耐碳青霉烯类肺炎克雷伯菌的耐药机制与分子流行病学特征

Antimicrobial resistance mechanism and molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae

目的了解耐碳青霉烯类肺炎克雷伯菌(CRKP)的碳青霉烯酶基因携带情况及分子流行病学特点,为医院感染控制和临床治疗提供实验室依据。方法收集山东省泰安市中心医院2014年1—11月临床分离的CRKP 13株,采用Walk Away 96 PLUS型全自动细菌分析仪进行细菌鉴定和药敏试验;改良Hodge试验及EDTA协同试验进行碳青霉烯酶表型的确认;采用多聚酶链反应(PCR)方法扩增相关碳青霉烯类耐药基因(bla KPC、bla IMP、bla VIM、bla GIM及bla NDM-1)并测序;采用脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)调查菌株的克隆相关性并进行流行病学比较。结果 13株CRKP主要来源于痰(7株,53.85%)及尿(4株,30.77%),对复方磺胺甲口恶唑、四环素的耐药率较低(均<40%),对其他抗菌药物(除阿米卡星)的耐药率均>70%,对碳青霉烯类药物的耐药率均为100%;13株细菌改良Hodge试验均为阳性,5株细菌EDTA协同试验阳性;经PCR测序确认,碳青霉烯酶基因中,bla KPC基因最常见(13/13),其次为bla NDM-1基因(5/13),未发现其基因bla IMP、bla VIM和bla GIM;PFGE聚类结果显示,13株肺炎克雷伯菌被分为5型,主要为C型(9/13),且均属于ST11,其他分别为ST37、ST626、ST628和ST668型。结论碳青霉烯酶基因bla KPC与bla NDM-1是引起该院肺炎克雷伯菌对碳青霉烯类抗生素耐药的主要原因,ST11是主要的克隆类型,医院需尽快强化医院感染预防控制措施。

Objective To investigate carbapenemase genes and molecular epidemiological characteristics of carbape-nem-resistant Klebsiella pneumoniae(CRKP),so as to provide laboratory basis for healthcare-associated infection(HAI)control and clinical treatment.Methods 13 strains of CRKP isolated from Taian City Central Hospital between January and November 2014 were collected,bacterial identification and antimicrobial susceptibility testing were performed by WalkAway 96 PLUS automatic bacterial analyzer;carbapenemase phenotypes were confirmed by modified Hodge test and EDTA-disk synergy test;carbapenem resistance genes(bla KPC,bla IMP,bla VIM,bla GIM,and bla NDM-1)were amplified by polymerase chain reaction(PCR),then sequenced;clone correlation of strains was investigated by pulsed-field gel electrophoresis(PFGE)and multilocus sequence typing(MLST),epidemiology comparison was performed.Results 13 strains of CRKP were mainly isolated from sputum(n=7,53.85%)and urine(n=4,30.77%),resistance rates to compound sulfamethoxazole and tetracycline were all low(all<40%),to the other antimicrobial agents(except amikacin)were all>70%,resistance rates to carbapenems were all 100%;13 strains were all positive for modified Hodge test,5 strains were positive for EDTA-disk synergy test;sequencing of PCR confirmed that bla KPC gene was most common among carbapenemase genes(13/13),followed by bla NDM-1 gene(5/13),while bla IMP,bla VIM,and bla GIM gene were not found;clustering analysis of PFGE showed that 13 strains of Klebsiella pneumoniae were divided into 5 types,mainly type C(9/13),all belonged to ST11,others were ST37,ST626,ST628,and ST668 respectively.Conclusion Carbapenemase genes bla KPC and bla NDM-1 are the main causes of the resistance of Klebsiella pneumoniae to carbapenems in this hospital,ST11 is the main clone type,hospital should strengthen the prevention and control of HAI as soon as possible.