2株产气肠杆菌临床株对厄他培南耐药机制的研究

目的研究2株产气肠杆菌临床株Ea293和Ea2880对厄他培南耐药的机制。方法采用微量肉汤稀释法测定最低抑菌浓度(MIC),应用K-B法检测菌株对美罗培南、亚胺培南及厄他培南的耐药性,改良Hodge试验检测碳青霉烯酶;聚合酶链反应(PCR)扩增碳青霉烯酶基因(KPC,IMP-1、2组,VIM)和广谱及超广谱β-内酰胺酶基因(TEM,SHV,CTXM-1、2、9组ESBI.s),并进行序列分析;十二烷基硫酸钠一聚丙烯酰胺凝胶电泳(SDS-PAGE)进行细菌外膜蛋白分析,并对外膜蛋白基因OmpE36进行PCR扩增及序列分析。结果药物敏感试验显示,产气肠杆菌Ea293和Ea2880均对厄他培南耐药。PCR扩增4种碳青霉烯酶基因和5种广谱及超广谱β-内酰胺酶基因,仅Ea2880的SHV和CTX-M-9组ESBLs基因阳性,SHV基因的PCR产物测序为SHV-11型广谱酶。细菌外膜蛋白SDS-PAGE结果显示,与碳青霉烯类抗生素敏感的产气肠杆菌Eal885相比,Ea293和Ea2880均缺失42 kD左右的条带,即OmpE36。PCR扩增外膜蛋白基因OmpE36,Ea2880可见预期片段,DNA序列分析显示其与GenBank公布的产气肠杆菌标准株ATCC 13048的相似性为87%,氨基酸序列的相似性亦为87%;而Ea293未扩增出预期片段。结论产气肠杆菌临床株Ea293与Ea2880对厄他培南耐药的主要原因可能是外膜蛋白基因OmpE36缺失,同时Ea2880可能还有CTX-M-9组ESBLs参与。

Study on resistant mechanism of two clinical strains of Enterobacter aerogenes to ertapenem

Objective To study resistant mechanisms of two clinical strains of Enterobacter aerogenes （Ea293 and Ea2880）to ertapenem. Methods The minimum inhibitory concentrations （MICs） of antimicrobial agents were determined by microbroth dilution method, drug resistance of strains to imipenem, meropenem, and ertapenem were determined by K-B test; carbapenemase was confirmed by modified Hodge test, carbapenemase genes（KPC, IMP-1 group, IMP-2 group, VIM）, broad-spectrum and extended-spectrum β-lactamases genes （ TEM, SHV, CTX M- 1 group,CTX-M-2 group,CTX-M-9 group） were amplified by polymerase chain reaction （PCR）, and sequences were analysed; Outer membrane protein （Omp） was analyzed by sodium dodeeylsulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and the coding gene OmpE36 was amplified by PCR. Results Antimicrobial susceptibility test showed that Ea293 and Ea2880 were all resistant to ertapenerru Among amplified 4 carbapenemase genes and 5 broad spectrum and extended spectrum genes, only blaSHV and blaCTXM-9 group ESBLs in Ea2880 were positive, BlaSHV DNA sequence was SHVll type. SDS-PAGE showed that compared with ertapenem-sensitive isolate Ea1885, ertapenem-resistant isolate Ea293 and Ea2880 lacked the protein band with 42kD which might be the outer membrane protein gene OmpE36. OmpE36 was amplified by PCR, Ea2880 appeared the excepted bands, but Ea293 didn＇t. The similarity of DNA and amino acid sequences of OmpE36 of Ea2880 with the standard Enterobacteraero genes ATCCl3048 from GenBank were both 87%. Conclusion The resistance of clinical strains of Enterobacter aerogenes Ea293 and Ea2880 to ertapenem might be associated with the loss of outer membrane protein gene OmpE36. Furthermore,Ea2880 might be associated with production of CTX-M-9 group ESBLs.