CONTACT : STELLA MAGDALENA stella.magdalena@atmajaya.ac.id 83 Abstract Bacteria can interact with each or other microorganisms by releasing, sensing, and reacting to small diffusible chemical signal molecules to alter their community behavior. This process, termed quorum sensing, is influenced by density of other bacteria that present in the environment. One example as a result of this process is the formation of biofilm. Biofilm consists of bacterial communities that attach to a surface and envelope themselves in secreted polymers. This formation can be beneficial to pathogenic bacteria because they become highly resistant to antibiotics and human immunity. Thus, antibiofilm agents that can inhibit biofilm formation are needed. The objective of this study were to screen and evaluate bacteria from hot spring and crater lakes that have antibiofilm activity against pathogenic bacteria. In this study, 26 isolates were successfully obtained and tested for quorum sensing and quorum quenching activities. Based on the result, two isolates, which were KM16 and PAP26, were found to have quorum quenching activity. Further research showed that KM16 and PAP26 had antibiofilm activity against more than six pathogenic bacteria. From characterization of the bioactive compounds, it is known that different compound from KM16 and PAP26 have different activity against each pathogen. In molecular identification, isolates KM16 and PAP26 were identified as Bacillus subtilis and Pseudomonas sp. through molecular identification. ISSN : 2580-2410 eISSN : 2580-2119 Evaluation of the potentials of Bacillus subtilis KM16 and Pseudomonas sp. PAP 26 isolated from the hot spring and crater lakes as antibiofilm agents Stella Magdalena1*, Fabiola Giovani 2, Yogiara3 1Department of Food Technology, Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia 2Department of Biotechnology, Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia 3Department of Magister of Biotechnology, Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia Introduction Biofilm is microbial community embedded on inert or living surface and encaged in self-produced extrapolymeric substances (EPS) that contains proteins, polysaccharides, and extracellular DNA. The EPS provided structural strength and defense against environmental condition, host immunity, and antimicrobial agent (Davies, 2003; Singh et al., 2017). Most chronic infection is caused by biofilm-forming microorganisms and it is very difficult to eradicate them by only using conventional antibiotics or other antimicrobial therapy. In OPEN ACCESS International Journal of Applied Biology Keyword antibiofilm, quorum quenching, quorum sensing, Bacillus. Article History Received 31 March 2021 Accepted 03 July 2021 International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Applied Biology, 5(1), 2021 84 biomedical, biofilm can be formed on inanimate surface such as medical device, catheters, and living-tissue associated infection. Human immune system cannot correctly kill pathogenic bacteria or fungal and therefore, they may cause damage to encircling tissue (Taraszkiewicz et al., 2013). Biofilm can also contaminate food processing and attach to water pipe, henceit may cause food-borne and water-borne disease (Kokare et al., 2009). Several strategies that can be used to inhibit biofilm formation are to interfere with quorum sensing mechanism and using antibiofilm agents. Quorum sensing can influence gene expression that is responsible for biofilm formation, virulence, sporulation, and pathogenicity. This process, alters wide-scale behavior of population in response to cell density. Therefore, mechanism that can interfere with quorum sensing mechanism, quorum quenching, is needed (Brackman & Coenye, 2015). Thermophilic bacteria are a type of bacteria that can survive at high temperature environment, as in hot spring and crater lake. These bacteria have tremendously gained popularity in pharmaceutical and many industries because they can produce heat-stable bioactive molecule, for example, thermostable protease that does not denature at high temperature, but remains active at such temperature (Panda et al., 2013). Furthermore, antibiofilm activity of bacteria from hot spring and crater lakes in Indonesia has n ot been much explored. The purpose of this study was to screen and evaluate bacteria from hot spring and crater lakes that have antibiofilm activity against pathogenic bacteria. Materials and Methods Water Samples and Isolation of Bacteria Water samples were obtained from hot spring and crater lakes at Mount Pancar, Bogor, Indonesia (Table 1). The media used for isolation were Luria Broth (LB) (10g tryptone, 5 g yeast extract, and 10 g NaCl, and 1000 