| Objective:1.To study the clinical distribution,resistance status,virulence characteristics and molecular epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae(CR-hvKP).2.To investigate the differences in resistance and virulence phenotypes between a carbapenem-resistant hypervirulent Klebsiella pneumoniae(CR-hvKP)produced by conjugation experiment and its parent strains,clinical strain,and the fitness cost of it.3.Study of the genomic and plasmid formation processes in carbapenem-resistant hypervirulent Klebsiella pneumoniae(CR-hvKP)produced by conjugation experiment to reveal its molecular evolutionary mechanism.Method:1.From January 2018 to December 2021,233 carbapenem-resistant Klebsiella pneumoniae strains were isolated from clinical specimens of patients attending medical treatment in a tertiary teaching hospital.After detection of relevant virulence and drug resistance genes by PCR,Klebsiella pneumoniae containing four virulence genes(rmpA2,terW,silS and iutA)were defined as Klebsiella pneumoniae containing virulence plasmid.From these,124 strains were screened from blaKPC-2 gene positivity carbapenem-resistant hypervirulent Klebsiella pneumoniae(CR-hvKP).Their clinical information characteristics,drug sensitivity and carriage of drug resistance virulence genes were analysed.Some ST11 CR-hvKP were stratified by year,then they were tested for serum resistance,biofilm formation and Galleria mellonella larval infection assay to analyse virulence characteristic.Affinity was compared by pulsed-field gel electrophoresis(PFGE).2.The hvKP strain AP8555 and CRKP strain S270 were used as donor-recipients for conjugation experiment.The size and location of the virulence plasmid and the blaKPC-2 containing plasmid in the conjugant were confirmed by southern blot.The conjugant S270v and its parental and clinical strain JX-CR-hvKP-4 were compared in the drug sensitivity test and virulence-related tests(string test,CPS quantification test,biofilm formation and Galleria mellonella larval infection assay).The conjugant was also studied in fitness cost tests(growth curve measurements,pairwise competition assay,plasmid stability experiment).3.Whole genome sequencing of the strains in the second part was performed to elucidate the plasmid recombination process and the mechanism of the conjugant evolution into clinical ST11 CR-hvKP.Results:1.Among the CRKP strains in this study,CR-hvKP strains were 135,of which ST11 type accounted for 91.86%.ST11 CR-hvKP were mainly from neurosurgery,with specimens mainly derived from sputum,and the mean age of patient was 55.2±16.4 years.They showed 100%resistance to cephalosporins,fluoroquinolones and carbapenems,and some antimicrobials.The detection of drug resistance genes was consistent with that.Besides,they carried virulence genes such as rmpA,rmpA2,terW,silS,iutA,iucA,iorN,iroB,peg-344.The virulence related test comfirmed virulence differences between them.PFGE analysis revealed three types,with type A predominating.2.The study showed that a significant plasmid recombination event occurred in the conjugation experiment.Southern blot results suggested that the conjugant S270v contained a virulence plasmid marked by rmpA2 and a drug-resistance plasmid marked by blaKPC-2,but with some changes in plasmid location.Drug sensitivity tests suggested that the conjugant was multi-drug resistance strain.The conjugant and parental CRKP strain were negative in the string test,the parental hvKP and clinical CR-hvKP strains were positive.The results of the CPS quantification test and biofilm formation were consistent with the string test.Galleria mellonella larval infection assay confirmed the conjugant to be hypervirulent strain.the conjugant strain imposed a fitness cost,but its plasmid stability was high.3.Whole-genome sequencing suggested that the virulence plasmid of the conjugant was formed after deleting transfer-related fragments of the virulence plasmid in the parental hvKP strain and involved a loss of the iron uptake system-related gene iroBCDN and the CPS synthesis regulatory gene rmpA,the acquisition of the silver resistance gene silABCEFGPR on the recipient bacteria.Analysis of drug-resistant plasmids in the conjugant showed that drug-resistant plasmids integrated with some fragments on the chromosome of the recipient bacteria to form drug-resistant plasmids of experimental CR-hvKP strain.In addition,it was found that the 5596 bp size plasmid in the parental CRKP was susceptible to loss during conjugation experiment but was also available during the evolution to the clinical CR-hvKP strain.Conclusion:1.ST11 CR-hvKP is prevalent in our hospital.It exhibits multiple resistance to clinical antibiotics,coupled with its high virulence characteristic,which will pose a significant challenge to clinical anti KP treatment..2.The CR-hvKP strain produced by the conjugation experiment exhibited multiple drug resistance.Compared to clinical CR-hvKP,it need some evolutionary.The virulence test comfirmed that it was hypervirulent strain.The conjugant had a fitness cost in the environment.The stability of its plasmid are high,which helps it to spread and evolve.3.The whole-genome sequencing suggested a mechanism for the formation of the experimental CR-hvKP strain:a new virulence plasmid was formed after deleting transfer-related fragments of the original virulence plasmid,drug-resistant plasmids were integrated with some fragments on the chromosome of the recipient bacterium to form resistance plasmids in the conjugant. |
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