is a questionnaire comprised of 8 items. the questionnaire rates 4 the responses of sleepiness in 8 daily situations ranging from 0 to 3, giving a total score of 0 (no daytime sleepiness) to 24 (the most excessive daytime sleepiness). the score equal to or greater than 10 is the cutoff point for excessive daytime[95,96]. sleep and health questionnaire the sleep and health questionnaire (shq) comprised of 16 questions that were grouped into 5 factors “self-reported breathing disturbances, functional impact of sleepiness, roommate-observed breathing disturbances, driving impairment, and insomnia”[97]. most of the responses to the questionnaire utilized either a 5-point frequency scale “never”, “rarely”, “sometimes”, “frequently” and “always” ; or by the use of a 6-point likert scale which graded the severity of the symptoms “1–2 points (not affected); 3–4 points (mild); 5 points (moderate); 6 points (severe)”[97]. itch medical outcome study (itch mos) the itch medical outcome study (itch mos) was developed from the medical outcomes study sleep questionnaire[98]. the itch mos instrument contained 10 questions assessing the effect of itch on sleep disruption, sleep latency and daytime somnolence[46]. validated questionnaires for quality of life and sleep assessment combine kidney disease quality of life short form (kdqol) kidney disease quality of life short form (kdqolsf) is one most valid and reliable questionnaire for assessment of the qol of ckd patients. it encompassed 3 domains: “kidney disease component score (kdcs) comprising of effect of kidney disease, symptoms, work status, burden of kidney disease, sleep, cognitive function, sexual function, social support, quality of social interaction, patient satisfaction and dialysis staff encouragement”. the physical component score (pcs) included “physical functioning, role functioning, general health perceptions and pain”, while mental component score (mcs) consist of “energy/ fatigue, social function, role emotional and emotional wellbeing”[99]. short-form health survey (sf-12 and sf-36) the short-form health survey (sf-12) is one of the most widely used tools for assessing health-related quality of life, it is originally developed from the medical outcomes study (mos) 36-item short-form health survey sf-36[100]. the sf-12 is a health-related quality of life questionnaire containing 12 questions measuring 8 health domains to assess physical and mental health. “physical health-related domains include general health (gh), physical functioning (pf), body pain (bp) and role physical (rp). mental health-related scales include vitality (vt), social functioning (sf), role emotional (re), and mental health (mh)”[101]. who-quality of life bref (whoqol-bref) whoqolbref is a 26-item instrument with 4 domains: “psychological health (6 items), physical health (7 items), social relationships (3 items) and environmental health (8 items). it also encompasses qol and general health items. each individual response is scored from 1 to 5 and then transformed linearly to a 0–100-scale”[102]. management of sleep disturbance among ckd-ap patients as sleep disturbance in ckd patients was mainly caused by ckd-ap, therefore the primary objective is mainly to treat the ckd-ap and eventually improves sleep quality. however, due to the refractory nature of ckd-ap, no absolute treatment is available for its management. so far, no reports on improving sleep quality using pharmacological or non-pharmacological treatment among ckd-ap patients. nevertheless, for dialysis patients the therapeutic treatment options for sleep disturbance is available, these options include pharmacotherapy with hypnotic agents[103], pharmacotherapy with wide range of the rapeutic agents for treatment and relief of ckdap[104] and improvement of sleep; cognitive behavioral therapy[105] e.g., relaxation[106] and sleep hygiene[107]. non-benzodiazepine hypnotics are considered to be alternative hypnotic agents in dialysis centers due to no physical dependence, good effects, no active metabolites and no or least adverse effects of inducing sleep apnea[108–110]. for non-pharmacological interventions, acupressure is applied at specific meridians or acupoints in traditional chinese medicine to improve sleep quality[111–114]. unlike pharmacological and other interventions, acupressure is a non-invasive therapy that has low risk of side effect profile[115]. conclusion in conclusion, acupressure and zolpidem tablets were able to improve sleep quality among ckd-ap patients on hemodialysis. with an overall improvement in sleep quality among ckd-ap patients observed in both control and intervention group. healthcare practitioners should consider acupressure therapy as an alternate method to improve the quality of sleep among ckdap patients on hemodialysis. however, more studies are needed to establish suitable data using specific tools to determine ckd-ap and sleep quality for quantitative analysis. ckd-ap should be treated with equal importance as other complications to improve patient’s quality of life, and to avoid secondary infections due to persistent scratching for relieve of the itch. conflict of interest the authors declare that there is no conflict of interest in this work. authors contributions the literature review and manuscript writing were performed by i-ur and t-mk. effect of chronic kidney... 5 reference 1. pisoni rl, wikström b, elder sj, et al. pruritus in haemodialysis patients: international results from the dialysis outcomes and practice patterns study (dopps). nephrol dial transpl 2006; 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burkholderia; paenibacillus; resistance; secondary metabolite; tropical peat swamp forest received: 18th march 2020 accepted: 20th april 2020 published online: 25th april 2020 citation: ong k-s, letchumanan v, law jw-f, et al. microbes from peat swamp forest — the hidden reservoir for secondary metabolites?. prog mircobes mol bio1 2020; 3(1): a0000077. https://doi.org/10.3687/pmmb.a0000077 introduction antimicrobials have been used for generations as prophylaxis to prevent initial or recurrence of infection, and as agents to destroy, inhibit or prevent pathogenic action of microbes[1]. over time, use of antimicrobials has created an inevitable selective pressure leading to the evolution of microbes to resist the action of antimicrobials. microbes can gain resistance towards antimicrobials intrinsically through mutations or by acquiring the ability via conjugation[2]. this phenomenon is further exacerbated by the extensive use of antimicrobials to control infections which has unprecedentedly accelerated the process and emergence of resistant microbes[3]. this is an alarming issue as the rapid emergence of antimicrobialresistant pathogens limits treatment options and increases mortality. according to the director general of the world health organization (who), we are heading towards a post-antibiotic era in which common infections and injuries could once again kill[4]. therefore, there is an urgent need for alternative measures to tackle the crisis of antimicrobial resistance. one of the methods is by bioprospecting for new antimicrobials from untapped resources such as the tropical peat swamp forests where extreme conditions and low nutrients promote competition among microbes. the review will discuss the nature of tropical peat swamp forests, taxonomy and production of secondary metabolites of both burkholderia and paenibacillus, as well as discuss the future prospects of isolating antimicrobial-producing microbes from tropical peat swamp forests. tropical peat swamp forests tropical peat swamp forests (tpsfs) are unique wetland ecosystems periodically flooded by fresh water copyright @ 2020 by ong k-s and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: kuan-shion ong, tropical medicine and biology multidisciplinary platform, monash university malaysia, jalan lagoon selatan, bandar sunway, 47500, subang jaya, selangor. kuanshion@gmail.com 2 from rainfall[5]. out of 30–45 million hectares of global wetlands in the world, about 18.1 million hectares are tpsfs widely distributed around southeast asia[6]. tpsfs contribute to almost 20% of the total global terrestrial organic carbon and form one of the largest terrestrial organic carbon sinks. carbon is stored in the forest biomass with trees up to 70m tall, but most of it is sequestered peat layers up to 25m deep. disturbance of tpsfs due to drainage and fire causes the release of greenhouse gases such as methane and carbon dioxide to the atmosphere. burning of tpsfs results in about 25% of the total global greenhouse gas emissions from deforestation and forest degradation which result in an estimated 3% of total global anthropogenic greenhouse gas emissions. furthermore, tpsfs hold a key role in regional hydrology (movement and distribution of water). this is because peat can hold 5–10 times its weight in water. this is important as the water stored can act as a buffer in reducing water velocity thus minimizing the impact of downstream flooding[5]. tpsfs grow on a substrate formed by the accumulation of layers of peat (partially decomposed organic matter) up to 25m deep. they are usually dome-shaped due to the buildup of peat, and are permanently waterlogged with a ph range of 2.9 to 4.5[8]. the leaching of tannic acids (20.2 ± 2.3 mg/l) from the leaves of endemic plants causes the dark brown, acidic water in tpsfs[9]. the dark brown water reduces light penetration and impedes photosynthesis by algae, which together with the slow flow rates and high temperatures create an anoxic environment, while toxic secondary compounds leach from the leaf litter which then reduces the microbial decomposition of organic matter (decay rates of 0.0006–0.0016 k day-1 for endemic plants), hindering nutrient recycling thus creating a highly concentrated carbon reservoir[5]. in addition, tpsfs are known to be ombrotrophic, receiving nutrients and water solely from rainfall and dust. the lack of nutrient input and slow decomposition rate results in a low nutrient environment in tpsfs[8]. bacterial community in tpsfs it was previously thought that such extreme environmental conditions (acidic, waterlogged and low nutrient availability) meant low bacterial diversity. however, metagenomic studies have revealed the complexity of the genetic information of the bacterial community and high diversity[10]. kanokratana et al. (2011)[10] deduced that bacteria constituted the most abundant microbial group in a thailand tpsf. from the bacterial sequences identified, proteobacteria was the largest species group (37.9% of total bacteria), which comprised mostly alpha-proteobacteria, followed by acidobacteria (35.0% of total bacteria). other key minor bacterial phyla include verrumicrobia (5.7%), planctomycetes (9.6%), actinobacteria (2.5%), bacteroidetes (1.1%), nitrospirae (1.8%), firmicutes (0.4%) and others unclassified bacteria (6.0%). moreover, the bacterial population (determined using next generation sequencing) in a malaysian tpsf showed a similar pattern[11] as the thailand tpsfs and was consistent with a previous study conducted by jackson et al. (2009)[12] which showed that tpsfs are dominated by proteobacteria and acidobacteria (more than 50% of the total bacteria population). antimicrobial-producing bacteria from tpsfs the slow degradation of organic matter in tpsf results in low levels of nutrients which creates a highly competitive environment[13]. consequently, it is likely that bacteria would produce secondary metabolites such as antimicrobial compounds to secure their niche and resources. such phenomena are consistent with the isolation of antimicrobial producing bacteria from two different tpsf: the southeast pahang and selangor tpsf. these antimicrobial producing isolates were identified via a polyphasic taxonomic approach to be novel species from the genus burkholderia and paenibacillus, namely burkholderia paludis sp. nov. (from pahang tpsf)[14] and paenibacillus tyrfis sp. nov. (from selangor tpsf)[15]. burkholderia and their secondary metabolites the burkholderia genus consists of a group of ubiquitous bacteria that occur in aquatic environments, soil, plant rhizospheres and animals. burkholderia are mesophilic gram-negative rods, oxidase positive, motile microorganisms. burkholderia can be characterized phenotypically by their pigmentation, presence of hydroxyl fatty acids of 14, 16 and 18 carbon atoms, possession of distinct polar lipids, and by having q8 cellular respiratory quinones[14]. the genus can be divided into three groups: burkholderia sensu stricto, paraburkholderia and caballeronia[16]. burkholderia sensu stricto is a group of closely related burkholderia species that share a high degree of 16s rrna (98–100%) and reca (94–95%) gene sequence similarity which makes them difficult to be differentiated using conventional molecular techniques. to differentiate different species of burkholderia sensu stricto, multilocus sequence analysis (mlsa) is usually adopted as the technique provides the discriminatory power needed for both identification and differentiation[17]. burkholderia sensu stricto species have diverse ecological roles and have been used in biocontrol and bioremediation. several burkholderia sensu stricto species can be used for biocontrol agents as they can produce secondary metabolites to repress soil borne pathogens. some burkholderia sensu stricto species can act as plant growth promoters. they can also be used for bioremediation of recalcitrant xenobiotics, for instance, burkholderia xenovorans can degrade chlorinated toxic phenolic compounds commonly found in pesticides and herbicides[18]. in contrast, nearly all paraburkholderia species (e.g. paraburkholderia bryophila, paraburkholderia tropica and paraburkholderia nodosa) and caballeronia (e.g. caballeronia ginsengisoli, caballeronia terrestris and caballeronia humi) are plant growth promoters as they are able to fix nitrogen and supply nutrients to their plant hosts[16, 19]. many secondary metabolites with antimicrobial activity are produced by the burkholderia species have been identified. they usually possess antifungal and/ or antibacterial activity (table 1). microbes from peat... 3 ong k-s et al. compounds burkholderia species bioactivity references 2-pyrrolidone-5-carboxylic acid burkholderia sp. hd05 antifungal zhang et al. (2019)[20] bis-(2-ethylhexyl) phthalate burkholderia gladioli or1 antibacterial bharti et al. (2015)[21] burkholdines burkholderia ambifaria 2.2n antifungal tawfik et al. (2010)[22] cepacidin a burkholderia cepacia antifungal lee et al. (1994)[23] cepacins a and b burkholderia cepacia sc 11 antibacterial parker et al. (1984)[24] cepafungin burkholderia sp. antifungal shoji et al. (1990)[25] cepalycin burkholderia cepacia antifungal abe and nakazawa (1994)[26] enacyloxins burkholderia ambifaria ammd antibacterial mahenthiralingam et al. (2011)[27] gladiolin burkholderia gladioli anti-mycobacterium song et al. (2017)[28] icosalide burkholderia gladioli antibacterial dose et al. (2018)[29] iminopyrrolidines burkholderia plantari #9424 icmp antibacterial mitchell and teh (2005)[30] occidiofungin burkholderia contaminans ms14 antifungal lu et al. (2009)[31] phencomycin burkholderia glumae 411gr-6 antibacterial han et al. (2014)[32] pyochelin burkholderia paludis antibacterial ong et al. (2017)[33] pyrazoles derivatives burkholderia glumae #3729 icmp antibacterial mitchell et al. (2008)[34] pyrrolnitrin burkholderia cepacia antifungal, antibacterial el-banna and winkelmann (1998)[35] vietnamycin burkholderia vietnamiensis antibacterial rowe et al. (2016)[36] xylocandin burkholderia cepacia antifungal meyers et al. (1987)[37] content which ranges from 39 to 59 mol%, and genome size ranges from 3.02 mbp (eg. paenibacillus darwinianus) to 8.82 mbp (eg. paenibacillus mucilaginosus)[38,39]. this group of bacteria can be isolated from a variety of environments, mainly from soil. they are often associated with humans, animals, and plants. the majority of the paenibacillus spp. are producers of antimicrobial compounds and enzymes that are useful for bioremediation (table 2). furthermore, some of these compounds can be utilized as bio-fertilizers for plant growth promotion or bio-pesticides against root pathogens[39]. paenibacillus and their secondary metabolites the genus paenibacillus comprises aerobic/facultative anaerobic and endospore-forming bacteria, with a majority of them typically showing gram-positive cell wall structures[38,39]. this genus was initially included in the genus bacillus based on morphological characteristics prior to reclassification. the genus paenibacillus — which means “almost a bacillus” was then proposed by ash et. al (1993)[40] using phylogenetic classification[39]. the paenibacillus spp. have mk-7 as major quinone, anteiso-c15:0 as major cellular fatty acid, dna g + c compounds paenibacillus species bioactivity references paenibacillin paenibacillus polymyxa osy-df antibacterial he et al. (2008)[41] paenicidin a paenibacillus polymyxa nrrl b-30509 antibacterial lohans et al. (2012)[42] penisin paenibacillus sp. a3 antibacterial baindara et al. (2016)[43] polymyxin paenibacillus polymyxa antibacterial nation and li (2017)[44] colistin paenibacillus polymyxa antibacterial tambadou et al. (2015)[45] octapeptin paenibacillus tianmuensis antibacterial qian et al. (2012)[46] paenibacterin paenibacillus tiaminolyticus osy-se antibacterial huang et al. (2014)[47] pelgipeptin paenibacillus elgii b69 antibacterial ding et al. (2011)[48] gavaserin paenibacillus polymyxa antibacterial pichard et al. (1995)[49] fusaricidins paenibacillus polymyxa kt-8 antibacterial kajimura and kaneda (1997)[50] table 1. antimicrobials produced by burkholderia species. table 2. antimicrobials produced by paenibacillus species. 4 future perspectives in tpsf microbial cultivation thus far, only two bacteria (burkholderia paludis sp. nov and paenibacillus tyrfis sp. nov) with antimicrobialproducing ability from tpsf have been successfully cultivated and identified. these bacteria such as burkholderia from proteobacteria and paenibacillus from firmicutes are common phyla of bacteria dominating the tpsfs. hence, what needs to be improved in order to isolate the uncommon bacteria with antimicrobialproducing ability from tpsf? the possible reasons for isolating those common bacteria are the suitability of media used and the incubation period. the use of alternative culture media the failure in cultivating peat-inhabiting bacteria using culture dependent techniques is often due to the usage of conventional media such as nutrient and tryptone soy media. such media contain near-neutral ph with high mineral salt content that do not simulate the acidic, low nutrient conditions of tpsfs[8]. besides that, it favors fast growing bacteria and these fast-growing bacteria will outgrow the other slow growing bacteria[51]. therefore, there is a need to use diluted acidic media with low salt content such as mm1, medium m1, medium m2, medium m31, nitrate mineral salt media, peat extract medium and r2a in order to cultivate various peat-inhabiting bacteria at the same time suppressing the fast-growing bacteria (table 3). table 3. examples of media with low salt content. type of media media compositions references mm1 100 mm nacl, 10 mm (nh4)2so4, 5 mm mgso4, 1 mm cacl2, 8mm kh2po4, 16 mm k2hpo4 and micronutrient mehta and rosato (2005)[52]; schulte and bonas (1992)[53] medium m1 0.25 g/l kno3, 0.1 g/l kh2po4, 0.1 g/l mgso4, 0.02 g/l cacl2.2h2o, 0.1 g/l yeast extract, 0.005 g/l na2moo4 and 0.05% (w/v) carbon source dedysh et al. (2006)[54] medium m2 0.1 g/l (nh4)2so4, 0.1 g/l mgso4, 0.02 g/l cacl2.2h2o and 0.05% (w/v) carbon source dedysh et al. (2006)[54] medium m31 0.1 g/l kh2po4, 20 ml hutner’s basal salt, 1 g/l n-acetylglucosamine, 0.1 g/l peptone and 0.1 g/l yeast extract kulichevskaya et al. (2012b)[55] nitrate mineral salt media 1 g/l kno3, 1 g/l mgso4.7h2o, 0.717 g/l na2hpo4.12h2o, 0.272 g/l kh2po4, 0.2 g/l cacl2.6h2o and 0.005 g/l ferric ammonium edta dedysh and dunfield (2011)[56] peat extract medium 500 ml of supernatant (400 g of wet peat mixed with 200 ml of distilled water) and 500 ml of base medium m2 dedysh et al. (2006)[54] r2a 0.5 g/l yeast extract, 0.5 g/l proteose peptone, 0.5 g/l casamino acid, 0.5 g/l dextrose, 0.5 g/l soluble starch, 0.3 g/l sodium pyruvate, 0.3 g/l kh2po4 and 0.05 g/l mgcl2 dedysh et al. (2006)[54]; edenborn and sexstone (2007)[57]; taylor et al. (2002)[58] based on other studies on northern wetlands, several types of bacteria were isolated using the minimal media shown in table 3. for example, an acidophilic methaneoxidizing bacterium was isolated using minimal mineral medium containing vitamin mixture with methane as sole carbon source. in another study conducted by dedysh et al. (2006)[54], alpha-proteobacteria, beta-proteobacteria, gamma-proteobacteria, actinobacteria, firmicutes and bacteroidetes were isolated using medium m1, medium m2 and diluted r2a media. however, acidobacteria and plactomycetes were not found in the same study which might due to the reason that some acidobacteria such as granulicella species are inhibited by the presence of phosphates found in most minimal media[59]. therefore, agar selection is one of the main criteria leading to successful cultivation of peat-inhabiting bacteria. prolonged incubation time most peat inhabiting bacteria are slow growing even under optimal growth conditions. these bacteria are usually fastidious facultative anaerobes such as methanotrophs, acidobacteria and planctomycetes. in a study conducted by kulichevskaya et al. (2012a)[60], colonies of telmatocola sphagniphila (planctomycetes) were developed after 4 weeks of incubation using modified medium m2 supplemented with trace element and vitamins under 5% co2 (v/v) condition. telmatobacter bradus which is a facultative anaerobe belonging to the phylum acidobacteria requires 4 weeks to grow using medium m2[59]. in another example, telmatospirillum sibiriense which is an acidotolerant facultative anaerobic, only had observable colonies after 5 months of incubation on n-free minimal media supplemented with a reducing agent[61]. this also indicates that the presence of reducing agents might promote the growth of fastidious facultative anaerobes. however, there is no published result on the successful isolation of strict anaerobes from either the northern wetlands or tpsfs, which suggest the need for other reducing agents to be included in the culture media[51]. furthermore, a more stringent method should be applied during sampling collection where peat samples are to be placed immediately in anaerobic conditions prior to sample transportation. this reduces the exposure of oxygen to the anaerobic bacteria at the same time simulating the actual anoxic conditions in the tpsf. to sum up, there is a need to incubate the peat culture for a prolonged duration with anoxic conditions, minimal nutrients, and with appropriate supplements in order to isolate anaerobes from tpsf. genomic approach to discover bioactive secondary metabolites production whole genome sequencing of bacteria has become increasingly common for routine use in microbiological microbes from peat... 5 laboratories. subsequently, a large quantity of dna sequence data from different microorganisms is currently available in public databases. as a result, this creates a path for uncovering novel natural products from microbes by utilizing new bioinformatics tools[62]. for instance, many recent studies have been performing whole genome sequencing of drug-prolific producers such as the streptomyces spp.[63–69] for further investigation of their secondary metabolite production ability this could also be useful for the prediction of novel products of nonribosomal peptide synthetases (nrpss) and polyketides synthases (pkss) through application of various sequence analysis tools[62]. similarly, this strategy has been applied for burkholderia and paenibacillus[70,71,72]. by taking the genus burkholderia as an example, the determination of genome sequences of these bacteria has essentially created a route for in silico structural prediction, wet lab experimental design, and execution. genome-guided approaches, which are made possible through accessibility of extensive genome sequence data coupled with genomemining technologies, have warrant the discovery of structurally and functionally diverse natural products from numerous burkholderia strains[71, 73,74]. conclusion tropical peat swamp forests are indeed a promising environment to source for secondary metabolites. however, culture-dependent methodologies should be scrutinized to ensure cultivation of rare bacterial species with important ecological and commercial roles which have never been captured before. besides, whole genome sequencing of the bacteria in the near future may allow further understanding of the antimicrobial synthesizing capability of these bacteria. nevertheless, efforts should be made to culture microbes from different genera with similar potential to discover new bioactive secondary metabolites. conflict of interest the authors declare that there is no conflict of interest in this work. author contributions k-so performed the literature search, critical data analysis and performed the writing of this review. technical support and proofreading were contributed by jw-fl and vl. k-so, cmy and s-ml founded the review writing project. acknowledgments the authors would like to thank school of science and tropical medicine and biology multidisciplinary platform, monash university malaysia for their support. references 1. banin, e, hughes, d, and kuipers, op. bacterial pathogens, antibiotics and antibiotic resistance. fems microbiol rev 2017; 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80 were significantly over-expressed, while 94 were underexpressed (adj. p-value < 0.1; log2 fold change ≤ -1 or ≥ 1). fifteen mirnas were significantly differentially expressed only in braf v600e-positive ptc, and eight of these were validated in tcga thca dataset (hsa-mir-212, -132, -135b-3p/5p, -200b, -200a-3p/5p, -27a-3p/5p, -29a and -1296). subsequent analysis revealed significant enrichment of cancer-related pathways including proteoglycans in cancer, ecm-receptor interaction and mapk pathways in braf v600e-positive ptc. using the mirnaseq and in silico validation using tcga thca study, we identified eight mirnas that were differentially expressed in ptc tissues with braf v600e. this study also complemented the existing knowledge about deregulated mirnas in ptc development. keywords: microrna; papillary thyroid cancer; braf v600e, next-generation sequencing. received: 9th october 2019 accepted: 10th november 2019 published online: 18th march 2020 citation: mohamad yusof a, tieng fyf, muhammad r, et al. in-depth characterization of mirnome in papillary thyroid cancer with braf v600e mutation. prog microbes mol biol 2019; 2(1): a0000043. https://doi.org/10.36877/pmmb. a0000043 introduction the incidence rate of thyroid cancer had risen significantly in many countries worldwide in the past decade. in 2012, there were approximately 230,000 and 70,000 new cases of thyroid cancer among women and men, respectively [1,2]. according to the united states cancer statistics 2019, 52,070 new thyroid cancer cases with 2,170 deaths were estimated by 2019 in both sexes [3]. most thyroid cancers originated from follicular epithelial cells, which were further divided into well-differentiated papillary thyroid carcinoma and follicular carcinoma, poorly differentiated carcinoma and anaplastic carcinoma [4–6]. papillary thyroid carcinoma (ptc) is the most prevalent histological type that contributed to 85 to 90% of reported cases [7]. several of its clinicopathological features are correlated to poor prognosis in ptc patients, which include the male gender, larger tumour size, older age, extrathyroidal extension, thyroid capsule invasion, copyright 2019 by mohamad yusof a et al. and hh publisher. this work under licensed under the creative commons attributionnoncommercial 4.0 international lisence (cc-by-nc 4.0) *correspondence: nurul-syakima ab mutalib, ukm medical molecular biology institute (umbi); syakima@ppukm.ukm.edu.my 2 lymph node metastasis as well as braf mutation [8]. although ptc patients had a high survival rate [1,9], these prognostic factors had been shown to affect the overall survival among ptc patients [10]. mirnas are small non-coding rnas that comprise of 19 to 22 nucleotides, which negatively regulate gene expression. each mirna can take part in many cellular pathways, and thus, mirnas are involved in many different diseases [11] including thyroid cancers [12–15]. in addition, it was reported that different histopathological types of thyroid tumours have discrete mirna profiles [16]. mir-146b [17,18], mir-221 and mir-222 are among the commonly upregulated mirnas in papillary thyroid carcinoma [16,19–23]. these non-coding rnas are promising biomarkers to identify aggressive ptc cases [17,24]. braf is a serine-threonine kinase that is activated by ras binding and protein recruitment to the cell membrane [25,26]. activation of mek along with the mapk signalling pathway is activated by braf phosphorylation [27]. the most frequent genetic changes in ptc are point mutations of braf which are observed in 35 to 70% of ptc cases [26,28]. more than 95% of braf mutations detected in thyroid cancers are thymine to adenine transversion at position 1799 (t1799a), resulting in the substitution of valine by glutamate at residue 600 (v600e) [29–31]. various studies had shown that the braf v600e mutation was associated with lymph node metastasis, therefore, it had received the attention as a diagnostic and prognostic molecular marker in recent years to improve the diagnosis and identify individuals at increased risk of ptc recurrence [30,32]. in the past few years, the evolution of next-generation sequencing technologies has allowed global expression profiling of mirnas [33] and the discovery of novel human mirnas [14,34]. in this study, we aimed to identify the differentially expressed mirnas in ptcs with braf v600e using small rna sequencing. to achieve this objective, five fresh-frozen tumour tissues paired with their normal adjacent thyroid tissues were included in the study. the mutational analysis of braf v600e gene and differentially expressed mirnas were performed using sanger sequencing and small rna sequencing, respectively. the results were validated with an in silico approach using the dataset obtained from the cancer genome atlas (tcga) thca. materials and methods clinical specimens five pairs of tumour-adjacent normal fresh frozen tissues were collected from patients diagnosed with ptc from the ukm medical centre (ukmmc). this study was approved by the universiti kebangsaan malaysia research ethics committee (ukmrec; ukm 1.5.3.5/244/umbi-2015-002). informed consent was obtained from all the study participants. the tissues were dissected, snap-frozen and stored in liquid nitrogen. all samples were cryosectioned and stained using haematoxylin and eosin and the percentage of tumour cells and normal cells contents were assessed by a pathologist. only tumour samples with at least 80% cancerous cells and normal adjacent thyroid tissues with less than 20% necrosis were selected for further analysis. nucleic acid isolation all fresh frozen tissues were subjected to nucleic acid extraction using allprep dna/rna/mirna universal kit (qiagen, hilden, germany) according to the manufacturer’s recommendations. the integrity of rna was assessed using agilent bioanalyzer 2100 (agilent technologies, ca, u.s.), while the quality of dna was assessed using 1% agarose gel. the quantity and purity of rna and dna were assessed using qubit 2.0 fluorometer (thermo scientific, ma, u.s.) and nanodrop 2000c spectrometer (thermo scientific, ma, u.s.), respectively. braf v600e genotyping pcr amplification of genomic regions of interest was performed using braf v600e forward primer 5’-tgcttgctctgataggaaaatg-3’ and braf v600e reverse primer 5’-agcatctcagggccaaaaat-3’ [35]. amplification was performed in a reaction volume of 25 μl containing 50 ng dna template, 10 μm each primer (integrated dna technologies, ia, u.s.),10x pcr gold buffer without mgcl2, dntp mix (10 mm), mgcl2 solution (25 mm) , amplitaq gold® (5u/μl) (applied biosystems, ca, u.s.) and nuclease-free water. pcr conditions were as follows; 95°c for 4 minutes; 35 cycles of 95°c for 45 seconds, 50°c for 30 seconds and 72°c for 1 minute; 72°c for 5 minutes; and held at 4°c. pcr products were visualized by electrophoresis on 1.5% agarose gel with an expected size of ~ 228 bp. pcr purification was conducted using qiaquick pcr purification kit (qiagen, hilden, germany) as per manufacturer’s instruction. subsequently, dna sequencing was performed using abi prism 3130xl genetic analyzer (applied biosystem, ca, usa). library preparation and mirna sequencing rna samples from tumour samples and their adjacent normal tissues were processed into libraries using truseq small rna sample prep kit (illumina, ca, usa). briefly, 3′ and 5′ adapters were sequentially ligated to the ends of small rnas fractionated from 2 μg of total rna, and reverse transcribed to generate cdna. the cdna was amplified using a common primer complementary to the 3′ adapter, and a primer containing 1 of 48 index sequences. samples were size-selected (140–160 bp fragments) on a 6% polyacrylamide gel, purified, quantified and pooled for multiplexed sequencing. the resulting pooled libraries were normalized to 2 nm and were hybridized to oligonucleotide-coated single-read flow cells for cluster generation using hiseq® rapid sr cluster kit v2 on hiseq 2500 (illumina, ca, usa). subsequently, the clustered pooled mirna libraries were sequenced on the hiseq 2500 for 50 sequencing cycles using hiseq® rapid sbs kit v2 (50 cycle) (illumina, ca, usa). in-depth characterization... 3 bioinformatics and statistical analyses pre-processing of data was executed in basespace software (illumina, ca, usa), and fastq files were generated. mirna analysis app version 1.0.0 was used for determination of differentially expressed mirnas using the workflow described by cordero et al, 2012 [25]. briefly, the pipeline includes 3’ end adapter removal using cutadapt, annotation to mirbase v21, mapping using shrimp aligner and differential analysis of mirnas using deseq2. benjamini and hochberg’s [36] correction was applied to ensure a false discovery rate (fdr) less than 0.1 and absolute log2 fold change ≤ -1 or ≥ 1 were considered for further analysis. heatmaps were created using genee from the broad institute (http://www.broadinstitute.org/ cancer/software/gene-e). mirna target prediction via diana-tarbase v7.0 [37] and pathway enrichment analysis was performed using diana-mirpath v3.0 [38]. other statistical analyses were performed using graphpad prism 6 unless stated otherwise. in silico validation we used the tcga-generated level 3 mirna sequencing data from thca project [24]. these data were accessed from ‘https://tcga-data.nci.nih.gov/tcga/dataaccessmatrix. htm’ on april 13, 2016. the normalised expression (reads per million or rpm) of all mirnas was log2-transformed and used for fold change calculation. we then performed the students’ unpaired t-test with benjamini hochberg false discovery rate (fdr) multiple testing correction and log2 fold change calculation using bioconductor version 3.1 (biocinstaller 1.18.2) [39] in r version 3.2.0 (r development core team, 2008). results demographic data all patients in the small discovery set were women with a mean age of 51.6 years. majority of the tumours were located in the right lobe of the thyroid and all tumours were larger than 1 cm. all of the patients had lymph node metastasis at diagnosis. validation cohort from tcga thca studies included 229 braf v600epositive ptc, 132 braf v600e-negative ptc and 59 unpaired normal thyroid samples. mirnaseq analysis with the rapid run mode using hiseq rapid sbs kit v2, we achieved an average of 5.6 m reads per sample with q30 (3,779,968 to 8,308,285 reads in each sample). the percentage of mapped reads in normal samples were significantly lower than in tumour samples (supplementary table 1). figure 1 illustrated the quality control statistics of mirnaseq experiment for braf v600e-positive versus adjacent normal samples. in figure 1(a), approximately 45 to 75% of the reads were identified as isomir (known precursor) and 25 to 55% were the already known mature mirnas. figure 1(bd) illustrated the principle component analysis (pca) plots of mirnas family, mature mirnas and precursor mirnas expression profiles. pca analysis revealed that samples formed distinct clusters, withall tumour and normal samples clustering based on their respective groups. differentially expressed mirnas there were 174 mirnas significantly differentially expressed in ptc braf v600e-positive versus their normal adjacent thyroid tissues. eighty mirnas were upregulated, while 94 mirnas were downregulated (log2 fold change ≤ -1 or ≥ 1; fdr p-value < 0.1). the volcano plot (figure 2) illustrated the significantly differentially expressed mirnas. in addition, the unsupervised hierarchical clustering analysis and heatmap illustrated in figure 3, clearly demonstrated the difference of the mirna expression profiles between tumour and normal samples. mohamad yusof a et al. figure 1. quality control statistics of mirnaseq experiment. (a) distribution of mirna sequences among the subcategories in braf v600e ptc and their adjacent normal. (b-d) principle component analysis (pca) plots of mirnas family, mature mirna and precursor mirnas. the plots showed that global mirnas family, mature mirnas and precursor mirnas expression pattern clearly differentiate the samples according to respective group 4 in-depth characterization... figure 2. mirnas with statistical significance after a t-test are shown in red and blue on a volcano plot above. those with no significance are shown in black. the red dots represent significantly upregulated mirnas while blue dots represented significantly downregulated mirnas. figure 3. hierarchical clustering and heatmap representation of differentially expressed mirnas in discovery set. the list of differentially expressed mirnas were filtered using fdr-adjusted p value <0.1, absolute log2 fold change ≥1 or ≤-1. in braf v600e-positve ptc versus normal-adjacent tissues, mirnaseq revealed 174 differentially expressed mirnas. ninety-four (94) were under-expressed while 80 were over-expressed. 5 mohamad yusof a et al. pathway enrichment analysis of the deregulated mirnas a single mirna might play specific roles in the pathogenesis of ptc; however, the mirna pathway as a whole might also be of importance. therefore, dianamirpath [37], a web-based computational tool, was used to identify pathways that were potentially altered by the expression of multiple mirnas, and to incorporate mirnas into molecular pathways. based on the 80 upregulated mirnas, there were 44 significantly enriched pathways identified by the meta-analysis algorithm using “pathway union” option (p-value < 0.05) (figure 4a). thirty-eight out of the 80 upregulated mirnas were involved in the proteoglycans in the cancer pathway, targeting 176 genes. figure 4b illustrated the hierarchical clustering analysis and the heatmap derived from the 44 significantly enriched pathways. the number of mirnas and experimentally validated predicted targets involved in each pathway were tabulated in table 1. we also performed the same analysis on 94 significantly downregulated mirnas. only five significantly enriched pathways were identified: fatty acid biosynthesis, fatty acid metabolism, ecm-receptor interaction, fatty acid elongation and proteoglycans in cancer pathway with fdr adj. p-value < 1e-325, 2.57e-07, 7.70e-05 and 0.000352, respectively. tcga thca in silico validation to validate the observed significance of the 174 mirnas in braf v600e-positive ptc as compared to their normal adjacent thyroid tissues, we analysed the expression of these mirnas in tcga thca dataset. comparison between braf v600e-positive ptc versus normal thyroid tissues revealed 123 significantly upregulated and 319 downregulated mirnas (supplementary table 2). to identify mirnas which were deregulated only in braf v600e-positive ptc, the intersection of the differentially expressed mirnas was performed (figure 5). since tcga thca dataset did not comprehensively annotate the mirnas based on 3p and 5p arm, the intersection was performed without taking into account the arm annotation. from the intersection, 15 mirnas were deregulated in braf v600e-positive ptc versus normal thyroid tissues in both and tcga thca datasets and the current study. from these 15 mirnas, eight mirnas were in concordance with tcga thca data expression levels which included hsa-mir-212, hsa-mir-132, hsa-mir135b-3p and 5p, hsa-mir-200b, hsa-mir-200a-3p and 5p, hsa-mir-27a-3p and 5p, hsa-mir-29a and hsamir-1296 (log2 fold change 1.59, 1.43, 2.36, 1.72, 2.12, 1.31, 1.52, 1.60, 1.69 and -1.12; adj. p-value < 0.1), respectively (figure 6). figure 4. pathway enrichment analysis of 80 significantly upregulated mirnas in braf v600e-positive ptc and mirnas versus pathway heatmap (clustering based on significance levels) using the diana mirpath v3.0 interface. (a) a list of 44 significant kegg targeted pathways with their log10 p-value. (b) hierarchical cluster and heatmap. dark red indicate lower significant values. the attached dendograms on both axes represent the hierarchical clustering results for mirnas and pathways respectively. mirnas are clustered together by exhibiting similar pathway targeting patterns and pathways are clustered together by related mirnas. 