The Detection Of TetA And TetB Genes And Research Of Its Genetic Environments In Escherichia Coli Isolates From Chickens

To explore the resistance mechanisms of the Escherichia coil isolates form chickens to tetracyclines and the diffusion mechanism of tetracycline resistance gene (tet gene). The susceptibilities of 32 clinical E. coil isolates form chickens to tetracycline, doxycycline and other 10 kinds of antimicrobials were determined according to CLSI2008 recommended method; 6 pairs of specific primers were designed and divided into two groups for the PCR analysis of tet gene among the 32 clinilcal E. coil. The isolates containing combinations of genes tet A and tetB as donor strain in typical were selected to study the genetic environment of tet A and tetB by the cloning method. An E. coli isolate containing tetA gene and an Escherichia coli isolate containing tetB gene as the test strain, pBluescript Ⅱ SK(+) as the vector, the plasmid and vetor were digested by the following two enzymes separately:EcoR I and Hind Ⅲ.Then the two digestion products were connection by T4 ligase and cloned into DH5α competent cells, blue-white screening conducted by LB/AMP/DOX/X-gal/IPTG plate, selection of white colonies as positive recombinant plasmid clones. Before sequencing by vector primers, the inserted fragment was confirmed through digestion and electrophoresis. By ERIC-PCR for 32 isolates were genotyped to analyze the clone relationship between them.Results:25 of 32 clinical E.coli isolates form chickens contained tet gene. MIC determination of 32 chicken E.coli to tetracycline, doxycycline, ceftazidime, ceftiofur, kanamycin, amikacin, tetracycline, doxycycline, florfenicol, enroflorxacin the minimum inhibitory concentration star and 10 kinds of antibiotics ciprofloxacin by microdilution method showed that 84%(25/32) of the isolates were multi-drug resistant. PCR analysis in 32 chicken E. coil showed that tetA gene were detected in 25 strains, the positive rate was 78%, tetB gene were detected in 7 strains, the positive rate was 22%.In No.27 strains, tetA and tetB existed at the same time, the positive rate was 3%. No any tet gene was detected in 7 strains, the detection rate was 84%.Sequencing analysis revealed that the gene sequence of the full-length 6159bp was fragment of tetA, and the length of 3764bp was fragment of tetB. The tetA gene fragment from left to right was:tetA gene, reverse pecM membrane anchor protein gene, transposase tnpA, tnpA, reverse ibfA transposase gene. The tetB gene fragment from left to right was:tetB gene, reverse tetR gene, putative protein, putative protein, putative protein. The 32 isolates were genotyped by Eric-PCR, 15 kinds of genotypes were determined, described as A, B, C, D, E, F to O respectively. The obvious connection among the bacterial genotypes and drug-resistance patterns and carrying tetracycline resistant genotype can not be found.