| Objective:1) To survey the popular characteristic and distribution of plasmid-mediated quinolone resistant genes qnr?aac(6')-Ib-cr and qepA about Escherichia coli and Klebsiella pneumoniae which were isolated from patients in ICU of Renmin hospital of Wuhan university between January2012to December2013.2) To know the resistance feature to quinolones of qnr?aac(6')-Ib-cr and qepA genes positive isolates.3) To explore the transmission type and drug-resistance mechaniams of the qnr? aac(6')-Ib-cr and qepA positive isolates, and research the influence to antibiotic resistance of plasmid-mediated quinolone resistant gene in Escherichia coli and Klebsiella pneumoniae of ICU.Methods:1) Escherichia coli and Klebsiella pneumoniae of quinolone resistant isolates were collected from January2012to December2013in ICU of Renmin hospital of Wuhan University(excluded the repeat strains from the same position of the same patient);2) All strains isolates were identified by Phoenix100and statistics drug sensitivity;3) qnr?aac(6')-Ib-cr and qepA genes positive strains were detected by PCR and seguerncing;4) The agar dilution method to detect qnr?aac(6')-Ib-cr and qepA genes positive strains to the MIC of ciprofloxacin and Levofloxacin;5) For qnr gene positive strains, intI genes were examined by PCR;Compared the MIC of Ciprofloxacin to the bacterias with Levofloxacin by t-test method in qnr?aac(6')-Ib-cr and qepA positive group respectively;6) Processing to plasmid conjugation test to explore whether qnr?aac(6')-Ib-cr and qepA genes located in plasmids or not by PCR;7) To process the drug-resistance of qepA positive strains with CCCP reversal test;8) To analys the consanguinity of chromosome DNA of qepA-positive strains by ERIC-PCR technique.Results:1) Among239Escherichia coli and Klebsiella pneumoniae which were isolated from ICU in Renmin hospital of Wuhan university,108were Escherichia coli,131were Klebsiella pneumoniae;108(45.2%) of them were qnr,63(26.4%) of them were aac(6')-Ib-cr and17(7.1%) of them were qepA positive strains; One Klebsiella pneumoniae carried rmtB of the17qepA positive strains, the remaining strains rmtB gene was negative;2) To compare the resuilt of sequerncing in NCBIGenBank, the consanguinity was100%with the bank;3)108Escherichia coli were susceptible completely to imipenem and meropenem, the resistance rate to Cefoperazone/sulbactam and Piperacillin/tazobactam were about10%, but resistance to some other drugs, to Ciprofloxacin and Levofloxacin which achieved70%and65.5%respectively;131Klebsiella pneumoniae of the resistance rate to imipenem and meropenem were1.5%,because2strains were KPC,and to Ciprofloxacin and Levofloxacin were37.2%and27.1%respectively;4) The MIC of qnr?aac(6')-Ib-cr and qepA genes positive isolates to Ciprofloxacin markedly higher than Levofloxacin;5)96of108qnr positive isolates were I type integron-positive isolates;6) The plasmid conjugation experiment is successful for12qnr and5qepA positive strains by randomly choosing,and10aac(6')-Ib-cr positive strains carried out plasmid conjugation experiment,8of them were successful.7) CCCP can reverse the MIC of5qepA positive strains and their conjugations to hydrophilic quinolone, made Ciprofloxacin MIC reduced3?4and3-5dilution ratio respectively.8) The result of ERIC-PCR revealed this3of5qepA positive strains belong to the same genotypes.Conclusion:1) The prevalence of qnr?aac(6')-Ib-cr and qepA genes were existed in ICU of Renmin hospital of Wuhan university;The detection rate of aac(6')-Ib-cr was the highest;2) These Escherichia coli and Klebsiella which existed resistance gene were the multiple resistance,and the trend was increase by yaerly;3) qnr positive stranis can carry several resistance genes cause seriously multi-and cross-drug resistance;4) Conjugation experiments showed that the qnr?aac(6')-Ib-cr and qepA-carrying plasmids can transferable horizontally;5) The resistance of qepA to quinolones may be through the efflux pump mechanism which mediated by plasmid.6) The same clong Escherichia coli was existed in ICU. |
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