本文關鍵詞： 鮑曼不動桿菌 MLST分型 β-內酰胺酶 外排泵系統(tǒng) 轉座子　出處：《上海交通大學》2015年碩士論文　論文類型：學位論文

【摘要】：目的:了解近年來上海仁濟醫(yī)院神經(jīng)外科病房分離鮑曼不動桿菌的耐藥性變遷;對菌株進行分子流行病學調查,分析菌株基因同源性,了解本院神經(jīng)外科分離鮑曼不動桿菌的主要流行克隆,為臨床治療和控制院內感染提供依據(jù);檢測β-內酰胺酶基因、主動外排系統(tǒng)結構基因和調控基因在菌株當中的分布,并對主要外排系統(tǒng)結構基因進行基因表達量分析,探討鮑曼不動桿菌對碳青霉烯類的耐藥機制。通過檢測blα_(oxa-23)耐藥基因所在的轉座子結構,并對它的上載質粒進行測序分析,深入了解質粒結構特征,更好得解釋blα_(oxa-23)耐藥基因的播散機制,對正確控制傳播具有重要的意義。方法:1、收集2011年7月至2013年6月上海仁濟醫(yī)院神經(jīng)外科病房分離鮑曼不動桿菌共87株。瓊脂稀釋法檢測菌株對臨床12種抗菌藥物的最低抑菌濃度(minimum inhibitory concentration,MIC),分析三年來本院神經(jīng)外科分離鮑曼不動桿菌的藥物敏感性和耐藥性變遷。2、采用多位點序列分型(multi-locus sequence typing,MLST)技術鑒定87株鮑曼不動桿菌的基因型別,分析菌株流行特征。3、聚合酶鏈反應(Polymerase Chain Reaction,PCR)檢測β-內酰胺酶基因與RND家族3個主要外排系統(tǒng)Ade ABC、Ade IJK及Ade FGH的結構基因與調控基因攜帶情況,比較其在亞胺培南耐藥組(imipenem-resistant Acinetobacter baumannii,IRAB)和亞胺培南敏感組(imipenem-sensitive Acinetobacter baumannii,ISAB)中的分布差異,用實時熒光定量聚合酶鏈反應(Realtime-PCR,RT-PCR)檢測主要外排泵結構基因ade B、ade J和ade G的基因表達水平。4、選取blα_(oxa-23)耐藥基因檢測陽性的耐碳青霉烯類鮑曼不動桿菌(carbapenemsresistant Acinetobacter baumannii,CRAB),PCR法檢測blα_(oxa-23)基因所在的轉座子類型,通過質粒抽提、Southern雜交等方法確定blα_(oxa-23)耐藥基因所定位的質粒,并對質粒進行測序分析。結果:1、上海仁濟醫(yī)院神經(jīng)外科病房連續(xù)三年分離鮑曼不動桿菌對頭孢菌素類、氟喹諾酮類、氨基糖苷類和碳青霉烯類等抗菌藥物耐藥性高,耐藥率呈逐年上升趨勢。87株鮑曼不動桿菌對米諾環(huán)素的耐藥率最低,為33.2%;對亞胺培南和美羅培南的耐藥率分別為74.7%和80.5%;其余抗菌藥物的耐藥率均在70%以上;對磺胺甲VA唑/甲氧芐啶全耐藥。另外,CRAB菌株往往同時對頭孢菌素類、氨基糖苷類、氟喹諾酮類等抗菌藥物耐藥。2、MLST分型結果顯示87株鮑曼不動桿菌可以分為6種ST型,分別是ST 208型(45株)、ST 191型(15株)、ST 540型(8株)、ST 195型(2株)、ST 381型(2株)和ST 368型(1株)。ST 208型是主要的流行型別。除ST 381型外,其余5種型別均屬于CC 92克隆群,具有基因同源性,提示神經(jīng)外科存在鮑曼不動桿菌克隆流行。3、本研究檢測了OXA-23、OXA-24、OXA-51、OXA-58、Amp C、IMP-1及VIM-1/2型等耐藥基因,發(fā)現(xiàn)所有菌株均攜帶OXA-51基因,未檢測到OXA-24、OXA-58、VIM-1和VIM-2型基因。β-內酰胺酶基因Amp C、OXA-23和IMP-1的檢出率分別為93.1%、93.1%和83.9%。Ade ABC外排泵系統(tǒng)的ade B、ade S和ade R基因檢出率分別為96.6%、96.6%和83.9%。Ade IJK及Ade FGH外排泵系統(tǒng)的ade J、ade L、ade F、ade G和ade H基因檢出率都大于90%。經(jīng)統(tǒng)計學分析,β-內酰胺酶基因Amp C、OXA-23和外排泵基因ade B與ade G在IRAB組和ISAB組中的分布差異具有統(tǒng)計學意義。RT-PCR結果顯示IRAB組ade B、ade J和ade G基因平均表達量是ISAB組的5.254、1.263和6.387倍,經(jīng)統(tǒng)計學分析,ade B和ade G基因表達量在IRAB組和ISAB組中的分布差異具有統(tǒng)計學意義。4、PCR結果顯示blα_(oxa-23)基因位于Tn 2006和Tn 2008轉座子上,Southem雜交證實blα_(oxa-23)基因位于質粒上。結論:近年來上海仁濟醫(yī)院神經(jīng)外科病房分離鮑曼不動桿菌對常用抗菌藥物的耐藥性高,且有逐年上升趨勢,需引起醫(yī)務人員高度重視。鮑曼不動桿菌對米諾環(huán)素耐藥率稍低,其次為亞胺培南和阿米卡星,神經(jīng)外科可根據(jù)藥敏結果選擇上述抗菌藥物。ST 208型是神經(jīng)外科感染鮑曼不動桿菌的主要流行型別,特別是IRAB菌株間具有較高同源性,科室內存在交叉感染可能性,臨床應采取切實有效的感染控制措施來阻斷耐藥菌株傳播。產(chǎn)生Amp-C、OXA-23型β-內酰胺酶與Ade ABC、Ade FGH主動外排泵系統(tǒng)過表達在鮑曼不動桿菌對碳青霉烯類耐藥中發(fā)揮重要作用。攜帶blα_(oxa-23)耐藥基因主要位于Tn 2008轉座子上,可通過上載不同的質粒在菌株中播散,導致CRAB菌株廣泛傳播。
