Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. We examined a unique collection of V. cholerae clinical and environmental isolates of widespread geographic distribution recovered over a 60-year period to determine their evolutionary genetic relationships based on analysis of two housekeeping genes, malate dehydrogenase (mdh) and a chaperonin (groEL). In addition, the phylogenetic distribution of 12 regions associated with virulence was determined. Comparative sequence analysis of mdh revealed that all V. cholerae O1 and O139 serogroup isolates belonged to the same clonal lineage. Single-strand conformational polymorphism (SSCP) analysis of these O1 and O139 strains at groEL confirmed the presence of an epidemic clonal complex. Of the 12 virulence regions examined, only three regions, Vibrio seventh pandemic island 1 (VSP-I), VSP-II, and RS1, were absent from all classical V. cholerae isolates. Most V. cholerae El Tor biotype and O139 serogroup isolates examined encoded all 12 virulence regions assayed. Outside of V. cholerae O1/O139 serogroup isolates, only one strain, VO7, contained VSP-I. Two V. cholerae El Tor isolates, GP155 and 2164-78, lacked both VSP-I and VSP-II, and one El Tor isolate, GP43, lacked VSP-II. Five non-O1/non-O139 serogroup isolates had an mdh sequence identical to that of the epidemic O1 and O139 strains. These isolates, similar to classical strains, lack both VSP-I and VSP-II. Four of the 12 virulence regions examined were found to be present in all isolates: hlyA, pilE, MSHA and RTX. Among non-O1/non-O139 isolates, however, the occurrence of the additional eight regions was considerably lower. The evolutionary relationships and multilocus virulence gene profiles of V. cholerae natural isolates indicate that consecutive pandemic strains arose from a common O1 serogroup progenitor through the successive acquisition of new virulence regions.

The Vibrio seventh pandemic island-II is a 26?9 kb genomic island present in Vibrio cholerae El Tor and O139 serogroup isolates that shows homology to a 43?4 kb genomic island in V. vulnificus Vibrio cholerae is the aetiological agent of the deadly diarrhoeal disease cholera. In this study the 7?5 kb Vibrio seventh pandemic island-II (VSP-II) that is unique to V. cholerae El Tor and O139 serogroups was analysed and it was found to be part of a novel 26?9 kb genomic island (GEI) encompassing VC0490-VC0516. The low-GC-content VSP-II encompassed 24 predicted ORFs, including DNA repair and methyl-accepting chemotaxis proteins, a group of hypothetical proteins and a bacteriophage-like integrase adjacent to a tRNA gene. Interestingly, V. cholerae ORFs VC0493-VC0498, VC0504-VC0510 and VC0516, which encodes an integrase, were homologous to Vibrio vulnificus strain YJ016 ORFs VV0510-VV0516, VV0518-VV0525 and VV0560, which also encodes an integrase, respectively. Some ORFs showed amino acid identities greater than 90 % between the two species in these regions. In V. vulnificus strain YJ016, a 43?4 kb low-GC-content (43 %) GEI encompassing ORFs VV0509-VV0560 was identified and named V. vulnificus island-I (VVI-I). The 52 ORFs of VVI-I included a phosphotransferase system gene cluster, genes required for sugar metabolism and transposase genes. There was synteny and homology between the 59 region of V. cholerae VSP-II and the 59 region of V. vulnificus VVI-I; however, VVI-I contained an additional 31?5 kb of DNA between VV0526 and VV0560 in strain YJ016. A second V. vulnificus strain, CMCP6, did not contain the 43?4 kb VVI-I; in this strain two ORFs were found between the 59 and 39 flanking genes VV10636 and VV10632, showing 100 % identity to VV0508 and VV0561, respectively, which flank VVI-I. INTRODUCTIONThe genus Vibrio, which belongs to the Gram-negative c-Proteobacteria, is ubiquitous in marine and estuarine environments. There are 71 recognized species within the genus Vibrio, of which 12 species are pathogenic to humans, in the current List of Bacterial Names with Standing in Nomenclature (Euzéby, 1997;Farmer et al., 2003). Vibrio cholerae is the most important human pathogen belonging to the genus since it is the aetiological agent of the deadly diarrhoeal disease cholera. Cholera is endemic in many areas of the world, particularly those areas where clean drinking water is absent. V. cholerae pathogenic isolates contain two main virulence factors, cholera toxin (CT) and toxin co-regulated pilus (TCP), both of which help facilitate cholera spread and pathogenesis (Sears & Kaper, 1996;Taylor et al., 1987). CT, the main cause of the explosive diarrhoeal disease, is encoded on a filamentous bacteriophage CTXw (Waldor & Mekalanos, 1996). The receptor for CTXw on the V. cholerae cell, TCP, which is an essential intestinal colonization factor, is also encoded on a mobile genetic element named the Vibrio pathogenicity island (VPI) (Waldor & Mekalanos, 1996;Kovach et al., 1996;Karaolis et al., 1998;Taylor et al., 1987). To dat...

Pathogenicity islands are large chromosomal regions encoding virulence genes that were acquired by horizontal gene transfer and are found in a wide range of pathogenic bacteria. In toxigenic Vibrio cholerae isolates the receptor for the cholera toxin encoding filamentous phage CTXP, the toxin-coregulated pilus, is part of the Vibrio pathogenicity island (VPI). In this paper, we show that the VPI can be transferred between O1 serogroup strains, the predominant cause of epidemic cholera, via a generalized transducing phage CP-T1. ß