Faculty of Biological Sciences

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    Whole Genome and Phenotypic Approaches to Study Vibrio cholerae Pathogenicity and Survival Mechanisms
    (2025-12-15) BFH2305MS102F
    ii Abstract Despite Vibrio cholerae causing epidemic and pandemic cholera has been well-established in brackish water environments, the aquatic reservoir is often debated because the bacterium is rarely, if ever, isolated from surface water by culturing methods. Non-culturable state has been proposed as a survival strategy for V. cholerae wherein it can change shape from curved rod to coccoid, as detected by Direct Fluorescent Antibody (DFA) assay. Recent studies showed V. cholerae O1 cells in the aquatic habitat, persisting mostly as coccoid cells in clusters of biofilms comprising non-culturable cells that can regain culturalabity upon animal challenge to initiate seasonal cholera. In this study, we have attempted to isolate and detect the bacterium from water samples collected fortnightly from household ponds and nearby canals in the coastal area of Noakhali, Bangladesh. Serotyping and molecular approaches were employed to identify V. cholerae O1. Polymerase Chain Reaction (PCR) was employed to detect the virulence- specific genes and Multilocus Sequence Typing (MLST) of the bacterium. Whole Genome Sequencing (WGS) was performed for further characterization of the isolated V. cholerae strains. In addition, a laboratory microcosm was constructed to observe the impact of temperature on growth response of the bacterium. Of 11 V. cholerae isolated from water samples collected during May - October, 2024, nine proved positive for serogroup O1, as they carried specific genes: ompW, rfbO1, tcpA, toxR, viuB, but, all were non-toxigenic having no ctxA encoding cholera toxin subunit A. Two isolates were non-O1/non-O139 having ompW only. Furthermore, MLST analysis confirmed these nine V. cholerae O1 as sequence type 69 (ST-69), whereas the two non-O1/non-O139 as ST-982. WGS data showed the presence of pathogenicity and related genes, including acfA, acfB, acfC, acfD, hlyA, makA, nanH, tagA, toxR, vasX, tcp etc., but lacked the CTX prophage. Some of these isolates also had multiple antimicrobial resistance genes for ampicillin, ceftazidime, and sulfamethoxazole suggesting multidrug resistance. Biofilm assays revealed that V. cholerae O1 isolates having all biofilm genes (including mshABCDEFGHIJKMN, omp U) failed to form a typical biofilm ring on a borosilicate glass tube, suggesting additional regulatory or structural factors that may influence the biofilm development. V. cholerae non-O1/non-O139 isolates lacked the key biofilm genes mshD and ompU and displayed no ring formation. Microcosm study revealed that at 37ºC, V. cholerae O1 grew actively isolates grew actively up to 49 days, whereas non O1/ non O139 strains became non-culturable after 35 days. In Microcosm at 4ºC, O1 cells failed to retain active growth state after 7 days, whereas the non-O1/non-O139 were active until 14 days. The overall results presented in this study might reflect the pathogenicity status, aquatic reservoir and possible transition from toxigenic to non-toxigenic strains, including the growth response and mechanism of the survival of V. cholerae O1 in the aquatic environment of the coastal areas of Noakhali, Bangladesh.