Comparison of the ICEs characterized in this study with other known elements provided further evidence for the presence of extensive genetic recombination amongst SXT/R391 ICEs, which lead to three major molecular snapshots. Firstly, none of the ICEs analyzed here displays identical gene organization patterns in all variable regions tested as those of the previously reported ICEs. The results reinforce the finding yielded from the phylogenetic analysis in that these ICEs may represent Adriamycin manufacturer a novel cluster in the SXT/R391 family, which could be shaped by the ecological environment in the Yangtze River Estuary, China. Secondly, distinct mosaic accessory gene structures with diverse origins are present in the

ICEs characterized in this study. For example, the ICEs derived from aquatic products share accessory genes with those of clinical, environmental and aquaculture environmental origins in different parts of the world. On the other hand, similar foreign DNA

appears to be captured by the ICEs in different environments. Finally, even within one hotspot, mosaic gene structures are present in some ICEs, such as the hybridized HS1 sequence in ICEVpaChn3. In addition, our results also demonstrated self-transmissibility of antibiotic resistance mediated by ICEVchChn6 and ICEVpaChn1 MI-503 manufacturer from V. cholerae, V. parahaemolyticus to E. coli via conjugation, respectively. Methods Bacterial isolation, screening and identification of ICEs-positive strains Bacterial isolation was carried out according to the instructions of the China Government Standard (GB17378-2007) and the Standard of the Bacteriological Analytical Manual (BAM) of U.S. Food and Drug Administration (8th Edition, Revision A, 1998). Pure cultures of Vibrio isolates grown on

selective thiosulfate citrate bile sucrose (Beijing Luqiao technology Co. Ltd., China) agar plates were picked, and transferred into sterile 96-well microtiter plates according to the instruction of the BAM. Bacterial cells in each row (12 wells) were combined and harvested for genomic DNA extraction and Ribonuclease T1 PCR-based screening of the conserved essential integrase gene (int) of SXT/R391-related ICEs. The isolates in the int gene-positive samples were further individually screened by PCR using the lysis buffer for microorganism to direct PCR kit (TaKaRa Biotechnology Co. Ltd. Dalian, China). Strain taxonomy was carried out by conventional biochemistry tests and 16S rRNA gene amplification and sequencing with the primer pair 27F and 1492R [46] (Table 2). Serotypes were identified using the V. cholerae and V. parahaemolyticus specific diagnostic antiserum kits (Tianjin Biochip Co. Ltd., Tianjin, China). Toxin-related genes were detected by PCR using the primers previously described [47, 48] and listed in Table 2. PCR conditions Genomic DNA was prepared using MiniBest bacterial genomic DNA extraction kit ver.2.