碳青霉烯类耐药的肠杆菌科细菌耐药机制研究  

Antimicrobial resistant mechanisms of carbapenem-resistant Enterobacteriaceae

作　　者：汪玥[1] 孙自镛[1] 陈中举[1] 王斌[1] 田磊[1] 朱旭慧[1] 李丽[1] 张蓓[1] 朱琴[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院检验科,武汉430030

出　　处：《中华检验医学杂志》2012年第4期339-344,共6页Chinese Journal of Laboratory Medicine

基　　金：国家“十一五”计划资助项目（NO.2009ZX10004-107）

摘　　要：目的探讨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。方法收集华中科技大学同济医学院附属同济医院2007年1月至2010年6月临床分离非重复肠杆菌科细菌5604株，其中美罗培南抑菌环直径≤21mm的肠杆菌科细菌100株。药物敏感性试验筛选出碳青霉烯类耐药菌株，采用聚合酶链反应（PCR）对碳青霉烯酶基因和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。结果共检出CRE11株，其中克雷伯菌属细菌7株。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，最低抑菌浓度美罗培南8—64mg／L，亚胺培南4—64mg／L，厄他培南4～64mg／L，对氨基糖苷类和氟喹诺酮类抗菌药物的敏感性则变化较大。PCR检出IMP-4阳性菌株6株，KPC-2阳性菌株3株，其中1株ST476型肺炎克雷伯菌同时携带blaIMP-4和blaKPG-2。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。MLST分型发现2株肺炎克雷伯菌同属于ST476型。SDS—PAGE提示1株产酸克雷伯菌（Kox656）存在外膜孔道蛋白（OmpK35和OmpK36）双缺失。结论本院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，IMP-4是其主要酶型。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，应重视医院细菌耐药性特点及耐药菌感染的临床流行病学资料，从而为临床合理用药提供参考。Objective To investigate the antimicrobial resistant mechanisms of carbapenem- resistant Enterobacteriaceae （CRE）, construct monitoring system of acquired carbapenemase. Methods Totally 5604 clinical isolates of Enterobacteriaceae in Tongji Hospital were collected from January 2007 to June 2010, including 100 isolates of which zone diameters of meropenem were not larger than 21 mm. Antibiotic susceptibility was performed to select CRE. Then, carbapanemase gene and genetic structure screenings were performed by polymerase chain reaction （PCR）. 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 were examined by sodium dodecyl sulfate polyaerylamide gel electrophoresis （SDS-PAGE）. Results Eleven isolates of CRE were collected and confirmed, most of them were Klebsiella spp. （7/11）. Susceptibility of antimicrobial agents indicated that all these strains resistant to most antimicrobials. The minimal inhibitory concentration （MIC） range of meropenem were 8 - 64 mg/L, imipenem were 4 - 64 mg/L, ertapenem were 4 - 64 mg/L. However, susceptibilities of aminoglycosides and fluoroquinolones were significantly different. PCR results showed that six isolates were blaiMp.4 positive and three isolates were blaKpc-2 positive, including one isolate of K. pneumoniae （ST476） carrying both blaiMp4 and blaxpc-2 genes. Genetic structure of carbapenemase genes were analyzed, suggesting that blaKPc-2 located in an integrated structure of a Tn3-based transposon and partial Tn4401 segment. PFGE showed that most CRE contained three or more plasmids. Two isolates of K. pneumoniae were assigned to the same sequence type, ST476, by MLST. SDS-PAGE indicated that only one isolate （Kox656） lacked two out membrane proteins （ OmpK35 and OmpK36）. Conclusions The most common carbapenemase-resistant Enterobacteriaceae was K. pne

关 键 词：肠杆菌科细菌 质粒 细菌外膜蛋白质类 

分 类 号：R446.5[医药卫生—诊断学]