The Study On Carbapenems Resistance,Molecular Epidemiology And Molecular Mechanisms Of Carbapenem Resistance Formation And Development In Hypervirulent Klebsiella Pneumoniae

Objective:1.To study the molecular characteristics of carbapenem-resistant Klebsiella pneumoniae and the prevalence of hypervirulent strains.2.To study the resistance mechanism of carbapenemase,PMQR genes and Omp K36 gene in hv KP strains and assess the relationship with the virulence characteristics.3.To investigate the genomics and molecular evolutionary mechanism of carbapenems-resistant hypervirulent Klebsiella pneumoniae.4.To study the molecular mechanism of carbapenems resistance development of hypervirulent Klebsiella pneumoniae and its influence on virulence.Method:1.A total of 1055 Klebsiella pneumoniae isolates of various samples from inpatients were collected during January 2014 to December 2018 from the First Affiliated Hospital of Nanchang University.CR-KP strains were screened by antibiotic susceptibility testing.Primer design according to the reference:polymerase chain reaction?PCR?and sequencing technologies were employed to screen hv KP and CR-hv KP strains.The clinical information and drug resistance genes of the strains were analyzed.The difference of antibiotic susceptibility was compared.We used PCR method to detect the major carbapenemase genes by previously described primers;S1-PFGE and southern blot hybridization was used to determine the location of plasmids.Multilocus sequence typing?MLST?and pulsed-field gel electrophoresis?PFGE?methods were used for molecular typing.Plasmid conjugation methods were performed to confirm the transferability of plasmids.2.We analyze the carbapenemase plasmid of CR-hv KP strains which were screened from the strains collected in the first part.Virulence genes and drug resistance genes were detected by PCR.PFGE,MLST and wzi typing were used for molecular typing of CR-hv KP strains.Southern blot was used to determine the location of resistance and virulence plasmids.Some strains were screened to identify the subtypes of the outer membrane protein Omp K36 gene.The differences of drug resistance and virulence among different subtypes were analyzed.The virulence of K1-hv KP strains was evaluated by phagocytosis assay,srum resistance assay,crystal violet assay,and mouse lethality assay.PMQR genes,gyr A and par C genes were detected by PCR and sequencing.PFGE was used to analyze the molecular typing of K1-hv KP strains and their relationship with PMQR carriers and virulence.3.Three representative CR-hv KP strains were screened from the first and second parts,and two of them were sequenced for genome analysis.We analyzed the virulence plasmid and drug-resistant plasmid and compared with the hv KP strains which have been sequenced.The virulence was assessed by phagocytosis assay,srum resistance assay,crystal violet assay,and mouse lethality assay.The possible molecular evolution mechanism of CR-hv KP strain was explored by genomics analysis.4.We selected a hv KP wild strain BD2411 to form a CR-hv KP strain Tfp NDM-hv KP by carbapenemase plasmid transformation.Genomics and transcriptomics were used to analyze the change before and after carbapenemase plasmid transformation.The virulence change of BD2411 and Tfp NDM-hv KP strains was evaluated by phagocytosis assay,srum resistance assay,crystal violet assay,and mouse lethality assay.Results:1.In the past five years,the detection rate of CR-KP has been reached to 24.93%,while the CR-hv KP was accounted for 15.97% among the CR-KP strains.The resistance rate of CR-hv KP to aminoglycoside antibiotics such as amikacin,tobramycin and gentamicin was significantly lower than that of CR-KP?P <0.05?,but the resistance rate of CR-hv KP to piperacillin/tazobactam was significantly higher than that of CR-KP?P < 0.05?.CR-KP strains in our hospital mainly carry bla KPC-2 gene,accounting for 71.1%,but bla NDM-1,bla NDM-5,bla NDM-7and bla IMP-4 genes were also prevalent.CR-KP strains producing NDM-1carbapenemase were mainly prevalent