【作者】 胡付品；

【导师】 朱德妹； 李光辉； 徐晓刚；

【作者基本信息】 复旦大学 ， 内科学， 2010， 博士

【摘要】 肠杆菌科细菌包括大肠埃希菌、肺炎克雷伯菌、弗劳地柠檬酸杆菌和阴沟肠杆菌等,是引起医院感染最常见的病原菌。据历年上海地区和CHINET全国细菌耐药性监测结果显示,肠杆菌科细菌占所有革兰阴性菌的60-70%。碳青霉烯类抗生素包括亚胺培南、美罗培南和厄他培南等是临床治疗肠杆菌科细菌尤其是产超广谱β-内酰胺酶(ESBLs)及AmpC酶等多重耐药菌株引起感染的最有效的抗菌药物。然而,随着碳青霉烯类抗生素在临床上的广泛应用,耐药肠杆菌科细菌(Carbapenem-Resistant Enterobacteriaceae, CRE)正在医院内悄悄出现。如在大肠埃希菌和肺炎克雷伯菌中的耐药率也已经由早年的零上升到1%；在肠杆菌科的其他一些菌属已经有7.0%的耐药率。在非发酵菌的某些菌属的耐药率也已经上升为20%-30%。由于大多数对碳青霉烯类抗生素耐药的细菌同时也对许多临床常用的抗生素耐药,成为泛耐药菌株,对病人的生命构成极大的威胁。该类药物在临床的应用受到严峻的挑战。对2005年-2009年华山医院所有分离的CRE菌株临床资料的分析结果发现,同一病床的不同时间入院患者均能分离到CRE菌株,如神经外科病房的某一床位,2005年8月、2006年1月、2007年3月和2007年9月分别从四位不同的患者分离到碳青霉烯类抗生素耐药的菌株,包括3株弗劳地柠檬酸杆菌和1株肺炎克雷伯菌;同一患者亦能分离到不同菌属的CRE菌株,如鲍曼不动杆菌和肺炎克雷伯菌组、弗劳地柠檬酸杆菌和肺炎克雷伯菌组、肺炎克雷伯菌和大肠埃希菌组等。因此,我们推测CRE菌株在医院范围内广泛播散的机制主要有以下两种：①水平传播：携带碳青霉烯酶如KPC-2型酶的质粒在不同细菌间进行直接转移；使敏感株成为耐药株；②克隆传播：CRE菌株通过各种方式在不同患者间克隆传播而导致医院感染暴发流行的发生。尽管CRE菌株已逐年增加且呈暴发流行的趋势,但目前国内的的研究大多集中于耐药机制的研究,对该类菌株如何在不同种属细菌间广泛播散的传播机制尚缺乏深入的研究,对该类菌株引起的医院感染的危害性亦未得到足够的重视。Kochar等人通过加强手卫生和环境表面消毒成功减少碳青霉烯类抗生素耐药的肺炎克雷伯菌的传播。但有关此方面的研究国内尚未见报道。由于CRE菌株引起的感染大多为重症感染且目前临床并无有效的治疗药物可供选择,尤其是对于CRE菌株引起的中枢神经系统感染更是如此。与往年相比,2009年碳青霉烯类抗生素耐药肺炎克雷伯菌的发生率在快速的上升,如再不采取有效的医院感染控制措施加以控制,该类菌株引起的大爆发流行所给患者带来的灾难性后果将不可避免。因此,了解CRE菌株在不同种属细菌间广泛播散的机制,采取有效的措施控制该类菌株引起的医院感染的发生及暴发流行已是迫在眉睫。本课题旨在通过分子生物学技术与临床流行病学调查相结合的方式揭示CRE菌株在不同种属细菌间广泛播散的机制,为采取行之有效的医院感染控制措施以及时遏制该类菌株引起的持续感染和暴发流行提供实验室和临床依据,这对促进临床抗感染治疗的合理用药以及患者的康复将产生积极的影响,同时也将带来极大的社会效益和经济效益。本研究内容共包括以下四部分。细菌产生碳青霉烯酶是CRE菌株对碳青霉烯类耐药的主要机制之一,文献报道,该类菌株还可产生超广谱β内酰胺酶(Extended-Spectrumβ-lactamases, ESBLs)和/或AmpC酶合并外膜孔蛋白缺失等。为检测肠杆菌科细菌中的碳青霉烯酶,美国临床实验室标准化研究所(Clinical Laboratory Standard Institute, CLSI)于2009年推荐了改良Hodge试验,用于肠杆菌科细菌中碳青霉烯酶的检测。该法最初是由Wavell hodge建立的一种用于检测产青霉素酶淋病奈瑟菌的试验。之后多位学者用此方法或进行改良用于铜绿假单胞菌和鲍曼不动杆菌中金属酶的检测以及大肠埃希菌和肺炎克雷伯菌中的CMY-1型AmpC酶的检测。华山医院于2005年首次出现CRE菌株,随后我们通过细菌耐药监测系统连续对此类菌株的出现进行了监测。2005年1月-2009年12月间我们共分离到220株对碳青霉烯类抗生素耐药的肠杆菌科细菌,包括克雷伯菌属161株(其中肺炎克雷伯菌158株,产酸克雷伯菌3株)、柠檬酸杆菌属41株(其中弗劳地柠檬酸杆菌35株,其他柠檬酸杆菌6株)、大肠埃希菌6株、阴沟肠杆菌4株、奇异变形杆菌3株、粘质沙雷菌2株、产气肠杆菌1株、支气管博得特菌1株和斯氏普罗威登菌1株。纸片扩散法药敏试验结果显示,2005年-2008年华山医院临床分离的肺炎克雷伯菌对亚胺培南、美罗培南和厄他培南等抗菌药的耐药率在2%以下,但2009年快速上升至16%左右；弗劳地柠檬酸杆菌2005年对碳青霉烯类抗生素的耐药率在10%,2006年快速上升至45%左右,2007年-2009年维持在35%左右。琼脂稀释法检测2005年-2009年中的78株CRE菌株的药敏试验结果显示,对亚胺培南、美罗培南、厄他培南和粘菌素的敏感率分别为16.4%、17.9%、1.3%和96.0%。上述药物对78株CRE菌株的MIC50/MIC90分别为32/256 mg/L、64/>256mg/L、128/>256mg/L和1/1mg/L。改良Hodge试验对78株CRE菌株中的碳青霉烯酶进行了检测。结果显示：93.6%(73/78)的菌株为阳性,提示这些菌株可能产碳青霉烯酶。采用PCR法对78株CRE菌株进行了各种碳青霉烯酶基因、ESBLs和质粒AmpC酶基因的检测及DNA测序结果显示,33.3%(26/78)的菌株产KPC-2型碳青霉烯酶,且其中分别有7.7%(2/26)、7.7%(2/26)、3.8%(1/26)的菌株同时伴有VIM-1、IMP-2和IMP-1型金属酶。6.4%(5/78)、5.1%(4/78)、3.8%(3/78)、6.4%(5/78)和12.8%(10/78)的菌株分别产VIM-1、IMP-1、IMP-2、GIM和OXA-69型碳青霉烯酶基因。多重PCR检测结果显示,20.5%(16/78)的菌株产OXA-23like、OXA-51like或OXA-58like型碳青霉烯酶。