儿童肠杆菌科细菌碳青霉烯类耐药的基因型研究

RESEARCH ON GENOTYPES OF CARBAPENEM-RESISTANT ENTEROBACTERIACEAE IN CHILDREN

目的:研究儿童碳青霉烯类耐药肠杆菌科细菌(CRE)耐药的基因型。方法:收集20株临床分离非重复CRE,采用梅里埃公司的GN-13药敏卡进行药敏试验筛选出CRE,改良Hodge试验及EDTA协同试验进行碳青霉烯酶表型确认,PCR扩增碳青霉烯酶基因、β-内酰胺酶基因,PCR阳性克隆测序分析,结果与NCBI Blast比对,确认基因亚型。结果:20株CRE以肺炎克雷伯菌为主占60%,其次是粘质沙雷菌占20%。改良Hodge试验阳性70%,EDTA纸片增效试验阳性85%,IMP基因阳性95%。16株CRE同时扩增出碳青霉烯酶基因blaimp-4,β-内酰胺酶基因blashv、blaTEM3种基因,18株同时扩增出两种基因。所有菌株未检出KPC、GES、SME、NMC、IMC、NDM-1、VIM、SIM型基因、CTX-M-1、CTX-M-13基因。结论:儿童CRE以肺炎克雷伯菌主,自儿童粘质沙雷菌中检测出IMP-4基因,多数CRE菌株携带IMP和TEM、SHV基因,产IMP-4型碳青霉烯酶是儿童临床分离CRE耐药的主要原因。

Objective：To study the genotypes of Carbapenem-Resistant Enterobacteriaceae（CRE）in children.Methods：20separated and unrepeated clinical CRE strains were collected to screen for carbapenemresistant enterobacteriaceae by using GN-13 test kit（BioMerieux）.Modified Hodge and EDTA synergistic experiment was utilized to confirm carbapenemase phenotype;PCR was perfomed to do carbapenemase andβ-lactamase genes amplification and sequencing.The sequencing analysis results were verified by NCBI blast to confirm its subgenotype.Results：60% of the 20 CRE strains were klebsiella pneumoniae,and 20%of 20 CRE strains were Serratia marcescens.Modified Hoggle Experiment got 70% positive,EDTA experiment got 85% positive,and IMP gene got 95%positive.Carbapenemase gene bldimp-4,β-lactamase bldshvand bldTEM were found through the amplification of 16 CRE strains.Carbapenemase gene bldimp-4andβ-lactamase bldTEM,two types of genes were found by the amplification of 18 CRE strains.KPC,GES,SME,NMC,IMC,NDM-1,VIM and SIM types of carbapenemase genes,and CTX-M-1、CTX-M-13typeβ-lactamase can not be detected in any strain of CRE.Conclusion：Majority of children CRE is klebsiella pneumoniae.IMP-4genes were detected in Children Serratia marcescens.Most strains of CRE carry carbapenemase IMP,β-lactamase TEM and SHV.Production of IMP-4type carbapenemase is the main reason why CRE resists to treatment.