6 in-depth characterization... figure 5. venn diagram of the differentially expressed mirnas in discovery and in silico validation dataset. from the intersection, there were 15 mirnas that were differentially expressed in braf v600e-positive ptc versus normal. from these 15 mirnas, eight deregulated mirnas from discovery data were in concordance with tcga thca data (seven were upregulated and one was downregulated). figure 6. box plot of eight concordance mirnas. all mirnas were significantly expressed (adjusted p-value <0.1; log2 fold change ≤-1 or ≥1). 7 mohamad yusof a et al. table 1. number of mirnas and experimentally validated targets involved in each of the 44 enriched pathways. kegg pathway adj p-value no. of genes no. of mirnas prion diseases <1e-325 18 8 micrornas in cancer <1e-325 140 12 oocyte meiosis <1e-325 83 19 tgf-beta signaling pathway <1e-325 62 19 thyroid hormone signaling pathway <1e-325 95 20 ecm-receptor interaction <1e-325 50 21 colorectal cancer <1e-325 59 21 prostate cancer <1e-325 81 21 fatty acid biosynthesis <1e-325 7 23 pathways in cancer <1e-325 303 23 fatty acid metabolism <1e-325 36 24 cell cycle <1e-325 109 24 protein processing in endoplasmic reticulum <1e-325 138 24 hepatitis b <1e-325 118 25 chronic myeloid leukemia <1e-325 70 27 glioma <1e-325 58 28 p53 signaling pathway <1e-325 66 29 viral carcinogenesis <1e-325 177 31 lysine degradation <1e-325 39 33 hippo signaling pathway <1e-325 119 35 adherens junction <1e-325 66 37 proteoglycans in cancer <1e-325 173 38 foxo signaling pathway 1.75e-14 104 22 bacterial invasion of epithelial cells 2.16e-14 65 21 bladder cancer 3.91e-13 34 18 endometrial cancer 4.77e-12 47 18 small cell lung cancer 2.19e-10 72 17 transcriptional misregulation in cancer 5.84e-10 133 17 melanoma 9.28e-10 56 20 signaling pathways regulating pluripotency of stem cells 7.09e-09 98 14 thyroid cancer 1.00e-08 26 16 endocytosis 1.43e-08 159 16 pancreatic cancer 2.29e-07 62 13 focal adhesion 6.68e-07 142 12 pi3k-akt signaling pathway 1.00e-06 182 14 ubiquitin mediated proteolysis 2.39e-06 105 14 non-small cell lung cancer 5.32e-06 48 14 renal cell carcinoma 1.47e-05 58 13 estrogen signaling pathway 2.93e-05 60 8 other types of o-glycan biosynthesis 0.000125 21 11 central carbon metabolism in cancer 0.000955 50 9 neurotrophin signaling pathway 0.001194 75 8 shigellosis 0.002684 50 10 steroid biosynthesis 0.009698 13 12 8 in-depth characterization... discussion there was various type of braf mutations reported for malignant tumours including ptc such as braf v600e, braf v600d, braf v600q, braf v600v and braf v600l [40,41]. for our study, we only focused on braf v600e mutation as it was the most common braf mutation in ptc, which comprised more than 90% of cases [42]. many studies had demonstrated an association of this braf mutation with the aggressive clinicopathological characteristics of ptc such as extrathyroidal invasion, lymph node metastasis and recurrence of ptc [8,43]. it was suggested that the involvement of braf v600e mutation in the activation of ras/raf/mapk pathway could result in higher deregulation of mirna expression [44]. while previously published studies utilized microarray and real-time pcr, we used small rna deep sequencing to determine the deregulation of mirnas in braf v600epositive ptc patients in an unbiased manner. there were more downregulated mirnas in the tumour samples as compared to their adjacent normal thyroid tissues (80 upregulated versus 94 downregulated mirnas). deregulation of hsa-mir-146b-5p, hsa-mir-146b-3p, hsamir-222-3p, hsa-mir-221-3p, hsa-mir-204-5p and hsamir-7-5p were further reconfirmed in this study, signifying that dysregulation of these mirna was common in ptc versus normal thyroid tissues. the association between braf v600e status and mirna expression in ptc had been controversial. a large-scale analysis of tgca data had demonstrated that the braf v600e mutation was one of the key drivers of ptc [24]. other studies had shown that downregulation of hsa-mir-7-5p and hsa-mir-204-5p in ptc were associated with the braf v600e mutation [23,44]. there were however contradictory findings that showed that braf v600e mutation is not related with the aggressiveness of ptc and thus, cannot serve as prognosis marker for ptc [45–47]. mir-200a and mir-200b were the members of mir200 family. upregulation of these two mirnas was observed in ptc [48], follicular thyroid carcinoma (ftc) and follicular adenoma [16], while being downregulated in anaplastic thyroid carcinoma [49]. it was suggested that mir-200b downregulates the tumour suppressor genes [48]. these mirnas were shown to play a crucial role in tumour cell metastasis progression or epithelial-mesenchymal transition (emt) [50] and inhibit angiogenesis [51]. hsa-mir200a and hsa-mir-200b were also upregulated in thyroid cell-derived cell lines and tissues with braf v600e [16,48]. in this study, mir-200a and mir-200b were upregulated in braf v600e-positive cases in both discovery and tcga thca datasets, suggesting their relation to braf v600e mutation. among the significantly enriched pathways in braf v600e-positive ptc were proteoglycans in cancer, cell cycle pathway and ecm-receptor interaction pathway. there were 173 genes with 38 mirnas in proteoglycans in cancer, 109 genes with 24 mirnas in cell cycle pathways and 50 genes with 21 mirnas involved in the ecmreceptor interaction pathway. proteoglycans (pgs) are key molecular constituents of the ecm and cell surfaces and play important roles in integrating signals from growth factors, chemokines and integrins, cell to cell interactions as well as matrix adhesion [52,53]. pgs act in a contextdependent manner; some have proand anti-angiogenic activities, while others can directly stimulate cancer growth by controlling key signalling pathways [52,54,55]. aberrant accumulation of pgs in human thyroid cancer was first reported in 1984 [56]. using the glycoproteomics approach, arcinas and colleagues studied the expression of proteoglycans in various thyroid cancer cell lines. two transmembrane heparan sulfate proteoglycans, syndecan-1 and syndecan-4, were uniquely expressed in ftc-133 and xtc-1 respectively. in addition, a gpianchored proteoglycan, glypican-1, was identified in three thyroid cancer cell lines (ftc-133, xtc-1 and dro-1). the extracellular heparan sulfate proteoglycan basement membrane-specific core protein or perlecan was only detected in aro, a dedifferentiated thyroid cancer cell line [57]. numerous studies support the important role of proteoglycans as mirna targets in cancer progression [58,59]. deregulation of mirnas results in atypical expression patterns of proteoglycans and their biosynthetic enzymes, thus leading to abnormal cell proliferation, apoptosis, adhesion, migration, invasiveness and epithelial-to-mesenchymal transition [60–62]. therapeutic strategies targeting the microrna– proteoglycan are emerging for cancers such as in melanoma and medulloblastoma [60,63,64]. while the relationship between mirnas expression and ecmreceptor interaction pathway in regards to braf v600e has been reported [65,66], there is currently no published evidence linking mirna regulation to proteoglycans in thyroid cancers, especially in the braf v600e-positive ptc. in order to validate our results in a larger dataset, we re-analysed tcga thca dataset containing 229 braf v600e-positive ptc, 132 braf v600e-negative ptc and 59 normal thyroid tissues. from the intersection of our discovery data with the tcga data, 15 mirnas were significantly deregulated in braf v600e-positive as compared to normal thyroid tissues and eight mirnas were in concordance in term of expression levels. the remaining seven mirnas showed the opposite trend of expression. the discrepancies could be due to the fact that tcga thca datasets used were from unmatched samples. secondly, at the time of the data retrieval, tcga thca had not yet annotated the mirnas according to their 3p and 5p arm and thus, the expression level between these two arms could not be differentiated. nevertheless, we were able to reconfirm the expression of commonly deregulated mirnas in ptcs and provide a new list of mirnas related to braf v600e. conclusion in conclusion, our study illustrated the interplay between braf v600e status and differentially expressed mirnas in ptc. this information would add to the understanding of the molecular mechanisms of mirnas in braf v600e-positive ptc. although these findings were needed to be validated in larger sample size, they 9 could serve as a basis for the identification of a potential diagnostic or prognostic biomarker of ptc. in addition, functional studies to clarify further the mechanisms of mirna regulation in braf v600e-positive ptc were warranted. declaration of interest the authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. funding this research was funded by the fundamental research grant scheme (frgs) from the ministry of education malaysia (frgs/1/2014/skk01/ukm/03/1). author contributions a mohamad yusof, ns ab mutalib involved in the specimen collections, library preparation and sequencing, data analyses, acquisition of data and drafting the manuscript. fyf tieng performed the tcga analyses. s. saidin performed the braf v600e genotyping. i. mohamed rose assessed tumour percentage of the tissues. sn abdullah suhaimi and r muhammad were thyroid surgeons involved in specimen retrieval. i ismail and r jamal provided critical review on the manuscript. all authors read and approved the final 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is it really orchestrating in thyroid cancer? oncotarget, 2010; 1(8): 751–756. 66. geraldo mv, kimura et. integrated analysis of thyroid cancer public datasets reveals role of post-transcriptional regulation on tumor progression by targeting of immune system mediators. plos one, 2015; 10(11): e0141726. doi:10.1371/journal. progress in microbes and molecular biology review article 1 an insight of vitamin e as neuroprotective agents hui-min yap1* and kwan-liang lye1 1department of biomedical sciences faculty of medicine and health sciences, universiti putra malaysia, 43400 upm serdang, selangor darul ehsan, malaysia abstract: nervous system is the network of nerve cells that transmits nerve impulses throughout the body. it is rich in both unsaturated fats and irons, making it predominantly susceptible to oxidative stress and damage. oxidative stress reflects the disruption of the redox balance between the formation and clearance of highly free radicals, for instance reactive oxygen species (ros) and reactive nitrogen species (rns). oxidative stress will further damage the cell lipid, protein and dna. oxidative stress has a role in the modulation of critical cellular functions, such as apoptosis program activation, ion transport and calcium mobilization which lead to cell death. many studies were conducted to prevent neuronal cell death caused by oxidative stress through administration of free radical scavenging antioxidant, such as vitamin e. vitamin e is known as a chain-breaking antioxidant that showed the capability to increase the viability of neuronal cells that had undergone glutamate injury by inhibiting glutamate-induced pp60 (c-src) kinase activation. vitamin e occurs in 8 forms, namely α-, β-, γand δ-tocopherols and α-, β-, γ-and δ-tocotrienols. tocotrienols differ from tocopherols by possessing an unsaturated isoprenoid side chain instead of a saturated phytyl tail. tocotrienols, compared to tocopherols, are lightly studied due to the abundance of α-tocopherol in the human body and its antioxidant properties. nevertheless, recent studies showed that α-tocotrienol is more effective in preventing lipid peroxidation compared to α-tocopherol. furthermore, tocotrienol was discovered to protect neuronal cell through antioxidant-independent activities. the tocotrienol-rich fraction (trf) is an extract that consists of 75% tocotrienol and 25% α-tocopherol. trf was reported to possess potent antioxidant, anti-inflammation, anticancer and cholesterol-lowering properties. thus, this writing highlights the significant neuroprotective effects of tocotrienol and tocopherol. keywords: neuroprotective agents; vitamin e; oxidative stress; tocotrienols; tocopherols. received: 10th march 2020 accepted: 12th april 2020 published online: 21th april 2020 citation: yap, h-m. & lye, k-l. an insight of vitamin e as neuroprotective agents. prog mircobes mol bio1 2020; 3(1): a0000071. https://doi.org/10.3687/pmmb.a0000071 introduction antioxidant it known that aerobic organisms have developed a series of defense mechanisms, which involve antioxidants, in response to free radical production in order to maintain free radicals’ level compatible with cellular functions and metabolic processes[1]. antioxidant defense mechanism can be classified into enzymatic and non-enzymatic. the enzymatic defense mechanism includes superoxide dismutase (sod), gsh peroxidase (gpx) and catalase (cat), whereas non-enzymatic antioxidant defenses include vitamin e, ascorbic acid (vitamin c), glutathione (gsh) and other antioxidants[2]. glutathione (gsh) glutathione (gsh) is the main thiol antioxidant and redox buffer of the cell[3,4]. it is a tripeptide comprised of glutamate, glycine and cysteine. it is synthesized in the cytosol by 2 enzymes that utilize atp, that is gsh synthetase and gamma-glutamylcysteine (γ-glucys) synthetase[5]. the gamma-glutamylcystein synthetase forms dipeptide gamma-glutamylcysteine by utilizing cysteine and glutamate as substrates. gamma-glutamylcysteine is then merged with glycine in a reaction catalyzed by gsh synthetase thus forming gsh. gsh production is controlled by feedback inhibition of the γ-glucys synthetase reaction by the end product gsh[6]. total gsh in the cells can be free or bound to protein. the free gsh is present in reduced form, which will be converted to the oxidized form (gssg) during oxidative stress and can be restored to the reduced form by the action of glutathione reductase (gr)[7]. the oxidation-reduction pathway of gsh is shown in figure 1. copyright @ 2020 by yap and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: hui-min yap, department of biomedical sciences faculty of medicine and health sciences, universiti putra malaysia, 43400 upm serdang, selangor darul ehsan, malaysia; huimin050686@gmail.com. 2 figure 1. oxidation-reduction pathway of reduced glutathione (gsh) and oxidized glutathione (gssg). gsh exerts its protection against oxidative stress by several approaches. firstly, gsh directly scavenges reactive hydroxyl free radicals, ros and radical centers on dna and other biomolecules. also, gsh functions as the cofactor for several detoxifying enzymes, for instance glutathione-s-transferases (gsts) and glutathione peroxidase (gpx), which are vital in protection against oxidative stress. gpx is an enzymatic antioxidant which is predominantly responsible for the intracellular reduction of hydrogen peroxide (h2o2) to water with the support of gsh as electron donor[8]. thus, gsh is oxidized to glutathione disulfide (gssg) through gpx activity and quickly restored to gsh by the reaction catalyzed by glutathione reductase (gr). whereas, glutathione-s-transferases conjugate gsh to free radicals, for instance hydrogen peroxides, thus reducing the deleterious interactions between free radical and essential cellular components[9,10]. furthermore, gsh provides reducing capacity for the formation of deoxynucleotides by ribonucleotide reductase, the reduction of dehydroascorbate to ascorbate and restoration of vitamin e from radical form[2,11]. also gsh has been suggested to function as neurohormone based on the presence of extracellular gsh in brain, the release of gsh from brain slices upon stimulation, the specific binding of gsh to extracellular receptors and the induction of sodium currents in neocortex, and the stimulation of a signal cascade in astrocytes[12,13]. furthermore, some studies suggested that astrocytes support neuronal cells by means of providing gsh and cysteinylglycine (cysgly), which is the gsh precursor to neuronal cells[6]. vitamin e vitamin e is a lipid-soluble vitamin vital for human nutrition and health. the term ‘vitamin e’ was first introduced by evans and bishop (1922)[14] in describing a dietary factor in rat reproduction. the vitamin e family includes 8 different isomers, namely α-, β-, γ-, and δ-tocopherols and α-, β-, γ-, and δ-tocotrienols. tocopherols and tocotrienols, as a group known as tocochromanols comprise of a chromanol ring system and a polyprenyl side chain. the 8 isomers of vitamin e vary in the degree of antioxidant and biological activities. all tocochromanols are amphipathic molecules, with the lipophilic isoprenoic side chain of tocochromanol is anchoring the membrane lipids, whereas the polar chromanol ring is exposed to the membrane surface[15]. vitamin e is the major component that present amongst the lipid elements of cell membranes and lipoproteins[16]. vitamin e is exclusively synthesized by photosynthetic eukaryotes and other oxygenic photosynthetic organisms for instance cyanobacteria. therefore, vitamin e is ingested along with fat-containing food, like nut oil seeds, egg yolk, vegetable oils, margarine, soya bean, wheat, avocados and germ[15]. vitamin e has numerous biological functions. the pharmacologic use of vitamin e, in doses 10 to 50 times the daily requirement, was recommended in 1947 for the treatment of an array of cardiovascular disorders[17]. the chain-breaking antioxidant properties of vitamin e was detected in the 1950s and consequently proved to be useful in preventing lipid peroxidation by scavenging chain-carrying peroxyl radicals and generates an induction period[18,19,20]. furthermore, studies reported that severe vitamin e deficiency in human will leads to neuromuscular abnormalities because of free radical damage to the nerve cells[21,22]. vitamin e deficiency seldom occurs in human as a result of dietary deficiencies but occurs as a result of genetic abnormalities in the α-tocopherol transfer protein (α-ttp)[23]. vitamin e also possesses non-antioxidant functions, with vital role in cellular signaling by regulating protein kinase c[24]. moreover, vitamin e in combination with selenium were exhibiting ability to prevent loss of spermatogenesis in males[25]. some studies also indicated that vitamin e prevents most of the glutamate-induced neuronal cell death[26]. furthermore, dietary of vitamin e can enhance immune responses in numerous animal models[27]. metabolism of vitamin e the hydrophobic nature of vitamin e make it preferentially located in oil storage organs, fat deposits and in cell membranes. it is transported around the body as an element of plasma lipoproteins. after ingestion of dietary vitamin e, it will be absorbed into the enterocyte, followed by packaging into chylomicrons. these nascent chylomicrons are then secreted into the lymphatic circulation. during the chylomicron catabolism in the circulation, the absorbed vitamin e is transferred to circulating lipoproteins and drained into the bloodstream. the high-density lipoprotein (hdl) in the bloodstream donates apolipoprotein c-ii (apocii) and apolipoprotein e (apoe) to the nascent chylomicron and thus converts it to a mature chylomicron. lipoprotein lipase (lpl) is bound to the endothelial lining of capillary walls. during the lipolysis by lpl, various form of vitamin e could be transferred to tissues. also, vitamin e could be exchanged between hdls and other circulating lipoproteins, which could deliver vitamin e to the peripheral tissues. the resultant chylomicron remnant from lipolysis are primarily taken up by the liver through the chylomicron remnant receptors[28]. in liver, remnant chylomicron-associated vitamin e is incorporated into nascent very-low density lipoproteins (vldl) via the action of α-ttp[29]. one of the vital determinants of vitamin e biological activity is the affinity of its analogues for α-ttp. the α-ttp has higher preference to α-tocopherol compared to other vitamin e isomers[30]. when the vldl are secreted into the plasma circulation, vldl are converted into intermediate density lipoprotein (idl) and low density an insight of vitamin e... 3 yap et al. lipoprotein (ldl) via the action of lpl[31,32]. vitamin e is then transferred from plasma to cells through uptake facilitated by receptor-mediated lipoprotein endocytosis, lipid transfer proteins and lipases, and selective lipid uptake[33]. studies demonstrated that ldl receptor were facilitating the tissue incorporation of plasma vitamin e as part of ldl, while lpl and phospholipid transfer protein enable the tissue incorporation of plasma vitamin e as part of triglyceride-rich lipoprotein[34–36]. furthermore, idl and ldl have ldl receptor-binding domains which allow receptor-mediated lipoprotein endocytosis to facilitate uptake of vitamin e into the peripheral tissue[37]. the pathways of vitamin e absorption and distribution are depicted in figure 2. figure 2. pathways for vitamin e absorption and distribution. vitamin e is one of the most vital lipid-soluble antioxidants that protects membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction[31]. as an antioxidant agent, vitamin e does not work independently in scavenging free radicals. it is a part of the redox antioxidant system. vitamin e is efficiently reduced from its free radical form (tocotrienoxyl or tocopheroxyl) back to its reduced native form via enzymatic or non-enzymatic mechanisms. vitamin c can directly restore vitamin e and thiol antioxidant, for instance gsh, and indirectly restore vitamin e via redox antioxidant network. this system maintains the concentration of vitamin e radicals low. hence, the loss or consumption of vitamin e is prevented[38]. isomer of vitamin e tocopherol tocopherol contains a chromanol ring and a saturated phytyl side chain[39]. the structural formulae of tocopherols are shown in figure 3. tocopherol is primarily found in sunflower and olive oils. among 8 isomers of vitamin e, α-tocopherol was firstly derived from wheat germ oil and named in 1936 by evan et al.[40]. the α-tocopherol have the highest bioavailability among the isomers because of the recognition of α-ttp[41]. the core function of α-tocopherol is terminating the chain reaction of lipid peroxidation to inhibit cell membrane and ldl from oxidative disintegration[42]. tocopherol also provides protection against peroxynitrite-induced lipid oxidation. other than antioxidant function, vitamin e has functions in cell signaling activities, for instance regulation of protein kinase c, inhibition of cyclooxygenase-2 activity and modulation of phospholipase a2 activity were due to the present of α-tocopherol. the α-tocopherol could dilate blood vessels and interferes with aggregation of platelets[43]. osakada et al.[44] reported that 1-10 µm α-tocopherol effectively protects striatal neurons against cytotoxicity induced by a l-buthionine-s,r-sulfoximine (bso) via the reduction of oxidative stress. study indicated that α-tocopherol can effectively relieve neuronal damage induced by oxygencentered free radicals[45]. also, α-tocopherol functions in regulating inflammation by reducing the release of cytokine interleukin-1β (il-1β) via inhibition of 5-lipoxygenase pathway[45]. figure 3. structural formulae of tocopherols. tocotrienol tocotrienols vary from tocopherols by having 3 double bonds in the hydrophobic tridecyl side chain[42]. figure 4 depicted the structural formulae of tocotrienols. tocotrienols are rich in barley oil and palm oil. more than 95% of studies on vitamin e focusing on α-tocopherol due to its richness in the human body and its antioxidant functions. nevertheless, recent studies exhibited that tocotrienol possesses healthpromoting properties such as vital neuroprotective effect, cholesterol lowering and anti-cancer properties that are usually not displayed by tocopherols[46]. even though tocotrienols have low bioavailability, its antioxidant activity is higher than tocopherols[47]. the α-tocotrienol exhibited better peroxyl radical scavenging potency than α-tocopherol in liposomal membrane[48]. the unsaturated side chain of tocotrienol allowing even distribution of tocotrienol in the membrane bilayer that further enhance the interaction of chromanol ring of α-tocotrienol with lipid radicals. tocotrienols also moves between lipid vesicles much faster than α-tocopherol. furthermore, the chromanoxyl radical of α-tocotrienol (α-tocotrienoxyl) was to be recycled in membranes and lipoproteins more rapidly compared to α-tocopheroxyl radical[49,50]. figure 4. structural formulae of tocotrienols. 4 funtions of vitamin e antioxidant vitamin e efficiently inhibits lipid peroxidation and scavenges the chain-propagating peroxyl radical. the scavenging outcome of α-tocotrienol was 1.5fold higher than α-tocopherol in liposomes[49]. moreover, α-tocotrienol was 6.5 times more effective in protecting cytochrome p-450 against oxidative damage. the tocotrienol-rich fraction (trf) from palm oil is significantly more effective than α-tocopherol in inhibiting oxidative damage in rat brain mitochondria induced by ascorbate-fe2+, the free radical initiator azobis (2-amidopropane) dihydrochloride (aaph) and photosensitization[51]. furthermore, palm trf at micromolar concentration providing better protection against copper-induced oxidation of plasma low density lipoprotein and also lipid peroxidation in human umbilical vein endothelial cells (huvec), as compared with α-tocopherol[52]. moreover, the efficacy of α-tocotrienol in protection against fe2+ nadphinduced lipid peroxidation in rat liver microsome was 40 times higher than α-tocopherol[49]. this could strongly suggest that α-tocotrienol has greater scavenging effect compared to α-tocopherol. neuroprotection recent studies demonstrated that vitamin e have health benefit properties which go beyond their known antioxidant activity. studies indicated that α-tocotrienol prevented both oxidative stress-dependent and oxidative stress-independent apoptosis, whereas δand γ-tocotrienol only inhibited oxidative stress-dependent apoptosis. this displays that neuroprotective effect of α-tocotrienol could be mediated via non-antioxidant antiapoptotic actions in addition to its antioxidant property[53]. moreover, nanomolar concentrations of α-tocotrienol could block glutamate-induced neuronal cell death, while α-tocopherol did not exhibit this property[54]. furthermore, nanomolar concentration of α-tocotrienol could protect glutamate-induced cell death in mouse neuroblastoma ht4 cell via inhibition of 12-lipoxygenase and phospholipase a2 activation that further interfere the state of phosphorylation[26,54,55]. additionally, tocotrienols effectively inhibited the activation of pp60 c-src kinase, a kinase that centrally involved in glutamate-induced cell death[26]. for neuroprotection properties, studies reported that other sources (e.g. microbial resources) were also demonstrating strong antioxidants[56–66] and neuroprotective properties[67–72], for instance radical scavenging and metal chelating potentials[73–80]. other beneficial properties of vitamin e numerous studies indicated that tocotrienols could suppress proliferation and induce apoptosis of several tumor cells such as breast, liver, lung, colon, stomach, skin, pancreas and prostate cancer cells[81–87]. the γ-tocotrienol and δ-tocotrienol were reported to have anti-tumor activity in breast cancer cell irrespective of estrogen receptor status[88,89]. the γ-tocotrienol also prevents cholesterol synthesis by suppressing 3-hydroxy-3-methylglutaryl-coa reductase activity via a post-transcriptional mechanism[90]. the cardioprotective effects of tocotrienol are also facilitated via their ability to suppress inflammation thus reduce the expression of adhesion molecules and monocyte-endothelial cell adhesion[91]. biomarker of neuronal cell injury the continuous supply of oxygen and glucose is extremely important for brain energy metabolism. the disruption of this supply for a few minutes can introduces a sequence of biochemical event that lead to cell swelling, leakage and damage leading to neuronal cell death[92]. intracellular components, such as neuron specific enolase (nse), can be detected in the extracellular fluid and celebrospinal fluid (csf) upon neuronal damage[93]. among various intracellular proteins, the concentrations of nse, s100β, glial fibrillary protein (gfap) and myelin basic protein (mbp) exhibited positive correlation to the severity of the brain damage[94]. the nse catalyzes the conversion of 2-phospho-d glycerate to phosphoenolpyruvate in glycolytic pathway and localized predominantly in neuronal cytoplasm[95]. the level of nse in the cerebrospinal fluid has been used as markers of neuronal damage in patients with a variety of neurologic condition including status epilepticus and metastatic lung cancer. furthermore, positive correlation was reported between the glutamate-induced changes of the neuron-specific enolase efflux fraction[96]. nse is highly expressed as a glycolytic enzyme to replenish the atp supply when energy depletion occurs, which could be due to neurotoxin agents for instance glutamate[97]. meanwhile, s100β is a calcium-binding protein localized in astrocytes. the s100β levels were increased after central nervous system lesions. furthermore, high level of nse and s100β were reported in the csf of infants and children after traumatic brain injury[98]. conclusion vitamin e, which made up of tocotrienols and tocopherol isomers, is a known chain-breaking antioxidant. studies demonstrated that vitamin e have health benefit properties beyond their known antioxidant activity. with the α-tocotrienol preventing both oxidative stressdependent and oxidative stress-independent apoptosis, while δand γ-tocotrienol only inhibited oxidative stress-dependent apoptosis. these findings demonstrated that neuroprotective effect of α-tocotrienol could be mediated via non-antioxidant anti-apoptotic actions in addition to its antioxidant property[53]. furthermore, trf and α-tocopherol at concentration of 100 to 300 ng/ml demonstrated minor prophylactic properties but significant recovery ability in improving the glutamate-injured cell viabilities in both mono-culture and co-culture model. trf at nanomolar concentration also exhibited better protection to neuronal cell against glutamate toxicity compared to α-tocopherol. therefore, the putative mechanism of trf and α-tocopherol action in protecting and recovering glutamate-injured cells was of great interest and warrant further research. more in vivo studies should be performed an insight of vitamin e... 5 to further understand the recovery mechanism of trf and α-tocopherol in a complete body system. conflict of interest the authors declare that there is no conflict of interest in this work. authors contributions the literature review and manuscript writing were performed by h-my and k-ll. reference 1. contestabile, a. oxidative stress in neurodegeneration: mechanisms and therapeutic perspectives. curr top med chem, 2001; 1(6): 553–568. 2. valko, m, leibfritz, d, moncol, j, et al. free radicals and antioxidants in normal physiological functions and human 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malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia 3institute of pharmaceutical sciences (ips), university of veterinary & animal sciences (uvas), pakistan 4biofunctional molecule exploratory research group (bmex), school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia abstract: almost 50–90% of chronic kidney disease patients undergoing haemodialysis have been reported to have chronic kidney disease-associated pruritus (ckd-ap). the intensity of ckd-ap may vary from a mild itch to an unbearable pruritic sensation which interferes with the patient’s quality of life. ckd-ap has become one of the upmost distressing cutaneous and most common symptom of chronic kidney disease which is often overlooked by nephrologists, primary care physicians, and other health-care professionals. typically sleep disorders, mental and social well-being have been correlated with chronic kidney disease patients. with that this article presents vital comprehensive review which includes epidemiology of chronic kidney disease in malaysia and pakistan, ckd-associated pruritus and other dermatological disorders associated with chronic kidney diseases, pathophysiology of ckd-associated pruritus, clinical features of chronic kidney disease-associated pruritus, diagnosis of ckd-associated uremic pruritus, differential diagnosis of ckd-associated uremic pruritus, assessment and quantification of pruritus severity. keywords: chronic kidney diseases (ckd); malaysia; pakistan; pathophysiology of ckd-associated pruritus received: 23th feb 2020 accepted: 23th march 2020 published online: 1st april 2020 citation: rehman iu and khan tm. epidemiology of chronic kidney diseases (ckd) in malaysia and pakistan, pathophysiology of ckd-associated pruritus and other ckd-associated dermatological disorders. prog microbes mol biol, 2020; 3(1): a0000063. https://doi.org/10.3687/pmmb.a0000063. introduction in malaysia, the prevalence of ckd is on the rise from 13,479 per million populations in year 2004 to 20,589 per million populations in year 2008[1]. in malaysia the exact estimation of ckd is unknown, nevertheless the expected incidence of chronic kidney disease in west malaysia is 9.07%[2]. additionally, its distribution stage-wise i.e. stage 1, 2, 3, 4, and 5 are 4.16%, 2.05%, 2.26%, 0.24%, and 0.36%, respectively[2]. according to the 21st report of malaysian dialysis and transplant registry, the number of patients on dialysis increased from 13,356 in year 2004 to 34,767 in year 2014[3]. in pakistan, chronic kidney disease is progressing, and multiple factors are responsible for this epidemic such as poor health care facility, deficient primary health care system, no proper health education, insufficient funding and higher prevalence of diabetes and hypertension[4]. definition and stages of chronic kidney disease (ckd) kidney disease outcome quality initiative (kdoqi) defines chronic kidney disease (ckd) as an immediate or continuing to decrease kidney function or efficiency for a duration exceeding three months[5]. the standards for assessing the disease initiation are mainly urinary outcome, proteinuria and hematuria[6,7]. in some cases, the initial presentations are temporary and can be solved by early initiation of drug therapy while in the majority copyright @ 2020 by rehman iu and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc4.0) *correspondence: inayat ur rehman, department of pharmacy, green campus, abdul wali khan university mardan, pakistan; inayat.rehman@monash. edu. 2 of conditions, the decrease in the creatinine clearance and accumulation of waste products like urea and uric acid occurs[7]. glomerular filtration rate (gfr) and creatinine clearance (cc) are two parameters used for estimation of kidney function[5,8]. “glomerular filtration rate (gfr) can be defined as the amount of blood that is filtered by bowman’s capsule per unit of time (ml/min/1.73m2)”. for a healthy human being the gfr values should be range from 120-130 ml/min/1.73m2[8]. listed here are two most common equations used in practice for estimating gfr based on serum creatinine (scrt). 1. cockcroft-gault equation[9] 2. modification of diet in renal disease (mdrd) equation[10] cockcroft-gault equation cc (ml/min) = ([140-age × weight] / [ 72 × scrt]) × 0.85 if female mdrd equation egfr (ml/min/1.73 m2) = 186 × [scrt] – 1.154 × [age] – 0.203 × [0.742 if female] note: for african/ black use the multiplication factor 1.21 stages of kidney diseases: on the basis of creatinine clearance (cc) or estimated glomerular filtration rate (egfr) kidney disease can be classified in five stages[5,11]: i. stage 1: normal or increased gfr i.e. 90 or more ml/minute/1.73m2 ii. stage 2: mild decrease in gfr i.e. 60–89 ml/ minute/1.73m2 iii. stage 3: moderate decrease in gfr i.e. 30–59 ml/ minute/1.73m2 iv. stage 4: severe decrease in gfr i.e. 15–29 ml/ minute/1.73m2 v. stage 5: kidney failure i.e. less than 15 ml/ minute/1.73m2or on dialysis creatinine is basically a by-product that forms from protein metabolism. when the kidney function starts deteriorating, clearance from kidney reduces which leads to an elevated serum creatinine, uric acid and urea[9]. majority of the patients at stage 4 and stage 5 of kidney diseases get frequent dialysis based on their renal reservoir. in general, most of the patients get dialysis three times a week. the main aim for performing dialysis is to eliminate the waste products from the blood such as urea, uric acid, nitrogen and excessive electrolytes. low gfr alone is not confirmatory for diagnosis of ckd, as it may be borderline normal or normal. in order to establish the diagnosis for ckd, the presence of one or more markers as listed is vital[12]. • histological abnormalities • urine sediment abnormalities • albuminuria (albumin excretion >30 mg/24 hr) • electrolyte and other abnormalities owing to tubular disorders • renal transplantation history • structural abnormalities detected by imaging epidemiology of ckd ckd is currently one of most serious health crises. epidemiological data suggests that ckd is a big threat globally for both developing and developed countries[13]. according to 2010 statistic of global burden of disease (gbd), among the directory causes of global deaths, ckd was categorized 27th in 1990 but due to its higher prevalence, it climbed to 18th in 2010[14]. the 2013 statistic by global burden of disease (gbd), comparing the mortality rate of ckd patients between 1990 and 2013, indicates mortality rate is increased by 134.6% in 2013 compared to 1990[15]. the prevalence of ckd is heterogeneous globally, with the incidence of ckd was higher in indo-asians population as compared to the european population. the statistics indicated ckd is more prevalent in asian countries as compared to the rest of the world. malaysia reported prevalence of 9.07%[2], while china and nepal reported prevalence of 10-19%[16]and 10-20%[17] respectively. whereas pakistan, bangladesh, and india reported prevalence of 20%[18-20]. one possible reasons for the increased in prevalence is the reduced access to preventative health care, which helps to reduce the progression of kidney diseases[21]. furthermore the global increase in ckd cases and its progression toward end-stage renal disease due to global increase of diabetes and hypertension pandemics [22–24]. chronic kidney diseases in malaysia in malaysia, the prevalence of ckd is increasing from 13,479 per million populations in 2004 to 20,589 per million populations 2008[1]. in malaysia the exact estimation of ckd is unknown, still the expected incidence of chronic kidney disease in west malaysia is 9.07%[2]. moreover, its distribution stage-wise i.e. stage 1, 2, 3, 4, and 5 are 4.16%, 2.05%, 2.26%, 0.24%, and 0.36%, respectively[2]. the 21st report of malaysian dialysis and transplant registry stated the number of patients on dialysis increased from 13,356 in 2004 to 34,767 in 2014[3], as depicted in figure 1. the acceptance rate and prevalence rate of dialysis are 210 and 1065 per million populations respectively[1]. the report stated that overall acceptance and prevalence rate of dialysis is almost doubled during the span of 10 years[1]. states in malaysia which are economically advanced have a much higher rate of dialysis treatment compared to those states which are economically less advanced. approximately 90% of new dialysis patients are accepted into haemodialysis center and the rest into the peritoneal dialysis program[1]. in 2013, the annual death rate on dialysis was reported as 11.3%, out of which 10.9% were of haemodialysis and 15.8% were of peritoneal dialysis[1]. diabetes is considered to be the typical cause of ckd, with reported cases of ckd due epidemiology of chronic... 