Analysis of clinical and environmental strains of nontoxigenic Vibrio cholerae for susceptibility to CTXPhi: molecular basis for origination of new strains with epidemic potential

Abstract

Toxigenic Vibrio cholerae strains are lysogens of CTXPhi, a filamentous phage which encodes cholera toxin. The receptor for CTXPhi 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 gene toxR, and the CTXPhi attachment sequence attRS, as well as for susceptibility of the strains to CTXPhi, to investigate the molecular basis for the emergence of new clones of toxigenic V. cholerae. DNA probe or PCR assays for tcpA, tcpI, acfB, toxR, and attRS 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 and attRS. An analysis of the strains for susceptibility to CTXPhi, using a genetically marked derivative of the phage CTX-KmPhi, 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 CTXPhi. 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 CTXPhi, leading to the origination of potential new epidemic clones.

FIG. 1

Susceptibility of TCP-positive and TCP-negative nontoxigenic V. cholerae O1 and non-O1 strains to CTX-KmΦ under in vitro laboratory conditions and in the intestines of infant mice. Strains SA-317, SA-406, SA-590, and SA-674 are TCP-positive V. cholerae O1 clinical strains isolated in Saudi Arabia; strains W-03, W-05, W-06, W-07, W-10, and W-21 are TCP-positive V. cholerae O1 clinical strains isolated in India; strain 55-V71 is a TCP-negative non-O1, non-O139 environmental strain isolated in Bangladesh. Values represent the averages of five independent observations.

FIG. 2

Southern hybridization analysis of genomic DNA or plasmids isolated from V. cholerae strains infected with CTX-KmΦ and the corresponding native strains. Total DNA or plasmids were digested with BglI (A) or BglII (B) and probed with an 850-bp PCR-generated probe for the zot gene. Lane 1, pCTX-Km linearized with BglI; lanes 2 and 7, total DNA from SA-317(pCTX-Km); lanes 3 and 8, total DNA from SA-317 (native); lanes 4 and 9, total DNA from SA-406(pCTX-Km); lanes 5 and 10, total DNA from SA-406 (native); lanes 6 and 11, total DNA and plasmid, respectively, from 55V-71(pCTX-Km); lane 12, total DNA from 55V-71 (native). Integration of CTX-KmΦ DNA into the chromosome of recipient V. cholerae O1 strains SA-317 and SA-406 is shown, whereas in non-O1 strain 55V-71, the phage genome is shown to be present in the RF. Numbers indicating the molecular sizes of bands correspond to a 1-kb DNA ladder (Bethesda Research Laboratories).

FIG. 3

BglI restriction patterns of rRNA genes of nontoxigenic V. cholerae O1 strains compared to those of selected toxigenic strains of V. cholerae O1 isolated from epidemic outbreaks of cholera. A Southern blot of BglI-digested genomic DNA was hybridized with the 7.5-kb BamHI fragment of the E. coli rRNA clone pKK3535. Lanes 1 and 2, TCP-negative CTX-KmΦ-resistant strains Fin-41642 and Fin-41643, isolated from seawater in Estonia and Latvia, respectively, in 1995; lanes 3 through 7, TCP-positive CTX-KmΦ-susceptible strains W-03, W-05, W-07, W-10, and W-21 respectively, isolated from patients in India in 1993; lanes 8 and 9, TCP-positive CTX-KmΦ-susceptible strains SA-317 and SA-674, respectively, isolated from patients in Saudi Arabia in 1997; lanes 10 through 15, epidemic strains of toxigenic V. cholerae isolated in different countries (the strains and their countries and years of isolation are as follows: AK-21445, Bangladesh, 1995; Syria-5, Syria, 1992; AF-9710, Bangladesh, 1990; 62-6-91, Tanzania, 1991; AG-19438, Bangladesh, 1991; and AH-806, Bangladesh, 1992). Numbers indicating molecular sizes of bands correspond to a 1-kb DNA ladder (Bethesda Research Laboratories).