神经外科鲍曼不动杆菌耐药性变迁及耐药机制研究

发布时间：2018-01-19 08:24

  本文关键词： 鲍曼不动杆菌 MLST分型 β-内酰胺酶 外排泵系统 转座子　出处：《上海交通大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:了解近年来上海仁济医院神经外科病房分离鲍曼不动杆菌的耐药性变迁;对菌株进行分子流行病学调查,分析菌株基因同源性,了解本院神经外科分离鲍曼不动杆菌的主要流行克隆,为临床治疗和控制院内感染提供依据;检测β-内酰胺酶基因、主动外排系统结构基因和调控基因在菌株当中的分布,并对主要外排系统结构基因进行基因表达量分析,探讨鲍曼不动杆菌对碳青霉烯类的耐药机制。通过检测blα_(oxa-23)耐药基因所在的转座子结构,并对它的上载质粒进行测序分析,深入了解质粒结构特征,更好得解释blα_(oxa-23)耐药基因的播散机制,对正确控制传播具有重要的意义。方法:1、收集2011年7月至2013年6月上海仁济医院神经外科病房分离鲍曼不动杆菌共87株。琼脂稀释法检测菌株对临床12种抗菌药物的最低抑菌浓度(minimum inhibitory concentration,MIC),分析三年来本院神经外科分离鲍曼不动杆菌的药物敏感性和耐药性变迁。2、采用多位点序列分型(multi-locus sequence typing,MLST)技术鉴定87株鲍曼不动杆菌的基因型别,分析菌株流行特征。3、聚合酶链反应(Polymerase Chain Reaction,PCR)检测β-内酰胺酶基因与RND家族3个主要外排系统Ade ABC、Ade IJK及Ade FGH的结构基因与调控基因携带情况,比较其在亚胺培南耐药组(imipenem-resistant Acinetobacter baumannii,IRAB)和亚胺培南敏感组(imipenem-sensitive Acinetobacter baumannii,ISAB)中的分布差异,用实时荧光定量聚合酶链反应(Realtime-PCR,RT-PCR)检测主要外排泵结构基因ade B、ade J和ade G的基因表达水平。4、选取blα_(oxa-23)耐药基因检测阳性的耐碳青霉烯类鲍曼不动杆菌(carbapenemsresistant Acinetobacter baumannii,CRAB),PCR法检测blα_(oxa-23)基因所在的转座子类型,通过质粒抽提、Southern杂交等方法确定blα_(oxa-23)耐药基因所定位的质粒,并对质粒进行测序分析。结果:1、上海仁济医院神经外科病房连续三年分离鲍曼不动杆菌对头孢菌素类、氟喹诺酮类、氨基糖苷类和碳青霉烯类等抗菌药物耐药性高,耐药率呈逐年上升趋势。87株鲍曼不动杆菌对米诺环素的耐药率最低,为33.2%;对亚胺培南和美罗培南的耐药率分别为74.7%和80.5%;其余抗菌药物的耐药率均在70%以上;对磺胺甲VA唑/甲氧苄啶全耐药。另外,CRAB菌株往往同时对头孢菌素类、氨基糖苷类、氟喹诺酮类等抗菌药物耐药。2、MLST分型结果显示87株鲍曼不动杆菌可以分为6种ST型,分别是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型是主要的流行型别。除ST 381型外,其余5种型别均属于CC 92克隆群,具有基因同源性,提示神经外科存在鲍曼不动杆菌克隆流行。3、本研究检测了OXA-23、OXA-24、OXA-51、OXA-58、Amp C、IMP-1及VIM-1/2型等耐药基因,发现所有菌株均携带OXA-51基因,未检测到OXA-24、OXA-58、VIM-1和VIM-2型基因。β-内酰胺酶基因Amp C、OXA-23和IMP-1的检出率分别为93.1%、93.1%和83.9%。Ade ABC外排泵系统的ade B、ade S和ade R基因检出率分别为96.6%、96.6%和83.9%。Ade IJK及Ade FGH外排泵系统的ade J、ade L、ade F、ade G和ade H基因检出率都大于90%。经统计学分析,β-内酰胺酶基因Amp C、OXA-23和外排泵基因ade B与ade G在IRAB组和ISAB组中的分布差异具有统计学意义。RT-PCR结果显示IRAB组ade B、ade J和ade G基因平均表达量是ISAB组的5.254、1.263和6.387倍,经统计学分析,ade B和ade G基因表达量在IRAB组和ISAB组中的分布差异具有统计学意义。4、PCR结果显示blα_(oxa-23)基因位于Tn 2006和Tn 2008转座子上,Southem杂交证实blα_(oxa-23)基因位于质粒上。结论:近年来上海仁济医院神经外科病房分离鲍曼不动杆菌对常用抗菌药物的耐药性高,且有逐年上升趋势,需引起医务人员高度重视。鲍曼不动杆菌对米诺环素耐药率稍低,其次为亚胺培南和阿米卡星,神经外科可根据药敏结果选择上述抗菌药物。ST 208型是神经外科感染鲍曼不动杆菌的主要流行型别,特别是IRAB菌株间具有较高同源性,科室内存在交叉感染可能性,临床应采取切实有效的感染控制措施来阻断耐药菌株传播。产生Amp-C、OXA-23型β-内酰胺酶与Ade ABC、Ade FGH主动外排泵系统过表达在鲍曼不动杆菌对碳青霉烯类耐药中发挥重要作用。携带blα_(oxa-23)耐药基因主要位于Tn 2008转座子上,可通过上载不同的质粒在菌株中播散,导致CRAB菌株广泛传播。
[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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