The Study Of MLST Genotype,Resistant Transmission And Pathogenic Mechanisms Of Carbapenemase-Producing Kleb,Siella Pneumoniae

Objectives1.To investigate the genotype,molecular epidemiology and resistance genes of carbapenem-resistant Klebsiella pneumoniae.2.To analysis the diversity of the genetic environment of the KPC-2-producing Klebsiella pneumoniaes3.To detect the capsular serotypes and virulence genes of the KPC-2-producing Klebsiella pneumoniaes and choose the hypervirulent and hyperresistant Klebsiella pneumoniaes.4.To study the roles of neutrophil apoptosis in hvKP infections.Methods1.sixty carbapenem-resistant Klebsiella pneumoniae clinical isolated from all specimens in the First Affiliated Hospital of Nanchang University from January 2012to June 2012 were collected.The modified hodge test and imipenem-EDTA double disk synergy test were used to screen out the carbapenem-resistant phenotypes.PCR analysis and gene sequencing were used to analyze the the carbapenemase genotypes(VIM?IMP?NDM?KPC?OXA-48?SPM?GIM).The plasmids of transconjugants were submitted to PCR-based replicon typing(PBRT)method.MLST were performed to analyze the molecular epidemiology of isolates.2.Thirty-five KPC-2-producing Klebsiella pneumoniaes clinical isolated from all specimens in the First Affiliated Hospital of Nanchang University from January2012 to December 2012 were collected.The genetic background of the blaKPC-2gene was investigated with a PCR mapping approach,the correlation of which was analyzed with MLST and plasmid replicon typing.3.Fiftyty-six KPC-2-producing Klebsiella pneumoniaes clinical isolated from all specimens in the First Affiliated Hospital of Nanchang University from January 2012to June 2013 were collected,Out of which were assessed for capsular serotypes and virulence genes.The vitro virulence was determined by serum resistance and neutrophil phagocytosis tests.4.The levels of apoptosis,ROS and Inflammatory cytokines(IL-1?,TNF-?)in neutrophils infected by hvKP were measured by flowcytometry.Results:1.Eighteencarbapenemase-producingK.Pneumoniaewerescreened,polymerase chain reactions showed 13 strains carrying KPC-2 gene,5 strains carrying IMP-4 gene,four strains carrying NDM-1 gene,1 strain carrying VIM-2 gene,five strains co-harboring IMP-4 and KPC-2 genes,the main carbapenemase genotype was KPC-2 gene;All the strains carried PMQR genes,ARDs genes and beta lactamase genes;All the strains were successfully transferred by conjugation except the IMP gens.blaKPC-2 was located on transferable?65 kb Inc L/M and?100 kb Inc A/C plasmids,respectively.On the other hand,blaNDM-1 was associated with a?70 kb Inc A/C plasmid.However,non-typable plasmids of?20 kb containing blaVIM-2were detected;The Eighteen carbapenemase-producing K.Pneumoniae had nine different MLST patterns and had various clonal groups,the main of which were ST395 and ST263.2.Thirty-five KPC-producing K.Pneumoniae had fifteen different MLST patterns and had various clonal groups,the main of which were ST11,ST395 and ST263.The KPC genes were successfully transferred by conjugation,which were located in the various-sized plasmids between 20 and 200 kb with the most prevalent types being?65 kb Inc L/M plasmids.The analysis of genetic environment of blaKPC-2 genes showed five distinct platforms(i-v).platform i mainly exist in the CC14 gene group,including ST14 and ST15,which were distributed in the A/C plasmid;platform ii were mainly distributed in ST23,ST101,ST263 and ST700,related to IncFII(K)plasmid;platform iii is widely distributed in our hospital,located in CC11 gene group including ST11,ST258 and ST340 belonging to L/M type plasmid,On the other hand,platform iv was associated with the Inc N plasmid,which belonged to ST37,ST392,ST340.However,platform v was only detected in the IncFIB plasmid,which belonged to ST86.3.Out of 56 KPC-2-producing Klebsiella pneumoniae strains,the main serotype is wzi47-K47,accounting for 66.1%,the two hypervirulent serotype were detected.the one hypervirulent and hyperresistant Klebsiella pneumoniae was found,which carried various virulence genes.Animal experiment through the abdominal infection showed the LD50(95%CI)was 2.13×10~1(7.2×10~0~1.21×10~2),which was lower significanly non-hvKP strains[LD50(95%CI)3.52×10~7(2.5×10~6~4.15×10~7)(P<0.05)].4.Compared with the non-hvKP group,hvKP infection can significantly decrease the level of neutrophil apoptosis and ROS,but increase the levels of the Inflammatory cytokines(IL-1?,TNF-?)(P<0.05).Conclusion:the main genotype of carbapenem-resistant Klebsiella pneumoniae is KPC-2,which were clonally related;the genetic environment of blaKPC-2 genes was different in the different STs strains.the one hypervirulent and hyperresistant Klebsiella pneumoniae was found;the decrease of neutrophil apoptosis may play a role in the hvKP infections.