VIM-2、SPM、NMC、IMI、IND、OXA-48、OXA-50、OXA-55、OXA-60和OXA-24like碳青霉烯酶基因检测结果全为阴性。PFGE同源性分析：按Tenover标准,25株弗劳地柠檬酸杆菌分为3种不同的DNA谱型(A-C),其中15株产KPC-2型酶的菌株为同一谱型(A),提示存在克隆菌株传播流行可能。43株肺炎克雷伯菌分为10个不同的DNA谱型(A-J),9株产KPC-2型酶菌株中7株为同一谱型(J),其余2株为同一谱型(I)；不产KPC-2型酶的CRE菌株间亦存在克隆菌株的传播流行。外膜孔蛋白分析：SDS-PAGE电泳结果显示,与敏感菌株相比,29.5%(23/78)的CRE菌株至少缺失OmpK35和OmpK36中的1条或两者全部缺失,60.3%(47/78)的菌株外膜孔蛋白均存在表达下降现象,提示外膜蛋白在CRE菌株对碳青霉烯类抗生素耐药过程中发挥了重要作用。接合试验：对产KPC-2型碳青霉烯酶基因的6株肺炎克雷伯菌(其中2株同时产VIM-1型金属酶)和3株弗劳地柠檬酸杆菌的接合试验结果显示,所有9株细菌均在选择性平板上获得转移接合子。药敏试验及β内酰胺酶基因PCR检测结果显示,供体菌中所含的ESBL基因均成功转移至受体菌中,转移接合子均表现为对第三代头孢菌素耐药。但KPC-2型碳青霉烯酶或VIM-1型金属酶基因均未能从供体菌转移至受体菌。转化试验亦未能将此两种碳青霉烯酶基因转移至受体菌,推测该耐药基因可能位于染色体上,或可能含有KPC-2型碳青霉烯酶基因的质粒过大而导致接合或转化试验失败。同一病床的不同时间入院患者均能分离到CRE菌株,同一患者亦能分离到不同种属的CRE菌株,以及在某些患者身上,经常看到这样一种现象：最初分离的肺炎克雷伯菌对碳青霉烯类抗生素是敏感的,但经过一段时间的治疗后变得对碳青霉烯类抗生素耐药。为在体外模拟体内菌株出现的上述现象,我们采用接合试验尝试对这一演变过程进行复制,以期能够初步解释CRE菌株在不同种属细菌间广泛播散的现象。本研究对同一患者临床分离的泛耐药鲍曼不动杆菌A1979和碳青霉烯类敏感的肺炎克雷伯菌K1980进行了接合试验,利用厄他培南和舒巴坦作为接合子菌株筛选药物,成功筛选到了鲍曼不动杆菌A1979和肺炎克雷伯菌K1980菌株的接合子,药敏试验结果证实接合子菌株对厄他培南、亚胺培南和美罗培南均表现为耐药。ERIC-PCR和PFGE试验均证实对碳青霉烯类药物抗生素耐药的接合子菌株与敏感的受体菌的ERIC-PCR和PFGE谱型完全一致。由于此次选择的供体菌鲍曼不动杆菌A1979其产金属酶的种类尚未知晓,因此接合子菌株中含有哪一种金属酶基因仍有待进一步的研究。在后续的进一步研究中,我们将选择更多的菌株进行类似的直接的接合试验。并对接合试验所获得的接合子菌株以及供体菌和受体菌中的耐药质粒DNA分别进行测序分析,以从分子生物学水平解释碳青霉烯酶在不同种属细菌间转移的机制。我们对神经外科病房某一床位进行了调查。该床位频繁地住过CRE菌株感染患者或定植者。采用含1片厄他培南(10μg)纸片的5毫升MH肉汤的增菌法从患者肛周标本、尿道口标本以及氧气湿化瓶中的水样标本中均分离到了对碳青霉烯类抗生素耐药的肺炎克雷伯菌。PFGE同源性分析证实分离自这3份标本中的3株菌株为同一PFGE谱型。同时采集该病房7位患者床头的氧气湿化瓶中的水样标本,结果发现从其中5名患者的湿化瓶水样中分离出了对碳青霉烯类抗生素耐药的菌株,包括2株肺炎克雷伯菌、3株鲍曼不动杆菌和1株嗜麦芽窄食单胞菌等。而对神经外科某一病房的环境标本及工作人员的一次集中采样筛选CRE菌株结果显示,病床左右栏杆及桌子台面的环境标本,护士的手上以及输液泵屏幕上均存在碳青霉烯类药物耐药的肺炎克雷伯菌。提示氧气湿化瓶和医院环境中存在的CRE菌株可能是在医院环境中引起各种感染尤其是呼吸道感染的关键传播源头之一。对78株CRE菌株感染患者的病史资料回顾性调查分析结果显示,发现多种抗菌药物的应用以及引流管包括导尿管和脑脊液引流管的留置可能是导致CRE菌株感染和难以清除的危险因素；CRE菌株携带或感染患者在不同床位和病房之间的频繁轮流更换可能是导致CRE菌株在医院范围内广泛播散的关键因素。通过上述研究,得出以下结论：1.药敏试验结果显示,自华山医院分离的肠杆菌科细菌,包括肺炎克雷伯菌、弗劳地柠檬酸杆菌等菌株对亚胺培南、美罗培南和厄他培南的耐药率高。其中CRE菌株绝大多数为对碳青霉烯类抗生素高度耐药株。亚胺培南、美罗培南和厄他培南对其的的MIC90均≥256 mg/L。这些CRE菌株绝大多数分离于神经外科病房患者,并以尿液标本与呼吸道标本的分离株为主。2.改良Hodge试验对78株CRE菌株中的碳青霉烯酶检测的结果显示,93.6%(73/78)的菌株为碳青霉烯酶产生株。与PCR法检测碳青霉烯酶基因结果相比,其检测灵敏度和特异性分别为97.9%、12.9%。3.PCR检测耐药基因结果及SDS-PAGE电泳分析外膜孔蛋白结果显示,60.3%(47/78)的菌株产碳青霉烯酶,其中33.3%(26/78)产KPC-2型酶。产生碳青霉烯酶是CRE菌株对碳青霉烯类抗生素耐药的主要机制之一。35.9%(28/78)的菌株至少丢失1条外膜孔蛋白,提示产生ESBLs和/或质粒AmpC酶且同时合并外膜孔蛋白丢失是导致CRE菌株对碳青霉烯类抗生素耐药的另一个重要的耐药机制。4.虽经多次接合试验和转化试验,但编码KPC-2型碳青霉烯酶的基因始终无法从供体菌转移至敏感菌中,但供体菌中的ESBLs基因却在接合的过程中进行了转移。推测编码KPC-2型碳青霉烯酶的基因可能位于染色体上,或与ESBLs基因分别存在于不同的耐药质粒上。5. PFGE同源性分析结果显示,碳青霉烯类抗生素耐药的弗劳地柠檬酸杆菌和肺炎克雷伯菌均存在克隆菌株在不同病房之间的流行和传播。6.含1片厄他培南(10μg)纸片的5毫升MH肉汤增菌法可提高从各类标本中筛选碳青霉烯类抗生素耐药菌株的检测率,可作为医院感染调查的推荐检测方法。7.流行病学调查研究结果显示,神经外科病房多数氧气湿化瓶中均检测到碳青霉烯类抗生素耐药的菌株,包括肺炎克雷伯菌、鲍曼不动杆菌和嗜麦芽窄食单胞菌等。病床周围、医护人员手部以及患者肛周均携带有CRE菌株。上述因素可能是导致CRE菌株在医院环境广泛播散引起各种感染尤其是呼吸道感染的关键传播源头。8.本研究调查初步结果显示,手卫生和加强医院环境消毒卫生将是阻断碳青霉烯类药物耐药菌株在医院环境内广泛播散的关键措施。及时采取有效地医院感染控制措施限制耐药菌株尤其是碳青霉烯类药物耐药菌株的播散将成为当务之急。更多还原

