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  本文選題：肺炎克雷伯菌　切入點：質(zhì)粒　出處：《浙江大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：最近十年,產(chǎn)碳青霉烯酶肺炎克雷伯菌成為”超級耐藥”細(xì)菌并迅速傳遍全球,史無前例。2009年報道的來自印度NDA-1金屬酶、2004報道的土耳其OXA-48D類酶以及2001年報道美國產(chǎn)生的A類KPC酶均分離自肺炎克雷伯菌,在短期內(nèi)均造成多地區(qū)的廣泛播散。同時,肺炎克雷伯菌是耐藥質(zhì)粒的“收藏家”,不僅增加自身的生存能力,而且還可以將耐藥質(zhì)粒傳播給其他菌種,造成耐藥基因的播散。自2007中國首次報道產(chǎn)KPC酶肺炎克雷伯菌后,已在局部地區(qū)造成流行。本論文主要目的是研究產(chǎn)KPC酶肺炎克雷伯菌在中國的流行狀況及質(zhì)粒介導(dǎo)的KPC酶基因傳播。 本研究于2010年在全國20個省市的61家醫(yī)院收集2971株非重復(fù)的肺炎克雷伯菌,并選擇20種常見抗菌藥物開展體外藥物敏感性試驗。對碳青霉烯類抗生素耐藥或中介的肺炎克雷伯菌進(jìn)行KPC型碳青霉烯酶檢測。改良Hodge確認(rèn)試驗和KPC基因特異引物PCR擴(kuò)增及測序用于細(xì)菌產(chǎn)KPC酶的確定。采用多位點序列分型(multilocus sequence typing, MLST)分型技術(shù)對產(chǎn)KPC酶肺炎克雷伯菌進(jìn)行同源性分析。 體外藥物敏感性試驗結(jié)果顯示,2971株肺炎克雷伯菌對亞胺培南、美洛培南和厄他培南的耐藥率分別為1.3%、1.9%和2.7%。肺炎克雷伯菌產(chǎn)ESBLs的檢出率為41.9%。75株碳青霉烯類抗生素耐藥或中介的肺炎克雷伯菌中有28株改良Hodge試驗陽性,KPC擴(kuò)增和測序發(fā)現(xiàn)有25株KPC-2基因陽性。產(chǎn)KPC-2酶肺炎克雷伯菌主要來自浙江省(20株),并在北京、遼寧、湖北、上海和江蘇各檢測到1株。2010年中國多地區(qū)產(chǎn)KPC-2酶肺炎克雷伯菌的檢出率0.84%。 25株產(chǎn)KPC-2酶肺炎克雷伯菌MLST分型檢測發(fā)現(xiàn),有17株為ST11,占68%；4株為ST494,4株為未知型,屬于新的ST型。北京、遼寧、湖北、上海和江蘇菌株均為ST11。4株肺炎克雷伯菌ST494來自浙江的同一家醫(yī)院。 本課題同時對來源于湖北一家省級兒童醫(yī)院分離的4株產(chǎn)碳青霉烯酶肺炎克雷伯菌進(jìn)行研究。采用PCR擴(kuò)增及測序檢測β內(nèi)酰胺酶基因、MLST和脈沖場凝脈電泳(PFGE)技術(shù)進(jìn)行細(xì)菌同源性分析,應(yīng)用質(zhì)粒PFGE、接合及轉(zhuǎn)化試驗、質(zhì)粒抽提、Southern雜交等技術(shù)進(jìn)行質(zhì)粒分析及β-內(nèi)酰胺酶基因在質(zhì)粒中的定位。 同源性分析顯示4株肺炎克雷伯菌PFGE圖譜條帶及數(shù)量相同,MLST分型均為ST439,提示4株細(xì)菌為同一克隆。質(zhì)粒圖譜結(jié)果表明4株細(xì)菌攜帶的質(zhì)粒大小在30到300kb這間,KPC-2基因位于30kb大小的質(zhì)粒上,IMP-4基因定位于300kb質(zhì)粒,CTX-M-15基因位于80kb大小的質(zhì)粒上,DHA-1基因分別位于80kb和300kb的2個質(zhì)粒上。本研究證實在肺炎克雷伯菌中同時產(chǎn)生A類碳青霉烯酶(KPC-2)、金屬碳青霉烯酶(IMP-4)、超廣譜p-內(nèi)酰胺酶(CTX-M-15)和質(zhì)粒介導(dǎo)的AmpC酶(DHA-1). 本課題還對分離自同一病人的1株嗜水氣單胞菌、2株大腸埃希菌和2株產(chǎn)氣腸桿菌進(jìn)行藥敏試驗、質(zhì)粒消除、PCR步移、質(zhì)粒分析等分子生物學(xué)技術(shù)研究,明確編碼KPC基因在不同質(zhì)粒中的定位及攜帶KPC基因的轉(zhuǎn)座子結(jié)構(gòu)。 結(jié)果發(fā)現(xiàn)5株臨床分離細(xì)菌均顯示對亞胺培南、美洛培南和厄他培南高水平耐藥,MIC值均32mg/L。通過質(zhì)粒消除試驗,嗜水氣單胞菌含KPC基因的質(zhì)粒被去除后,細(xì)菌對亞胺培南、美洛培南和厄他培南MIC值明顯降低。 PCR擴(kuò)增及測序證實在2株產(chǎn)氣腸桿菌均含有blaKPC-blaTEM-1,blaSHV-12和blaDHA-1,而它們的接合子中只含blaKPC-2和blaTEM-1。