04. Nisa.cdr Vol.15, No.2, June 2021, p 61-68 DOI: 10.5454/mi.15.2.4 Potential Z foonotic Faecal Bacteria rom Sunda Porcupine ( ) and Hystrix javanica Their Antimicrobial Resistance Profiles SARSA A. NISA , RIFKA A. N. SAFITRI , NURUL INAYAH , ACHIRUL NDITASARI , SUSIANA 1 1 2 3 PURWANTISARI , REJEKI S. FERNIAH , ANANG S. ACHMADI , TAUFIQ P. NUGRAHA , 1 1 2 2 AND SUGIYONO SAPUTRA * 3 1 Department of Biology, Faculty of Sains and Mathematics, Diponegoro University, Jalan Prof. Sudarto No.13, Tembalang, Semarang 50275, Indonesia; 2Museum Zoologicum Bogoriense, Research Center for Biology, Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor Km 46, Cibinong 16911, Indonesia; 3 Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor Km 46, Cibinong 16911, Indonesia. Sunda porcupine ( ) is one of the Indonesian endemic species which is often sought after for Hystrix javanica their meat. Although it is becoming increasingly popular for extreme culinary, information regarding biological risks arising is very limited. This study aimed to assess zoonotic bacteria carried from this wildlife potential faecal by Sunda porcupine and A total 22 faecal samples to investigate their antimicrobial resistance (AMR) profile. were collected from captive Sunda porcupine and and wo subjected to presumptive test for . TSalmonella Listeria samples (9%) were regarded as positive for which indicated by black colonies Salmonella was the presence of on x l dylose ysine eoxycholate (XLD) agar. Meanwhile, the presence for was positive in all samples (100%), Listeria indicated by olour change in medium from straw to black in solation ranswab®. In total, 3 bacterial c I T 6Listeria colonies were isolated a s t Most and subjected for ntimicrobial usceptibility esting (AST) by disk diffusion method. importantly, multidrug-resistant bacteria and resistance to third generation cephalosporins (ceftriaxone) were not observed, indicating low risk of AMR dissemination. Based on gene analysis and 16S rRNA phylogenetic tree construction from selected isolates, we identified several potential food-borne zoonotic pathogens, including Proteus mirabilis Shigella Klebsiella (XH3.3, H4.2, and E1.2) (XD8.2 and G11.3), and flexneri , quasipneumoniae similipneumoniae subsp. (XF4.2) Further research to confirm the pathogenicity of . those isolates is still needed but based on these results, we support the hypothesis that Sunda porcupine is potential as a reservoir of pathogenic bacteria. P crucial when this reventive measures are to prevent transmission processing bushmeat. r antimicrobial resistance Sunda porcupine, zoonosesKey words: , 16S RNA , Landak Jawa ( ) merupakan salah satu spesies endemik Indonesia yang sering dimanfaatkan Hystrix javanica dagingnya. Meski semakin populer untuk kuliner ekstrem, informasi mengenai risiko biologis yang timbul dari satwa liar ini masih sangat terbatas. Penelitian ini bertujuan untuk mengkaji bakteri potensial penyebab zoonosis yang dibawa oleh landak Jawa dan untuk mengetahui profil (AMR) pada isolat yang antimicrobial resistance didapatkan. Sebanyak 22 sampel feses diambil dari landak Jawa disertakan untuk uji presumtif dan Salmonella Listeria Salmonella . Dua sampel (9%) dinyatakan positif yang ditunjukkan dengan adanya pertumbuhan koloni hitam pada (XLD) agar. Sementara itu, keberadaan dinyatakan positif pada xylose lysine deoxycholate Listeria semua sampel (100%), ditunjukkan dengan munculnya warna hitam pada media Isolation Transwab®. Listeria Secara total, 36 koloni bakteri berhasil diisolasi dan diuji resistensinya menggunakan metode difusi cakram. Bakteri multidrug-resistant dan resistensi terhadap generasi ketiga sefalosporin (ceftriaxone) tidak terdeteksi yang mengindikasikan resiko rendah penyebaran AMR. Berdasarkan analisis gen 16S rRNA dan konstruksi pohon filogenetik isolat terpilih, kami mengidentifikasi beberapa bakteri yang potensial sebagai food-borne zoonotic pathogens flexneri, diantaranya dan , dan Proteus mirabilis Shigella (XH3.3, H4.2, E1.2) (XD8.2 G11.3) dan ). Penelitian lebih lanjut untuk mengkonfirmasi Klebsiella