【Abstract】 Enterobacteriaceae species including E. coli, Klebsiella pneumoniae, Citrobacter freundii and Enterobacter cloacae were the most frequent pathogens of causing nosocomial infections. According to surveillance of bacterial resistance in Shanghai and CHINET, Enterobacteriaceae species accounted for 60% to 70% among gram-negative bacilli. Carbapenems such as imipenem, meropenem and ertapenem showed strong activity against infections caused by multi-drug resistant Enterobacteriaceae, especially extended-spectrumβ-lactamases (ESBLs) and/or AmpC-producing isolates. However, with the widely use of carbapenems in clinic, the occurrence and increase of carbapenem-resistant Enterobacteriaceae (CRE) was unavoidable. The resistant rate of E.coli and K. pneumoniae to carbapenem increased to 1.0%, and in some Enterobacter spp. the resistant rate has already reached 7.0%. The resistant rate of some nonfermentative Bacilli was also about in 20%-30%. The carbapenems met an urgent challenge in clinic because most of carbapenems-resistant bacteria were also resistant to other antimicrobial agents.Among all the CRE isolates from 2005 to 2009 in Huashan Hospital, we often isolated CRE strain from different patients in the same bed in a ward. In Aug.2005, Jan 2006, Mar 2007, and Sep 2007,3 C. freundii isolates and 1 K. pneumoniae strain were collected from four different patients in a bed in a neurosurgery ward. Different species of CRE isolates were also isolated from the same one patient, including Acinetobacter baumannii and K. pneumoniae, C. freundii and K. pneumoniae, K. pneumoniae and E. coli. Therefore, we presumed there were two major mechanisms underlying the widespread dissemination of CRE isolates in hospital. One was horizontal transmission, plasmid harboring carbapenemase gene, such as KPC-2 type carbapenemase, was transmitted between different species directly. The other was clone transmission, CRE isolates were widely disseminated in different patients through various ways and caused outbreak. Currently, most of studies have focused on resistant mechanism and only few are associated with the mechanism for the widespread dissemination of CRE isolates between different species and nosocomial infection despite the frequency of CRE isolates is increasing year by year. A survey by Kochar revealed that improved decontamination of hands and environmental surfaces could reduce the spread of carbapenem-resistant K. pneumoniae successfully. However, such study has been not reported in