mL ddH2O) and Luria Agar (LA) (LB with 1.5% (w/v) bacteriological agar). A total 5 mL water sample was transferred into 250 ml conical flask containing 45 ml LB and incubated at 37°C, 70°C, and 80°C for 5 hours. The suspension was plated onto LA plates and incubated at 37°C overnight. Morphologically different colony that grew on LA plate was collected and inoculated by repeated streaking on the same medium. For short term preservation, isolates were streaked on LA and stored at 4°C. For long-term preservation, the culture was stored at -80°C in 15% (v/v) glycerol. Table 1. Water source and condition Source Location Condition (temperature; pH) Hot Spring Mount Pancar, Bogor, Indonesia - Merah Crater Lake Hitam Crater Lake Natural Crater Lake Mount Pancar, Bogor, Indonesia 67oC; 7 48oC; 7 43oC; 7 Quorum Sensing Assay Quorum sensing activity from bacteria sample was determined via Cross-feeding assay as sketched by Magdalena et al. (2020). N-acyl-homoserine lactone production was examined by streaking the isolate in parallel with a lane of the monitor strains Chromobacterium violaceum 026 (CV026) onto Brain Heart Infusion Agar (BHIA). BHIA medium incubated at 28°C for 48 hours. CV026 that showed production of violacein (purple International Journal of Applied Biology, 5(1), 2021 85 color) indicated a positive result of quorum sensing activity. Experiments were performed in triplicates. Quorum Quenching Assay The procedure used in this quorum quenching assay was agar well diffusion method which is a modification of the procedure of Soundari et al. (2014). Each isolate was grown in LB at 37°C until they reached absorbance value of 0.132 at λ=600 nm (McFarland 0.5). The inoculated LB was centrifuged twice at 12,000×g for 10 minutes and then filtered (0.22 µm) to obtain the cell-free supernatant. C. violaceum was grown in Brain Heart Infusion Broth (BHIB) at 28°C until it reached absorbance value of 0.5 at λ=600 nm (McFarland 0.5). One hundred μL of C. violaceum was spotted and streaked on BHIA using sterile cotton bud. Each well was made using cork borer. A total of 15 μL of cell-free supernatant was loaded into the well. LB was used as negative controls. The plates were incubated 28°C overnight and inhibition of purple pigment production was interpreted as positive results. Experiments were performed in triplicates. Biofilm Inhibition Assay The method used in this biofilm inhibition assay was the static biofilm assay which is a modification of the procedure of Magdalena et al. (2020). In this method, the biofilm inhibition was observed using 5% (v/v) concentration of cell-free supernatant. Pathogenic bacteria that were used in this assay were Acinetobacter baumanii ATCC 19606, Pseudomonas aeruginosa ATCC 1637, Escherichia coli ATCC 4157, Salmonella enterica ATCC 51741, Staphylococcus aureus ATCC 25923, Bacillus cepacia ATCC 25416, and Bacillus licheniformis ATCC 12759. The test bacteria were grown in LB at 37°C until they reached absorbance value of 0.132 at λ=600 nm (McFarland 0.5). In 96-well round bottom (U) microplates, 200 μL of tested bacteria were inoculated in each well along with 5% (v/v) cell-free supernatant. In this assay, 200 μL tested bacteria alone and 200 μL of uninoculated LB were used as negative control. The microplate was incubated at 37°C overnight. After incubation, the media and planktonic cells were discarded and the wells were rinsed twice using sterile distilled water and air-dried. Then, each well was stained with 200 μL of crystal violet and incubated for 30 minutes. The crystal violet was discarded and the wells were rinsed three times using sterile distilled water and air-dried. Afterwards, 200 μL of absolute ethanol was added to each well and incubated for another 30 minutes. Finally, the dissolved crystal violet was transferred to a new microplate and measured at λ=595 nm with Biorad 680 Microplate Reader. The percentage of biofilm inhibition was calculated using the following formula from Nikolić et al. (2014): % biofilm inhibition = (OD growth control − OD sample) OD growth control × 100 Characterization of Bioactive Compounds This method was adapted from the procedure of Jiang et al. (2011). Cell-free supernatant of each isolate was treated with proteinase-K (1 mg/ml), nuclease (100 μg/ml DNAse and 25 μg/mL RNAse), and NaIO4 (20 mM) separately and incubated in 37°C for 12 hour. After incubation, treated cell-free supernatants were then used in static biofilm inhibition assay with cell-free supernatant concentration of 5% (v/v). International Journal of Applied Biology, 5(1), 2021 86 Microscopic Observation and Biochemistry Tests For the microscopic observation the isolates were stained using Gram staining and observed under