Distribution Characteristics Of Virulence-related Genes And Integrons Of Vibrio Cholerae Strains

ObjectiveTo understand the distribution and characteristics of the virulence genes of strains isolated from different areas of Tianjin during the past decades, and explore prevalence of rule. To understand of the drug resistance of Vibrio cholerae strains isolated from Tianjin in 1995, to investigate characteristics of distribution and the genotypes of variable region resistant gene of three different integrations among 23 strains to disclose its role in the acquisition and dissemination of antibiotic resistance.MethodsPCR techniques were applied to detect ctxAB,and zot gene of CTXΦgenetic element, cri gene of TLC cluster of the 176 strains of Vibrio cholerae isolated from 1964 to 2008.we randomly selected six Vibrio cholerae strains from different ages and used PCR techniques to amplify ctxA, ctxB, cri, tcpA and toxR. genes, and then sequenced PCR products, using DNAStar software to Mosaic of sequences and bioinformatics analysis.first the antimicrobial susceptibility testing for Vibrio cholerae strains isolated from Tianjin in 1995 was performed using standard disk diffusion method (improved K-B) recommended by WHO method, according to the guidelines from The Clinical and Laboratory Standards Institute (CLSI) committee (2009) to judge which is resist, intermediate and susceptible.PCR techniques were applied to detectⅠ,Ⅱ,Ⅲclasses integrating enzyme gene and gene cassette of integrons.PCR product which variable area was positive was purified and then connected to the carrier pMD18-T simple, transformed into E.coli TOP 10, positive identification by polymerase chain reaction (PCR) after recombination, then sequencing analysis. Using BLAST to analyze, determine the sequence of variable area contains resistance genes box.according to PulseNet monitoring network which provided PFGE standard operating procedures of Vibrio cholerae, analysis of the homology between strains.Results131 strains of Vibrio cholerae carried ctxAB and zot of CTXΦgenetic element in 176 strains, the positive rates separately were 72.72%(128/176 strain), 73.30%(129/176 strain). there were 69.00%(69/100 strain) of Vibrio cholerae O1 strains isolated from 1964 to 1996 carrying cri gene, however the rate was only 1.33%(1/75 strain). Results of multiple sequence alignment among six EVC Vibrio cholerae strains compared with international standards strains N16961, showed that the identities of ctxA and tcpA sequences were 100%,ctxB,cri and toxR sequences were 99.5%-100%,98.4%-100% and 99.4%-100% respectively. according to the results of CLSI(2009):23 Vibrio cholerae strains were sensitive to ampicillin,penicillin,tetracycline,trimethoprim/trimethoprim-sulfamethoxazole, chloramphenicol. Integrase genes from three class integrons in Vibrio cholerae strains varied differently:integronⅠwas 82.61%, ingtegronⅡandⅢwere not detected. All of classⅠintegron carried one gene cassette encoding aadA1, mediated drug resistance of streptomycin and spectinmycin.The result of PFGE showed that part of tested strains isolated in 1995 could come from the same clone.ConclusionDetection and sequence of virulence-related genes can reflect the molecular characteristics of strains, and it is important to the molecular epidemiology of Vibrio cholerae research.It showed that the sensitive drugs, such asβ-lactams, tetracycline, trimethoprim-sulfamethoxazole, chloromycetin, could be used. The detected rate of integronⅠwas 82.61% of strains isolated in 1995. Antimicrobial susceptibility test results showed that the drug-resistant genes was consistent with its resistance phenotype of Vibrio choleiae strains, classⅠintegron was associated with resistance to streptomycin of tested strains. Pulse field gel electrophoresis results showed that part of them could come from the same clone.