产新德里金属β-内酰胺酶泛耐药鲍氏不动杆菌相关耐药机制与同源性分析

Related drug resistance mechanism of New Delhi metallo-β-lactamase-producing pandrug-resistant Acinetobacter baumannii and analysis of homology

目的了解产新德里金属β-内酰胺酶(NDM)泛耐药鲍氏不动杆菌相关耐药机制及同源性,为临床泛耐药鲍氏不动杆菌(PDRAB)感染治疗提供参考依据。方法收集2011年3月-2014年4月从临床感染标本检出PDRAB 427株,重新传种、采用VITEK-2Compact型全自动微生物进行菌株的鉴定;对16株产NDM PDRAB进行改良Hodge试验及亚胺培南-乙二胺四乙酸(EDTA)协同试验;PCR扩增blaNDM、blaKPC、blaIMP、blaOXA-40、blaDHA、blaGES、blaRMTB、膜孔蛋白(carO)基因并测序,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析blaNDM阳性菌株carO表达量;肠杆菌科基因间重复一致序列(ERIC)-PCR分析blaNDM阳性菌株基因同源性。结果 16株产NDM-ABA改良Hodge试验和亚胺培南-EDTA协同试验,均为阳性;在427株PDRAB中,16株携带blaNDM,1株携带blaGES,2株携带blaDHA,1株携带blaRMTB,未检测出blaKPC、blaIMP、blaOXA-40,产NDMAB中有7株carO基因缺失,缺失率为43.75%;SDS-PAGE结果显示,部分产NDMAB的CarO缺失;ERIC-PCR分型显示,16株产NDMAB主要可分成6个谱型,B型11株;A、C、D、E、F各1株。结论 PDRAB耐药机制可能与携带blaNDM和膜孔蛋白CarO缺失或改变联合作用有关。

OBJECTIVE To understand the related drug resistance mechanism of New Delhi metallo‐β‐lactamase （NDM ）‐producing pandrug‐resistant A cinetobacter baumannii and analyze the homology so as to provide guidance for clinical treatment of pandrug‐resistant A .baumannii （PDRAB） infection .METHODS A total of 427 strains of PDRAB were isolated from the clinical specimens from Mar 2011 to Apr 2014 ,then the strains were identified by using VITEK‐2 Compact automatic microorganism system ;the modified Hodge test and imipenem‐EDTA syner‐gistic test were performed for 16 strains of New Delhi metallo‐β‐lactamase‐producing PDRAB .The blaNDM , blaKPC ,blaIMP ,blaOXA‐40 ,blaDHA ,blaGES ,blaRMTB ,and porin （carO） genes were amplified by PCR and were sequenced ,the sodium dodecyl sulfate‐polyacrylamide gel electrophoresis （SDS‐PAGE） was carried out to analyze the expression quantity of the blaNDM‐positive strains ,and the homology of the blaNDM‐positive strains was analyzed by means of Enterobacteriaceae repetitive intergenic consensus sequences （ERICS ）‐PCR . RESULTS The modified Hodge test and imipenem‐EDTA synergic test demonstrated that carbapenemase was ex‐pressed in all the 16 strains of NDM‐producing A .baumannii .Of 427 strains of PDRAB ,16 harbored blaNDM gene ,1 harbored blaGES gene ,2 harbored blaDHA gene ,and 1 harbored blaRMTB gene ,while the blaK PC , blaIMP ,and blaOXA‐40 genes were not detected . Among the NDM‐producing A .baumannii strains , 7 （43 .75% ） lacked the carO gene ,as indicated by PCR amplification assay and DNA sequencing .The SDS‐PAGE analysis further proved that the expression of outer member porin CarO of NDMAB was partly missed .The 16 strains of NDM‐producing A .baumannii were classified mainly into 6 genotypes by the ERIC‐PCR ,inclu‐ding 11 strains of genotype B ;there were respectively 1 strain in genotypes A ,C ,D ,E ,and F .CONCLUSION The drug resistance mechanism of the PDRAB may be associated with the joint effect of harboring blaNDM gene and deficiency or change of the porin （carO） .