Project description:The environmental reservoirs for Vibrio cholerae are natural aquatic habitats, where it colonizes the chitinous exoskeletons of copepod molts. Growth of V. cholerae on a chitin surface induces competence for natural transformation, a mechanism for intra-species gene exchange. The antigenically diverse O-serogroup determinants of V. cholerae are encoded by a genetically variable biosynthetic cluster of genes that is flanked on either side by chromosomal regions that are conserved between different serogroups. To determine whether this genomic motif and chitin-induced natural transformation might enable the exchange of serogroup-specific gene clusters between different O serogroups of V. cholerae, a strain of V. cholerae O1 El Tor was co-cultured with a strain of V. cholerae O139 Bengal within a biofilm on the same chitin surface immersed in seawater, and O1-to-O139 transformants were obtained. Serogroup conversion of the O1 recipient by the O139 donor was demonstrated by comparative genomic hybridization, biochemical and serological characterization of the O-antigenic determinant, and resistance of O1-to-O139 transformants to bacteriolysis by a virulent O1-specific phage. Serogroup conversion was shown to have occurred as a single-step exchange of large fragments of DNA. Crossovers were localized to regions of homology common to other V. cholerae serogroups that flank serogroup-specific encoding sequences. This result and the successful serogroup conversion of an O1 strain by O37 genomic DNA indicate that chitin-induced natural transformation might be a common mechanism for serogroup conversion in aquatic habitats and for the emergence of V. cholerae variants that are better adapted for survival in environmental niches or more pathogenic for humans.
Project description:The spread of cholera in the midst of an epidemic is largely driven by direct transmission from person to person, although it is well-recognized that Vibrio cholerae is also capable of growth and long-term survival in aquatic ecosystems. While prior studies have shown that aquatic reservoirs are important in the persistence of the disease on the Indian subcontinent, an epidemiological view postulating that locally evolving environmental V. cholerae contributes to outbreaks outside Asia remains debated. The single-source introduction of toxigenic V. cholerae O1 in Haiti, one of the largest outbreaks occurring this century, with 812,586 suspected cases and 9,606 deaths reported through July 2018, provided a unique opportunity to evaluate the role of aquatic reservoirs and assess bacterial transmission dynamics across environmental boundaries. To this end, we investigated the phylogeography of both clinical and aquatic toxigenic V. cholerae O1 isolates and show robust evidence of the establishment of aquatic reservoirs as well as ongoing evolution of V. cholerae isolates from aquatic sites. Novel environmental lineages emerged from sequential population bottlenecks, carrying mutations potentially involved in adaptation to the aquatic ecosystem. Based on such empirical data, we developed a mixed-transmission dynamic model of V. cholerae, where aquatic reservoirs actively contribute to genetic diversification and epidemic emergence, which underscores the complexity of transmission pathways in epidemics and endemic settings and the need for long-term investments in cholera control at both human and environmental levels.
Project description:Vibrio metoecus is a recently described aquatic bacterium and opportunistic pathogen, closely related to and often coexisting with Vibrio cholerae. To study the relative abundance and population dynamics of both species in aquatic environments of cholera-endemic and cholera-free regions, we developed a multiplex qPCR assay allowing simultaneous quantification of total V. metoecus and V. cholerae (including toxigenic and O1 serogroup) cells. The presence of V. metoecus was restricted to samples from regions that are not endemic for cholera, where it was found at 20% of the abundance of V. cholerae. In this environment, non-toxigenic O1 serogroup V. cholerae represents almost one-fifth of the total V. cholerae population. In contrast, toxigenic O1 serogroup V. cholerae was also present in low abundance on the coast of cholera-endemic regions, but sustained in relatively high proportions throughout the year in inland waters. The majority of cells from both Vibrio species were recovered from particles rather than free-living, indicating a potential preference for attached versus planktonic lifestyles. This research further elucidates the population dynamics underpinning V. cholerae and its closest relative in cholera-endemic and non-endemic regions through culture-independent quantification from environmental samples.