[Abstract]:Objective: To investigate the Department of Neurosurgery of Shanghai Renji Hospital ward separated Bauman real changes of drug resistance of bacteria; molecular epidemiology investigation of strain, strain gene homology analysis, understand the main epidemic clone in our hospital department of Neurosurgery Bauman Acinetobacter, provide the basis for clinical treatment and control of nosocomial infection; detection of beta lactamase genes. The distribution of the active efflux system of structural and regulatory genes in these strains, and analyze the main efflux system structure of gene expression, to explore the real Bauman mechanism of carbapenem resistant bacillus. By detection of BL alpha _ (OXA-23) transposon structure resistance gene and the plasmid and uploaded on it the sequencing analysis, in-depth understanding of the plasmid structure, a better interpretation of BL alpha (OXA-23) _ dissemination mechanism of resistance genes, which has important significance to the correct control method of communication. 1, from July 2011 to June 2013 from the Department of Neurosurgery of Shanghai Renji Hospital ward Bauman Acinetobacter 87 strains. The minimum inhibitory concentration was detected by agar dilution method on 12 clinical antimicrobial agents (minimum, inhibitory, concentration, MIC) of three years in our hospital department of Neurosurgery Bauman real separation of drug sensitivity and drug resistance of.2 coli, using a number of sequence type (multi-locus sequence typing, MLST) genotype identification of 87 strains of Bauman Acinetobacter strains, analysis of epidemic characteristics of.3, polymerase chain reaction (Polymerase Chain Reaction, PCR) detection of beta lactamase 3 main genes and RND family Ade ABC exhaust system, structure and regulation of gene carriers Ade IJK and Ade FGH genes, compared to imipenem resistant group (imipenem-resistant Acinetobacter, baumannii, IRAB) and imipenem group (imipenem-sens Itive Acinetobacter baumannii, ISAB) the difference in the distribution, using real-time fluorescence quantitative polymerase chain reaction (Realtime-PCR, RT-PCR) detection of efflux pump gene ade ade J structure of B, ade and G gene expression levels of.4, select the BL alpha _ (OXA-23) positive drug resistance gene detection of carbapenem resistant Acinetobacter Bauman coli (carbapenemsresistant Acinetobacter baumannii, CRAB), BL alpha _ PCR detection method (OXA-23) gene where the transposon type, by plasmid extraction, Southern hybridization method to determine BL alpha _ (OXA-23) to locate the resistance gene plasmid and the plasmid was sequenced and analyzed. Results: 1, the Department of Neurosurgery of Shanghai Renji Hospital ward for three from Bauman Acinetobacter to cephalosporins, fluoroquinolones, aminoglycosides and carbapenem antibiotics such as high drug resistance, drug resistance rate is rising year by year.87 strains of Bauman Acinetobacter of minocycline 鐨勮，

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