the magnification of 100x10. The biochemistry tests used for this research were catalase test, triple sugar iron agar (TSIA) test, carbohydrate fermentation test (glucose, lactose, maltose, and mannitol), and citrate test. Molecular Identification of KM16 And PAP26 Genomic DNA was isolated using Wizard® Genomic DNA Purification Kit (Promega, Wisconsin, USA) and used as a DNA template in PCR. The identification of the isolates was conducted by amplifying the 16S rRNA gene using universal primer sequences 63F (5′- CAGGCCTAACACATGCAAGTC-3′) and 1387R (5′-GGGCGGWGTGTACAA GGC-3′) (Marchesi et al., 1998). The reaction mixture consisted of 0.5 μL DNA template, 2 μL of forward primer 63F, 2 μL reverse primer 1387R, 25 μL of Go Taq® Green (Promega) 2x, and 20.5 μL of ddH2O. Thermal cycling of 30-cycle PCR, included pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, and followed by a post-extension at 72°C for 20 minutes. PCR products were then analyzed by gel electrophoresis in 1% agarose gel at 90 Volt for 60 minutes using 1x TAE buffer, visualized under UV light with the help of FloroSafe DNA Stain (1st BASE), and recorded with Gel Doc instrument (BioRad, USA). The marker used was 100 bp DNA ladder (Promega). PCR results were sent to 1st BASE, Malaysia for sequence. The sequences were used to identify the isolates using BLAST (NCBI) and submitted to GenBank. Results And Disscussion Isolation of Bacteria In this research, water samples were collected from different crater lakes and hot spring at Mount Pancar, Bogor, Indonesia, using sterile glass container. The container then kept inside vacuum flask to maintain the water temperature. Bacteria were tested using broth media at diverse temperatures, but mostly at high temperature. In high temperature environment, most bacteria are believed to have characteristics of thermophile. This is because thermophilic bacteria have optimum growth temperature in the range of 45 - 80°C. Thermophilic bacteria are known to produce bioactive compound that can worked at high temperature (Taylor & Vaisman, 2010), such as antimicrobial agent and quorum sensing inhibitor is produced by hot spring cyanobacterial mats (Dobretsov, 2010). In this study, 26 bacterial isolates were retrieved. They were 8 isolates from Merah crater lake, 12 isolates from Hitam crater lake, 3 isolates from Natural crater lake, and 3 isolates from hot spring (Table 2). All isolates were tested for quorum sensing activity via cross feeding assay using C.violaceum 026 (CV026) as a detector. Table 2. Isolates from Mount Pancar, Bogor, Indonesia Source Number of Isolates Code of Isolates Merah crater lake 8 KM - 2, 7, 13, 14, 15, 16, 19, 22 Hitam crater lake 12 KH - 3, 5, 6, 8, 9, 10, 11, 17, 18, 20, 21, 23 Natural crater lake 3 KN - 1, 4, 12 Hot spring 3 PAP - 24, 25, 26 International Journal of Applied Biology, 5(1), 2021 87 Quorum Sensing Activity AHL-mediated quorum sensing control genes were responsible for colonization in eukaryotes in most bacteria species (Anbazhagan et al., 2012). CV026 is AHL negative mutant because the presence of mini-Tn5 mutagenesis in CviI (AHL synthase), hence it requires exogenous AHL to produce violacein (purple pigment) (Vasavi et al., 2013). Based on the result, none of the CV026 produced purple pigment when they were grown together with each isolates. CV026 detected wide range of AHL with N-acyl side chains ranging from C4 to C8 in length, but did not detect AHL with N-acyl side chain raging from C10 to C14 (Anbazhagan et al., 2012). This showed that all 26 isolates did not produce short chain AHL molecules. In spite of that, it did not rule out the possibility that these isolates can perform cell-to-cell communication. For example, wild type B. subtilis uses ComQXPA and Rap-Hpr quorum sensing systems to coordinate sporulation and competence. As gram-positive bacteria, B. subtilis uses small peptide as signal molecule (Kalamara et al., 2018). Thus, it cannot be detected by CV026. Another example is Pseudomonas species, P. aeruginosa, which uses LasI/LasR and RhII/RhIR quorum sensing system to control biofilm and generate extracellular enzymes. Although Pseudomonas as gram-negative uses AHL as their molecule signal (Umesha & Shivakumar, 2013), PAP26 is likely to produce long chain AHL molecules. Bacteria have differences in quorum sensing systems, including signal types, receptors, and signal transduction mechanisms) (Waters & Bassler, 2005). Although they are in the same Gram type of bacteria, there is specificity in terms of signal types and receptors structure, hence this can cause inhibition (quorum quenching) because homologous signal molecule interferes with signal binding to receptor and decrease receptor concentration (Dong et al., 2007). For example, Staphylococcus aureus has been divided into four groups based on the interaction between molecule signal and its receptor. Each group produced homologous AIP and only activated response in the same group member, but inhibited other group response (Umesha & Shivakumar, 2013). Therefore, quorum sensing activity could be used to inhibit quorum sensing of other bacteria. Quorum Quenching and Biofilm Inhibition Activity It was found out that bacterial isolates KM16 (Figure 1) and PAP26 had quorum quenching activity. Quorum quenching activity can be achieved by inhibiting signal synthesis, degradation of the signal molecule, and preventing signal molecule binding to transcriptional factors (Grandclément et al., 2016). Figure 1. Positive result of the quorum quenching assay of KM16 isolate (red arrow) The biofilm inhibition activity was performed using static biofilm inhibition assay to determine whether bacterial isolates KM16 and PAP26 can prevent biofilm formation of tested bacteria. Seven pathogen bacterial species were inoculated with 5% cell-free International Journal of Applied Biology, 5(1), 2021 88 supernatant (v/v) of KM16 and PAP26. Based on the result, bacterial isolates KM16 and PAP26 had antibiofilm activity towards several pathogenic bacteria (Table 3). KM16 showed the highest antibiofilm activity against A. baumannii with 82.29% activity. On the other hand, PAP26 showed the highest antibiofilm activity against E. coli with 84.09% activity. This activity can be influenced by polysaccharide, protein, or nucleic acid (DNA or RNA) compound in the isolate supernatant. Table 3. Biofilm inhibition activity using 5% (v/v) crude extract of bacterial isolates KM16 and PAP26 Pathogenic bacteria % inhibition KM16 PAP26 A. baumannii ATCC 19606 82.29 78.74 P. aeruginosa ATCC 1637 48.28 36.72 S. aureus ATCC 25923 41.49 44.87 E. coli ATCC 4157 78.52 84.09 S. enterica ATCC 51741 29.13 34.72 B. cepacia ATCC 25416 14.77 - B. licheniformis ATCC 12759 28.00 42.57 Characterization of Bioactive Compounds Afterward, cell-free supernatant from each isolates was pre-treated using NaIO4, proteinase-K, and nuclease. It is well known that proteinase-k, DNAse, and RNAse can degrade protein, DNA, and RNA. NaIO4 is capable to hydrolyze carbohydrate molecules by oxidizing the carbons bearing hydroxyl groups and cleaving the C-C bonds (Jiang et al., 2011). In Figure 2(a), reduction of biofilm activity against P. aeruginosa was shown after cell- free supernatant of KM16 was pre-treated with proteinase-K and nuclease. On the contrary, cell-free supernatant pre-treated with NaIO4 presented increasing activity. This can be caused by carbon and energy resources from breakdown of biopolymers used by pathogen to enhanced biofilm formation (Rabin et al., 2015). Figure 2. NaIO4, proteinase-K, and nuclease effects on (a) KM16 crude extract against P. aeruginosa, (b) PAP26 crude extract against S.aureus biofilm. In Figure 2(b), pre-treated cell-free supernatant of PAP26 with NaIO4 exhibited decline in antibiofilm activity against S. aureus. This suggested that antibiofilm compound consisted of DNA, RNA, protein, and polysaccharide (Table 4). Biofilm matrix components in P. aeruginosa consist of Psl and Pel proteins that enhance intercellular adhesion and also (a) (b) International Journal of Applied Biology, 5(1), 2021 89 function as a barrier for immune and antibiotic attacks. Alginate plays role in structural stability and protection of biofilm. eDNA is created from random chromosomal DNA that serves as a cell-to-cell component united in the matrix biofilm. Protein and proteinaceous components serve as adhesion molecules and structural strength in biofilm formation (Karygianni et al., 2020; Wei & Ma, 2013). Cell-free supernatant of KM16 produced protein and nucleic acid as antibiofilm agent, meanwhile PAP26 produce all three biomolecules. S. aureus produce adhesion factor, such as serine-aspartate-repeat (Sdr) family, accumulation-associated protein (Aap), and Autolysin (Atl). Polysaccharide intercellular adhesion (PIA) or PNAG together with other polymer such as teichoic acids and proteins form major part of EPS in Staphylococci. Bap is also involved in intercellular adhesion and biofilm formation. Controlled cell death in Staphylococci released DNA that was needed for biofilm formation (Otto, 2008). KM16 produced polysaccharide and protein as antibiofilm agent. On the other hand, PAP26 produced all three biomolecules. Some bacteria exopolysaccharides can inhibit and destabilize biofilm from other bacteria without bacteriostatic and bactericidal activities, for example, P. aeruginosa cells degraded biofilm formation by Staphylococcus epidermidis and