Molecular characterisation of rough variants of Vibrio cholerae isolated from hospitalised patients with diarrhoea.

Seven rough isolates of Vibrio cholerae isolated as the sole infecting agent from patients with cholera-like diarrhoea were examined for the presence of the regulatory element toxR and certain virulence-associated genes of the CTX genetic element and V. cholerae pathogenicity island (VPI). Multiplex PCR analysis with wb-specific genes of either O1 or O139 origin showed that six of the seven isolates produced an O1 wb-specific amplicon and the remaining isolate produced an O139-specific amplicon. Analysis of lipopolysaccharide profiles of smooth variants of V. cholerae revealed the presence of long repeated units of 'O' polysaccharide side chains but all the rough variants appeared to be devoid of the latter and possessed only core oligosaccharide. PCR amplification with primers specific to the ctxA, ctxB, tcpA, tagA, int, aldA, toxT, LJ, RJ and toxR genes revealed that six of the seven rough isolates were positive for these genes. One isolate was found to be negative for tagA and RJ, indicating the presence of an altered VPI. Each of these isolates showed media-dependent expression of cholera toxin (CT) and produced more toxin than the reference V. cholerae O1 El Tor strain VC20 or O139 strain SG24 under comparable conditions. Studies on the clonality of these isolates by the analysis of rRNA genes indicated their relatedness to strains of V. cholerae O1 El Tor or O139, isolated during the same time period.