基于耐药相关基因耐碳青霉烯类肺炎克雷伯菌亲缘关系分析

Genetic relationship with drug resistance-related genes in carbapenem-resistant Klebsiella pneumoniae

目的了解耐碳青霉烯类肺炎克雷伯菌耐药相关基因与可移动遗传元件存在状况和菌株之间的亲缘关系。方法收集2014年1-12月浙江省磐安县人民医院住院患者标本中分离20株耐碳青霉烯类肺炎克雷伯菌,用gyrA测序后BLASTn比对确认菌种,再采用聚合酶链反应(PCR)法分析40种β-内酰胺酶获得性耐药基因、两种膜孔蛋白基因、12种氨基糖苷类获得性耐药基因、1种喹诺酮类作用靶位基因、4种喹诺酮类获得性耐药基因、12种可移动遗传元件遗传标记,并对检测结果作样本聚类分析。结果 20株耐碳青霉烯类肺炎克雷伯菌对β-内酰胺类、氨基糖苷类、喹诺酮类耐药率很高,除未检出喹诺酮类药物获得性耐药基因外,其他基因均有阳性检出;共检出4种β-内酰胺类获得性耐药基因、两种膜孔蛋白基因、两种氨基糖苷类获得性耐药基因、1种喹诺酮类作用靶位基因、8种可移动遗传元件遗传标记,且阳性率较高;样本聚类分析提示20株菌可分为A与B两个簇群;A簇群5株,分别为1、2、3、4、16号株,为克隆传播;B簇群分B1、B2二亚簇群,B1亚簇群12株,分别为5、6、7、8、9、10、11、13、14、17、18、19号株,为克隆传播;B2亚簇群3株,分别为12、15、20号株,亦为克隆传播。结论肺炎克雷伯菌耐药表型与基因型结果相符,菌株呈3个克隆传播特征,为医院感染。

OBJECTIVE To investigate the genetic relationship between the drug resistance-related genes in carbapenem-resistant Klebsiella pneumoniae and the genetic markers of mobile genetic elements and the strains.METHODS A total of 20 strains of carbapenem-resistant K.pneumoniae were isolated from the specimens that were obtained from the patients who were hospitalized the Pan′an People′s Hospital from Jan 2014 to Dec 2014,then the strains were identified by gyrA sequencing and contrast to BLASTn,the polymerase-chain-reaction（PCR）method was employed to analyze 40 kinds of acquired resistance genes toβ-lactams,2kinds of porin genes,12 kinds of acquired resistance genes to aminoglycosides,1kind of target gene of quinolones,4kinds of acquired resistance genes to quinolones,and 12 kinds of genetic markers of mobile genetic elements,and the sample cluster analysis was performed for the detection results.RESULTS The drug resistance rates of the 20 strains of carbapenem-resistant K.pneumoniae toβ-lactams,aminoglycosides,and quinolones were very high.All of the drug resistance genes were detected positive except for the acquired resistance genes to quinolones.Totally 4kinds of acquired resistance genes toβ-lactams,2kinds of porin genes,2kinds of acquired resistance genes to aminoglycosides,1kind of target gene of quinolones,and 8kinds of genetic markers of mobile genetic elements were detected positive,and the positive rates were high.The sample cluster analysis indicated that the 20 strains could be divided into the cluster A and the cluster B;5strains（Strain No.1,2,3,4,and 16）belonged to the cluster A and were clonal transmission.The cluster B were divided into the subcluster B1 and the subcluster B2,12strains（Strain No.5,6,7,8,9,10,11,13,14,17,18,and 19）belonged to the subcluster B1 and were clonal transmission,and 3strains（Strain No.12,15,and 20）belonged to the subcluster B2 and were also clonal transmission.CONCLUSIONThe drug resistance phenotypes of the K.pneumoniae strains are in line with the genotypes;the strains are characterized by three clonal transmissions and are nosocomial infection.