3 to diabetes at 53% in 2004 and 61% in 2013[1]. end stage renal disease caused by diabetes has increased dramatically and is accountable for 50% incidents in dialysis patients. the malaysian national registry estimated the prevalence of end-stage renal disease in 2007 will be 680 per million population[25]. according to national renal rehman iu et al. registry malaysia (2015)[3], 32,026 patients were on dialysis; of whom 91% were on haemodialysis and 9% were on peritoneal dialysis. it has been estimated that by 2040, the number of patients with end stage renal disease would triple from existing 2014 cases[26]. ibrahim et al. (2011)[27] reported the prevalence of ckd-ap in malaysia by 64.2% and sleep disturbance with 61.7% in ckd patients. chronic kidney disease in pakistan in pakistan, chronic kidney disease is progressing with multiple factors responsible for this epidemic such as poor health care facility due to deficient primary health care system, no proper health education, insufficient government funding and higher occurrence of diabetes and hypertension marked as a high-risk factor for ckd[4]. in pakistan, the absence of periodically maintained central registry kidney diseases on a national level about its epidemiological and burden makes it extremely difficult to assess the cases of ckd, dialysis, mortality, and allocation of the fund in pakistan[4]. data from the dialysis registry of pakistan 2007–2008 indicated the number of ckd patients increased from 4,393 in 2006-2007 to 6,127 in 2007–2008[28]. while current data indicated 16,000 pakistanis suffer from ckd annually and pakistan is ranked the eighth in the world on basis of highest number of epidemiology and prevalent cases of ckd[29]. jessani et al. (2014)[19] reported that ckd prevalence in karachi is 12.5%. while luqman et al. (2012)[30] reported the prevalence of ckd in pakistan as 64%. ckd-associated pruritus (ckd-ap) and other dermatological disorders associated with chronic kidney diseases dermatological disorders are regularly observed in ckd patients. skin-associated problems like ckd-ap, xerosis, pallor, and hyperpigmentation are very common in ckd patients[31]. most often, in the case of renal failure, the skin becomes an excretory organ for the substances the kidney usually clears from the body. a study on dermatological problems associated with ckd indicated the prevalence of skin conditions observed were pallor 91.5%, xerosis 75.9%, pigmentary changes 65%, ckd-ap 60.2% and panniculitis 1.2%[31]. ckd-ap is a commonly found complication reported by majority of chronic kidney disease patients[32]. the prevalence of ckd-ap was found to be more in haemodialysis patients (68%) than in peritoneal dialysis patients (38%) [33]. the occurrence of ckd-ap in ckd patients differ extensively from 22% to 90%[34-38]. normally, the whole body is affected while the back and forearms are most likely to get affected compared to other parts of the body. severe ckdap has a major impact on the patient’s life quality which leads to other disorders such as anxiety, disturbed sleep, and depression[39–43]. recently two studies (a japanese study and the dopps) demonstrated a relationship between ckd-ap and an amplified threat of mortality[34,38]. several studies report a considerable prevalence of ckd-ap among dialysis patients, ranging from 10% to 70% in pd patients and 20% to 90% in hd patients[44]. almost 50% or greater chronic kidney disease patients are reported of having the dermatological problem like dry skin and itching[45]. these associated dermatological problems have a significant impact on quality of life which negatively impacted on ckd patients’ physical and mental health[31]. pruritus is linked with some other metabolic changes which figure 1. statistic of increase in dialysis patients indicating an increase during december 2005december 2014 in malaysia. 4 trigger and potentiate it, such as xerosis, decreased transepidermal elimination of pruritogenic factors, hyperparathyroidism, hypercalcemia and hypophosphatemia, higher levels of histamine and transdermal mast cell proliferation, and uremic sensory neuropathy[46]. in parallel to internal factors, few external factors are also hypothesized to be related with pruritus, such as dehydration, excessive sweating, humid and hot weather, shower with cold/hot water and stress[46]. also ckd-ap patients could experience abnormal skin pigmentation and low platelet counts as a result of prolonged bleeding[47]. usually after a temporary relief from pruritus, the symptoms reappear within six months with much-intensified degree regardless of any demographic variables[46]. ckd-ap occurs intermittently in some cases that may last for few minutes while some patients suffer from lengthy phases of severe ckd-ap that may be present during both day and night[45]. ckd-ap onset, duration, and intensity can change over time and the itching is usually worsened at night time. most normally affected body parts by ckd-ap are the back, limbs, chest, and head, however, approximately 20–50% of patients experience a generalized ckd-ap[38]. pathophysiology of ckd-associated pruritus (ckd-ap) the exact mechanism associated with the pathophysiology of ckd-ap is poorly understood. several hypotheses are discussed in this article for the pathophysiology of ckdap. immune-mediated hypothesis ckd-ap is potentially due to dysregulated systemic inflammation[48-50]. in ckd-ap patient’s elevated levels of t-helper type-1 cells[49], interleukin-6[49], interleukin-2[50] and c-reactive protein[51] were observed. it is recommended that ckd-ap is associated with high white blood cell count, high ferritin level and low albumin level[51]. the immune hypothesis refers ckd-ap as overproduction of proinflammatory substances such as histamine (by mast cells), interleukin 2, tumor necrosis factor α and interferon γ by t helper 1 lymphocytes[52,53]; elevated level of inflammatory markers such as c-reactive protein and interleukin 6[50,54,55]. the elevated levels of serine protease and proteinase-activated receptor-2 could also play vital roles in the pathogenesis of ckd-ap[56]. xerosis hypothesis xerosis (dry skin), is another common dermatological condition reported in patients with ckd[57,58]. the reduction of the eccrine sweat glands size and the atrophy of the sebaceous glands are assumed as the main reasons for xerosis[59]. xerosis has been considered as a substantial contributor in ckd-ap severity[60]. histamine hypothesis the increased of mast cells and histamine levels[61–63], serotonin level[64], eosinophil’s and tryptase have been witnessed in patients with ckd-ap[61,62]. nevertheless, prasad et al. (2015) reported that despite increased mast cells and histamine levels there was no direct correlation between histamine level and ckd-ap. neuropathic hypothesis the neuropathic hypothesis suggests that the somatic and autonomic neuropathy caused by lesion can result in neuropathic itch[65,66]. it is recognized that neuropathic pain and pruritus shared the same neuronal pathway[67]. the involvement of neuropathic mechanisms in the mediation of pruritus is further demonstrated with gabapentin and pregabalin that are effective in the improvement of the patient with pruritus[68,69]. this also advocated that afferent c-terminal nerve fibers that are gaba-aminobutyric acid (gaba) dependent is involved in the ckdap[70]. opioid hypothesis the opioid hypothesis suggests that the imbalance of endogenous opioidergic system plays an important role in the pathophysiological mechanism of pruritus by the ability of μ receptor antagonists and ϰ receptor agonists in relieving itchiness[67,71,72]. the μ-opioid receptor activation is involved in the intervention of pruritus while ϰ-opioid receptor activation has an inhibitory effect on μ-opioid receptor both peripherally and centrally[67,72–74]. hyperparathyroidism hypothesis the hyperparathyroidism is expected to play a role in pruritus through inducing mast cell secretion[75]. secondary parathyroidectomy trigger elevation of divalent ions such as magnesium, phosphate, and calcium, this could result in micro-precipitation that is known to have affect the modulation of mast cells degranulation[76]. low serum phosphorus level was recently found to be significantly lower in haemodialysis patients with severe and frequent ckd-ap as compared to those without ckd-ap[77]. other factors other factors associated with ckd-ap include the production of pruritogenic substances such as abnormal growth, cytokines, and sprouting of “itch fibers” in the skin, also neuropathy that leads to the decreased of the threshold for itch sensation[78,79]. clinical features of ckd-ap clinical characteristic varies for each patient and over time among patients. as the ckd-ap onset, intensity and duration can change over time[37], and the itch is typically worsened at night time[45]. ckd-ap could be generalized or localized; however back, abdomen and forearms are most likely to be affected compared to other parts of the body[38,45]. other characteristics include: • worsen in itch intensity at night and cause sleep disturbance[34,38,45,80,81]. • xerosis (dry skin), skin scaling and epidermal cracking[60]. • elevated blood urea nitrogen (bun), calcium, phosphate, and magnesium and parathyroid hormone (pth), levels[34]. • fatigue and depression[38,82]. epidemiology of chronic... 5 diagnosis of ckd-ap ckd-ap is one of the most common complication reported by ckd patients on haemodialysis, so the diagnosis is easy unless there is other compelling evidence of other causes. the most common suggestive diagnosis characteristic of ckd-ap is its occurrence after the imitation of haemodialysis. also, elevated calcium, magnesium, phosphate, blood urea nitrogen (bun) and parathyroid hormone (pth) levels[34] were confirmed among ckd-ap patients on haemodialysis. differential diagnosis of ckd-ap several diseases could cause ckd-ap in patients with and without ckd. in order to confirm ckd-ap, a non-uremic cause of ckd-ap should be kept in thought among patients on haemodialysis with symptoms that are refractory to common treatments such as an oral antihistamines, gabapentin, analgesic agents and topical emollient[83]. assessment and quantification of ckd-ap severity ckd-ap has become one of the most distressing cutaneous, and the most common symptom of ckd that is commonly overlooked by nephrologists, primary care physicians, and other health-care professionals[46]. to assess the severity of ckd-ap, several instruments have been developed and these tools shall be illustrated in this review. the literature showed that the rating scales are commonly used for assessment and quantification of ckd-ap. the rating scales/tools includes verbal rating scale (vrs), visual analogue scale (vas), numerical rating scale (nrs), eppendorf itch questionnaire (eiq), dermatology quality of life questionnaire (dlqi) and 5ditch scale (5d-is), modified pruritus questionnaire for itch severity score, skindex( skindex-29 and skindex-16)[84–91]. visual analogue scale (vas) visual analogue scale is a unidimensional scale that is an easy and rapid tool commonly used for assessment of pruritus severity[71]. the vas consists of a 10 mm horizontal line indicating the severity of itch; “no itch” (o points) and ending with “worst itching”[92]. the evaluation of ckdap relying on a single measure is not sufficient, vas is a useful tool for the assessment of pruritus intensity but it does not provide any further information on other aspects of ckd-ap[93]. verbal rating scale (vrs) verbal rating scale (vrs) is another unidimensional tool for assessment of pruritus that assists the patient to verbally describe the degree of pruritus. this is possibly the most convenient method for assessment of pruritus; with four-point scale “none, mild, moderate and severe pruritus”[94] and five-item scale “none, mild, moderate, severe and very severe pruritus”[95] are used for assessment of pruritus. the variability in different versions of vrs point scale contributes toward a major limitation of this scale, thus making the comparison of these results very difficult[96]. however, vrs has the advantage as one of the most suitable tool for assessment of ckd-ap in certain populations such as elderly or patients with cognitive problems[96]. eppendorf itch questionnaire (eiq) the eppendorf itch questionnaire was developed by darsow et al. (2001)[97], for the exact characteristic of pruritus using a comprehensive list of sensory and affective descriptors, and also collects evidence on the impact of pruritus on quality of life[97]. dermatology quality of life questionnaire (dlqi) the dermatology life quality index, the first dermatology was by finlay and khan (1994) [98], and contains 10 questions concerning “symptoms and feelings, daily activities, leisure, work, and school, personal relationships and treatment” over previous one week. the responses for this tool are ranged from 0 to 3 ‘‘not at all’’, ‘‘a little’’, ‘‘a lot’’ or ‘‘very much’’ respectively. each response is scored from 0 to 3 and then summed up, score of 0 indicates no impairment of life quality while score of 30 indicates maximum impairment[98]. 5d-itch scale (5d-is) a 5d-itch scale is a multidimensional tool, that comprises of five domains, addressing the “duration, degree, direction, disability, and distribution of itching”[87]. the duration, degree and direction domains each included one item, while the disability domain had four items. all items of the first four domains were measured on a five-point likert scale (where 1 represented the lowest degree of pruritus, and 5 represented the highest degree). the 4th domain (disability) measured the effect of itching on daily activities, and its score was calculated by selecting the highest score. in the 5th domain, participants were asked to select which part of the body was most affected by ckd-ap, and participants could select as many body parts as they wished. if two body parts were affected, the score given was 1; 3–5 body parts affected was scored as 2, 6–10 body parts were scored as 3, 11–13 body parts were scored as 4, and 14–16 body parts were scored as 5. the overall score of the 5d-is was calculated with all the five domains; 5 indicates no pruritus; whilst a score of 25 indicates severe pruritus[87]. modified pruritus questionnaire for itch severity score the itch severity scale developed by[81] for evaluation of pruritus was modified by[88], as the questionnaire by yosipovitch et al. (2001)[81] has no method of scoring for quantification of symptom severity. the modified questionnaire by majeski et al. (2007)[88] consists of 7 components: “frequency, itch description, affected body surface area, intensity, the effect on mood, an effect on sexual desire/function and effect on sleep”. the responses to a different component of the questionnaire were separately summed and divided by a maximum score for the respective question. all the seven values achieved were then added and multiplied by 3 to get a total out of 21 scores. the yielding total scores ranging from 0 to 21[88]. rehman iu et al. 6 skindex questionnaire skindex is among the best dermatological instruments for the measurement of dermatological specific health related quality of life (qol)[89]. skindex was originally comprised of 61 questions that were later on divided into two brief versions i.e. skindex 16[91] and skindex 29[90]. the skindex-29 is instruments of choice in dermatology[99], it is comprised of 7 items addressing the symptoms domain, 10 items for the emotional domain, and 12 items for the functioning domain. the responses of all domains were transformed to a linear scale of 100, varying from 0 (no effect) to 100 (effect experienced all the time)[100]. whereas skindex-16 is a single page questionnaire, that is an accurate and sensitive measurement for patients experience and widely used for skin-related quality of life[101,102]. the skindex-16 comprised of 4 items related to symptoms, 7 items related to emotions and 5 items related to functioning scales and linear scale of 100, varying from 0 (no effect) to 100 (effect experienced all the time)[91]. conclusion researchers reported the prevalence of ckd-ap was slightly higher in pakistani patients when compared to malaysian patients. prevalence studies in pakistan reported rates of 64.0% to 77.7%[30,103,104] while malaysia studies reported rates of 58.6% to 64.2%[27,105]. the pruritus median score was also significantly higher in pakistani 10.0 [8.0–12.0] compared to malaysian patients 8.0 [6.0-9.0]; p=<0.001. the possible reasons for the variation in the prevalence of ckd-associated pruritus between malaysian and pakistani because patients in pakistan receive haemodialysis twice/week, whereas in malaysia, it is three times/ week. moreover, low to medium flux dialyzers were used in pakistan, whilst high flux dialyzer (which removes average-sized molecules more effectively — thus reducing the severity of uremic pruritus) was used in malaysia. ckd-ap is believed to be caused by middle-molecule uremic toxins which are not dialyzed properly when using low flux dialyzer[34]. globally the high-flux haemodialysis is the most commonly used blood purification method. but in developing countries such as pakistan, low-flux dialysis is the main method of extracorporeal blood purification therapy due to insufficient funds to purchase high flux dialyzers[106]. haemodialysis machines are also limited in pakistan as in 2004 there were 140 dialysis centers although in 2009 these machines increased to 175[107], alarmingly out of the available dialysis centers in pakistan, 10–15% are non-functional and the patients have limited access to treatment[107]. the pakistani patients were significantly younger than malaysian patients and had a shorter median duration of ckd and being on haemodialysis. this could be due to poorer management of chronic conditions (such as hypertension or diabetes mellitus) which predisposes an individual to renal damage at younger age. additionally, pakistani patients may have less knowledge of the complications of their chronic conditions and therefore may not take any preventive measures such as control of hypertension and dietary modifications[108]. malaysian population was graded higher when their knowledge was tested against diabetes and hypertension[109–112], these results were quite different for the pakistani population[113–116]. furthermore, awareness and attitude also have a decisive role in the actions of diabetic and hypertension patients. in pakistan, many factors may account for poorer outcomes during dialysis; such as malnutrition, late referral, anaemia and lack of qualified nephrologists at dialysis centers[117–120]. in conclusion, this review provided vital insight on the epidemiology of chronic kidney diseases (ckd) in malaysia and pakistan, together with the pathophysiology of ckd-associated pruritus and other ckd-associated dermatological disorders. both countries could improve the awareness of their populations and providing more support to the healthcare setting to increase the quality of life of ckd patients. authors contributions the literature review and manuscript writing were performed by i-ur and t-mk. conflict of interest the authors declare that there is no conflict of interest in this work. reference 1. national renal registry malaysia. 21st report of the malaysian dialysis and transplant 2013. 2014; available from: https://www. crc.gov.my/wpcontent/uploads/documents/report/21stdialysis_ transplant_2013.pdf [accessed 28th janurary 2016]. 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epidemiology of chronic... progress in microbes and molecular biology review article 1 decoding the mystery of how bacteria “talk”: among gramnegative microorganisms tan wen-si1,2*, jodi woan-fei law3, vengadesh letchumanan3, kok-gan chan4,5 1illumina singapore pte ltd, woodlands industrial park e1, singapore 2division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, kuala lumpur, malaysia 3novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength jeffrey cheah school of medicine and health sciences, monash university malaysia, bandar sunway, selangor darul ehsan, malaysia. 4division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, 50603 kuala lumpur, malaysia 5international genome centre, jiangsu university, zhenjiang 212013, pr china abstract : to date, microbial diversity is still the least well understood component of biodiversity. bacteria are the most abundant microorganisms where most species are often found ubiquitous. microorganisms such as bacteria are diverse in their impacts such as in spreading of infectious diseases or play a valuable role in biotechnological purposes. hence, it is interesting to gain a look upon the ways where bacteria regulate their daily processes in the environment. bacteria communicate with each other through extracellular signalling molecules or also known as autoinducers (ais) that are produced, detected and show response. this process is termed as quorum sensing (qs) which indicates that bacteria do communicate in order to perform various physiological activities. qs enable bacteria to have the advantages that are unattainable as individual bacterial cell. this review emphases on the characteristics of quorum sensing (qs) and its benefits in understanding different kind of bacterial qs-dependent activities. this fundamental insight from qs system will enable us to manage bacterial activities by targeting their communication circuit. keywords: communication; quorum-sensing; signalling molecules; n-acylhomoserine lactone received: 23rd september 2019 accepted: 24th october 2019 published online: 31st october 2019 citation: tan w-s, jodi w-f l, letchumanan v. decoding the mystery of how bacteria “talk”: among gram-negative microorganisms. prog microbes mol biol 2019; 2(1): a0000038 introduction bacteria are always recognized to exist as isolated and anti-social lives. they can be present as free-living, associated with decaying material or attached to surfaces of rocks, stones, sand grains or aquatic animals as part of biofilm[1–8]. researches over the past decades had disclosed that bacteria are able to communicate via chemical signalling system in order to communicate within species had led to the realization that bacteria are able to behave in a much more complex patterns[9]. specifically, bacteria release signaling molecules which are also known as autoinducers; then detect and respond to the accumulation of those molecules[10]. bacterial communication or coiled as “quorum sensing (qs)” by fuqua and colleagues to describe the process where bacterial communication achieved depending on the density of bacterial cell population where it is synchronize with the concentration of signal molecules in the extracellular environment[11,12]. in a simplest guise, qs benefits bacteria as a community rather than as an individual. this review emphases on the characteristics of quorum sensing (qs) and its benefits in understanding different kind of bacterial qsdependent activities. quorum sensing (qs) the paradigm shift of understanding the ability of bacteria in conducting complex patterns of co-operative behaviors had lead us into translating qs into four essential steps[13]. first, bacterial cell population density and concentration of autoinducers in the external environment increase simultaneously. once the bacteria sense the threshold, the signal will diffuse into bacterial cells and bind by receptor proteins which hence causing an activation of signal transduction cascade (figure 1). the autoinducer-receptor protein complex will bind with targeted promoter to induce an auto-regulatory mechanism to either up-regulate or down-regulate certain bacterial phenotypes[13,14]. copyright 2019 by tan w-s et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) *correspondence: wen-si tan, tmarilyn36@gmail. com. illumina singapore pte ltd, woodlands industrial park, 757716 singapore. 2 the evolution of qs was originated from the discovery of controlling luminescence by vibrio fischeri, a bacterium that forms a mutualistic light organ symbiosis with hawaiian bobtail squid (eupryma scolope)[15–17]. the cellpopulation density influenced luminescence portrayed by v. fischeri have been convincing many scientists to pursue on qs research for the past decade in defining the details of quorum regulation in this bacterium. experimental analysis on dramatic pattern of light production in the squid portray the findings that autoinducer played a role in induction of luciferase[13,15,16]. a minute amount of v. fischeri is harbored in the light organ of the squid during daytime. as bacterial incubation hour increases, the production of signaling molecules at its concentration threshold trigger the luciferase expression. this bioluminescence was needed by the squid to counter eliminate its shadow and avoid predation at night. “switching off” of luminescence occurred when the squid pumped out the bacteria pool from its light organ, hence bacterial population decreases discouraging the triggering of luciferase production due to insufficient signalling molecules production. the information emerged from v. fisheri system serves as a model for further discovery of quorum circuit in other species[17]. auto-induction of luminescence is now recognized as a qs model with wide applicability in applied research on gene regulation and host association of bacteria. qs indeed not only involved in luminescence but also regulates a vast array of phenotypes. qs facilitates bacteria for adaptability and survival where it also appears as bridge for interaction of several different bacterial species with eukaryotic hosts. however, the life-threatening ability phenotypes that are regulated by qs causes many concerns. biofilm formation, production of virulence factors and antibiotic resistance in several notorious pathogens such as pseudomonas aeruginosa[18], burkholderia cepacia[19], vibrio cholera[20], streptococcus mutans[21], clostridium difficile[22], and erwinia carotovora[23] were reported to be regulated by qs to attack different hosts ranging from human, plants and aquatic animals. subsequently, the study into both qs systems and qs signal molecules are essential in various field from biotechnology, pharmaceutical and to agricultural industries, particularly targeting qs for establishment of novel antibacterial measures[13]. qs mechanisms have derived to three tracks; (i) n-acylhomoserine lactone (ahl)-based signalling system of gram-negative bacteria (ii) oligopeptide-based system in gram-positive bacteria and (iii) shared furanone-based system between gram-negative and gram-positive bacteria [24]. qs signal molecules are structurally diverse and ranged according to the needs of the bacterium itself. there are various types of qs signalling molecules discovered and documented such as the ahl, 2-alkyl4(1h)-quinolones (ahqs), autoinducer peptides (aip), dsf, palmitate methyl ester (pame) and diketopiperazines (dkp) but they did share similarity such as small (< 1000da) organic molecules or peptides with 5-20 amino acids and are highly diffusible[13,25–29]. of all the signalling molecules documented and reported, ahl received the utmost attention by most research institutions. ahl is basically a group of signalling molecules which employed by most gram-negative bacteria[14]. quorom sensing of gram-negative bacteria quorum size is sensed by gram-negative bacteria through ahls production that accumulates in their surroundings as the cell population increases. to date, there are more than 100 species of bacteria reported to portray qs properties. these bacteria are found ranging from marine, soil and freshwater environment to plants and animals. their presence play roles involving pathogens, symbiont, extremophiles and plant-growth promoting bacteria that varies according to its environment[30]. many of these bacteria are able to produce multiple ahls and contain more than one ahl synthases[12,13]. some of the examples of ahl-dependent qs systems with the phenotypes controlled are summarized in table 1. however, there are still an astronomical number of bacteria that are yet to be discovered and characterized whether they do communicate interor intraspecies. many researches should be carried on to understanding qs deeply; it will be the foundation for various areas in biotechnology, pharmaceutical and agricultural industries in such that qs is particularly the target of interest in antimicrobial purposes. decoding the mystery... figure 1. two qs regulatory components in gram-negative bacteria: luxr (transcriptional activator protein) and luxi (autoinducer synthase). signal molecules accumulated in a cell-density-dependent manner until a threshold level is reached. no gene expression is driven at low bacterial cell density but at high bacterial cell density, gene expression will be activated. 3 ahl as signalling molecules ahl signalling is highly conserved among the proteobacteria and received the absolute attention in which intensive studies had been carried out[51]. a homoserine lactone ring with its βand γ-positions remained unsubstituted, but has an α-position, the n-acylated with a fatty acid chain. this ring is highly conserved in all the ahls documented and characterized (figure 2)[51]. there are several structural differences that influence the characteristics of an ahl molecule which are (i) acyl side chain range commonly from 4 to 18 carbons where ahl usually carries an even number carbon chain (ii) reduction or oxidation carbonyl or presence of hydroxyl group at third carbon of the acyl side chain (iii) there is also possible for presence of unsaturated ahls where double bond occurred in the 5 and 7 positions of a long acyl chain (12-14 carbons)[13,52]. the minimum acyl chain to be function as signal molecule is 4 carbons as in existence with lactone ring itself will be hydrolysed at phs above 2 where 70% of n-propionyl-homoserine lactone is hydrolysed at ph 6[52]. the shortest naturally occurring ahls was produced by several gram-negative bacteria such as vibrio harveyi, pseudomonas aeruginosa and aeromonas hydrophila. ahl molecules was also found to interact with some other molecules such as cysteine, biotin and fluorescence that bring an effect on the binding affinity of modified ahl to its native ahl-receptor[53]. besides that, alkali-driven rearrangement reaction can occur in 3-oxo-ahls that lead to formation of corresponding tetramic acids, iron chelating compounds and antibacterial activities. in short, differences in substitution at predetermined sites on the ahl molecule confer its specificity and affect the functions to the cells figure 2. general structure of n-acyl homoserine lactone (ahl). r represents the fatty acid acyl side chains[51]. tan w-s et al. table 1. some examples of phenotypes controlled in ahls-dependent qs systems in gram-negative bacteria. microorganisms major ahl(s) phenotypes references acinetobacter baumannii 3-hydroxy-c12-hsl biofilm, virulence niu et al. 2008[31] aeromonas hydrophila c4-hsl biofilms, exoproteases, motility, virulence jahid et al. 2013[32] aeromonas salmonicida c4-hsl extracellular protease swift et al. 1997[33] agrobacterium tumefaciens 3-oxo-c8-hsl plasmid conjugation wang et al. 2014[34] burkholderia cepacia c6-hsl, c8-hsl biofilms, virulence riedel et al. 2001[35] burkholderia glumae c8-hsl protein secretion, oxalate production, swarming, virulence nickzad, et al. 2015[36] burkholderia pseudomallei c8-hsl, c10-hsl, 3-hydroxy-c8-hsl, 3-hydroxy-c10-hsl, 3-oxo-c14-hsl virulence, exoproteases ulrich et al. 2004[37] chromobacterium violaceum c6-hsl exoenzymes, cyanide, pigment mcclean et al. 1997[38] enterobacter agglomerans 3-oxo-c6-hsl pectinase expression chalupowicz et al. 2009[39] erwinia carotovora 3-oxo-c6-hsl biofilm, virulence joe et al. 2015[40] nitrosomonas europaea 3-oxo-c6-hsl emergence from lag phase burton et al. 2005[41] pantoea stewartii 3-oxo-c6-hsl exopolysaccharides chug et al. 2015[42] pseudomonas aeruginosa c4-hsl, c6-hsl, 3-oxo-c12-hsl biofilms, exoenzymes, exotoxins, swarming, virulence jakobsen et al. 2013[43], jimenez et al. 2012[44], williams 2007[13] rhizobium leguminosarum c6-hsl, c8-hsl rhizome interaction lithgow et al. 2000[45] serratia marcescens 3-oxo-c6-hsl, c6-hsl, c7-hsl, c8-hsl biofilm formation, sliding motility and prodigiosin produciton horng et al. 2002[46], rice et al. 2005[47] vibrio fischeri 3-oxo-c6-hsl bioluminescence ruby et al. 1998[17] vibrio harveyi 3-hydroxy-c4-hsl bioluminescence, virulence cao and meighen, 1989[48], manefield et al. 2000[49] yersinia pseudotuberculosis 3-oxo-c6-hsl motility and clumping atkinson et al. 2008[50] 4 ahl diffusion and transportation through membrane is highly related with its structural features. ahls tagged as amphipathic molecules, are wonted to diffuse freely from the internal cell environment to the external cell environment, and vice versa, demonstrated with v. fischeri and e. coli using a 3h-labelled derivative[54]. however, this demonstration only proved using a short chain ahl, 3-oxo-c6hsl. the hydrophobic characteristic is influence by the acyl side-chain length, the number of in-saturations and the nature of the c3 substituent (h, o or oh). on the other hand, the rate of ahl diffusion is correlated with the nature of acyl chain. if a long acyl chain ahls diffuse through cell membrane, it would diffuse slower as compared with a shorter acyl chain ahls[55]. commonly, short acyl side chains are usually those ahl with acyl side chain lesser than c8 and can be passively diffused in and out the bacterial cell. it is to facilitate the transport of long acyl side chain ahl, an active transport mechanism. the presence of an active efflux of 3-oxo-c12-hsl in p. aeruginosa was evidenced, where the short-chain c4-hsl freely diffuses across the cell membrane[56,57]. another active efflux is evidenced in burkholderia pseudomallei. gram-negative qs circuits the qs system in gram-negative bacteria consist of signalling molecules (autoinducers), autoinducer synthase (luxi), lux-r type regulators and target genes[58]. the signaling circuits composed of luxi/luxr appear to be the standard communication mechanism in gram-negative bacteria, where qs system resembling the canonical. v. fischeri circuit have been shown to be the model system in controlling gene expression in over 100 species of gram-negative bacteria[59]. commonly, the acylated hsl synthesized by the responsible enzyme luxi-like protein; a cognate luxr-like protein that will recognize the hsl autoinducer and activation of transcription downstream target genes occurred subsequently[60]. the mode of action of the luxi/luxr pairs is highly conserved across all cases. the coupling of acyl-side chain of a specific acylacyl carrier protein (acyl-acp) from the fatty acid biosynthetic machinery with the homocysteine moiety of s-adenosylmethionine (sam) is produced by luxi homologue. the coupling process forms a ligated intermediate which then convert to form acyl-hsl and methylthioadenosine (mta). the luxr homologue, on the other hand function by binding their substrate, autoinducer and activating the transcription of targeted dna. the luxr homologue consist of an amino-terminal region that binds to autoinducers and the c-terminal domains responsible for oligomerization and promoter dna binding[60–62]. rather delicate signalling specificity exists in luxi/luxr type circuits and specificity inherent stems from a high selectivity of the luxr proteins to its signalling molecules. as evolutionary goes by, more regulatory complexity has been added to the basic backbone of qs circuit, such as the use of multiple ahl autoinducers and luxr proteins that can act either parallel or in series[63]. this can be seen in the plant phytopathogen, the gene regulation of ralstonia solaacearum luxi/luxr like autoinduction system (soli/ solr) are regulated by phca and also rpos[64]. next, the opportunistic pathogen p. aeruginosa employ two pairs of luxi/luxr homologues (lasi/lasr, rhli/rhlr) and function in tandem to control the virulence factors production[65]. recently qscr, identified as third luxr homologue was found from the complete genome sequence of p. aeruginosa. however, there are yet indication of cognate luxi homologue that could be responsible in producing the autoinducer in which qscr can respond[66]. in fact, level of communication complexity layered to the luxi/luxr backbone circuit highly dependent on the nature of bacteria. apparently the complex interconnected network could serve for precise timing of the expression of various qs controlled phenotypes[60]. qs has been potentially responsible in managing various bacterial physiological activities such as expression of virulence factors, biofilm formation and swarming. hence, by aiming bacterial communication circuit would be a novel way either in interfering its virulence or to enhance them for biotechnological purposes. employment of ahl biosensors the discovery of vast diverse ahl qs system has been rendered possible by adopting bacterial biosensors capable in sensing the ahls production in a rapid manner. the bacterial biosensors contain defective luxi protein which led them in disability in producing their own ahls[67]. however, these biosensors carry a functional luxr-family protein cloned with a cognate target promoter that up-regulates the transcription of reporter genes that exhibits phenotypes such as green-fluorescent protein, bioluminescence and purple violacein pigmentation. specificity in sensing exogenous ahl by bacterial biosensors strongly relies on the luxr family protein and hence it is essential to carry out the detection with several biosensors. there are several biosensors available in detecting short and medium acyl chain ahls (acyl chain range within c4 to c8 in length). chromobacterium violaceum (cv026), a gram-negative water and soil bacterium is commonly employed to serve this type of detection[38]. cv026 was developed after mini-tn5 transposons insertion into cvii ahl synthase gene while the ability to induce purple pigmentation via cvir was retained. this mini-tn5 mutant forms white colony and will only be able to turn purple in the presence of exogenous ahls. cv026 is incapable to detect any ahls with acyl chains of c10 or longer and all 3-hydroxy-ahls. several biosensors available in detecting short and medium acyl chain ahls rely on a plasmid construct harboring luxcdabe operon and the host e. coli was commonly used for the cloning of plasmids because e. coli is not able to produce any ahls. genetically modified e. coli carrying ahl sensors plasmids; psb401, psb536 and pal101 containing fusion of luxri’::luxcdabe, ahyri’::luxcdabe, rhlri’::luxcdabe respectively are able to exert bioluminescence in presence of ahl molecules[68]. other than short and medium length acyl chain ahls, there are also some biosensors available in detecting long acyl chain ahls (length of c10 and above). one of the biosensors is genetically modified e. coli harboring psb1075 and pkdt17 both containing same fusion of lasri’::luxcdabe but psb1075 luminesce[68] while pkdt17 responds through standard β-galactosidase acdecoding the mystery... 