Analysis of Clinical and Environmental Strains of Nontoxigenic Vibrio cholerae for Susceptibility to CTXΦ: Molecular Basis for Origination of New Strains with Epidemic Potential

Abstract

Toxigenic Vibrio cholerae strains are lysogens of CTXΦ, a filamentous phage which encodes cholera toxin. The receptor for CTXΦ for invading V. cholerae cells is the toxin-coregulated pilus (TCP), the genes for which reside 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 genetoxR, and the CTXΦ attachment sequence attRS, as well as for susceptibility of the strains to CTXΦ, to investigate the molecular basis for the emergence of new clones of toxigenicV. cholerae. DNA probe or PCR assays for tcpA,tcpI, acfB, toxR, andattRS revealed that 6.85% of the strains, all of which belonged to the O1 serogroup, carried the TCP pathogenicity island,toxR, and multiple copies of attRS, whereas the remaining 93.15% of the strains were negative for TCP but positive for either one or both or neither of toxR andattRS. An analysis of the strains for susceptibility to CTXΦ, using a genetically marked derivative of the phage CTX-KmΦ, 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 lysogens. 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 CTXΦ. The results of this study further indicated that strains belonging to the O1 serogroup of V. cholerae are more likely to possess the TCP pathogenicity island and hence to be infected by CTXΦ, leading to the origination of potential new epidemic clones.

Comparative analysis of cytotoxin, hemolysin, hemagglutinin and exocellular enzymes among clinical and environmental isolates of Vibrio cholerae O139 and non-O1, non-O139

“…2) which controls the coordinated expression of genes associated with pathogenicity in toxigenic V. cholerae O1 and O139. Previously, Mekalanos [17] had reported the absence of TCP in non-O1 strains which do not produce CT. Three clinical isolates of O139 and four O139 strains isolated from lake water during the O139 epidemic in Calcutta were positive for both ctx and tcpA gene in PCR assays. All the O139 strains yielded O139 rfb-speci¢c amplicon while non-O1, non-O139 strains did not yield either O1 rfb or O139 rfb (Fig.…”

Molecular analysis of the rstR and orfU genes of the CTX prophages integrated in the small chromosomes of environmental Vibrio cholerae non‐O1, non‐O139 strains

“…Thus, it appears that the acquisition of vibrio pathogenicity island (VPI) containing the TCP biosynthesis gene clusters is the initial event for the origination of toxigenic V. cholerae. However, existence of alternate mechanism for CTXφ infection without involving TCP has also been indicated for non-O1, non-O139 strains (Faruque et al, 1998). Nucleotide sequence analysis of the rstR allele of environmental CTX prophages indicated that all these were of similar type.…”

Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia, Italy

“…The receptor for this phage is the toxin-coregulated pilus (TCP) (essential for the bacterial colonization of human and animal intestine). Faruque et al (1998b) reported that the tcpA gene, which is part of a larger genetic element called the TCP pathogenicity island, could be of phage origin and it has been speculated that it could be transferred by transducing phages (Gosh et al 1997;Faruque et al 1998a, b). Expression of CT and TCP is coregulated by the regulatory proteins ToxR and ToxS (Faruque et al 1998a;Gosh et al 1997;Pfau and Taylor 1998;Wong et al 1998;Skorupski and Taylor 1999).…”

“…Furthermore, no other information is available on the presence of V. cholerae virulence genes in other Vibrio species. As it has been suggested that V. cholerae strains may arise from non-toxigenic V. cholerae O1 strains positive for TCP and ToxR by infection with CTXf (Faruque et al 1998b) and virulence genes reported in other Vibrio strains (Manning et al 1999), a need was identified for an investigation into the dissemination of these genes among other Vibrio species.…”