产KPC酶肺炎克雷伯菌耐碳青霉烯类药物的分子机制研究

Molecular mechanism of carbapenem resistance in KPC producing Klebsiella pneumoniae

目的研究临床分离肺炎克雷伯菌对碳青霉烯类药物的分子耐药机制,为临床合理用药提供理论及实验依据。方法收集2020年1月—2020年12月皖南医学院弋矶山医院临床分离非重复肺炎克雷伯菌株923株,利用全自动微生物分析系统进行菌种鉴定及药敏分析,并用16S rRNA PCR扩增产物测序验证菌种,K-B法复核碳青霉烯类药物耐药情况;胶体金法对耐碳青霉烯类肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumonia,CRKP)菌株进行碳青霉烯酶的表型检测,并用碳青霉烯酶PCR扩增结果进行验证;同时利用全自动微生物分析系统对产KPC酶CRKP产超广谱β-内酰胺酶(extended-spectrumβ-lactamases,ESBLs)进行检测,并用ESBLs耐药相关基因PCR扩增结果进行验证;多位点序列分型技术(MLST)对产KPC酶CRKP进行序列分型(ST)。结果923株肺炎克雷伯菌中256株(27.74%)为CRKP,其中183株携带bla_(KPC),ST分型为ST11型和ST12型,产KPC酶CRKP携带3种ESBLs基因bla_(TEM-1)、bla_(SHV-11)和bla_(CTX-M-1)。结论CRKP产碳青霉烯酶以KPC酶型为主,bla_(KPC)可合并3种ESBLs的产生。

Objective To study the molecular resistance mechanism of clinical isolates of Klebsiella pneumoniae to carbapenems,and to provide theoretical and experimental basis for clinical rational drug use.Methods 923 strains of non-repetitive Klebsiella pneumoniae were collected from Yijishan Hospital of Wannan Medical College from January 2020 to December 2020.The bacteria identification and drug sensitivity analysis were carried out by using automatic microbial analysis system,and the PCR amplification products of 16S rRNA were sequenced to verify the strains.The K-B method was used to check the carbapenem resistance,the colloidal gold method was used to detect the phenotype of carbapenemase of carbapenem-resistant Klebsiella pneumoniae(CRKP),and the results were verified by PCR amplification of carbapenemase.At the same time,automatic microbial analysis system was used to detect extended-spectrumβ-lactamases(ESBLs)of KPC producing CRKP.The results were verified by PCR amplification of ESBLs resistance related genes.Finally,the KPC producing CRKP were sequenced by MLST.Results Among 923 strains of Klebsiella pneumoniae,256 strains(27.74%)were CRKP,and 183 of them carried bla_(KPC).The ST types were ST11 and ST12.The KPC producing CRKP carried three ESBLs genes bla_(TEM-1),bla_(SHV-11),and bla_(CTX-M-1).Conclusion The carbapenemase produced by CRKP was mainly KPC,and KPC producing CRKP could also produce three kinds of ESBLs.