our country. Infections caused by CRE isolates, especially bacterial meningitis, have usually been very serious and antibiotic choices may be quite limited. Compared with previous years, the frequency of carbapenems-resistant K. pneumoniae increased dramatically in 2009. We presumed the outbreak of CRE isolates would be unavoidable in hospital environment if the effective actions of infection control were still lack. Therefore, it was urgent to study the mechanism of CRE dissemination between different species and take effective infection control actions immediately to limit the occurrence and outbreak. In order to provide a basis for controlling outbreak and spread of CRE isolates, our study aims to reveal the mechanism of CRE dissemination between different species through molecular and epidemiological study. These may have a positive impact on improving clinical therapy, rational drug use and rehabilitation of patients. This study included four parts as follows.Carbapenemase-producing was the main resistant mechanism of CRE isolates to carbapenems. Some studies also reaveled that extended-spectrum P-lactamases (ESBLs) and/or AmpC-producing coupled with outer membrane porin loss in CRE isolates is another main resistant mechanism to carbapenems. In 2009, CLSI recommends modified Hodge testing (MHT), which initially used to detect penicillinase-positive Neisseria gonorrhoeae by Wavell Hodge, for detecting carbapenemase-producing isolates among Enterobacteriaceae. Then, MHT was used to detect metallo-β-lactamase in Pseudomonas aeruginosa and A. baumannii and CMY-1 type AmpC in K. pneumoniae and E. coli.In 2005, the first CRE strain was isolated in Huashan Hospital. Up to Dec.2009, 220 CRE isolates were collected by the surveillance of bacterial resistance, including K. pneumoniae (158), K. oxytoca (3), C.freundii (35), Citrobacter spp. (6), E. coli (6), Enterobacter cloacae (4), Proteus mirabilis (3), Serratia marcescens (2), Enterobacter aerogenes (1), Bordetella bronchiseptica (1) and Providencia stuartii (1). The result of antimicrobial susceptibility testing indicated that the frequency of K. pneumoniae isolates resistant to imipenem, meropenem and ertapenem was all below 2%, but in 2009 quickly increased to 16%. The frequency of C. freundii resistant to carbapenems was about 10% in 2005, but increased to 45% in 2006, and 35% during 2007 to 2009. The antimicrobial susceptibility testing of 78 CRE isolates from Jan. 2005 to Apr.2009 by agar dilution indicated that the susceptibility rate of imipenem, meropenem, ertapenem and colistin was 16.4%,17.9%,1.3% and 96.0%, respectively. The MIC50/MIC90 of