in burn wards,showing high levels of carbapenem resistance?MIC > 32 ug/ml?.In addition,most of them occurred in patients with deep second-degree burn.Moreover,some strains could transfer the carbapenemase genes by plasmid conjugation,with the conjugation rate as high as 44.44%.Southern blot showed that bla NDM-1 gene was located in a 46 Kb Inc X3 type plasmid.ST11 and ST17 were the main epidemic clones of NDM-1-producing CR-KP strains,and most of them have Omp K36 gene deletion or decreased expression.Thirty-five strains of CR-hv KP were string test positive,and hypervirulent serotypes such as K1,K2,K5 and K57 were also found in capsule serotyping.88.1% of the patients were infected and the mortality rate was as high as 47.6%.2.Thirty-five CR-hv KP strains isolated from the first part were mainly carrying the bla KPC-2 gene,while only 2 strains carried the bla NDM gene.Most strains co-carried with ESBL and PMQR genes.In addition,CR-hv KP strains mostly carried virulence genes,such as rmp A,iro N,rmp A2,aerobactin,wca G.ST11 was the main type in the CR-hv KP strains,accounting for 85.7%.Hypervirulence ST typing such as ST23,ST65 also existed.PFGE showed that there were five types,of which type A accounts for 85.71%.The number of plasmids carried by CR-hv KP was significantly higher than that of CS-hv KP strain.Southern blot showed that 26 CR-hv KP strains co-habored both bla KPC-2-carrying plasmid and p LVPK-like virulence plasmid.Furthermore,we found that three CR-hv KP strains carried a hybrid virulence-and resistance-encoding plasmid,harbouring both the virulence gene rmp A2 and the carbapenemase gene bla KPC-2.In view of the dual function of Omp K36 protein on virulence and drug resistance,80 Klebsiella pneumoniae strains were genotyped by Omp K36,which were divided into four types: A,B,C and D,accounting for 42.5%,7.5%,32.5%,and 15%,respectively.The carrying rates of rmp A,iro B,rmp A2,aerobactin and all S genes in Omp K36 C type were significantly higher than those of non-C type strains.21.25% of the strains showed hypermucoviscosity.The positive rate of hypermucoviscosity in type C strains was significantly higher than that of non-type C strains?50% vs 7.4%,P < 0.001?.48.75% of the strains were liver abscess-related capsular serotypes,including K1,K54,K20.Moreover,the resistance rate of type C strains was significantly lower than that of non-type C strains,especially in the positive rate of bla KPC-2 gene.In clinical factors,community-acquired bloodstream infection was the main cause in patients infected by Omp K36 C type strains?46.2% vs.11.1%,P = 0.0002?,usually with no underlying diseases?46.2% vs.7.4%,P<0.0001?.The serum resistance and neutrophil phagocytosis of Omp K36 C type strains was significantly higher than those of non-C type strains?P=0.0195 and < 0.001?,but there was no significant difference in biofilm formation?P=0.1348?.Furthermore,we also found that51.42% of K1-hv KP strains carried PMQR genes,mainly qnr S1.And 14.3% of them co-carried ESBL genes.PMQR positive K1-hv KP strains were mainly ST23,but also in ST11,ST700 and ST25 clones.3.Three representative CR-hv KP strains?Kp1500,NUHL24835,NUHL30457?were screened.CR-hv KP strain Kp1500 was formed by a ST11 c KP strain carrying a p LVPK-like virulence plasmid,exhibiting serum resistance and anti-neutrophil phagocytosis,with an LD50 of only 1.2×102 cfu/ml and a Tn4401 transposon structure carrying bla KPC-2.CR-hv KP strain NUHL24835 was developed from a hv KP strain carrying NDM-5 carbapenase plasmid.Genomics showed that it carried an Inc X3 type plasmid.Though lack of p LVPK-like virulence plasmid,it carried ICEKp1 element containing several virulence factors.It showed high virulence characteristics such as serum resistance and low LD50.CR-hv KP strain NUHL30457 was formed by a ST65 K2 hv KP strain co-carrying carbapenemase plasmids and p LVPK-like virulence plasmid.Genomic sequencing revealed that it carries three drug-resistant plasmids?p30547-2,p30547-3,and p30547-4?and a virulence plasmid?p30547-1?.The