【作者】 汪玥；

【导师】 曹文静； 孙自镛；

【作者基本信息】 华中科技大学 ， 临床检验诊断学， 2011， 硕士

【摘要】 [目的]研究碳青霉烯类耐药肠杆菌科细菌(CRE)的耐药机制,建立获得性碳青霉烯酶流行监测体系。[方法]收集同济医院2007年1月至2010年6月分离的美洛培南抑菌环直径≤21mm的肠杆菌科细菌,测定抗菌药物敏感性,筛选出碳青霉烯类耐药菌株,进行酶表型和基因型筛查;对整合子插入序列和碳青霉烯酶基因附属结构进行分析;采用脉冲场凝胶电泳(PFGE)和Southern印迹杂交方法分析耐药菌质粒;采用多位点序列分型(MLST)方法对菌株进行分型及分析同源性;采用PCR和十二烷基磺酸钠-聚丙烯凝胶电泳(SDS-PAGE)方法对菌株的外膜孔道蛋白进行分析。[结果]收集并证实CRE 11株,主要是肺炎克雷伯菌(7/11)。抗菌药物敏感性试验显示所有菌株对大多数抗菌药物耐药,美洛培南8~64μg/ml,亚胺培南4~64μg/ml,厄他培南4~64μg/ml,对氨基糖苷类和氟喹诺酮类抗菌药物的敏感性显著不同。PCR检测耐药基因,检出IMP-4阳性菌株6株,KPC-2阳性菌株3株,其中一株肺炎克雷伯菌(Kpn6617)同时携带blaIMP-4和blaKPC-2。整合子插入序列均不含有碳青霉烯酶编码基因。对基因附属结构进行分析,结果显示blaKPC-2基因位于由Tn3转座子和Tn4401部分片段构成转座子上。PFGE显示大多数CRE含有3个或3个以上质粒。SDS-PAGE检测外膜孔道蛋白的表达,结果显示仅Kox656存在外膜孔道蛋白(OmpK35和OmpK36)双缺失。对肺炎克雷伯菌进行MLST分型,Kpn6617和Kpn6099均属于ST476型。通过接合试验证明,Kpn6617中携带blaIMP-4质粒具有转移性。进一步研究发现,不同菌株携带blaKPC-2的质粒具有同源性,而携带blaIMP-4的质粒不完全同源。[结论]我院CRE主要是肺炎克雷伯菌,产生碳青霉烯酶是细菌耐药的主要原因,blaIMP-4是主要的碳青霉烯酶酶型。发现一株罕见的同时携带blaKPC-2和blaIMP-4的碳青霉烯类耐药肺炎克雷伯菌,属于ST476型,其产生机制可能是由于blaIMP-4编码质粒转移至ST476型产KPC-2酶肺炎克雷伯菌中造成。肠杆菌科细菌中碳青霉烯酶基因的出现和传播对治疗和感染控制造成巨大威胁,应引起人们的警觉。更多还原

【Abstract】 Objectives: To investigate the antimicrobial resistant mechanisms of CRE, construct monitoring system of acquired carbapenemase.Methods: Clinical isolates of Enterobacteriaceae in our hospital which zone diameters of meropenem were not larger than 21mm were collected from January 2007 to June 2010. Antibiotic susceptibility was performed to select CRE. Then, phenotype and gene screenings were performed. Integron insertion sequence and genetic structure of carbapenemase genes were also detected by PCR method. Pulsed-field gel electrophoresis (PFGE) and Southern blot were used to analyze the plasmids of CRE. Multilocus sequence typing (MLST) was used to determine the genotypes and homology of these isolates. In addition, out membrane proteins (OMPs) were examination by PCR and SDS-PAGE.Results: 11 isolates of CRE were collected and confirmed, mostly were Klebsiella pneumoniae (7/11). Susceptibility of antimicrobial agents indicated that all these strains resistant to most antimicrobials, including carbapenems (meropenem 8~64μg/ml, imipenem 4~64μg/ml, ertapenem 4~64μg/ml). However, susceptibilities of aminoglycosides and fluoroquinolones were significantly different. Using PCR method to detect resistant genes, the result showed that 6 isolates were IMP-4 positive and 3 isolates were KPC-2 positive, including one isolate carrying both blaIMP-4 and blaKPC-2 genes. Integron insertion sequences were amplified and sequenced. All of them did not include carbapenemase encoding genes. Genetic structure of carbapenemase genes was analyzed, suggesting that blaKPC-2 located in an integration structure of a Tn3-based transposon and partial Tn4401 segment. PFGE showed that most CRE contained three or more plasmids. Out membrane proteins were detected by SDS-PAGE, indicating that only one isolate, Kox656, lacked OMPs (OmpK35 and OmpK36). Kpn6617 and Kpn6099 were assigned to a novel sequence type, ST476, by MLST。Besides, blaKPC-2 encoding plasmid in Kpn6617 was transmissible. Further evidence showed the homology of blaKPC-2-harbouring plasmids among KPC-2 positive isolates. However, the probing data for blaIMP-4 suggested a diversity of blaIMP-4-harbouring plasmids.Conclusions: The most common carbapenemase-resistant Enterobacteriaceae was K.pneumoniae in our hospital. Producing carbapenemases, which type mostly IMP-4, was the most reason that bacteria resistant to carbapenem. A novel carbapenemase-resistant K.pneumoniae encoding both blaKPC-2 and blaIMP-4 was detected, probalbly due to transmission of the blaIMP-4-harbouring plasmid into KPC-2-producing K.pneumoniae (ST476). The concomitant presence of these genes is alarming and poses therapeutic as well as infection control problems.更多还原