The Mechanism Of Spread And Diffusion Of Tetracycline Resistance Genes In Escherichia Coli Isolates From Duck

Objective: To elucidate the resistance molecular mechanisms of E.coli isolates from duck to tetracyclines and the transfer and spread mechanisms of tetracycline resistance genes (tet genes).Methods: The drug-resistant phenotypes of the E.coli isolates ( n =48) from duck against 7 antibacterial agents including tetracycline, oxytetracycline, doxycycline, florfenicol, enrofloxacin, amikacin, cefotaxime were determined using the broth microdilution method according to CLSI2008; Primer pairs of 6 tet genes, divided into two groups, were designed for the multiplex PCR reactions to detect the various tet genes in 48 tested isolates and furtherly to analyse their combination as well as distribution characteristics.6 primer pairs in two groups were as follows: Group I: tetB, tetC, tetD; GroupII: tetA, tetE, tetM. A conjugation assay was performed to observe plasmid transfer of resistance and tet genes.Some selected isolates and rifampin-resistant E.coli C600 were respectively used as the donor strains and recipient strain. PCR mapping and the method of cloning genes were used for tetA and tetM environment research: for tetM, three pairs of specific primers UM500、CM、DM500a and DM500b were designed for the PCR of upstream of tetM gene, ORF of tetM gene and the downstream gene with the transconjugants and 20 isolates plasmids as templates sequence,;the method of cloning genes was used for the tetA environment: two zygotes strains containing tetA gene for the test.using pBluescriptⅡSK (+) as a carrier plasmid,transconjugants and vector plasmid were digested by the following four programs:○1EcoR I and Xho I double digestion.○2EcoR I and SalI double digestion.○3EcoR I single digestion.○4BamH I single digestion. using T4 ligase enzyme for the ligation of both digestion products , were then cloned into DH5a competent cells. using LB / AMP / DOX / X-gal / IPTG plates for blue-white screening, white colonies were positive selection of recombinant clone, the methods of Digestion and electrophoresis was used to confirm the correct insert, the insert fragment was sequenced with vector universal primers M13。ERIC-PCR was used for genotyping and analysis of the clone relationship among 20 isolates.Results:All 48 E.coli isolates were resistant to oxytetracycline and tetracycline with resistance rate of 100% , and 36 of 48 isolates were resistant to doxycycline with resistance rate of 75%, the resistance rate for florfenicol, cefotaxime and enrofloxacin was 79.17%、60.4% and 27.08%, sensitive to amikacin, most of the strains showed multiple drug resistance characteristics. All the isolates showed positive results of tet gene and the positive rate was 100%. Four tet genotypes and three genotype conbinatios were found among the tested isolates.Besides one isolate which contained tetA and tetC, 21 and 26 isolates coharbored respectively tetA+B+C and tetA+B+C+M genes. The four tet genes can be transfered into recipients through conjunction.5 ,5and 4 strains of 7 transconjugants acquire resistance to oxytetracycline, tetracycline and doxycycline, respectively. 4 tet genes in 3 donor strains were not transfered simultaneously into recipients.The results of sequence analysis are as follows: We obtained upstream and downstream sequences of tetM which was total 2632bp, both upstream and downstream sequences are transposon Tn916, and two transconjugants and the corresponding donor strain results are the same, 6 isolates Tn916 positive, accounting for 30% of tested strains; tetA and its upstream and downstream sequences was about 11.9kb, upstream sequence were some molecular chaperones, dnaJ, stbE and stbD, the end of which was the Xho I restriction site, tetA and tetR genes were together in Tn1721 and the downstream of which were tnpA, Tn21 and the integron Int I,and the end of the downstream sequence was EcoR I restriction site, two zygote have the same test results. Using ERIC-PCR for genotyping for 20 strains, there were six genotypes, respectively, described as A, B, C, D, E, and F-type. There are no obvious relations among the genotype, resistance patterns of bacteria and carrying tetracycline resistance genotype.Conclusion: Tetracyclines resistance in E.coli isolates from duck is mediated two mechanisms:efflux and ribosomal protection. Tet genotypes were mainly 3 and 4 tet gene combinations. 4 tet genes can be completely or partially transferred into recipients to impart some of them resistance to tetracyclines.Tetracycline resistance genes carried by transposons Tn916, Tn1721, Tn21 and together the integron IntI can be transferred between different E. coli clones in ducks.