耐碳青酶烯类肠杆菌科细菌的耐药机制研究

Molecular Epidemiology and Resistance Mechanism of Carbapenem-resistant Enterobacteriaceae

目的研究ICU收集的耐碳青酶烯类肺炎克雷伯菌、大肠埃希菌、褪色沙雷菌分子流行病学特征及耐药机制。方法用脉冲场凝胶电泳(PFGE)和肠杆菌科基因间重复一致性序列-PCR(ERIC-PCR)分析耐药株的分子流行病学特征,采用特异性PCR和序列分析、接合试验、质粒提取和转化试验、质粒消除试验、外膜蛋白分析技术来分析介导碳青酶烯类耐药的分子机制。结果 12株肺炎克雷伯菌分属2个流行克隆型、13株大肠埃希菌属于5个流行克隆型、3株褪色沙雷菌属于2个流行克隆型;药物敏感试验显示,分离株对亚胺培南和美罗培南的MIC分别为16～128μg/ml和8～256μg/ml;所有菌株均扩增出介导碳青酶烯类耐药的KPC-2基因;3株褪色沙雷菌接合试验获得成功,部分菌株进行质粒提取、质粒分子杂交、质粒转化和质粒消除试验显示,KPC-2定位于约为56kb的质粒上;外膜蛋白比较分析显示部分耐药菌株发生外膜蛋白改变。结论医院ICU分离出耐碳青酶烯类肺炎克雷伯菌、大肠埃希菌和褪色沙雷菌;产KPC-2是介导对碳青酶烯类药物耐药的主要原因;部分菌株外膜蛋白改变可能参与耐药;垂直传播以及通过质粒的水平传播可能是耐药株在ICU流行的主要方式。

OBJECTIVE To investigate the molecular epidemiology and resistance mechanism of carbapenemresistant Enterobacteriaceae isolates including Klebsiella pneumoniae, Escherichia coli and Serratia marcescens collected from intensive care units （ICUs）. METHODS Pulsed-field gelelectrophoresis （PFGE） and enterobacterial repetitive intergenic consensus-PCR （ERIC-PCR） were perfomed to analyze the molecular epidemiology of carbapenem-resistant isolates. Specific PCRs and DNA sequer/eing, conjugation experiments, plasmid DNA extraction, plasmid transformation assays, elimination of plasmid from isolates, and SDS-PAGE of outer membrane proteins （OMPs） were performed to confirm genotype of carbapenemase and its transmission mechanism. RESULTS PFGE indicated that K. pneumoniae and S. marcescens isolates belonged to two different clones, E. coli isolates belonged to five clones. The MICs of both imipenem and meropenem were 16 to 128μg/ml and 8 to 256 μg/ml . PCR and DNA sequencing suggested that all isolates encoded KPC-2. Conjugation experiments with E. coli （EC600） were succeeded in S. marcescens, and plasmid DNA extraction, plasmid transformation assays, and elimination of plasmid from some isolates all comfirmed that KPC-2 encoded on a 56 kb plasmid. OMPs analysis revealed that there were alterations in isolates of E. coli and S. marcescens. CONCLUSIONS Carbapenem-resistant K. pneurnoniae, E. coli and S. marcescens are isolated in our hospital. Production of KPC-2 earhapenemase mainly contributes to reduced susceptibility of carbapenem in Enterobacteriaceae isolates. The alterations in OMPs may as a cofactor in high-level drug-resistance in E. coli and S. rnarcescens. Vertical and horizontal plasmid-mediated transmission are probably the principal epidemical mechanism.