碳青霉烯类耐药鲍曼不动杆菌耐药机制研究

发布时间：2018-05-20 16:00

本文选题：鲍曼不动杆菌 + 同源性分析　；参考：《上海交通大学》2015年博士论文

【摘要】：目的本研究通过了解我院鲍曼不动杆菌感染情况、耐药特征与流行趋势,研究重症监护病房临床菌株与环境菌株耐药特征,检测耐碳青霉烯类鲍曼不动杆菌水解酶及外排泵基因,检测主动外排系统结构基因与调控基因在亚胺培南耐药组(imipenem-resistant Acinetobacter baumannii IRAB)与敏感组(imipenem-sensitive Acinetobacter baumannii ISAB)中分布差异,对主要外排基因进行表达量分析,并研究外膜蛋白Omp34介导鲍曼不动杆菌对碳青霉烯类耐药,深入探讨鲍曼不动杆菌对碳青霉烯类药物的耐药机制。方法1、本实验收集2012年7月到10月我院临床分离鲍曼不动杆菌共78株,并采用ERIC-PCR及PFGE技术对该78株临床分离株进行同源性分析,了解本院鲍曼不动杆菌是否存在克隆株的播散流行。2、同期采用琼脂稀释法检测神经外科重症监护病房(Intensive Care Unit,ICU)27株临床菌株和28株环境菌株对常用抗菌药物的最低抑菌浓度;用肠杆菌科基因间重复序列PCR(Enterbacteriat Repetitive Intergenic Consensus,ERIC-PCR)、多位点序列分型(Multi Locus Sequence Typing,MLST)及脉冲场凝胶电泳(Pulsed Field Gelelectrophoresis,PFGE)技术对共分离的55株鲍曼不动杆菌进行基因分型,分析其可能的传播途径。3、采用聚合酶链反应(Polymerase Chain Reaction,PCR)检测78株临床菌株β-内酰胺酶基因(OXA-23、OXA-24、OXA-51、OXA-58、IMP-1、VIM-1、VIM-2、Amp-C)及RND家族3个主要外排系统Ade ABC、Ade IJK及Ade FGH结构基因(ade B、ade J、ade F、ade G、ade H)与调控基因(ade R、ade S、ade L)的携带情况,比较其在IRAB及ISAB中分布差异,进一步使用Realtime-PCR对其主要外排基因进行表达量分析,初步探讨鲍曼不动杆菌对碳青霉烯类药物的耐药机制。4、研究通过采用双重同源重组技术在鲍曼不动杆菌ATCC 19606中构建Omp34基因的缺失突变株(ATCC 19606-△Omp34),采用十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-Polyacrylamide Gel Electrophoresis,SDS-PAGE)蛋白电泳分析外膜蛋白的表达,PCR法和基因测序进一步验证Omp34基因敲除。比较野生株和敲除株对常用抗菌药物尤其是碳青霉烯类的抑菌圈大小,以探讨外膜蛋白Omp34在介导鲍曼不动杆菌对碳青霉烯类耐药中的作用。结果1、临床分离78株鲍曼不动杆菌只对米诺环素耐药率稍低,为30.9%;其次为亚胺培南,耐药率为53.1%;对其它抗菌药物耐药率均超过60%;对头孢西丁、复方新诺明耐药率最高,分别为97.5%、93.8%。78株鲍曼不动杆菌ERIC-PCR结果显示可分为A、B、C、D、E、F 6型,A型62株,为主要克隆株,其中A型又分为A1型46株,A2型16株,B型7株,C型6株,D、E、F型各1株。PFGE主要分为7型,A型56株(包括A1、A2、A3三个亚型),B型7株,C型4株,D型5株,E型3株,F型2株、G型1株。2、临床分离27株鲍曼不动杆菌除对替加环素、粘菌素全部敏感,对其他14种抗菌药物普遍耐药,米诺环素耐药率稍低,为25.9%。其次为头孢哌酮/舒巴坦与亚胺培南,耐药率分别为51.9%、59.3%,对其他抗菌药物的耐药率均超过70%,对复方新诺明全部耐药。环境分离28株鲍曼不动杆菌相比临床分离菌株更为敏感,替加环素和粘菌素全部敏感,对美罗培南和头孢哌酮/舒巴坦的耐药率均为3.6%,对与亚胺培南耐药率为7.1%,对米诺环素、哌拉西林/他唑巴坦耐药率均为10.7%,对头孢西丁耐药率最高,为92.9%。27株临床分离鲍曼不动杆菌ERIC-PCR基因分型主要为A、B、C三型,其中A型20株,B型6株,C型1株。28株环境分离鲍曼不动杆菌主要可分为6型,A型13株,B型4株,C型2株,D型4株,E型2株,F型3株。27株临床分离鲍曼不动杆菌MLST分为5个ST型(ST 208,1-3-3-2-2-97-3;ST 368,1-3-3-2-2-140-3;ST 191,1-3-3-2-2-94-3;ST 195,1-3-3-2-2-96-3;ST 540,1-3-3-2-2-160-3)与4个新ST型。其中ST208是在神经外科ICU中分布最为广泛的基因型,占到了菌株数的66.7%,环境菌株中MLST主要有2个ST型(ST 208和ST229)。27株临床分离菌株PFGE可分为5型,其中A型23株(包括A1型15株,A2型7株,A3型1株),B型1株,D型2株,E型1株,28株ICU环境分离株主要分为9型,其中A型15株(包括A1型7株,A2型2株,A3型6株),B型2株,C型1株,D型2株,E型3株,F型2株,G、H、I型各1株。3、在78株鲍曼不动杆菌中,均检测到OXA-51,未检测到OXA-24、OXA-58、VIM-1、VIM-2基因。Amp C、OXA-23、IMP-1基因的检测率分别为84.6%、74.3%、56.4%。66株检测到Amp-C,其中42株为IRAB,22株ISAB。58株OXA-23阳性包括42株IRAB,12株ISAB。44株检测到IMP-1,其中23株为IRAB,19株为ISAB。加入外排泵抑制剂PAβN,78株鲍曼不动杆菌分离株中,41株(52.5%)对亚胺培南的MICs有了4~32倍的降低,可认为外排泵表型阳性。在Ade ABC外排系统中,ade B、ade S、ade R基因的检出率为77%、77%、73%。60株ade B阳性菌株中,39株为IRAB,19株为ISAB。在78株临床菌株中,检测到ade J基因72株(92%),其中42株为IRAB,28株为ISAB。ade L、ade F、ade G和ade H的检出率分别为94%、97%、90%和92%。70株ade G阳性菌株包括42株IRAB和26株ISAB。对8株IRAB和8株ISAB进一步使用实时荧光定量PCR对主要外排基因ade B、ade J和ade G进行表达量分析,经统计分析ade B和ade G在IRAB和ISAB中表达量差异具有统计学意义。4、通过PCR试验成功构建ATCC19606-△Omp34基因敲除株,并通过基因测序验证。SDS-PAGE蛋白电泳显示,敲除株较野生株在34k Da处有一个蛋白缺失,说明Omp34基因敲除成功。药物敏感性试验结果显示野生株和敲除株对碳青霉烯类抗生素的耐药性没有显著改变。结论我院神经外科ICU鲍曼不动杆菌对替加环素和粘菌素全部敏感,对米诺环素耐药率稍低,其次为头孢哌酮/舒巴坦和亚胺培南。临床可根据药敏结果选择合适抗菌药物。A型克隆株为我院鲍曼不动杆菌主要流行株,临床菌株与环境菌株具有较高同源性,且同时分布于其他多个科室,科室间存在交叉感染的可能性,需采取切实有效的措施及时进行干预。产生β-内酰胺酶Amp-C、OXA-23与主动外排系统Ade ABC、Ade FGH过表达在鲍曼不动杆菌碳青霉烯耐药中发挥重要作用。药物敏感性实验结果显示野生株和敲除株对碳青霉烯类抗生素的耐药性没有显著改变,提示Omp34外膜蛋白低表达仅仅是引起碳青霉烯类耐药的辅助因素。
