Sequencing And Structural Genomics Analysis Of Multi-drug Resistant Plasmids P1512-KPC And P92944-TEM

Enterobacteriaceae was one of the most common pathogen isolates in clinic.In recent years,the significant increasing in the prevalence of resistant strains,even multi-resistant strains,belonging to Enterobacteriaceae,have raised huge concerning around the world.According to the data from the China Antimicrobial Resistance Surveillance System（CARSS）in 2017.In China,among the Enterobacteriaceae clinical isolates,the resistance rate of Escherichia coli isolates to the third-generations cephalosporins and carbapenems were 54.2%and 1.5%,respectively;the resistance rate of Klebsiella pneumoniae to the third-generation cephalosporins and carbapenems were 33.0%and 9.0%,respectively.While,in 2013,the resistance rate of K.pneumoniae to the carbapenems was only 4.9%[1].Plasmid,a closed circular double-stranded DNA molecule,was free from the chromosome and was self-replicating in their hosts.They confer some biological characters,such as drug-resistance and virulence,for their host bacteria,which facilitate the survival of the host bacteria under specific environmental conditions.Moreover,most of the plasmids can be transferred into other bacteria and confer the corresponding phenotype for their recipient.Therefore,plasmids carrying drug-resistance genes play an important role in the generation and spread of bacterial resistance.In this study,in two multi-drug resistant K.pneumoniae and E.coli clinical isolates,the molecular mechanisms of multi-drug resistance mediated by plasmids were explored.Sequencing and comparative genomics analysis of K.pneumoniae plasmid p1512-KPC carrying blaKPC-2The carbapenem-resistant K.pneumoniae 1512 strain was isolated from the blood sample of a patient after liver transplanting operation in a teaching hospital in Guangzhou.Plasmid pl512-KPC from the K.pneumoniae 1512 strain carried the resistance genes coding the extended-spectrum β-lactamases,carbapenems and 16S rRNA methylase（conferring aminoglycoside resistance）.Moreover,plasmid 1512 can be transferred into the recipient bacteria by electroporation and conferred the corresponding phenotype for their recipientor.In addition,plasmid p1512-KPC contained two replicons repA and repB,which belong to the IncFII incompatible group and the Rep₃ family unknown incompatible group,respectively.In the GenBank database,plasmid p1512-KPC was the second reported plasmid containing these two replicons（the first one was the plasmid p283747-KPC isolated from K.pneumoniae 283747）.In this paper,the structural characters and the genetic environment of drug-resistance genes of p1512-KPC were confirmed and the structural genomics analysis of the mobile elements in p1512-KPC was also carried out.Additionally,the comparative genomics analysis of p283747-KPC and p1512-KPC was performed in detailed,which will deepen the understanding to the diversity and evolutionary of the plasmid simultaneously carrying the IncFII replicon and the Rep₃ family replicon repB.Sequencing and structural genomics analysis of multi-drug resistant IncY-FIA plasmid p92944-TEMPlasmid p92944-TEM was isolated from a clinically isolated XDR（extensively-drug-resistant）E.coli 92944.This plasmid contained the new-identifiedβ-lactamase gene,named blaTEM-237,and other ten classes of drug-resistance genes,such as cephalosporins,quinolones,aminoglycosides,and tetracyclines resistance.Plasmid p92944-TEM can also be transferred to the recipient bacteria by electroporation and make recipientor to express the corresponding resistance phenotype.Additionally,plasmid p92944-TEM contains two replicons belonging to the IncY and IncFIA incompatible group and it is the first reported IncY-FIA type plasmid.Notably,plasmid p92944-TEM carried a phage-derived element that is highly homologous to phage P1 and P1-like phage P7.In this study,a deep structural genomics analysis of this multi-drug resistant IncY-FIA plasmid carrying the phage-derived element was performed.Plasmid p92944-TEM is a new example that the phage-derived element bearing plasmid as a vehicle to mediate the spread of various resistance genes.Simultaneously,it is the first identified plasmid concurrently containing replicons from the IncY and IncFIA incompatibility groups,which could provide the reference for the following study of IncY-FIA plasmid.