大肠埃希菌GyrA突变、超广谱β-内酰胺酶及整合子检测分析

Detection of GyrA Mutation,ESBLs and Integron in Escherichia coli

目的研究大肠埃希菌(ECO)GyrA突变、产超广谱β-内酰胺酶(ESBLs)基因型及整合子的存在情况。方法 8株ECO在2008年分别分离自同一起由猪霍乱沙门菌引起的食物中毒事件中8例腹泻患者的粪便;使用K-B法进行药物敏感试验,并检测是否产ESBLs;PCR法扩增ECO对环丙沙星耐药相关基因gyrA,ESBLs基因blaTEM、blaSHV和blaCTX-M,3类整合酶基因(intⅠ1,intⅠ2,intⅠ3)和第Ⅰ类整合子可变序列;对PCR产物进行测序分析;使用脉冲场凝胶电泳(PFGE)对ECO进行分子分型。结果 2株菌在GyrA的QRDR区83位Ser→Leu和87位Asp→Asn,同时产TEM1和携带第Ⅰ类整合子;1株菌在QRDR区83位Ser→Leu,同时产TEM55;1株菌在QRDR区83位Ser→Leu;2株菌分别产TEM1和TEM55;整合子所携带的耐药基因盒为aacA4、cmlA1、dfrA1和aadA1;PFGE将8株菌分为8个PFGE型。结论 GyrA突变、产ESBLs和携带整合子是ECO产生耐药的重要机制。

OBJECTIVE To investigate the GyrA mutation, extended-spectrum beta-lactamases （ESBLs） and integron in Escherichia coli. METHODS Eight E. coli isolates from the feces of eight diarrhea patients caused by Salmonella choleraesuis were collected in 2008. KB method was used to perform the in vitro antimicrobial susceptibility testing for all isolates, and extended-spectrum beta-lactamase （ESBLs） production was tested for all isolates according to CLSI criteria. PCR were used to amplify ciprofloxacin resistance associated gene, gyrA, and ESBLs genes, blaTEM, blaSHV and blaCTX-M, and integrase genes, int I 1, int I 2 and int I 3, and variable sequence of class 1 integron. PCR products were sequenced and analyzed. Pulsed-field gel electrophoresis （PFGE） was performed for all isolates. RESULTS Five out of eight isolates were showed multidrug resistant. Of the 8 isolates, 2 isolates had the changes, 83 site Ser→Leu and 87 site Asp→Asn, inside the quinolone resistance- determining region （QRDR） of GyrA, and producing TEM1 and containing class 1 integrons; 1 isolate had the change, 83 site Ser→Leu, in the QRDR and producing TEM55; 1 isolate had the change, 83 site Ser→Leu, in the QRDRI 2 isolates produced TEM1 and TEM55 respectively. The cassettes of class 1 integrons were the following： aacA4, cmlA1, dfrA1 and aadA1. PFGE analyses showed eight PFGE patterns were obtained. CONCLUSION GyrA mutation（s）, ESBLs-producing and integron-containing are the major mechanisms for E. coli to develop the drug resistance.