The impact of biofilm formation on the survival of Vibrio cholerae in aquatic environment and its role in pathogenicity

Vibrio cholerae, the causative agent of epidemic cholera, has been established as an autochthonous flora of the aquatic environment, persisting predominately as nonculturable cells within clusters of biofilms and in association with plankton. Although biofilm has been proposed to serve as an important reservoir for V. cholerae, very little is known about the factors responsible for biofilm formation in the natural aquatic ecosystem, including its role in the active growth and survival, especially how the bacterium turns actively growing to initiate the seasonal outbreaks of cholera. Laboratory microcosm prepared with water collected from estuarine ecosystem of Mathbaria, Bangladesh showed slower biofilm formation and extended culturability for 68 days at 4°C, as compared to microcosms that were maintained at higher temperatures (30°C, 37°C, and 45°C), suggesting biofilm formation to be temperature dependent and negatively linked to loss of culturability. Mathbaria water (MW) microcosm supported the active growth of V. cholerae O1 for an extended period up to 54 days under higher pH (8.0-9.0), whereas microcosms having pH 6 and pH 7 supported the active growth for only 26 days and 40 days, respectively. Instant ocean (IO; salinity 1%) microcosms maintained at room temperature supported the active growth of V. cholerae for 54 days, while the IO microcosms with 0%, 2%, and 4% salinity supported the active growth for only 26 days. The biofilm formation was robust in Luria Bertani (LB) broth with 2% salinity and pH range 7.0-9.0 in presence of calcium chloride (10mM) and magnesium chloride (40mM). MW microcosm supplemented with dehydrated shrimp chitin chips (MW-CC) as the single source of nutrient supported both active growth of toxigenic V. cholerae O1 for up to six months and biofilm formation by the bacteria. V. cholerae O1 cells in chitinassociated biofilms remained metabolically active even in a high acidic environment without losing either viability or virulence. Biofilm-bound coccoid, nonculturable V. cholerae O1 cells maintained in MW microcosm for 495 days were able to be culturable upon passage through rabbit ileal loop (RIL). Genetic screening by polymerase chain reaction (PCR) of V. cholerae cells in MW microcosm revealed loss of several virulence and related genes including ctxA, ace, zot, rstR, tcpA, acf, toxT and two of four mshA genes. The mutant strains lacking mshA genes produced significantly less amount of biofilm in the air-water interface of the borosilicate glass tubes when compared with the parent strain. Pulsed-field gel electrophoresis (PFGE) analysis of the genomic DNA (NotI-digested) of the mshA-mutant daughter strains revealed them to be clonal, as they had identical PFGE pattern, but they differ from the parent strain. Results obtained from the field-based investigations in estuarine ecosystem of Mathbaria, Bangladesh have shown the presence of non-culturable V. cholerae cells as a component of the bacterial community of which only a negligible proportion could be enumerated when conventional culture methods were employed. In conclusion, V. cholerae biofilms formed in situ in response to physicochemical, and related biotic and abiotic factors of aquatic environment thus serve as an important reservoir where the bacterium can take refuge to, and persist as dormant cell until the favorable season returns for them to be actively growing again to increase the probability of infecting human and spreading cholera epidemics. Dhaka University Institutional Repository