【作者】 李浩；

【导师】 李连青；

【作者基本信息】 山西医科大学 ， 免疫学， 2006， 硕士

【摘要】 目的分析太原地区产ESBL大肠埃希菌流行趋势和耐药机制,为指导临床合理使用抗生素,减少耐药菌株在本地区扩散及流行病研究提供实验依据和指导。方法1对临床分离的耐药菌株进行纸片扩散法ESBL表型筛选,将筛选出68株ESBL表型阳性菌重新进行生化鉴定、药敏实验。2用PCR方法对产ESBL大肠埃希菌进行分型并测序。3用三维实验、多重PCR的方法对耐头孢西丁的产ESBL大肠埃希菌进行AmpCβ-内酰胺酶的检测。4 SDS-PAGE对部分菌株外膜蛋白进行分离鉴定。5 ERIC—PCR的方法流行病学分型。6 PCR方法对整合酶及其基因盒进行分析并测序。结果1临床收集耐药大肠埃希氏菌株中,通过ESBL表型确证试验共筛选出68株ESBL阳性菌株。其中门诊病人10株,住院病人58株。其中外科病房分离占2/3。2 PCR结果TEM型67株为阳性,SHV型04LE030阳性,测序为SHV12, CTX-M2、OXA1、OXA2、OXA10引物PCR电泳未见阳性。CTX-M1阳性21株,CTX-M9阳性48株。3 AMPC三维实验04LE030弱阳性,多重PCR结果04LE030阳性带在400BP左右,测序为DHA1。4 SDS-PAGE可见04LE048出现OmpF缺失出现大分子蛋白条带。5.ERIC—PCR的方法显示不同医院菌株流行病学分型有差异。6整合酶ⅠPCR阳性39株,整合酶ⅡPCR阳性1株,其基因盒有两种结构,均包括氨基糖苷类抗生素和磺胺类抗生素的耐药的基因。结论1产ESBL大肠埃希菌引起的感染以泌尿系统和呼吸道感染为主,住院病人以外科病房为主,门诊病人泌尿系统老人多见。2本地区产ESBL大肠埃希菌以CTX-M9群、CTX-M1群为主。表型筛选时可使用头孢噻肟和氨曲南,确证可只使用头孢噻肟和头孢噻肟+棒酸。单头孢他定和头孢他定+棒酸表型确证试验阳性的大肠高度怀疑为SHV型。3本地区同时产ESBL和AmpC大肠埃希菌还不多见;头孢西丁完全耐药(直径=6mm)更多还原

【Abstract】 Objective:Investigate the mechanisms of drug-resistance and epidemiology of Escherichia coli.These strains producing extended-spectrumβ-lactamases come from taiyuan. With a view to provide the experimental reference and guideline for judicious use of antibiotics.Methods:1.ESBLs-producing strains were confirmed by the NCCLS phenotypic confirmatory test using disc agar diffusion method (Kirby-Bauer diffusion test).2. PCR amplification of ESBLs genes, then direct analysis DNA sequencing.3.Using three-dimension method and mutiPCR detection of AmpC-β-lactamases . The strains were cefoxitin drug-resistance.4.Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolation and analysis outer membrane protein of some strains.5. Useing ERIC2-PCR analysis epidemiological investigations. 6.PCR amplification of integrase and Genecassettes genes, then direct DNA sequencing analysis.Results1. 68 ESBLs-producing strains were confirmed by the NCCLS phenotypic confirmatory test using Kirby-Bauer diffusion test.10 strains isolation from outpatient ,other isolation from inpatients ,surgery ward accounting for 2/3.2 .The PCR results of gene in plasmid DNA showed that TEM positive was 67 strains, The sequence showed 100% amino acid identity to TEM-1. SHV positive was 04LE30, The sequence showed identity to SHV-12. CTX-M2、OXA1、OXA2、OXA10 all are negative. CTX-M 1 showed 21 strains positive .CTX-M 9 positive was 48 strains.3 .Detection three-dimension method of AmpC-β-lactamases ,04LE30 was positive,mutiPCR show a bind at 400bp ,The sequence showed identity to DHA1.4. Omp profile analysis showed that OmpF expression presented diminution or deletion in some ESBL-producing strains.04LE048 OmpC expression presented deletion and has a new更多还原