【作者】 齐艳；

【导师】 俞云松；

【作者基本信息】 浙江大学 ， 临床检验诊断学， 2012， 博士

【摘要】 肺炎克雷伯杆菌属革兰阴性杆菌,常存在于人体上呼吸道和肠道,当机体抵抗力降低时可引起肺部感染、泌尿道感染、甚至血液感染等。肺炎克雷伯菌引起的医院感染率近期逐年增高,已成为临床分离及医院感染的重要致病菌之一。碳青霉烯类抗生素是抗菌谱最广,抗菌活性最强的一类β-内酰胺抗生素,因其具有对β-内酰胺酶稳定以及毒性低等特点,已经成为治疗严重细菌感染最主要的抗菌药物之一。但是,随着碳青霉烯类抗生素在临床上使用的增加,碳青霉烯类抗生素的耐药性也不断增加。近年来,碳青霉烯类耐药肠杆菌科细菌(carbapenem-resistant Enterobacteriaceae,CRE)呈世界性流行,在我国流行情况也日益严重。其耐药的重要机制就是细菌产生碳青霉烯酶(carbapenem hydrolyzing (3-lactamases, carbapenemases)。碳青霉烯酶是指能分解包括碳青霉烯类抗生素在内的几乎所有p内酰胺类抗生素的一大类p内酰胺酶,包括Ambler分子分类中的A类、B类、D类3类酶,其中A类酶大多数能被克拉维酸抑制,属于Bush分类中的2f亚群,主要见于肠杆菌科细菌,近来也在非发酵菌中有报道。KPC(Klebsiella pneumoniae carbapenemase, KPC)型碳青霉烯酶属于功能分类法的2f组,分子分类法的A类,是一种由质粒介导的丝氨酸p-内酰胺酶,以丝氨酸作为活性位点,是目前引起肠杆菌科细菌对碳青霉烯类耐药的重要原因。自2001年美国首次报道第一株产KPC酶肺炎克雷伯菌后,产KPC型碳青霉烯酶肺炎克雷伯菌在全球范围快速播散,且在世界各地多个医院报道出现院内爆发流行。现已报道过产KPC酶肺炎克雷伯菌的国家横跨美洲、欧洲和亚洲等十几个国家和地区。此外,菌种也不再局限于肺炎克雷伯菌中,产KPC酶的其他肠杆菌科细菌日益增多,包括产酸克雷伯菌、大肠埃希菌、阴沟肠杆菌、肠炎沙门菌古巴血清型、弗劳地枸橼酸杆菌、粘质沙雷菌和奇异变形杆菌等,最近甚至还在铜绿假单胞菌、荧光假单胞菌和鲍曼不动杆菌等非发酵细菌中也发现了该酶。由于产KPC酶细菌对临床应用的青霉素类、头孢菌素类、碳青霉烯类以及氨曲南等多种抗生素耐药,以及常常同时携带有其他耐药基因,因此多重耐药菌株的不断增加常导致临床抗菌药物治疗的失败和病程迁延,给临床治疗带来严峻挑战。国内外研究发现,KPC酶的快速传播与其编码基因定位在转座子移动元件上密切相关,推测产KPC酶菌株在医院范围内广泛播散的机制主要通过水平传播和克隆传播两种形式导致医院感染暴发流行的发生。因此,急需成功有效的院内感染控制措施,以阻断KPC酶的流行、减少患者的感染率、缩短住院时间和节约宝贵的医疗资源。2007年,浙江省报道了全国第一例产KPC酶肺炎克雷伯菌,随后产KPC酶肺炎克雷伯菌在浙江省及华东地区等省市快速蔓延,可能与东部地区经济发达、医疗水平较高及抗生素的使用有关,给碳青霉烯类抗生素的使用及感染治疗造成严重威胁。近来,产KPC酶菌株在全国也有逐渐增多的趋势并在多个地方造成流行。本研究收集了来自全国5个省份9个城市13家医院95株碳青霉烯类抗生素耐药并且产KPC-2型碳青霉烯酶肺炎克雷伯菌,对这些菌株的耐药性、超广谱β-内酰胺酶及质粒介导的AmpC酶基因型、同源性及分子分型和碳青霉烯酶编码基因周围序列进行系统深入的研究,旨在阐明产KPC酶肺炎克雷伯菌的分子流行、耐药特性和传播机制,明确产KPC酶肺炎克雷伯菌株在我国主要流行的克隆分型和携带KPC酶编码基因的转座元件。研究内容包括以下四部分。1.碳青霉烯类抗生素耐药肺炎克雷伯菌的耐药性研究利用Etest法测定12种抗生素对95株肺炎克雷伯菌的最低抑菌浓度(MIC)。发现95株肺炎克雷伯菌对亚胺培南、美罗培南和厄他培南均显示出从低到高度的耐药性(MIC值为3->32mg/L)；对头孢他啶、头孢吡肟、头孢西丁、哌拉西林/三唑巴坦和头孢哌酮/舒巴坦均产生很高的耐药性；对环丙沙星和阿米卡星两种抗生素却显示了不同程度的敏感和耐药,MIC值分别在0.38->32和1->256mg/L范围内变化。但是,这95株碳青霉烯类耐药肺炎克雷伯菌对多粘菌素和替加环素则均显示了较好的敏感性,MIC值范围52mg/L。2.碳青霉烯类耐药肺炎克雷伯菌同源性研究利用脉冲场凝胶电泳(PFGE)技术和多位点序列分型(MLST)方法对95株碳青霉烯类耐药肺炎克雷伯菌进行同源性和分子分型研究。PFGE分析结果发现95株肺炎克雷伯菌属于9个流行克隆株。其中,B、c和E克隆是我国产KPC-2肺炎克雷伯菌主要流行的克隆,分别在杭州、宁波、南京、合肥等多家医院流行。；MLST分型发现我国产KPC酶肺炎克雷伯菌的序列型共7个,分别是ST11、ST15、ST23、ST349、ST351、ST438和ST439,分布在浙江、上海、江苏、安徽、河南等省市地区。在我国主要流行的序列型为ST11(61/95),和欧美国家主要流行的ST258仅有一个管家基因(tonB)的差别,属于同一个克隆复合体CC258,具有较近的亲缘关系。同时发现有其他的序列型包括ST15和ST23等出现在同一家医院或地区,呈现局部流行的态势,需引起我国医院感染控制相关部门的高度重视。本研究从95株肺炎克雷伯菌中挑选出14株分别代表不同ST型和不同地区的菌株,应用质粒抽提实验、接合实验、转化实验、Southern杂交等进行质粒同源性分析及碳青霉烯酶基因定位研究。这14株菌株中,编码blaKPC基因的质粒均可成功转至受体菌,大小介于40-180kb之间,经HindⅢ内切酶酶切质粒表现出多样性。MLST和PFGE分型结果一致,ST11的8株细菌的PFGE分型结果的相似性>80%。3.超广谱p-内酰胺酶及质粒介导的AmpC酶基因型检测利用特异性引物,运用PCR方法扩增超广谱β-内酰胺酶及质粒介导的AmpC酶编码基因,包括blaCTX-M, blaTEM, blaSHV,blaVEB和6组质粒介导的AmpC酶编码基因(MOX, CIT, DHA, ACC, EBC, FOX),并对PCR阳性产物进行克隆测序。研究结果发现广谱p-内酰胺酶blaTEM-1出现在43株肺炎克雷伯菌中,而blaVEB未在任何一株细菌中检出。在61株序列型为ST11的菌株中,有38株细菌检测到超广谱p-内酰胺酶编码基因,包括blaCTX-M-14或blasHV-12;5株细菌检测到质粒介导的AmpC酶编码基因blaDHA-1；其中有3株细菌同时检测到超广谱β-内酰胺酶及质粒介导的AmpC酶编码基因。此外,在32株序列型为ST15,ST23,ST349,ST351和ST439的菌株中,有9株细菌检测到超广谱β-内酰胺酶编码基因(blaCTX-M-3,blaCTX-M-14或blaSHV-12);17株细菌检测到质粒介导的AmpC酶编码基因blaDHA-1;其中有4株细菌同时检测到超广谱β-内酰胺酶及质粒介导的AmpC酶编码基因。但是,在2株序列型为ST438的菌株中,未检测到任何超广谱β-内酰胺酶及质粒介导的AmpC酶编码基因。4.KPC酶编码基因周围序列分析利用一系列特异性引物,运用PCR方法扩增KPC酶编码基因周围序列,对PCR产物进行克隆测序。我国曾报道KPC基因位于一个由Tn3转座子和Tn4401转座子共同整合而成的复杂结构上,这一结构包含Tn3转座酶基因、Tn3熔解酶基因、插入序列ISKpn8,blaKPC-2编码基因和部分ISKpn6序列。