quasipneumoniae similipneumoniae subsp. (XF4.2 patogenisitas isolat masih diperlukan tetapi berdasarkan hasil ini, kami mendukung hipotesis bahwa landak Jawa berpotensi sebagai reservoir bakteri patogen. Tindakan pencegahan sangat penting dilakukan untuk mencegah penularan terutama pada saat memproses daging hewan liar ini. Kata kunci: r landak Jawa, zoonosis antimicrobial resistance16S RNA , , MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone 21-87907604; : +62- Fax: +62- 21-87907612; sugiyono.saputra@lipi.go.id E-mail: disease transmission. The existence of wild animals that should be in nature and far from human life, now carried in the form of exotic food and medicine, which can accelerate the transmission of zoonotic diseases (Broad 2020). The spillover events occur due to human-animal Bushmeat hunting and wildlife trade for consumption are contributing factors to zoonotic interactions during hunting, carrying, storing, raising, selling, killing, and consuming wild animals (Plotkin 2020). This unsustainable wildlife hunting practices mainly operated in Asia, Africa and South America, leading to a significant extinction threat to several wild terrestrial mammals . (Ripple 2016)et al. Bushmeat from wild mammals, has been used for consumption in Indonesia for decades. One of the wild mammals that is becoming more popular for consumption is Sunda porcupine ( ) Hystrix javanica which is an endemic Indonesian wildlife that belongs to the Rodentia rder . The o (Lunde and Aplin 2008) distribution region of these animals includes Sumatra, Kalimantan, Java, and Bali . (Lunde and Aplin 2008) Sunda porcupine have spines as their defence system (Myers . 2017)et al , nocturnal and herbivorous as in their natural habitat, they eat plant parts such as tubers, bamboo shoots, seeds, fruit, tubers, rhizomes, young shoots, and bark . Sunda porcupine meat (Farida 2015) have long been consumed as a source of protein for extreme culinary. They believe that consuming porcupine meat is preventing from osteoporosis and improving body vitality, while the liver and bile can cure asthma, and the thorns can be used to treat toothaches and ulcers ; (Farida 2007; Farida 2015 Wardi et al. 2011). The international conservation status based on the IUCN Red List of Threatened Species classified Sunda porcupine to least concern or not threatened. In Indonesia, this wildlife is protected under the Regulation of the Minister of Environment and Forestry of the Republic of Indonesia Number: P.106/MENLHK/SETJEN/KUM.1/12/2018. Despite they have protected status, illegal hunting and trade of Sunda porcupine are still rampant . (Inayah 2016) Additionally, biological risk information including transmission of zoonoses and antimicrobial resistance that may result from the hunting and trade this wild animal is not well recorded. In this preliminary study, we aimed to assess potential zoonotic bacteria arising from Sunda porcupine faecal samples by culture- dependent approach. Faeces and intestinal contents are often contaminated the meat during slaughtering and processing activities. The presence of antimicrobial resistance will also be investigated by characterizing their phenotypic resistance to several antimicrobial categories. This assessment will be useful for prevention measures for tackling emerging and re- emerging infectious diseases from wildlife. MATERIALS AND METHODS Sampling was conducted Animals and Sampling. at the small mammal research facility in Research Center for Biology, Indonesian Institute of Sciences. A total 22 Sunda porcupine (aged 2-3 years) were used as the object of study. Prior sampling, Sunda porcupine had no receive any special medical treatment for the last three months with usual diet include leaves and tubers. Sampling was performed using non-invasive methods without making direct contact to each individual by swabbing fresh faecal samples in the morning and transfer directly into Rappaport- Vassiliadis enrichment broth (RV) Salmonella (Himedia, India) and Isolation Transwab® Listeria (Medical Wire, UK) for the bacterial