5 tivity upon exposure of exogenous ahls[69]james. on the other hand, the p. aeruginosa pao1 m71lz could be utilized in detecting particularly c12 3-oxo-hsl and this biosensor is a lasi genomic knock-out mutant under control of rsal promoter with transcriptional fusion of lasr::lacz [70]. in order to detect ahls with 3-hydroxyl group attached, a bacterial biosensor known as p. fluorescens 2-79 could be employed. strain 2-79 employed genetically linked phzi/r qs system that regulates expression of phzabcdefg operon[71]. the strain 2-79 biosensor basically was developed from wildtype p. fluorescens 1855 that harbors two plasmid system; (i) psf105 carrying phzr gene regulates by trc promoter (ii) psf107 harbors phzr-phza divergent that regulates by dual promoter region and fuse with two different reporters, uida and lacz. the sensing of exogenous ahls could be easily detected via β-glucuronidase and β-galactosidase activity. there are some other biosensors that have been eveloped to detect broad range of ahls such as agrobacterium tumefaciens wcf47, biosensors to detect uncommon ahls, such as sini/r-based biosensors to detect any ahls with longer than 12 carbon length acyl chain, and there is also biosensors with gene encoding for the green fluorescent protein allowing detection of ahls at single-cell level[67]. although a negative results usually indicates that the no ahls are produced by the tested bacterial strain, but this could be due to the biosensors used could not detect novel ahls or the ahls produced are in low concentration and below a threshold that biosensors could barely detect[67]. hence, other methods such as thin layer chromatography (tlc) or high-resolution tandem-mass spectrophotometry could be used in detecting the ahls. other signalling molecules besides ahls as qs signalling molecules, there are presence of other signalling molecules being reported. some of the well-documented intercellular signalling molecules are the members of a family of quinolone compounds termed 4-hydroxy-2-alkylquinolines (haqs)[72]. the transcriptional regulator mvfr controls the synthesis of haqs, which leads to modulation of several genes expression in the production of anthranilic acid and its conversion to 4-hydroxy-2-heptylquinoline (hhq). the molecule 3,4-dihydroxy-2-heptylquinoline or known as pseudomonas quinolone signal (pqs) is produced from the conversion of hhq via pqsh action. however, the production of mvfr and pqsh are tightly control by lasr to intertwine with ahl-based pathway. the signalling of pqs is incorporated in the ahl qs pathway that is governed by las and rhl systems and known to be upregulated in cystic fibrosis patients during lung infections[73]. plant pathogen xanthomonas campestris and x. fastidiosa utilize a new type of communication language in regulating their virulence factors[74]. this signalling molecule is known as diffusible signalling factor (dsf) which was later identified as unsaturated fatty acid, cis-11-methyl-2-dodecenoic acid. three major qs components are needed in this qs pathway: rpff, rpfc and rpfg where they are involve in catalyzes, perception and transduction of the signalling molecules. the dsf-based qs mechanisms have been expanding and found to be utilized in other microorganisms such as xylella fastidiosa[75], stenotrophomonas maltophila[76] and burkholderia cepacia[76]. gramnegative bacteria are also capable in producing other types of signaling molecules; the iron mediated oxetane ring containing bradyoxetin namely 2-4-[[4-(3-aminooxetan2-yl)phenyl](imino)methyl]phenyl oxetane-3ylamine and 3-hydroxypalmitic acid methyl ester (3-oh pame) by bradyrhizobium japonicum[77] and ralstonia solanacearum[78]. both these signalling molecules are involved in protruding symbiotic relationship with higher organism, the plant. while majority gram-negative bacteria uses ahls as autoinducers, gram-positive bacteria uses post-translationally modified autoinducing peptide (aip) molecules for qs[63]. the aip is secreted through an atp binding cassette (abc) transporter protein[63]. gram-positive bacteria employ the two-component qs systems for aip detection where it involved a membrane-bound histidine kinase receptor and a cognate cytoplasmic response regulator. the two component system is regulated by a series of auto-phosphorylation cascade. similar with the ahl-based qs, the concentration of secreted aip increases parallel with increasing cell density. several peptide-based qs systems include the agrc/agra system of staphylococcus aureus in regulating virulence[79], the comd/come system of streptococcus pneumoniae in controlling bacterial competence[80], the comp/coma system of bacillus subtilis in regulating dna uptake and sporulation[81] and fsrb/fsrd system of enterococcus faecilis for conjugation [82]. conclusion in summary, bacteria have adapted quorum sensing systems to enable them for 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fingerprinting and antibiotic resistance profiling. antonie van leeuwenhoek. 2008; 94(3):377. tan w-s et al. progress in microbes and molecular biology 1 review article the bioprospecting of anti-vibrio streptomyces species: prevalence and applications loh teng-hern tan1,2, learn-han lee1,4*, bey-hing goh3,4* 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. 2institute of biomedical and pharmaceutical sciences, guangdong university of technology, guangzhou 510006, pr china. 3biofunctional molecule exploratory (bmex) research group, school of pharmacy, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. 4health and well-being cluster, global asia in the 21st century (ga21) platform, monash university malaysia, bandar sunway 47500, selangor, malaysia abstract: vibrio sp. has been a major pathogen that resulted in difficult to treat infections, and greatly impacting the aquaculture industry. thus, more effective approaches are needed to overcome this problem. bacteria of the genus streptomyces is a group of prolific producers for various bioactive compounds. streptomyces species with antibacterial activity against vibrio sp. have been reported from numerous studies, indicating that streptomyces could be a good candidate for treatment of vibrio infections. this review aims to provide an overview on the distribution of the streptomyces with anti-vibrio activity from diverse geographical locations. furthermore, this review also highlighted that streptomyces sp. can be a great source for anti-vibrio agents to control vibriosis, such as in the aquaculture settings. keywords: streptomyces, vibrio sp., secondary metabolites, antibacterial, biocontrol agent received: 18th may 2019 accepted: 15th june 2019 publish online: 05th july 2019 * correspondence: bey-hing goh, goh.bey.hing@ monash.edu. school of pharmacy; learn-han lee, lee. learn.han@monash.edu, leelearnhan@yahoo.com. jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500 bandar sunway, selangor darul ehsan, malaysia. citation: tan lth, lee lh, goh bh, the bioprospecting of anti-vibrio streptomyces species: prevalence and applications. prog microbes mol biol 2019; 2(1): a0000034 introduction seafood is rich in nutritional values, serving as a healthy food choice for major protein source in human diet. for the past decades, the accelerated growth in commercial aquaculture for total seafood supply is growing in folds in order to satisfy the increased demand for seafood globally [1]. however, seafood is prone to various contaminants, such as pathogenic microorganisms which include bacteria, viruses, fungi and parasites [2-7]. these pathogens are posing high risk for seafood and water borne illnesses in consumers [8-11]. this is because seafood can be a vehicle for pathogens. vibrio sp., which is one of the genera from bacteria kingdom [12], has been associated with gastroenteritis and wound infections in human [13], such as v. vulnificus [14], v. parahaemolyticus [15] and v. cholerae [16]. foodborne diseases active surveillance network (foodnet) reported that in the year 2018, vibrio sp. have inflicted 537 cases of infections with 1.1 incidence per 100,000 population in united states. foodnet also indicated that the number of vibrio infection cases have increased significantly by 109% in 2018 when compared with previous reported cases within the year 2015 to 2017 [17]. furthermore, vibrio sp. have also inflicted several major outbreaks worldwide [18-20]. for instance, the biggest outbreak of cholera was reported in haiti on october 2010 with more than seven thousand deaths recorded for the first time in more than a century [21]. besides causing infections in human, vibrio species is also a great threat towards aquaculture by causing vibriosis that hampers the fishery industry growth and causes serious economic losses globally. the etiological agents of vibriosis include v. harveyi, v. alginolyticus, v. anguillarum, v. salmonicida, v. mimicus and v. parahaemolyticus [22]. these pathogens have been reported to cause mortalities up to 100% in aquaculture. for example, the v. harveyi has caused mass mortality of black tiger shrimp penaeus monodon by causing luminous vibriosis [23-25]. another species v. mimicus is also responsible for epidemic in catfishes in china with high mortality rate between 80 to 100% [26]. consequently, copyright 2019 by tan lth et al. and hh publisher. this work under licensed under the creative commons attribution-noncommercial 4.0 international lisence (cc-by-nc 4.0) 2 antibiotics are used as prophylactic measures or to treat the established infections in the culture systems due to the immense impact of vibriosis in aquaculture. however, antibiotic resistant strain of pathogens are emerging due to the routine and uncontrolled usage of antibiotics and leading to therapeutic failure of existing antibiotic [27]. therefore, it necessitates the search for more effective alternatives to overcome this problem. in this regards, recent efforts have been evidenced in bioprospecting for natural products derived from plant [28-32], animal [33] or microbial origins [34] with promising antimicrobial effects to facilitate future development of new strategies against the antibiotic resistant strains of vibrio. the interest on the discovery of bioactive compounds from microbial origin is increasingly attractive towards the researchers, especially from the extreme environments. this is because that the sea and soil microbiota are frequently exposed to the complex, fluctuating and competitive environments which is believed to be the driving forces for metabolic pathway adaptation and lead to production of valuable metabolites [35-39]. the extremely diverse and unsurpassed richness of the secondary metabolism exhibited by streptomyces has made these filamentous bacteria to serve as a rich bioresource for valuable bioactive compounds [35, 40-43]. ever since the discovery of streptomycin as the first therapeutically beneficial antibiotic in 1944 [44], streptomyces species have been known to synthesize enormous amount of bioactive secondary metabolites, including antibiotics, antitumor agents, antiparasitic, immunosuppressive agents and industrially important enzymes [34, 45, 46]. the genus streptomyces is ubiquitously found in soil. in fact, they are also found to inhabit in wide range of niches such as in the aquatic environments, marine dwelling animals [47] and as symbionts of plants [48] and insects [49]. therefore, we attempted to evaluate the potential of streptomyces as a source of antibiotics against the antibiotic-resistant strains of vibrio. this review discusses the current knowledge on the streptomyces as a promising biocontrol agent of vibrio and assesses their distribution, isolation, secondary metabolites production. figure 1 depicts the potential of streptomyces bacteria as a source for anti-vibrio metabolites and their application in aquaculture. the bioprospecting of anti-vibrio... figure 1. the summary of the potential of streptomyces bacteria as a source for anti-vibrio biocontrol agent and its application as probiotic in aquaculture. 1. streptomyces sp. has been isolated from different ecosystems, including both terrestrial and aquatic environments. the percentage on the pie chart illustrates the proportion of streptomyces strain with anti-vibrio activity from respective ecosystem. (percentage of isolation from specific sources from both ecosystems are provided in the text) 2. the anti-vibrio active metabolites produced not only exhibits direct killing or growth inhibitory effect against vibrio pathogens, specific mechanisms are also demonstrated such as anti-virulence, anti-biofilm and anti-quorum sensing activity against vibrio pathogens. 3. streptomyces also exhibits the potential to be used as biocontrol agent in aquaculture for prevention of vibriosis. vibrio sp. and virulence factors being one of the six genera for the family vibrionaceae, the genus vibrio are gram-negative, halophilic and curved-rod in shape [50]. they are ubiquitous inhabitants of the warm coastal and estuarine waters as well as in the gut of filter-feeding shellfish. there are at least 12 species of vibrio have been known to be pathogenic and cause foodborne diseases in human [51]. besides capable to cause massive pandemics resulting in many cases of infections and deaths worldwide, some of the vibrios are known to be pathogenic to aquatic organisms, such as finfish, shellfish and corals [52]. virulence factors are the unique molecular features possessed by pathogen for colonisation, nutrient acquisition, infection and damage to a host [53]. studies have identified numerous virulence factors from the genus vibrio, including pathogenic v. cholerae, v. parahaemolyticus and v. vulnificus. for example, the cholera toxin (ctx), a well-known virulence factor or enterotoxin produced by v. cholerae [54], the thermostable direct hemolysin (tdh) and the tdh-related hemolysin (trh) in v. parahaemolyticus [15] and capsule polysaccharide (cps) in v. vulnificus [55]. all these virulence factors are attached on the surface of the cells or are secreted into the extracellular environment. to transport these virulence factors, specific secretion system is essential to facilitate the delivery of the effector virulence factors into host cells from the bacterial cells. for instance, the type iii secretion systems (t3ss1 and t3ss2) are the well characterized systems play significant roles in the pathogenicity of vibrio pathogens. studies demonstrated that regulation of virulence gene expression could be the 3 critical aspect for pathogenicity. to illustrate, a higher expression of those virulence genes could render a bacterial strain to be virulent, but this may not often be the case. as pathogenicity of bacteria is not always dependent on the presence of virulence genes [58]. quorum sensing (qs) is a machinery adapted by bacteria to coordinate the expression of certain genes, including those encoding virulent phenotypes, through the mediation of small signalling molecules [56]. for examples, the n-acylhomoserine lactone and the multi-channel qs systems are the two common qs systems acquired by the vibrio bacteria [57, 58]. there are many strategies used to control vibrio infections, including the antibiotics, water disinfectants, vaccines, immunostimulants, bacteriophages and probiotics in aquaculture [59-61]. despite that, new antibiotics or chemotherapeutic approaches are needed to cope with the ever-increasing evidences of antibiotic resistance among the vibrio sp. besides that, inhibition of the virulence factors of the pathogens is an alternative to kill the vibrio pathogens such as the disruption of bacterial cell-to-cell signalling or quorum sensing and the development of antagonistic compounds (antivirulence therapy) that specific targeting the virulence machinery of vibrio pathogens. hence, it is important to fully understand the virulence regulation mechanism (described earlier) in order to identify better therapeutic targets for prevention of outbreaks caused by vibrio pathogens. in this review, streptomyces bacteria is suggested as the promising candidate for the management of vibrio pathogens based on the potential of streptomyces bacteria in the production of anti-vibrio compounds and its application in aquaculture. emergence of antibiotic resistant vibrio sp. given the excessive use of antibiotics for the past few decades, the emergence and the ever-increasing prevalence of antimicrobial resistant pathogens is of a great concern in global health [27, 62-64]. today, many of antibiotics have been totally restricted in agriculture and aquaculture of developed countries due to the enormous detrimental impacts on the environment [65, 66]. despite that, the unrestricted use of antibiotics remains in countries with growing scale of agriculture and aquaculture industries such as china, chile and thailand. the antibiotics were used prophylactically by most of the farmers from aquaculture and agriculture settings to prevent or treat disease outbreaks, particularly infections caused by vibrio bacteria. for instance, excessive and frequent use of antibiotics as preventive management was observed from shrimp farming in thailand [62]. a total of 86% of the shrimp farmers from thailand were reported highly dependent on antibiotic use as a preventive measure, 14% of the farms even used antibiotics in a daily basis. norfloxacin, oxytetracycline, enrofloxacin and sulphonamides were the commonly used antibiotics in shrimp farms [62]. frequent use of antibiotic is also widely evident in other regions, including mexico [67], italy [27], philippines [68] and china [69]. undoubtedly, the enormous misuse of antibiotics has resulted the ever-increasing reports of multi-drug resistant vibrio species in aquaculture settings and marine environments [70]. for instance, a recent study showed the presence of vibrio sp. resistant to β-lactam and tetracycline in the hemolymph of litopenaeus vannamei shrimp [71]. furthermore, a plasmid mediated tetracycline resistant v. parahaemolyticus was isolated from shrimps infected with acute hepatopancreatic necrosis disease (ahpnd), indicating the presence of antibiotic resistance that can potentially be transferred through transposition, conjugation and plasmid uptake to other bacterial species in the same environment [72]. the disease ahpnd, also known as early mortality syndrome, is one of the major threats to shrimp farming. the disease has caused severe mortality up to 100% in aquaculture of p. vannamei and p. monodon [73, 74]. recently, castillo et al. (2015) [75] reported a draft genome sequence of v. parahaemolyticus strain vh3 isolated from farmed amberjack in greece. the strain vh3 was found to possess multidrug resistance efflux pumps and antibiotic resistant genes for fluoroquinolones and tetracycline [75]. moreover, v. parahaemolyticus has also been reported to be resistant to numerous classes of antibiotics such as penicillins (ampicillin), aminoglycosides (amikacin, kanamycin, streptomycin), cephalosporins (cefotaxime, ceftazidime, cefazolin) [76, 77], quinolones (ciprofloxacin, nalidixic acid), macrolides (azithromycin, erythromycin) and chloramphenicol [78, 79]. besides the antibiotic resistance incidences occur in aquaculture, there are enormous number of literatures focus on the antibiotic resistance of v. cholerae [80, 81], the causative agent of cholera which is an infectious diarrheal disease associated with hypovolemic shock and rice watery stools. this bacterium appears to be a reemerging problem to human worldwide, causing many disease outbreaks in which constant monitoring for their ever-changing antibiotic resistance profile is required. over the years, multidrug resistant v. cholerae has been reported from many regions of the world especially the under-developed and developing countries, including bangladesh [82], india [83], africa [84], haiti [85] and vietnam [86]. reports have shown that clinical isolates of v. cholerae have become resistant to numerous antibiotics including tetracycline [87], ampicillin [88], nalidixic acid [89], streptomycin, sulphonamides, trimethoprim, gentamicin [90] and ciprofloxacin [89]. v. cholerae is a naturally competent bacterium containing a highly diverse genome (genomic plasticity), readily taking up external dna and possibly recombine into their genome [91]. the antibiotic resistance in v. cholerae was attributed to target modification or acquisition of resistance gene cassettes from mobile genetic elements (mge). both integrative conjugative elements (ice) and superintegron are known to be the major source of conferring antibiotic resistance in v. cholerae. for instance, the sxt element, an ice responsible for gene translocation, is found in v. cholerae encoding various antibiotic resistance genes such as chloramphenicol, sulphamethoxazole, trimethoprim and streptomycin [92]. in fact, these sxt and closely-related elements are present in almost all v. cholerae clinical isolates and some environmental isolates from asia and africa [93]. a group of researcher has confirmed that the sxt elements were the vectors of genes conferring multidrug resistance in chinese epidemic o1 v. cholerae to tetracycline and trimethoprimsulfamethoxazole [94]. taken together, the resistance development limits the useful lifespan of antibiotic and tan lth et al. 4 results in the requirement for a constant introduction of new antibacterial compounds [95, 96]. streptomyces sp. as potential source for anti-vibrio agent the genus streptomyces (phylum: actinobacteria) are soildwelling gram-positive bacteria with high g+c (70%) genomic content. they have characterized filamentous growth involving tip extension and filamentous branching which eventually form network of filaments named as substrate mycelium [97, 98]. interestingly, streptomyces possess a remarkably complex developmental cycle [99]. under environmental stress and solid cultivation condition, they are capable to switch from vegetative phase (substrate mycelium) into a reproductive sporulation phase (aerial hyphae mycelium) [100]. the secondary metabolites are produced at the end of the active vegetative growth and during the dormant or reproduction stage [101]. more than 70 years ago, streptomycin was discovered as the first therapeutically beneficial antibiotic produced by s. griseus [44]. today, streptomyces bacteria remain to be prolific sources of novel secondary metabolites with diverse range of biological activities such as antibacterial, antitumor, antiviral, antifungal, immunosuppressive activity, antifeedant, insecticidal and neuroprotective activity [45, 102]. numerous studies have also described the production of valuable enzymes and compounds by streptomyces with industrially and clinically importance [45, 103, 104]. the enormous biosynthetic capabilities of streptomyces have made them an irreplaceable resource for microbial natural products in microbial world [105]. the streptomyces derived secondary metabolites are structurally diverse and based on different backbone structures, including polyketides, β-lactams, peptides and pyrroles [42, 101]. for example, the bioactive compounds include glycopeptides (vancomycin, teicoplanin, telavancin) [106], angucycline (tetrangomycin, landomycin, urdamycin) [107], tetracycline (chlortetracycline, oxytetracylcine, demeclocycline) [108], phenazine (saphenamycin, endophenazine, phenazinomycin) [109], macrolide (erythromycin, spiramycin, oleandomycin) [110], aminoglycoside (streptomycin, kanamycin, tobramycin) [111], benzoxazolophenanthridine (jadomycin) [112] and oligosaccharides (flambamycin, avilamycin, curamycin) [113]. majority of the streptomyces derived secondary metabolites are known to be antibiotics, given that they are needed for inhibiting the growth of other competing microorganisms present in the same environment [114]. the production of secondary metabolites also involves in the symbiotic interactions between the streptomyces and the plants. there are strains of saprophytic streptomyces colonize the plant roots and even in the plant tissues. the antibiotics produced by streptomyces protect the host plant from potential pathogens while the symbionts provide nutrients for streptomyces development [48]. terrestrial soils are the classical habitats of streptomyces sp. but current evidences indicate that streptomyces can be isolated from marine soils as well [42, 115]. these soils are known to be complex environments with many stressors such as diverse and variable nutrient availability, huge fluctuation in temperature, ph and salinity [116]. as a group of non-motile microorganism, streptomyces species requires to evolve and adapt for the survival in the diverse environmental challenges. bentley et al. (2002)[117] explained the large genome (>8mbp) of streptomyces sp. that encoding regulators, transport proteins and enzymes render them to be resistant to those environmental stressors. in 2001, streptomyces coelicolor a3(2) was reported to possess a more than 8 mega base pairs linear chromosome as the first and largest ever sequenced microbial genome [117]. a large proportion of the genome was shown to contain regulatory genes which are likely to be involved in detection of, and response to extracellular stimuli and stresses [117]. furthermore, approximate 23 cluster of the genes consisted of 4.5% of the total genome were found to be encoded for the biosynthetic enzymes that produce wide range of secondary metabolites. ikeda et al. (2003)[118] further revealed a larger secondary metabolic gene cluster covering approximately 6% of the genome found in s. avermitilis atcc31267. the genome of streptomyces is significantly larger as compared to the recent reported 5.1 mega base pairs chromosome from the genus bacillus. also as one of the best characterized bacterial genera, the genus bacillus has been extensively exploited for biotechnological use in the food and pharmaceutical production [119]. in the light of the expanding knowledge of microbial genetics and genomics, genome mining has revealed the potential of streptomyces sp. in synthesizing a large diversity of compounds that have yet to be identified via the detection of numerous cryptic novel secondary metabolite biosynthetic gene clusters [120, 121]. overall, these interesting features of streptomyces have demonstrated that this genus is a very good candidate for bioprospection of bioactive compounds with antibacterial properties [122], especially in anti-vibrio activity as the main focus of this review. studies of streptomyces with antivibrio activity up to the year 2015, based on the data reported from 64 studies (table 1), there are around 128 strains of streptomyces exhibited antibacterial activity toward vibrio sp. two and 3 strains of streptomyces were shown to exhibit antivirulence and antibiofilm activity against vibrio sp., respectively. table 1 tabulates the number of streptomyces strains with anti-vibrio activity with different stages of work performed ranging from the preliminary screening stage to an in-depth characterization of a streptomyces strain exhibiting anti-vibrio activity. based on these studies, streptomyces strains with anti-vibrio activities have been isolated from diverse ecosystems ranging from terrestrial to marine environments, and from marine organisms to aquatic plants. as depicted in table 1, 80% of the studies revealed streptomyces strains with anti-vibrio activities were isolated from aquatic environments while the remaining 20% of the studies showed streptomyces with anti-vibrio activities were derived from terrestrial origin. majority of the studies (48.3%) isolated streptomyces with anti-vibrio activity from marine and mangrove sediment, followed by marine organisms such as sponges, coral and fishes (21.7%), terrestrial soils (18.3%), aquatic plants (6.7%), water (3.3%) and terrestrial plants (1.7%). among the 128 strains of streptomyces with antibacterial activity the bioprospecting of anti-vibrio... 5 against vibrio sp., 116 strains (90%) were isolated from aquatic environment. this data suggests that marine ecosystem could be more preferable source for isolation of streptomyces with anti-vibrio activity as compared to the samples collected from terrestrial regions. despite that, it cannot be disregarded that terrestrial soil could be a potential source for streptomyces strains with anti-vibrio activity. in fact, some interesting streptomyces strains with anti-vibrio activity were reported from terrestrial soils [123, 124]. table 1. different isolation sources of streptomyces with anti-vibrio activity. source of isolation country locations number of streptomyces with anti-vibrio activity isolated the identified streptomyces sp. with anti-vibrio activity references marine sediment india andaman island 6 streptomyces sp. mks-09 (s. xantholiticus) streptomyces sp. mks-13 (s. aureofascicus) streptomyces sp. mks-17 (s. galtieri) streptomyces sp. mks-24 (s. vastus) streptomyces sp. mks-35 (s. galbus) streptomyces sp. mks-39 (s. rimosus) [125] sediment from coastal area of thondi, palk bay (lat. 9o45’n, long. 79 o3’e) 1 streptomyces sp. s8-08 (s. albus dq333301.1 99%) [126] chennai coast area, tamilnadu 1 streptomyces ecr3 [127] vellar estuary, tamilnadu 3 streptomyces sp. f1 streptomyces sp. f2 streptomyces sp. f3 [128] ns 1 streptomyces sp. isolate 6 [129] royapuram, muttukadu, mahabalipuram seashores, adyar estuary 2 streptomyces sp. c11 streptomyces sp. c12 [130] near-sea shore sediment from palk bay, (lat. 9 o44’10”n, long. 79 o10’45”e) southeast coast of thondi, tamilnadu 1 streptomyces sp. (99% s. fradiae bdms1) [131] visakhapatnam, india 1 streptomyces sp. ks1908 [132] andaman and nicobar islands (11o38’42.8”, 92o42’30.7”) 5 streptomyces sp. niot-vkkma02 (100% s. griseus) streptomyces sp. niot-vkkma26 (100% s. venezuelae) [133] bay of bengal 1 streptomyces sp. lcj94 [134] bay of bengal (lat. 11o42’23.15”n, long. 79o46’57.97”e) 1 streptomyces sp. ss7 [135] saltpan soil sample from parangipettai potnovo (lat. 11o30’n, long. 79o46’e) cuddalore district, tamilnadu 1 streptomyces sp. dptd215 (98% s. noursei ay999827) [136] versova coast, mumbai (lat. 19 o28’26.32”n, long. 72o48’07.21”e) 1 streptomyces sp. mvcs6 (kc292198) [137] versova coast, mumbai (lat. 19o08’26.12”n, long. 72o48’07.41”e) 1 streptomyces sp. mvsc13 (kc292199) [138] china submarine sediment from sanya bay (109o32’e, 18o11’n), northern south china sea 1 streptomyces sp. scsio 01689 (98.3% s. sanyensis) [139] sediment from shrimp farms hainan island, china marine 7 streptomyces sp. a03, a05 (s. cinerogriseus majority antagonistic to vibrio sp.) streptomyces sp. a26, a42 (s. griseorubroviolaceus) streptomyces sp. a41 (s. lavendulae) streptomyces sp. a45 (s. roseosporus) streptomyces sp. b15 (s. griseofuscus) [140] vietnam sediment from shrimp culture pond in thua thien hue 1 streptomyces sp. a1 hm854225 [141, 142] korea seaweed rhizosphere and sediment (10m depth) from coast of korea 1 streptomyces sp. pk288-21 (99% s. atrovirens dq026672.1) [143] egypt coastal lagoon sediment from sinai peninsula 1 s. ruber erkh2 [144] cuba near-shore sediment from matanzas, villa clara, cienfuegos and ciego de avila, central provinces of cuba. 3 [145] tan lth et al. 6 australia queensland, (lat. 21o43’09”s, long. 149o25’54”e) 3 streptomyces sp. cls-28 streptomyces sp. cls-39 streptomyces sp. cls-45 [146] mangrove sediment/ rhizophere soil/estuarines india mangalavana, narakkal, puthuvyppu, (9o55’10o10’n and 76o10-76o20’e ns ns [147] sundarbans, india and bangladesh ns ns [148] velar estury, tamilnadu, india (lat. 11.4900on long.79.7600oe) 1 streptomyces sp. ma7 [149] east coast region, pichavaram mangrove forest (lat. 11.43on, long. 79.77oe) tamilnadu, india 2 streptomyces sp. ecr64 streptomyces sp. ecr77 (accession number kf158225) (s. labedae) [150-152] bonnie camp & kalash, (lat. 21o51’05.823” n, long. 88o38’27.021” e) & (lat. 22o00’25.599” n, long. 88o42’13.948” e), sundarbans, india 3 streptomyces sp. sms_7 (closely related to s. tendae atcc19812t) streptomyces sp. sms_su13 (96.59% similarity to s. labelae nbrc 15864t, s. variabilis nbrc 12825t, s. erythrogriseus lmg 19406t) streptomyces sp. sms_su21 (99.75% similarity to s. griseorubens nbrc 12780t) [153] water sample india aquaculture water from vellore, tamilnadu 4 ns [154] seawater from visakhapatnam 1 s. rochei mtcc 10109 [155] marine sponges india marine sponges (callyspongia diffusa, mycale mytilorum, tedania anhelans, dysidea fragilis) from vizhinjam port, (lat. 8o22’30”n, long. 76o59’,16”e) south west coast india. 10 streptomyces sp. aqbcd03 streptomyces sp. aqbcd11 streptomyces sp. aqbcd24 streptomyces sp. aqbmm35 streptomyces sp. aqbmm49 streptomyces sp. aqbta66 streptomyces sp. aqbdf81 [156-158] kovalam coast, west coast of kerala (8o23’n, 76o57’e). ns ns [47] china mycale sp. from sea area of gulei port, fujian, china (lat. 23.74, long. 117.59) 3 hns054 (99% s. labedae) hns049 (s. microflavus) hns056 (s. flaveus) [159] egypt red sea 1 streptomyces sp. hc9 (accession number jq929061) 97% streptomyces rochei sbpl-21 [160] marine corals india mucus of coral, a. digitifera from hare island (9o12’n,79o5’e), gulf of mannar, tamilnadu 6 streptomyces sp. ca3 (99.8% s. akiyoshiensis fj486367.1) streptomyces sp. ca4 (96.7% streptomyces sp. eu523135.1) streptomyces sp. ca5 streptomyces sp. ca9 streptomyces sp. ca15 streptomyces sp. ca18 (96.7% streptomyces sp. eu523135.1) [161] china gorgonian coral (e. aurantiaca, m. squamata, m. flexuosa, s. suberosa, v. umbraculum) from sanya coral reef conservation (18o11’n, 109 o25’e), south china sea 3 streptomyces sp. zxy018 streptomyces sp. zxy077 streptomyces sp. zxy090 [162] lu hui tou fringing reef 3 scsio 11527 (s. fimicarius isp5322 100%) scsio 11469 (s. rutgersensis nbrc 12819 100%) scsio 11531 (s. variabilis nbrc 12825 99.859%) scsio 11717 (s. viridodiastaticus nbrc 13106 100%) [163] fishes india ornamental fish, chaetodon callare (red tail butterfly), archamia fucata (orange-lined cardinal) from vizhinkam port, india vizhinjam port, (8o22’30”n, 76 o59’16”e) southwest coast of india 7 aqbcc06 aqbcc 20 aqbcc 24 aqbcc 40 aqbcc 51 aqbcc 54 aqbcc 75 [164] marine epinephelus diacanthus (grouper), estuarine oreochromis mossambicus (tilapia), fresh-water cyprinus carpio (common carp) from vizhinjam, veli, centre for aquatic and research extension ns streptomyces sp. [165] red snapper from tamilnadu ns ns [166] the bioprospecting of anti-vibrio... 7 sea plants and animals in the intertidal zones china shark (mustelus manazo) local market, marine plant animal sea hare (aplysia dactylomela), sea anemone (actiniaria) and sea plant (ulva lactuca, enteromorpha, gracilaria verrucosa) from xiamen island 5 streptomyces sp. a4 streptomyces sp. a9 streptomyces sp. a16 streptomyces sp. a18 streptomyces sp. a29 [167] marine algae/seeweeds india intertidal rocky surfaces of muttom coast, south west coast of india (lat. 8o7’15”n, long. 77o1’e) 25 aqb.skku 8 (s. coelicolor) aqb.skku 10 (s. autotrophicus) aqb.skku 18 (s. pedanensis) aqb.skku 20 (s. deccanensis) aqb.skku 25 (s. vinaceus) aqb.skku 37 (streptomyces nov. sp.) [168-170] terrestrial soil sediment iran grassland, orchards, vegetable fields from kerman, hormozgan, sistan and baloochestan, south and south east provinces of iran 1 streptomyces sp. 419 [171] india rhizosphere soil from shikaripura, karnataka 3 srdp-s-03 srcp-s-05 srdp-2-30 [172] rhizosphere soil from thirthahalli, shivamogga, karnataka, india 1 srdp-07 [173] similipal biosphere reserve (21o28’ to 22o08’ n, 86o04’ to 86o37’ e) 1 streptomyces sp. ss2 [174] forest soils from western ghats region, kanyakumari district (lat. 8o03’ to 8o35’n, long. 77o15’ to 77o36’ e) 3 streptomyces sp. eri-1 streptomyces sp. eri-3 streptomyces sp. eri-26 [124, 175] thailand agricultural soil from sakonnakhon province 1 streptomyces sp. no.87 [176] chile desert soil (salt falt, zero vegetation cover, hyper-arid) from atacama desert (salar de atacama, laguna de chaxa) (23o17’s, 68o10’w) 1 streptomyces leeuwenhoekii sp. nov. c34t dsm42122 [123] chili field soil from chittagong, bangladesh 1 streptomyces sp. mu9 [177] terrestrial plant australia snakevine plant (kennedia nigriscans) from aboriginal community of manyallaluk, se of katherine, nothern territory (14o16’352” s, 132o49’750”e) 1 streptomyces sp. nrrl30562 [178] *ns – not specified tan lth et al. there were significant lower number of studies reported streptomyces with anti-vibrio activity from the terrestrial environments as compared to the much higher number of studies on the marine streptomyces. the higher isolation rate of streptomyces strains from marine environment could be due to the recent interest of researchers toward the marine natural products discovery as many novel bacteria genus and species with production of novel compounds have been identified from the marine environment [179-181]. in another context, these phenomena seem to imply that the resources which can be accessed easily had been exhausted as extensive studies on the terrestrial soil derived microorganisms were observed over the years. the recurrent isolation and screening of the predominant species from the environments have resulted in rediscovery of known compounds which is a major problem faced in drug discovery. as reported, similar well-known and structurally-related antibacterial compounds were discovered from the streptomyces isolated from different terrestrial environments [182]. to support the hypothesis that marine streptomyces is a better source for anti-vibrio activity, comparison between the efficacy of the metabolites produced by the anti-vibrio streptomyces isolated from respective environments were performed. to render easier inter-study comparison, the anti-vibrio activity of the streptomyces from each study was represented by the highest inhibition zone reported. the efficacy of the anti-vibrio metabolites produced by streptomyces isolated from respective source was obtained based on the median inhibition zone of respective site of isolation. these antivibrio streptomyces strains were then categorized into four different groups based on their source of isolations. according to table 2, it shows that the strength of antivibrio activity displayed by each group and the ranking is as follow, mangrove sediment (21.0 mm) > marine organisms (plants and animals) (18.0 mm), terrestrial soil (18.0 mm) > marine sediment (15.01 mm). the antivibrio streptomyces isolated from the respective isolation source with differential strength against vibrio sp. were discussed as follow. 8 table 2. comparing the anti-vibrio efficacy of streptomyces from different environment sources. source of isolation median of inhibition zone (mm) terrestrial soil 18.00 (n = 7) marine sediment and water 15.01 (n = 10) mangrove soil 21.00 (n = 3) marine organisms 18.00 (n = 8) terrestrial environments the number of undiscovered antimicrobials from streptomyces and the estimated number of antibiotics still be discovered from actinobacteria could be well above 105 as predicted with the use of mathematical models [183]. furthermore, new species of streptomyces are being identified every day, indicating that our knowledge on this genus is still far from exhaustive. hence, continuous effort has to be put into the exploitation of streptomyces from terrestrial regions by taking advantage of underexplored ecological niches as demonstrated by several groups of researchers that discovered streptomyces with anti-vibrio activity. this study identified a total of 11 strains of streptomyces with anti-vibrio activity were found from different types of terrestrial soils and an endophytic streptomyces isolated from a terrestrial plant [178]. the streptomyces with anti-vibrio activity were isolated from a wide variety of the terrestrial soils ranging from the commonly accessible agriculture soils [176], forest soils [124], grassland and orchard soils [171, 177] to the more extreme environments such as the hyper arid desert soil [123] and arctic sediments [184]. the detailed locations for the sources of isolation of the streptomyces with anti-vibrio activity were described in table 1. rateb et al. (2011)[123] reported a desert soil derived streptomyces strain c34 produced rare 22-membered macrolactone polyketides, known as chaxalactins a-c (1-3) with anti-vibrio activity. a recent report determined that this streptomyces strain c34 represents a new species and named as streptomyces leeuwenhoekii sp. nov., a strain showing high potential for drug discovery with total genome size of around 7.86mb [185]. the site of isolation of this novel species of streptomyces is from the hyper-arid and high-attitude atacama desert located in chile (23o17’s, 68o10’w). the three macrolactone polyketides including the chaxalactins a (1), b (2) and c (3) displayed a minimum inhibitory concentration of 12.5, 20 and 12.5μg/ml against the pathogen v. parahaemolyticus nctc10441 [123] which was isolated from the feces sample of a patient with gastroenteritis. the molecular structures of the chaxalactins were depicted in figure 2. meanwhile, an endophytic streptomyces nrrl30562 isolated from snakevine plant (kennedia nigriscans) was found to produce newly described antibiotics, named as munumbicin b (4), c and d [structures of munumbicin c and d have not been elucidated]; that displayed antibacterial activity against v. fischeri pic345 with inhibition zones measured at 16mm, 9mm and 12mm respectively at 10μg concentration [178]. the bioprospecting of anti-vibrio... figure 2. chemical structures of bioactive compounds with anti-vibrio activities. 