Thereafter, genomic sequencing of the non-O1, non-O139 V. cholerae strain AM-19226 revealed that V. cholerae carry T3SS genes related to V. parahaemolyticus T3SS2 in VPI-2 [8]. Additionally, in the infant mouse model T3SS in V. cholerae is needed for efficient intestinal colonization; the effector proteins have

already been characterized [9-11]. Therefore, in addition to CT, T3SS in V. cholerae is another possible virulence determinant. The T3SS gene cluster is distributed among various non-O1, non-O139 strains [8, 12] and a phylogenetic analysis of T3SS-related genes implied horizontal gene transfer of a T3SS gene cluster among Vibrio species [13, 14]. Up to now, however, there has been no experimental evidence of horizontal transfer of the T3SS-related genes. We herein examined the distribution of T3SS-related genes among various serogroups of V. cholerae isolates and found that the cassette this website of T3SS-related genes was transferrable among V. cholerae isolates by transformation, and that these subsequently integrated into a VPI-2. V. cholerae strains used in this study were isolated from natural surface water (environmental; 110 isolates) and diarrhea patients (clinical;

14 isolates) in Bangladesh. These V. cholerae isolates were obtained from the culture collection Small molecule library purchase of the International Center for Diarrhoeal Disease Research, Bangladesh. All 124 isolates, which were primarily confirmed as cholera toxin gene (ctxAB) negative V. cholerae serogroups non-O1/non-O139, were screened by PCR assays with three sets of primer pairs (T3SS-1, T3SS-2 and T3SS-3; Table 1) to detect T3SS-related genes. The primer pair of T3SS-1 amplified a target gene of A33_1670, which encodes structural protein. The primer pairs of T3SS-2 and T3SS-3 targeted genes for translocated effector proteins of A33_1684 and A33_1697, respectively. All primers were designed in the conserved sequence of each gene. The PCR conditions were as follows: after initial denaturation at 95°C for 2 mins,

25 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 20 s and extension at 72°C for 1 mins; and final extension at 72°C Arachidonate 15-lipoxygenase for 3 mins with TaKaRa Ex Taq (Takara Bio, Shiga, Japan). The amplified fragments were detected by agarose gel electrophoresis after staining with ethidium bromide. Strains producing the three amplicons from the three primer pairs were defined as positive for T3SS-related genes. Subsequently, strains positive for T3SS-related genes were serogrouped by slide agglutination using a panel of specific antisera for each serogroup of V. cholerae. To evaluate the genetic similarity between T3SS-related gene regions, a PCR-RFLP analysis was performed with the positive strains identified as described above. Because the long length of the whole locus precluded its amplification with one primer pair, it was divided it into seven regions (ca. 5–10 kb) to ensure successful amplification with seven sets of primer pairs (RFLP-1 to RFLP-7; Table 1).