2株大腸埃希菌中檢測到blaKPC-2和blasHV-12,而它們的轉(zhuǎn)化子或接合子中只檢測到blaKPC-2。臨床分離的嗜水氣單胞菌和它的接合子只含blaKPC-2,而質(zhì)粒消除的嗜水氣單胞菌blaKPC-2檢測陰性。 質(zhì)粒電泳圖譜分析發(fā)現(xiàn)所有5株臨床細(xì)菌都含有3-5個大小不等的質(zhì)粒。分子雜交表明KPC基因位于嗜水氣單胞菌和大腸埃希菌的30kb可接合或轉(zhuǎn)化的質(zhì)粒上,而2株產(chǎn)氣腸桿菌攜帶KPC基因的質(zhì)粒大小不同,分別為200kb和170kb左右。對嗜水氣單胞菌轉(zhuǎn)化子進(jìn)行PCR步移法及測序獲得一個11467bp核苷酸片段,結(jié)構(gòu)依次為Tn3的轉(zhuǎn)座酶和解旋酶、部分缺失的TEM、ISKpn8、KPC-2、 ISKpn6-like、KorC、KlcA和有缺失的起始復(fù)制蛋白基因。該結(jié)構(gòu)同時還在其他4株細(xì)菌中檢測到。 本研究首次在氣單胞菌屬細(xì)菌中檢測到KPC-2型碳青霉烯酶,并發(fā)現(xiàn)不同菌種細(xì)菌攜帶相同的質(zhì)粒,包含KPC-2基因的相同轉(zhuǎn)座子結(jié)構(gòu)出現(xiàn)在不同菌種的不同質(zhì)粒上。
[Abstract]:The last ten years, carbapenemase producing Klebsiella pneumoniae resistant bacteria become "super" and quickly spread throughout the world, India NDA-1 metal There was no parallel in history. from.2009 was reported in 1993, Turkey OXA-48D enzyme 2004 reports and reports of America 2001 produced a class KPC enzyme were isolated from Klebsiella pneumoniae, in the short term due to widely disseminated multiple regions. At the same time, Klebsiella pneumoniae plasmid "collectors", not only increase the survival ability, but also can be transmitted to other strains of resistant plasmids, caused by the spread of resistant genes. Since 2007 China reported for the first time in KPC producing Klebsiella pneumonia, has caused popular in the local area. The main purpose of this thesis is KPC gene and plasmid mediated spread of epidemic situation of KPC producing Klebsiella pneumoniae in China guide.
This study in 2010 61 hospitals in 20 provinces and cities nationwide collection of 2971 strains of non Klebsiella pneumoniae repeat, and select 20 kinds of antibiotics in vitro drug sensitivity test. The detection of KPC carbapenemases of Klebsiella pneumoniae resistant to carbapenems or modified Hodge intermediary. To confirm amplification and sequencing test and KPC gene specific primer PCR was used to determine the KPC enzyme producing bacteria by multilocus sequence typing (multilocus sequence, typing, MLST) homology analysis on KPC producing Klebsiella pneumoniae typing technique.
