血流感染产碳青霉烯酶大肠埃希菌的耐药基因及同源性研究
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摘要
目的探讨血流感染中耐碳青霉烯类大肠埃希菌耐药机制和分析同源性。方法收集温州医科大学附属第一医院2015年6月-2016年5月门诊及住院患者血流感染中分离出的大肠埃希菌,共216株,常规方法进行细菌分离培养,进行菌种鉴定和药敏试验,对碳青霉烯类耐药株进行改良Hogde试验和金属酶初筛试验,聚合酶链式反应(Polymerase Chain Reaction,PCR)方法检测碳青霉烯酶耐药基因,测序确定其基因型,采用多位点序列分型(Multilocus Sequence Typing,MLST)和脉冲场凝胶电泳(Pulsed Field Gel Electrophoresis,PFGE)对碳青霉烯类耐药株进行同源性分析。结果在216株血流感染大肠埃希菌中,有6株耐碳青霉烯类抗菌药物,占2.8%,其中3株亚胺培南耐药株改良Hodge试验阳性,1株金属酶初筛试验阳性,3株厄他培南耐药株则均为阴性。3株亚胺培南耐药株中1株携带blaNDM-5基因,2株携带blaKPC-2基因。6株碳青霉烯类抗菌药物耐药大肠埃希菌临床分离株中,其克隆分型分别为ST648、ST5150、ST131、ST964、ST2003和ST38。PFGE共分为5个型别,其中C1与C6为同一型别,其余4株为不同型别。结论本研究中6株耐碳青霉烯类大肠埃希菌的主要耐药机制为产KPC-2酶和NDM-5酶,MLST和PFGE的结果显示属于不同的克隆型。
OBJECTIVE To investigate the resistance mechanism and homology of E.coli isolates producing carbapenemases associated with bloodstream infection(BSI).METHODS A total of 216 E.coli isolates isolated from the patients with BSI at the First Affiliated Hospital of Wenzhou Medical University from Jun.2015 to May 2016 were collected,and routinely cultured.The identification and antimicrobial susceptibility testing for E.coli isolates were performed.The modified Hodge test was used for detecting carbapenemases and EDTA-disk synergy test for detecting metallo-β-lactamases among carbapenem-resistant E.coli isolates.The genes responsible for carbapenem resistance were detected by polymerase chain reaction(PCR)and DNA sequencing.Multilocus sequence typing(MLST)and pulsed field gel electrophoresis(PFGE)were used for determining the homology of the carbapenemresistant E.coli isolates.RESULTS Among the 216 E.coli isolates,6(2.8%)were resistant to carbapenems.Three isolates with resistance to imipenem were positive in the modified Hodge test,and 1 strain was positive in preliminary screening for metallo-β-lactamase,while 3 isolates with resistance to ertapenem were negative.Among the three imipenem-resistant strains,one carried blaNDM-5 gene and two carried blaKPC-2.The six carbapenem-resistant E.coli isolates belonged to ST648,ST5150,ST131,ST964,ST2003 and ST38,respectively.Five PFGE types were found among the six carbapenem-resistant E.coli isolates,with E.coli C1 and E.coli C6 being the same PFGE pattern.CONCLUSIONProduction of KPC-2 and NDM-5 was the main resistance mechanism for the six carbapenem-resistant E.coli isolates,and the results of MLST and PFGE showed that they belonged to different cloning types.
