Abstract: Cholera is a waterborne-disease caused by Vibrio cholerae Biofilm inhibition has become a new approach to overcome this disease. Several publication reported marine microorganisms have been known to produce many bioactive compounds against pathogenic bacteria. The objective of this research was to obtain isolate marine bacteria with the ability to inhibit Vibrio cholerae Biofilm formation. The antibiofilm activity was determined by measuring the optical density of crystal violet which was used to stain the Vibrio cholerae Biofilm. Eleven, out of 238 isolates from several shores in Jakarta showed antibiofilm activity. Three of the eleven isolates with higher activity (KMC10, BMA1 and BLC1) were further identified using PCR and DNA sequencing of 16S rRNA gene analysis. KMC10 showed 96% similarity with Pseudoalteromonas nigrifaciens, BMA1 showed 96% similarity with Exiguobacterium sp., while BLC1 showed 99% similarity with Vibrio gallicus. Further studies are needed to characterize the bioactive compound as potential agent for cholera treatment.
INTRODUCTION
Cholera is one of waterborne-disease caused by Vibrio cholerae. Seven cholera pandemics have been reported in recent years. All of these pandemics have killed thousands of people every year. V. cholerae have biofilm-forming ability which plays a role in colonization of gut surface and serves as a protective matrix. Therefore, it is able to increase V. cholerae resistance against antibiotics, chlorines and environmental stresses. Biofilm-associated V. cholerae are more resistant to acid shock than planktonic V. cholerae and has been hypothesized to enhance survival during passage through the acidic gastric environment (Van Dellen and Watnick, 2006).
V. cholerae is a natural inhabitants of both freshwater and marine environment. They are Gram negative, motile and acid intolerant bacteria which produce cholera enterotoxin (CTX). They produce biofilm which is regulated by Vibrio polysaccharide (vps) gene. Expression of the vps genes is positively regulated by the transcriptional regulators VpsR and VpsT but negatively regulated by the HapR regulator protein (Beyhan et al., 2007).
The majority of natural products of marine bacterial origin have arisen from a small number of taxonomic groups that include Streptomyces, Alteromonas, Pseudomonas, Vibrio, Agrobacterium and the cyanobacteria (Toledo et al., 2006). Hence, aquatic environment especially marine become very potential to look for biofilm-inhibiting microorganisms.
Biofilm is an Extracellular Polymeric Substance (EPS) matrix which consist of polysaccharides, proteins, nucleic acids, lipids, phospholipids and humic substances (Simoes et al., 2010). Polysaccharides and proteins are the fundamental elements that determines the stability of biofilms. Biofilm-forming has been used by many bacteria to increase survival in environments and some of them used it for host-infection.
New approach to penetrate the biofilm layer is needed to find effective medical treatment for infected patients. Indonesia as a maritime country has a high potential source of antibiofilm-producing microorganisms. The objective of this research was to obtained marine bacteria from marine environment with the ability to inhibit biofilm specifically produced by V. cholerae.
MATERIALS AND METHODS
Collection of samples: Marine sediments and seawater were collected from several shores in Jakarta, Indonesia. V. cholerae were used from our previous study.
Bacterial isolation: For Marine sediment, 1 g of the sediment was included into 9 mL of 0.85% saline solution. While the seawater sample was taken as much as 1 mL in to vial. Each of them were gradually diluted until concentration achieved 10-3. The dilution of 10-1-10-3 were spread into Marine Agar 2216 medium (Difco) and Luria Agar (Oxoid) and incubated at 30°C overnight. Suspected colonies were picked randomly with distinct morphology. Isolates were picked based on morphological observation and subcultured in new agar medium.
Antibiofilm assay: This procedure is modification of antibiofilm assay method according to Djordjevic et al. (2002). V. cholerae cultures was grown in Thiosulfate Citrate Bile-salt Sucrose (TCBS) (Oxoid) Agar medium at 37°C for 24 h. The colony was inoculated into reaction tube containing Brain Heart Infusion (BHI) (Oxoid) broth and incubated at 37°C for 16 h. Marine bacteria cultures were refreshed in LA medium and were inoculated in BHI broth at 37°C for 24 h. The inoculated bacteria were centrifuged at 12 xg for 5 min and the supernatant were collected.
As much as 500 μL of supernatant of each marine bacteria cultures were added into V. cholerae biofilm in BHI medium and were incubated for an hour in room temperature. The medium of biofilm culture were washed out and one milliliter of crystal violet solution was added to visualize the biofilm. After 45 min of incubation, the vial tubes were rinsed with distilled water for 5 times and being air-dried. The optical density was then measured with spectrophotometer at 595 nm. Positive isolates were kept for further analysis.
