Accessory colonization factor is located immediately adjacent to and downstream of TCP cluster. These genes (acfA, B, C, D) are involved in bacterial colonization and pathogenesis. The aim of this study was to analyze the ACF cluster prevalence rate and gene content in clinical isolates of Vibrio cholerae. Materials and Methods: All of the 21 V. cholerae isolates used in this study were collected during 2011-2012 outbreaks in Iran. All of the isolates were screened by biochemical tests and

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... rmed by specific PCR for 16srRNA-23srRNA intergenic space. The gene content of ACF cluster in the isolates was analyzed using 4 primer pairs with overlapping sequences and then subjected into Restriction Fragment Length Polymorphism (RFLP) assay. Results: Among the 21 V. cholerae isolates, all of them (100%) were identified as V. cholerae O1 Inaba, 20 (95%) isolates were determined with El Tor biotype specificity, and 1 isolate (5%) appeared as Classical biotype. A total of 18 strains (85.8 %) contained a complete set of ACF-associated genes, 3 strains (14.2 %) were negative for ACF cluster, and all of the strains showed similar RFLP pattern to each other and to V. cholerae ATCC 14035. Conclusion: The results showed that O1 Inaba was the dominant serotype and positive for ACF cluster in pathogenic V. cholerae isolates collected during 2011-2012 in Iran. The presence of some ACF negative strains with potentially pathogenic characteristics proposed that other colonization factors might have been involved in causing pathogenicity and diarrhea in these strains.