此外,我国产KPC酶细菌中KPC基因周围结构也存在两种变异型,变异1型和变异2型。变异1型结构发现在插入序列ISKpn8和blaKPC-2编码基因之间出现一段不完整的blaTEM基因编码序列；变异2型结构则没有发现Tn3转座酶基因和Tn3熔解酶基因编码序列。本研究中95株肺炎克雷伯菌中KPC酶编码基因序列与我国报道的该基因周围结构一致,仅有2070bp大小的结构和国外报道相似,包括KPC基因和部分ISKpn6序列。有33株细菌与原始结构一致,12株与变异1型结构一致,50株与变异2型结构一致。结论：1.碳青霉烯类抗生素耐药肺炎克雷伯菌在体外药敏实验中,对多种抗生素耐药率较高,对多粘菌素和替加环素显示了较好的药物敏感性,临床可以综合考虑病人临床表现及感染部位药物富集浓度来进行联合治疗。2.我国产KPC酶肺炎克雷伯菌的主要流行的序列型为ST11,与世界流行的ST258同属克隆复合体CC258,呈现世界范围的大流行趋势,需引起各国医院感染控制相关部门的高度重视。3.碳青霉烯酶KPC-2的产生是肺炎克雷伯菌对碳青霉烯耐药的重要原因,同时携带多种耐药基因,导致临床对多种抗生素耐药。4.我国产KPC酶菌株中KPC编码基因周围这种由2个转座子整合而成的复杂转移单元很好地揭示了国内KPC-2基因的传播途径以及遗传背景。更多还原

【Abstract】 Clinically-significant Gram-negative species remain mostly Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Carbapenem molecules are often the last resort for treating infections due to multidrug resistant isolates. In Enterobacteriaceae, resistance to carbapenems may result from combined mechanisms of resistance associating β-lactamases with weak (if any) intrinsic carbapenemase activity and decreased outer membrane permeability, or from true carbapenemases. Reports of carbapenemases have been increasing over the last few years. This phenotypic grouping of enzymes is a heterogeneous mixture of β-lactamases belonging to molecular Ambler class A (penicillinases), class B (metalloenzymes) and class D (oxacillinases).Carbapenem-hydrolyzing Klebsiella pneumoniae carbapenemase (KPC)-type enzymes (partially inhibited by clavulanic acid) have been identified mostly in Klebsiella pneumoniae. KPC are a group of carbapenemases which belong to Bush group2f, molecular class A. They are capable of hydrolyzing carbapenems, penicillin, cephalosporin, and aztreonam. Threrfore, treatment of infection caused by these pathogens is thus a considerable challenge for clinicians. KPC enzymes differ from the other2f enzymes by two specific characteristics:they are found on transferable plasmids and they are able to hydrolyse the aminothiazoleoxime cephalosporins such as cefotaxime. Plasmid-encoded class A enzyme KPC-1from a K. pneumoniae isolate that confers high-level resistance to carbapenems by itself.KPC was first identified in a K. pneumoniae isolate from North Carolina, and since then the enzyme has been found most frequently in K. pneumoniae. Recently however, it has been detected in multiple genera and species of the Enterobacteriaceae, including Salmonella enterica, K. oxytoca, Enterobacter spp., Citrobacter freundii, E. coli, Serratia marcescens, Proteus mirabilis and Morganella morganii, even in the non-lactosefermenting bacteria isolates of Pseudomonas aeruginosa, Pseudomonas putida and Acinetobacter baumannii. To date, eleven different variants (KPC-2-KPC-12) of the KPC enzyme have been reported, with KPC-2and KPC-3reported the most frequently. Of note, re-sequencing of the blaKPc1gene revealed it to be identical to blaKPc-2-Reports are now appearing that KPC-producing isolates have disseminated worldwide including France, Israel, Greece, Colombia, Argentina, China, Brazil, the U.K., Canada and Poland. Clonal dissemination of KPC-producing K. pneumoniae isolates is often observed, indicating that there are major