Sequence analysis of Vibrio cholerae orfU and zot from pre-CTXΦ and CTXΦ reveals multiple origin of pre-CTXΦ and CTXΦ

Summary A multiplex PCR was developed to detect pre‐CTXΦ and CTXΦ in Vibrio cholerae. A total of 115 V. cholerae were tested, of which 42 V. cholerae O1 and 18 V. cholerae O139 contained CTXΦ. Six V. cholerae O139 contained only pre‐CTXΦ and three V. cholerae O1 and 23 V. cholerae O139 contained both pre‐CTXΦ and CTXΦ. None of the V. cholerae non‐O1 and non‐O139 that were tested had pre‐CTXΦ or CTXΦ. Results of Restriction Fragment Length Polymorphism (RFLP) analysis revealed the V. cholerae isolates possessed single or multiple copies of pre‐CTXΦ and CTXΦ, always proceeded by a tandemly arranged RS1 element. Comparative nucleotide sequence analyses of the core region genes, orfU and zot, of 15 V. cholerae showed pre‐CTXETΦ and CTXETΦ lineage with V. cholerae El Tor and pre‐CTXClassΦ, pre‐CTXCalcΦ, and CTXCalcΦ with classical V. cholerae O1 and O139. Two distinct types of ctxB were detected in V. cholerae O139. Multi‐locus Sequence Typing (MLST) of seven V. cholerae housekeeping genes indicated clonal origin, irrespective of the presence of pre‐CTXΦ and/or CTXΦ.