S.aureus. Therefore, Pel, Psl, and alginate do not only facilitate adhesive molecules to form biofilm, but also have antibiofilm properties (Rendueles et al., 2013). Table 4. Characterization of bioactive compounds of KM16 and PAP26 against seven tested bacteria Pathogen Bacterial Isolates KM16 PAP26 A. baumannii Polysaccharide − − Protein √ √ Nucleic acid √ √ P. aeruginosa Polysaccharide − √ Protein √ √ Nucleic acid √ √ S. aureus Polysaccharide √ √ Protein √ √ Nucleic acid − √ E. coli Polysaccharide √ √ Protein − √ Nucleic acid √ √ S. enterica Polysaccharide √ √ Protein √ − Nucleic acid √ − B. cepacia Polysaccharide √ Protein − Nucleic acid √ B. licheniformis Polysaccharide √ − Protein √ √ Nucleic acid √ √ *(√) = present, (-) absent, (■) not tested due to not having antibiofilm activity International Journal of Applied Biology, 5(1), 2021 90 There are three modes of action that are involved in polysaccharide antibiofilm activity, which are modifying abiotic and biotic surface properties, acting as signaling molecules that modulate gene expression, and acting as competitive inhibitor in carbohydrate-protein interaction. Biosurfactants change surface characteristic (wettability and charge of the surface), thus influencing interaction between bacteria and surface. Antibiofilm polysaccharides also alter physical properties of cell surface. For example, B. licheniformis reduced cell surface hydrophobicity, hence reducing P. aeruginosa colonization. Bacterial polysaccharides also caused down regulation of several genes that are responsible for biofilm formation, such us adhesion factor. This mechanism brings advantage to bacteria in bacteria competition and biofilm regulation (Rendueles et al., 2013). Protein acted as antibiofilm in the form of enzyme that degrade EPS matrix component and object that was trapped in EPS matrix. Negative charge of eDNA can act as repulsive force in initial attachment (Rabin et al., 2015). eDNA also can bind to bacteria adhesive structure and inhibit cell attachment (Berne et al., 2010). sRNAs can interfere translation process by binding to ribosome and promote mRNA degradation using RNase. sRNA can also terminate premature transcription by binding to a nascent mRNA (Mika & Hengge, 2013). Identification of Bacteria Bacterial isolate identification assay was performed by microscopy, biochemistry, and molecular assay (Table 5). Microscopy observation was done with Gram staining. From the result, each isolate had different gram type but similar morphology. Based on biochemistry assay, it was known that both isolates had completely different substrate preference. Table 5. Bacterial isolate identification assay of KM16 and PAP26 Bacterial identification with 16S rRNA showed that KM16 and PAP26 were identified as 99% B. subtilis and Pseudomonas sp. B. subtilis is gram positive and catalase positive Bacterial isolate identification assay Isolate KM16 PAP26 Microscopy Gram + - Shape Bacil Bacil Biochemistry Citrat - + TSIA Slant Acid Alkaline Butt Acid Alkaline Gas - - H2S - - Glucose + - Lactose - - Maltose + - Mannitol + - Catalase + + Molecular Identification Genus B. subtilis Pseudomonas sp. Accession KU877820.1 KU877821.1 Identity 99% 99% International Journal of Applied Biology, 5(1), 2021 91 bacilli. B. subtilis can utilize citrate and produce acid from glucose, sucrose, maltose, and manitol fermentations. However, B. subtilis cannot produce acid from lactose (Saleh et al., 2014). On the other hand, KM16 fermented lactose, but could not utilize citrate. Previous study showed that extracellular α-amylase from B. subtilis induced biofilm inhibition and degradation by disrupting exopolysaccharide in methicillin-resistant Staphylococcus aureus (MRSA) and P. aeruginosa (Kalpana et al., 2012). Cyclic lipopeptide (LP) biosurfactants produced by Bacillus, also modified bacterial surface hydrophobicity and affected the development of flagella. Thus, it demonstrated significant anti-adhesive and antibiofilm activity (Moryl et al., 2015) Pseudomonas sp. is a gram negative, rod shaped, catalase positive bacteria, and naturally found in soil and water ecosystem. Most species do not require polysaccharide as carbon source. The genus Pseudomonas comprises species that are capable of living under diverse environmental conditions. Pseudomonas species are capable to biofilm formation and often resistant to antibiotics, disinfectants, detergents, heavy metals, and organic solvents (Rocha et al., 2019). The mechanisms of antibiofilm activity on Pseudomonas remain unclear. Conclusions Twenty six isolates were successfully isolated from crater lakes and hot spring at Mount Pancar, Bogor, Indonesia. 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