9 besides the direct antagonism of streptomyces against vibrio sp. by direct killing of the microorganism or impeding microbial growth, the bioactive products derived from streptomyces sp. also exhibited activity that interferes with the expression of pathogenic traits of vibrio pathogens [186, 187]. augustine et al. (2012)[184] reported the 20% culture supernatant of strains streptomyces a733 and a745 isolated from arctic sediment (ny-alesund, an island in svalbard archipelago (79o55’n, 11o56’e) reduced the biofilm formation of v. cholerae o1 mcv09 by 88% and 80% respectively. furthermore, an antivirulence compound, known as guadinomine b (5) was produced by a strain streptomyces sp. k01-0509 isolated from soil sample collected from the amami oshima, kagoshima, japan [186]. the guadinomine b (5) was reported to be potent in inhibiting the type iii secretion system (ttss) of gram-negative bacteria, including most of the pathogenic vibrio sp. that utilize this apparatus for protein secretion and translocation as their primary virulence mechanism with ic50 at 14nm [188]. another study showed that the soil-derived s. mobaraensis dsm40847t from mobara city, japan, produced endoprotease inhibitors that against cysteine protease papain which is known to be virulence factors involved in bacterial pathogenicity [187]. the endoprotease inhibitor was known as streptomyces papain inhibitor (spi) which exhibits inhibitory effect on the growth of wide range of gram-positive and gramnegative bacterial pathogens. the addition of 10µm of spi was shown to be bactericidal toward v. cholerea serotype o1 (atcc 14035). this study suggested that spi could be a potential novel broad-spectrum antimicrobial agent for clinically relevant infectious diseases [187]. aquatic environments marine environments are the largest source of microbes and new secondary microbial metabolites. the marine sources ranging from the seashore soil sediments to the depths of 10,000 metres [189] are rich sources of microbes. furthermore, marine environment contains wide range of distinct microorganisms that are not present in terrestrial environment [45, 190, 191]. this may be attributed to the extremely different physical and chemical conditions as compared to the terrestrial conditions. it has been suggested that marine actinobacteria exhibit distinct characteristics from those terrestrial counterparts and therefore produce more potentially novel bioactive compounds [190, 192]. thus, marine environment is a potential source for isolation of novel actinobacteria in which increasing evidences on the discovery of novel antibiotic and industrially important enzyme from marine actinobacteria [193-196]. likewise, 80% of the reviewed studies demonstrated the isolation of streptomyces with anti-vibrio activity from aquatic environments such as marine sediments, marine invertebrates and mangrove ecosystems. marine sediments based on the literatures collected, a total of 38 strains of streptomyces with anti-vibrio activity were isolated from marine sediments of diverse geographical locations. these diverse geographical locations include coastal lagoon sediment [144], near-shore sediment [131], shrimp culture pond [146] and submarine sediment (45m underwater) [139]. streptomyces species found in virgin soil is expected to produce broad-spectrum antimicrobial compounds, hence rendering them to be successful in outcompeting others and effectively colonize the newly formed soil. mitra et al. (2011)[197] suggested that the specific area favorable for obtaining maximum number of isolates with broadspectrum activity in an estuarine setting is limited to the narrow band between the mean high and low tide marks. in brief, the samples collected from sites influenced by tides were suggested to exhibit a high antagonistic potential [198]. mitra et al. (2008)[198] believed that antibacterial compound is required to aid in colonizing a newly formed top soil during the transition periods between high and low tides, thereby the periodic oscillations of dry and wet conditions trigger more antagonistic activity of actinobacteria. in agreement with observations of mitra et al. (2011)[197], several marine sediment derived streptomyces strains exhibiting broad antibacterial spectrum and surfactants producing ability were isolated from area constantly affected by tidal gradient in minnie bay, a & n islands, india [199]. furthermore, the high nutrient availability and osmotic flux in the sampling site could be another reason for the broad-spectrum activities exhibited by these strains. for example, the ethyl acetate extract of the streptomyces sp. niot-vkkma02 displayed the maximum inhibitory activity against a classical o1, hypertoxigenic strain v. cholerae 569b (mtcc3904) with 20 mm inhibition zone measured at concentration of 50 μg [199]. furthermore, studies demonstrated the purified dopa melanin produced by streptomyces sp. mvsc13 and mvcs6 isolated from the marine sediment of versova coast, mumbai, india (lat. 19o28’26.32”n, long. 72o48’07.21”e) exhibiting strong antibacterial activity against several fish and human vibrio pathogens [137, 138]. specialized media (tyrosine asparagine medium) was employed to cultivate streptomyces sp. mvsc13 and mvsc6 for the production of dopa melanin which displayed good activity against vibrio sp. fpo5 (from infected region of carassius auratus, 16s rrna 98% v. parahaemolyticus) (15±0.01mm), v. fluvialis rmmh10 (12±0.02mm), v. splendidus rmmh11 (9±0.02mm), v. parahaemolyticus rmmh12 (15±0.03) [137, 138]. moreover, a new pyranosesquiterpene compound (6) was discovered from a strain streptomyces sp. scsio 01689 isolated from submarine sediment, located 45m underwater of northern south china sea (18o11’n, 109o32’e). the isolation of streptomyces sp. scsio 01689 and the preparation method for its production of cyclic peptide type compounds were patented [139]. the patent disclosed the cyclic peptide type compounds, pyranosesquiterpene compound (6), cyclo(d)-pro-(d)-ile (7), cyclo(d)-pro-(d)-leu (8) and cyclo(d)-trans-4-oh-pro-(d)-phe (9) exhibited potent anti-vibrio activity, specifically against v. anguillarum with mic measured at >100, 0.05, 0.04 and 0.07 μg/ml. besides that, you et al. (2007)[200] indicated the metabolite of streptomyces sp. a66 isolated from marine sediment was found to be effective in reducing the development of antibiofilm in vibrio sp. the strain attenuated the biofilm formation of v. harveyi with 99.3% of inhibition rate and 74.6% of degradation rate at concentration of 2.5% (v/v) [140]. another study also indicated that the antibiofilm activity of streptomyces sp. a66 involved in the inhibition of the quorum sensing system of vibrio sp. by reducing the n-acylated homoserine lactones activity tan lth et al. 10 [200]. the n-acylated homoserine lactones are responsible for the coordination of virulence expression in response to density of surrounding bacterial population [200]. mangrove environment mangroves are located along the intertidal zones of estuaries, backwaters, deltas, marshes and mudflats along the tropical and subtropical regions. mangrove ecosystem is a unique ecological niche which contains highly productive and diverse microbial community [201-204]. similarly, the mangrove environment has been known to be potent reservoir for isolation of antibiotic-producing actinobacteria [205]. eccleston et al. (2008)[206] revealed that the ecology has great impact on the diversity of actinobacteria. higher population of actinobacteria was isolated from mangrove mud sediments than the benthic communities associated with littoral sand sediments, freshwater creek and lake habitats. eccleston et al. (2008)[206] suggested the low numbers of actinobacteria from freshwater habitats and littoral sand sediments could be attributed to the low organic nutrient levels as compared to high nutrient habitats such as mangrove mud [206]. accordingly, hong et al. (2009)[35] also demonstrated the abundance of bioactive strains is correlated with ecological influences. a low number of bioactive strains was recorded from soil containing more sand and less organic matter while rhizosphere soil was rich source of bioactive strains [35]. by comparing the different isolation sources, the data showed that the streptomyces strains derived from mangrove soil displayed the strongest antibacterial activity against vibrio sp. with the highest median inhibition zone (21.0 mm), followed by marine sediment (15.0 mm), marine organisms (18.0 mm) and terrestrial soil (18.0 mm). this observation suggests that mangrove environments provides a better site for isolation of streptomyces strains with 39.9% higher anti-vibrio activity than those from marine sediment and water. mohana and radhakrishnan (2014)[149] reported an antivibrio streptomyces sp. strain ma7 from mangrove rhizosphere sediment collected from vellar estuary region at parangipettai, tamilnadu, india (11.4900on; 79.7600oe). strain ma7 displayed antibacterial activity towards several vibrio sp. pathogens including v. mimicus, v. cholerae o1, v. cholerae o139 and v. parahaemolyticus. the methanol extract of strain streptomyces sp. ma7 exhibited strong antibacterial activity against v. parahaemolyticus with 21 mm inhibition zone measured at concentration of 250 μg [149]. furthermore, an aliphatic compound named as n-isopentyltridecanamide was identified from the ethyl acetate extract of strain streptomyces ecr77 (16s rrna 99% s. labedae) isolated from the mangrove sediment of east coast region, pichavaram mangrove forest (lat. 11.43on, long. 79.77oe). the ethyl acetate extract of streptomyces ecr77 showed the maximum inhibitory activity against v. cholerae, v. parahaemolyticus and v. alginolyticus with inhibition zones 13.66±0.47mm, 9.66±0.94 and 16.33±0.47mm measured at 25 μl concentration [152]. similarly, sengupta et al. (2015)[153] isolated three mangrove derived anti-vibrio streptomyces in sundarbans, they displayed high antibacterial activity against v. cholerae (mctc 3906) with the inhibition zone measured more than 25 mm and minimum inhibitory concentration at 50 μg/ml. marine animals and plants streptomyces species is also found to form symbioses with other organisms, most notably plants and invertebrates. in many cases, streptomyces species showed protective mutualistic symbioses with the host in which the host provides nutrients and protections for the bacteria while the bacteria produce antibiotics to protect host from pathogens [49, 207]. researches have indicated marine invertebrates which are sessile, such as sponges and corals are great sources of marine bioactive metabolites. these bioactive metabolites in these marine organisms were produced by the marine bioactive metabolite producing microorganisms as symbiotic relationships. for instance, theopaulauamide, an antifungal bicyclic glycopeptide isolated from palauan sponge, theonella swinhoei has been confirmed to be originated from a novel delta-proteobacterium known as candidatus entotheonella palauensis, served as one of the first experimental evidences for microbial derived compounds from sponge [208]. there has been an increasing evidence of sponges and corals as the potential sources for isolation of streptomyces with anti-vibrio activity. the comparison made earlier revealed that the streptomyces isolated from marine organisms, such as sponges and corals, represent alternative sources for anti-vibrio streptomyces. these streptomyces were isolated from marine sponges such as the callyspongia diffusa, mycale mytilorum, tedania anhelans and dysidea fragilis collected from vizhinjam port (lat. 8o22’30”, long. 76o59’16”e) located at southwest coast of india [157]. the ethyl acetate extracts of these streptomyces strains exhibited diverse strength of antibacterial activity toward both human and fish vibrio pathogens such as the v. harveyi, v. parahaemolyticus and v. alginolyticus with maximum inhibition zone measured up to 30 mm at 50 μg concentration [156]. su et al. (2014)[159] reported the isolation of streptomyces sp. hns054 (16s rrna 99% similarity to s. labedae) from marine sponges, mycale sp. collected from gulei port, fujian, china (lat. 23.74, long. 117.59) exhibiting antibacterial activity against both v. parahaemolyticus and v. diabolicus, with 10-15 mm inhibition zone observed against v. parahaemolyticus. the study suggested that streptomyces sp. strain hns054 may play an important in conferring a chemical defensive mechanism to protect the sponges from pathogenic vibrio sp. which are associated with mortality of marine animals [159]. the detection of these streptomyces strains with secondary metabolite production further support the facts that sponges or marine invertebrates are important source of biologically active compounds [209, 210]. coral is also a potential source to isolate streptomyces sp. with genetic capacity to produce diverse potentially bioactive molecules which may contribute to the chemical defense of coral holobionts [162, 163]. there were 4 studies (6%) reported the isolation of streptomyces with anti-vibrio activity from different species of corals, including the acropora digitifera [211], melitodes squamata [212], porites lutea, galaxea fascicularis [213], sarcophyton glaucum [160]. li et al. (2014)[213] reported a total of four different species of streptomyces with anti-vibrio activity in the coral the bioprospecting of anti-vibrio... 11 samples collected from lu hui tou fringing reef (18o13’n, 109o28’e). the ethyl acetate extracts of these streptomyces showed different degree of anti-vibrio activity against both pathogenic v. coralliilyticus atcc baa-450 isolated from diseased coral pocillopora damicornis and v. alginolyticus serotype xii atcc 17749 isolated from spoiled horse mackerel which caused food poisoning. the highest anti-vibrio activity was displayed by streptomyces sp. scsio11717 (16s rrna 100% s. viridodiastatitus nbrc13106) with zone of inhibition of 12.3±2.5mm measured at 20 mg/ml as compared to the standard drug, ciprofloxacin (20 mg/ml) with 15±1mm against the pathogenic v. alginolyticus [213]. furthermore, streptomyces sp. scsio 11527 (16s rrna 100% s. fimicarius) with anti-vibrio activity isolated from coral galaxea fascicularis was positive for pks-ii gene with 90% similarity to ketoacyl synthase from s. argillaceus, suggesting its potential in producing anthracycline-related compounds [163]. this finding was supported with one of the previous study demonstrated the production of nanaomycins a (10) and d (11) by streptomyces rosa var. notoensis os-3966 isolated from a soil sample collected at nanao-shi in noto peninsula, japan [214]. the study showed that both nanaomycins a (10) and d (11), anthracycline/anthraquinone antibiotics exhibited strong inhibitory activity against both marine pathogens, v. alginolyticus 138-2 and v. parahaemolyticus k-1 [214]. besides marine sponges and corals, seaweed is also another source for anti-vibrio streptomyces. there were 3 studies reported the presence of streptomyces with antivibrio activity from seaweeds collected from intertidal rocky surfaces at muttom coast, southwest coast of india (8o7’15”n, 77o1’e) [170]. according to hollants et al. (2013) [215], the macroalgal-bacterial interactions are not unusual. in fact, it has been evidenced that the production of antimicrobial compounds by the microorganism is to protect the algae surface from pathogens, herbivores and fouling organisms. interestingly, a strain streptomyces sp. strain aqb.skku20 derived from seaweed was expressing antagonistic activity towards vibrio sp. after the exposure to ethidium bromide, suggested that the mutations induced by ethidium bromide stimulates antibiotic production [168]. furthermore, study also indicated that streptomyces with anti-vibrio activity isolated from seaweeds could be used as probiotics and biocontrol agents against vibriosis in aquaculture [169]. this study demonstrated that the incorporation of the anti-vibrio strains of streptomyces in the probiotic feed resulted in higher percentage of survival rate of macrobrachium rosenbergii prawn juveniles with no external disease manifestations after challenged with pathogenic v. vulnificus at 105 cfu/ml which caused up to 79.2% mortality in control group with no streptomyces as probiotic [169]. application of anti-vibrio compounds producing streptomyces sp. in aquaculture streptomyces sp. constitute a group of industrially and clinically important microorganisms [40, 42, 115, 216] that produce valuable compounds including antibiotics [41], antitumor agents, antiparasitic, immunosuppressive agents and enzymes [45]. being the fact having an immense potential for bioactive secondary metabolites production, streptomyces has the advantage of producing potential antagonistic and antimicrobial compounds can be valuable as biocontrol agent against vibrio pathogens in aquaculture [217]. the production of antagonistic compounds renders streptomyces sp. capable to compete for nutrients and attachment sites in the host. for example, streptomyces sp. was reported to produce siderophores which could influence the growth of pathogenic vibrio sp. [140]. siderophores are ferric ion-specific chelating agents which aiding the streptomyces sp. to compete for iron in the aquatic environment [218]. studies have indicated that the intracellular iron concentration is essential for biofilm formation and development in bacteria and also the vibrio sp. [219-221]. mey et al. (2005)[221] revealed the wild-type v. cholerae suffered poor biofilm formation in iron-deficient medium and also elucidated the role rhyb gene in iron homeostasis to biofilm formation as the rhyb mutant v. cholerae was unable to form wild-type biofilm in low-iron medium. biofilm formation plays many imperative roles in vibrio sp. for their survival, virulence and environmental stressors resistance [53]. biofilms serve to render vibrio sp. more protected and less susceptible to antimicrobial agents and hence difficult to control. the discovery of streptomyces strains with ability to produce siderophores is providing a new approach in controlling vibrio sp. in aquaculture settings as biofilms are considered a reservoir for some pathogenic vibrio sp. that can cause detrimental effects on the cultured livestock in aquaculture. moreover, studies also revealed the production inhibitory compounds with anti-quorum sensing [200] and anti-virulence activities [186] targeting vibrio sp. by streptomyces sp. these promising anti-vibrio activities also further strengthen the view of the applicability of streptomyces in aquaculture as an alternative biocontrol agent against vibrio sp. [217]. conclusion there is an urgent need to search for new therapeutic drugs, especially antibiotics due to the rapid increase of resistance in vibrio sp. pathogens to the major frontline antibiotics. thus, extensive effort is required by the researchers focusing on the screening and isolation of promising strains of streptomyces with antimicrobial properties. the information and knowledge obtained in this review could help in selecting the potential sources of isolation and as a guide for future bioprospectors in finding antibiotic-producing streptomyces, especially against vibrio spp. based on the findings of this review, mangrove sediment could be a better source for streptomyces with anti-vibrio activity. nevertheless, there is still limited studies on the investigation of the exact antibacterial mechanisms of these streptomyces derived bioactive metabolites against the vibrio pathogens. therefore, future studies on the elucidating the antibacterial mechanisms of these streptomyces are warranted. as a whole, these anti-vibrio streptomyces represent a valuable source for future development of clinically important drugs to treat infections caused by v. cholerae, v. parahaemolyticus and v. vulnificus in clinical settings as well as to be applied as probiotics to control vibriosis in aquaculture. tan lth et al. 12 author contributions the literature review and manuscript writing were performed by lt-ht, l-hl and b-hg. l-hl and b-hg founded the research project. conflict of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. acknowledgments this work was supported by the monash university malaysia ecr grant (5140077-000-00), mosti escience fund (02-02-10-sf0215 and 06-02-10-sf0300). reference 1. hixson sm, fish nutrition and current issues in aquaculture: the balance in providing safe and nutritious seafood, in an environmentally sustainable manner. j aquac res dev 2014; 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73(9): 5706-5719. tan lth et al. pmmb 2023, 6, 1; a0000324. doi: 10.36877/pmmb.0000324 http://journals.hh-publisher.com/index.php/pmmb review article probiotics: comprehensive exploration of the growth promotion mechanisms in shrimps joanna xuan hui goh1, loh teng-hern tan1,2, jodi woan-fei law1, kooi-yeong khaw3, gokhan zengin4, kok-gan chan5,6, vengadesh letchumanan1, learn-han lee1*, bey-hing goh3,7* article history 1novel bacteria and drug discovery (nbdd) research group, microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor, malaysia; joanna.vetpharm@gmail.com (jxhg); tenghern@gmail.com (lt-ht); jodilaw48@gmail.com (jw-fl); lvengadesh@yahoo.com (vl) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia. 3biofunctional molecule exploratory (bmex) research group, school of pharmacy, monash university malaysia, bandar sunway 47500, selangor, malaysia; khaw.kooiyeong@monash.edu (k-yk) 4department of biology, science faculty, selcuk university, campus, 42130 konya, turkey; gokhanzengin@selcuk.edu.tr. (gz) 5division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, kuala lumpur 50603, malaysia; kokgan@um.edu.my (k-gc) 6international genome centre, jiangsu university, zhejiang 212013, china. 7college of pharmaceutical sciences, zhejiang univeristy, hangzhou 310058, china. *corresponding author: bey-hing goh, school of pharmacy, monash university malaysia, selangor darul ehsan, malaysia; beyhing@gmail.com (b-hg); learn-han lee, novel bacteria and drug discovery research group, microbiome and bioresource research strength, jeffrey cheah school of medicine and health sciences, monash university malaysia, selangor darul ehsan, malaysia; leelearnhan@yahoo.com (l-hl) received: 01 november 2022; received in revised form: 30 december 2022; accepted: 02 january 2023; available online: 04 january 2023 abstract: as feed accounts for a significant proportion of a farm’s expenditure, animal nutrition is one of the key profit determinants. attributed to the size-dependent market value, enhancing shrimps' growth is essential to maximize profit. despite not being the best option, antibiotics are often used as growth-promoting agents in farming. although this trend is less explicit in aquaculture, increasing production yield is paramount, especially when intensive aquafarming compromises animal growth and increases disease prevalence. however, the environmental and clinical pitfalls of indiscriminate antibiotic usage are surfacing. fortunately, increasing evidence demonstrated probiotics as a safer, more sustainable, and environmental-friendly substitute for antibiotics. nonetheless, most studies are observational, and the growth-promotion mechanisms of these agents are yet to be elucidated. mailto:joanna.vetpharm@gmail.com mailto:tenghern@gmail.com mailto:jodilaw48@gmail.com mailto:lvengadesh@yahoo.com mailto:gokhanzengin@selcuk.edu.tr mailto:kokgan@um.edu.my pmmb 2023, 6, 1; a0000324 2 of 86 in this light, this review aims to decipher the growth promotion mechanisms of probiotics in shrimps based on the primary works conducted. evidently, probiotic treatment modulates the gut microbiome composition. the growth promotion effect of probiotics is partly mediated through the production of bioactive compounds such as short-chain fatty acids, vitamins, and polyamines. besides, elevated digestive enzyme activities following the introduction of probiotics may help enhance digestibility and utilization. histological changes at the hepatopancreas and intestine were evident. furthermore, probiotics may reinforce the protective mechanisms in the gut and strengthen immune function. treated shrimps demonstrate better appetite and exhibit superior metabolic and growth-related genes profile. contrasting these recognized mechanisms with antibiotics helps construct the initial framework for designing high-quality probiotics for growth enhancement in farmed animals. keywords: growth; probiotic; feed additives; mechanism; shrimp; antibiotic 1. introduction in recent years, progress in the aquaculture industry has been gaining increasing momentum. according to a recent report released by the fisheries department of the food and agriculture organization (fao), global aquaculture production rises by 6.1% annually, with the highest production centred in asia [1]. although crustaceans only account for 7.5% of the total global production by weight, it is characterized by a high unit value, which amounts to 24.5% of the total global production value [1, 2]. shrimp aquaculture, which has emerged as a promising economic endeavour, has been progressively intensified in many developing countries [1]. over the past two decades, crustacean production has increased by 9.9% annually, achieving 8.4 million tonnes in 2017. among the shrimp species cultured, the pacific white shrimp (litopenaeus vannamei) recorded the highest production rate, which is followed by the black tiger shrimp (penaeus monodon) and the giant freshwater prawn (macrobrachium rosenbergii) [1, 2]. despite the promising development, further intensification of shrimp farming often reaches a bottleneck where the high stocking density significantly increases the risk of disease transmission and severely decimates the production yield [3-6]. attributed to the high unit price of shrimp and the size-dependent market value, optimizing the growth of shrimp within the shortest production frame became a pivotal factor in maximizing revenue. a higher production yield would compensate for the untoward losses to disease episodes and sustain the culture production [7]. the application of antibiotics at subtherapeutic doses for disease control and growth enhancement has been a time-honored convention in farming practice [8-10]. data from the state of the world’s antibiotics 2015 revealed that 65% of the 100,000 tonnes of antibiotics produced globally were capitalized for animal production [11]. however, the negative impacts of antibiotic use are gradually surfacing. the detection of high antibiotic residue levels in the farm wastewater and sediment of shrimp ponds poses a threat to the surrounding marine or coastal ecosystems [12-15]. antibiotic use exerts a selective pressure on resistant bacteria, pmmb 2023, 6, 1; a0000324 3 of 86 which creates a risk for the transference of antimicrobial resistance genes (args) to other bacteria via horizontal gene transfer mechanisms such as transformation, conjugation, or transduction [14, 16]. this gradually precipitates the emergence of multi-antibiotic-resistance bacteria pathogenic to other animals and humans [17-24]. besides, the residual antibiotic detected in animal flesh is another alarming concern for public health, particularly when the concentration of antibiotics exceeds the maximum residue limit [25, 26]. the increasing awareness regarding the detrimental consequences of indiscriminate antibiotic use has increased the demand for antibiotic-free products from sceptical consumers. in some countries, drastic antibiotic use restrictions have been reflected in banning certain antibiotics or stringently controlling their application for limited indications [27, 28]. the environmental hazards and health threats accompanying antibiotic application hampered its continuous use in farming. therefore, the quest for a safer and sustainable alternative to antimicrobial growth promoters (agps) is an exigency to safeguard the animal production yield and to forestall the aggravation of antimicrobial resistance (amr) development. at this juncture, a mounting body of research evaluates the effectiveness of probiotics as a potential candidate to replace antibiotics in farming [29-31]. on average, metaanalysis results revealed that probiotic treatment improved the feed conversion ratio (fcr) of 49 studies and the specific growth rate (sgr) of 60 studies by 19% and 14%, respectively [32]. several studies reported that probiotics demonstrated comparable growth promotion effects to antibiotics [33-35]. moreover, besides the growth promotion effects, probiotics also elevate the resistance to disease and environmental stressors, enhance the animal's immune function and ameliorate the quality of rearing water [36-39]. more often than not, the growth enhancement effects of probiotics are typically reported as a mere ‘positive side effect’ to its primal role in disease control. acknowledging the dire need to enhance aquaculture yield, the growth enhancement effects of probiotics should, instead, be maximally harnessed in the current farming practice to increase farm production. probiotics use in aquaculture could be a boon to the farming industry. therefore, further research is warranted to investigate how probiotics can be more efficiently integrated with the feed additives commonly applied in farms. in this light, this review aims to compile the possible probiotic mechanisms contributing to the growth of shrimps. deciphering the mechanisms of probiotics about the action of agps also helps to develop better alternatives to agps. addressing this knowledge gap will shed light on the positive traits of probiotics that facilitate the growth of livestock. understanding these crucial factors will be critical for developing probiotics tailored for the growth enhancement of shrimps. pmmb 2023, 6, 1; a0000324 4 of 86 2. antimicrobial growth promoters antibiotic has been an indispensable confederate in farming practice [40]. it is still the mainstay for disease management among husbandry animals, especially during the early breeding phases [41]. antimicrobial agents can be administered via different routes, including direct application to water, incorporated into feed, or injected intramuscularly [14, 41]. sulphonamides, tetracyclines, quinolones, chloramphenicol, and nitrofurans have been extensively exploited for aquaculture use [25, 42]. usage of oxytetracycline, sulphadiazine, florfenicol, amoxicillin, oxolinic acid, sulphamethoxazole, trimethoprim, and erythromycin have also been recorded [43, 44]. nevertheless, it is difficult to estimate the total annual global antibiotic use in shrimp farming alone. this is attributed to the discrepancies in farming modes, climates, disease risks, antibiotic limits, and the regular shifts between the diverse farmed species [43, 45]. moreover, the policies vary significantly between countries, and there is little detail regarding each region's indication and antibiotic usage pattern [46]. although using antimicrobials at subtherapeutic doses to boost growth performance has been discouraged for animals intended for food supply, some farmers still embrace antibiotics for their growth-promoting effects in aquaculture [17]. on average, antibiotics use could increase feed utilization by 2% to 5%, translating to an estimated growth improvement ranging from 4% to 8% [47]. for example, over eight weeks, the sgr and weight gain rate (wgr) in l. vannamei receiving daily supplementation of 0.3% florfenicol to the basal diet increased by 2% and 7%, respectively, when compared to the untreated control [35]. in another experiment, daily inoculation of oxytetracycline directly to the rearing water at a concentration of 4 mg/l resulted in a significantly 8% higher development rate of l. vannamei larvae after nine days of treatment [34]. the growth promotion effect may differ according to animal species, antibiotic selection, and treatment regimens. in recent years, increasing reports unveiled environmental and food safety concerns regarding antibiotics usage [48, 49] [50-53]. ironically, in contrast to the expected decline in antibiotic use following the heightened consciousness of their negative implications, antibiotic consumption for aquacultural purposes is still threading on an increasing trend [54]. judging from the continuously increasing demand for food production, the global antimicrobial utilization intended for food production is forecasted to exceed 100,000 tonnes by 2030 if no proper substitute for agp for farming is sought [41]. therefore, it is crucial to decipher the mechanisms of antibiotics in enhancing the survival and growth of animals in the quest for a desirable replacement compound. 2.1. mechanisms of agps for a long time, the growth promotion effect of antibiotics was attributed to the suppression of subclinical infections [55]. the bacteriostatic or bactericidal effect against opportunistic pathogens helps mitigate disease occurrence in intensive farming [56]. although pmmb 2023, 6, 1; a0000324 5 of 86 the antimicrobial effect at a subtherapeutic dose was speculative [57], this rationale has invariably augmented the unwarranted use of prophylactic antibiotics to overcome the sanitary shortcomings in crowded farming sites [17]. the remarkable growth-promoting effect remains a subject of interest that intrigues many researchers. the underlying mechanism of antimicrobial growth promoters is yet to be fully elucidated. several hypotheses were proposed to explain the growth-promoting phenomenon of antibiotics. however, there is still ongoing debate regarding the plausibility of these hypotheses [57]. further research is warranted to fill the knowledge gap. nonetheless, the mechanisms can be broadly classified into two categories: bacterial-centric and host-associated factors (figure 1). rather than treating these factors as exclusive events, it is highly probable that these two mechanisms complement each other and dually contribute to animal growth. these two factors are further compounded by other external factors such as hygiene, stress, and diet [58]. figure 1. the bacterial-centric and host-centric growth-promoting mechanisms of agp. an underlying assumption for the bacterial-centric hypothesis is that normal gut microbiota suppresses animal growth [57]. this notion is well promulgated by the fact that agps do not promote the growth of germ-free mice. meanwhile, depression of growth becomes evident following the inoculation of bacteria to germ-free mice [57]. in this sense, the growth promotion mechanism can be expounded through the concept of ‘dysbiosis’. dysbiosis is a term that describes the compositional shift of bacteria distribution in the gut induced by a disruption to the gut microbial homeostasis leading to metabolic and functional changes. antibiotic was believed to be an intervening factor that disrupts the homeostatic balance [59]. it causes a significant alteration of gut microbiota composition in the treated animals. the growth promotion effect becomes evident when microorganisms with better pmmb 2023, 6, 1; a0000324 6 of 86 metabolizing potential gain predominance in the gastrointestinal tract [60]. increasing evidence demonstrated the involvement of gut microbiota in energy conversion and metabolic processes [61]. the gut microbiota composition indirectly influences the host's metabolic activity. this idea has been well supported by several studies involving mice models [62-68]. another possible explanation within this context is that antibiotics caused a reduction in the bacteria population residing in the gastrointestinal tract. this may reduce in proportion the growth-suppressing toxins or nutrient-destructing metabolites such as biogenic amines and ammonia secreted by the gastrointestinal bacteria. in this sense, agps are sometimes regarded as growth-permitting instead of growth-promoting agents [57]. scaling down the population of competing microorganisms also spares the nutrients and increases the energy sources available for the host cells. besides, suppressing the pathogenic strains within the gut also indirectly lowers the incidence of intestinal infections [69-72]. repressing subclinical infections eradicates the unnecessary energy drainage through the immune function and conserves the energy store to favour growth [73]. from the host-centric perspective, the immunomodulation mechanism represents a convincing hypothesis supporting the growth promotion effect of agps [57]. different antibiotics exert different extents of inhibitory effects on the immune system. for instance, the immunomodulatory effect of florfenicol appears to be less pronounced than for oxalinic acid and oxytetracycline [56]. agp is believed to benefit the animal by limiting the immune activation in response to inflammation which is often obligatorily associated with disease states [74, 75]. this results in the suppression of pro-inflammatory cytokines, thus preventing the initiation of acute-phase response, which is essentially an energy-demanding catabolic process. the acute phase response should be avoided at all costs as its activation is accompanied by metabolic alteration that leads to reduced feeding and nutrient assimilation, which severely compromises animal growth [76, 77]. some highly penetrative antibiotics can accumulate in the phagocytic cells reaching up to 10or even 100-fold the ambient concentration [57]. this discrepancy in the immunomodulatory effects between different classes of antibiotics may stem from the differences in the diffusing potential of each antibiotic into the phagocytic cells [74]. to illustrate, clindamycin, macrolides, and quinolones can efficiently diffuse into the phagocytes; whereas aminoglycosides and beta-lactams have limited penetrating potential [78, 79]. niewold [57] propounded an excellent reference to the intra-phagocytic accumulating potential and the phagocytic inhibitory effect for several antibiotics. accumulated antibiotics drive the intracellular killing of pathogens and partly attenuate the innate immune response. notably, the application of antimicrobial agents has been found to impair several downstream immunological cascades such as chemotaxis, phagocytosis, respiratory burst, and cytokine production [58, 80]. for example, rifamycin was reported to dampen the stress-induced inflammation of the intestinal mucosa in the mice model [81]. in-vitro studies using immortalized keratinocytes (hacat) also demonstrated that low doses of doxycycline at 0.3 µg/ml resulted in the significant 68.7% suppression of interleukin (il-8) release when induced by lipopolysaccharide (lps). a similar trend is noted for other pro-inflammatory pmmb 2023, 6, 1; a0000324 7 of 86 cytokines such as tumour necrosis factor-alpha (tnf-α) and il-6 in cells treated with lowdose doxycycline [82]. controlling cytokine production is likely to have a pronounced effect on growth due to its impact on metabolic homeostasis. compelling evidence shows that proinflammatory cytokines may systematically alter lipid and amino acid intake and metabolism rates [77]. to sum up, downregulating the immune response has a far-reaching effect on growth. attenuating the immunological cascade would limit the catabolic expenditure in maintaining an immune response. thereby, more resources can be channelled for anabolic activities directed toward growth [58, 83]. moreover, dampening the immune system also reduce the accumulation of immune cells in the mucosa. since the intestinal mucosal is a dynamic layer modulating nutrient absorption, metabolic and immunological functions [84], the thinning of the intestinal wall helps facilitate the absorption of nutrients [85, 86]. 