Clinical and environmental isolates of Vibrio cholerae serogroup O141 carry the CTX phage and the genes encoding the toxin-coregulated pili

Abstract

We report sporadic cases of a severe gastroenteritis associated with Vibrio cholerae serogroup O141. Like O1 and O139 serogroup strains of V. cholerae isolated from cholera cases, the O141 clinical isolates carry DNA sequences that hybridize to cholera toxin (CT) gene probes. The CT genes of O1 and O139 strains are carried by a filamentous bacteriophage (termed CTX phage) which is known to use toxin-coregulated pili (TCP) as its receptor. In an effort to understand the mechanism of emergence of toxigenic O141 V. cholerae, we probed a collection of O141 clinical and environmental isolates for genes involved in TCP production, toxigenicity, virulence regulation, and other phylogenetic markers. The collection included strains isolated between 1964 and 1995 from diverse geographical locations, including eight countries and five U.S. states. Information collected about the clinical and environmental sources of these isolates suggests that they had no epidemiological association. All clinical O141 isolates hybridized to probes specific for genes encoding CT (ctx), zonula occludens toxin (zot), repetitive sequence 1 (RS1), RTX toxin (rtxA), the major subunit of TCP (tcpA), and the essential regulatory gene that controls expression of both CT and TCP (toxR). In contrast, all but one of the nonclinical O141 isolates were negative for ctx, zot, RS1, and tcpA, although these strains were positive for rtxA and toxR. The one toxigenic environmental O141 isolate was also positive for tcpA. Ribotyping and CT typing showed that the O141 clinical isolates were indistinguishable or closely related, while a toxigenic water isolate from Louisiana showed a distantly related ribotype. Nonclinical O141 isolates displayed a variety of unrelated ribotypes. These data support a model for emergence of toxigenic O141 that involves acquisition of the CTX phage sometime after these strains had acquired the pathogenicity island encoding TCP. The clonal nature of toxigenic O141 strains isolated from diverse geographical locations suggests that the emergence is a rare event but that once it occurs, toxigenic O141 strains are capable of regional and perhaps even global dissemination. This study stresses the importance of monitoring V. cholerae non-O1, non-O139 serogroup strains for their virulence gene content as a means of assessing their epidemic potential.

FIG. 1

Genomic organization of the pTLC (TLC) element, the CTX prophage, and the RTX gene cluster on the V. cholerae chromosome. The pTLC element is 842 bp upstream of the CTX element whereas the RTX gene cluster is 693 bp downstream of the CTX element. Open and filled boxes represent ORFs that are oriented left to right and right to left, respectively. The pTLC element is tandemly duplicated on the chromosome. In El Tor strains, the first copy contains an insertion sequence that shares sequence similarity to IS911. The CTX prophage is also commonly tandemly duplicated in toxigenic strains. In classical strains, the RTX gene cluster is disrupted by a deletion that removes part of rtxA and rtxB and all of rtxC.

FIG. 2

Examples of BglI ribotypes of V. cholerae O141 and serotype O1 and O139 reference strains. Lanes (strain designation, O serogroup, and ribotype): B, O79, O1, unnamed ribotype; C, 1083/30, O1, unnamed type; D, 2722/33, O1, unnamed type; E, ADC 1125/9, O139, unnamed type; F, NIH 178, O139, unnamed type; G, 3176-78, O141, ribotype E; H, 609-84, O141, type B; I, 2527-86, O141, type A; J, F2031, O141, type C; K, 574-94, O141, type D; L, 930122, O141, type F; M, CH236, O141, type G; N, 827-95, O141, type H; O, 834-95, O141, type I. Lanes A and P are 1-kb molecular size standards.