本文選題：鮑曼不動桿菌 + 同源性分析��； 參考：《上海交通大學(xué)》2015年博士論文

【摘要】：目的本研究通過了解我院鮑曼不動桿菌感染情況、耐藥特征與流行趨勢,研究重癥監(jiān)護(hù)病房臨床菌株與環(huán)境菌株耐藥特征,檢測耐碳青霉烯類鮑曼不動桿菌水解酶及外排泵基因,檢測主動外排系統(tǒng)結(jié)構(gòu)基因與調(diào)控基因在亞胺培南耐藥組(imipenem-resistant Acinetobacter baumannii IRAB)與敏感組(imipenem-sensitive Acinetobacter baumannii ISAB)中分布差異,對主要外排基因進(jìn)行表達(dá)量分析,并研究外膜蛋白Omp34介導(dǎo)鮑曼不動桿菌對碳青霉烯類耐藥,深入探討鮑曼不動桿菌對碳青霉烯類藥物的耐藥機(jī)制。方法1、本實驗收集2012年7月到10月我院臨床分離鮑曼不動桿菌共78株,并采用ERIC-PCR及PFGE技術(shù)對該78株臨床分離株進(jìn)行同源性分析,了解本院鮑曼不動桿菌是否存在克隆株的播散流行。2、同期采用瓊脂稀釋法檢測神經(jīng)外科重癥監(jiān)護(hù)病房(Intensive Care Unit,ICU)27株臨床菌株和28株環(huán)境菌株對常用抗菌藥物的最低抑菌濃度;用腸桿菌科基因間重復(fù)序列PCR(Enterbacteriat Repetitive Intergenic Consensus,ERIC-PCR)、多位點序列分型(Multi Locus Sequence Typing,MLST)及脈沖場凝膠電泳(Pulsed Field Gelelectrophoresis,PFGE)技術(shù)對共分離的55株鮑曼不動桿菌進(jìn)行基因分型,分析其可能的傳播途徑。3、采用聚合酶鏈反應(yīng)(Polymerase Chain Reaction,PCR)檢測78株臨床菌株β-內(nèi)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個主要外排系統(tǒng)Ade ABC、Ade IJK及Ade FGH結(jié)構(gòu)基因(ade B、ade J、ade F、ade G、ade H)與調(diào)控基因(ade R、ade S、ade L)的攜帶情況,比較其在IRAB及ISAB中分布差異,進(jìn)一步使用Realtime-PCR對其主要外排基因進(jìn)行表達(dá)量分析,初步探討鮑曼不動桿菌對碳青霉烯類藥物的耐藥機(jī)制。4、研究通過采用雙重同源重組技術(shù)在鮑曼不動桿菌ATCC 19606中構(gòu)建Omp34基因的缺失突變株(ATCC 19606-△Omp34),采用十二烷基硫酸鈉-聚丙烯酰胺凝膠(SDS-Polyacrylamide Gel Electrophoresis,SDS-PAGE)蛋白電泳分析外膜蛋白的表達(dá),PCR法和基因測序進(jìn)一步驗證Omp34基因敲除。比較野生株和敲除株對常用抗菌藥物尤其是碳青霉烯類的抑菌圈大小,以探討外膜蛋白Omp34在介導(dǎo)鮑曼不動桿菌對碳青霉烯類耐藥中的作用。結(jié)果1、臨床分離78株鮑曼不動桿菌只對米諾環(huán)素耐藥率稍低,為30.9%;其次為亞胺培南,耐藥率為53.1%;對其它抗菌藥物耐藥率均超過60%;對頭孢西丁、復(fù)方新諾明耐藥率最高,分別為97.5%、93.8%。78株鮑曼不動桿菌ERIC-PCR結(jié)果顯示可分為A、B、C、D、E、F 6型,A型62株,為主要克隆株,其中A型又分為A1型46株,A2型16株,B型7株,C型6株,D、E、F型各1株。PFGE主要分為7型,A型56株(包括A1、A2、A3三個亞型),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、臨床分離27株鮑曼不動桿菌除對替加環(huán)素、粘菌素全部敏感,對其他14種抗菌藥物普遍耐藥,米諾環(huán)素耐藥率稍低,為25.9%。其次為頭孢哌酮/舒巴坦與亞胺培南,耐藥率分別為51.9%、59.3%,對其他抗菌藥物的耐藥率均超過70%,對復(fù)方新諾明全部耐藥。