3. probiotics probiotics are live microbes introduced deliberately to improve the health of the targeted host [87, 88]. since their inception to farmed animals as feed supplements in the 1970s, the growth promotion and disease resistance effects have encouraged their continuous implementation in farming [89]. this trend is gradually expanded to the aquaculture industry. besides the disease control and growth enhancement effect, when administered in adequate amounts, specific probiotic strains can modulate the host’s gut microbial composition, improve water quality, elevate the immune function and increase the animals' survival rate [90-97]. these positive reports fueled new interest in research. increasing evidence suggests that probiotic represents a safer and more sustainable alternative to antimicrobial agents in farming [29, 36, 37, 98]. more than 20 genera of microorganisms have been studied for their growth promotion effect in shrimp models. among the probiotics, bacillus sp. was the most studied and widely applied genus in shrimp farming. table 1 presents a list of microorganisms showing promising growth promotion effects in shrimps. the growth promotion effect was evaluated using parameters such as sgr, average daily growth (adg), weight gain rate (wgr), fcr, and feed efficiency (fe) [99, 100]. the majority of the strains demonstrated multifaceted functions. they represent the up-and-coming candidates to replace agps. further research, particularly deciphering the mechanism underlying the growth promotion effect of probiotics, is warranted before large-scale commercial implementation. pmmb 2023, 6, 1; a0000324 8 of 86 table 1: microorganisms demonstrating significant growth promotion effect when introduced as probiotics in shrimps. probiotics with growth promotion effect method of administration dosage frequency and duration of trial shrimp species treated ref. genus species bacteria aeromonas bivalvium feed additive 107 cells/g diet daily for 28 d l. vannamei [101] alteromonas sp. water additive 106 cfu/ml daily for 18 d p. monodon [102] arthrobacter sp. water additive 105-107 cfu/ml every 5 d for 24 d l. vannamei [103] bacillus amyloliquefaciens water additive (mixture) 109 cfu/ml once weekly l. vannamei [104] coagulans water additive 107 cfu/ml daily for 35 d l. vannamei [105] feed additive 107-109 cfu/g diet daily for 56-90 d l. vannamei m. rosenbergii [95] [106] [107] cereus water additive 106 cfu/ml every 14 d for 110 d l. vannamei [108] feed additive 104 cfu/g diet daily for 28 d m. rosenbergii [109] 0.1-0.4 %/100 g diet daily for 90 d p. monodon [92] licheniformis water additive 104-109 cfu/ml once weekly/ daily for 8 d l. vannamei [110] [104] feed additive 106-109 cfu/g diet daily for 60-90 d l. vannamei m. rosenbergii p. monodon [111] [112] [113] megaterium water additive (mixture) 2 ml/10 l of water once weekly for 9 mo l. vannamei [114] pmmb 2023, 6, 1; a0000324 9 of 86 water additive 109 cells/ml, 10 ml to 90 l of water every five days for 60 d p. monodon [115] feed additive 109 cfu/g diet 104-108 cfu/g diet every five days for 60 d daily for 28-90 d p. monodon l. vannamei [116] [115] [111] polymyxa feed additive (mixture) 108 cfu/g diet daily for 90 d l. vannamei [111] pumilus water additive 106 cfu/ml every three days for 18 d p. monodon [117] subtilis water additive 109 cfu/ml 109 cfu/l once weekly for 120 d every three days for 14 d l. vannamei [104] [118] 2 ml/10 l of water once weekly for 9 mo [114] feed additive 104-1012 cfu/kg diet daily for 28-98 d l. vannamei [119] [120] [116] [121] [122] [123] [124] [125] [33] 3%, 107 cfu/g probiotics m. rosenbergii [126] [127] [90] 5 g/kg feed p. monodon [113] thuringiensis feed additive (mixture) 108 cfu/g diet once-daily for 90 d l. vannamei [111] pmmb 2023, 6, 1; a0000324 10 of 86 bifidobacterium bifidum enrich live feed (rotifer) (mixture) 0.43 mg/ml (6h) 109 cells/g probiotics daily for from mysis i to postlarvae 5 l. vannamei [128] water additive (mixture) 109 cells/l of water [128] clostridium butyricum feed additive 108-14 cfu/g diet daily for 42-60 d l. vannamei m. rosenbergii marsupenaeu s japonicus [129] [130] [131] [132] [133] enterobacter hominis feed additive 107 cfu/g diet daily for 28 d l. vannamei [134] enterococcus faecium enrich live feed (rotifer) (mixture) 0.43 mg/ml (6h) 109 cells/g probiotics daily for from mysis i to postlarvae 5 l. vannamei [128] water additive (mixture) 1g/l of water 109 cells/g probiotics l. vannamei [128] 107 cfu/ml added = approximately 200 µl/100 postlarvae twice daily p. monodon [135] feed additive 107 cfu/g feed daily for 28 d l. vannamei [136] 107 cfu/ml added = approximately 200 µl/100postlarve p. monodon [135] halomonas aquamarine water additive 106 cfu/ml, 0.1% v/v for 12 d l. vannamei [137] pmmb 2023, 6, 1; a0000324 11 of 86 sp. enrich live feed (artemia) 300 mg/l (24h) 8 naupii/ml/day for 15 days [138] water additive 107 cfu/ml [139] feed additive 107 cfu/g feed daily for 42 d fenneropenae us chinensis [140] lactobacillus acidophilus enrich live feed (rotifer) (mixture) 0.43 mg/ml (6h) 109 cells/g probiotics daily for from mysis i to postlarvae 5 l. vannamei [128] water additive 107 cfu/ml 1 g/l of water 109 cells/g probiotics daily for 35 d [105] [128] feed additive (mixture) 5 g/kg feed daily for 60 d p. monodon [113] coagulans feed additive 108 cfu/g feed daily for 56 d l. vannamei [95] fermentum water additive (mixture) 2 ml/10 l of water once weekly for 9 mo [114] delbrueckii enrich live feed (rotifer) (mixture) 0.43 mg/ml (6h) 109 cells/g probiotics daily for from mysis i to postlarvae 5 l. vannamei [128] water additive (mixture) 1 g/l of water 109 cells/g probiotics [128] pentosus feed additive 107-109 cfu/g feed daily for 28-56 d l. vannamei [136] [125] plantarum enrich live feed (rotifer) (mixture) 0.43 mg/ml (6h) 109 cells/g probiotics daily for from mysis i to postlarvae 5 l. vannamei [128] water additive (mixture)3 1 g/l of water 109 cells/g probiotics l. vannamei [128] pmmb 2023, 6, 1; a0000324 12 of 86 2 ml/10 l of water once weekly for 9 mo l. vannamei [114] 109 cfu/l daily for 90 d m. rosenbergii [141] feed additive 107 -1012 cfu/g feed daily for 21-90 d l. vannamei m. rosenbergii [142] [143] [144] rhamnosus enrich live feed (rotifer) (mixture) 0.43 mg/ml (6h) 109 cells/g probiotics daily for from mysis i to postlarvae 5 l. vannamei [128] water additive (mixture) 1 g/l of water 109 cells/g probiotics daily for 45 d l. vannamei [128] sporogenes enrich live feed (artemia) 107 cfu/l (12h) m. rosenbergii [145] feed additive (mixture) 3-4% in the diet, 107 cfu/g probiotics daily for 60 -90 d m. rosenbergii [126] [90] 5 g/kg feed daily for 60 d p. monodon [113] lactococcus lactis feed additive 108 cfu/g diet daily for 56 d l. vannamei [146] [33] nitrobacter sp. water additive (mixture) 2 ml/10 l of water 106 cells/ml 3 mg/l every 16 d for 12 weeks, then 5 mg/l till the end of the culture once weekly for 9 mo l. vannamei [114] [147] nitrosomonas sp. water additive (mixture) 2 ml/10 l of water l. vannamei [114] pmmb 2023, 6, 1; a0000324 13 of 86 2ml/10 l of water once weekly for nine months 106 cells/ml 3 mg/l every 16 d for 12 weeks, then 5 mg/l till the end of the culture [147] pediococcus acidilactici water additive 106 cfu/ml every 14 d for 110 d l. vannamei [108] feed additive (mixture) 108 cfu/g diet daily for 60 d m. rosenbergii [148] pentosaceus feed additive 108 cfu/g diet daily for 65 d l. vannamei [149] pseudomonas aestumarina feed additive 105 cfu/g diet once-daily for 28 d l. vannamei [119] sp. water additive (mixture) 109 cfu/ml daily for 15 d l. vannamei [104] psychrobacter sp. water additive 105 cfu/ml once weekly l. vannamei [150] rhodopseudomo nas palustris water additive 107 cfu/ml daily for 35 d l. vannamei [105] roseobacter gallaeciensis feed additive 105 cfu/g diet once-daily for 28 d l. vannamei [119] shewanella algae water additive 105 cfu/ml, 0.1% v/v for 12 d l. vannamei [137] haliotis feed additive 107 cells/g diet daily for 28 d l. vannamei [101] sp. feed additive 107 cells/g diet daily for 56 d l. vannamei [96] streptococcus phocae feed additive 107 cfu/ml of probiotics 200 mg/100 post larvae p. monodon [135] water additive 107 cfu/ml added =approximately 200 µl/100 post larvae twice daily [135] pmmb 2023, 6, 1; a0000324 14 of 86 salivarius enrich live feed (rotifer) (mixture) 0.43 mg/ml (6h) 109 cells/g probiotics daily for from mysis i to postlarvae 5 l. vannamei [128] water additive (mixture) 1g/l of water 109 cells/g probiotics l. vannamei [128] streptomyces fradiae water additive 109 cells/ml, 10 ml to 90 l of water every 5 d for 60 d p. monodon [115] feed additive 109 cells/g feed every five days for 60 d p. monodon [115] vibrio alginolyticus feed additive 105 cfu/g diet once-daily for 28 d l. vannamei [119] yeast debaryomyces hansenii feed additive (mixture) 108 cfu/g diet once-daily for 90 d l. vannamei [111] rhodotorura sp. feed additive (mixture) 108 cfu/g diet once-daily for 90 d l. vannamei [111] saccharomyces cerevisiae feed additive 4% in the diet, 107 cfu/g probiotics daily for 60 days 108 cfu/g diet 3-4% diet, 107 cfu/g 10-40 g/kg diet daily for 60-90 d m. rosenbergii [148] [126] [127] [151] [90] 5 g/kg feed daily for 60 d p. monodon [113] *mixture means the probiotics contain more than one microorganism. pmmb 2023, 6, 1; a0000324 15 of 86 4. mechanism of probiotics in promoting animal growth the growth promotion effect of probiotics is postulated to be driven by several factors. the mechanisms may vary from strain to strain, and the exhibited effect may vary when introduced to different animals [152]. based on the empirical observational approaches, several events demonstrated strong correlations to growth. this includes the alteration to the gut microbiota composition, elevation of enzymatic activities, modification of the hepatopancreatic and intestinal morphology, enhancement of immune function, and modification of genes expression [90, 134, 153-155]. in startle contrast to agps, probiotic administration was found to strengthen the immune function of the animals. besides, probiotics also help to increase animals’ resistance to environmental stressors, including ammonia and oxidative stress, by ameliorating the water quality. this implies that more underlying mechanisms that may contribute to animal growth are yet to be elucidated. this review synthesizes a summary of the underlying mechanisms of probiotics in promoting animal growth based on the available studies involving shrimp models. the growth promotion mechanisms of probiotics are then compared and contrasted with agps to generate new insights to propel the pursuance of better growth-promoting agents for shrimp farming (table 2). 5. modulating the gut microbiome gut microbiota composition is recognised as one of the key determinants for the normal function and maintenance of the digestive tract structure of the host [156, 157]. due to their pivotal roles and the high collective metabolic activity in the gut, the indigenous microbiota is also regarded as another virtual organ within the gut [158, 159]. these microbes co-evolve with the host along the long evolutionary process. a mutualistic relationship exists between these bacteria and the host [160-162]. to illustrate, the gut microbiota plays vital roles adjunctive to the gut, particularly in homeostasis maintenance, immune regulation, energy distribution, nutrient absorption, and storage [163-166]. in this regard, the involvement of the gut microbiota is likely indispensable to the growth promotion effect of probiotics. probiotics increase the proportion of beneficial microbes in the gut and help maintain a healthy and functioning gut microbiota. pmmb 2023, 6, 1; a0000324 16 of 86 table 2: compare and contrast the mechanisms of agps and probiotics in promoting animal growth. mechanisms affecting the growth of animals antimicrobial growth promoters probiotics gut microbiota • reduce the gut microbiota diversity • reduce the abundance of gut bacteria • increase the proportion of bacteria that promotes growth • increase the gut microbiota diversity • increase the ratio of beneficial microbes to pathogenic microbes without significant changes to the total abundance of gut bacteria • establish a healthy and functioning gut microbiota enzymatic activity • no significant alteration • enhance the enzymatic activities • enhance nutrient digestibility gastrointestinal tract morphology • reduce the muscularis wall thickness • increases the number of b cells in the hepatopancreas • lower the degree of atrophy and necrosis in the hepatopancreas and midgut (during infection) • increase epithelial integrity • increase the size of epithelial cell • increase the villus number and height increase the surface area of the inner surface of the intestine immune system • attenuate the immune system • limit the immune activation in response to inflammation • impair immunological events such as chemotaxis, phagocytosis, respiratory burst and cytokine production • prime the immune system • strengthen the protective mechanism • elevate the inhibitory capacity against pathogens pmmb 2023, 6, 1; a0000324 17 of 86 5.1. increasing the proportion of beneficial bacteria it is well established that dietary factors could modulate an organism's gastrointestinal microbial community composition [8, 96, 164, 167-174]. alteration to the gut microbiota composition of aquatic animals following probiotic supplementation has been well demonstrated [38, 61, 175-177]. contrary to the action of antibiotics in reducing the bacteria population and diversity in the gut [10, 178, 179], probiotics often only alter the composition of the gut microbiota without significantly affecting the total abundance of the gut microflora [153]. the gut microbiome of probiotic-treated groups generally demonstrates higher species richness and biodiversity [61, 94]. this is reflected in the higher number of operational taxonomy units (otus) as well as the higher abundance-based coverage estimators (ace), shannon, chao-1, and mcintosh indexes in the probiotic treatment groups [94, 134, 180]. the diversity of the microbial community could also be analysed based on the variety of carbon sources available in the gastrointestinal tract of shrimps using the biolog-eco technique. this parameter can also be employed to index the aerobic metabolism rate [134, 180]. using this method, zuo et al. [134] demonstrated the elevated intestinal microbiome activity through the significant increase in average colour change rate per hole (awcd) in cohorts supplemented with probiotics lactobacillus and enterobacter hormaechei. polymerase chain reaction denaturing gradient gel electrophoresis (pcr-dgge) analysis also revealed the incorporation of bacillus spp. probiotics into the feed enriched the individual variation and total diversity of intestinal bacteria in kuruma shrimps (marsupenaeus japonicus) [181]. this result is consistent with the single-strand conformation polymorphism (sscp) fingerprint analysis which shows higher intestinal bacteria diversity in l. vannamei following the administration of bacillus spp. probiotics [61]. interestingly, some probiotic strains seem to exert a selective action towards different microbial species. probiotics introduced were observed to increase the abundance of beneficial bacteria in the gut and suppress the growth of pathogenic strains. notably, adding a probiotic mixture of bacillus subtilis and saccharomyces cerevisiae together with prebiotics (mannan oligosaccharides and ß-glucan) into the shrimp diet significantly increased the lactococcus count in the gastrointestinal tract by 11% and depressed the pathogenic vibrio population by 32% when compared with the untreated group [182]. along this line, a shift in the microbiota composition of l. vannamei was evident through the increment in beneficial bacteria pseudoalteromonas sp. proportion and depression of vibrio sp. in the group fed 2% dietary yeast (s. cerevisiae) culture [183]. this is consistent with the findings of nimrat et al. [153], who noted the addition of bacillus spp. and yeast probiotic mix to l. vannamei increases the ratio of beneficial bacteria species such as bacillus spp. and debaryomyces hansenii without altering the total number of culturable heterotrophic bacteria in the gut. this implies that probiotics can enhance the gut ecosystem without adversely affecting the equilibrium of the natural microflora. similarly, wei et al. [96] also reported that beneficial pseudomonas sp. increased proportionally. in contrast, pathogenic species such as bacteroides and escherichia shigella decreased in abundance following the eight-week probiotic shewanella sp. dietary pmmb 2023, 6, 1; a0000324 18 of 86 supplementation trial. this particular activity is harnessed when the probiotics are introduced to enhance the proliferation of beneficial strains, suppress the growth of pathogenic strains and mitigate the risk of animals succumbing to infectious diseases. this protectivemechanism demonstrated by probiotics contrasts with antibiotics, in which the bactericidal activity may create free ecological spaces for other opportunistic pathogens to thrive post-treatment [56, 178]. in this regard, the colonisation of probiotics is a plus point as these beneficial microbes readily occupy the ecological niches and mitigate the colonisation of opportunistic strains, thus prolonging the desirable effects of the treatment [33] (see section 5.3). 5.2. establishment of a healthy gut microbiota from another perspective, it is postulated that probiotics promote the growth of shrimps by establishing a healthy gut microflora [39]. although positive correlations between several bacteria phyla and the health indices of the host have been identified [184], hitherto, there is no definite microbiota pattern that can conclude the ‘desired microbiota’ which favours animal growth. several significant hurdles stumble research in this aspect. firstly, because only a tiny fraction of bacteria is currently culturable under lab environment, a substantial fraction of the intestinal microbiota remains unknown. although advances in molecular techniques offer descriptive data, without representative culturable strains to support further studies, the internal processes, mechanisms, and interactions between the microbiota and the host remain speculative [185]. secondly, the gut microbiota constitutes a dynamic and highly complex ecosystem involving the interplay of a wide array of bacteria species. these bacteria interact with one another differently, some strains antagonize the growth of another, while others support the growth of others [134, 186]. thirdly, the gut microbiota composition is further compounded by external factors such as individual variations, developmental stages, feeding, stress, and environmental fluctuations [58, 187-190]. regardless, a healthy microbiome can generally be characterised as a healthy and functioning core comprising a stable yet flourishing blend of microbe species actively involved in physiological regulatory pathways and could ably resist any external or internal perturbations [188, 191-194]. recent findings suggest the involvement of commensal microbiota in modulating the host’s metabolism, digestibility, and immune response [184, 195-197]. this further reinforces the idea that the gut microbiota is closely associated with regulating the host’s growth performance [61, 180, 198]. this notion justifies that applying probiotic supplements can modulate gut microbiota composition to enhance the attainment of better health status and boost the growth performance of cultured shrimps [180, 198]. the microbiota modulated by agps could not provide a good reference for the ‘desired microbiota template’; similar to the case of probiotics, antibiotics also resulted in inconsistent effects on the gut microbiota despite the proven growth promotion effect [56, 58]. however, significant changes in gut microbiota composition following the addition of probiotics is discernable when compared to the untreated controls [38, 175-177]. luis-villaseñor et al. [61] show that shrimps treated with two different probiotics exhibited a high percentage similarity (73%) in gut microbiota composition. still, both only show a 24% similarity to the pmmb 2023, 6, 1; a0000324 19 of 86 untreated control group [61]. this indirectly implies that empirical observational studies may offer glimpses of the desired gut microbiota pattern that reinforces growth. studies consistently showed that proteobacteria is the most abundant phylum in shrimps’ intestines, followed by firmicutes, bacteriodetes, and actinobacteria [94, 96, 176, 180, 198-201]. αproteobacteria, γ-proteobacteria, and flavobacteria are the dominant classes identified in shrimps regardless of the treatment type [180]. interestingly, duan et al. [180] discovered that probiotics exert a dose-dependent influence on gut microbial composition. a higher clostridium butyricum dietary supplementation enriched the proteobacteria phylum and firmicutes, whereas a lower probiotic dose increased the dominance of bacteriodetes and firmicutes. in contrast to the untreated counterparts, the probiotic-treated group demonstrated a broader diversity of microbes [186]. xie et al. [94] also showed that introducing different graded probiotics contributed to the selection of unique bacterial compositions. clearly, it is rather difficult to reconcile the complex microbiome analysis currently accumulated in the arsenal. the advancement in metagenomics and bioinformatics will eventually foster the pursuit of a unifying principle from the infinite paradigms of microbiota resulting from different probiotic treatment regimes. at this juncture, the microbiota of healthy shrimps with high growth rates at different growth phases or stocking densities could serve as good references [200, 201]. otherwise, insights could also be drawn from other animal models or clinical trials. an increasing number of studies relate the weight changes to the proportion of two prominent phyla in the gut, namely bacteriodetes and firmicutes. a higher ratio of firmicutes to bacteroidetes is hypothesised to contribute to weight gain through the shift in metabolic potential viz the increment in calories and fats absorption [202-205]. this result has also been consistently demonstrated in mice models [67, 206]. nevertheless, a better understanding of the gut microbiome and its interaction with the host is warranted so that suitable probiotic supplementation could be designed to shape the desired microbiota to optimise animal health and growth [61, 188]. although it is unlikely to draw a simplistic conclusion on the ‘desired microbiota that promote growth’ based on the varying results gathered from the available studies, the approach presented may help pave a preliminary path that could guide future research. 5.3 establishment of a functioning gut microbiota the protective mechanism of the gut is strengthened via the introduction of probiotics. probiotics competitively exclude opportunistic pathogens from adhering to the gut lining and reduce the availability of space, nutrients, and energy that supports the proliferation of pathogenic strains [207, 208]. for example, yeast strains, such as d. hansenii, demonstrated higher dominance traits in the gut and competitively excluded other strains [153, 209, 210]. besides, studies consistently show that introducing bacillus probiotics substantially reduced the vibrio count in the digestive tract of shrimps compared to the probiotic-free group. this corroborated with the growth suppression of potential pathogens such as desulfobulbus sp. pmmb 2023, 6, 1; a0000324 20 of 86 and desulfovibrio sp. when beneficial bacteria such as lactobacillus sp., lachnospiraceae sp., and lachmoclostridium sp. were enriched following supplementation of c. butyricum [180]. the correlation of this protective effect to growth performance and survival rate postinfection has also been established [211, 212]. moreover, some probiotic strains are equipped with the potential to antagonise pathogenic microbes through quorum quenching [213, 214]. probiotics suppress the virulence expression of pathogens through the degradation of signalling molecules such as n-acyl homoserine lactone (ahl) [109, 215-217]. quantitative polymerase chain reaction (qpcr) results showed that administering pseudomonas sp. probiotics effectively lower the toxin-coding gene piravp copies in shrimps [218]. in addition, some bacteria also secrete inhibitory compounds such as siderophores, proteases, lysozymes, hydrogen peroxide, organic acids, antibiotics, and bacteriocins that antagonise the growth of pathogens [31, 37, 113, 212, 219, 220]. also, probiotics promote the re-establishment of normal flora in shrimps, particularly when dysbiosis is prevalent at the subclinical stages [188, 221]. in this sense, probiotics strengthen the barrier effects of gut microbiota against pathogenic microorganisms (see section 8.2) and act as an additional line of defence to preserve the epithelial integrity of the gut [222, 223]. this protective effect potentially mitigates the risk of infection, reducing energy's unnecessary dissipation in eliciting an immune response to combat diseases [61]. in this sense, the energy obtained is reserved for growth and other basal life processes. although a direct comparison between probiotics and antibiotics is lacking, won et al. [33] reported that shrimps fed probiotics demonstrated comparable survival rates to that fed oxytetracycline, which is significantly higher than the cumulative survival rates of the untreated control. similarly, l. vannamei larvae exposed to bacillus probiotic strains and commercial probiotics showed better development rates than the control group. on top of that, mixing both bacillus strains yc3-b and c2-2 at a 1:1 ratio resulted in a significantly better developmental rate when compared to the larvae exposed to antibiotics oxytetracycline [34]. 6. secretion of bioactive compounds probiotic application stimulates the secretion of a wide range of bioactive compounds such as short-chain fatty acids (scfas), vitamins, polyamines, and exopolysaccharides [224, 225]. these bioactive molecules promote the functional maturation of the intestine, enhance protein and nucleic acids biosynthesis, improve nutrient absorption and facilitate cell differentiation [153, 226]. bacillus sp. [227-231], streptomyces sp. [232-234], lactic acid bacteria [207, 235-237], as well as yeast [238], are some common probiotic examples known for their proliferate secretion of bioactive compounds. these compounds serve as growth factors to stimulate animal growth [8, 239-244]. they may act as critical supplementary sources of beneficial dietary compounds and constitute part of the sustenance for the animal. probiotics can be introduced as aquafeed additives to improve feed value [207, 245]. several studies demonstrated the efficiency of probiotic supplementation in lowering the fcr, which correlated with significant weight improvement of shrimps [95, 114, 240, 246, 247]. pmmb 2023, 6, 1; a0000324 21 of 86 6.1. short-chain fatty acids like most organisms, shrimps lack the necessary enzymes to digest resistant starches and complex polysaccharides. they, therefore, depend on the gut microbiome to decompose fibrous nutrient or complex carbohydrate molecules into pyruvate and acetyl-coa via the glycolytic pathway or the phosphoketolase route [199, 248] (figure 2). these ‘intermediate substrates’ are later converted into scfas through other gut processes mediated by different microbes [195, 199, 248-252]. scfas are carboxylic acids with less than six carbons at the aliphatic tails. they are the primary end products of bacterial fermentation of the non-digestible dietary carbohydrates ingested by the host [195, 253, 254]. however, the exact species facilitating each scfa production pathway in shrimp is yet to be identified to allow targeted control of the metabolic profile of shrimp through the introduction of specific probiotic strains. figure 2. formation of scfas and their effects on shrimps. pmmb 2023, 6, 1; a0000324 22 of 86 scfas are important bioactive molecules produced from bacteria fermentation. it is well known that scfas play important roles in several physiological processes, including metabolism and immune defences [255]. scfas can mediate physiological activities through the modulatory effect on the digestive tract or directly affect the metabolism rate [256, 257]. scfas are akin to the link between the gut microflora, diet, and host metabolism [258]. they provide energy sources, help maintain gastrointestinal homeostasis, and act as immune modulators and anti-inflammatory agents [195, 259]. in this sense, scfas constitute a pivotal point in explaining the growth promotion effects of probiotics (see figure 2). as described in section 5.1, probiotic introduction alters the gut microbiota composition, subsequently altering the host's scfa content and metabolic profile. scfas such as acetate, butyrate, propionate, and their salts have common existence in the shrimps’ intestines [199, 260]. kyoto encyclopaedia of genes and genomes (kegg) pathway integrity and genes enrichment analysis mapped on pyruvate metabolism revealed that acetate was the primary type of scfa found in the gastrointestinal tract of l. vannamei [199]. this corroborated the findings of duan et al. [129]. scfas can be perceived as a form of energy recovered by the gut microbiota for host absorption. scfas are easily absorbed and constitute an essential energy source to drive cellular processes such as chemotaxis, cell proliferation, and differentiation [225, 254, 255, 261-263]. the proliferation of the epithelial cells lining the intestinal mucosa and the increment in cell size, villus height, and villus number thus, contribute to a larger surface area for better nutrient absorption [86, 133, 239, 264, 265]. furthermore, scfas can act as signal transduction molecules that modulate mucin expression and the tight junctions of the epithelial cells, thereby improving gut permeability and enhancing nutrient absorption [195, 225, 253, 266]. moreover, the reduction of intestinal ph induced by scfas also significantly improves the enzymatic activities of amylase, pepsin, trypsin, and lipase compared to the untreated control, thus implying better nutrient digestive ability [267]. significant growth augmentation was discernible with increasing scfa concentration [268, 269]. scfas effectively improve the feed efficiency (fe), protein efficiency rate (per), digestibility, nitrogen retention, weight gain, development, and survival rate of shrimps. the positive results seem to be consistently proven across different shrimp species tested, including l. vannamei [268-271], p. monodon [272, 273], and m. rosenbergii [274]. for example, butyrate increased the bioavailability of several nucleotide derivatives and essential amino acids [239]. the provision of scfas as fuel may lower amino acid and glucose oxidation, thereby conserving energy for growth and other physiological processes [239]. besides that, scfa is intimately involved in regulating lipid metabolism and maintaining intestinal health [275, 276]. evidence suggests that scfa can activate the amp-activated protein kinase (ampk) directly or indirectly [277]. ampk serves as an energy gauge and a chief regulator for cellular metabolic homeostasis [278, 279]. upon activation, ampk inactivates the enzymes such as acetyl-coa carboxylase (acc) and 3-hydroxy-3-methylglutaryl-coa (hmg-coa), which catalyse the fatty acid and cholesterol biosynthesis, thus limiting the energy-consuming biosynthesis pathways and promoting the atp-producing catabolic processes [277, 280, 281]. pmmb 2023, 6, 1; a0000324 23 of 86 dissociation of scfas lowers the ph, alters the transport mechanism, and affects the chelating potential of minerals. thus, scfas elevate dietary minerals like calcium, phosphates, and other trace elements available to the shrimp. precipitation is reduced when these acids chelate with mineral ions, thereby increasing the absorption of minerals in the intestine [239, 254, 269]. the calcium and zinc levels increase favours the calcium/calmodulindependent protein kinase/ampk (ca2+/camkkß/ampk) pathway in shrimps and promotes growth [282]. in addition, the proliferation of opportunistic pathogens may also be suppressed by the acidic environment created by scfas [239, 283]. scfas can diffuse through the bacterial cell wall and dissociate to release protons, thereby triggering the efflux mechanisms to expel the excess intracellular protons in the pathogenic bacteria. this culminates in cell exhaustion, thus resulting in lower growth and even the death of pathogenic bacteria [129, 284, 285]. in other words, a low ph microenvironment in the gut antagonises the proliferation of pathogens such as vibrio sp. and stabilizes the gut microbiota to promote animal health and growth performance [184, 195, 239, 268, 269]. besides, the oxidative actions triggered by the binding of scfa to its respective receptor for metabolic activities resulted in a low oxygen environment that restrained the growth of pathogens [243, 286]. the introduction of acetate [269], poly-ßhydroxybutyrate [274], and sodium propionate [268, 269] significantly reduce the vibrio count in the gastrointestinal tract of shrimps. an intriguing relationship exists between scfas and guts microbial composition, where scfas could modify the intestinal microbiota composition and vice versa [268, 269]. last but not least, scfas also improve the immunity of shrimps by regulating the immune genes and augmenting the immune components [195, 239] (see section 9). for example, 60 days of propionic acid supplementation up-regulated the expression of prophenoloxidase (po), crustin, penaeidin-3a (pen-3a) and lysozyme in the hepatopancreas of l. vannamei [287]. likewise, the dietary inclusion of a 2% organic acid blend significantly increased the shrimp’s survival rate post-challenged with vibrio harveyi. treated shrimps demonstrated a lower degree of hepatopancreatic damage when infected with v. harveyi, which corresponds to a higher po activity [270, 273]. although the mode of action of scfas in modulating the shrimps' immunity is yet to be verified by further mechanistic studies, the results available support scfas as effective immune stimulators for aquatic animals [239, 254, 288, 289]. the significantly elevated serum agglutination titre in l. vannamei fed propionate and butyrate further attested to the immunomodulatory effect of scfas [268]. to sum up, scfas are important bioactive molecules for shrimps in mediating energy production, controlling digestive function, modulating gut microbial composition, dictating disease resistance, and regulating the immune response. interestingly, the introduction of sodium propionate significantly suppressed the expression of the myostatin (mstn) gene and elevated the expression of the appetite-related gene, ghrelin (ghrl) (see section 10) and the growth-regulating genes such as growth hormone (gh) and insulin-like growth factor (igf1) [254] which favour shrimps’ growth. irrefutably, scfa is one of the critical substrates affecting shrimps’ survival rate and growth performance. the growth promotion effect of pmmb 2023, 6, 1; a0000324 24 of 86 probiotics can be explained through these bioactive molecules as probiotic inclusion has been proven to positively increase the scfa concentration in the intestine [155, 188, 290, 291]. therefore, scfa-producing bacteria could be harnessed to enhance shrimps' growth and survival rate. 6.2. vitamins another essential type of compound produced by probiotics is vitamins. for example, probiotic bacteria c. butyricum can directly produce vitamin b in the intestinal tract [129, 292]. vitamins are groups of heterogenous compounds crucial for the growth and well-being of an organism, including shrimps. unlike the major nutrient sources such as proteins, lipids, or carbohydrates, vitamins are only required in trace amounts. the vitamin requirement of shrimps is affected by multiple factors, including animal species, culture system, growth rate, physiological condition, nutrient composition, and feeding behaviour [293-295]. although the vitamin requirements of shrimp seemed to vary between studies, likely due to the various factors described above, the significance of vitamins to penaeid shrimps has long been established [295, 296]. vitamin availability is closely associated with aquatic animals’ growth performance [31, 297]. for example, vitamin b is key player in metabolism and antioxidative mechanisms [293, 298-300]. the b complexes, such as vitamin b1 (thiamine), vitamin b5 (pantothenic acid), vitamin b6 (pyridoxine) and vitamin b12 (cobalamin), are also intimately involved in protein, lipid and carbohydrate metabolism [244, 295, 301, 302]. vitamin b1 acts as a co-factor that catalyze the cleavage of α-keto acids such as pyruvic acid in producing energy-storing molecules, adenosine triphosphate (atp) [244, 303]. vitamin b6 participates in several metabolic reactions by acting as a prosthetic group of enzymes in the form of pyridoxal phosphate [301]. elevation of the glutamic pyruvic transferase (gpt) and the glutamic oxaloacetic transferase (got) activities following vitamin b6 supplementation also attested to its role in regulating protein metabolism [302, 304]. several reports corroborated the dose-dependent effects of vitamin b6 on the growth performance of shrimps [301, 302]. a similar trend was reported for vitamin b9 (folic acid), vitamin b12 (cobalamin), vitamin c and vitamin e [305-308]. a significantly higher growth rate of p. monodon was attained in groups supplemented with vitamin b9 compared to the non-supplemented group [306]. vitamin b9 is a precursor for the active tetrahydrofolate coenzymes, which are essential for nucleotide and amino acid metabolism reactions [306, 309]. vitamin c is a powerful antioxidant, potent immunomodulator, and haematological booster for shrimps [305, 310-314]. vitamin e is an effective antioxidant, offering protection against the ascorbic acid-driven lipid peroxidation in cellular membranes, muscles and hepatopancreas [308]. clearly, vitamins are micronutrients essential for the shrimp’s proper growth and survival. in addition to that, dietary vitamin supplementations can effectively suppress infectious diseases in the treated cohort. although the information on shrimps is meagre, the results cross-referenced from other species demonstrated the phenomena. for instance, dietary inclusion of vitamin c at 1,000 mg/kg of feed to three-day-old hatchings of the mrigal pmmb 2023, 6, 1; a0000324 25 of 86 carp (cirrhinus mrigala) significantly flattened the mortality curve when challenged with 105 aeromonas hydrophila cells per fish at the end of the four-month trial. moreover, vitamin c supplementation was found to quicken the phagocytic infiltration rate, thus resulting in minimal lesion at the injection site and culminating in complete resolution on day nine following the challenge test [315]. similarly, supplementing vitamin c was found to effectively reduce the mortality rate of wuchang bream (megalobrama amblycephala) [316] and striped catfish (pangasianodon hypophthalmus) [317] when confronted with a. hydrophila. besides, cholecalciferol, the inactive form of vitamin d3, has been proven as an ideal feed additive for atlantic salmon (salmo salar), particularly to harness its effect in resisting aeromonas salmonicida infections [318]. vitamin e dietary inclusion in the form of α-tocopheryl acetate increases the immune response and resistance of the parrotfish (oplegnathus fasciatus) against vibrio anguillarum infection [319]. this result corroborates with the findings of chen et al. [320], where adding 300 mg of α-tocopheryl acetate and 6% fish oil enhances the resistance of chinese mitten crab (eriocheir sinensis) to a. hydrophila. most importantly, vitamin e supplementation was found to improve the specific growth rate of the crab substantially. although only required in trace quantity, inadequate vitamin supply can negatively affect animal development and may indirectly impact the production cost [237, 321]. vitamins, including water-soluble and fat-soluble vitamins, are considered essential for the health maintenance of shrimps. previous works studied the vitamins required and the suggested dietary intake for different shrimp species [295, 296, 322-324]. the recommended dosage and deficiency signs for each vitamin required by penaeid shrimps are deciphered in figure 3. vitamin deficiency may lead to reduced appetite, poor feed conversion efficiency, growth reduction, swollen hepatopancreas, decreased activity, body discolouration, improper molting, poor healing, increased susceptibility to stress and infectious diseases, as well as high mortality rates among the shrimps [261, 293, 298, 308, 323]. it is important to note that crustaceans have limited physiological ability to synthesise vitamins [31, 325]. besides being supplemented through dietary intake, vitamins are supplied by the mutualistic biota residing in the gut and the rearing water [224, 306, 326]. however, information on the gut microbiota production of vitamins in shrimps is scanty [244, 306]. pmmb 2023, 6, 1; a0000324 26 of 86 figure 3. the function, recommended dosage and deficiency signs of vitamins required by penaeid shrimps. acknowledging the vitamin synthesizing capacity of the gastrointestinal bacteria as a determining factor for the bioavailability of vitamins [295], probiotics can, thus, be perceived as a supplementary source of vitamins for the optimal growth of the animals. it is proven that introducing probiotic strains such as lactobacillus sp., bifidobacterium sp., and propionibacterium sp. can significantly elevate the production of vitamins [236, 327-329]. these bioactive molecules serve as growth factors for the gut microbial community and render a mutualistic effect to the host when the shrimps also take up the exogenous vitamins released by the microbes. in this sense, it may