plasmid p30547-4 was Inc N replicon typing,mainly carrying bla NDM-1 and qnr S1 genes,and p30547-3 was Inc FII?K?replicon typing,mainly co-harboring bla CTX-M-65,bla TEM-1,and bla KPC-2 genes.The plasmid p30547-1 was Inc HIB replicon typing,highly homologous to the virulence plasmids in hv KP strain.In addition,NUHL30457 showed hypervirulence such as serum resistance,anti-neutrophil phagocytosis and low LD50.there was a cross between CR-hv KP,c KP and hv KP in homology.4.The NDM-5 carbapenemase plasmid was transformed into a wild hv KP strain to form a CR-hv KP strain Tfp NDM-hv KP.Despite its increased fitness cost and reduced serum resistance,the Tfp NDM-hv KP strain maintained hypervirulence phenotype such as hypermucoviscosity,anti-neutrophil phagocytosis and low LD50.Genomic sequencing showed no significant changes in genome after carbapenemase plasmid transformation.However,RNA-seq had shown 683 genes with differential expression,of which 107 were up-regulated and 576 were down-regulated.The transcriptomic results were validated through the RT-q PCR analysis of a subset of differentially expressed genes.Gene groups with functions relating to carbohydrate metabolism and multidrug efflux system were enriched in genes with increased expression,and those relating to capsule biosynthesis and virulence factors were accumulated in the genes with decreased expression.The down-regulated CPS-related genes were mainly located in the KP1₃714-26 gene cluster,with KP1₃714?wzc/ptk?,KP1₃718?wzb/tyrosine kinase?,KP1₃720?wza/capsule polysaccharide export protein?,and KP1₃726?orf/glycosyltransferases?genes.While the virulence genes with decreased expression were mostly located on the p LVPK-like virulence plasmid,with the yad A?-11.25?and iut A?-10.17?genes decreased significantly.Conclusion:1.CR-KP strains mainly produced KPC carbapenemase,but accompanied with the prevalence of NDM and IMP carbapenemase in this area.The high-level carbapenem-resistance of CR-KP strains in burn ward was mainly due to NDM-1carbapenemase combined with the outer membrane protein Omp K36 loss or decreased expression.The isolation rate of CR-hv KP strians showed an increasing trend,some with hypervirulence capsular serotypes and a high mortality rate.However,the susceptible to aminoglycoside is relatively higher.2.CR-hv KP strains mainly carried blaKPC-2gene,accompanied with multi-drug resistant genes.They also harbored several virulence genes and plasmids.ST11 was the main type which co-carried both virulence plasmids and resistance plasmids.Furthermore,we also found CR-hv KP strains carried a hybid plasmid co-harbouring both the virulence gene and the carbapenemase gene.3.Bloodstream infection Klebsiella pneumoniae can be divided into four types by the Omp K36 gene.Among them,type C strains were significantly higher in virulence genes,hypermucoviscosity,hypervirulence capsular serotypes,serum resistance and anti-neutrophil phagocytosis than other non-C types,while the non-C types strains were more resistant to antibiotic than the C type strains.4.Most K1-hv KP strains harboured PMQR genes,with qnr S1 as the dominant.The qnr S1 gene could be co-transferred with ESBL gene.5.We analyzed the reasons of CR-hv KP formation from the perspective of genomics for the first time.The transfer of virulence plasmid and resistance plasmid may play an important role in the formation of CR-hv KP strain.6.CR-hv KP strain was successfully constructed by plasmid transformation for the first time in vitro.Although the virulence of hv KP strain could be reduced after plasmid transformation,it could still maintain hypervirulence.7.Although the virulence of hv KP can be reduced after plasmid introduction,it can still maintain high virulence.The expression of gene groups with functions relating to carbohydrate metabolism and multidrug efflux system could be increased after plasmid introduction,while the expression of CPS synthesis and virulence genes could be decreased,leading to the formation of drug resistance and the virulence decrease.