enrichment stage (Kartika 2017)et al. . This procedure has been approved by animal ethics commission, Indonesian Institute of Sciences (B-12220/IPH/KS.02.04/IPH/IX/2019). Presumptive Test and Bacterial Isolation. Presumptive test for and were Salmonella Listeria selected in this study because these bacteria widely known as foodborne pathogens and contaminant of animal products. After inoculating faecal samples, RV Salmonella enrichment broth was incubated at 42˚C for 24 hours, followed by subsequent inoculation to xylose lysine deoxycholate (XLD) (Himedia, India). agar XLD gar is a selective medium that primarily used for a isolation and differentiation of andSalmonella Shigella Shigella. colonies maybe difficult to differentiate with other Gram-nagative bacteria, thus presumptive test was not performed. In the other hand, typical colony growth with black spot in the center on XLD gar media is easy to observe, indicating the a presence of . Meanwhile,Salmonella Isolation Listeria Transwab® After is used for the detection of . Listeria incubation at 37˚C for 24 hours color change from straw to black is an indication the growth of Listeria spp in the sample and the results were regarded as . positive. The isolation process was continued by using Columbia agar supplemented with dehydrated horse blood (5%) to detect hemolysis activity. A clear zone that appears below or around the colonies indicate beta hemolytic activity Preservation of isolates were . conducted using Mueller Hinton Broth media added with 20% glycerol and t en stored at 80˚C. h - Salmonella enterica enterica Listeria subsp. JCM 1652 and monocytogenes JCM 7671 were used as positive controls during the isolation process. 62 NISA ET AL . Microbiol Indones Volume 15, 2021 Microbiol Indones 63 A n t i m i c r o b i a l S u s c e p t i b i l i t y Te s t i n g . Antimicrobial susceptibility testing was performed using the disk diffusion method based on protocol from the Eur op ea n Committe e on An timicr obial Susceptibility Testing (EUCAST) . (EUCAST 2020) Mueller Hinton Agar (MHA) was used by inoculating bacterial inoculum with turbidity standard of 0.5 McFarland or equivalent to 1.5×108 CFU/mL. Antimicrobial disc (Oxoid, USA) used in this study i n c l u d e 3 0 μ g e a c h o f a m i k a c i n ( A K , aminoglycosides , ) (AMC, amoxicillin-clavulanic acid penicillin-beta lactamase inhibitors , ) (FOX, cefoxitin second generation cephalosporins , ) (CROceftriaxone third generation of cephalosporins). The inhibition zone diameter (IZD) in agar media was indicated by the formation of a clear zone around the antibiotic disc. Interpretation of IZD results is species-specific and antimicrobial-specific, however, in order to describe potentially resistant isolates, we interpreted IZD results (resistant, intermediate or sensitive) using breakpoints for Enterobacterales according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guideline and zone diameter interpretive standards breakpoints for veterinary pathogens according to Clinical & Laboratory Standards Institute (CLSI, 2015). We then categorised resistance cut off for amoxicillin-clavulanic acid and amikacin when with IZD ≤14 cm while for cefoxitin and ceftriaxone when IZD ≤17 cm. Multidrug- resistant bacteria are defined when bacterial isolates have resistance to one agent in three or more antimicrobial categories (CLSI 2015). Escherichia coli ATCC 25922 and ATCC 25923 Staphylococcus aureus were used as control bacteria. Molecular Identification Based on the 16S rRNA. Extraction of bacterial DNA using Chelex® 100 sodium form 10% (Sigma Aldrich) according to the protocol from . Amplification Saputra (2017)et al. of the 16S rRNA gene was performed on a Veriti ™ 96- Well Fast Thermal Cycler (Applied Biosystems, USA) using Bioline MyTaq ™ Red Mix (Meridian Bioscience) using universal primer 27F and 1492R. PCR products were subjected to Sanger dideoxy sequencing (1st BASE, Singapore). The DNA sequences obtained using primer 27F were analyzed using the MEGA X program and alignment of the bases sequences were performed on