coli 803 strain. Mating assays were performed by mixing equal volumes of overnight cultures
of donor and recipient strains. Briefly, the cells were harvested by centrifugation and resuspended in a 1/20 volume of LB broth. Cell suspensions were poured onto LB agar plates and incubated at 37°C for 6 h. The cells were then resuspended in 1 ml of LB medium, and serial dilutions were plated onto appropriate selective media to determine the numbers of donors, recipients, and exconjugants. Frequency of transfer was expressed as the number of exconjugant PD-1/PD-L1 Inhibitor 3 cells per donor cells in the mating mixture at the time of plating. V. cholerae O139 MO10 , V. cholerae E4:ICEVchInd1 , V. cholerae O1 VC20 , V. cholerae N16961 , V. cholerae O1 CO840 , V. cholerae APR-246 O1 VC7452, VC15699, and VC9258 isolated in India (Maharashtra) , and E. coli AB1157:R391  were appropriately used as negative or positive controls for class 1 integrons, ICE, tcpA, and rstR detection, CTXΦ array and ribotype analysis. Molecular biology procedures Bacterial
DNA for PCR analysis was prepared with a Wizard Genomic DNA Purification kit (Promega). Amplicons to be sequenced were directly purified from PCR or extracted from agarose gel by Wizard SV Gel and PCR Clean-up System (Promega) according to the manufacturer’s instructions. DNA sequences were IPI-549 determined by BMR Genomics (Padova, Italy). Class 1 integron detection was performed by PCR amplification with specific primer pairs as previously described . ICEs of the SXT/R391 family were screened by PCR analysis, using 17 specific primer pairs previously described by our group [25, 26]. int SXT, prfC/SXTMO10 right junction, floR, strA, strB, sul2, dfrA18, dfrA1, rumAB operon, traI, traC, setR,
and Hotspots or Variable Regions s026/traI, s043/traL, traA/s054, s073/traF during and traG/eex were screened. A second set of 15 primer pairs designed on the specific sequences of ICEVchInd5  were used to detect ICEVchInd5 and ICEVchAng3 specific Hotspots and Variable Regions. All PCR reactions were set in a 50-μl volume of reaction buffer containing 1 U of Taq polymerase as directed by the manufacturer (Promega). Ribotype analysis Ribotyping of V. cholerae strains was performed by BglI restriction of chromosomal DNA with fluorescent-labeled 16S and 23S DNA (Gene Images 3540 RPn3510, Amersham) generated by reverse transcriptase polymerase chain reaction of ribosomal RNAs, as already described . CTX array analysis and ctxB, tcpA, rstR biotype characterization The structure of CTX array was determined by multiple PCR analysis (Table 2) and by Southern Blot hybridization. The genetic structure of the two CTX prophage arrays described in Figure 1 was determined using the primers described in Table 2.