The composition and gene arrangement of the CTX genetic element were compared in 36 Vibrio cholerae isolates obtained during 2004-2006 from Iran. Long-PCR amplification of the CTX genetic element, using primers targeting ig1 and attB2, revealed three PCR products of c. 6.9, 5.6 and 2.6 kb, respectively. Southern blot hybridisation revealed that 30%, 17% and 53% of the isolates had one, two and three copies of the zot gene, respectively. PCR analysis of internal regions showed that isolates with three copies of the CTX genetic element carried one complete (6.9 kb) and two truncated CTX elements (each of 5.6 kb). In contrast, isolates with one or two copies of CTX carried the complete 6.9-kb CTX element. Pulsed-field gel electrophoresis revealed two pulsotypes among the isolates, with 75% of the isolates belonging to pulsotype 2. The pulsotype 2 isolates had varying CTX genomic arrangements, whereas the pulsotype 1 isolates had a homogeneous CTX arrangement. Thus, variations in the content, arrangement and copy number of the CTX genetic element may occur in isolates belonging to the same clone.

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF)

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.

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