【作者】 杜娟；

【导师】 赵生芳； 段金菊；

【作者基本信息】 山西医科大学 ， 药剂学， 2009， 硕士

【摘要】 鲍曼不动杆菌是目前引起医院内感染的重要病原菌。随着广谱抗菌药物的广泛应用,多重耐药的鲍曼不动杆菌比例不断上升,并在全球多处的重症监护病房发生爆发流行,成为抗感染治疗的棘手问题。根据美国院内感染监测数据(NNIS)以及中国院内感染病原菌调查显示,不动杆菌属在医院内感染中占第4位,成为仅次于铜绿假单胞菌的又一个重要的非发酵病原菌。自1991年美国首例碳青霉烯类耐药的不动杆菌属(CRA)报道以来,世界各地陆续出现此类菌株。近两年来,CRA已成为国际社会讨论的热点话题之一。因为一旦亚胺培南耐药,就意味着对现有的常用广谱抗菌药物均耐药,即泛耐药株(PDRA)。由PDRA引起的感染,常常无药可用,病死率高。不动杆菌的耐药机制复杂,包括产生灭活酶,使抗菌药物失活;改变作用靶部位,从而逃避抗菌药物的作用;改变外膜孔蛋白结构和数量,降低了细菌外膜药物的通透性;激活外排泵系统,进一步降低了细菌胞内抗菌药物的浓度等。近年来山西医科大学第二医院不动杆菌尤其是鲍曼不动杆菌的分离率不断增加,耐药率也不断上升,本研究筛选2007年7月到2008年5月临床分离的多重耐药鲍曼不动杆菌,并对其耐药性、同源性及主要β-内酰胺酶基因型进行研究。1、多重耐药鲍曼不动杆菌的筛选及耐药性研究菌株来源:连续收集山西医科大学附属第二医院2007年7月到2008年5月临床分离的多重耐药鲍曼不动杆菌菌株。方法:采用K-B纸片法,以大肠埃希菌ATCC25922、鲍曼不动杆菌ATCC19606为质控菌,筛选多重耐药不动杆菌。采用琼脂稀释法测定12种抗菌药物的MIC值。抗菌药物结果按2007年CLSI标准判定。结果:筛选出60株多重耐药鲍曼不动杆菌,它们对12种抗菌药物的抗菌活性中,亚胺培南、美罗培南的耐药率分别为45.2%、33.9%,含酶抑制剂的复合制剂头孢哌酮/舒巴坦、哌拉西林/他唑巴坦的耐药率分别为33.3%、59.0%,米诺环素和多粘菌素E分别为16.1%、9.7%,其它抗菌药物的耐药率均在70%以上。2、多重耐药鲍曼不动杆菌同源性研究菌株来源:连续收集山西医科大学附属第二医院2007年7月到2008年5月临床分离的多重耐药鲍曼不动杆菌菌株。方法:利用脉冲场凝胶电泳(PFGE)技术对我院多重耐药鲍曼不动杆菌进行同源性研究,并对其流行情况、克隆分型进行了分析。结果:60株菌株可分为5个克隆株,其中A、B为主要克隆株。A克隆有3个亚型共28株, B克隆有1个亚型共9株, C克隆有2个亚型共4株, D、E各有3和2株,另外还有14株散发菌株。克隆株在病房内、病房间传播成为我院鲍曼不动杆菌分离率上升,菌株不断增加的主要原因。3、多重耐药鲍曼不动杆菌主要β-内酰胺酶基因型研究菌株来源:连续收集山西医科大学附属第二医院2007年7月到2008年5月临床分离的多重耐药鲍曼不动杆菌60株。方法:利用三维实验、双纸片协同实验,进行各种β-内酰胺酶检测。利用PCR扩增、序列分析明确β-内酰胺酶基因型,PCR扩增主要β-内酰胺酶编码基因包括:①ESBLs基因:TEM型、SHV型、PER型、CTX-M-1型、CTX-M-9型、GES型、VEB型;②染色体携带的AmpC酶基因:ADC;③碳青霉烯酶基因:OXA-23型、OXA-24型、OXA-58型、IMP型、VIM型、SIM-1。接合实验、质粒抽提、电转化等明确β-内酰胺酶基因定位。结果:三维实验阳性,提示菌株可能产β-内酰胺酶。60株多重耐药鲍曼不动杆菌PCR扩增后,blaTEM基因阳性的有42株,blaPER有20株阳性,2株菌株携带SHV-2基因;1株菌株CTX-M-1组引物扩增得到阳性产物,序列分析证实为blaCTX-M-3;blaADC1-7基因阳性的有57株,blaADC8基因阳性的有2株;blaOXA-23基因阳性的有15株。对产CTX-M-3型ESBLs的菌株成功进行接合试验,并对其原始菌和接合菌抽提了质粒,国内未见相关报道。产SHV型的两株菌株虽然成功抽提了质粒,但接合实验虽进行多次仍未成功,转化实验也未成功。其它菌株反复多次接合试验、抽提质粒以及电转化均未成功。以上研究表明:1、我院鲍曼不动杆菌多重耐药现象严重,对多粘菌素E的耐药率最低,对头孢哌酮/舒巴坦的耐药率相对较低。2、我院多重耐药的鲍曼不动杆菌的分离率较高,达34%;菌株可分为5个克隆株,其中A、B为主要克隆株。提示我院有多重耐药鲍曼不动杆菌小型爆发流行的可能,因此,控制院内感染至关重要。3、表型检测显示所有菌株金属酶检测试验阴性;头孢曲松、头孢西丁三维实验提示菌株产β-内酰胺酶。4、我院多重耐药鲍曼不动杆菌主要的ESBL基因型是blaTEM和blaPER,同时检测到SHV型基因;碳青霉烯酶基因型为blaOXA-23,全部为CARB,提示我院的β-内酰胺酶的基因型分布与国内其他地区的分布情况存在很大差异。5、我院多重耐药的鲍曼不动杆菌同产多种β-内酰胺酶基因型的菌株较多,提示临床治疗应针对这种耐药分布合理选用抗菌药物。6、对产CTX-M-3型ESBLs的菌株成功进行了接合试验,并对其原始菌和接合菌抽提了质粒。质粒介导的鲍曼不动杆菌的耐药性可以在不同种属间广泛传递、导致耐药性播散的可能,造成医院感染的流行,给临床抗感染治疗构成很大威胁。更多还原

【Abstract】 Acinetobacter baumannii is an important pathogen which caused the nosocomial infection. With the use of a broad range of antimicrobial agents, the proportion of the multi-drug resistant Acinetobacter baumannii strains is rising, and happened outbreak in many intensive care unit around the world. According to monitoring data of National nosocomial infection surveillance (NNIS) and nosocomial infection surveillance in Chinese, Acinetobacter spp. was the 4 most frequently