本文選題：碳青霉烯酶　切入點(diǎn)：肺炎克雷伯菌　出處：《吉林大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:研究本院肺炎克雷伯菌產(chǎn)生的碳青霉烯酶的基因分型及菌株的同源性,為臨床治療碳青霉烯類(lèi)耐藥肺炎克雷伯菌(CarbapenemresiSTant Klebsiella pneumonia,CRKP)提供理論依據(jù),同時(shí)為防控碳青霉烯類(lèi)抗生素耐藥的肺炎克雷伯菌的發(fā)生及流行提供基礎(chǔ)材料。方法:收集吉林大學(xué)中日聯(lián)誼醫(yī)院2015年臨床分離的碳青霉烯類(lèi)抗生素耐藥的肺炎克雷伯菌,采用VITEK2 Compact全自動(dòng)細(xì)菌鑒定儀進(jìn)行菌種鑒定,應(yīng)用M-H瓊脂稀釋法測(cè)定頭孢噻肟、頭孢他啶、頭孢吡肟、阿莫西林/克拉維酸、厄他培南、亞胺培南、美羅培南、環(huán)丙沙星、阿米卡星和多粘菌素E的最小抑菌濃度,應(yīng)用改良Hodge試驗(yàn)檢測(cè)菌株是否產(chǎn)碳青霉烯酶,同時(shí)應(yīng)用EDTA雙紙片協(xié)同試驗(yàn)檢測(cè)菌株是否產(chǎn)金屬β-內(nèi)酰胺酶。應(yīng)用聚合酶鏈?zhǔn)椒磻?yīng)(Polymerase chain reaction,PCR)技術(shù)進(jìn)行常見(jiàn)的β-內(nèi)酰胺酶耐藥基因的檢測(cè),包括碳青霉烯酶基因bla_(KPC),B類(lèi)金屬酶基因bla_(IMP)、bla_(VIM)、bla_(NDM),超廣譜β-內(nèi)酰胺酶(extended-spectrum beta-lactamase,ESBLs)基因bla_(CTX-M),廣譜β-內(nèi)酰胺酶基因bla_(TEM)、bla_(SHV),最終應(yīng)用基因測(cè)序技術(shù)確定基因分型。本實(shí)驗(yàn)應(yīng)用脈沖場(chǎng)凝膠電泳(Pulsed field gel electrophoresis,PFGE)和多位點(diǎn)序列分析(Multilocus sequence typing,MLST)對(duì)菌株進(jìn)行同源性分析和基因分型。結(jié)果:1.本研究共收集12株碳青霉烯類(lèi)耐藥的肺炎克雷伯菌菌株,瓊脂稀釋法藥敏試驗(yàn)結(jié)果顯示,12株細(xì)菌均對(duì)頭孢噻肟、頭孢他啶、頭孢吡肟、阿莫西林/克拉維酸、厄他培南、亞胺培南、美羅培南耐藥,對(duì)環(huán)丙沙星的耐藥率為25%(3/12),中介率為16.7%(2/12),敏感率為58.3%(7/12),對(duì)阿米卡星的耐藥率為33.3%(4/12),中介率為8.3%(1/12),敏感率為58.3%(7/12),對(duì)多粘菌素e的敏感率為100%。2.12株肺炎克雷伯菌中有8株菌改良hodge試驗(yàn)陽(yáng)性及7株edta雙紙片協(xié)同試驗(yàn)陽(yáng)性。3.耐藥基因檢測(cè)結(jié)果顯示12株實(shí)驗(yàn)菌中7株菌都攜帶bla_(NDM-1)基因,未檢出其他β-內(nèi)酰胺酶耐藥基因。4.7株碳青霉烯類(lèi)耐藥的肺炎克雷伯菌mlST共5個(gè)型,JL-12和JL-132株菌為ST11,JL-14為ST307,JL-6為ST2233,JL-3及JL-52株為ST2232、JL-1為ST2231,其中ST2231、ST2232、ST2233為新型,已被pubmlST數(shù)據(jù)庫(kù)收錄。5.7株肺炎克雷伯菌分為4種pfge型(a-d組),a組有2株,為JL-12、JL-13,b組有3株,為JL-1、JL-3,JL-5,c組為JL-14,d組為JL-6。結(jié)論:1.我院分離的碳青霉烯類(lèi)抗生素耐藥的肺炎克雷伯菌對(duì)其他β-內(nèi)酰胺類(lèi)抗生素全部耐藥,對(duì)多粘菌素e全部敏感,部分菌株對(duì)環(huán)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環(huán)丙沙星作為聯(lián)合治療碳青霉烯類(lèi)抗生素耐藥的肺炎克雷伯菌感染的選擇藥物。2.我院分離的肺炎克雷伯菌對(duì)碳青霉烯類(lèi)抗生素耐藥的主要機(jī)制是產(chǎn)生碳青霉烯酶,本研究檢出的7株耐藥基因全部為金屬酶β-內(nèi)酰胺酶NDM-1基因(bla_(NDM-1)),這不同于我國(guó)主要流行的KPC-2基因型。3.本研究PFGE分型共有4個(gè),其中A組有2個(gè)、B組有3個(gè)菌株具有同源性,與MLST分型結(jié)果一致,本院碳青霉烯類(lèi)抗生素耐藥的肺炎克雷伯菌存在爆發(fā)流行的克隆,除了發(fā)現(xiàn)MLST分型ST11這種在我國(guó)廣泛流行的基因型外,也同時(shí)發(fā)現(xiàn)ST2231、ST2232、ST2233三種新基因型的出現(xiàn),說(shuō)明我國(guó)碳青霉烯類(lèi)抗生素耐藥的肺炎克雷伯菌的克隆存在地域差別。
[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紺，

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