GenBank® DNA using the Basic Local Alignment Search Tool (BLAST) menu. Phylogenetic tree construction was made by neighbor joining method with 1000 bootsrap value. RESULTS Presumptive Test for and Salmonella Listeria. Presumptive test for on XLD gar showed Salmonella a that 2 out of 22 samples tested positive (F2 and H3), which was indicated by the growth of typical black or black spots colonies. A total of 18 bacterial isolates were successfully purified and preserved. For Listeria presumptive test, all samples (100%) were regarded positive which was indicated by color change in the Listeria Isolation Transwab® from straw to black. A total of 18 isolates were successfully purified and preserved, with four isolates showing hemolytic activity (E5.1, G8.2, G11.1, and G11.3) in Columbia agar supplemented with 5% dehydrated horse blood. Presumptive tests and all isolates collected in this study are presented in Table 1. Distribution of Inhibition Zone Diameter (IZD). Antimicrobial susceptibility testing only showed that one isolate that showing no inhibition zone diameter (XG10.1) and regarded as cefoxitin resistant strain. Four isolates with inhibition zone diameter <10 was observed. Resistance to third generation of cephalosporins (ceftriaxone) was not detected. The distribution of all isolates with the inhibition zone diameter against four can be seen in antimicrobials Figure . 1 Interpreted according to IZD breakpoint from EUCAST and CLSI, only two isolates that showed resistance to two antimicrobials tested (AMC and FOX), while five isolate showed resistance to one antimicrobials (AK or FOX). Resistance to all antimicrobial (multidrug-resistant bacteria) tested in this study was not observed. Th of e resistance profile the seven bacterial isolates from the Sunda porcupine are presented in Table .1 Identified Bacteria and Their Susceptibility against Antibiotics. Seven bacterial isolates were selected based on colony appearance for bacterial identification using 16S rRNA gene. At least ~700-800 bp of nucleotides were generated for BLAST analysis in GenBank . Percentage of identity of those isolates ® can be seen in Table 2 which also include inhibition zone diameter results All s. even isolates were sensitive to amikacin, amoxicillin-clavulanic acid, cefoxitin and ceftriaxone, except for (XD8.2) which Shigella flexneri was resistant to cefoxitin The phylogenetic tree . constructions of seven isolates compared with reference strains are presented in Figure .2 64 NISA ET AL . Microbiol Indones DISCUSSION To the best of our knowledge, this is the first study in Indonesia to assess potential zoonoses from faecal samples of Sunda porcupine. This preliminary study has three main findings: 1) proportion of presumptive test for and which regarded as Salmonella Listeria positive were 9% (2/22) and 100% (22/22), respectively; 2) several potential zoonotic pathogens was observed, including (n=3), Proteus mirabilis S h i g e l l a f l e x n e r i K l e b s i e l l a ( n = 2 ) a n d quasipneumoniae similipneumoniae subsp. (n=1); and 3) multidrug-resistant bacteria and resistance to the latest generation antimicrobial used in this study (ceftriaxone) was not detected. Porcupine is one of the popular bushmeat not only in Southeast Asian countries (Engel and Ziegler 2020) but also in several African countries (Bachand . et al 2012, Friant . 2015). Brush-tailed porcupine et al ( ) is the most preferred animal for Atherurus africanus their meat in Nigeria and consumed regularly up to once per week (Friant . 