同时产ArmA型16S rRNA甲基化酶及KPC-2型β-内酰胺酶泛耐药阴沟肠杆菌的研究

目的 探讨上海交通大学医学院附属瑞金医院分离的5株泛耐药阴沟肠杆菌产碳青霉烯酶及16S rRNA甲基化酶的情况及两者之间的相关性.方法 用E test法检测5株泛耐药阴沟肠杆菌对10种抗菌药物的MIC值.PCR扩增16S rRNA甲基化酶基因armA、mtA、rmtB、rmtC、rmtD、npmA,β-内酰胺酶基因TEM、SHV、CTX-M-1、CTX-M-2、CTX-M-8、CTX-M-9、CTX-M-25、PER-1、VEB-1.鸟枪克隆法克隆碳青霉烯酶基因,并对克隆片段进行测序.质粒接合试验验证碳青霉烯酶基因及16S rRNA甲基化酶基因是否具有转移性.脉冲场凝胶电泳(PFGE)法对5株菌进行分子分型.等电聚焦电泳法检测β-内酰胺酶等电点.Southern杂交法对耐药决定因子进行定位.结果 5株阴沟肠杆菌对10种抗菌药物的MIC值均＞32 mg/L.克隆的碳青霉稀酶基因为blaKPC-2,酶编码基因上游为一转座酶基因,两侧为复制靶位,下游为ISKpn6的插入序列,该序列包括一个重复序列和tnpA转座子,酶编码基因位于54.2 kb的一个非接合性大质粒上.等电聚焦电泳显示5株菌均产4种β-内酰胺酶(TEM-1,pI5.4；KPC-2,pI6.7；SHV-12,pI8.2；CTX-M-14,pI8.4).16S rRNA甲基化酶基因位于接合性质粒上,而blaKPC-2基因则位于非接合性质粒上,5株菌PFGE型别一致.结论 5株泛耐药阴沟肠杆菌对碳青霉烯类耐药由产碳青霉烯酶KPC-2所介导.blaKPC-2与armA型16S rRNA甲基化酶基因由两条不同的质粒编码,不存在相关性.临床医院应加强监控,以防止交叉感染.

Co-production of carbapenem-hydrolyzing enzyme KPC-2 and ArmA 16S rRNA methylase in pandrugs resistant Enterobacter cloacae

Objective To investigate the production of carbapenemase and 16S rRNA methylase in five isolates of pan-drugs resistant E.cloacae recovered in Ruijin hospital.Methods MICs of the five isolates to 10 antibiotics were determined by E test.Six kinds of 16S rRNA methylase genes and a series of β- lactamase genes were amplified by PCR.Shotgun cloning was performed to detect carbapenem resistance determinant.The conjugal transfer of carbapenemase gene and 16S rRNA methylase gene was performed in broth culture with E.coli J53 as the recipient.Pulsed-field gel electrophoresis (PFGE) was carried out to analyse the genotyping.IEF was performed to detect β-1actamases.Southern blot was performed to determine the location of carbapenem resistance determinant.Results The MICs of 10 antibiotics were ＞32 mg/L.Four β-1actamases with pIs of 5.4 ( TEM-1 ),6.7 ( KPC-2 ),8.2 ( SHV-12 ),8.4 (CTX-M-14) were determined.The insertion sequence in the recombinant plasmid was blaKPC-2 flanked by a transposon.blaKPC-2 was located on a large non-conjugative plasmid whereas armA was located on an other conjugative plasmid.PFGE patterns of 5 isolates were identical.Conclusion KPC-2 was responsible for carbapenem resistance in pandrugs resistant Enterobacter cloacae.There was no relationship between blaKPC-2 and armA.Although pandrug resistant Enterobacteriaceae remain rare,the emergence of this group of organism merits monitoring.