| Objective:To investigate the antibiotic resistance and virulence genes and homology of carbapenem-resistant Klebsiella pneumoniae bloodstream infection strains isolated from a tertiary hospital so as to provide evidence for clinical treatment and prevention and control of patients with nosocomial infection.Methods:Thirty-one carbapenem-resistant Klebsiella pneumoniae strains were continuously collected from inpatients with bloodstream infection in a tertiary hospital from May 2018 to August 2018.The initial identification and antimicrobial susceptibility testing of these strains were carried out by the VITEK-2 Compact（Bio Mèrieux,Missouri,France）automatic system,and the final identification of the strains was conducted by the method of Average Nucleotide Identity（ANI）.Carbapenemase genes were confirmed by PCR amplification and sequencing.The next-generation sequencing technology platform was subjected to obtain the whole genome sequencing data of all strains.Then,the antimicrobial resistance genes,virulence genes,capsular serotype genes and multi-locus sequence typing（MLST）of the strains were analyzed by using BLAST,Res Finder,Plasmid Finder and VFDB softwares.The homology of the strains was analyzed by MLST and pulsed-field gel electrophoresis（PFGE）as well as Bio Numberic（7.6）software.The number and size of plasmids carried by these strains were detected by S1 nuclease electrophoresis（S1-PFGE）.Liquid mating conjugation experiments were subjected to detect the transfer properties of plasmids.Results:After sequence analysis by ANI value comparison of of all strains,3 strains of Klebsiella quasipneumoniae and 1 strain of Klebsiella michiganensis were initially misidentified as Klebsiella pneumoniae by VITEK-2 Compact system,that is,there were only 27 strains of Klebsiella pneumoniae among the 31 strains.All 31 strains were resistant to penicillins,third-generation cephalosporins and carbapenems,and these strains showed high resistance rates to aminoglycosides,quinolones and other antibiotics also.The results of PCR amplification and sequencing showed as follows:（1）Out of 27Klebsiella pneumonias,26 strains carried bla KPC-2genes and the other one harbored bla NDM-1gene;（2）3 Klebsiella quasipneumoniae strains carried bla NDM-5,bla IMP-4and bla KPC-2genes,respectively;（3）1 Klebsiella michiganensis strain habored bla NDM-1gene;（4）In addition,these strains also carried other antibiotic resistance genes（beta-lactams,aminoglycosides,quinolones,and fosfomycin）.Except for two strains（FK7 and FK31）that can not be typed,the remaining 29 strains were divided into 8 capsular serotypes,and K64 and K47 capsular serotypes were dominant,accounting for 32.26%（10/31）and25.81%（8/31）,respectively.All of 31 strains carried aerobicin iut A genes and enterobactin genes,while only some strains carried salmonin and yersinomycin genes.ST11 and ST15Klebsiella pneumoniae strains were dominant,accounting for 58.06%（18/31）and 9.68%（3/31）,respectively.And the PFGE band profiles of the 31 strains were catgorized into 9groups from A to I.The group A was dominant,accounting for 58.06%（18/31）,which were mainly bla KPC-2-haboring Klebsiella pneumoniae ST11 strains.The S1-PFGE profiles revealed that each strain carried 1 to 5 plasmids with a size ranging from 28.8 kb to 310 kb.In addition,the analysis of high-throughput sequencing data of 31 strains demonstrated that bla KPC-2genes were mainly located on the Inc FII plasmids,while all bla NDMgenes were located on the Inc X3 plasmids.The liquid mating assays verified that the bla KPC-2gene-bearing plasmids in the Klebsiella pneumoniae FK8 and FK18 strains were successfully transferred to Escherichia coli J53Azi R.Conclusions:（1）The molecular mechanism of carbapenem resistance of Klebsiella bloodstream infection isolates in this study is related to the bla KPC-2,bla NDM-1,bla NDM-5and bla IMP-4genes.Moreover,the multidrug-resistant phenotypes of these strains are also consistent with the multidrug resistance genes they carry.（2）Different species of Klebsiella or the same klebsiella carrying different carbapenemase resistance genes were different in carrying some virulence gene.The reasons for this need to be further studied（3）To the best our knowledge,co-harboring bla NDM-1,bla TEM-1Band bla SHV-12genes in a Klebsiella michiganensis bloodstream infection isolate was firstly reported in the worldwide.In addition,the current VITEK-2 Compact automatic system can not distinguish Klebsiella pneumoniae from Klebsiella quasipneumoniae or Klebsiella michiganensis,which should be noted. |
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