isolated pathogens causing nosocomial infection. Since 1991 ,the first carbapenem- resistant Acinetobacter (CRA) has been reported in the United States , such strains have emerged one after another around the world. Recently, CRA has become one of the hot topics in the international community. Once imipenem-resistant clone was found, it means that those resistant to all classes of antimicrobial agents available ,that is, the pandrug-resistant Acinetobacter (PDRA). Infection caused by PDRA usually cannot use any classes of antimicrobial agents available, and the fatality rate was high.Acinetobacter have complex resistance mechanisms, including generating of inactivated enzyme to inactivate of antimicrobial agents; changing the target site to escape the role of antimicrobial agents; changing the outer membrane pore protein structure and quantity to reduce permeability of the bacterial outer membrane; activating the efflux pump systems to decrease the bacterial intracellular concentration of antibiotics. In recent years, the isolation rate and resistance rates of Acinetobacter baumannii increases gradually in the second hospital of Shanxi Medical University rising. This study screened multi-drug resistant Acinetobacter strains from July 2007 to May 2008 , analyzing their resistance, homology and the majorβ-lactamase genotype.1. The screen of multi-drug resistant A. baumannii and Minimal Inhibitory Concentrations determinationStrain Source: collect the multi-drug resistant A. baumannii which were isolated from July 2007 to May 2008 from the second hospital of Shanxi Medical University. All strains were identified by VITEK-2.Methods: Muti-resistant A. baumannii were screened by using K-B method. The MICs of 12 antimicrobials to muti-resistant A. baumannii were detected by agar dilution.Results: The resistant rates to imipenem and meropenem were 45.2% and 33.9%, and Cefoperzone/sulbactam and piperacillin/sulbactam were 33.9% and 59.0%, respectively. The strains were resistant to minocycline and polymyxin E of 16.1 and 9.7%, respectively. The resistant rates to other antimicrobial agents were more than 70%.2. The relateness of multi-drug resistant A. baumanniiStrain Source: The multi-drug resistant A. baumannii which were isolated from July 2007 to May 2008 from the second hospital of Shanxi Medical University.Methods: Pulse field gel electrophoresis (PFGE) was performed to analyze the relateness of 60 isolates of multi-drug resistant A. baumannii.Results:Total of 60 strains belonged to the popular 5 clones. Clone A had 28 strains, Clone B had 9 strains. Clone C, Clone D and Clone E had 4 strains,3 strains and 2 strains respectively. We found that clones A and B were the dominant isolates