Sequencing and comparative genomics analysis of Klebsiella pneumoniae plasmid p1512-KPC

Abstract:

[Objective] Comparative genomics analysis of the fully sequenced plasmid p1512-KPC from the multi-drug resistant clinical Klebsiella pneumoniae 1512 isolate.[Methods] We used 16S rRNA gene sequencing to identify the bacterium.Seven housekeeping genes (gapA,infB,mdh,pgi, phoE,rpoB,and tonB) were used for the multilocus sequence typing (MLST) scheme.Resistance genes were screened by PCR amplification.The plasmid was transferred into recipient Escherichia coli EC600 using conjugal transfers and electroporation experiments.The activity and type of carbapenemase were detected using Carba NP,and the minimum inhibitory concentration (MIC) was determined using VITEK 2 compact system.Sequencing and bioinformatics analysis were used to characterize the plasmid p1512-KPC.[Results] The 1512 isolate was an ST11 multi-drug resistant K.pneumoniae strain showing Ambler class A carbapenemase.PCR demonstrated strain 1512 harbored bla_KPC-2,dfrA1 and sul1 genes.The bla_KPC-2 gene was located in non-self-transmissible plasmid p1512-KPC.Sequencing and bioinformatics analysis revealed that p1512-KPC,117.69 kb in length,harbored IncFⅡ replicon and replicon repB belonging to Rep_3 family unknown incompatibility groups.In addition,p1512-KPC contained bla_KPC-2,bla_CTX-M-65, bla_TEM-1 and rmtB genes,among which bla_KPC-2,bla_CTX-M-65 and bla_TEM-1 were carried by ΔTn6296,Tn6367 and ΔTn2 respectively.[Conclusion] The resistance of K.pneumoniae 1512 to penicillin,cephalosporin,carbapenem,monobactam and aminoglycoside were mediated by non-self-transmissible plasmid p1512-KPC.Moreover,the comparative genomics analysis provided a deeper insight into the diversification and evolution of those plasmids that harbored IncFⅡ replicon and replicon repB belonging to Rep_3 family unknown incompatibility groups.