【作者】 杨青；

【导师】 俞云松；

【作者基本信息】 浙江大学 ， 内科学， 2003， 硕士

【摘要】 一、课题背景 鲍曼不动杆菌广泛分布于自然界、医院环境和人体的皮肤表面，是引起医院感染的重要病原菌，主要引起医院获得性肺炎，尤其在免疫受损、气管切开、使用呼吸机病人中。鲍曼不动杆菌对各种抗菌药物均有一定的耐药性，临床治疗困难，碳青霉烯类抗生素对不动杆菌有很好的抗菌活性。但随着碳青霉烯类抗生素的广泛应用，对其耐药性日益增多。细菌对碳青霉烯类耐药的机制主要有：碳青霉烯酶的产生、外膜孔蛋白的丢失和主动外排。近年来在临床常见病原菌铜绿假单胞菌、鲍曼不动杆菌、肠杆菌科细菌中有关获得性碳青霉烯酶的报道有所增加。由这些产酶株感染造成的暴发流行，使治疗陷入无药可用的境地，病死率很高。 碳青霉烯酶是指对亚胺培南或美罗培南等碳青霉烯类抗生素有较强水解能力的一类β内酰胺酶，它属于Ambler分子分类中的A类、B类、D类酶。其中B类为金属酶，能水解除氨曲南以外的所有β-内酰胺类抗生素，可由染色体、质粒或转座子介导，由后者编码的获得性金属酶可见于铜绿假单胞菌、不动杆菌、肠杆菌科细菌；A类酶能被克拉维酸抑制，种类较少仅见于一些肠杆菌科细菌，属于Bush分类 浙江大学2003届硕士学位论文中的Zf亚群；D类酶属于Bllsh分类中的Zd亚群，仅见于鲍曼不动杆菌，对碳青霉烯类抗生素水解作用相对较弱。 为了解碳青霉烯类抗生素耐药鲍曼不动杆菌耐药特点及流行情况，我们分析了sl株亚胺培南耐药鲍曼不动杆菌的同源性及对14种抗菌药物的耐药性，并对其所产碳青霉烯酶进行初步研究。二、材料与方法 收集浙江大学医学院附属第一医院（浙一医院）2000年 10月至 2002年 9月临床分离的亚胺培南耐药鲍曼不动杆菌46株，浙江大学医学院附属邵逸夫医院（邵逸夫医院）2002年8月临床分离的亚胺培南耐药鲍曼不动杆菌5株，采用浓度梯度法 （Etest）测定最低抑菌浓度（MIC人 脉冲场凝胶电泳0FGE）技术分析同源性；通过等电点聚焦电泳（IEF）、三维试验、2－颈基丙酸抑制试验、聚合酶链反应（PCR）、克隆测序等方法分析碳青霉烯酶类型。三、结果1．药敏结果： sl株鲍曼不动杆菌对亚胺培南、美洛培南均耐药；对头抱派酮／舒巴坦敏感率最高为82％，其次为氨书西林／舒巴坦为40％，头抱毗胎、头抱他陡、头抱噬厉有66～74％处于中介。其余抗菌药物耐药率高达94％～100％。L 染色体DNA同源性分析及碳青霉烯酶初步研究 浙一医院46株菌株 PFGE图谱分为 A、B、C三型，A型是主要流行株（44株），主要集中在重症监护病房（ICU），可分为 4个亚型 AI、AZ、A3、A4。B型 1株来自血液科病房，C型 1株来自 ICU。46株菌株中有 44株产碳青霉烯酶，其中 16号 3 浙江大学2003届硕十学位论文菌株（PFGE为B型）等电点有6．7、72两个条带，OXA－23组引物PCR反应阳性，pI6．7条带与文献报道的OXA《3等电点一致；30号菌株0FGE为C型）等电点有6．4、6．8两个条带，OXA－23组引物PCR反应阳性，产物经克隆测序与OXA－23相比在 22位插入 1个丙氨酸，为一种新的碳青霉烯酶（OXA49，GenBank登录号为AY288523人其余42株（AI～A4型）等电点均有6．4、7．0两个条带，均不被克拉维酸、氯哇西林及2－疏基丙酸抑制，PCR方法也未能检出OXA－23、OXA．24系列的基因。邵逸夫医院5株细菌，l株与浙一医院AZ型条带完全一致，另4株同时产OXA上3型碳青霉烯酶和 PER刁型超广谱 p－内酚胺酶（ESBLS人均来自普外科病房，PFGE为D型。四、结论＊）碳青霉烯类耐药鲍曼不动杆菌的流行主要为医院感染所致。口） 该流行株呈多重耐药，头抱呢酮／舒巴坦、氨芋西林／舒巴坦可作为这部分菌株感 染的治疗选择。o）引株菌株中 49株菌株均产碳青霉烯酶，但不属于金属酶，仅 6株产 OXA型碳 青霉烯酶，5株为 OXA－23，l株产一种新的碳青霉烯酶 OXA－49（GenBank登 录号为AY288523人其他43株可能产…6．4或7．0的碳青霉烯酶。K） 首次在浙江省内发现同时产 O皿23型碳青霉烯酶和 PER八型 ESBLS的鲍曼不 动杆菌。更多还原

