Aims:  The objective of this study was to investigate the molecular diversity of CTX genetic element within toxigenic Vibrio cholerae genomes and to determine the genetic diversity of V. cholerae population collected in a 6‐year period (2004–2009) in Iran. Methods and Results:  The results of mismatch amplification mutation assay (MAMA)‐PCR and sequencing showed cytosine nucleotide in positions 203 and 115 in all 50 El Tor V. cholerae strains, which is the same as classical ctxB sequence. One strain yielded amplicons with both El Tor and classical biotype primers in MAMA‐PCR indicative of presence of two copies of CTX phages with different genotypes (rstRET ctxBclass and rstRET ctxBET) integrated within the genome of this isolate, which suggested the integration of two different CTX phages at different occasions or point mutation in one copy of CTX. Sequencing and PCR analysis indicated the presence of hybrid CTX genotype (rstRET ctxclass) in 70·6% of the isolates; however, only El Tor RS1 phage has been integrated in flanking to the CTX phages with different genotypes. Conclusions:  Enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR) and ribosomal gene spacer‐PCR (RS‐PCR) showed a relatively homogenous population in different years. Our findings indicate that sequence analysis of RS and ctxB regions has more discriminative power than restriction‐based methods. Significance and Impact of the Study:  Investigating the molecular diversity of CTX prophage among V. cholerae strains helps to establish a new valuable database of genetic information about isolates, which is of great importance for epidemiologic studies in Iran and other countries encountering cholera epidemics.

Aims:  To investigate the molecular basis for serotype variation in Vibrio cholerae O1 and the genetic relatedness amongst different serotypes isolated from 2004 to 2008 in Iran. Methods and Results:  Despite the presence of all three serotypes of V. cholerae O1 (Ogawa, Inaba and Hikojima) in Iran in the last decade, the Inaba strains have been the dominated serotype. Sequence analysis of wbeT determined only a single substitution of G for A at position 295 in all Inaba strains resulting in a replacement of serine to proline. No difference was found in the copy numbers and profile of IS1004 between the classical and El Tor V. cholerae O1 strains, supporting the clonality amongst the isolates obtained over 5 years in Iran. In addition, Southern blots of HpaII‐digested chromosomal DNAs of our Ogawa and Inaba isolates showed the presence of an incomplete copy of IS1004 for all isolates. Conclusions:  IS1004 profiling can be a reliable method for analysis of clonal dissemination of V. cholerae. The results indicated that specific point mutation at a particular position within the wbeT of V. cholerae O1 strains in Iran may occur which, in turn, may result in serotype switching. Significance and Impact of the Study:  Understanding the molecular basis for serotype conversion of V. cholerae and their genetic relatedness could give insights for the incoming cholera epidemic prediction and control.

Cholera toxin (CT) is the major virulence factor produced by Vibrio cholerae. Several genomic arrangements within the CTX cassette have been elucidated in V. cholerae. Previously, it was shown that three different CTX cassette arrangements, one complete CTX cassette (arrangement A), one complete and two incomplete CTX cassettes (arrangement B), and two complete CTX cassettes (arrangement C), exist within V. cholerae isolates. In the present study, the level of CT expression by V. cholerae isolates carrying different CTX cassette arrangements was evaluated. Real-time quantitative PCR analysis showed unequal production of CT mRNA in V. cholerae isolates with different CTX arrangements. V. cholerae with the CTX arrangement C expressed more CT mRNA than isolates with the other CTX arrangements. In addition, CT mRNA was expressed more in the isolates with CTX arrangement B than in those with arrangement A. Overall, these results suggest that the arrangement and number of regulatory elements (rstA) within the CTX cassette could affect the level of expression of CT.

The emergence of a single environmental isolate in our study containing the pathogenicity genes amongst the diverse non-pathogenic environmental isolates needs to be further studied in the context of V. cholerae pathogenicity sero-coversion.