Sequencing And Genomics Analysis Of Multidrug Resistant Plasmids PBTR-CTXM And PTEM-2262

The multidrug-resistance（MDR）plasmids are widely encountered in Enterobacteriaceae species.They mediate the horizontal transfer of many resistance genes and lead to the upsurge of MDR strains.This study mainly explores the resistant phenotype of clinical MDR isolates,the transferability of MDR plasmids and their structural and comparative genomics analysis,and the plasmid-mediated horizontal transfer mechanism of resistance genes.The main contents are divided into two parts,one is structural genomics of IncFII-FIA-FIB multidrug resistant plasmid pBTR-CTXM,and the other is comparative genomics analysis of pTEM-2262,an MDR plasmid harboring two unclassified replicons.In the first part,the aims were to investigate the genomic characterization of the MDR plasmid pBTR-CTXM（GenBank accession number MF156697）assigned into IncFII-FIA-FIB incompatibility group and the plasmid-mediated horizontal transfer mechanism of resistance genes of Escherichia coli BTR.The transferability of plasmid pBTR-CTXM was confirmed by conjugation experiments and electroporation experiments.The minimal inhibitory concentration（MIC）values were tested by VITEK 2 Compact system.The complete nucleotide sequence of pBTR-CTXM was determined using next-generation sequencing technology from a mate pair library.Structural genomics of pBTR-CTXM was analyzed subsequently.The result showed bla_CTX-M-15,mph（A）,erm（B）,and tetA（B）genes were located on pBTR-CTXM with 144 939 bp in length.Owing to the lack of some genes of conjugal transfer region,pBTR-CTXM could be conjugatively mobilized to the recipient strain E.coli EC600 by pNDM-BTR,a conjugative plasmid existed in the E.coli BTR.It could transfer into E.coli DH5αthrough electroporation and belonged to the mobilizable plasmid.Plasmid pBTR-CTXM carried three replicons RepFII,RepFIA,and RepFIB and belonged to IncFII-FIA-FIB incompatibility group.In total,three plasmids were included in the study of this part,namely the first completely sequenced IncFII-FIA-FIB plasmid pRSB107（GenBank accession number AJ851089）,pBTR-CTXM（this study）,and pBTR-CTXM（GenBank accession number JX127248）which showed the highest sequence homology（86%+99%）to pBTR-CTXM.Plasmid pBTR-CTXM possessed typical backbones of IncFII-FIA-FIB plasmids and a MDR region which was comprised of a novel composite transposon Tn6492,the Tn2 remnant,the Tn10 remnant,the ISEcp1-bla_CTX-M-15-Δorf477 unit and some insertion sequences（IS）elements.The novel composite transposon Tn6492 was generated from complex transposition and homologous recombination events of IS26,In54,the chrA-orf98 region,IS26-mph（A）-mrx-mphR（A）-IS6100 unit,ΔTn6295,and Tn6415.In conclusion,the resistance for Monobactam,part of Cephalosporins,Penicillins（not including enzyme inhibitor）,Macrolide,and Tetracycline were mediated by plasmid pBTR-CTXM in strain BTR and the electroporant BTR-CTXM-DH5α.The novel composite transposon Tn6492,the Tn10 remnant,and the ISEcp1-bla _CTX-M-15-Δorf477 unit mediated the horizontal transfer of resistance genes and the antibiotic resistance of E.coli BTR.In the backbones of pRSB107,pBTR-CTXM,and pRSB225,there were two‘hotspots’that were located between pemK and ahr,and between orf249 and orf165 integrated a vast majority of accessory modules,particularly mobile elements harboring resistance genes,which promoted the dissemination of MDR genes among different bacterial species.The second part aimed to genetically characterize the MDR plasmid pTEM-2262（GenBank accession number MG387191）that could not be classified into any known incompatibility group from the clinical Citrobacter freundii isolate 2262 and demonstrate that repA and repB was prerequisite in the replication of pTEM-2262.C.freundii strain 2262 was isolated from the urine specimen of an 89-year-old male patient with cerebral infarction in a public hospital in Taian city,China,in 2016 and was assigned to the novel sequence type 171with new alleles of five housekeeping genes（clpX:86,dnaG:69,fadD:94,lysP:80,and mdh:76）confirmed by MLST.The pTEM-2262,a conjugative plasmid,could transfer from C.freundii 2262 into E.coli EC600 through conjugation.Antimicrobial susceptibility test showed that strains 2262 and 2262-TEM-EC600 were both resistant to ampicillin,ampicillin/sulbactam,piperacillin,ciprofloxacin,tetracycline and gentamicin.Additionally,strain 2262 was resistant to tobramycin,cefuroxime,cefuroxime axetil,cefazolin,cefotetan,ceftazidime,cefepime,ceftriaxone,imipenem,meropenem,aztreonam,trimethoprim/sulfamethoxazole and nitrofurantoin.The complete nucleotide sequence of pTEM-2262 was determined by next-generation mate-pair sequencing.The result showed pTEM-2262 was 262 833 bp in length and harbored two unclassified replicons,repA and repB.To determine whether repA and repB were necessary for pTEM-2262 replication,the repA and repB deletion mutants of pTEM-2262 were constructed,respectively,using Scarless Cas9Assisted Recombineering（no-SCAR）system.The results indicated that repA but not repB was essential for pTEM-2262 replication.Bioinformatics analyses demonstrated that the MDR region of pTEM-2262 had a complex mosaic structure that was composed of In37,ΔTn2,Tn6545,ΔTn6415,the truncated fosA island,and the truncated IS26-tetA（D）-tetR（D）-IS26 unit.BLAST analysis revealed four plasmids（pKrDSM16656L,pKPC_CAV1321-244,p705SK3₁,and pCW001）belonged to this incompatibility group in GenBank.However,the detailed structural characterization and genomic comparison of this incompatibility group have not been reported.Comparative genomic analysis of pTEM-2262and the other four previously sequenced plasmids carrying the repA and repB were performed.The five analyzed plasmids possessed conserved backbones,while massive and different accessory modules were primarily integrated at two‘hotspots’that were located between orf597 and orf504,and between orf393 and orf405.This is the first study to thoroughly investigate the detailed structure and genomic comparison of this unknown incompatibility group.These findings should serve to further the understanding of the horizontal transfer of multidrug resistance genes.Future routine surveillance and molecular epidemiological studies of pTEM-2262-like plasmids should be performed to further contribute to the understanding of the evolution and diversification of this unknown incompatibility group plasmid.