Analysis of clinical and environmental strains of nontoxigenic Vibrio cholerae for susceptibility to CTX Phi: Molecular basis for origination of new strains with epidemic potential

Toxigenic Vibrio cholerae strains are lysogens of CTX Phi, a filamentous ph age which encodes cholera toxin. The receptor for CTX Phi for invading V. c holerae cells is the toxin-coregulated pilus (TCP), the genes for which res ide in a larger genetic element, the TCP pathogenicity island. We analyzed 146 CTX-negative strains of V. cholerae O1 or non-O1 isolated from patients or surface waters in five different countries for the presence of the TCP pathogenicity island, the regulatory gene toxR, and the CTX Phi attachment sequence attRS, as web as for susceptibility of the strains to CTX Phi, to investigate the molecular basis for the emergence of new clones of toxigeni c V. cholerae, DNA probe or PCR assays for tcpA, tcpI, acfB, toxR, and attR S revealed that 6.85% of the strains, all of which belonged to the O1 serog roup, carried the TCP pathogenicity island, toxR, and multiple copies of at tRS, whereas the remaining 93.15% of the strains were negative for TCP but positive for either one or both or neither of toxR and attRS. An analysis o f the strains for susceptibility to CTX Phi, using a genetically marked der ivative of the phage CTX-Km Phi, showed that all TCP-positive CTX-negative strains and 1 of 136 TCP-negative strains were infected by the phage either in vitro or in the intestines of infant mice, The phage genome integrated into the chromosome of infected V. cholerae O1 cells forming stable lysogen s, Comparative analysis of rRNA gene restriction patterns revealed that the lysogens derived from nontoxigenic progenitors were either closely related to or distinctly different from previously described clones of toxigenic V . cholerae, To our knowledge, this is the first demonstration of lysogenic conversion of naturally occurring nontoxigenic V. cholerae strains by CTX P hi. The results of this study further indicated that strains belonging to t he O1 serogroup of V, cholerae are more likely to possess the TCP pathogeni city island and hence to be infected by CTX Phi, leading to the origination of potential new epidemic clones.