碳青霉烯类耐药肺炎克雷伯菌的耐药性及毒力特征的分子生物学研究

Molecular biological study on antimicrobial resistance and virulence characteristics of carbapenem-resistant Klebsiella pneumoniae

目的了解宁夏医科大学总医院碳青霉烯类耐药肺炎克雷伯菌(CRKP)的分子流行病学及毒力特征。方法收集2015年1月—2019年12月分离的CRKP 103株,采用改良碳青霉烯酶灭活试验(mCIM)联合EDTA改良碳青霉烯酶灭活试验(eCIM)检测产碳青霉烯酶表型,PCR分子生物学技术检测碳青霉烯酶等耐药基因以及荚膜血清型、毒力基因,多位点序列分型(MLST)和脉冲场凝胶电泳(PFGE)对碳青霉烯类耐药高毒力肺炎克雷伯菌(CR-hvKP)进行同源性分析。结果103株CRKP对临床常见抗菌药物表现为不同程度的耐药,mCIM联合eCIM试验结果显示94株CRKP产碳青霉烯酶,其中67.0%产金属酶,33.0%产丝氨酸碳青霉烯酶。耐药基因检出率依次为blaNDM 50.5%(52/103)、bla_(KPC)33.0%(34/103)、bla_(OXA-48)22.3%(23/103)、bla_(IMP)10.7%(11/103)。18种ST型中以ST11型35.3%(36/102)及ST76型22.5%(23/102)为主。CR-hvKP 10株,其中仅4株为荚膜血清K1型、10株均为菌毛黏附基因fim-H和铁载体相关基因ent-B携带者。10株CRhvKP菌株PFGE同源性分析显示以D族群居多,亲缘性接近;未发现同一病区同一时间段的暴发流行。结论该院CRKP菌株以产NDM-5型碳青霉烯酶为主,ST11型为优势流行株。已发现9.7%的CRKP为CR-hvKP菌株,应当引起临床和院感相关部门的重视,防止暴发流行。

Objective To understand the molecular epidemiology and virulence characteristics of carbapenem-resistant Klebsiella pneumoniae(CRKP)in the General Hospital of Ningxia Medical University.Methods A total of 103 CRKP strains isolated from January 2015 to December 2019 were collected.Modified carbapenem inactivation method(mCIM)combined with EDTAmodified carbapenem inactivation method(eCIM)were used to detect the carbapenemase-producing phenotype.PCR technique was used to detect carbapenemase-resistant genes,capsule serotype,and virulence genes.The homology of carbapenemresistant hypervirulent K.pneumoniae(CR-hvKP)was analyzed by multi-locus sequence typing(MLST)and pulsedfield gel electrophoresis(PFGE).Results The 103 CRKP strains showed variable levels of resistance to commonly used antibiotics.Phenotyping based on mCIM and eCIM tests showed that 94 CRKP strains were carbapenemase producers,of which 67.0%produced metallo-beta-lactamase and 33.0%produced serine carbapenase.The prevalence of resistant genes was 50.5%(52/103)for blaNDM,33.0%(34/103)for blaKPC,22.3%(23/103)for blaOXA-48,and 10.7%(11/103)for blaIMP.MLST identified 18 ST types among the CRKP strains,mainly including ST11(36/102,35.3%)and ST76(23/102,22.5%)types.Ten CRKP strains were identified as CRhvKP,all of which harbored fimbrial adhesion related gene fimH and siderophore enterobactin gene(entB).Only 4 of the 10 CR-hvKP strains were capsular serotype K1.PFGE-based homology analysis showed that majority of the 10 CR-hvKP strains were clsoely related and belonged to cluster D.No outbreak was found in the same ward in the same time period.Conclusions The CRKP strains in this hospital mainly produce NDM-5 carbapenemase,and ST11 is the dominant prevalent strain.About 9.7%of the CRKP strains were hypervirulent strains,which should be addressed carefully in clinical settings to prevent a potential outbreak.