imipenem, meropenem, ertapenem and colistin against 78 CRE isolates was 32/256 mg/L,64/>256 mg/L,128/>256 mg/L and 1/1 mg/L. The result of MHT indicated that 93.6%(73/78) of these CRE isolates were carbapenemase-producing.The result of detection of carbapenemases, ESBLs and plasmid-borne AmpC gene by PCR method and DNA sequencing indicated that 33.3%(26/78) were KPC-2 type carbapenemase-producing isolates, in which 7.7%(2/26),7.7%(2/26),3.8% (1/26) were coupled with VIM-1, IMP-2, IMP-1 metallo-β-lactamase, respectively. In addition,6.4%(5/78),5.1%(4/78),3.8%(3/78),6.4%(5/78),12.8%(10/78) were VIM-1, IMP-1, IMP-2, GIM, OXA-69 type carbapenemase-producing isolates, respectively. The result of multiple PCR analysis indicated,20.5%(16/78) were OXA-23-like, OXA-51-like or OXA-58-like carbapenemase-producing isolates. VIM-2, SPM, NMC, IMI, IND, OXA-48, OXA-50, OXA-55, OXA-60 and OXA-24-like type carbapenemases genes were all negative.According to Tenover criteria,25 C. freundii isolates belonged to 4 different DNA patterns and in which 64.0%(16/25) isolates were the same genotype. The 43 strains of K. pneumoniae belonged to 10 different DNA patterns,6 KPC-2 type carbapenemase-producing isolates were the same genotype and other 3 isolates were different. Interestingly, some KPC-2 type carbapenemase negative isolates were also clonally related.Compared with carbapenem-susceptible Enterobacteriaceae,29.5%(23/78) CRE isolates presented one single band, probably corresponding to OmpK35 or OmpK36 porin, suggesting that they have lost at least one porin.60.3%(47/78) CRE isolates were down-regulation of the OmpK35 or OmpK36 porin.The result of conjugation experiment with 6 strains of KPC-2 type carbapenemase-producing K. pneumoniae (including 2 strains of KPC-2 type carbapenemase-producing coupled with VIM-1 type metallo-β-lactamase-producing K. pneumoniae) and 3 strains of carbapenem-resistant C. freundii indicated that transconjugants of all 9 isolates were obtained from selective plate. The result of antimicrobial susceptibility testing andβ-lactamase gene detection by PCR method indicated ESBL or AmpC gene were successfully transferred from donor to recipient by conjugation. Transconjugants were all resistant to third cephalosporins. However, KPC-2 type carbapenemase gene and VIM-1 type metallo-β-lactamase gene were both not transferred. The transformation testing of KPC-2 type carbapenemase gene and VIM-1 type metallo-β-lactamase gene also failed. We presumed that the carbapenemase gene may locate on chromosome or the plasmid bearing KPC-2 type carbapenemase gene was too large to be transferred.The same CRE strain was