浙江省丽水地区碳青霉烯类耐药肠杆菌目细菌多黏菌素耐药性及mcr-1基因背景分析

Polymyxin resistance and the genetic background analysis of mcr-1 gene of carbapenem-resistant Enterobacteriacae strains in Lishui area of Zhejiang province

目的了解丽水地区碳青霉烯类耐药肠杆菌目细菌(CRE)的多黏菌素耐药情况及其耐药机制,为临床CRE抗感染治疗和防控提供实验依据。方法收集丽水市中心医院2010—2018年临床分离的CRE菌株,采用多位点序列分型(MLST)技术分析菌株的克隆群,采用微量肉汤稀释法检测菌株对碳青霉烯类和多黏菌素的最低抑菌浓度(MIC),利用PCR法检测碳青霉烯耐药基因和多黏菌素耐药基因mcr-1及其周围基因结构,并测序确认。以转化或接合试验分离纯化耐药质粒,利用复制起始子分析法鉴定质粒类型,纯化提取质粒并以二代测序(NGS)验证。结果共分离CRE菌株223株,其中多黏菌素MIC≥4μg/mL的耐药菌11株,且均为bla_(NDM)基因阳性。多黏菌素耐药菌中8株为mcr-1阳性且均呈低水平耐药(4μg/mL≤MIC≤8μg/mL)。4株菌获得mcr-1基因阳性的接合/转化子,其中IncⅠ2、IncⅩ4型质粒各2例,mcr-1基因分别位于IS Apl1-mcr-1-PAP2和ParA-hyp-hyp-mcr-1-PAP2结构。结论丽水地区CRE菌株对多黏菌素耐药主要呈mcr-1阳性并呈低水平耐药,首次发现bla_(NDM)和mcr-1双阳性的多黏菌素耐药性CRE菌株,需加强对其监测。

Objective To investigate the polymyxin resistance of carbapenem-resistant Enterobacteriacae(CRE)and its resistance mechanism in Lishui area of Zhejiang province,so as to provide experimental basis for clinical anti-infection treatment,prevention and control of CRE.Methods The CRE strains isolated from Lishui Municipal Central Hospital between 2010 and 2018 were collected,and the clone groups of the strains were analyzed by the multilocus sequence typing(MLST).The minimum inhibitory concentration(MIC)of the strains against carbapenems and polymyxin was detected by the microbroth dilution method.The carbapenem resistance genes,polymyxin resistance gene mcr-1 and its surrounding gene structures were detected by PCR,and confirmed by the sequencing.The drug-resistant plasmids were isolated and purified by the transformation or conjugation tests,and the types of plasmids were identified by the replication initiator analysis.The plasmids were extracted and purified,and verified by the next-generation sequencing(NGS).Results A total of 223 CRE strains were isolated,and all were positive for the bla_(NDM) gene.Eleven out of 223 strains were resistant to polymyxin with MIC≥4μg/mL.Among the polymyxin-resistant strains,8 were positive for mcr-1 and showed low-level resistance(4μg/mL≤MIC≤8μg/mL).Four strains obtained mcr-1-positive conjugants/transformants,including 2 cases of IncⅠ2 and 2 cases of IncⅩ4 plasmids,with the mcr-1 gene located in IS Apl1-mcr-1-PAP2 and ParA-hyp-hyp-mcr-1-PAP2 structures,respectively.Conclusion The CRE strains against polymyxin in Lishui area of Zhejiang province are mainly mcr-1 positive and show low-level resistance to polymyxin.The polymyxin-resistant CRE strains with both bla_(NDM) and mcr-1 positive are found for the first time in Lishui area,indicating that monitoring CRE strains needs to be strengthened.