Diverse CTX Phages among Toxigenic Vibrio cholerae O1 and O139 Strains Isolated between 1994 and 2002 in an Area Where Cholera is Endemic in Bangladesh

Abstract

PCR surveillance of the rstR genes of CTX phages in Vibrio cholerae O1 and O139 showed no relationship between the incidence of disease and changes in the rstR but showed variations in their presence in O1 and O139 strains and the occurrence of multiple types in a few strains.Of the 209 currently recognized serogroups of Vibrio cholerae, only strains belonging to serogroups O1 and O139 can cause cholera. Two major virulence gene clusters are now known to carry key virulence genes that are essential for the pathogenicity of V. cholerae O1 and O139. These gene clusters include the CTX prophage (14), which carries the ctxA and ctxB genes (the genes that encode cholera toxin [CT], which is responsible for severe diarrhea), and the toxin-coregulated pilus (TCP) pathogenicity island, which carries genes for the biosynthesis of the TCP, required for colonization of the small intestinal epithelium (7).The approximately 7-kb CTX genome consists of the core and the RS2 region. The core region encodes proteins needed for the assembly and secretion of viral particles (Psh, Cep, pIII CTX , Ace, and Zot) and also encodes CT, which is not necessary for phage morphogenesis (3), while the RS2 region represents a site-specific recombination system that allows lysogenic phage to integrate at a specific site on the host chromosome (14). The RS2 region of CTX prophage encodes proteins required for replication (RstA), phage integration (RstB), and regulation (RstR) of the lysogeny of CTX (14). An antirepressor, rstC, is carried by a satellite phage, RS1, often present adjacent to the CTX prophage in toxigenic V. cholerae O1 El Tor and O139 strains (1, 5).Diversity of the CTX phage repressor rstR has been described previously, and this diversity constitutes heteroimmunity among diverse CTX phages (8, 2). The difference in the rstR gene is also the only known genetic difference between any two different CTX phage types. The existence of at least four different rstR genes carried by different CTX phages, namely, CTX ET , CTX class , CTX Calc , and CTX Env , has been recognized (8, 2, 10). The epidemiological significance of the diversity of CTX phages is not clearly known, but at least two periods of explosive resurgence of cholera have been associated with strains showing changes in the rstR type of CTX phages. The first was the resurgence of V. cholerae O139 in August 1996 in Calcutta, India, which continued for a year (8, 9, 13), and the second was the resurgence of strain O139 in March to April of 2002 in Dhaka, Bangladesh (6). On the basis of their rstR genes and other phenotypic traits, genetic hybrids of classical and El Tor biotypes that cause cholera have been shown to exist, and these hybrids have been designated the Matlab variants of V. cholerae (11). To further document the distribution and temporal changes in the CTX phage contents of epidemic strains, we conducted a surveillance of CTX phage types by analyzing the types of rstR genes carried by a large collection of toxigenic V. cholerae strains.We selected every 10th con...

Characterization of Vibrio cholerae O1 El Tor Biotype Variant Clinical Isolates from Bangladesh and Haiti, Including a Molecular Genetic Analysis of Virulence Genes

“…These base transitions, which result in amino acid conversions of H 39 Y and T 68 I, respectively, are completely conserved within each biotype, allowing them to be used as reliable markers for biotype characterization. These clinical El Tor O1 strains were found to contain the classical biotype cholera toxin B subunit gene (ctxB) and have since been termed El Tor variants (34,39). These El Tor variants have become a major focus of research, since other El Tor variants also have been identified in Asia and Mozambique (29,34,35,38) and recently have been a focus of attention in Haiti (1,6,7,8,9).…”

Cholera Outbreaks Caused by an Altered Vibrio cholerae O1 El Tor Biotype Strain Producing Classical Cholera Toxin B in Vietnam in 2007 to 2008

“…No DNA fragment was amplified from the Vietnamese isolates with the classical rstR-specific primer set (rstRclaF/rstAR). For additional confirmation of the absence of other types of rstR, namely, rstR Cal (O139 type) and rstR env (environmental type), we used PCR primers rstRCalF/rstRCalR and rstREnvF/rstREnvR, and no PCR product was identified from the Vietnamese isolates (11). Presence and the location of rstC (RS1).…”

Isolation of Vibrio cholerae O1 strains similar to pre-seventh pandemic El Tor strains during an outbreak of gastrointestinal disease in an island resort in Fiji

“…All strains were tested for susceptibility to polymyxin B (50 U), chicken red cell agglutination (CCA) and sensitivity to group IV classical and group 5 El Tor phages as described in World Health Organization (1987). To complement the biotype characterization by genetic traits, PCR assays targeted to detect tcpA (classical and El Tor variant) (Keasler & Hall, 1993) and the rstR gene encoding the phage transcriptional regulator were performed using procedures described elsewhere (Davis et al, 1999;Kimsey et al, 1998;Nusrin et al, 2004).…”