often isolated from different patients in the same bed, and different species of CRE isolates were also isolated from the same one patient. We also found some K. pneumoniae strains developed carbapenem resistance during therapy. We tried to reproduce in vitro the phenomenon observed in vivo and hoped to explain the mechanism of wide CRE dissemination between different species. The transfer experiment of carbapenemase gene was performed by conjugation. The donor and recipient were pan-drug resistant A. baumannii, and carbapenems-susceptible K. pneumoniae, respectively. Transconjugants were obtained after selection on ertapenem and sulbactam, respectively. The result of antimicrobial susceptibility testing indicated that transconjugants were resistant to imipenem, meropenem and ertapenem. ERIC-PCR and PFGE demonstrated that transconjugants and recipient were the same genotype. Because the type of metalloenzyme gene was unknown in donor, the resistant mechanism of transconjugants to carbapenems still needs further research. We should select more pan-drug resistant isolates for reproduction in vitro the phenomenon observed in vivo. The resistant plasmids of transconjugants, donor and recipient should be sequenced in order to reveal the transfer mechanism of carbapenemase.An epidemiological survey was conducted for different patients who infected or colonizated by CRE isolate in the same one bed in a neurosurgery ward. The specimen were cultured in 5 mL MH broth containing one piece of ertapenem (10μg). We isolated the CRE from rectal swab sample, meatus urinarius and the water in oxygen filter system. PFGE demonstrated that three CRE isolates were the same genotype. After that, we isolated 5 carbapenems-resistant isolates, including 2 K. pneumoniae isolates,3 A. baumannii isolates and 1 S. maltophilia isolate from the water in five oxygen filter system in this ward. The results of screening CRE isolates in specimens from hospital environment and medical staff in neurosurgery ward indicated that the handrails of bed, the hands of nurse and the screen of infusion pump were all positive for carbapenems-resistant K. pneumoniae. Therefore, we presumed that the CRE isolates in oxygen filter system and the hospital environment may the key source of various infections, especially respiratory tract infections.The retrospective review of the medical history of patients with CRE infection or colonization indicated that prior antimicrobial agents use, presence of a variety of catheters may contribute to CRE infection and difficult to eradicate. One of the key factors for the spread of CRE isolates in hospital was the