Characterization Of Resistance Plasmids In KPC-producing Klebsiella Pneumoniae

Klebsiella pneumoniae is one of the major opportunistic pathogen attacking immunocompromised individuals and causes various nosocomial infections.Carbapenemases are specific?-lactamases with ability to hydrolyze carbapenems.Three major molecular classes of carbapenemases,namely A,B and D,are recognized.K.pneumoniae carbapenemase?KPC?is a class A?-lactamase that has disseminated worldwide among Enterobacteriaceae,Pseudomonas,and Acinetobacter species,with K.pneumoniae being the most common species harboring bla_KPC genes.The aim of this research is to characterize the detailed molecular mechanisms of the multidrug-resistance K.pneumoniae isolates from China.Chapter One:This study aimed to genetically characterize two fully-sequenced novel IncFII-type multidrug resistance?MDR?plasmids,p0716-KPC and p12181-KPC,recovered from two different clinical K.pneumoniae isolates.p0716-KPC and p12181-KPC had a very similar genomic content.The backbones of p0716-KPC/p12181-KPC contained two different replicons?belonging to a novel IncFII subtype and the Rep₃ family?,the Inc FIIK and IncFIIY maintenance regions,and conjugal transfer gene sets from IncFIIK-type plasmids and unknown origins.p0716-KPC and p12181-KPC carried similar three accessory resistance regions,namely?Tn6209,a MDR region,and the bla_KPC-2 region.Resistance genes bla_KPC-2,mph?A?,strAB,aacC2,qacE?1,sul1,sul2,and dfrA25,which are associated with transposons,integrons,and insertion sequence-based mobile units,were located in these accessory regions.p0716-KPC carried two additional resistance genes:aphA1a and bla_TEM-1.Together,our analyses showed that p0716-KPC and p12181-KPC belong to a novel IncFII subtype and display a complex chimeric nature,and that the carbapenem resistance gene bla_KPC-2 coexists with a lot of additional resistance genes on these two plasmids.Chapter Two:This study dealt with detailed genomic characterization of two multidrug resistant?MDR?plasmids p675920-1 and p675920-2 from a single clinical K.pneumoniae isolate 675920.p675920-1 was essentially a hybrid of the IncFII plasmid pHN7A8 and the IncR plasmid pKPC-LK30,and functioned as an IncFII plasmid with inactivation of the IncR replication gene.The backbone of p675920-2 was a hybrid of a novel replicon,three maintenance regions?22.0-,2.7-,2.6-kb in length,respectively?as found in pKPYL2,p10164-3 and pK1HV,respectively,and the entire 25.9-kb conjugal transfer region of pKPYL2.p675920-1 and p675920-2 carried a large number of resistance genes,which contributed to resistance to at least seven classes of antibiotics??-lactams,quinolones,aminoglycosides,fosfomycins,sulphonamides,trimethoprims,and tetracyclines?and one kind of heavy mental?mercury?.All of these resistance genes are associated with mobile elements such as insertion sequences,insertion sequence-based transposition units,and transposons,which constituted a total of three novel MDR regions,two in p675920-1 and another in p675920-2.Coexistence of two MDR plasmids p675920-1 and p675920-2 made K.pneumoniae 675920 tend to become extensively drug-resistant.Chapter Three:A total of 51 bla_KPC-carrying K.pneumoniae strains were isolated from 2012 to 2016 from five Chinese hospitals.K.pneumoniae 1068,20049,12139 and64917 are selected to characterize the bla_KPC-carrying plasmids in these strains.High-throughput sequencing showed that the 1068,20049,12139 and 64917 isolates contained the bla_KPC-2-carrying plasmids p1068-KPC,p20049-KPC,p12139-KPC and p64917-KPC,respectively.The six KPC-encoding plasmids p1068-KPC,p20049-KPC,p12139-KPC and p64917-KPC?sequenced in this study?and p675920-1 and pCT-KPC slightly differed from one another due to deletion and acquisition of various backbone and accessory regions.Two major accessory resistance regions,which included the bla_KPC-2 region harboring bla_KPC-2?carbapenem resistance?and bla_SHV-12??-lactam resistance?,and the MDR region carrying rmtB?aminoglycoside resistance?,fosA3?fosfomycin resistance?,bla_TEM-1B??-lactam resistance?and bla_CTX-M-65??-lactam resistance?,were found in each of these six plasmids and exhibited several parallel evolution routes.The pCT-KPC-like plasmids were present in all the 51 K.pneumoniae isolates,all of which belonged to CG258.There was clonal dissemination of K.pneumoniae CG258 strains,harboring bla_KPC-2-and rmtB-carrying IncFII-family pKPC-LK30/pHN7A8 hybrid plasmids,among multiple Chinese hospitals.In concluding,all of the bla_KPC-carrying plasmids in this study belong to IncFII group,and display complex chimera structures.Each of these plasmids harbours a lot of resistance genes.The accumulation of various antibiotic resistance genes on a plasmid have resulted from complex horizontal genetic transfer events under selection pressure of multiple antibiotics.Occurrence of such a MDR plasmid in a bacterium will make it tend to become extensively drug-resistant,thus resulting in limited choice of antibiotics for treatment and increased risks of death.