2015). The activities et al related to this bushmeat consumption which include hunting, trade, and meat processing has led to a higher risk of zoonotic bacteria transmission (Plotkin 2020). It is reported that were observed on the dorsal Salmonella skin of Sunda porcupine ( and Prawira 2018)et al. carcass of Brush-tailed porcupine (Bachand . et al Table 1. Presumptive test results resistance 22 f samples and profile of isolated bacteria from aecal Sample ID Presumptive Salmonella Presumptive Listeria Isolate code Hemolysis activity Colony appearance Resistance profile D6 - + XD6.2 milky white - XD6.3 yellow - D7 - + - - - - D8 - + D8.1 - white - XD8.2 white FOX E1 - + E1.2 - milky white - E4 - + XE4.2 yellow - E5 - + E5.1 + transparent white, clear zone AK XE5.1 yellow - E6 - + E6.3 - white - XE6.2 yellow - E7 - + E7.1 - transparent white - E8 - + E8.1 - milky white AK F1 - + - - - - F2 + + F2.2 - white - XF2.2 white - F4 - + F4.2 - milky white - XF4.2 yellowish black - G4 - + G4.1 - transparent white AMC, FOX XG4.4 white - XG4.5 yellowish white - G5 - + - - - - G8 - + G8.2 + yellowish white, clear zone - XG8.2 yellow - G9 - + XG9.3 white - G10 - + G10.1 - transparent white AK G10.2 - white AK XG10.1 yellow AMC, FOX G11 - + G11.1 + milky white, clear zone - G11.2 - transparent white - G11.3 + white, clear zone - H1 - + H1.3 - white - XH1.2 yellowish white - H2 - + XH2.3 white - H3 + + H3.3 - transparent white, - XH3.2 black spot - XH3.3 black - H4 - + H4.2 - white - XH4.1 white Note: AK amikacin, AMC amoxicillin-clavulanic acid, FOX cefoxitin. Volume 15, 2021 Microbiol Indones 65 Fig 1 The distribution of inhibition zone diameter (IZD, cm) of bacterial isolates from Sunda porcupine for four antimicrobial tested. Fig 2 Phylogenetic tree construction from isolates XG4.4, XD8.2, XH3.3, XF4.2, H4.2, E1.2, and G11.3, was carried out using the neighbor joining method with 1000 bootsrap value. 2012) in Indian porcupine , while was observedListeria ( ) .Hystrix indicus (Sarangi and Panda 2013) Based on our study, it can be assumed that Sunda porcupine has the potential as a natural reservoir for Salmonella. Percival and Williams (2014) states that natural reservoir of is very diverse, including farm Salmonella animals, domesticated animals and wild animals, including poultry, pigs, cattle, birds, dogs, rodents, turtles and cats. Consumption of contaminated poultry and meat products is the most common source of transmission. Meanwhile, the presumptive test Listeria showed positive results for all samples, showing that Listeria may presence in Sunda porcupine faecal samples. and are among common Salmonella Listeria 66 NISA ET AL . Microbiol Indones cause of foodborne zoonotic diseases, as reported by European Food Safety Authority (EFSA 2020). Three species of pathogenic bacteria were identified, including (XH3.3, H4.2, and P. mirabilis E1.2) (XD8.2 and G11.3), S. flexneri and K. quasipneumoniae subsp. similipneumoniae (XF4.2), while the status of potential pathogens of another identified isolates ( XG.4.4) Pseudomonas xiamenensis was unknown (Lai and Shao 2008). Evidence from other studies regarding zoonotic potentials of these bacteria were presented in Table 2. One of the most cases of infection in humans are caused by Proteus mirabilis . According to(Jacobsen and Shirtliff 2011) Nemati (2013), is pathogenic in humans P. mirabilis because it can cause urinary tract infections. The pathogenicity of is supported by its unique P. mirabilis virulence factors, such as adhesin, flagella, toxins, quorum-sensing, enzymes, and immune invasion. Together with other species such as Shigella S. dysenteriae, S. boydii S. sonnei, S. flexneri and are responsible for diarrheal diseases and dysentery occurring globally and may be associated with life- threatening complications (Niyogi 2005). The spread of can occur by the faecal-oral route, where Shigella infected animals excrete along with their Shigella feces, then pollute the environment (Said and Marsidi 2017). Several strains according to Klebsiella Simmons and Gibson (2012) have been known to be pathogenic and capable of causing infection in humans, t h i s i n c l u d e K . q u a s i p n e u m o n i a e s u b s p . similipneumoniae that cause infections in human and dogs (Brisse . 2014). spp. is an et al Klebsiella opportunistic pathogenic bacterium from the Enterobacteriaceae family that can cause respiratory infections, urinary tract infections and nosocomial infections and are commonly found as microflora in the mouth, skin, intestines of humans and animals (Brisse et al. 2006, Paran . 