frequent exchange of patients with CRE infection or colonization between different beds or wards. Conclusions:1. The result of antimicrobial susceptibility testing indicated that most of CRE isolates including K. pneumoniae and C.freundii in Huashan Hospital were highly resistant to imipenem, meropenem and ertapenem. The MIC90 of imipenem, meropenem and ertapenem was all above 256 mg/L. Most of CRE isolates were isolated from patients in neurosurgery. The main source of specimens was urine and respiratory tract.2. The results of detection of carbapenemase in 78 CRE isolates by MHT indicated, 93.6%(73/78) were carbapenemase-producing isolates. Compared with PCR method, the sensitivity and specificity of detection of carbapenemase by MHT were 97.9% and 12.9, respectively.3. The results of resistance gene detection and outer membrane porin analyzed by SDS-PAGE indicated,60.3%(47/78) were carbapenemases-producing isolates in which 33.3%(26/78) isolates producing KPC-2 type carbapenemase. Production of carbapenemases is the main mechanism for carbapenem resistance in Enterobacteriaceae isolates; 35.9%(28/78) isolates have lost at least one porins. Production of ESBL and/or plasmid-borne AmpCβ-lactamases coupled with porin loss is another important resistant mechanism.4. Although several conjugation and transformation experiments were performed, KPC-2 type carbapenemase still did not transfer from donor to recipient except for ESBLs. We presumed that the KPC-2 type carbapenemase may locate on chromosome or the plasmid bearing KPC-2 type carbapenemase gene is too large to be transferred from donor to recipient.5. The results of PFGE suggest clone spread of CRE isolates between different wards, including both C.freundii and K. pneumonia.6. Compared with direct method,5 mL of MH broth containing 10μg of ertapenem can be used in epidemiologic study because it may increase the detection rate of CRE isolates in all kinds of specimens.7. Many oxygen filter systems harbor carbapenems-resistant isolates, including K. pneumoniae, A. baumannii and S. maltophilia. The CRE isolates were also isolated in environmental surfaces of bed, nursing staff’s hands and the rectal in patient. All of above factors may the key source of resulting in various infections, especially respiratory tract infections.8. Our research indicated that improved hand hygiene and environmental surface cleaning may be the key procedure to limit the spread of carbapenems-resistant isolates. It is urgent to take effective procedures to control infections due to carbapenem-resistant or carbapenemase-producing Enterobacteriaceae in hospital environment.更多还原