international clones circulating. Transposons and IS elements are likely associated with the dissemination and spread of bla^pc among different bacterial species. Meanwhile, the horizontal transmission of KPC-harboring plasmid was also observed in a Michigan hospital in the United States and2nd Affiliated Hospital of Zhejiang University in China, respectively. The rapidly spread of this enzyme in the world partial because of clinical detection of carbapenemase producers remains difficult based on a simple phenotypic analysis of antibiotic susceptibility testing. A PCR-based technique may be used for detection of known carbapenemases.KPC has been emerging increasingly, and it makes the resistance mechanism a significant public health concern for their broad-spectrum activity and mobility. Reports appear that KPC-producing isolates have disseminated worldwide including more than ten countries across Asia, America and Europe. Recently, it has been proved ST258is the most frequent clone contributing the worldwide spread of KPC-producing K. pneumoniae, which has been identified in Poland, Norway, Sweden, Israel, Finland, Italy, Germany, Denmark, Hungary, Korea and especially the United States. In2007, KPC-producing K. pneumoniae was firstly reported in Zhejiang province, China. Recently, KPC-producing isolates are on the emerging in many places in China. However, little is known about the sequence type of KPC-producing K. pneumoniae spread in China. The aim of this study was to analyze the molecular epidemiology of KPC-producing K. pneumoniae circulating in China on the basis of sequence typing. In the study, ninety-five carbapenem-resistant KPC-2-producing K. pneumoniae isolates from13hospitals of nine cities covering five provinces in China were analyzed.1. Antimicrobial susceptibility testing and Mininal Inhibitory Concentrations determinationThe MICs of antimicrobial agents were determined by Etest (bioMerieux, France) according to the manufacturer’s instructions and were interpreted as recommended by the Clinical and Laboratory Standards Institute (CLSI) update of June2010guidelines. All isolates exhibited resistance to imipenem, meropenem and/or ertapenem, with MICs ranging from3to>32mg/L. In addition, no susceptible isolates was detected in antimicrobial susceptibility testing to ceftazidime, cefepime, cefoxitin, piperacillin-tazobactam and cefoperazone-sulbactam. These isolates were demonstrated variable susceptibilities to ciprofloxacin and amikacin. Moreover, all isolates were susceptible to colistin (MIC≤2mg/L) and tigecycline (MIC≤2mg/L).2. The relatedness of carbapenem-resistant K. pneumoniae PFGE and MLST analysisPFGE analysis of the95clinical isolates of KPC-producing K. pneumoniae showed nine clones including A、B、C、D、E、F、G、H and I. Among these clones, clone B、 C and E were the dominant clones and spread widely among hospitals in Hangzhou, Ningbo, Nanjing and Hefei cities. MLST showed seven STs based on the analysis of the seven housekeeping genes among the95clinical isolates of KPC-producing K. pneumoniae according to the MLST database of the K. pneumoniae. The prevalent clone was