烧伤患者产NDM-1型金属β-内酰胺酶肺炎克雷伯菌的碳青霉烯类抗生素耐药机制研究  

Research on the mechanism of the New Delhi metallo-β-lactamase-1 possessing Klebsiella pneumoniae resistant to carbapenems among burn patients

作　　者：杜芳玲 梅艳芳[1] 刘盼盼[1] 万腊根[1] 魏丹丹[1] 曹先伟[1] 文江雄 向天新[1] 刘洋[1] 

机构地区：[1]南昌大学第一附属医院检验科,330006

出　　处：《中华检验医学杂志》2018年第1期29-34,共6页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金（81560323）;江西省重点研发计划项目（20171BBG70053）;江西省科技厅基金项目（20151BAB215028,20161BAB205247）;江西省教育厅重点项目（GJJ160029）

摘　　要：目的探讨烧伤患者感染肺炎克雷伯菌对碳青霉烯类抗生素高水平耐药的分子机制。方法采用回顾性分析方法，收集南昌大学第一附属医院2014年7月至2015年6月烧伤患者分离的非重复碳青霉烯类耐药肺炎克雷伯菌（CR－KP）18株。测定菌株最低抑菌浓度（MIC），对菌株进行特异性PCR扩增和序列分析、质粒接合试验、Southern杂交以及外膜蛋白分析。多位点序列分型（MLST）分型技术和脉冲场凝胶电泳（PFGE）分析菌株同源性。结果药敏试验显示所有碳青霉烯类耐药肺炎克雷伯菌（CR－KP）具有多重耐药，对哌拉西林/他唑巴坦、头孢曲松、左氧氟沙星、环丙沙星、庆大霉素、妥布霉素、头孢替坦、头孢他啶、头孢吡肟、氨曲南、亚胺培南、美罗培南全部耐药，耐药率100%（18/18）；对替加环素、复方新诺明、阿米卡星对耐药率分别为0%（0/18）、61.1%（11/18）、72.2%（13/18）。接合试验可使受体菌大肠埃希菌J53对碳青霉烯的MIC值从≤0.0625或0.125 μg/ml上升到16或32 μg/ml。特异性PCR扩增和序列分析发现18株产NDM－1菌株，5株KPC－2菌株。接合试验证实blaNDM－1可以通过质粒传播。Southern杂交显示blaNDM－1基因定位于46Kb大小的质粒上，质粒复制子分型显示为IncX3型。PFGE共7种细菌基因型，而MIST分型发现18株CR－KP分别属于ST11、ST395、ST17、ST37、ST263、ST14和ST76型。外膜蛋白的SDS－PAGE和ompK35/36基因序列分析发现ST11、ST395和ST37型CR－KP菌株OmpK36膜孔蛋白缺失，而其他ST分型菌株OmpK36膜孔蛋白表达量降低。结论本院烧伤患者分离的肺炎克雷伯菌对碳青酶烯类药物高水平耐药的主要机制是质粒介导的NDM－1型金属β－内酰胺酶的产生合并OmpK36膜孔蛋白表达降低或缺失，并且发现了同时携带NDM－1和KPC－2种碳青霉烯酶的肺炎克雷伯菌。ObjectiveTo investigate the antimicrobial resistant mechanisms of high level carbapenem resistant Klebsiella pneumoniae infection of burn patients.MethodsA retrospective study was conducted on totally 18 non-repetitive high level CR-KP which were isolated from burn patients hospitalized between July 2014 and June 2015. MIC of antibiotics were determined by using the GN13 cards and agar dilution method. The specific PCR and DNA sequence analysis were performed to confirm the β-lactamase type. Plasmid conjugation transfer experiments and southem hybridization were applied to study the mode of carbapenem resistance transmission. Outer membrane proteins （Omps） were isolated and examined by PCR and （sodium dodecyl sulfate polyacrylamide gel electrophores） SDS-PAGE. Pulsed-field gel electrophoresis （PFGE） and Multilocus sequence typing （MLST） was used to determine the genotypes.ResultsSusceptibility of antimicrobial agents indicated that all these strains with multiple drug resistance. The resistance rate to piperacillin/tazobactam, ceftriaxone, levofloxacin, ciprofloxacin, gentamicin, tobramycin, cefotetan, ceftazidime, cefepime, aztreonam, imipenem, and meropenem was 100% （18/18）. Moreover, the resistance rate of CR-KP isolates to amikacin was 72.2% （13/18）, compound sulfamethoxazole was 61.1% （11/18）, tigecycline was 0% （0/18）. Conjugation study with Escherictda coli J53 resulted in the transfer of significant reduced carbapenem susceptibility from donors （MICs increased at least 8-fold）. By PCR, eighteen strains of Klebsiella pneumoniae carried NDM-1 gene, 5 strains carried KPC-2 gene. The blaNDM-1 was transferable by plasmids. Southern blot hybridization indicated that the blaNDM-1 gene was located on plasmid in size of 46 kb. The plasmid belonged to incompatibility group IncX3. Seven types of CR-KP were detected by PFGE. In addition, MLST assigned them to sequence type（ST）11, ST395, ST17, ST37, ST263, ST14 and ST76 types. SDS-PAGE and ompK35/36 genes sequence analysis of O

关 键 词：克雷伯菌 肺炎 流行病学 分子 碳青霉烯类 抗药性 细菌 

分 类 号：R446.5[医药卫生—诊断学] R644[医药卫生—临床医学]