2019). et al Based on antimicrobial susceptibility testing and interpretation according to EUCAST and CLSI, resistance to ceftriaxone was not detected in all 36 isolates while resistance to AMC, FOX and AK was detected in seven isolates. Multidrug-resistant bacteria were not observed. This result indicates that antimicrobial resistance may not disseminate in Sunda porcupine, although accumulated evidence has revealed the presence of antimicrobial resistance in wildlife due to contaminated environment (Allen et al. 2010). Resistance to third generation cephalosporins is a major concern globally, mainly conferred by extended-spectrum b-lactamases (ESBLs) which commonly found in birds, mammals, livestock, and companion animals . (Vittecoq 2016)et al. This study has several limitations. The method used was based on culture dependent method which only showing presumptive results for and Salmonella Listeria. We were also unable to recover those bacteria due to having issues with purification, preservation, and the complexity of nutrient requirements, especially for . Further, detection on XLD Listeria Salmonella agar after enrichment step may give false positive results because the growth of bacteria in the Proteus Table 2 antimicrobial susceptibility testing results from seven bacterial isolatesBacterial identity and Isolate code Species Identity (%) Accession Inhibition Zone Diameter (mm) Potential zoonotic pathogens status AMC FOX CRO AK XH3.3 Proteus mirabilis 99.87 NR_113344.1 24,44 24,44 31,79 18,83 Yes, commonly detected in broiler chicken (Jamaluddin et al. 2018) H4.2 Proteus mirabilis 99.87 NR_113344.1 22,76 22,63 28,88 18,35 E1.2 Proteus mirabilis 99.88 NR_113344.1 26,86 25,02 27,51 22,31 G11.3 Shigella flexneri 99.98 NR_026331.1 20,73 21,31 27,04 19,11 Yes, causing foodborne diseases (Bintsis 2017) XD8.2 Shigella flexneri 100 NR_026331.1 20,14 7,82* 29,12 20,89 XF4.2 Klebsiella quasipneumoniae subsp. similipneumoniae 99.77 NR_134063.1 23,28 22,84 28,12 21,21 Yes, detected in food animals with other Klebsiella species (Davis and Price 2016) XG4.4 Pseudomonas xiamenensis 99.79 NR_043533.1 30,65 24,52 35,82 31,29 Unknown, regarded as denitrifying bacterium isolated from activated sludge (Lai and Shao 2008) Note: AK amikacin, AMC amoxicillin-clavulanic acid, FOX cefoxitin, CRO ceftriaxone. *Resistance to AK, AMC, FOX and CRO was not detected. Only XD8.2 that considered as cefoxitin resistant. Volume 15, 2021 Microbiol Indones 67 medium was resemble to colonies. Salmonella Additionally, not all collected isolates were identified, therefore, the diversity of culturable faecal bacteria from Sunda porcupine was not fully understood. Based on presumptive test results and bacterial culture, we strengthen the hypothesis that the Sunda porcupine has the potential as a reservoir zoonotic of bacteria. Several identified isolates reveal that they may be regarded as foodborne zoonotic pathogens, including from genus , and Proteus, Klebsiella Shigella. Due to low exposure to antimicrobials Sunda porcupine in this study may not play as an important host for antimicrobial resistance. Further research is needed such as identification of collected bacteria all and confirm their pathogenicity. etection ation test for D of virulence genes can be and resistance genes combined to complement the detection results through a culture dependent method so that risk factors for the spread of zoonotic diseases can be understood more comprehensively. ACKNOWLEDGMENT This research was supported by DIPA Research Center for Biology 2018 and DIPA Research Center for Biotechnology 2019, Indonesian Institute of Science (LIPI). AUTHOR CONTRIBUTIONS The contribution of each author in this article is SS as the main contributor who is responsible for the formulation and overall research objectives. SAN, RANS contributed to laboratory investigations and NI and collected the data. SAN SS contributed to , AN and analyzed the data. SAN, SP, RSF, and SS ASA, TPN were responsible for original draft writing, and SAN and SS contributed to reviewing and editing the manuscript. 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