产碳青霉烯酶KPC-2的肺炎克雷伯菌和铜绿假单胞菌的分离与耐药性研究

摘要

目的：研究临床分离的1株肺炎克雷伯菌（ KPN）和1株铜绿假单胞菌（ PAE）对碳青霉烯类抗生素的耐药机制。方法利用种属特异基因PCR扩增、MALDI－TOF质谱技术及细菌16S rDNA测序鉴定细菌种属；采用VITEK 2 Compact系统检测细菌对各类抗生素的最低抑菌浓度（ MIC）；应用改良型Carba NP法、质粒抽提、电转化实验、耐药基因PCR扩增及测序，分析细菌编码的β－内酰胺酶基因。结果临床分离的1株KPN和1株PAE及其相应的转化子均产A类酶，PCR扩增测序显示PAE含有blaKPC－2型碳青霉烯酶编码基因，KPN含有blaKPC－2型碳青霉烯酶编码基因和blaSHV型超广谱β－内酰胺酶编码基因。二者的转化子只有blaKPC－2编码基因。结论临床分离的耐碳青霉烯类抗生素的KPN和PAE均携带A类2f组型碳青霉烯酶blaKPC－2编码基因且位于质粒上，而KPN所含的超广谱β－内酰胺酶blaSHV编码基因可能位于不同质粒上，或存在于该菌的染色体上。%Objective To investigate the carbapenem-resistant mechanism of one strain of Klebsiella pneumoniae( KPN) and one strain of Pseudomonas aeruginosa( PAE) .Methods The identity of the isolates was confirmed by using MALDI-TOF mass spectrometry, 16S rDNA and special gene amplification .The minimum inhibitory concentrations (MICs) of the antimicrobial agents were determined by VITEK 2 Compact System .CarbaNP Antimicrobial susceptibility testing , plasmid extraction, electroporation experiment , PCR amplification, and cloning and sequencing were carried out to analyze the en-coding gene of β-lactamases.Results The types of β-lactamases of the KPN were blaKPC-2 and blaSHV, confirmed by se-quencing of the PCR products , and that of the PAE were blaKPC-2 .Only blaKPC-2 was displayed in both transformants .All of the results of CarbaNP were type A .Conclusion Both strains of KPN and PAE resisting to carbapenem produce a plasmid-mediated carbapenemase blaKPC-2 , which belongs to Bush group 2f, class A β-lactamase.The extended-spectrum β-lacta-mases gene encoding blaSHV of the KPN might be located in the chromosome or not in the plasmid carrying with blaKPC-2 .