Study On Molecular Characteristics,Carbapenem Resistance Mechanism And Evolution-related Fitness Cost Of Hypervirulent Klebsiella Pneumoniae

Objective: To investigate the clinical distribution,antimicrobial resistance rates,and molecular epidemiological characteristics of hypervirulent Klebsiella pneumoniae(hv KP).To analyze the resistance mechanism and virulence characteristics of carbapenem-resistant hypervirulent K.pneumoniae(CR-hv KP).To explore the main evolutionary pathway of CR-hv KP formation and evolution-related fitness cost.Methods:(1)During 2015 to 2017,a total of 2677 K.pneumoniae isolates continuous isolated every September were collected from 34 hospital in Anhui,China.Among them,1494 strains were selected after the duplicate and data unintegrity isolates were removed and re-identified using MALDI-TOF MS.Hv KP were screen out using string test and virulence genes detection.The minimal inhibitory concentration(MIC)of common antimicrobial agents were detected using ager dilution method.The data were analyzed using WHONET 5.6software,and the CR-hv KP isolates were screen out among them.Both multilocus sequence typing and capsular serotyping were determined,and the relationship for each sequence type were analyzed using e BURST software.(2)The homology of CR-hv KP isolates were detected using pulsed field gel electrophoresis(PFGE).The resistance mechanisms were tested using Multiplex-PCR and q RT-PCR.Both carbapenem resistant plasmid and virulence plasmid were detected using S1-PFGE combined with Southern blot hybridization.The virulence phenotypes of CR-hv KP were established using the serum killing and Galleria mellonella lethality assays.(3)The chromosome and plasmid genome characteristics of a ST23K1 hv KP(hv KP01)and a ST11K47 carbapenem resistant K.pneumoniae(CRKP07)were determined by whole genome sequencing and bioinformatics software such as BRIG/Snapgene.Two CR-hv KP variants loading double plasmid system were constructed by using the above strains as parents to cross-introduce their plasmids,and the stability of exogenous plasmids were analyzed.Growth curves,MIC values of major antimicrobial agents,biofilm formation,yield of capsular polysaccharide,and virulence phenotypes of parental and CR-hv KP variants were compared.Fitness cost for each variant were evaluated by pairwise competitive assays.q RT-PCR assay was used to compare the expression levels of major virulence factors and drug resistance genes between parental and variants.Results:(1)Of the 1494 isolates,164(11.0%)were hv KP.During 2015 to 2017,the detection rates of hv KP among K.pneumoniae were 11.3%,10.0% and 11.6%respectively.The patients infected with hv KP were mainly male(61.6%),with an average age of 60.3±17.6 years.The main clinical departments of the patients were intensive care unit(21.3%),respiratory medicine department(13.4%)and neurosurgery department(6.7%).Hv KP isolates was mainly isolated from sputum(66.1%),pus and secretions(10.8%)and blood(6.5%).All hv KP were divided into 35 ST types and 13 capsular serotypes.ST types were mainly ST23(23.3%),ST86(11.0%)and ST65(11.0%),while K1(32.9)and K2(28.0%)were dominant.There was a corresponding relationship between STs and capsular serotypes.The virulence gene detecting rates of hv KP was high,and the resistance rates of hv KP to carbapenem antibiotics were 6.7 ~ 7.3%.Notably,CR-hv KP isolates were detected every year.(2)Of the 164 hv KP isolates,12(7.3%)were CR-hv KP.Among them,ST11K47 were dominant(58.3%),and 3 CR-hv KP isolates belonged to ST11K47 were homologous.Production of KPC-2(75.0%)and NDM-1(16.7%)type carbapenemases were the mainly resistance mechanism of CR-hv KP isolates,andall carbapenem resistance genes were loaded by plasmids with the molecular weight of 28.8 ~ 78.2kb.Ten(83.3%)of CR-hv KP harbored a virulent plasmid with the molecular weight greater than 200 kb.The serum resistance level and ability of the Galleria mellonella larval lethality for CR-hv KP isolates were various.(3)One bla KPC-2-postive plasmid with the molecular weight of 43 334 bp was harbored by CRKP07,while a p LVPK-like plasmid with the molecular weight of225 890 bp was harbored by h KP01.Both CRKP-p LVPK-like and hv KP-pbla KPC-2had been constructed successfully,and the retentive rates of exogenous plasmid were,after continuous passage for 7 days.Compared with parental generation,the colony of CRKP07-p LVPK-like showed hypermucoviscosity phenotype.The ability of biofilm formation,serum resistance level,and the lethality rates of Galleria mellonella larvae were increased,while the MIC to meropenem and the expression level of bla KPC-2 were decreased.On the other hand,stable carbapenem resistance phenotypes were obtained by hv KP01-pbla KPC-2.But the hypermucoviscosity phenotype,serum resistance level,the lethality rates of Galleria mellonella larvae,and the expression level of iuc A and rmp A2 were decreased.The competitive index of two variants were <1,and the competitive growth kinetics curves of them demonstrated a decreasing trend.Conclusions:(1)The detection rate of hv KP among K.pneumoniae was approximately 11.0%.The hv KP isolates mainly isolated from sputum,pus/secretions and blood,and the major molecular characteristic of them were ST23K1,ST86K2,and ST65K2.Although hv KP showed low resistance ratio to common antimicrobial,the CR-hv KP isolates had been emergence,mainly belong to ST11K47 and a clonal transmission trend was existed.(2)The mainly resistance mechanism of CR-hv KP isolates was the production of carbapenemases.Harboring both p LVPK-like virulence plasmid and carbapenemase-positive plasmid was the major molecular characteristic of CR-hv KP isolates.(3)Twoevolutionary pathways could generate CR-hv KP variants,one was p LVPK-like plasmid obtained by CRKP belonged to ST11,the other was pbla KPC-2 plasmid obtained by hv KP belonged to ST23.The fitness cost was exhibits in both variants.Antagonism relationship between p LVPK-like and pbla KPC-2 was exist in CR-hv KP isolate.