23株碳青霉烯耐药大肠埃希菌blaNDM基因分子流行病学

Molecular epidemiology of bla gene in 23 strains of carbapenem-resistant Escherichia coli

目的分析大肠埃希菌中blaNDM基因的分子流行病学特征,及其遗传背景,为临床防控提供理论支持。方法收集2015-2019年丽水市中心医院临床分离非重复碳青霉烯耐药大肠埃希菌,梅里埃Vitek 2 Compact系统鉴定菌株。微量肉汤稀释法联合K-B纸片法确定药物敏感性,聚合酶链式反应(PCR)检测常见碳青霉烯类、超广谱-β内酰胺类和喹诺酮类耐药基因。多位点序列分型(MLST)明确各菌株ST型,并结合BacWGST数据库中92株产新德里金属-β-内酰胺酶(NDM)大肠埃希菌构建进化树分析。接合和转化实验纯化耐药质粒并确定质粒转移性,复制起始子分型(PBRT)鉴定质粒类型,特异性PCR检测blaNDM转座结构,最终从菌株,质粒,转座结构分析blaNDM基因流行特征。结果共分离23株NDM型大肠埃希菌,对β-内酰胺类(除氨曲南),喹诺酮类,氨基糖苷类(除阿米卡星)抗菌药物高度耐药,仅对米诺环素、阿米卡星、磷霉素,多黏菌素B、替加环素敏感,PCR检出blaNDM-5、blaNDM-1、blaCTX、blaTEM、blaSHV、qnrA、qnrS、qnrD、acc(6)-Ib-cr多种耐药基因。系统进化分析将14种ST型菌株,归为8类克隆群。PBRT检测到IncX3、IncF两种质粒,IncX3型占95.6%。转座结构分析显示blaNDM-1基因均位于IncX3型质粒相同转座结构中,具有一定的地区特异性,而blaNDM-5基因则在IncX3、IncF型质粒上均有,主要位于(ISAba125-IS5-blaNDM-5-bleMBL-trpF-dsbC-IS26)转座结构中,部分转座子缺失ISAba125序列。结论blaNDM基因分离自14种ST型大肠埃希菌,位于IncX3、IncF质粒的3种转座结构中,主要通过IncX3型质粒转移传播。

OBJECTIVE To analyze the molecular epidemiological characteristics and genetic background of the blaNDM gene in Escherichia coli,and to provide theoretical support for clinical prevention and control.METHODS Non-repetitive carbapenem-resistant Escherichia coli strains isolated in Lishui Municipal Central Hospital from 2015 to 2019 were collected and identified by the Mérieux Vitek 2 Compact system.The drug sensitivity of the strains were determined by microbroth dilution method combined with K-B disk diffusion method,and drug-resistant genes of carbapenems,extended-spectrum-beta lactams and quinolone were detected by PCR assay.Multilocus sequence typing(MLST)was used to identify the ST types of each strain,and 92 E.coli strains producing NDM in BACWGST database were constructed for phylogenetic analysis.Conjugation and transformation experiments were used to purify drug-resistant plasmids and determine the transferability of the plasmids.The plasmid type was identified by PCR-based replicon typing(PBRT),and blaNDM transposition structure was detected by specific PCR.Finally,the epidemic characteristics of blaNDM gene from strains,plasmids,and transpositions were analyzed.RESULTS A total of 23 strains of NDM type E.coli were isolated,which were highly resistant toβ-lactams(except aztreonam),quinolones,and aminoglycosides(except amikacin),and only sensitive to minocycline,amikacin,fosfomycin,polymyxin B and tigecycline.blaNDM-5,blaNDM-1,blaCTX,blaTEM,blaSHV,qnrA,qnrS,qnrD and acc(6)-Ib-cr resistance genes were detected in these strains by PCR assay.In phylogenetic analysis,14 ST strains were classified into 8 clones.Two plasmids of IncX3 and IncF were detected by PBRT,and IncX3 type accounted for 95.6%.Analysis of the transposition structure showed that the blaNDM-1 gene was located in the same transposition structure of the IncX3 plasmid,which showed a certain region specificity.While the blaNDM-5 gene was found both on the IncX3 and IncF plasmids,and mainly located in(ISAba125-IS5-blaNDM-5-bleMBL-trpF-dsbC-IS26)transposition structure,but some transposons were missing IsAba125 sequence.CONCLUSION The blaNDM gene was isolated from 14 strains of ST-type E.coli,which located in the three transposition structures of IncX3 and IncF plasmids,and mainly transferred by IncX3 type plasmids.