【作者】 蔡加昌；

【导师】 王选锭； 陈功祥；

【作者基本信息】 浙江大学 ， 呼吸内科， 2010， 博士

【摘要】 目的：本研究通过对86株碳青霉烯类抗生素耐药或敏感性降低的肠杆菌科细菌进行β-内酰胺酶、外膜蛋白和外排泵检测,旨在阐明肠杆菌科细菌对碳青霉烯类抗生素耐药的分子机制；通过碳青霉烯酶基因周围序列分析,探讨碳青霉烯耐药基因的传播机制。方法：琼脂稀释法测定抗生素对细菌的最低抑菌浓度(MIC)。脉冲场凝胶电泳和肠杆菌基因间重复性一致序列-PCR (ERIC-PCR)分析菌株之间的同源性。等电聚焦电泳、PCR扩增和序列分析确定菌株所产β-内酰胺酶的类型。质粒接合试验、质粒消除试验和Southern blot杂交验证碳青霉烯耐药基因是否位于质粒上。提取细菌的外膜蛋白作十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),检测外膜蛋白表达情况。羰酰氰氯苯腙(CCCP)抑制试验检测细菌中是否存在针对碳青霉烯类抗生素的外排泵。用限制性内切酶EcoRI和HindⅢ对碳青霉烯酶基因编码质粒进行酶切分型。对部分碳青霉烯酶基因编码质粒进行测序,分析碳青霉烯酶基因周围DNA序列的基因结构。结果：86株菌株对碳青霉烯有不同程度的耐药,亚胺培南、美罗培南和厄他培南的敏感率分别为43.0%、44.2%和1.2%,其它β-内酰胺类抗生素和环丙沙星的耐药率和耐药水平均很高,多粘菌素B的敏感率最高(92.9%),阿米卡星次之(80.2%)。同一医院内的菌株多数具有同源性,存在克隆传播现象。82株菌株产KPC-2型碳青霉烯酶,5株产IMP型金属β-内酰胺酶(其中3株同时产KPC-2),2株检测不到碳青霉烯酶。TEM-1、SHV-11／-12和CTX-M-14／-M-3等β-内酰胺酶的检出率非常高,大部分菌株同时产多种酶。大多数菌株可通过接合试验将碳青霉烯耐药性传递给大肠埃希菌。Southern blot杂交显示blaKPC-2基因位于50kb的可接合质粒上。4株肺炎克雷伯菌(包括2株检测不到碳青霉烯酶的菌株)由于ompK36基因中存在插入序列或碱基突变而缺失OmpK36膜孔蛋白,一株阴沟肠杆菌缺失一个38kDa的外膜蛋白,弗劳地柠檬酸杆菌的两个外膜蛋白表达量均明显下降。CCCP不能降低亚胺培南对细菌的MIC值。82株产KPC-2菌株的blaKPC-2基因编码质粒可根据EcoRI和HindⅢ的限制性酶切图谱分为11种类型(Ⅰ型-Ⅺ型),大部分质粒(62／82)为Ⅰ型,其次为Ⅴ型(8／82)。选取Ⅰ型(pKP1)和V型(pKP20)质粒各一个进行测序,pKP1质粒获得7788bp的DNA序列,pKP20为6000bp。两个质粒的blaKPC-2基因周围序列中均含有多个转座相关元件。pKP1质粒依次编码Tn3转座酶、Tn3解离酶、ISKpn8转座酶、KPC-2和ISKpn6-like样转座酶等蛋白。pKP20质粒依次编码IS26转座酶、Tn3解离酶截短蛋白、ISKpn8转座酶、KPC-2、ISKpn6样转座酶截短蛋白和IS26转座酶等,其中Tn3解离酶截短蛋白、ISKpn8转座酶、KPC-2和ISKpn6样转座酶截短蛋白基因等部分与pKP1质粒相同(共3354bp)。根据pKP1和pKP20质粒的DNA序列设计引物对其它80个blaKPC-2基因编码质粒进行PCR扩增发现,大部分质粒(69／82)的PCR扩增结果与pKP1质粒相同,8个Ⅴ型质粒的PCR结果与pKP20质粒相同。除3个质粒外,其余质粒均具有共同的3354bp序列。另外2个质粒中检测到了2种变异体。结论：同一家医院内碳青霉烯类抗生素耐药的肠杆菌科细菌主要以克隆株的形式传播。碳青霉烯酶尤其是KPC-2的产生是肠杆菌科细菌对碳青霉烯耐药的主要原因,碳青霉烯酶合并膜孔蛋白缺失或低表达可引起碳青霉烯高水平耐药,超广谱β-内酰胺酶合并OmpK36缺失也可导致肺炎克雷伯菌对碳青霉烯耐药或敏感性下降。相同的blaKPC-2基因编码质粒在不同菌种中广泛传播。blaKPC-2基因位于约50kb质粒上的复合转座子中。多数blaKPC-2基因位于相同的转座子结构中,另外还检测到了四种变异体。更多还原