Project description:These experiment were performed to show a serogroup conversion in Vibrio cholerae from O1 to O139. For this purpose, V. cholerae O1 WT = A1552 was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (2 ug each) from strain VCO139-Kan was added after 24h and the cells grwon further for 24h. The VCO139-Kan strain is a MO10 derivative (both O139 serogroup) which harbors a Kanamycin cassette in the O139 region (as part of the operon between wbfA and wbfB w/o own promotor) for better selection. Transformants were selected on LB+Kan plates. Two groups of transformants were gained: Group I had a full exchange of the O1 region by the O139 region (clones serogroup-converted: SGC#1-3); the crossovers for the homologous recombination event had occurred within or upstream of the gmhD gene and in most instances within or downstream of the homolog gene of VC0271. This implies an exchange of an at least 33 kb spanning O1 genomic region by more than 42 kb of the O139 region. Group II had only half of the O139 region transfered and therefore half of the O1 region kept (clones HSGC#4-6). We analyzed their genotype and found that all of them had undergone a homologous recombination event with one crossover in or upstream of the gmhD gene and the second one inside the VC0254 and IS1358 gene. The transformation experiment was done three independent times (I - III). Three clones from group I and group II were selected from each experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone.
Project description:Vibrio cholerae, the etiologic agent of cholera, is autochthonous to various aquatic environments, but despite intensive efforts its ecology remains an enigma. Recently, it was suggested that copepods and chironomids, both considered as natural reservoirs of V. cholerae, are dispersed by migratory waterbirds, thus possibly distributing the bacteria between water bodies within and between continents. Although fish have been implicated in the scientific literature with cholera cases, as far as we know, no study actually surveyed the presence of the bacteria in the fish. Here we show for the first time that fish of various species and habitats contain V. cholerae in their digestive tract. Fish (n = 110) were randomly sampled from freshwater and marine habitats in Israel. Ten different fish species sampled from freshwater habitats (lake, rivers and fish ponds), and one marine species, were found to carry V. cholerae. The fish intestine of Sarotherodon galilaeus harboured ca. 5 x 10(3)V. cholerae cfu per 1 gr intestine content-high rates compared with known V. cholerae cfu numbers in the bacteria's natural reservoirs. Our results, combined with evidence from the literature, suggest that fish are reservoirs of V. cholerae. As fish carrying the bacteria swim from one location to another (some fish species move from rivers to lakes or sea and vice versa), they serve as vectors on a small scale. Nevertheless, fish are consumed by waterbirds, which disseminate the bacteria on a global scale. Moreover, V. cholerae isolates had the ability to degrade chitin, indicating a commensal relationship between V. cholerae and fish. Better understanding of V. cholerae ecology can help reduce the times that human beings come into contact with this pathogen and thus minimize the health risk this poses.
Project description:Vibrio cholerae O1 exists as two major serotypes, Inaba and Ogawa, which are associated with the O antigen of the lipopolysaccharide and are capable of unequal reciprocal interconversion. The 20-kilobase rfb regions encoding O-antigen biosynthesis in strains 569B (Inaba) and O17 (Ogawa) have been cloned in Escherichia coli K-12 and the nucleotide sequences have been determined. Besides several base substitutions and a small deletion in the 569B sequence relative to O17, there is a single nucleotide change resulting in a TGA stop codon within the gene for the 32-kDa RfbT protein. We have demonstrated that rfbT is responsible for serotype conversion (Inaba to Ogawa). The construction of a specific rfbT mutation in the Ogawa strain O17, and the ability of the gene from O17 to complement Inaba strains to Ogawa, confirmed rfbT as the gene required for the Ogawa serotype. By Southern hybridization and sequencing of PCR products of a number of strains, we have shown that the changes observed in one Inaba strain (569B) are not conserved in other Inaba strains. This may explain why some Inaba strains are able to convert to Ogawa whereas others are not. The protein encoded by rfbT has been identified and expressed in E. coli K-12 using a phage T7 expression system. Amino-terminal analysis of partially purified protein has identified the translational start of the protein. Primer extension studies have enabled the 5' end of the mRNA to be defined. It exists as a separate transcript from the rest of the rfb region, and the distinctive G + C content of rfbT suggests that it has been acquired from a non-Vibrio source.