In vitro drug sensitivity test showed that 2971 strains of Klebsiella pneumoniae resistant to imipenem, meropenem and ertapenem detection rates were 1.3%, 1.9% and 2.7%. ESBLs producing Klebsiella pneumoniae was 41.9%.75 strains of carbapenem resistant or intermediate Klebsiella pneumoniae in 28 strains the modified Hodge test positive, KPC amplification and sequencing of KPC-2 gene of 25 strains were found. KPC-2 producing Klebsiella pneumoniae (20 strains) from Zhejiang Province, and in Beijing, Liaoning, Hubei, Shanghai and Jiangsu each detected detection rate of 1 strains of.2010 in Chinese KPC-2 producing Klebsiella pneumoniae bacteria 0.84%.
MLST genotyping of 25 strains of KPC-2 producing Klebsiella pneumoniae showed that 17 strains were ST11, accounting for 68%. 4 strains of ST494,4 were unknown type, belonging to the new ST type. The isolates from Beijing, Liaoning, Hubei, Shanghai and Jiangsu were ST11.4 strain Klebsiella pneumoniae ST494 from the same hospital in Zhejiang.
This paper makes a study on the source for a Hubei Provincial Children's Hospital of 4 strains of carbapenemase producing Klebsiella pneumoniae. At the same time using PCR amplification and sequencing of beta lactamase gene, MLST and pulsed field gel electrophoresis (PFGE) technique for analysis of bacterial origin, application of plasmid PFGE junction and the transformation test, plasmid extraction, Southern hybridization of plasmid analysis and beta lactamase genes in the plasmid localization.
Homology of 4 strains of Klebsiella pneumoniae PFGE band and the same number of analysis showed that the MLST type was ST439, suggesting that the 4 strains of bacteria were the same clone plasmid. Results showed that 4 strains of bacteria carrying plasmid size from 30 to 300KB, the plasmid KPC-2 gene is located on the size of 30KB, IMP-4 gene located in the 300KB plasmid, plasmid CTX-M-15 gene is located in 80Kb size, 2 plasmid DHA-1 gene located at 80Kb and 300KB. This study also confirmed to produce class a carbapenemase in Klebsiella pneumoniae (KPC-2), metal carbapenemase (IMP-4), extended spectrum lactamases (p- CTX-M-15) and plasmid mediated AmpC enzyme (DHA-1).
The subject is also isolated from the same patient of 1 strains of Aeromonas hydrophila, drug sensitivity test, 2 strains of Escherichia coli and 2 strains of Enterobacter aerogenes plasmid elimination, PCR step, study on molecular biology technology analysis of transposon plasmid, clear structure encoding KPC gene localization in different plasmid and carrying the KPC gene.
The results showed that 5 strains of clinically isolated bacteria were shown to imipenem, meropenem and ertapenem high level resistance, MIC values were 32mg/L. by plasmid elimination test, plasmid Aeromonas containing KPC gene has been removed, the bacteria to imipenem, meropenem and ertapenem MIC decreased significantly.
PCR amplification and sequencing in 2 strains of Enterobacter aerogenes contain blaKPC-blaTEM-1, blaSHV-12 and blaDHA-1, and their transconjugants only contained blaKPC-2 and blaTEM-1.2 strains of Escherichia coli were detected in blaKPC-2 and blasHV-12, and transformed them or zygote was only detected in blaKPC-2. clinical isolates of Aeromonas hydrophila and the zygote contains only blaKPC-2, and the plasmid elimination of Aeromonas hydrophila blaKPC-2 was negative.
Plasmid electrophoresis analysis showed that all 5 strains of clinical bacteria contain 3-5 sizes of the plasmid. The molecular hybridization indicated that the plasmid KPC gene in Aeromonas hydrophila and Escherichia coli 30KB can be joined or transformed, and plasmids of 2 strains of Enterobacter aerogenes carrying KPC gene is not the same, and 200KB respectively. About 170kb. On Aeromonas hydrophila transformant PCR walking method and sequenced a 11467bp nucleotide fragment structure, followed by Tn3 transposase and helicase, deletion of TEM, ISKpn8, KPC-2, ISKpn6-like, KorC, KlcA and a lack of initiation of replication protein gene. The structure is also the other 4 bacterial strains were detected.
In this study, KPC-2 carbapenems were detected for the first time in Aeromonas spp.. It was found that the same transposon structure of different bacterial strains contained the same KPC-2 transposon structure on different plasmids.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R563.1

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本文編號：1589665