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

Abstract

The ctxAB genes encoding cholera toxin, reside in the genome of a filamentous bacteriophage CTXphi. The presence of CTX prophage in non-epidemic environmental Vibrio cholerae strains is rare. The CTX prophage, the lysogenic form of CTXphi in V. cholerae, is comprised of the 'RS2' and the 'Core'. Analysis of the rstR gene present in the RS2 region of the CTX prophage revealed the presence of new alleles of the prophages in four environmental non-O1, non-O139 strains VCE22 (O36), VCE228 (O27), VCE232 (O4) and VCE233 (O27), and the CTX prophages are located in the small chromosomes. Phylogenetic analysis based on the nucleotide sequences of the rstR and orfU (present in the core) genes of these prophages placed them in a single unique cluster, which is distally located compared with that of epidemic V. cholerae O1 strains. Further analysis indicated that the genome of the prophage present in the strain VCE22 is devoid of the ctxAB genes, called pre-CTX prophage and the strain also possess the toxin-coregulated pilus protein coding gene tcpA of classical type, another important pathogenicity determining locus of the epidemic V. cholerae strains. Comparative analysis of the nucleotide sequences of the rstR and orfU genes indicated that the pre-CTX prophage of VCE22 might be the progenitor of new alleles of the CTX prophages present in these environmental strains.

Vibrio cholerae O1 clinical strains isolated in 1992 in Kolkata with progenitor traits of the 2004 Mozambique variant

“…Among the constituent genes of the CTX prophages, heterogeneity at the nucleotide level of rstR, the phage (Davis et al, 1999;Kimsey & Waldor, 1998). A PCR-based assay has been developed by exploiting the variations that exist between the rstR loci of the CTX Cl and CTX El phages (Bhattacharya et al, 2006). PCR-based screening was carried out using allele-specific rstR against clinical V. cholerae strains isolated in 1992 in Kolkata.…”

“…The bacterial strains were maintained in nutrient agar stabs and stored in the dark. Fifteen V. cholerae O1 strains including VC20 (Bhattacharya et al, 2006), were considered in this study. Reference V. cholerae O1 strains of classical (569B, O395) and El Tor (N16961) biotypes were included as a comparison.…”

“…Purified genomic DNA was subjected to ethanol precipitation, airdried, reconstituted in sterile water and stored at 4 uC. The DNA was then used in the PCR assay to detect the rstR alleles (Bhattacharya et al, 2006). Detection of 474 or 501 bp amplicons corresponded to the presence of rstR alleles of classical (rstR Cl ) or El Tor (rstR El ) types, respectively.…”

“…Previously, we have reported on the variant traits of V. cholerae O1 El Tor strain VC20 that possessed both classical and El Tor alleles of rstR, one of the constituent genes of CTX prophage (Bhattacharya et al, 2006); the strain was isolated in 1992 from clinical cases. Detection of such a variant prompted us to revive archived V. cholerae O1 strains to screen them for traits similar to that of the Mozambique variant isolated previously.…”

Genetic Analysis of CTX Prophage and Antibiotic Resistance Determinants in Vibrio cholerae O1 Belonging to the Atypical El Tor Biotype from Kelantan, Malaysia

“…The type of rstR gene in each isolate was determined using a set of allele-specific forward primers (rstR El Tor , rstR Classical , rstR Calcutta and rstR Environment ) and a common reverse primer, rstRR (Nusrin et al, 2004;Bhattacharya et al, 2006). A list of all primers used in this study is presented in Table 1.…”