be reasonable to relate the growth promotion effect of probiotics to the cultivation of a conducive gastrointestinal environment that favours the smooth functioning of all metabolic activities via the production of vitamins and other important bioactive molecules [224]. since vitamins are among the valuable outputs of probiotics [330], the deliberate inclusion of probiotic strains capable of synthesizing the essential vitamins will foster farmed animals' healthy development [295, 331]. furthermore, exploiting probiotic use can help ferment the animal feed in-situ and fortify the feed with essential vitamins. tapping on the vitaminsparing effects of probiotics in culture systems may, in turn, substantially lower the feed expenditure without compromising the survival and growth rate of shrimps [294]. leblanc et al. [244] detailed a concise list of microbial strains comprising the food-grade probiotics and the commensal strains and their range of vitamin production. an abundance of in-vitro studies demonstrated the vitamin biosynthesizing potential of various microbial strains isolated from multiple sources (see table 3). however, most studies evaluated the vitamin content in the fermented microbial media under well-controlled laboratory conditions. pmmb 2023, 6, 1; a0000324 27 of 86 in contrast, the in-vivo production of the specific vitamin by probiotics or gut microbes in shrimps is largely understudied. the high vitamin production in-vitro does not necessarily guarantee similar efficacy for vitamin synthesis under treacherous condition in the animal’s gastrointestinal tract. this is an evident research gap awaiting urgent attention. moreover, as intriguing as the idea might suggest, the practicality of identifying an ideal probiotic strain carrying all the desired genes for vitamin biosynthesis is equally challenging. providentially, the biosynthetic pathways of vitamins have been actively studied. the genes, enzymes, and precursors involved in the production of vitamins have also been progressively unveiled through recent studies [332-334]. this knowledge could be integrated into bioengineering the ideal strain carrying all necessary genes and demonstrating stellar probiotic characteristics to support animal growth. suitable probiotic strains with prominent vitamin synthesising ability can be screened and introduced to farms to improve the growth and well-being of shrimps. table 3: microbial strains identified with the capacity to biosynthesise vitamins. vitamin strains identified with the capacity to biosynthesise vitamin details/ insights references b1 (thiamine) lactobacillus acidophilus cscc2400 in-vitro: vitamin b1 concentration progressively increases by 1.7-fold in the fermented soymilk. strain demonstrated high potential to deglycosylate isoflavone glucoside in the media. [335] bifidobacterium infantis in-vitro: 48h fermentation in soymilk increases the concentration of vitamin b1 by 15%. [336] bifidobacterium longum in-vitro: 48h fermentation in soymilk increases the concentration of vitamin b1 by 12%. [336] b2 (riboflavin) enterococcus faecium c43 lactococcus lactis subsp. lactis c173 lactococcus lactis subsp. lactis c195 in-vitro: the strains produce 230 ng/ml, 223 ng/ml and 175 ng/ml of vitamin b2, respectively strains simultaneously produce vitamin b9 strains demonstrated good probiotic characteristics. [237] lactococcus lactis info: under normal conditions, l. lactis does not accumulate vitamin b2 extracellularly. [337] pmmb 2023, 6, 1; a0000324 28 of 86 the jc017 mutant strain screened using the microfluidic droplet technology can produce vitamin b2 extracellularly. in-vitro: strain produces 0.82 mg/l of vitamin b2 when cultured in a medium containing 0.5% of glucose. lactobacillus brevis atcc367 lactobacillus crispatus st1 lactobacillus delbrueckii subsp. bulgaricus 2038 lactobacillus fermentum ifp 3956 lactobacillus plantarum jdmi lactobacillus plantarum subsp. plantarum st-iii lactobacillus reuteri dsm 20016 lactobacillus reuteri jcm 1112 lactococcus lactis subsp. lactis cv 56 lactococcus lactis subsp. lactis i11403 lactococcus lactis subsp. lactis kf 147 lactococcus lactis subsp. cremoris nz9000 lactococcus lactis subsp. cremoris nz9000 lactococcus lactis subsp. cremoris a176 lactococcus lactis subsp. cremoris mg 1363 leuconostoc citereum km20 leuconostoc mesenteroides mesenteroides atcc 8239 leuconostoc mesenteroides mesenteroides j18 pediococcus pentosaceus atcc 25745 info: -the strains listed were predicted using the prokaryotic operon database to carry all four genes required for the biosynthesis of vitamin b2 (riba, ribb, ribg and ribh). [338] pmmb 2023, 6, 1; a0000324 29 of 86 lactococcus lactis info: (biosynthetic) the overexpression of all four biosynthetic genes: riba, ribb, ribg and ribh, are required to achieve a substantial production of vitamin b2. [339] bifidobacterium infantis in-vitro: 48h fermentation in soymilk increases the concentration of vitamin b2 by 13%. [336] bifidobacterium longum in-vitro: 48h fermentation in soymilk increases the concentration of vitamin b2 by 21%. [336] bacillus subtilis candida famata ashbya gossypii info: supplying glycine enhances the production of vitamin b2 by c. famata and a. gossypii supplying hypoxanthine enhances the production of vitamin b2 by a. gossypii. [340] b9 (folate) lactococcus lactis subsp. lactis c173 lactococcus lactis subsp. lactis c195 in-vitro: -the strains produce 595 ng/ml and 58 ng/ml of vitamin b9, respectively. strains simultaneously produce vitamin b2 both strains demonstrated good probiotic characteristics. [237] lactobacillus plantarum lactobacillus sakei in-vitro: out of the 180 lactobacillus strains isolated from japanese prickles, only one l. plantarum and two l. sakei strains produce a high level of vitamin b9 extracellularly (>100 µg/l) after 24h of fermentation. [235] streptococcus thermophilus bifidobacterium animalis enterococcus faecium in-vitro: s. thermophilus was the most dominant producer of vitamin b9; it increases the folate level by 3.54.3-fold. the combination of s. thermophilus and bifidobacterium animalis increases the vitamin b9 level by 6-fold. [341] pmmb 2023, 6, 1; a0000324 30 of 86 streptococcus thermophilus lactococcus lactis leuconostoc lactis leuconostoc paramesenteroide in-vitro: s. thermophilus fermentation is sensitive to the ph level of the fermentation medium. s. thermophilus produced the highest folate/biomass (214 µg/l/od600). l. lactis produces the highest level of vitamin b9 in anaerobic conditions (291 µg/l). [342] streptococcus thermophilus bifidobacterium longum lactobacillus acidophilus lactobacillus delbrueckii ssp. bulgaricus in-vitro: lactic acid bacteria produce higher vitamin b9 in reconstituted milk compared to complex media. all strains produce the maximum vitamin b9 levels after 6h of fermentation. vitamin b9 productions by s. thermophilus and l. acidophilus are most stable; they decline by approximately 8% in week 2 and about 12% in week 3. [343] b12 (cobalamin) lactobacillus reuteri species with proven probiotic properties in-vitro: supply the required precursor compounds [δaminolevulinic acid (ala) and 5,6dimethylbenzimidazole (dmb)] production to enhance the production of vitamin b12. produces the active forms of vitamin b12: (i) α-(5,6-dimethylbenzimidazolyl)-cobinamide cyanide (ii) cyanocobalamin [327] propionibacterium spp. (eg. p. freudenreichii) info: -two-step production; genus necessitates both the anaerobic (step 1) and aerobic (step 2) conditions to produce vitamin b12 enzymes necessary. genes associated (coding the): cbi (step 1), cob (step2). [328] propionibacterium freudenreichii rhodopseudomonas protamicus propionibacterium shermanii info: when glucose is the main component of the [344] pmmb 2023, 6, 1; a0000324 31 of 86 culture medium, the strains produced 206, 135 and 60 mg/l of vitamin b12, respectively. propionibacterium freudenreichii info: a classical dairy microorganism that can be used for fermentation to produce vitamin b12 for feed application. [345] euglena bioassay method to determine vitamin b content in samples (in-vitro and in-vivo). [346] klebsiella sp. in-vitro: klebsiella sp. utilize methanol as a sole carbon source for the production of vitamin bi2 supplying organic nutrients like peptone, yeast extract, and vitamin enhances the production of vitamin bi2. [347] selenamonas ruminantium peptostreptococcus elsdenii butyrivibrio jibrisolvens in-vitro: e.coli cup-plate assay: among the 21 microbial species isolated from the cow’s rumen, s. ruminantium is the most prolific producer of vitamin b12, followed by p. elsdenii. o. malhamensis assay: assay reflects the actual activity of vitamins in animal. results suggest that the relative proportion of vitamin b12 to the analogues under the pure culture conditions was not as high as that in the rumen. [348] c (ascorbic acid) gluconobacter info: strain developed for the biosynthesis of vitamin c intermediate (2-keto-l-gulonic acid). [334] a escherichia coli mutant (contains genes coding for the four key enzymes involved in the ß-carotene biosynthesis: in-vivo (mice): detection of ß-carotene-producing bacteria and ßcarotene in the faeces demonstrate the persistence of the probiotic strains in the intestine. [349] pmmb 2023, 6, 1; a0000324 32 of 86 geranylgeranyl pyrophosphate, lycopene cyclase, phytoene desaturase and phytoene synthase from erwinia herbicola) e (tocopherol) lactobacillus rhamnosus wq2 in-vitro: strain produced a high level of α-tocopherol (376.6 µg/g). strain is able to increase the antioxidant capacity of the media within a short incubation time. the antioxidant capacity highly correlates to the bacterial proteolytic activity. [335] k (menaquino nes) bacillus subtilis in-vitro: the production of vitamin k parallel increases in proportion with the number of cells in the first 8h. the vitamin k extracellular secretion increases rapidly after 10h (1.7%) and reaches a plateau after 32h (31%). [350] bacteroides ovatus enterobacter agglomerans enterococcus faecalis escherichia coli prevotella buccae staphylococcus capitis staphylococcus epidermidis staphylococcus haemolyticus staphylococcus warneri in-vitro: strains produce vitamin k. [351] bacteroides sp. citrobacter freundii enterococcus faecium serratia marcescens staphylococcus capitis staphylococcus warneri in-vitro: strains produce vitamin k. [351] pmmb 2023, 6, 1; a0000324 33 of 86 6.3. polyamine polyamines are small polycationic molecules composed of multiple amine groups on an aliphatic hydrocarbon backbone [352, 353]. these compounds are essential metabolites ubiquitously found in almost all living organisms. some standard polyamines include spermine, spermidine, and putrescin [352-354]. these polyamines have distinct valences and different molecular structures and assume different functional roles. moreover, the respective concentration of each polyamine varies in other organisms. for instance, spermine is absent in the fungal saccharomycotina subphylum. in contrast, the concentrations of spermine and spermidine are high in eukaryotes, whereas putrescine is the dominant polyamine identified in escherichia coli bacteria [352]. for the case of shrimp, gas chromatography-mass spectrometry (gc-ms) revealed that polyamines such as spermidine, n, n'-bis(3aminopropyl)-l,3-propanediamine (bap) and 3,3'-diaminodipropylamine (dad) were present in the white shrimp (penaeus setiferus). however, the non-detection of common polyamines such as putrescine and 1,3-diaminopropane can be attributed to the rapid carbon flux through these intermediates, or possibly the bap and dad are selectively derived from dietary sources [355]. unfortunately, information related to polyamine in shrimps is relatively limited, although the presence and significance of polyamines in shrimps have been established. polyamines are vital components to ensure normal cellular processes such as stress response, genetic expression, cell division, differentiation, growth, and survival [352, 354, 356, 357]. in this regard, polyamine levels within cells are tightly regulated through a complex mechanism which includes the mediation of catabolism and the de novo biosynthetic pathways of polyamines to meet the cellular demand [352, 358]. sugiyama et al. [359] comprehensively analyzed polyamines' biosynthesis and transport mechanism in dominant bacteria identified in the human gastrointestinal tract. natural polyamines are formed through a series of precisely regulated energy-dependent reactions driven by several key enzymes such as ornithine decarboxylase, s-adenosylmethionine decarboxylase, spermidine synthase and spermine synthase [352, 354, 357, 360, 361]. in some bacteria and plant species, an alternative pathway, namely the arginine decarboxylase pathway that involves another two enzymes, polyamine oxidase, and spermidine-spermine acetyltransferase, directs the conversion of spermine to putrescine [352, 354]. besides that, regulating the transportation of the extracellular polyamine across the plasma membrane through passive diffusion or distinct polyamine transporters is essential to ensure proper cellular function because polyamines are also supplied through extracellular sources such as a gut microbial pool or via dietary inclusion [358, 362]. therefore, this implies that probiotic strains could be another novel source to supply polyamine in-situ to support growth and cellular function. yeast is an important dietary source for polyamine supply [360, 363]. polyamine production varies between different yeast species. d. hansenii is one of the strains known for its distinct potential in secreting polyamines. the concentration of spermidine, spermine and putrescine measured in d. hansenii are significantly higher than that in s. cerevisiae and saccharomyces boulardii [226, 364]. reyes-becerril et al. [365] studied pmmb 2023, 6, 1; a0000324 34 of 86 the polyamine-producing capacity of 13 different d. hansenii strains isolated from diverse sources. high-pressure liquid chromatography (hplc) analysis revealed two strains, d. hansenii cbs004 and d. hansenii l2, isolated from marine water and citrus fruit, respectively, as the most prolific producers of polyamines among the 13 strains studied. furthermore, tovar et al. [226] noted the strong adhesion potential of d. hansenii to the intestinal mucus, thus demonstrating the probiotic efficiency of the strain. the inclusion of 1.1% of d. hansenii in the diet, which corresponds to an approximately 106 cfu/g of diet, was shown to improve the survival rate of sea bass (dicentrarchus labrax) larvae by 10% as well as lower the malformation rate by 14%. most importantly, the final average weight of the treated cohort is two times higher than the untreated group [364]. this finding corroborates the effect of d. hansenii on gilthead seabream (sparus aurata) [366] and longfin yellowtail (seriola rivoliana) [367]. these effects of d. hansenii are likely attributed to the polyamine metabolites produced by the live yeast in the intestinal tract [364]. this postulation could likely be proven by comparing the growth parameters in animals fed with probiotic supplements (live yeast strains with polyamines production capacity) and those provided with inactive yeast supplements [368]. although the growth-related effects of polyamine are yet to be fully deciphered, its primal role in regulating the broad range of cellular processes, ranging from cell growth to differentiation, mrna transcription to protein translation, has been established [352, 354, 356]. increasing evidence points to the involvement of polyamines in ca2+ signalling, which is vital for regulating the stimuli of growth factors and hormones on the cell surface receptor. besides, polyamine was also known to play a role in the formation of the cytoskeleton by facilitating the conversion of tubulin to microtubules and actin polymerization. spermine and spermidine are known to induce cytokinesis [361]. some studies also identified the involvement of polyamine in the nervous system, particularly in the mediation of synaptic function [354]. the rat model demonstrated that polyamine putrescine is rapidly absorbed and converted into succinate, which serves as a form of instant energy supplied to the intestinal cells [369]. at the molecular level, polyamines help to stabilise the cell membrane, proteins, and nucleic acid structures, including dna and rna [352-354, 370, 371]. under physiological ph, polyamines are positively charged. as polycations, polyamines bind electrostatically with the negatively charged macromolecules, thus stabilizing the structural conformation and protecting them from enzymatic degradation or thermal denaturation [358, 361]. besides, polyamines also participate as substrates and stimulate dna, rna, and protein synthesis, to facilitate cell division [226, 358]. the polyamine levels strongly correlate with the proliferative activity of cells, where the uptake and utilization of exogenous polyamine are significantly higher in rapidly proliferating cells compared to the quiescent cells. it was also noted that maintaining the polyamine concentration is indispensable for cells to proliferate [354]. on the contrary, genetic studies revealed that the deletion of genes involved in the polyamine metabolic negatively affects cell proliferation and survival rate [352]. sustained depletion of polyamines limits the formation of hypusine (an amino acid component of the pmmb 2023, 6, 1; a0000324 35 of 86 initiation factor eif-5a, whose precursor is spermidine), thus blocking the subsequent mrna translation pathways such as initiation and elongation of the new peptide chain on the ribosome [361]. depleting polyamine through mutating its biosynthetic pathways resulted in retarded growth of the organisms [372]. besides, polyamine deficiency was reported to culminate in severe hypoplasia in the intestinal mucosa [362]. under extreme cases, dysregulation of polyamine levels can negatively impact energy homeostasis and disrupt the regulation of lipids and glucose [358]. the implication of polyamines is not solely limited to the growth and survival of cells but is also expressed in catalysing the maturation of the gastrointestinal tract. to illustrate the mrna expression of enzymes like alkaline phosphatase, aminopeptidase, lipase, maltase, and trypsin showed that the pancreas and intestine of sea bass fed with yeast probiotics developed at a significantly higher rate as compared to the group devoid of this supplement [364]. similarly, the dietary inclusion of purified spermine significantly enhances the intestinal maturation of sea bass [373]. this phenomenon is further evidenced by direct polyamine supplementation to neonatal mice that appreciably improved intestinal health and increased the proportion of beneficial microbes in the gut [374]. additionally, polyamines have been regarded as essential players in regulating paracellular permeability. polyamines enhance the epithelial integrity through their stimulative effectiveness in producing intercellular junction proteins like e-cadherin, occludens-1, occluding, and zonula [358, 375]. therefore, it can be inferred that polyamines play vital roles in promoting the development of the intestinal mucosa and stimulating the maturation of the larvae's digestive organs, which enhances the animal's growth and survival [360]. last but not least, polyamines appear to exert an influence on the immune system [360, 368]. for example, the polyamine secreted by the d. hansenii l2 strain is postulated to be the cause of elevating the immune function of gilthead seabream (s. aurata) [366]. evidence demonstrates that extracellular polyamine concentrations are significantly elevated during inflammation due to the secretion from the damaged cells or excretion during tissue regeneration [354]. these extracellular polyamines suppressed the production of inflammatory cytokines and elevated the release of cytokines that promotes healing [376, 377]. moreover, polyamines exert an antioxidant effect by acting as scavengers for reactive oxygen species [378, 379]. in this regard, polyamine levels can be used as surrogate markers to determine the growth condition of the organism. in one of the few works studying polyamines in crustaceans, stuck et al. [380] concluded that expressing the polyamine level as a ratio to dna constitutes an effective method to more accurately reflect the nutritional status of l. vannamei postlarval. this is because dna is never catabolised even under prolonged starvation and can therefore compensate for the effect of differential catabolism during starvation. in another experiment, watts et al. [381] noted that the polyamine level at the head of l. vannamei is significantly higher than the tail and more appropriately reflects the development and growth status of the animal. results showed that the polyamine to dna ratio is highly dependent on feeding. the parameter is significantly lowered in the starved group compared to the fed group and is immediately upregulated upon resuming feeding [380]. the extent of pmmb 2023, 6, 1; a0000324 36 of 86 probiotics’ contribution to polyamine levels in shrimp is yet to be further assessed. these studies can be applied to optimized future studies to verify the effect of polyamines conferred by probiotics in shrimps. besides d. hansenii, the introduction of probiotics bifidobacterium spp. also elevated the polyamine content in the intestinal lumen [382]. intriguingly, bifidobacterium spp. lack the homologs of enzymes for polyamine biosynthesis. it is postulated that bifidobacterium spp. acidifies the intestinal microenvironment by producing lactate or acetate and thus stimulates the autochthonous microbiota to synthesise polyamines. [383]. the involvement of the intestinal microbial in polyamine production is further strengthened when the gastrointestinal organ maturation effect is only evident and when polyamines are administered orally and not via other routes [384]. furthermore, this notion is further vindicated that this putrescineenhancing effect is completely eliminated when the animal is co-treated with arginine and antibiotics [385]. recent findings further deciphered the mechanism of this novel pathway of polyamine production. polyamine putrescine is produced from arginine by transforming the reactive intermediate, agmatine, in an acidic environment. kitada et al. [383] tested 91 different combinations of strains and concluded that e. coli, enterococcus faecalis, and bifidobacterium spp. formed an excellent combination to induce putrescine production. the three strains act as the acid tolerant-arginine supplier, energy cum agmatine deiminase system provider and acidic environment creator, respectively. this may imply that the direct polyamine biosynthesis ability of strains is not a mandatory prerequisite for probiotic selection. right combinations of strains that facilitate the polyamine production in-vivo can too generate polyamines. to illustrate, clostridium sp., enterococcus sp., lactobacillus sp., lactococcus sp. and streptococcus sp. are a few prevalent examples of commensal genera identified for their arginine deiminase pathway to degrade arginine anaerobically but lack the essential polyamines biosynthetic enzymes [358, 386]. combining these strains with those that supply the required enzymes complement the activities between strains and can orchestrate the production of polyamines in-vivo. in other words, probiotics could be an effective means to optimize the polyamine composition in the gastrointestinal tract by modulating the biochemical reactions between the gut microbial community. 7. increasing the digestive enzyme activity enhancing the digestive enzyme activities represents another convincing pro-growth rationale for probiotics. a recent quantitative analysis by fernandes et al. [104] demonstrated the significance of probiotics in elevating the total enzymatic activity in the gastrointestinal tract of l. vannamei. at the end of the 120-day culture, lipase activity significantly increased by 58%, protease activity increased by 49%, and amylase activity increased by 34% in the shrimps treated with the salt pan bacteria compared to the untreated shrimps. pearson's correlation analysis further revealed a strong positive correlation between the enzymatic activities and the final yield of shrimps. this result is congruent with the findings of ziaeinejad et al. [387], in which the administration of bacillus spp. probiotics significantly increased the total protease, lipase, and amylase activity and recorded 8 to 22% higher wet pmmb 2023, 6, 1; a0000324 37 of 86 weight compared to the control group. similarly, gamboa‐delgado et al. [388] reported that the weight gain of l. vannamei increases with lipase and chymotrypsin activities. intriguingly, the range of enzymatic activity greatly varies between probiotics, even among strains from the same genus. for instance, among the three promising actinomycete strains investigated, the ability to degrade many molecules, such as lipids, proteins, and carbohydrates, varies between strains [389]. in-vivo investigation revealed that the salt pan bacteria probiotics increased the cellulase activity in l. vannamei, but the 10% increment compared to the control group is not statistically significant. in contrast, commercial probiotics increased cellulase activity significantly by 30% [104]. the outcome may vary depending on the probiotic strain selected. in this regard, in-vitro screening for the enzymatic activity of probiotic strains will provide a helpful prediction of the in-vivo effect of probiotics in enhancing digestive function [104, 222, 390-392]. additionally, it is important to note that the dose and duration of probiotic supplementation may also affect the results [393]. notably, although the significance of each enzymatic elevation differs at different stages of growth, zhou et al. [394] reported that adding bacillus coagulans increases the lipase, protease, and amylase activity in l. vannamei. it could be inferred that probiotic strains exhibiting different enzymatic properties can be tailored in the treatment regime to complement the digestive function of the animal at specific life phases. bacillus sp. [212, 240, 387, 394-396], enterobacter sp. [134], lactobacillus sp. [134], pediococcus sp. [149] are some examples of probiotics that have been reported to increase the enzymatic activity in shrimps. some earlier works have covered a concise list of probiotics and their respective enzymatic augmentation effects in aquatic species. probiotic strains, particularly bacillus sp. and yeast, are renowned for their ability to synthesize a wide array of extracellular enzymes such as amylase, cellulase, chitinase, lipase, phytase, and protease [153, 397-399]. certain unique strains thrive in extreme niches, such as the hypersaline bacteria and streptomyces spp. isolated from mangrove soils are excellent sources of exogenous enzymes that work efficiently under extreme conditions [104, 400-404]. these extracellular enzymes supplemented by probiotics may function optimally at broader ph and salinity range, thereby extending the period of enzymatic digestion to facilitate more efficient hydrolysis of substrates [405, 406]. to date, a lack of study distinguishes whether the activity arising from the exogenous enzymes is directly contributed by probiotics or indirectly through inducing the endogenous enzymes produced by the host [222, 395]. usually, enzymatic activity improvement is assayed using the pooled gastrointestinal samples or whole-body extract of shrimps [33, 104, 182, 211, 387, 393, 395, 407]. ziaei-nejad et al. [387] reported that bacillus sp. probiotic additives significantly elevated the total enzymatic activities in the indian white shrimps(fenneropenaeus indicus) reared in earthen ponds. however, the colonisation rate of the probiotic bacteria detected in the digestive tract is very low. this may suggest that although these exogenous enzymes secreted by probiotics may only constitute a minor contribution to the overall digestive activity in shrimps, the presence of probiotics may likely enhance the microbial activity in the gut and stimulate the secretion of endogenous enzymes [90, 134, 387, 395, 408]. this speculation pmmb 2023, 6, 1; a0000324 38 of 86 is well supported by the recent work of zuo et al. [134], in which the epithelium cells in the midgut of l. vannamei treated with probiotics revealed an actively secreting state under the observation of an electron microscope. this phenomenon is congruous to the increment of digestive enzyme activities in the shrimps throughout probiotic treatment. the digestive enzymes identified in shrimps include proteases (aminopeptidase, carboxypeptidase, chymotrypsin, metalloprotease, trypsin), lipase, esterase, and carbohydrase (amylase, cellulase and chitinase) [222, 238, 407, 409, 410]. despite being an omnivorous animal, shrimp has a limited capacity to digest some forms of complex carbohydrates like starch due to the lack of specific enzymes such as the α-1,6-glucosidase. supplying this enzyme that is not naturally present in the host via the introduction of probiotics may aid in the digestion of the α-1,6-glycosidic bond of amylopectin and thus facilitate the digestion of those inherently indigestible nutrients [406]. arellano-carbajal and olmos-soto [411] isolated a bacillus sp. strain from the marine environment, endowed with the capacity to synthesise high levels of thermostable α-1,4-glucosidase and α-1,6glucosidase in a relatively short time. although in-vivo data is lacking, it is a promising candidate for shrimp probiotics. this is because the enzymes function at an optimal temperature and ph similar to the shrimps' enzymes [411, 412]. similarly, ochoa-solano and olmos-soto [406] also demonstrated the capacity of several bacillus strains in producing α1,4-glucosidase, α-1,6-glucosidase and α-galactosidase through the quantitative assay using substrates such as amylose, amylopectin, melibiose, and raffinose. another promising probiotic strain for shrimp culture is lactobacillus casei which produces the extracellular enzyme inulinase. inulinase works by hydrolysing the ß-2,1-glycosidic linkages of prebiotic inulin in the gastrointestinal tract to release small fructooligosaccharides or fructose, which can become an energy source for the host [413]. the idea is further supported by the findings of shiau and peng [414], in which complex carbohydrates such as starch is proven to be better feed options for p. monodon. they resulted in higher feed efficiency and protein efficiency, which resulted in higher weight gain and better survival rates compared to the group fed simple sugars like glucose. in this regard, introducing carefully selected probiotic strains that are prolific producers of carbohydrase will further improve the nutritional value of the animal feed by augmenting the assimilation of carbohydrates in shrimps. from another perspective, these enzymes derived from probiotics can degrade the feed's antinutritive factors, thereby enhancing shrimp's receptivity to plant-substituted fish meal [208, 406]. this is an important property to be harnessed when corn, soybean, and wheat are becoming increasingly popular aquafeed ingredients due to their lower prices and easier accessibility [415-417]. supplementing these feed substitutes with probiotic strains that actively secrete enzymes such as cellulase, galactosidase, glucosidase, glycosidase, and pectinases will significantly improve the energy intake from the diet substituted with plant-based ingredients [415, 418-420]. moreover, the improved digestibility mediated by higher enzymatic activity also maximizes feed utilisation. for instance, lactobacillus and enterobacter hominis supplementation resulted in higher carbon utilization ability in l. vannamei [134]. increased awcd measurement reflected the relative increment of carbon sources, including pmmb 2023, 6, 1; a0000324 39 of 86 amines, amino acids, carbohydrates, and polymers available to l. vannamei juveniles following the probiotic intervention [155]. an oxygen bomb calorimeter can be employed to measure the energy content of shrimps [421]. several studies consistently demonstrated the substantial improvement of energy utilization parameters (feeding rate, absorption rate, conversion index, excretory and metabolic rate) in shrimps fed the diet incorporated with probiotics [421, 422]. in this regard, probiotics can significantly improve feed efficiency and increase feed expenditure savings in shrimp farming [73, 406, 420]. in addition, the elevated digestive enzyme activities in the intestinal canals of shrimps induced by probiotics led to improved nutrient digestibility. the enzyme catalyses the breakdown of complex food molecules into smaller, more digestible forms that could be readily taken in. take the example of protein; higher protease activity hydrolyses peptide bonds and liberates free amino acids that can readily be assimilated [152]. higher levels of alanine, arginine, isoleucine, and proline were detected in m. rosenbergii fed with the probiotic-supplemented diet compared to the control group fed with a basal diet. both essential (arginine, histidine, isoleucine, leucine, lysine, threonine, and tyrosine) and nonessential amino acids (alanine, glutamine, glycine, proline, and serine) are essential for animal development as each amino acid perform specific functions. these amino acids are building blocks for synthesizing new proteins [90]. notably, a higher crude protein level is detected in m. japonicus fed with c. butyricum probiotic [155]. this finding agrees with seenivasan et al. [421], who also reported that the protein composition, amino acid, lipid, carbohydrate and ash content are significantly higher in m. rosenbergii treated with 2% lactobacillus sporogenes and s. cerevisiae probiotics. in an experiment in which chromic acid (cr2o3) was employed as an indicator for digestibility, b. subtilis probiotic has been found to significantly increase the apparent digestibility coefficient (adc) of amino acids in l. vannamei [124]. besides, several studies demonstrated that probiotic application could effectively increase the protein efficiency ratio (per) and lower the fcr [422, 423]. indirectly, by increasing the enzymatic activity, probiotics inflect shrimps' metabolic function, which translates to a better growth profile. the exogenous enzyme activities of probiotics could therefore serve as valuable indicators for nutrient digestibility, feed utilization, and growth index of shrimps [424, 425]. interestingly, antibiotics seemingly do not demonstrate this boosting effect on enzymatic activity. in a recent publication, probiotic-supplemented diets significantly improved lipase activity in l. vannamei compared to the control group and the group fed with antibiotic oxytetracycline [33]. however, information on shrimps is somewhat limited. an early study using rainbow trout (oncorhynchus mykiss) shows no apparent increase in lipid digestibility following the administration of oxalic acid, oxytetracycline, and chloramphenicol [426]. on the contrary, the treatment of antibiotics (ampicillin trihydrate, streptomycin sulphate, and antifungal nystatin) significantly diminished the cellulase, chitinase, lipase, and protease activity in northern krill (meganyctiphaunes norvegica). the lower enzymatic activity corresponded with the decline in bacteria counts in the hepatopancreas and stomach of animals reflected in the lower acridine orange direct count pmmb 2023, 6, 1; a0000324 40 of 86 (aodc). this finding surmises a commensal relationship between gut bacteria and the digestive enzymatic activities in the host organism [179]. 8. modifying the gastrointestinal morphology when examining the growth promotion effects of probiotics, gut physiology could not be neglected. the digestive tract of decapod crustaceans can be arbitrarily divided into three parts, namely the foregut, midgut, and hindgut [427, 428]. histological studies revealed that significant morphological changes typically occur in the midgut region of shrimps following probiotic administration [162]. probiotics improve the architecture of the hepatopancreas (see section 8.1) and intestine (see section 8.2), thereby facilitating digestion and assimilation of nutrients in the animal. strengthening the gut function protects the animal from opportunistic disease, safeguards animal health, and improves growth performance. 