Molecular analysis of toxigenic vibrio cholerae strains isolated in Bangladesh during 1961-1996: relationship between continual emergence of new toxigenic clones and epidemics of cholera

Abstract

Objective: Analyze virulence-associated genes, and study clonal relationships among toxigenic Vibrio cholerae strains isolated in Bangladesh during 1961-1996 to establish whether seasonal epidemics are caused by repeated emergence of the same strains or by diverse clones of toxigenic V cholerae. Methodology: Three hundred seventy-eight V. cholerae isolates obtained from cholera patients or from surface water were included in the study. Molecular analysis of virulence-associated gene clusters, including the CTX genetic element, tcpA, tcpl, and ToxR was performed using specific probes or PCR assays. Comparative analysis of serotype-specific rfb gene clusters in selected V. cholerae 01 and O139 strains was performed by multiple PCR assays using primers corresponding to defined regions of the rfb genes. Clonal relationships among strains were studied by computer-assisted numerical analysis of restriction fragment length polymorphisms in conserved rRNA genes. Induction and propagation of the lysogenic bacteriophage-encoding cholera toxin (CTX?) were studied both in animal models and under laboratory conditions. Results: Analysis of toxigenic V. cholerae strains isolated during 1961-1996 revealed clonal diversity among the strains isolated during different epidemics. This study demonstrated the transient appearance and disappearance of more than 6 different clones among classical vibrios, at least 5 clones of El Tor vibrios, and 3 different clones of V. cholerae 0139. Different clones of strains belonged to different ribotypes and often to different CTX genotypes resulting from differences in the copy number of CTX element and variations in the integration site of CTX in the chromosome. Studies on the induction of lysogenic CTX? revealed that 37.95% of the strains tested could be induced with mitomycin-C to produce infectious extracellular CTX? particles which infected recipient strains under conditions conducive to the expression of tcp genes.