OBJECTIVE To investigate the resistance mechanism and homology of E.coli isolates producing carbapenemases associated with bloodstream infection(BSI).METHODS A total of 216 E.coli isolates isolated from the patients with BSI at the First Affiliated Hospital of Wenzhou Medical University from Jun.2015 to May 2016 were collected,and routinely cultured.The identification and antimicrobial susceptibility testing for E.coli isolates were performed.The modified Hodge test was used for detecting carbapenemases and EDTA-disk synergy test for detecting metallo-β-lactamases among carbapenem-resistant E.coli isolates.The genes responsible for carbapenem resistance were detected by polymerase chain reaction(PCR)and DNA sequencing.Multilocus sequence typing(MLST)and pulsed field gel electrophoresis(PFGE)were used for determining the homology of the carbapenemresistant E.coli isolates.RESULTS Among the 216 E.coli isolates,6(2.8%)were resistant to carbapenems.Three isolates with resistance to imipenem were positive in the modified Hodge test,and 1 strain was positive in preliminary screening for metallo-β-lactamase,while 3 isolates with resistance to ertapenem were negative.Among the three imipenem-resistant strains,one carried blaNDM-5 gene and two carried blaKPC-2.The six carbapenem-resistant E.coli isolates belonged to ST648,ST5150,ST131,ST964,ST2003 and ST38,respectively.Five PFGE types were found among the six carbapenem-resistant E.coli isolates,with E.coli C1 and E.coli C6 being the same PFGE pattern.CONCLUSIONProduction of KPC-2 and NDM-5 was the main resistance mechanism for the six carbapenem-resistant E.coli isolates,and the results of MLST and PFGE showed that they belonged to different cloning types.
引文
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[11]孙鹏飞,夏文颖,王珏,等.ST131型产NDM-1型碳青霉烯酶大肠埃希菌的耐药机制研究[J].临床检验杂志,2018,36(9):655-659.
[12]Peirano G,Bradford PA,Kazmierczak KM,et al.Global Incidence of carbapenemase-producing Escherichia coli ST131[J].Emerg Infec Dis,2014,20(11):1928-1931.
[2]CLSI:Performance Standards for Antimicrobial Susceptibility Testing,21th Informational Supplement.CLSI document M100-S22[S].Wayne,PA:CLSI,2012.
[3]李军,邹明祥,王海晨,等.耐碳青霉烯类肠杆菌科细菌耐药机制的研究[J].中华医院感染学杂志,2016,26(21):4801-4804.
[4]Queenan AM,Bush K.Carbapenemases:the versatile betalactamases[J].Clin Microbiol Rev,2007,20(3):440-458.
[5]赵晓杰,邓丽华,施德仕,等.碳青霉烯类耐药肺炎克雷伯菌分子流行病学和耐药基因分析[J].国际检验医学杂志,2015,36(11):1530-1532.
[6]Satlin MJ,Chen L,Patel G,et al.Bacteremia due to carbapenem-resistant Enterobacteriaceae(CRE):a multicenter clinical and molecular epidemiologic analysis in the Nation's Epicenter for CRE[J].Antimicrob Agents Ch,2017,61(4):e02349-16.
[7]李玉雪,郭玉梅.耐碳青霉烯类肺炎克雷伯菌的耐药表型及同源性分析[J].中华医院感染学杂志,2018,28(17):2572-2575.
[8]Hu X,Xu X,Wang X,et al.Diversity of New Delhi metallo-bata-lactamase-producing bacteria in China[J].Int J Infect Dis,2017,55:92-95.
[9]闫文娟,李轶,王山梅,等.耐碳青霉烯类抗菌药物大肠埃希菌耐药机制的研究[J].检验医学,2016,31(1):56-60.
[10]Chitnis AS,Caruthers PS,Rao AK,et al.Outbreak of carbapenem-resistant Enterobacteriaceae at a long-term acute carehospital:sustained reductions in transmission through active surveillance and targeted interventions[J].Infect Centrel Hosp Epidemiol,2012,33(10):984-992.
[11]孙鹏飞,夏文颖,王珏,等.ST131型产NDM-1型碳青霉烯酶大肠埃希菌的耐药机制研究[J].临床检验杂志,2018,36(9):655-659.
[12]Peirano G,Bradford PA,Kazmierczak KM,et al.Global Incidence of carbapenemase-producing Escherichia coli ST131[J].Emerg Infec Dis,2014,20(11):1928-1931.