環(huán)境分離28株鮑曼不動桿菌相比臨床分離菌株更為敏感,替加環(huán)素和粘菌素全部敏感,對美羅培南和頭孢哌酮/舒巴坦的耐藥率均為3.6%,對與亞胺培南耐藥率為7.1%,對米諾環(huán)素、哌拉西林/他唑巴坦耐藥率均為10.7%,對頭孢西丁耐藥率最高,為92.9%。27株臨床分離鮑曼不動桿菌ERIC-PCR基因分型主要為A、B、C三型,其中A型20株,B型6株,C型1株。28株環(huán)境分離鮑曼不動桿菌主要可分為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株臨床分離鮑曼不動桿菌MLST分為5個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)與4個新ST型。其中ST208是在神經(jīng)外科ICU中分布最為廣泛的基因型,占到了菌株數(shù)的66.7%,環(huán)境菌株中MLST主要有2個ST型(ST 208和ST229)。27株臨床分離菌株P(guān)FGE可分為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環(huán)境分離株主要分為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株鮑曼不動桿菌中,均檢測到OXA-51,未檢測到OXA-24、OXA-58、VIM-1、VIM-2基因。Amp C、OXA-23、IMP-1基因的檢測率分別為84.6%、74.3%、56.4%。66株檢測到Amp-C,其中42株為IRAB,22株ISAB。58株OXA-23陽性包括42株IRAB,12株ISAB。44株檢測到IMP-1,其中23株為IRAB,19株為ISAB。加入外排泵抑制劑PAβN,78株鮑曼不動桿菌分離株中,41株(52.5%)對亞胺培南的MICs有了4~32倍的降低,可認(rèn)為外排泵表型陽性。在Ade ABC外排系統(tǒng)中,ade B、ade S、ade R基因的檢出率為77%、77%、73%。60株ade B陽性菌株中,39株為IRAB,19株為ISAB。在78株臨床菌株中,檢測到ade J基因72株(92%),其中42株為IRAB,28株為ISAB。ade L、ade F、ade G和ade H的檢出率分別為94%、97%、90%和92%。70株ade G陽性菌株包括42株IRAB和26株ISAB。對8株IRAB和8株ISAB進(jìn)一步使用實時熒光定量PCR對主要外排基因ade B、ade J和ade G進(jìn)行表達(dá)量分析,經(jīng)統(tǒng)計分析ade B和ade G在IRAB和ISAB中表達(dá)量差異具有統(tǒng)計學(xué)意義。4、通過PCR試驗成功構(gòu)建ATCC19606-△Omp34基因敲除株,并通過基因測序驗證。SDS-PAGE蛋白電泳顯示,敲除株較野生株在34k Da處有一個蛋白缺失,說明Omp34基因敲除成功。藥物敏感性試驗結(jié)果顯示野生株和敲除株對碳青霉烯類抗生素的耐藥性沒有顯著改變。結(jié)論我院神經(jīng)外科ICU鮑曼不動桿菌對替加環(huán)素和粘菌素全部敏感,對米諾環(huán)素耐藥率稍低,其次為頭孢哌酮/舒巴坦和亞胺培南。臨床可根據(jù)藥敏結(jié)果選擇合適抗菌藥物。A型克隆株為我院鮑曼不動桿菌主要流行株,臨床菌株與環(huán)境菌株具有較高同源性,且同時分布于其他多個科室,科室間存在交叉感染的可能性,需采取切實有效的措施及時進(jìn)行干預(yù)。產(chǎn)生β-內(nèi)酰胺酶Amp-C、OXA-23與主動外排系統(tǒng)Ade ABC、Ade FGH過表達(dá)在鮑曼不動桿菌碳青霉烯耐藥中發(fā)揮重要作用。藥物敏感性實驗結(jié)果顯示野生株和敲除株對碳青霉烯類抗生素的耐藥性沒有顯著改變,提示Omp34外膜蛋白低表達(dá)僅僅是引起碳青霉烯類耐藥的輔助因素。
[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R446.5

【參考文獻(xiàn)】

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