上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（临床研究） • 上一篇    下一篇

肺炎克雷伯菌对碳青霉烯类抗生素的耐药机制研究

刘婧娴，俞静，李媛睿，刘瑛   

  1. 上海交通大学 医学院附属新华医院检验科临床微生物室， 上海 200092
  • 出版日期:2016-01-28 发布日期:2016-02-26
  • 通讯作者: 刘瑛, 电子信箱: lywjw0129@163.com。
  • 作者简介:刘婧娴（1988—）， 女， 硕士生; 电子信箱: ljxwwwyy@163.com。

Study on resistance mechanism of Klebsiella pneumoniae towards carbapenem

LIU Jing-xian, YU Jing, LI Yuan-rui, LIU Ying   

  1. Department of Clinical Microbiology, Clinical Laboratory, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
  • Online:2016-01-28 Published:2016-02-26

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摘要：

目的 探讨临床标本中分离的耐碳青霉烯类肺炎克雷伯菌(CRKP)的耐药机制。方法 收集2010年1月—2013年12月临床标本中分离的CRKP。采用Vitek-2 Compact全自动微生物分析系统进行鉴定并联合纸片扩散法（K-B法）进行药物敏感性试验，MicroflexTM MALDI-TOF MS进行菌种复核。改良Hodge试验筛查碳青霉烯酶表型；PCR方法检测blaKPC、blaGES、blaIMP、blaVIM、blaNDM-1和blaOXA-48等碳青霉烯酶基因及AmpC酶基因，并对阳性扩增产物进行基因测序；SDS-PAGE检测菌株外膜蛋白Ompk35和Ompk36的表达情况；对携带碳青霉烯酶基因的菌株进行质粒转移接合试验。 结果 75株CRKP中，有71株改良Hodge试验阳性，其中69株携带blaKPC-2基因合并Ompk36变异（68株）或Ompk36缺失（1株）；1株携带blaIMP-4 基因合并Ompk35和Ompk36变异，1株携带blaNDM-1和blaCIT-1基因合并Ompk35和Ompk36变异。4株改良Hodge试验阴性的菌株中，3株DHA-1基因阳性合并Ompk36缺失（1株）或Ompk36变异（2株），另1株未检测到耐药基因但Ompk36缺失。仅4株产碳青霉烯酶菌株（4/71）转移接合试验成功。结论 临床标本分离的CRKP对碳青霉烯类抗生素的主要耐药机制为产KPC-2酶合并外膜蛋白表达异常。需加强院内感染预防控制措施，防止此类菌株的播散。

关键词: 肺炎克雷伯菌, 碳青霉烯类抗生素, 耐药机制, 碳青霉烯酶, AmpC酶, 外膜蛋白

Abstract:

Objective To investigate the resistance mechanism of Klebsiella pneumoniae towards carbapenem. Methods Carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from clinical samples between January, 2010 and December, 2013 were collected and identified by the automated microbial analysis system Vitek-2 Compact. Combined with disk diffusion method (K-B method), drug sensitivity test was conducted. The identification results were verified by MicroflexTM MALDI-TOF MS. Modified Hodge test was performed to screen the phenotype of carbapenemase. Carbapenemase genes such as blaKPC, blaGES, blaIMP, blaVIM, blaNDM-1, and blaOXA-48 and AmpC β-lactamases genes were detected by PCR and DNA sequencing was performed for positive amplified products. SDS-PAGE was adopted to detect the expressions of outer membrane proteins Ompk35 and Ompk36 and the plasmid conjugation experiment was conducted for strains carrying cabapenemase genes. Results For 75 CRKP strains, 71 of them were positive towards modified Hodge test. Among 71 strains, 69 of them carried blaKPC-2 gene with abnormal expression (68 strains) or no expression (1 strain) of Ompk36, 1 strain carried blaIMP-4 gene with abnormal expressions of Ompk35 and Ompk36, and 1 strain carried blaNDM-1 and blaCIT-1 genes with abnormal expressions of Ompk35 and Ompk36. For 4 strains that were negative towards modified Hodge test, 3 of them carried DHA-1 gene with abnormal expression (2 strains) or no expression (1 strain) of Ompk36, and 1 strain did not carry any drug resistant genes with no expression of Ompk36. The plasmid conjugation experiment of only 4 carbapenemase-producing strains was successfully. Conclusion The major resistance mechanism of Klebsiella pneumoniae isolated from clinical samples towards carbapenem is the production of KPC-2 carbapenemase and abnormal expressions of outer membrane proteins. Measures for preventing and controlling the nosocomial infection should be enhanced to avoid the dissemination of these strains.

Key words: Klebsiella pneumonia; , carbapenem, antibiotic resistance mechanism, carbapenemase, AmpC β-lactamases, outer membrane porin