[Abstract]:Objective to study the drug resistance characteristics and epidemic trend of Acinetobacter in our hospital, to study the resistance characteristics of clinical strains and environmental strains in ICU, to detect the gene of Acinetobacter spp. of Acinetobacter Bauman and the efflux pump gene, and to detect the resistance of active outer row system structure gene and regulatory gene in imipenem (imipenem) in Bauman. The distribution difference between the imipenem-resistant Acinetobacter baumannii IRAB and the sensitive group (imipenem-sensitive Acinetobacter baumannii ISAB), the expression of the main outer row genes, and the study of the outer membrane protein Omp34 mediating the resistance of Acinetobacter Bauman to carbapenems, and the study of Acinetobacter Bauman to carbapenems Method 1. 1. We collected 78 strains of Acinetobacter Bauman in our hospital from July 2012 to October, and analyzed the homology of the 78 strains of clinical isolates by ERIC-PCR and PFGE techniques. To find out whether the Acinetobacter of Acinetobacter in our hospital had the spread of.2 in the clone, and the agar dilution method was used to detect the Department of Neurosurgery at the same time. The minimum inhibitory concentration of 27 strains of Intensive Care Unit and 28 strains of environmental strains to common antimicrobial agents; PCR (Enterbacteriat Repetitive Intergenic Consensus, ERIC-PCR) by Enterobacteriaceae (Enterbacteriat Repetitive Intergenic Consensus, ERIC-PCR), multipoint sequence typing (Multi Locus) and pulsed field gel electrophoresis Pulsed Field Gelelectrophoresis (PFGE) technique was used to genotyping 55 strains of Acinetobacter Bauman. The possible transmission route.3 was analyzed. Polymerase chain reaction (Polymerase Chain Reaction, PCR) was used to detect the beta lactamase gene of 78 strains of clinical strains (OXA-23, OXA-24, OXA-51, absent, excluded) and family 3 Ade ABC, Ade IJK and Ade FGH structure genes (ADE B, ADE J, ADE F) and the distribution differences between them, and the analysis of the expression of the main outer row genes, and the preliminary study of Acinetobacter Bauman on carbapenems Drug resistance mechanism.4, the deletion mutant of Omp34 gene (ATCC 19606- Delta Omp34) was constructed by double homologous recombination technology in Acinetobacter Bauman ATCC 19606 (ATCC 19606- Delta Omp34), and the expression of outer membrane protein was analyzed by electrophoresis of sodium alkyl sulfate polyacrylamide gel (SDS-Polyacrylamide Gel Electrophoresis, SDS-PAGE), PCR Omp34 gene knockout was further verified by method and gene sequencing. The antimicrobial activity of wild plants and knockout strains to the commonly used antibiotics, especially carbapenems, was compared to explore the role of outer membrane protein Omp34 in the resistance of Acinetobacter Bauman to carbapenems. Results 1, 78 strains of Acinetobacter in clinical isolates were only resistant to minocycline. The rate was slightly lower, 30.9%, followed by imipenem, the drug resistance rate was 53.1%, the drug resistance rate of other antibiotics exceeded 60%, and the drug resistance rate of cefoxitin was the highest, which was 97.5%. The 93.8%.78 strain of Acinetobacter Bauman showed that it could be divided into A, B, C, D, E, F 6, A type 62, and A type was also divided into A1 46 strains, A 16 strains of type 2, 7 strains of B type, 6 strains of type C, 1 strains of D, E and F, 56 strains of A type (including A1, A2, A3 three), B type 7, 5, 4, 5, 3, polymyxin are all sensitive to tegacycline and polymyxin, and the resistance rate of minocycline is a little. Low, 25.9%. was followed by Cefoperazone / sulbactam and imipenem, the drug resistance rate was 51.9%, 59.3% respectively, and the resistance rates of other antibiotics were all over 70%, and all were resistant to compound sulfamethoxazole. 