A search for cholera toxin (CT), toxin coregulated pilus (TCP), the regulatory element ToxR and other virulence factors in non-01/non-0139 Vibrio cholerae

Twenty-four selected non-O1/non-O139 Vibrio cholerae strains were examined for the presence of virulence associated genes like ctxA, tcpA, toxR and the repetitive sequence (RS element). Seventeen of these were isolated from diarrhoeal stool samples while the remaining seven were of local environmental origin. Nine and four respectively of these strains were positive for ctxA and tcpA by Multiplex PCR analysis. The majority (16 out of 18 tested) of the strains (including the four tcpA + strains) contained toxR sequences as determined by another PCR assay. The presence of RS element was demonstrable in ctxA(+) strains only. Interestingly, three of these non-O1/non-O139 strains were shown to contain all the three virulence associated genes (ctxA, tcpA and toxR) as well as the RS element. Two of these belonged to serogroups 037 (V2) and 064 (CG15) while the third one (V315-1) was untypable. These three strains also produced cholera toxin, expressed toxin coregulated pilus (TCP) and/or TcpA related antigens when grown under appropriate culture conditions. Southern hybridization analysis of their chromosomal DNA fragments using DNA probes representing ctxA, zot, ace and RS element revealed that the strains V2 and CG15 contained, at least, two complete copies of the CTX genetic element, while the strain V315-1 had three or more copies of the same. Presence of the RS element in these strains led to tandem duplication of the CTX genetic element in the chromosome of V2 and V315-1, but not in CG15 where the copies were likely to be present at different loci. These results also indicate the presence of additional copies of incomplete 'core region' with zot and ace genes, but not ctxA, in strains V2 and CG15. The significance of these results in terms of the pathogenic and epidemic potential of V. cholerae strains is discussed. (C) 1997 Academic Press Limited.