【Abstract】 1. BackgroundAcinetobacter baumannii can be found in natural environment, hospital surroundings, and skin of human body. It is an important opportunistic nosocomial pathogen and particularly important in hospital acquired pneumonia, especially in immuno-compromised patients and those with tracheotomy or mechanical ventilation. A. baumannii isolates are usually multiresistant, and their infections are complicating therapy. Carbapenems have good antibacterial activity against A. baumannii. However, carbapenem-resistant isolates are increasingly isolated as carbapenems are used widely in clinical practice. Mechanisms of carbapenems resistance include: production of carbapenemase, loss of outer membrane porin, alteration of target, and efflux system. In recent years, the reports about acquired carbapenemase in frequently isolated pathogens such as P. areruginosa, A. baumannii, and Enterobacteriaceae has been increasing. The outbreak caused by these strains makes the situation worse. Few effective antimicrobial agents are available for these infections with high mortality.Carbapenemases may be defined as β -lactamases that significantly hydrolyze atleast imipenem or/and meropenem. They belong to Ambler molecular class A, class B and class D. The class B enzymes are metalloenzymes and hydrolyze virtually all β-lactams except aztronam, whose genes are chromosome, plasmid or integron located. The acquired metalloenzymes are plasmid or integron encoded in Psedomonas aeruginosa A. baumannii, and Enterobacteriaceae. Class A, clavulanic acid-inhibited carbapenemases, have only been reported in rare Enterobacterial isolates. They belong to the group 2f as defined by Bush et al. The class D carbepenemases belonging to the group 2d are from A. baumannii isolates, and hydrolyze imipenem and meropenem weakly.We carried out this study to demonstrate the resistance pattern and prevalence of carbapenems-resistant A. baumannii in the First hospital and Sir Run Run Shaw Hospital, affiliated to the college of medicine, Zhejiang University. We described the resistance pattern of 51 strains of imipenem-resistant A. baumannii to 14 antimicrobial agents and analyzed the homology between these strains and studied the carbapenemases produced.2. Materials and methodsThe homology of 51 strains of imipenem-resistant A. baumannii collected from clinical specimens was analyzed by pulsed-field gel electrophoresis (PFGE). The minimal inhibitory concentrations(MICs) of these strains were detected by Etest strips, and the carbapenemases produced by these strains were typed by isoelectric focusing (IEF) electrophoresis, three-dimensional test, 2-mercaptopropanoic acid inhibited assays.The encoding genes were analysed by polymerase chain reaction (PCR), cloning and sequencing.3. ResultsAll 51 strains were multi-drug resistant and also highly resistant to meropenem. Themost active agents against these strains were cefoperazone/sulbactam and ampicillin/sulbactam with susceptibility rate of 82% and 40%, respectively. About 66%~74% of these strains were intermediate to cefepime, ceftazidime and cefotaxime. All strains were highly resistant to other antimicrobial agents tested.Forty-six strains isolated from the First Hospital were classified into Type A, B and C based on PFGE pattern. Most of type A strains were isolated from ICU and were the dominant strains including subtypes A1, A2, A3 and A4. Only 1 strain isolated from hematology department belongs to Type B, and another strain isolated from ICU belongs to Type C. Forty-four strains produced carbapenemases. One strain ( PFGE type B) had 2 bands on IEF electrophoresis. The pIs of which were 6.7 and 7.2 respectively. The band with pI of 6.7 was OXA-23. Another strain(PFGE type C) produce a new carbepenemase(OXA-49,GenBank accession No.AY288523) being a point-mutant of OXA-23. The other 42 strains produced 2 β-lactamases bands with pIs of 6.4 and 7.0 that couldn’t be inhibited by clavulanic acid, cloxacillin and 2-mercaptopropanoic acid, OXA-23, OXA-24 typ更多还原