Molecular Characteristics Of Resistance And Virulence Of Klebsiella Pneumoniae With Hypermucoviscous Producing MCR-8.2 And NDM-1

Objectives:Our study aims at clarifying the molecular mechanism of Klebsiella pneumoniae KP18-29 producing extensively drug-resistant and virulence.An further investigation of the characteristics of resistant,hypermucoviscous,and virulence of ST11 carbapenem-resistant Klebsiella pneumoniae KP18-29 that is resistance to tigecycline and polymyxin,with hypermucoviscous phenotype.Methods:（1）The minimum inhibited concentration（MIC）of 18 antibacterial drugs commonly used in clinical treatment of enterobacteriaceae bacterial infections of KP18-29 were determined by micro broth dilution and agar dilution.（2）Whole-genome sequencing was used to using Illumina Miseq and PacBio RSII platform to analyze the resistance and virulence genes of KP18-29;（3）The hypermucoviscous phenotype was performed by using the string test;Mouse model of sepsis was used to assess the hypervirulent phenotype of KP18-29.（4）The transferability of mcr-8.2 gene and its function in mediating polymyxin resistance were determined ’by conjugation assay.（4）The structural information of the MCR-8.2 protein were investigated by the expression and purification.Results:（1）Klebsiella pneumoniae KP18-29 is a extensively drug-resistant CRKP that borderline susceptible to fosfomycin and is resistant to all other test drugs including polymyxin and tigecycline;The positive result of the string test shows that KP18-29 has hypermucoviscous phenotype.（2）Whole-genome sequencing revealed that KP18-29 MLST was determined ST11 and Serotype was confirmed K60;The genome sequence of strain KP18-29 was completed and contains a 5,360,130 bp circular chromosomal and four plasmids of 278,636 bp,91,072 bp,64,394 bp,44,148 bp;The chromosomal and plasmid carries 30 resistance genes including mcr-8.2,tet（A）gene variant and blaNDM-1.（3）The plasmid p18-29mcr-8.2 carrying the mcr-8.2 gene is conjugative plasmid that may have developed from a pKp_Goe₄14-3-like plasmid that contains the conjugative transfer loci tra-trb with a pMCR8₀95845-like plasmid of mcr-8.2-carrying segment by homologous recombination.（4）KP 18-29 did not harbor the pLVPK-like virulence plasmid or ICEKp elements,but 81 putative virulence genes were detected on KP18-29 chromosome,including Klebsiella pneumoniae strains harboured several conserved virulence genes,namely entB,fimABCDEFGHIK,iroE and mrkABCDFHIJ;The results of Mouse model of sepsis revealed that hypermucoviscous Klebsiella pneumoniae KP18-29,which the lack of the pLVPK-like virulence plasmid,was less virulent than the pLVPK-like virulence plasmid-carrying strain,but was more virulent than the non-hypermucoviscous Klebsiella pneumoniae strain.（5）The target protein fragment of MCR-8.2 was detected in the precipitation of the Star-expressing strain,but the target protein was not found in the Eluation.Conclusion:The observation that an ST11 type CRKP in China prevalent clone of the conjugative mcr-8.2-carrying plasmid was for the first time found.In addition,a novel conjugative plasmid with the mcr-8.2 gene was identified.And a ST11 CRKP strain that is resistant to both polymyxin and tigecycline and has a extensively drug-resistant and hypermucoviscous phenotype were analyzed by research.