8.1. hepatopancreas hepatopancreas, also known as the midgut gland, is a vital organ that sits in the cephalothorax region of the crustacean, connected right behind the stomach in the gastrointestinal tract. it is relatively large and typically constitutes 2 to 6% of the total body weight of a decapod [428]. the hepatopancreas is a versatile organ that plays essential roles in the digestive system through the regulation of numerous processes such as steroid hormone synthesis, digestive enzymes secretion, carbohydrate and lipid metabolism, nutrient absorption, storage, and distribution [427-432]. besides, the hepatopancreas also acts as the main detoxification organ for shrimps, particularly during the rapid growth phase [432]. these biological processes are closely associated with growth performance. the role of the hepatopancreas in shrimp is akin to the combined functions of both pancreas and liver in mammals [38, 432]. hence, the health of the hepatopancreas is the main index in the growth profile of the animal. the hepatopancreas is primarily composed of five different types of epithelial cells, namely embryonalzellen/ embryonic cells (e cells), fibrenzellen/ fibrillar cells (f cells), blasenzellen/ blister/ vesicular/ extrusion cells (b cells), restzellen/ resorptive/ reabsorption cells (r cells) and midget/ basal cells (m cells) [270, 427, 433-436]. meanwhile, m cells may not be present in some shrimp species [437] (see figure 4). each cell type has unique features and functions (see table 4). the small cuboidal e cells widely distributed at the distal tubules are undifferentiated cells responsible for epithelial renewal through mitotic divisions [427, 437, 438]. e cells serve as precursors for other cell types in the hepatopancreas. the f cells further differentiate into b cells. the cells become more differentiated, moving from the distal to the proximal tubules [432, 435, 439]. f cells have a distinctive fibrillar appearance, a characteristic large oval nucleus at the centre, and well-developed reticulum and ribosomes [182, 438]. silva et al. [437] further discovered that f cells not only secrete digestive enzymes but are also involved in the production of protective mucus. besides, the f cells are involved in detoxification, protein synthesis, nutrient absorption, metabolism, and storage [427, 432, 440]. b cells are the largest epithelial cell type distributed primarily in the middle and proximal pmmb 2023, 6, 1; a0000324 41 of 86 regions of hepatopancreatic tubules. b cells are characterised by a distinctively large vacuole accompanied by several pinocytic apical vacuoles and a round basal nucleus in the cytoplasm. they are the primary producers of endogenous digestive enzymes in shrimps. also, b cells assume the roles of intracellular digestion, nutrient accumulation, and distribution [182, 427, 432, 435, 437]. r cells can be recognised through their prismatic appearance with multiple small vacuoles in the granular cytoplasm. they are ubiquitously distributed throughout the hepatopancreatic tubules but mainly concentrated in the proximal region. r cells are the main storehouses for lipids, glycogen, and trace elements. they are also involved in absorbing and metabolizing nutrients and detoxifying heavy metals [432, 435, 437, 440]. since r cells serve as the primary energy storehouse in the hepatopancreas, it is often used as an indicator to assess the nutrition status of shrimp. a higher expression of r cells may imply higher energy utilization and nutrition value [441, 442]. m cells are triangular basal cells with a rounded nucleus containing several nucleoli [437]. detection of m cells at the embryonic zone suggests that m cells have their origin independent of e cells [440]. they are few but sparsely distributed across the hepatopancreatic tubules, usually located close to r and b cells. unlike the b cells, f cells and r cells, m cells do not have microvilli, and their apex does not extend to the lumen [427, 437]. m cells are believed to function as a storage reserve for organic substances [435]. following a 5-week supplementation with bacillus aqahbs001, a healthy hepatopancreas structure demonstrating a complete set of epithelial cells such as f, b, and r cells was evident in l. vannamei [123]. moreover, the probiotic treatment resulted in a comparatively higher number of b cells in the hepatopancreatic tubules than in the control set [443]. a similar observation was reported in juvenile l. vannamei following a 10-day feeding of probiotics (mixture of s. cerevisiae and lactobacillus acidophilus) at a 1:1 ratio with two different doses of 108 and 109 cfu per kilogram of diet. both probiotic mixtures not only resulted in a significant increase in b cell count in the hepatopancreatic tubules but also substantially reduced the hepatopancreas pathology in shrimps infected with acute hepatopancreatic necrosis disease (ahpnd) [444]. figure 4. longitudinal and lateral cross-section illustration of hepatopancreatic tubules. pmmb 2023, 6, 1; a0000324 42 of 86 table 4: five different types of epithelial cells in the hepatopancreas. hepatopanc reatic epithelial cells other names features distributi on across tubule functions e cell -embryonalzellen embryonic cell cuboidal shape small undifferentiated cells cytoplasm presents intense basophilia mainly in the distal region mitotic division renewal of epithelial tissue precursor cell for other cell types in the hepatopancreas f cell fibrenzellen fibrillar cell fibrillar appearance -prismatic/triangular/cylindrical shape contain a large amount of welldeveloped endoplasmic reticulum and ribosomes large oval nucleus at the centre cytoplasm presents intense basophilia with microvilli apex extends to the lumen mainly in the middle and distal region secrete a digestive enzyme secrete protective mucus absorb nutrient synthesise protein store nutrients differentiate into b cells detoxify organic xenobiotics and heavy metals b cell blasenzellen blister cell vesicular cell extrusion cell largest epithelial cell globular shape large vacuole small pinocytic apical vacuoles round basal nucleus acidophilic cytoplasm with microvilli at the apical region of the cytoplasm apex extends to the lumen mainly in the proximal and middle region also, in the distal region secrete digestive enzymes intracellular digestion nutrient accumulation transport digested materials r cell restzellen resorptive cell reabsorption cell elongated/ prismatic shape contain multiple small vacuoles round nucleus granular cytoplasm with microvilli apex extends to the lumen across tubule absorb nutrient metabolize nutrient synthesise lipoprotein storage of lipids and glycogen sequester trace elements (copper, magnesium, phosphorus, sulphur and zinc) detoxification (i.e. heavy metals) m cell midget basal cell triangular/rounded shape round nucleus containing several nucleoli -cytoplasm presents intense basophilia without microvilli -remain in contact with the basal lamina -restricted at the basal lamina -apex does not extend to the lumen across tubule located close to r and b cells few in number storage of organic reserve pmmb 2023, 6, 1; a0000324 43 of 86 besides, the histopathology studies showed that exposing p. monodon to probiotic bacillus thuringiensis did not negatively impact the internal organs. the hepatopancreatic epithelial cells retain normal nuclei and vacuoles [445]. this may imply that the probiotics are safe and non-toxic to the shrimps. in addition, lesser necrotic tubules and a remarkably lower degree of atrophy were observed in the hepatopancreatic tissues of shrimps treated with probiotic streptomyces spp. (n7 and rl8) when compared to the untreated counterpart. shrimps treated with l. acidophilus presented less intense hemocytic inflammation and multifocal necrosis at the hepatopancreatic tubules after being challenged with vibrio spp. [446]. this also corroborated with wee et al. [109], who noted higher epithelial integrity in the hepatopancreas of m. rosenbergii treated with bacillus cereus. although there was slight sloughing of the hepatopancreatic tubules in the treatment group, most of the epithelial cells remained intact. the shrimps exposed to probiotics demonstrated higher hepatopancreatic integrity post-infection with a. hydrophila than the control set, which demonstrated significant haemocyte infiltrations accompanied by major sloughing and highly necrotic tissues [109]. similar trends have also been noted in shrimps treated with synbiotics (coadministration of probiotics with prebiotics) [182, 447]. in this light, the health of the hepatopancreas constitutes one of the major elements dictating the growth performance of shrimp. selecting the right probiotic strain could positively reinforce the hepatopancreas' functionality and help secure a better growth rate and farm harvest [432]. 8.2. intestine the effect of probiotics is also manifested through the changes to the intestinal morphology of shrimps. most importantly, the cell surface protein's function in mediating probiotics' effects has been established as the shrimps fed the probiotics deprived of surface protein did not demonstrate significant improvement in the intestinal environment [162]. the surface proteins likely execute the modulatory effect on the intestine upon binding to the intestinal epithelial cells and the gastrointestinal mucins [162, 448, 449]. the most evident morphological alteration in the shrimps’ intestine induced by probiotics is reflected in the architecture of the intestinal villi. the introduction of bacillus coagulans [95], b. subtilis [123], mixed probiotics (bacillus spp. + lactobacillus sp.) [94] and s. cerevisiae [183] consecutively demonstrated a consistently increasing trend in the villus height of the enterocytes. probiotic supplementation improved the size of the intestinal epithelial cells, which is reflected in the increased thickness of the intestinal wall and the width of the mucosa [123]. higher villus number, submucosa, and lamina propria were reported following an eight-week feeding of bacillus lichenformis. the disparity against the control group became evident in the fourth week of the trial [450]. shrimps fed probiotics lactococcus lactis, and pediococcus pentosaceus exhibited a significantly thicker muscular layer at the midintestine compared to the control group and the antibiotic oxytetracycline-treated group. the same experiment also affirms the importance of strain selection as bacillus spp. probiotics did not induce any statistically significant alteration to the intestinal muscular layer in l. vannamei [33]. higher intestinal height correlates with animal weight gain [451]. pmmb 2023, 6, 1; a0000324 44 of 86 in addition, the electron microscopic view discloses the epithelial cells in the probiotic group presented an active secretory state. high-density granules were noted in the cytoplasm of the probiotic-treated cohort [134]. these results correlated with the higher digestive enzyme activities observed following probiotic interventions (see section 7). furthermore, small clusters of bulges can be observed on the inner surface of the intestine. a higher fold depth and fold density were apparent in the probiotic treatment groups when contrasted with the non-treated group [134]. a more significant number of smaller crypts implied by probiotic treatment fabricate a larger surface structure of the intestine for nutrient assimilation and significantly improve the absorptive capacity of the intestine [452]. since the intestine functions through a luminal absorption mechanism, these modifications induced by probiotics bolster the digestive and absorptive efficiency of the organ. it is often neglected that the intestine also plays an essential role in regulating immune homeostasis and growth [154, 167, 176, 222, 453]. patrolled by immune proteins and further barricaded by a stable microbiome, the structural integrity across the large surface area of the intestine forms a protective barrier against inflammation and pathogen invasion [453]. from another perspective, intestinal mucosal is the first line of defence against pathogens. pathogenic bacteria may invade the digestive system by triggering the onset of mechanisms that foster the permeability of the gut mucosal layer to acquire passage [140, 454-456]. in this context, probiotics represent a promising strategy to mitigate infectious diseases [264, 457, 458]. probiotics are postulated to effectuate their protective effect against infectious diseases by strengthening the protective mechanisms in the gut, thus enhancing the immune function (see section 9) and maintaining the intestinal mucosal integrity. a histopathology study revealed a significantly healthier intestinal tissue in the probiotic cohort is discernible post-infected by vibrio parahaemolyticus [162]. the qualitative morphological disparity between treatment groups becomes evident when examined under electron microscopy. transmission electron microscopy (tem) images showed a higher mucosal layer density and better epithelial cell integrity in the probiotic-treated group [134]. besides, dietary supplementation of halomonas sp. to chinese white shrimp (fenneropenaeus chinensis) resulted in a regular and compact arrangement of epithelial cells in the intestine, which is in startle contrast to the sparse and irregular epithelial cells arrangement observed in the untreated counterpart [140]. this is consistent with the effect of lactobacillus pentosus treatment in l. vannamei, in which the mucosal layer of the treated group is denser and has more blooms. in contrast, the untreated group demonstrated looser and thinner mucosae [162]. the results strongly supported the application of probiotics for improving the mucosal structure, thereby fortifying the epithelial barrier integrity to guard against the invasion of opportunistic pathogens. securing the health of the animal allows more resources to be invested for growth rather than being depleted to sustain the defence activities. clearly, a better growth profile could be attributed to probiotics via the improvement in the intestinal microstructure. pmmb 2023, 6, 1; a0000324 45 of 86 9. priming the immune function strengthening the immune function has a far-reaching effect on the animals' health and growth. intriguingly, probiotics exhibited a unique spectrum of immunomodulatory properties not seen in antibiotic treatments. in recent years, probiotics have been exploited for their immunostimulatory effects and stress-relieving properties in aquatic animals [31, 264, 459-461]. probiotics represent a promising alternative to chemotherapeutics for prophylaxis against infectious diseases affecting shrimps [461]. similar to the concept of vaccination, probiotics can act as immunostimulants to boost the animals' immune systems and augment their resistance to infectious diseases and environmental stressors [462-466]. although one may argue that stimulating the immune function may be an energy-draining exercise, in the desired scenario, immunostimulants should not trigger a massive immune reaction in the host. on the contrary, immunostimulants expedite the innate immune response by enhancing the recognition and elimination mechanisms of a wide array of foreign substances and infectious agents [461]. this is achieved by stimulating the immune function via the introduction of pathogen-associated molecular patterns (pamps). the cell surface components of probiotic bacteria, such as peptidoglycans, lipopolysaccharides (lps), lipoteichoic acid, and glucans, act as immunostimulants and form complexes with the pattern recognition protein (prps) in the host. 11 prrs have been identified in shrimps [467]. each prr exhibits different binding specificity and effector functions [467, 468]. nonetheless, the formation of the pamp-prr complex constitutes a key event to activate the immune response, leading to the upregulation of immune gene expression and thereby improving immune function [123, 469-471]. this mechanism aids the animal to appear in the best state of defence. therefore, upon pathogen invasion, the animal can elicit an immediate defence to clear the pathogen [472-474]. figure 5. the innate immune system of shrimps. the modulatory effects of probiotics have been bracketed. pmmb 2023, 6, 1; a0000324 46 of 86 shrimps lack an adaptive immune system, in which their immunity is heavily dependent on the innate immune response that plays a pivotal role in driving the defence mechanisms [475]. although the existence of immune memory in shrimps remains controversial due to the lack of memory cells and immunoglobulin (antibody) production, recent findings proved that some forms of immune memory do exist in shrimps [476-478]. ideas such as ‘immune priming’, ‘quasi-immune response’, and ‘trained immunity’ further supported the rationale of this argument [477, 478]. the prophenoloxidase (propo) system is one of arthropods' most well-defined defence mechanisms (see figure 5). briefly, the pampprp complex triggers the serine protease cascade and catalyses the proteolytic cleavage of propo, which eventually culminates in the release of the terminal enzyme po. the released po actively mediates several critical reaction pathways, including melanin synthesis, cytotoxic reactant liberation, phagocytosis, encapsulation, and nodule formation [479-484]. notably, the serine protease, peroxynectin, and propo expressions in shrimps-fed probiotics are substantially higher than those in the control group and antibiotic oxytetracycline-treated group [33]. the activity of immune enzymes, such as po and lysozyme, was also significantly elevated following probiotic feeding [33, 129, 134, 485]. lysozyme is an effective antibacterial agent that kills the invading pathogens by disrupting the peptidoglycan, which leads to the building up of osmotic pressure within the pathogen and eventually ruptures the cell [486]. besides, the introduction of probiotics such as bacillus sp. has been shown to elevate the phagocytic activities in different shrimp species, such as p. monodon [487] and l. vannamei [123]. the immunostimulatory effect of probiotics effectively reduces the mortality rates of shrimps when confronted by common pathogens such as a. hydrophila [488], v. parahaemolyticus [95, 123, 182, 218, 443, 489], v. harveyi [35, 212, 490], vibrio alginolyticus [125, 491, 492], pseudomonas aeruginosa [493] and white spot syndrome virus (wssv) [134]. in proportion to the increment of phagocytic activity, the liberation of reactive oxygen species (ros) such as reactive oxygen intermediate (roi), singlet oxygen (o2·), hydroxyl ions (oh-), hydroxyl radical (·oh), hydrogen peroxide (h2o2) and superoxide anion radical (o2 -·) may be the culprits for oxidative damage in the body [38, 494]. fortunately, probiotic addition also induced the secretion of antioxidative enzymes such as catalase (cat), superoxide dismutase (sod) and glutathione peroxidase (gpx) to enhance the clearance efficiency of pathogens and safeguard the general wellbeing of the animal [35, 123, 495]. to illustrate, duan et al. [129] demonstrated that dietary inclusion of c. butyricum probiotic effectively improved the total antioxidant capacity (t-aoc) of l. vannamei. the antioxidative mechanisms of probiotics have been clearly detailed in the recent work of wang et al. [496]. additionally, the introduction of c. butyricum probiotic significantly increased the inducible nitric oxide synthase (inos) and heat shock protein (hsp) gene expression of l. vannamei under ammonia stress. hsp70 is a vital regulator for signalling pathways and protein homeostasis, thus rendering a protective effect to shrimps against environmental stressors [497]. on top of that, priming the immune system also diminishes the shrimps’ susceptibility to environmental stressors, including ammonia and oxidative stress [96, 99, 129, pmmb 2023, 6, 1; a0000324 47 of 86 290, 450, 498]. the synergistic improvement in both the antioxidant and innate immune defence systems irrefutably promotes shrimps' health and growth performance. in general, the immunomodulatory effect of probiotics is clearly established. however, discrepancies in results may be discernible between studies which are likely attributed to the differences in study designs, probiotic strains, dose, duration of treatment, shrimp species, growth phase, and environmental factors [95]. augmenting disease resistance is critical to secure optimal growth as the shrimps affected by diseases typically display symptoms such as highly necrotic tissues, inactivity, anorexia, and poor growth [495]. careful consideration needs to be taken when designing probiotics as immunostimulants. proper mitigation is necessary to establish a more robust immune function of shrimp while avoiding the overactivation of the immune response, which may lead to the direct opposite effect [75, 461]. overstimulation of the immune system will cause detrimental consequences such as unregulated generation of immune effectors leading to the depletion of immune components and eventually culminating in immune exhaustion. besides, prolonging the immune response unnecessarily may incur a massive energy cost to regenerate the immune components, which may draw resources away from sustaining other physiological processes and thus negatively impact animal growth [461]. clearly, a rigorous testing protocol is important to assess the large assortment of immune indices pertinent to the specific shrimp species targeted. long-term data covering the entire production phase is also critical to evaluate the suitability of probiotics as immunostimulants [461]. despite the complexity of the immune system, probiotics can be exploited as an effective means to enhance growth if properly utilized. 10. altering the growth-related genes expression in the context of decapod crustaceans, the shrimps’ development is an intermittent process interrupted by the recurring moult cycle [499]. the muscle tissues in shrimps undergo frequent dramatic remodelling across each moult cycle. the shrimp experiences a characteristic deliberated atrophy during the pre-moult phase, followed by significant muscle growth in the early post-moult phase to fill up the available space before the hardening of the exoskeleton [499-501]. regulation of the moulting process is greatly dependent upon the coordination of growth-related hormones, such as 20-hydroxyecdysone (20e) and crustacean hyperglycaemic hormone (chh) [502, 503]. endogenous factors such as the inherent hereditary genetic and hormonal factors also contribute to the variation in growth [504]. irrefutably, the investigation of the influence of probiotics on the metabolism and growth profile of shrimp remains incomplete without looking into their effect on these pathways. dietary supplementation of probiotics represents a novel approach to intervening with the metabolic function of aquatic animals to promote growth [504-507]. growth-related gene expressions can therefore serve as surrogate markers for growth status. their expression levels in tissue samples can be quantitated using real-time polymerase chain reaction (rtpcr) [504, 508]. duan et al. [180] investigated the effect of probiotic supplementation on the expression levels of immune and digestive-related genes in l. vannamei. results show that the expression levels are higher in the group fed with c. butyricum than in the control group pmmb 2023, 6, 1; a0000324 48 of 86 [180]. besides, the introduction of c. butyricum remarkably up-regulated the genes in the mechanistic target of the rapamycin (mtor) pathway, which includes the target of rapamycin (tor), eukaryotic translation initiation factor 4e-binding protein (4e-bp), eukaryotic translation initiation factor 4e (eif4e1α) and (eif4e2) in l. vannamei. the mtor pathway is a key regulatory centre for growth through the mediation of the moulting process, protein synthesis, nutrient transport, immune response, cell proliferation, autophagy and cell apoptosis [130, 509-516]. notably, these changes correlated with the significant improvement of growth parameters like weight gain, sgr and fcr in the probiotic treatment group. by contrast, the control and cell-free fermentation supernatant group treatment group with low expression of eif4e1α and eif4e2 portrayed a poorer growth profile. the results imply the significance of the mtor pathway associated with growth performance [130]. kegg enrichment analysis revealed that administering probiotic strain with a high adhesive ability to the intestinal mucosa significantly increased the differential expression of proteins involved in metabolic regulation, immune processes, and cell signalling pathways. immune pathways were activated, such as endoplasmic protein processing, oxidative phosphorylation, mitogen-activated protein kinase (mapk), and mtor signalling pathway [162]. moreover, feeding l. vannamei with c. butyricum-enriched diet also significantly increases immune deficiency (imd) and toll genes expression [129]. imd and toll pathways are two primary components of the shrimps’ immunity which mediate the antagonising action against the gram-negative and gram-positive bacteria via the production of various antimicrobial peptides (amps) [517]. besides, cell signalling machinery for calcium, oxytocin, wingless-related integration site (wnt), and forkhead box protein o (foxo) signalling pathway in l. vannamei was enhanced [162]. du et al. [162] substantiated the involvement of cell surface proteins in mediating the modulatory effects of probiotics in shrimps by demonstrating the comparatively insignificant alteration to the proteomic and histological parameters when lithium-chloride (licl)-treated probiotics were introduced to the shrimps instead. licl was applied to detach the cell surface proteins non-covalently bound to the probiotic cell surface. however, documentation on the molecular mechanisms of probiotics in shrimps is somewhat limited. from another perspective, growth hormone (gh) is one of the rate-limiting enzymes that play a pivotal role in glycolysis, lipolysis, protein synthesis, cell proliferation and immune response regulation [461, 504, 518-521]. the effect of gh is mediated through the expression of insulin-like growth factor-1 (igf-1) [505, 508]. igf-1 positively regulates animal growth through the direct stimulation of cell proliferation and differentiation [504]. functional studies associated igfs with metabolic control in crustaceans [522]. igf-1 is a primary indicator gene for somatic growth [523]. the positive correlation between igf-1 and animal growth rate has consistently been proven [523-525]. through a series of periodic dietary restrictions and reintroduction of diet, montserrat et al. [526] demonstrated the association between igf and the nutritional status of the animal. although molecular data on the effect of probiotics in the shrimp model is lacking, information gathered from other aquatic species suggests that gene expression modulation can be regarded as a highly probable growth pmmb 2023, 6, 1; a0000324 49 of 86 promotion effect of probiotics in shrimps. for instance, adding l. rhamnosus to the rearing water yielded a remarkable elevation of igfs expression and increased the average body weight of treated a. ocellaris by 3-fold [525]. long-term dietary supplementation of l. rhamnosus likely accelerates growth by modulating growth-promoting factors such as igfs, retinoic acid receptor γ (rarγ) or peroxisome proliferator receptors [527]. although reports gathered from other aquatic species can be used for cross-references in the speculation of the molecular mechanisms of probiotics in shrimp, it should be noted that the growth promotion effect in relation to myostatin (mstn) expression in the vertebrates may not be directly modelled to shrimps. mstn, also known as growth differentiation factor8 (gdf8), a member of the transforming growth factor-ß (tgf-ß), is typically noted as a negative growth regulator in the vertebrates due to its recognised function in inhibiting myoblast proliferation [528, 529]. the administration of lactobacillus sp. and bacillus spp. probiotics have been reported to lower the level of mstn while increasing the growth of d. labrax [524] and sea bream (s. aurata) [525], respectively. unlike mammals, mstn can be found not only in muscular tissues but also widely expressed in non-muscular tissues, including eyestalk, thoracic muscle, heart, gill, abdominal ganglion, abdominal muscle, intestine, hepatopancreas and swimming leg of shrimps. the highest expression of mstn concentrates in the heart and abdominal muscle [499-501, 530, 531]. the ubiquitous expression of mstn in different tissues may suggest that the mstn ortholog in shrimp may have other functions as that in the vertebrates. the mstn ortholog in shrimp was found to perform the opposite role to those in the vertebrates [499-501]. a decline in mstn transcripts in the abdominal, pereiopod, and pleopod muscles of shrimps was detected with the introduction of 20-hydroxyecdysone (20e), thus propounding the involvement of mstn in the moult cycle [501]. results also imply that mstn expression may be regulated by hormones [499, 501]. significant elevation of mstn is common in the early post-moult phase and gradually down-regulated following the moulting phase [499]. increased expression of mstn was found to positively regulate the growth of l. vannamei [530], p. monodon [500], freshwater shrimp macrobracium nipponense [499], and f. chinensis [532]. on the contrary, reduced expression of mstn featured a significant stagnated growth pattern [500]. thereby, it is essential to note the different functions of orthologs when mapping findings across species. recent studies through the genome-wide association approach successfully identified several novel genes and single nucleotide polymorphisms (snps) associated with the growth trait of shrimps, namely the l. vannamei class c scavenger receptor (lvsrc) [533], l. vannamei insulin growth factor binding gene-1 (lvigfb1) [534], l. vannamei myostatin/growth differentiation factor (lvmstn/gdf11) [530], ras-related protein (rap-2α) and the delta type protein kinase c (pkcδ) [535]. future research can investigate the effect of probiotics on these growth-related genes to validate the correlation between gene expression and the growth promotion effects of probiotics (see section 13). pmmb 2023, 6, 1; a0000324 50 of 86 11. increasing feed intake growth is an index for a multitude of parameters. besides the immune status, nutrition is an indispensable factor. another growth promotion factor of probiotics may stem from their effect in encouraging feed intake by shrimps. probiotics have been reported to stimulate the appetite of animals, thereby allowing more resources to be allocated for growth [208, 211]. however, the reason behind the improved appetite is yet uncertain. the increased appetite may probably be associated with the effect of probiotics in improving nutrient digestibility (see section 7) [156, 208]. otherwise, increased feeding may arise from the improved attractiveness and taste of feed supplemented with probiotics [208]. huynh et al. [536] surmised that some attractive compounds produced by synbiotics trigger increased feed intake. recently, proton magnetic nuclear resonance (1h-nmr) based metabolomic analysis identified that three metabolites out of the total 22 compounds detected were significantly higher in concentration in the hepatopancreas of shrimps treated with synbiotics compared to that of the untreated cohort. these three compounds are betaine (an organic osmolyte), inosine monophosphate (imp) (a nucleotide), and valine (an essential amino acid) [537]. interestingly, betaine is a common chemoattractant applied to prompt feeding in aquaculture. thus, synbiotics/probiotics can act as betaine enhancers and initiate shrimps' feeding response. furthermore, betaine plays a central role in dna methylation, modulating gene expression, and regulating cell proliferation and differentiation [537, 538]. from another perspective, it is also hypothesised that the secretion of antinutritive compounds, scfas (see section 6.1), and vitamins (see section 6.2) by the probiotics may contribute to a better appetite [208]. although in-vivo studies in shrimps are limited, da silva et al. [269] demonstrated that scfa introduction resulted in a substantial increase in feed intake, phosphorous availability, and higher apparent gross energy in shrimps. in another experiment, scfa, specifically sodium propionate, was observed to demonstrate a remarkable dose-dependent effect in elevating the expression of ghrelin (ghrl) in zebrafish [254]. ghrl is a gene encoding the orexigenic ghrl hormone that stimulates feed intake and regulates weight gain [539]. however, the up-regulation of ghrl may not always correspond to higher feed intake [523]. to illustrate, the report from santos et al. [523], which portrayed the upregulation of the ghrl gene in zebrafish following the 30-day administration of transgenic phytase-expressing probiotics, recorded significant growth enhancement in zebrafish without significantly increasing feed intake. this may be attributed to the multifunctional facets of ghrl. ghrl is not only an orexigenic hormone (appetite stimulant); it is also actively involved in homeostasis regulation and other physiological processes [523, 540]. most importantly, it should be noted that not all probiotics can lead to elevation of ghrl gene expression, better appetite, or increased feed intake. for example, falcinelli et al. [541] recorded the appetite suppression effect of probiotic lactobacillus rhamnosus, which could be harnessed for managing glucose intolerance. therefore, the selection of good probiotic strains for the intended result should be treated with circumspection. future research can also look into the effects of probiotics in regulating the expression of other pmmb 2023, 6, 1; a0000324 51 of 86 appetite-related factors in shrimps, including neuropeptides (npy) such as moult-inhibiting hormone-like neuropeptide (rmih-b) and cholecystokinin (cck) [542-544]. 12. environmental bioremediation as far as aquaculture is concerned, the quality of the rearing environment is a key index for growth. water quality parameters such as temperature, salinity, ph, dissolved oxygen, and chemical composition of water are essential factors in the health status of shrimps [93, 189]. fluctuations of these variables might inflict stress upon the animals, alter their normal physiological functioning and ultimately affect the growth performance of the animals [164, 168, 545-547]. environmental changes can impose selection pressure on microbial populations in culture water, which indirectly mould the gut microbiota of aquatic animals [548-550]. notably, zhao et al. [168] documented that water quality accounts for nearly one-fifth of the variations of gut microflora in shrimps. this suggests that besides the direct modulation of the water quality measures to favour the growth of shrimp, probiotics also indirectly aid in disease control by dictating the bacteria composition in water [37, 551]. the growth promotion effect of probiotics with regard to water quality could be comprehended through the involvement of the microbial population in manipulating the nitrite and ammonia levels in the rearing water, which are common end products of nitrogen catabolism. certain species of bacteria are endowed with the nitrifying potential to convert nitrite and ammonia into nitrate, which is a less toxic form [147, 241, 552, 553]. in this regard, gram-positive bacteria often demonstrated better efficiency in the decomposing organic matter when compared to their gram-negative counterparts [241, 554]. this nitrification process also indirectly improved the ph level of water through the secretion of hydrogens as byproducts [147, 210, 555]. besides, this process also facilitates the turnover of carbon and nitrogenous compounds and increases nutrient availability to support growth [556]. stemming from the aspects narrated, ameliorating water quality may account for the growth promotion phenomenon evinced by probiotics. 13. future perspectives as one of the fastest-growing industries worldwide, aquaculture is a key insurer for global food security [557]. besides addressing the food security crisis, a steady supply of aquaculture produce would eventually culminate in a promising economic prospect for the global aquaculture industry. creative strategies have been applied to intensify aquaculture production to meet the global demand. however, the progressive development of the amr catastrophe resulted in the exigency for an alternative to the antimicrobial growth-promoting agent. nevertheless, the state-of-the-art probiotic application as growth promoters in aquaculture witnesses several critical knowledge gaps. to illustrate, a vast array of the available studies involving probiotics were products of the empirical observational approach, whereas reports on the mechanistic molecular studies in shrimps are meagre [239]. only a handful of studies concerned genomic studies in aquaculture animals. in recent years, genome-association studies are gradually replacing marker-assisted selection (mas) in pmmb 2023, 6, 1; a0000324 52 of 86 selecting quality crops and animals for breeding. gene-assisted selection (gas) offered an added edge to mas due to its higher accuracy and efficiency in selecting beneficial growth traits in animals [535, 558-563]. several genes associated with body weight, metabolism, immunity regulation, moulting cycle, apoptosis, cell migration, and cytoskeletal arrangements have been listed in section 10. since the heritability factor can account for 24 to 52% of growth traits, therefore, introducing probiotics inducible to the growth-related genes to the livestock bred from selective broodstock would synergistically boost farm production yield [535, 562, 564, 565]. from another outlook, the progressive unveiling of the knowledge regarding the gut microbiome and its interaction with host physiology will increase the appreciation for probiotic application in aquaculture. over the past few decades, the slow implementation rate of probiotics in aquaculture may partly be ascribed to the paucity of technological advances and research expertise [566]. only a tiny fraction of bacteria in the biosphere can be cultivated using conventional culturing techniques [567-569]. since there is a tremendous underestimation of the gut microbiome diversity, many key players in the gut still exist in the dark, and their actual roles and relations to the growth performance of the host are yet to be disclosed. nonetheless, the popularization of molecular-based methods and their increasing affordability reignited great research interest in this arena. next-generation sequencing (ngs), 16s rrna sequencing, denaturing gradient gel electrophoresis (dgge), temperature gradient gel electrophoresis (tgge), fatty acid methyl ester (fame) analysis and terminal restriction fragment length polymorphism (t-rflp) are some prominent examples of the culture-independent techniques [570-573]. revealing the microbiome diversity extends the frontiers of human understanding in the microscopic province. when viewed concurrently through the lenses of other omics such as metabolomics, proteomics, transcriptomics, and metagenetics, new insights will surface [573]. mastering the mechanics of the microbiome will undoubtedly pave the way for more sophisticated research endeavours. when the technical hurdles are triumphed over by technological advancement and assiduous research effort, probiotics can be effectively tailored as growth promoters in aquatic livestock. unravelling the mechanisms of probiotics' growth promotion effect helps clarify the selection criteria when shortlisting putative strains as growth promoters. in other words, the growth-promoting factors elaborated on in this review could form the basis for the growth-promoting probiotic strain selection. for instance, the ability to secrete metabolites and digestive enzymes of probiotic strains could be screened through in-vitro assays. strains exhibiting antimicrobial resistance could be excluded for further testing. small-scale in-vivo pilot runs can focus on studying the effects of probiotics on gut microbiota, immunity, midgut histology, digestive enzyme activities and growth-related genes expression to provide a preliminary abstraction of the growth-promoting effect of promising probiotic strain [222, 566, 574, 575]. this approach could effectively increase the efficiency of designing growth promoters for shrimps. thereby, less promising candidates could be eliminated, whereas more potential strains could proceed for larger-scale validation [576]. besides the widely applied probiotics such as firmicutes, bacteroidetes and proteobacteria, the actinobacteria, pmmb 2023, 6, 1; a0000324 53 of 86 marine purple non-sulphur photosynthetic bacterium, salt pan bacteria, and other bacteria from varying sources are also potential candidates for aquaculture application lie in wait for further bioprospection [96, 104, 577-586]. 14. conclusion introducing probiotics opens a new vista for promoting animal growth and curbing infectious diseases in the aquaculture industry. although large-scale implementation data is still lacking, studies have attested to probiotics' positive effects in improving shrimps' growth performance and survival rate. different probiotics may trigger animal growth through distinct mechanisms (see figure 6). this paper condenses the outcomes of empirical observational studies on probiotics revolving around shrimp models. the most notable changes induced by probiotics are reflected in the gut microbiota. probiotics introduction helps increase the microbiota population's diversity and suppress the pathogenic strains. probiotics also contribute to the growth promotion effect by aiding the establishment of healthy and functioning gut microbiota. the results of probiotics are closely associated with the secretion of bioactive compounds such as scfas, vitamins, and polyamines. these bioactive compounds act as growth factors for the animal. besides supplying exogenous enzymes, the administration of probiotics has been noted to stimulate the secretion of endogenous digestive enzymes. this is consistent with the histological changes in the hepatopancreatic tubules. morphological changes reflected in the midgut region, particularly the increase in intestinal villi height and numbers, increased the total surface area for nutrient absorption. in contrast to agps, probiotics could act as immunostimulants that prime the immune system in the animal and confer a better resistance to infectious disease. mitigating the risk of illness helps to conserve energy directed for growth. additionally, probiotic additives tend to increase feed intake in animals. a higher nutrient input also contributed to animal growth. body metabolism, in particular, the catabolic activities, are postulated to increase, which is coherent with the increase in the expression of growth-related genes and proteins. last but not least, several probiotic strains are equipped with environmental ameliorative effects. probiotics modulate the microbial composition in the water. this indirectly mediates critical parameters such as ammonia and nitrate, thus relieving the chemical stresses inflicted on the shrimps to create a conducive environment to support animal growth. to summarise, the factors contributing to growth discussed in this review should not be viewed as fragmented events. condensing the constellation of results generated from the multitude of observational studies regarding the effects of probiotics in shrimps helps to offer a better picture of the growth promotion effect of probiotics. the information provides a more coherent overview of the potentialities of probiotic application in aquaculture and dictates the knowledge gap in state-of-the-art research. understanding the growth promotion effect of probiotics about the critical features of agps helps to quicken the quest for alternative growth-promoting agents that will be a boon to the aquaculture industry. pmmb 2023, 6, 1; a0000324 54 of 86 figure 6. mechanisms of probiotics in promoting animal growth. author contributions: conceptualization, jgxh; formal analysis, jgxh; data curation, jgxh; writing— original draft preparation, jgxh; writing—review, editing and proof, jgxh, lt-ht, jw-fl, k-yk, gz, vl, l-hl and b-hg. funding: this work was supported by the ministry of education fund (frgs/1/2019/wab09/musm/02/1) and jeffrey cheah school of medicine and health sciences (jcsmhs) strategic grant 2021 (grant code: stg-000051). conflicts of interest: the authors declare no conflict of interest. references 1. fishstatj fao. a tool for fishery statistics analysis. fao fisheries and aquaculture department, fips– statistics and information: rome, italy 2017. 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