中国碳青霉烯耐药肠杆菌科菌流行病学及耐药机制研究

发布时间：2018-04-03 01:00

本文选题：肠杆菌科菌　切入点：碳青霉烯类药物　出处：《北京协和医学院》2015年博士论文

【摘要】：[目的]研究碳青霉烯耐药肠杆菌科菌的感染危险因素、临床预后、分子流行病学以及耐药机制。[方法]收集2004-2012年我国34家教学医院的342株碳青霉烯耐药肠杆菌科菌。采用病例-对照研究分析碳青霉烯耐药肠杆菌科菌感染的危险因素和临床预后。采用对倍琼脂稀释法测定抗菌药物的最低抑菌浓度；多位点序列分析(MLST)和脉冲场凝胶电泳(PFGE)分析CRE的同源性。采用改良的Hodge试验检测菌株产的碳青霉烯酶；聚合酶链式反应(PCR)检测包括碳青霉烯酶基因在内的多种p内酰胺酶基因；对PCR阳性产物进行序列测定以确定基因型别；接合试验检测耐药基因的可移动性；质粒提取试验分析菌株携带的质粒；Southern杂交进行碳青霉烯酶基因定位；全质粒序列测序和分析进行碳青霉烯酶基因环境研究：十二烷基磺酸钠一聚丙烯酰胺凝胶电泳(SDS-PAGE)分析菌株的外膜孔通道蛋白。基因克隆技术研究marR基因突变对大肠埃希菌碳青霉烯耐药的影响。[结果]CRE感染的临床治疗失败率76.7%。“感染CRE前2个月内广谱抗生素使用大于7天”是CRE感染的独立危险因素(OR= 19.088, P=0.006)。CRE敏感性较高的药物包括多粘菌素B(总体敏感率96.1%),替加环素(总体敏感率84.2%)和阿米卡星(总体敏感率55.5%)。342株CRE菌株中241株(70.5%)携带碳青霉烯酶基因,其中173株携带blaKPC-2基因,26株携带blaNDM-1基因,42株携带blaIMP-type基因。产KPC-2酶的菌株在近年间的数量上升迅速,产NDM-1酶的菌株在2010年开始出现,近年来也呈现增长趋势。产KPC-2酶的CRE菌株主要分布于浙江省、北京市和江苏省。多位点序列分析发现152株产碳青霉烯酶的肺炎克雷伯菌中105株(69.1%)为ST11型。29株产碳青霉烯酶的大肠埃希菌属于17种ST型别。不同的碳青霉烯酶基因经Southern杂交证实位于不同大小的质粒上。全质粒测序的结果显示转座子、插入序列以及整合子等多种可移动元件在碳青霉烯酶基因的传播中扮演重要作用。外膜蛋白分析显示外膜蛋白缺失为不产碳青霉烯酶CRE的主要耐药机制。MarR突变分析研究显示MarR的Gln42Arg突变可引起MarR功能失活,从而提高MarA的表达,进而提升大肠埃希菌对碳青霉烯类药物的耐药性。[结论]本研究是一项全国多中心的连续9年CRE流行病学和耐药机制研究。阐明了CRE感染的高治疗失败率和危险因素,分子流行病学显示产碳青霉烯酶菌株是主要的CRE类型,且ST11型为中国主流的碳青霉烯耐药肺炎克雷伯菌克隆型。产碳青酶烯酶以及外膜蛋白缺失在CRE耐药机制中起主要作用。质粒、转座子、插入序列以及整合子等多种可移动元件在碳青霉烯酶基因的传播中扮演重要作用。marR基因突变在大肠埃希菌中能导致碳青霉烯耐药。
[Abstract]:[objective] to study the risk factors, clinical prognosis, molecular epidemiology and drug resistance mechanism of carbapenem resistant Enterobacteriaceae.Methods 342 strains of carbapenem resistant Enterobacteriaceae were collected from 34 educational hospitals in China from 2004 to 2012.Case-control study was used to analyze the risk factors and clinical prognosis of carbapenem-resistant Enterobacteriaceae infection.The minimal inhibitory concentration of antimicrobial agents was determined by para-Agar dilution method, and the homology of CRE was analyzed by multilocus sequence analysis and pulsed field gel electrophoresis (PFGE).The improved Hodge test was used to detect carbapenase, polymerase chain reaction (PCR) was used to detect many kinds of plactamases including carbapenem genes, and the positive products of PCR were sequenced to determine the genotypes.The conjugation test was used to detect the mobility of drug resistance genes, and the plasmids carried by the strains were analyzed by Southern blotting to locate the carbapenem gene.Sequencing and analysis of the whole plasmid sequence for carbapenem gene environment: sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the outer membrane pore channel protein of the strain.The effect of marR gene mutation on carbapenem resistance of Escherichia coli was studied by gene cloning technique.[results] the clinical treatment failure rate of CRE infection was 76. 7%."use of broad-spectrum antibiotics more than 7 days before CRE infection" is an independent risk factor for CRE infection, OR = 19.088. The drugs with higher P=0.006).CRE sensitivity include polymyxin B (total sensitivity rate 96.1g), tegicycline (overall sensitivity rate 84.2%) and amitine (total sensitivity rate 84.2%).Caraxine (total sensitivity rate of 55.5%, #number0# strains of CRE strain, 70.5%) carried carbapenase gene,Among them, 173 strains carried blaKPC-2 gene, 26 strains carried blaNDM-1 gene and 42 strains carried blaIMP-type gene.The number of strains producing KPC-2 enzyme increased rapidly in recent years. The strains producing NDM-1 enzyme began to appear in 2010 and also showed an increasing trend in recent years.KPC-2-producing CRE strains were mainly distributed in Zhejiang, Beijing and Jiangsu provinces.Multilocus sequence analysis showed that 105 strains of Klebsiella pneumoniae producing carbapenem belonged to 17 types of St type, which belonged to ST11 type. 29 strains of Escherichia coli.Different carbapenase genes were identified to be located on plasmids of different sizes by Southern hybridization.The results of whole plasmid sequencing showed that transposon, insertion sequence and integron play an important role in the transmission of carbapenase gene.The analysis of outer membrane protein showed that the absence of outer membrane protein was the main drug resistance mechanism of non-carbapenase CRE. Marr mutation analysis showed that the Gln42Arg mutation of MarR could cause MarR function inactivation and thus increase the expression of MarA.Furthermore, the resistance of Escherichia coli to carbapenems was enhanced.[conclusion] this study is a national multi-center 9-year CRE epidemiology and drug resistance mechanism study.The high treatment failure rate and risk factors of CRE infection were elucidated. Molecular epidemiology showed that carbapenem producing strain was the main type of CRE, and ST11 type was the dominant clone of Klebsiella pneumoniae in China.Carbogenase production and absence of outer membrane proteins play a major role in the mechanism of drug resistance of CRE.Plasmid, transposon, insertion sequence and integron play an important role in the transmission of carbapenase gene. The mutation of Marr gene can lead to carbapenem resistance in Escherichia coli.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R446.5

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