耐碳青霉烯类肠杆菌的耐药机制及基因分型的研究

发布时间：2018-04-08 23:13

本文选题：耐碳青霉烯类肠杆菌　切入点：KPC　出处：《大连医科大学》2015年硕士论文

【摘要】：目的:由于临床上抗生素的不合理使用,使得耐碳青霉烯肠杆菌逐年增多。研究我院耐碳青霉烯类肠杆菌科细菌的耐药机理,以及基因分型,进行分子流行病学调查,为我院预防和控制耐碳青霉烯类肠杆菌的感染和暴发流行及指导临床合理用药提供实验依据。方法:筛选2011年1月到2014年11月大连医科大学附属第一医院,对亚胺培南、美罗培南、厄他培南耐药的97株肠杆菌科菌株(即耐碳青霉烯肠杆菌CRE)。用西门子Microscan Walkway96SI全自动药敏检测系统进行细菌鉴定,常规药敏检测,对初筛的产碳青霉烯酶菌株进行改良Hodge实验;提取细菌基因组DNA,PCR扩增碳青霉烯酶基因(KPC、VIM、IMP、GES、OXA-48等);对整合子、ISCR1耐药元件及其携带的可变区耐药基因、复杂性整合子进行检测,PCR阳性产物测序(可变区阳性产物先用限制性内切酶Hinf I酶切,选取不同类型的酶切图谱的产物进行测序),从而了解我院CRE整合子、ISCR1携带耐药基因的类型及分布特征;对临床分离的耐碳青霉烯肺炎克雷伯菌、大肠埃希菌,建立ERIC-PCR基因分型方法,进行分子流行病学研究,为临床控制细菌感染及院感监测提供指导依据。结果:1.97株耐碳青霉烯类肠杆菌中,包括69株肺炎克雷伯菌,20株大肠埃希菌,3株产酸克雷伯菌,2株奇异变形杆菌,1株产气肠杆菌,1株枸橼酸杆菌,1株聚团杆菌。2.39株细菌改良Hodge实验阳性。3.碳青霉烯酶基因扩增结果显示:32株细菌KPC基因阳性、阳性率为33%;21株细菌VIM基因阳性、阳性率为21.6%;9株细菌IMP基因阳性,阳性率为9.3%。GES、OXA-48基因均为阴性。4.97株CRE中,62株细菌Int I阳性,阳性率为63.9%;46株Int I可变区阳性, 阳性率为74.2%;int I可变区携带的耐药基因盒组合方式有5种:32株携带aad A1,2株携带dfr A15,6株aac6+arr3+drf27+aad A16,2株aac6,4株aad A2+aad B。5.56株细菌orf513阳性,阳性率为57.7%。其中16株ISCR携带的耐药基因阳性,阳性率为28.6%,ISCR可变区携带的耐药基因盒的组成方式为三种:2株肺炎克雷伯菌携带ISCR+qnr B6+qac E1+sul I,4株肺炎克雷伯菌携带ISCR+qnr A1+qac E1+sul I,其余10株携带ISCR+qnr B2+qac E1+sul I,包括7株肺炎克雷伯菌,一株大肠埃希菌,一株枸橼酸杆菌,一株产酸克雷伯菌。6.同时携带整合子和ISCR的18株细菌中,有三株肺炎克雷伯菌既携带整合子可变区又含有ISCR可变区,耐药基因组合方式为:int I+aad A1+qac E1+sul I+ISCR+qnr B2+qac E1+sul I。7.ERIC结果表示:耐碳青霉烯类肺炎克雷伯菌存在7个基因型在重症ICU和急诊科主要存在2种基因型,耐碳青霉烯酶大肠埃希菌存在8个基因型,均散在分布在临床各个科室。结论:1.本研究中产碳青霉烯酶菌株主要为肺炎克雷伯菌和大肠埃希菌,碳青霉烯酶基因主要为KPC型和VIM型。2.本研究中整合子主要介导对氨基糖苷类抗生素的耐药;ISCR主要介导喹诺酮类抗生素的耐药;复杂性整合子介导多重耐药。本研究中耐碳青霉烯类肠杆菌整合子、ISCR耐药元件阳性的菌株,并没有携带碳青霉烯酶基因。3.本研究中耐碳青霉烯类肠杆菌科细菌主要分布在重症医学科和急诊科,以耐碳青霉烯酶的肺炎克雷伯杆菌为局部流行爆发趋势。
[Abstract]:Objective: the clinical antibiotics, the carbapenem resistant Enterobacteriaceae increased year by year. The mechanism of carbapenem resistance in Enterobacteriaceae bacteria in our institute, and genotype, molecular epidemiology, and provide experimental basis for the prevention and control of carbapenem resistant Enterobacteriaceae the infection and outbreak and to guide clinical rational drug use. Methods: screening of the first Affiliated Hospital of Dalian Medical University from January 2011 to November 2014, to imipenem, meropenem, 97 strains of Enterobacteriaceae strains resistant to ertapenem (i.e. carbapenem resistant Enterobacteriaceae CRE). The bacteria were identified with the SIEMENS Microscan Walkway96SI automatic drug sensitive detection system, conventional drug susceptibility detection, modified Hodge experiment of carbapenemase Producing Strains Screening; bacterial genome DNA extracted from PCR carbapenemase genes were amplified by VIM (KPC, IMP, GES, OXA-48, etc. The integron, ISCR1); the resistance element and the variable region of drug resistance genes, complexity of integron were detected and sequenced PCR positive products (positive product of variable region first with restriction enzyme Hinf I digestion, product selection of different types of restriction maps were sequenced), so as to understand our CRE integron, ISCR1 carry the types and distribution characteristics of resistance genes; in clinical isolates of carbapenem resistant Klebsiella