肺炎克雷伯菌碳青霉烯酶基因型的研究

发布时间：2018-03-06 11:40

  本文选题：碳青霉烯酶　切入点：肺炎克雷伯菌　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究本院肺炎克雷伯菌产生的碳青霉烯酶的基因分型及菌株的同源性,为临床治疗碳青霉烯类耐药肺炎克雷伯菌(CarbapenemresiSTant Klebsiella pneumonia,CRKP)提供理论依据,同时为防控碳青霉烯类抗生素耐药的肺炎克雷伯菌的发生及流行提供基础材料。方法:收集吉林大学中日联谊医院2015年临床分离的碳青霉烯类抗生素耐药的肺炎克雷伯菌,采用VITEK2 Compact全自动细菌鉴定仪进行菌种鉴定,应用M-H琼脂稀释法测定头孢噻肟、头孢他啶、头孢吡肟、阿莫西林/克拉维酸、厄他培南、亚胺培南、美罗培南、环丙沙星、阿米卡星和多粘菌素E的最小抑菌浓度,应用改良Hodge试验检测菌株是否产碳青霉烯酶,同时应用EDTA双纸片协同试验检测菌株是否产金属β-内酰胺酶。应用聚合酶链式反应(Polymerase chain reaction,PCR)技术进行常见的β-内酰胺酶耐药基因的检测,包括碳青霉烯酶基因bla_(KPC),B类金属酶基因bla_(IMP)、bla_(VIM)、bla_(NDM),超广谱β-内酰胺酶(extended-spectrum beta-lactamase,ESBLs)基因bla_(CTX-M),广谱β-内酰胺酶基因bla_(TEM)、bla_(SHV),最终应用基因测序技术确定基因分型。本实验应用脉冲场凝胶电泳(Pulsed field gel electrophoresis,PFGE)和多位点序列分析(Multilocus sequence typing,MLST)对菌株进行同源性分析和基因分型。结果:1.本研究共收集12株碳青霉烯类耐药的肺炎克雷伯菌菌株,琼脂稀释法药敏试验结果显示,12株细菌均对头孢噻肟、头孢他啶、头孢吡肟、阿莫西林/克拉维酸、厄他培南、亚胺培南、美罗培南耐药,对环丙沙星的耐药率为25%(3/12),中介率为16.7%(2/12),敏感率为58.3%(7/12),对阿米卡星的耐药率为33.3%(4/12),中介率为8.3%(1/12),敏感率为58.3%(7/12),对多粘菌素e的敏感率为100%。2.12株肺炎克雷伯菌中有8株菌改良hodge试验阳性及7株edta双纸片协同试验阳性。3.耐药基因检测结果显示12株实验菌中7株菌都携带bla_(NDM-1)基因,未检出其他β-内酰胺酶耐药基因。4.7株碳青霉烯类耐药的肺炎克雷伯菌mlST共5个型,JL-12和JL-132株菌为ST11,JL-14为ST307,JL-6为ST2233,JL-3及JL-52株为ST2232、JL-1为ST2231,其中ST2231、ST2232、ST2233为新型,已被pubmlST数据库收录。5.7株肺炎克雷伯菌分为4种pfge型(a-d组),a组有2株,为JL-12、JL-13,b组有3株,为JL-1、JL-3,JL-5,c组为JL-14,d组为JL-6。结论:1.我院分离的碳青霉烯类抗生素耐药的肺炎克雷伯菌对其他β-内酰胺类抗生素全部耐药,对多粘菌素e全部敏感,部分菌株对环丙沙星及阿米卡星敏感。因此,除多粘菌素e外,也可以把阿米卡星及环丙沙星作为联合治疗碳青霉烯类抗生素耐药的肺炎克雷伯菌感染的选择药物。2.我院分离的肺炎克雷伯菌对碳青霉烯类抗生素耐药的主要机制是产生碳青霉烯酶,本研究检出的7株耐药基因全部为金属酶β-内酰胺酶NDM-1基因(bla_(NDM-1)),这不同于我国主要流行的KPC-2基因型。3.本研究PFGE分型共有4个,其中A组有2个、B组有3个菌株具有同源性,与MLST分型结果一致,本院碳青霉烯类抗生素耐药的肺炎克雷伯菌存在爆发流行的克隆,除了发现MLST分型ST11这种在我国广泛流行的基因型外,也同时发现ST2231、ST2232、ST2233三种新基因型的出现,说明我国碳青霉烯类抗生素耐药的肺炎克雷伯菌的克隆存在地域差别。
[Abstract]:Objective: To study the homology of carbapenemase in Klebsiella pneumoniae produced by genotype and strain, for the clinical treatment of carbapenem resistant Klebsiella pneumoniae (CarbapenemresiSTant Klebsiella, pneumonia, CRKP) to provide a theoretical basis, and provide basic materials for the occurrence and epidemic prevention and control of carbapenem antibiotics drug resistance of Klebsiella pneumoniae. Methods: Klebsiella pneumoniae clinical isolates collected from Jilin University in 2015 in China Japan Union Hospital of carbapenem resistant bacteria, were identified by VITEK2 Compact automatic bacterial identification instrument, determination of cefotaxime by M-H agar dilution method, ceftazidime, cefepime, amoxicillin / clavulanic acid, ertapenem, imipenem, meropenem, ciprofloxacin, MIC Amikacin and polymyxin E, modified Hodge test whether strains producing carbon black Penem enzyme, and application of EDTA double disk synergy test to detect whether strains of metallo beta lactamase producing. The polymerase chain reaction (Polymerase chain reaction, PCR) to detect common beta lactamase resistant gene technology, including carbapenemase gene bla_ (KPC), B metal enzyme gene bla_ (IMP), bla_ (VIM), bla_ (NDM), ESBLs (extended-spectrum beta-lactamase ESBLs) gene bla_ (CTX-M), ESBLs genes bla_ (TEM), bla_ (SHV), the final application of gene sequencing technology to determine the experimental application of genotyping. Pulsed field gel electrophoresis (Pulsed field gel electrophoresis, PFGE) and multilocus sequence analysis (Multilocus sequence, typing, MLST) of the strains homology analysis and genotyping. Results: 1. strains of Klebsiella pneumoniae were collected for the study of 12 strains of carbapenem resistant drug, agar dilution method 鏁忚瘯楠岀粨鏋滄樉绀，

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