产KPC酶肺炎克雷伯菌分子流行和质粒介导的KPC酶传播机制研究

发布时间：2018-03-09 18:10

本文选题：肺炎克雷伯菌　切入点：质粒　出处：《浙江大学》2013年博士论文　论文类型：学位论文

【摘要】：最近十年,产碳青霉烯酶肺炎克雷伯菌成为”超级耐药”细菌并迅速传遍全球,史无前例。2009年报道的来自印度NDA-1金属酶、2004报道的土耳其OXA-48D类酶以及2001年报道美国产生的A类KPC酶均分离自肺炎克雷伯菌,在短期内均造成多地区的广泛播散。同时,肺炎克雷伯菌是耐药质粒的“收藏家”,不仅增加自身的生存能力,而且还可以将耐药质粒传播给其他菌种,造成耐药基因的播散。自2007中国首次报道产KPC酶肺炎克雷伯菌后,已在局部地区造成流行。本论文主要目的是研究产KPC酶肺炎克雷伯菌在中国的流行状况及质粒介导的KPC酶基因传播。本研究于2010年在全国20个省市的61家医院收集2971株非重复的肺炎克雷伯菌,并选择20种常见抗菌药物开展体外药物敏感性试验。对碳青霉烯类抗生素耐药或中介的肺炎克雷伯菌进行KPC型碳青霉烯酶检测。改良Hodge确认试验和KPC基因特异引物PCR扩增及测序用于细菌产KPC酶的确定。采用多位点序列分型(multilocus sequence typing, MLST)分型技术对产KPC酶肺炎克雷伯菌进行同源性分析。体外药物敏感性试验结果显示,2971株肺炎克雷伯菌对亚胺培南、美洛培南和厄他培南的耐药率分别为1.3%、1.9%和2.7%。肺炎克雷伯菌产ESBLs的检出率为41.9%。75株碳青霉烯类抗生素耐药或中介的肺炎克雷伯菌中有28株改良Hodge试验阳性,KPC扩增和测序发现有25株KPC-2基因阳性。产KPC-2酶肺炎克雷伯菌主要来自浙江省(20株),并在北京、辽宁、湖北、上海和江苏各检测到1株。2010年中国多地区产KPC-2酶肺炎克雷伯菌的检出率0.84%。 25株产KPC-2酶肺炎克雷伯菌MLST分型检测发现,有17株为ST11,占68%；4株为ST494,4株为未知型,属于新的ST型。北京、辽宁、湖北、上海和江苏菌株均为ST11。4株肺炎克雷伯菌ST494来自浙江的同一家医院。本课题同时对来源于湖北一家省级儿童医院分离的4株产碳青霉烯酶肺炎克雷伯菌进行研究。采用PCR扩增及测序检测β内酰胺酶基因、MLST和脉冲场凝脉电泳(PFGE)技术进行细菌同源性分析,应用质粒PFGE、接合及转化试验、质粒抽提、Southern杂交等技术进行质粒分析及β-内酰胺酶基因在质粒中的定位。同源性分析显示4株肺炎克雷伯菌PFGE图谱条带及数量相同,MLST分型均为ST439,提示4株细菌为同一克隆。质粒图谱结果表明4株细菌携带的质粒大小在30到300kb这间,KPC-2基因位于30kb大小的质粒上,IMP-4基因定位于300kb质粒,CTX-M-15基因位于80kb大小的质粒上,DHA-1基因分别位于80kb和300kb的2个质粒上。本研究证实在肺炎克雷伯菌中同时产生A类碳青霉烯酶(KPC-2)、金属碳青霉烯酶(IMP-4)、超广谱p-内酰胺酶(CTX-M-15)和质粒介导的AmpC酶(DHA-1). 本课题还对分离自同一病人的1株嗜水气单胞菌、2株大肠埃希菌和2株产气肠杆菌进行药敏试验、质粒消除、PCR步移、质粒分析等分子生物学技术研究,明确编码KPC基因在不同质粒中的定位及携带KPC基因的转座子结构。结果发现5株临床分离细菌均显示对亚胺培南、美洛培南和厄他培南高水平耐药,MIC值均32mg/L。通过质粒消除试验,嗜水气单胞菌含KPC基因的质粒被去除后,细菌对亚胺培南、美洛培南和厄他培南MIC值明显降低。 PCR扩增及测序证实在2株产气肠杆菌均含有blaKPC-blaTEM-1,blaSHV-12和blaDHA-1,而它们的接合子中只含blaKPC-2和blaTEM-1。2株大肠埃希菌中检测到blaKPC-2和blasHV-12,而它们的转化子或接合子中只检测到blaKPC-2。临床分离的嗜水气单胞菌和它的接合子只含blaKPC-2,而质粒消除的嗜水气单胞菌blaKPC-2检测阴性。质粒电泳图谱分析发现所有5株临床细菌都含有3-5个大小不等的质粒。分子杂交表明KPC基因位于嗜水气单胞菌和大肠埃希菌的30kb可接合或转化的质粒上,而2株产气肠杆菌携带KPC基因的质粒大小不同,分别为200kb和170kb左右。对嗜水气单胞菌转化子进行PCR步移法及测序获得一个11467bp核苷酸片段,结构依次为Tn3的转座酶和解旋酶、部分缺失的TEM、ISKpn8、KPC-2、 ISKpn6-like、KorC、KlcA和有缺失的起始复制蛋白基因。该结构同时还在其他4株细菌中检测到。本研究首次在气单胞菌属细菌中检测到KPC-2型碳青霉烯酶,并发现不同菌种细菌携带相同的质粒,包含KPC-2基因的相同转座子结构出现在不同菌种的不同质粒上。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563.1

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