Identification of the isolates: Genomic DNA extraction were done using CTAB method (Marchesi et al., 1998) and continued with PCR amplification of 16S rDNA and DNA sequencing analysis using 63f and 1387r primer (Generay Biotech). With the total of 50 μL PCR reaction contained 25 μL GoTaq (Fermentas), 1 μL each of the primer (10 pmol μL-1, 2 μL template, 19 μL Nuclease Free Water. Condition of PCR were set for pre-denaturation of 5 min at 94°C and followed by 30 sec of denaturation at 94°C, 30 sec of primer annealing at 55°C, 1 min extension at 72°C and 20 min post-extension at 72°C as much as 30 cycles using C1000™ Thermal Cycler (Bio Rad, USA). PCR products were then analyzed by gel electrophoresis in 2% agarose gel, visualized under UV light and recorded with Gel Doc instrument (Bio Rad, USA). Purification of PCR products were done with QIA Quick PCR Purification kit (QIAGEN, USA). DNA sequencing was carried out in Macrogen, South Korea. Data analysis nucleotide sequence were analyzed using BLASTN from National Center for Biotechnology Information (NCBI) for identification. The sequences were submitted to GenBank.
RESULTS
Marine bacteria isolation: A total of 238 isolates microbes obtained from three shores in Jakarta. 97 isolates were obtained from Pantai Indah Kapuk, 88 isolates were from Pantai Ancol, 53 isolates were from Pantai Mutiara.
Screening of antibiofilm activity: From 238 isolates, eleven isolates shown positive results for antibiofilm activity assay. The biofilm ring formed by V. cholerae could be seen with crystal violet staining. Biofilm ring degradation were shown with the positive isolates (Fig. 1).
The antibiofilm activity was determined by the lower optical density of crystal violet. Eleven isolates were shown to have lower value compared to control. Three isolates with the highest antibiofilm activity were KMC10, BLC1 and BMA1 (Table 1).
| Fig. 1: | Visualization of V. Cholerae inhibited |
| Table 1: | Data for optical density of biofilm V. cholerae inhibited |
These isolates were continued for further molecular identification. Those three isolates were also tested for their antimicrobial activity using well-diffusion method. All of them showed negative results for the antimicrobial test.
Characterization of the isolate: Three of the eleven isolates (KMC10, BLC1 and BMA1) were identified using 16S rDNA amplification and DNA sequencing analysis. Based on BLASTn of 16S rDNA sequences, isolate KMC10 showed 96% similarity with Pseudoalteromonas nigrifaciens, BMA1 has 96% similarity with Exiguobacterium sp. and BLC1 has 99% similarity with V. gallicus. The results of DNA sequencing were submitted to GenBank.
DISCUSSION
In this study, V. cholerae were cultured at 16 hours incubation to form mature biofilm. The biofilm material which consist of EPS were stained with crystal violet. As a cationic dye, crystal violet will bind to matrix component because most of the EPS material are polyanionic due to the presents of either uronic acids or ketal-linked pyruvate (Sutherland, 2001).
Two of the positive isolates (P. nigrifaciens and V. Gallicus) are inhabitant of marine environment. Dheilly et al. (2010) found that Pseudoalteromonas sp. have antibiofilm activity against Paracoccus sp. and Vibrio sp. and also impaired biofilm formation of other human-pathogenic species.
Pseudoalteromonas sp. also known to produce many biologically active substance. They displayed antibacterial, bacteriolytic, agarolytic and algicidal activities (Holmstrom and Kjelleberg, 1999). Several species of Pseudoalteromonas has been known able to produce antifouling agents against common marine fouling organisms (Holmstrom et al., 1998).
V. gallicus is an alginolytic, facultatively anaerobic, non-motile bacteria isolated from the gut of an abalone (Sawabe et al., 2004). Vibrio sp. strain QY101 have been reported to possess antibiofilm activity by its exopolysaccharide (Jiang et al., 2011). The antibiofilm exopolysaccharides have displayed inhibitory activity towards biofilm formation of many Gram negative and Gram positive bacteria, including Pseudomonas aeruginosa. Though the EPS have not been tested on biofilm of V. cholerae, it did not rule out that there is potential for inhibition of biofilm-formed by closely-related bacteria.
Exiguobacterium sp. is a gram positive and facultative anaerobe bacterium which possesses high nuclease activity (Rodrigues et al., 2006). They are inhabitant of extreme environment such as hot springs, glacial water and permafrost and some strains are also isolated from marine (Lee et al., 2009). There has been no study due to its antibacterial activity but Exiguobacterium sp. was known able to degrade cellulose and hemicellulose (Vishnivetskaya et al., 2011). Hemicellulose like D-mannose is one of the component of V. cholerae biofilm (Wai et al., 1998).
V. cholerae have negative regulation of quorum sensing mechanism. In inactive state where density cell is low, it induces biofilm formation. To the contrary, in high-density cell the biofilm formation is repressed. Hence, biofilm-inhibiting strategy using antiquorum-sensing molecule might give less impact to biofilm-forming V. cholerae (Waters et al., 2008). There are multifactor that play a role in V. cholerae biofilm forming which should be considered for inhibition strategy, such as nutrient source, gene regulation and c-di-GMP regulation. This study showed that several genera of marine bacteria have exhibit antibiofilm activity against V. cholerae. Furthermore, the characterization of the isolates is needed to analyze the potential of antibiofilm molecules as new drugs for cholera treatment.