pneumoniae, Escherichia coli, established ERIC-PCR genotyping methods, molecular epidemiology, clinical control of bacterial infection and hospital infection monitoring provides guidance. Results: 1.97 strains of carbapenem resistant Enterobacteriaceae, including 69 strains of Klebsiella pneumoniae, 20 strains of Escherichia coli, 3 strains of Klebsiella oxytoca, 2 strains of Proteus mirabilis, 1 strains of Enterobacter aerogenes, 1 strains of Citrobacter strains, 1 strains of bacteria Bacillus.2.39 clusters modified Hodge test positive carbon.3. Carbapenem enzyme gene amplification results showed that 32 strains of bacteria KPC gene positive, the positive rate was 33%; 21 strains of bacteria VIM gene positive, the positive rate was 21.6%; 9 strains of bacteria IMP gene positive, the positive rate of 9.3%.GES and OXA-48 genes were negative.4.97 strains CRE, 62 strains of bacteria Int I positive, positive rate 63.9%; 46 strains of Int I variable region of positive, the positive rate was 74.2%; there are 5 kinds of resistance gene cassettes combinations with int I variable region: 32 strains carrying aad A1,2 strains carrying DFR aac6,4 strains of A15,6 strain aac6+arr3+drf27+aad A16,2 aad A2+aad B.5.56 bacterial strains were orf513 positive, the positive rate of 57.7%. among 16 strains of ISCR resistant gene carry a positive, positive rate was 28.6%. The composition of resistance gene cassette carrying the ISCR variable region into three kinds: 2 strains of Klebsiella pneumoniae carrying ISCR+qnr B6+qac E1+sul I, 4 Klebsiella pneumoniae strains carrying ISCR+qnr A1+qac E1+sul I, the remaining 10 strains carrying ISCR+qnr B2+qa C E1+sul I, including 7 strains of Klebsiella pneumoniae, a strain of Escherichia coli, a strain of Citrobacter, a strain of Klebsiella oxytoca.6. carrying integron and ISCR 18 strains of bacteria, three strains of Klebsiella pneumoniae carry integron variable region and a ISCR variable area resistance genes: int I+aad A1+qac combination E1+sul I+ISCR+qnr B2+qac E1+sul I.7.ERIC said: the results of carbapenem resistant Klebsiella pneumoniae are 7 genotypes in severe ICU and emergency department, there are 2 main genotypes of carbapenem resistant Escherichia coli in 8 genotypes were scattered in the distribution of the various clinical departments. Conclusion: 1. middle carbapenemase strains were mainly Klebsiella pneumoniae and Escherichia coli, carbapenemase genes were mainly KPC type and VIM type.2. integron in this study mainly mediated resistance to aminoglycoside antibiotics; ISCR mediated The resistance of quinolone antibiotics; complexity of integron Mediated Multidrug resistance. The study of carbapenem resistant Enterobacteriaceae strains resistant to ISCR integron positive elements, did not carry carbapenemase gene.3. in the study of carbapenem resistant Enterobacteriaceae bacteria mainly distributed in ICU and emergency department of the local epidemic outbreak trend to carbapenem resistant Klebsiella pneumoniae.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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