Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: Clonal relationships between clinical and environmental isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139 , and non-O1, non-O139 serogroups from both clinical and environmental sour ces, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolys in (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tc pA), and outer membrane protein (ompU), for genomic organization, and for t he presence of the regulatory protein genes tcpI and toxR in order to deter mine relationships between epidemic serotypes and sources of isolation. Whi le 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. Ail O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA,zot, and ace genes. Also, O1 strain VO3 was nega tive for the zot gene. All of the non-O1, non-O139 strains were negative fo r the ctxA, tot, ace, tcpA, and tcpI genes, and all of the non-Ol, non-O139 strains except strain VO26 were negative for ompU. Ail of the strains exce pt non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative fo r the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cho lerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 stra in, an O139 strain, and six non-O1, non-O139 strains, regardless of the sou rce of isolation, caused fluid accumulation after two to five serial passag es through the rabbit gut. Culture filtrates of all non-cholera-toxigenic s trains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKT medium by these strains. Studies of clonalit y performed by using enterobacterial repetitive intergenic consensus sequen ce PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-held gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O 139 strains belonged to different clones. The clinical isolates closely res embled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE anal yses, and individual strains derived from clinical and environmental source s produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V; cholerae, whe ther of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced b y toxigenic V; cholerae O1 and O139 strains. We also concluded that the aqu atic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O 139 serogroup strains.