产KPC型碳青霉烯酶大肠埃希菌分子流行病学及耐药传播机制研究

发布时间：2018-03-28 19:33

本文选题：碳青霉烯酶　切入点：大肠埃希菌　出处：《浙江大学》2015年硕士论文

【摘要】：碳青霉烯类抗生素是临床治疗多重耐药革兰阴性菌感染的重要药物,碳青霉烯耐药肠杆菌科细菌的流行给临床感染性疾病的治疗带来了很大难题,而耐药的主要原因即是细菌产碳青霉烯酶,其中在我国以KPC型碳青霉烯酶最为常见。本研究收集2012年7月至2014年1月杭州市中医院和浙江大学医学院附属第一医院临床共25株产KPC酶大肠埃希菌。利用K-B纸片法测试所有菌株对22种常用抗菌药物的敏感性；改良Hodge实验确认菌株产碳青霉烯酶,PCR扩增确认blaKPC基因；脉冲场凝胶电泳(PFGE)及多位点序列分型(MLST)技术进行菌株同源性分析。利用滤膜接合实验将临床菌株携带质粒接合转移至受体菌；碱裂解法提取质粒；细菌包埋S1核酸酶酶切再PFGE的方法判断质粒分子量大小；挑选代表菌株进行高通量测序及序列分析,注释携带有blaKPC基因的片段,并设计引物PCR扩增其余菌株,分析blaKPC基因的周围结构；PCR扩增筛查其它常见的耐药基因。 25株临床收集大肠埃希菌经确认均为产KPC酶菌株,编码有blaKPC-2基因,这些产KPC酶菌株对大多常用抗菌药物的耐药率很高,均表现为多重耐药菌。流行病学分析显示,产KPC酶菌株来源病人以中老年为主,送检的临床科室分布有一定的集中性,多集中在急诊科、ICU和老年病科。PFGE分型可以将25株菌株分为克隆A(4株)、克隆B(5株)和克隆C(2株),其余14株为散发克隆。MLST分型则分为8个ST型,其中以ST131型为主,共有14株,另外还包括ST167型3株,ST2003型3株,其余ST型各有1株,这些ST型之间同源性相距较远。临床分离大肠埃希菌往往携带多个质粒,质粒分子量大小在25kb-200kb之间,而且大多具有可接合性。bldKPC-2基因由质粒编码,高通量测序所获得的序列片段分析表明blaKPC-2基因位于一个Tn3-Tn4401复合转座子中,并且大部分菌株都拥有一致的周围结构。这些菌株还常共同编码blaCTX-M、blaTEM、blaSHV、qnr、 aac(6')-Ib及rmtB等耐药基因。产KPC酶大肠埃希菌已经出现院内流行的趋势,这些菌株往往由质粒编码blaKPC等多种耐药基因,耐药性的传播转移能力很强,急需加强医院感染控制及耐药性监测,以防止多重耐药菌的大范围播散。
[Abstract]:Carbapenem antibiotics are important drugs for the treatment of multidrug resistant gram-negative bacteria infection. The prevalence of carbapenem resistant Enterobacteriaceae bacteria has brought great difficulties to the treatment of clinical infectious diseases. The main reason of drug resistance is carbapenem produced by bacteria, of which KPC type carbapenem is the most common in China. From July 2012 to January 2014, 25 strains of Escherichia coli producing KPC enzyme were collected from Hangzhou Hospital of traditional Chinese Medicine and the first affiliated Hospital of Zhejiang University Medical College. K-B disk method was used to test the sensitivity of all strains to 22 commonly used antimicrobial agents. The modified Hodge assay confirmed that the blaKPC gene was confirmed by PCR amplification of carbapenem producing enzyme. Pulse field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to analyze the homology of the strains. The plasmids of clinical strains were transferred to the recipient bacteria by filtration membrane bonding experiment, and the plasmids were extracted by alkaline lysis. The method of bacterial embedding S1 nuclease digestion and PFGE was used to determine the molecular weight of plasmids, the representative strains were selected for high-throughput sequencing and sequence analysis, the fragments carrying blaKPC gene were annotated, and primers PCR were designed to amplify the other strains. The peripheral structure of blaKPC gene was analyzed by PCR to screen other common drug resistance genes. All 25 strains of Escherichia coli were confirmed to be KPC producing strains, encoding blaKPC-2 gene. The resistance rate of these KPC producing strains to most commonly used antimicrobial agents is very high, all of them are multidrug resistant bacteria. The patients from KPC producing strains were mainly middle and old people, and the clinical departments submitted for examination had a certain concentration. Most of the 25 strains were divided into four clones, four strains and five clones, and the other 14 strains were classified into 8 ST-types, including ST131 type (14 strains), sporadic clone and MLST type (14 strains), and the other 14 strains were classified into 8 ST-types, and the other 14 strains were classified into 8 ST-types, including ST131 type (14 strains), and the other 14 strains were classified as sporadic clones and MLST genotypes into 8 ST-types. In addition, there were 3 strains of ST167 type ST2003 and 1 strain of other ST-type. The homology of these ST-types was far away. Clinical isolates of Escherichia coli often carried multiple plasmids, and the molecular weight of the plasmids was between 25kb-200kb, and the molecular weight of the plasmids was in the range of 25kb-200kb. Moreover, most of the conjugable. BldKPC-2 genes were encoded by plasmids. Sequence analysis obtained by high throughput sequencing showed that the blaKPC-2 gene was located in a Tn3-Tn4401 complex transposon. And most of the strains have the same surrounding structure. These strains also coencode resistance genes such as blaCTX-Mnr, aac(6')-Ib, rmtB and so on. Escherichia coli producing KPC enzyme has become prevalent in hospitals. These strains are often encoded by plasmids and other multidrug resistant genes such as blaKPC. The ability of transmission and transfer of drug resistance is very strong. It is urgent to strengthen the control of nosocomial infection and surveillance of drug resistance. To prevent the wide spread of multidrug resistant bacteria.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

【共引文献】