28 strains of Acinetobacter Bauman were more sensitive than the clinical isolates, and all were sensitive to tigocycline and mycoplasma, and to meropenem and head. The resistance rate of piperazone / sulbactam was 3.6%, the resistance rate to imipenem was 7.1%, the resistance rate to minocycline, piperacillin / tazobactam was 10.7%, and the drug resistance rate to cefoxitin was the highest. The ERIC-PCR genotyping of Acinetobacter Bauman was mainly A, B, C three, of which 20 A, 6 B and 1.28 strains of C Acinetobacter Bauman can be divided into 6 types, 13 A, 4 B, 2 C, 4 D, 2 E, 3.27 strains of F, and 5 ST type (ST 208,1-3-3-2-2-97-3, ST) and 4 new types. The most widely distributed genotype in the Department of Neurosurgery, ICU, accounted for 66.7% of the number of bacteria plants. In environmental strains, MLST mainly has 2 ST types (ST 208 and ST229).27 strain, PFGE can be divided into 5 types, including 23 A type (including 15 strains of A1, 7 A2, 1 A3), 1, 2, 1, 28, and 28 isolates are mainly divided into 9 type. 15 middle A strains (including 7 A1, 2 A2, 6 A3), 2 B, 1 C, 2 D, 3 E, F 2, G, H, 1, respectively, were detected in 78 strains of Acinetobacter 22 strains of ISAB.58 strain OXA-23 positive included 42 strains of IRAB, 12 strains of ISAB.44 strains detected IMP-1, of which 23 were IRAB, 19 strains were added to the outer row pump inhibitor PA beta N, 78 strains of Acinetobacter Bauman isolates, 41 (52.5%) decreased the MICs 4~32 times of imipenem. The detection rates of B, ADE S and ade R were 77%, 77%, 39 of ADE B positive strains in 73%.60 strain, and 19 for ISAB. in 78 clinical strains, and 72 of ADE J genes (92%) were detected, 42 of which were 94%, 28 and 94% and 94%, respectively, including 42 strain and 26 plants. The expression of ADE B, ADE J and ade G in 8 IRAB and 8 strains of ISAB were analyzed by real time fluorescence quantitative PCR. AGE protein electrophoresis showed that the knockout plant had a protein deletion at 34k Da than the wild plant, indicating that the Omp34 gene knockout was successful. The drug sensitivity test showed that the drug resistance of the wild plants and the knockout strains to carbapenems did not change significantly. Conclusion the whole ICU Bauman Acinetobacter in Department of neurosurgery in our hospital has a total of tigocycline and polymyxin Sensitivity, the resistance rate to minocycline was slightly lower, followed by Cefoperazone / Shubatan and imipenem. The main epidemic strain of Acinetobacter Bauman in our hospital could be selected according to the drug sensitivity results. The clinical strain and the environmental strain had high homology, and they were distributed in many other departments. The possibility of cross infection requires effective and timely intervention. Beta lactamase Amp-C, OXA-23 and active efflux system Ade ABC, Ade FGH are overexpressed in the resistance to carbapenems of Acinetobacter Bauman. Drug sensitivity test results show that wild plants and knockout strains are resistant to carbapenems. There was no significant change in drug properties, suggesting that the low expression of Omp34 outer membrane proteins is only a cofactor for carbapenem resistance.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R446.5

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