ST11(61/95), which was detected in isolates from Hangzhou, Ningbo, Nanjing and Hefei. Furthermore, six types were identified:ST15, ST23, ST349, ST351, ST438, and ST439. To understand the significance of PFGE pattern similarities and differences,14KPC-2-producing K. pneumoniae isolates represented different STs and regions were selected for PFGE and plasmid analysis. The MLST data were in concordance with the results generated by PFGE and the isolates with ST11shared the similarity of>80%in PFGE patterns. Plasmids from14selected transformants had a diversity of HindⅢ restriction maps.3. PCR amplification and DNA sequencing of extended-spectrum P-lactamases (ESBL) and plasmid-mediated AmpC β-lactamase genesAll original isolates were screened by PCR with specific primers for blaCTX-M, blaTEM, blasHV, blaVEB and six families of plasmid-mediated AmpC β-lactamase genes (MOX, CIT, DHA, ACC, EBC, FOX). Sequencing confirmed the blaTEM gene as blaTEM-1in43isolates and the blaVEB gene was not detected in any of the K. pneumoniae isolates. Among the61ST11isolates,38isolates contained ESBL genes(blaCTX-M-3, blaCTX-M-14, and blaSHV-12), five isolates contained AmpC gene(blaDHA-1), and three of them contained both ESBL and AmpC genes. Simultaneously, of the32isolates with other STs (ST15, ST23, ST349, ST351and ST439),9contained ESBL genes (blaCTX-M-3, blaCTX-M-14, blaSHV-12),17contained an AmpC gene (blaDHA-1), and4contained both ESBL and AmpC genes. In the other two isolates with ST438, no ESBL and AmpC genes were detected.4. Genetic environment of blaKPC geneA series of primers were designed at the base of blaKPC-surrounding sequences and PCR experiments were performed according to previously reported conditions. The obtained amplification products were sequenced. As reported previously, the environment surrounding blaKPC-2in plasmid from China is composed of a partial Tn4401structure and a Tn3-based element with the gene order of Tn3-transposase, Tn3-resolvase,ISKpn8, the blaKPC-1gene, and the ISKpn6-like element. In addition, there are another two variants. In our study, it has been observed that all the three structures as reported appeared in the95isolates, including33isolates with the original structure,12isolates with the structure of variant1and50isolates with the structure of variant2.The above results showed:1. Carbapenem-resistant K. pneumoniae are resistant to most antimicrobial agents. However, isolates were susceptible to colistin and tigecycline. Clinicians could select antimicrobial agents in combination according to drug concentrations.2. The dominant clone of KPC-producing K. pneumoniae in China is ST11, which is closely related to ST258that has been reported worldwide.3. The ESBL and/or the plasmid-mediated AmpC genes were detected in six STs besides of ST438. However in KPC-2-producing K. pneumoniae, it is hard to distinguish the difference of the presence of resistant genes between the STs.4. The special environment surrounding blaKPC-2in plasmid from China is composed of a partial Tn4401structure and a Tn3-based element, which may account for the high mobility of this resistance mechanism.更多还原