【Abstract】 Objectives:Investigate the mechanisms of carbapenem resistance in 86 clinical isolates of Enterobacteriaceae by detection ofβ-lactamase, outer membrane proteins, and efflux pump. Investigate the transmission mechanisms of carbapenem resistance gene by analysis of genetic structures surrounding carbapenem resistance gene.Methods:Antibiotic susceptibilities were determined by agar dilution method. Pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) were performed to analyze the molecular epidemiology of isolates. Isoelectric focusing (IEF), PCRs, and DNA sequencing were carried out to confirm the genotype of P-lactamases. Conjugation experiments, elimination of plasmid, and southern blot were performed to confirm whether the carbapenem resistance gene was located on plasmid. Outer membrane proteins (OMPs) were extracted and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition assays by carbonyl cyanide m-chlorophenylhydrazone (CCCP) were performed to confirm whether the presence of efflux pump. Typing the carbapenem resistance gene encoding plasmids on the basis of the restriction maps of EcoRI and HindⅢ. Two representative plasmids were sequenced and the DNA sequence surrounding carbapenem resistance gene were analyzed.Results:Eighty-six isolates exhibited various level of carbapenem resistance. Susceptibility rate to imipenem, meropenem, and ertapenem were 43.0%,44.2%, and 1.2%, respectively. Resistance rate and resistance level to otherβ-lactams and ciprofloxacin were high, whereas polymoxin B showed strong activity against carbapenem-non-susceptible Enterobacteriaceae with susceptibility rate of 92.9%, followed by amikacin (80.2%). Major isolates from the same hospital were indistinguishable or closely related to each other. Eighty-two isolates produced KPC-2 carbapenemase,5 produced IMP metallo-β-lactamase (3 of them produced KPC-2 simultaneously), and 2 did not produced any carbapenemase. Severalβ-lactamase genes of blaTEM, blaSHV-11 or-12, and blaCTX-M-14 or -3 were prevalent in these isolates, and majority produced more than oneβ-lactamases. Reduced carbapenem susceptibility can be transferred from most isolates to E. coli (EC600) by conjugation. Southern blot analysis confirmed that blaKPC-2 was located on a ca.50kb conjugative plasmid. Four K. pneumoniae (including two isolates which did not produce carbapenemase) failed to express OmpK36 because of nonsense mutations or insertional inactivation by an insertion sequence in ompK36 gene. An E. cloacae lack a 38 kDa OMP, and expression of two OMPs were decreased in a C. freundii. CCCP can not reduced the minimum inhibitory concentrations values (MICs) of imipenem in Enterobacteriaceae.Eighty-two blaKPC-2-encoding plasmids can be divided into 11 types (type I to XI) according to the restriction maps of EcoRI and HindⅢ. The type I plasmid predominant (62/82), and 8 isolates carried type V plasmid. Two plasmids of pKP1 (type I) and pKP20 (type V) were sequenced. A 7788bp DNA sequence from pKPl and a 6000bp from pKP20 were obtained. Sequence analysis revealed several transposon-associated elements surround blaKPC-2. pKP1 encoded Tn3 transposase, Tn3 resolvase,ISKpn8 transposase, KPC-2, and ISKpn6-like transposase, while pKP20 encoded IS26 transposase, truncated Tn3 resolvase,ISKpn8 transposase, KPC-2, truncated ISKpn6-like transposase, and IS26 transposase. DNA sequence for truncated Tn3 resolvase,ISKpn8 transposase, KPC-2, and truncated ISKpn6-like transposase were the common segment in two plasmids (total 3354bp). A series of primers were designed according to the sequence of pKP1 and pKP20 to amplify the blaKPC-2-surrounding sequence from other 80 plasmids. Sixty-nine plasmids and pKPl shared the same PCR results, and 8 type V plasmids and pKP20 have the same PCR results. All blaKPC-2-encoding plasmids except 3 contained the common 3354bp segment. Another 2 variants were detected in 2 plasmids.Conclusions:Carbapenem-resistant Enterobacteriaceae clonally spread within the same hospital. Carbapenem resistance in Enterobacteriaceae was mainly due to production of carbapenemases, especially KPC-2. Carbapenemases production combined with reduced amounts of porins contributed to high-level resistance to carbapenems. Combination of extended-spectrumβ-lactamase and OmpK36 deficiency resulted in ertapenem resistance, and reduced imipenem and meropenem susceptibility in K. pneumoniae. The same blaKPC-2-encoding plasmids wide disseminated among different species. Majority of the blaKPC-2 genes were located on the same transposon. Four transposon variants were detected in other blaKPC-2-encoding plasmids.更多还原