Theoretically, one Ogawa strain may arise from the reversion of an original mutation, but the correction of the specific substitution

or deletion is necessarily a rare event [3, 22]. BIX 1294 mutations in rfbT were used to assess the clonal origin and dissemination of clinical Inaba isolates [24]. The serotype shift pattern of cholera in endemic areas Stem Cells inhibitor was also historically observed [25, 26] and indicated to be associated with high, but incomplete, cross-immunity between the Ogawa and Inaba serotypes [20]. Continuous surveys on the Inaba strains may reveal more mutations of the rfbT gene, and even clonality of the epidemic V. cholerae strains. In China the seventh cholera pandemic caused by O1 El Tor V. cholerae started in July 1961 [27]. Notifiable cases of cholera reported to the national disease surveillance and reporting system showed that there were serotype shifts during the years of El Tor biotype epidemics. In this study, diversity of the rfbT sequence CX-5461 supplier and the effect of the rfbT mutations on the serotyping were investigated. Characteristic mutations causing serotype shifts in different Inaba predominant epidemics were observed. Methods Bacteria strains, media and plasmids This study was conducted on 134 O1 El Tor and 1 O1 classical V. cholerae

strains isolated from different provinces in China from 1961 to 2008,together with 18 laboratory-collected O1 classical strains and 10 O1 El Tor strains isolated outside of China (Additional file 1: Table S1). All strains were recovered from −80°C laboratory stocks. Slide agglutination tests were used to serotype the strains using

anti-Ogawa and anti-Inaba monoclonal antibodies (S&A reagents lab, Bangkok, Thailand). Classical biotype strains were further confirmed using the Classical IV bacteriophage susceptibility assay [28] and the polymyxin B (50U) susceptibility assay with V. cholerae 569B and N16961 used as reference strains. The pBR322 plasmid was used as the cloning vector. Suicide plasmid pCVD442 was used to engineer mutations in host strains Protein kinase N1 via allelic exchange. Escherichia coli strain Top10 and SM10λpir were used as the recipient strains. All strains were grown in Luria-Bertani (LB) broth or Luria-Bertani (LB) agar plates at 37°C. Ampicillin was used at a final concentration of 100 μg/ml when necessary. PCR amplification and construction of complementary plasmid PCR amplification was carried out using standard protocols with rfbt-up (5′ GCG TCG ACG AAT CGG CAG TCG CAA CA 3′) and rfbt-dn (5′ CCC AAG CTT CAA AGC TAT ACT AAA CTG 3′) primers. A water-boiled template of each strain was used. The 1441 bp PCR products were purified with a QIAGEN PCR purification kit (Qiagen Inc., Hilden, Germany) and applied for commercial sequencing.