耐碳青霉烯肺炎克雷伯菌的耐药机制研究

Mechanisms of carbapenem-resistant Klebsiella pneumoniae

目的探讨2014年该院收集的耐碳青霉烯肺炎克雷伯菌(CRKP)的耐药机制。方法采用纸片扩散法或者Vitek 2Compact全自动药敏分析系统初筛亚胺培南非敏感菌株,琼脂稀释法确证亚胺培南非敏感肺炎克雷伯菌;通过Carba NP确证试验检测碳青霉烯酶表型;通过PCR扩增及测序检测碳青霉烯酶基因、超广谱β-内酰胺酶(ESBLs)基因、质粒携带的AmpC基因在菌株中携带情况;外膜蛋白SDS-PAGE分析菌株外膜蛋白的改变;外排泵抑制剂羰酰氰氯苯腙(CCCP)抑制试验检测不产碳青霉烯酶基因的肺炎克雷伯菌中是否存在针对碳青霉烯类抗菌药物的外排泵。结果琼脂稀释法验证30株为碳青霉烯非敏感菌株,其中19株为Carba NP表型试验阳性,其中18株产KPC-2,1株产NDM-1。11株Carba NP表型试验阴性的不产碳青霉烯酶基因的肺炎克雷伯菌的ESBLs、AmpC酶的携带率为blaCTX-M 72.7%,blaSHV 27.3%,blaTEM 72.7%,blaDHA 27.3%。SDS-PAGE结果显示编号2368、2875、3050的菌株OmpK36缺失,其余菌株未发现外膜蛋白缺失。CCCP存在时,11株不产碳青霉烯酶的肺炎克雷伯菌对亚胺培南、美罗培南和厄他培南的最小抑菌浓度值均明显降低。结论碳青霉烯酶产生,尤其是产KPC-2是该院CRKP的主要耐药机制,外排泵的高表达,外膜蛋白的缺失合并产AmpC酶也发挥了重要作用。

Objective To investigate the molecular mechanism of carbapenem-resistant Klebsiella pneumoniae （CRKP） isolated from our hospital in 2014. Methods Imipenem non-sensitive strains were screened by disk diffusion method or Vitek 2 Compact automatic susceptibility analysis system and confirmed by agar dilution method.Carbapenemases phenotypes was confirmed by Carba NP test.Carbapenem resistance genes,superwide-spectrum β lactamase （ESBLs） genes and plasmid-carrying AmpC gene were amplified by PCR and sequenced.The changing of outer membrane protein of the strain was analysised by SDS-PAGE methods.Efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone （CCCP） inhibition test was used to detected the role of efflux pump in carbapenem-resistance Klebsiella pneumoniae. Results A total of strains of carbapenem-resistant strains were confirmed by agar dilution method,and 19 strains producing KPC-2 carbapenemase strain of which were Carba NP phenotype positive 18 stranis producting KPC-2,1 stain producting NOM-1.The rate of blaCTX-M,blaSHV,blaDHA blaTEM in the strains which producing no carbapenemase were 72.7 %,27.3%,27.3% and 72.7%.The results of SDS-PAGE showed that only the OmpK36 protein of strain No.2368,No.2875,No.3050 were lost.In the presence of CCCP,the minimum inhibitory concentration （MIC） of Imipenem,Meropenem and Ertapenem in the 11 KPC2-producing strans was significantly descend. Conclusion The production of carbapenemase,especially KPC-2 is CRKP to carbapenem antibiotics in Klebsiella pneumoniae,followed by high expression of efflux pump and the production of AmpC.