and had spread widely among wards in the hospitals.3.Investigation of majorβ-lactamase genotype in multi-drug resistant A. baumannii Strain Source: Collect the multi-drug resistant A. which were isolated from July 2007 to May 2008 from the second hospital of Shanxi Medical University.Methods: Crudeβ-lactamase preparations were extracted and three-dimensional tests were performed to detectedβ-lactamase. Metallo-β-lactamaseproducing isolates were screened by the double-disk.β-lactamase genotype were analyzed by PCR. Conjugation test, plasmid extraction and electrotransformation were performed to locate theβ-lactamase gene.Results: Metallo-β-lactamase were not detected by the double-disk. Three-dimensional tests were positive, suggesting that the strains can produceβ-lactamases.42 multi-drug resistant A. baumannii contained blaTEM-1, blaPER has 20 positive. 2 strains and 1 strains carried blaSHV-2 and CTX-M-1 respectively. blaOXA-23 gene-positive was detect in 15 strains. And the 15 strains were CARB.The conjugation test of producing CTX-M-3-type ESBLs strains were performed successfully. There is no reports which we investigated on A. baumannii C in China. Although the strains blaSHV-1 extracted the plasmid , the conjugation test failed. Plasmid extraction, conjugation test, electrotransformation, and Sourthern blot were not successful in other strains.The above sresults shows:1. Multi-drug resistance was more and more serious. Colinstin remained good susceptibility against A. baumannii. The resistance rates to cefoperazone/sulbactam were low relatively.2. The isolate rates of multi-drug resistant Acinetobacter baumannii increased.Total of 60 strains belonged to the popular 5 clones and clones A and B were the dominant isolates.It suggested that there ia a small outbreak of multi-drug resistant Acinetobacter baumannii in our hospital.3. Metallo-β-lactamase were not detected by the double-disk. Three-dimensional tests were positive, suggesting that the strains can produceβ-lactamases.4. In our multi-drug resistant A. baumannii, blaTEM and blaPER are the main ESBL genotype. blaOXA-23 was main carbapenemases genotype,and the strains were CARB.It show that there was a diversity in the distribution of lactamase genotype between our hospital and other regions.5. There are strains producing the more than twoβ-lactamase genotypes. It suggested that we should choose the antibacterial agent reasonably to this situation.6.The conjugation test of producing CTX-M-3-type ESBLs strains were performed successfully. The plasmid-mediated resistance of A. baumannii can be transfered widely in different species, leading to disseminating resistance. It resulted in the prevalence of nosocomial infection .更多还原