Rearch On Biomarkers For Identify Hypervirulent Klebsiella Pneumonia And The Genome Of Klebsiella Pneumoniae

Objective:The study aims at screening the biomarkers of hypervirulent Klebsiella pneumoniae to provide evidence for clinical identification of hypervirulent K.pneumoniae.The whole genome sequencing is used to further clarify the biological characteristics of K.pneumoniae,including virulence,drug resistance and other aspects.The research may provide insight into the molecular pathogenic mechanism of K.pneumoniae.Methods:151 K.pneumoniae strains were isolated from an affiliated hospital of medical university during October 2016 to September 2018.Identification species were performed by MicroScan WalkAway 96plus automatic bacterial identification/antimicrobial sensitivity analyzer and matrix assisted laser desorption ionization time-of-flight mass spectrometer?MALDI-TOF MS?.The clinical data of the patients with the strains were inquired.Infection model of Galleria mellonella was successfully established to detect the virulence of the strains.Antimicrobial susceptibility was determined by MicroScan WalkAway 96plus automatic bacterial identification/antimicrobial sensitivity analysis system and Imipenem and Meropenem were rechecked with K-B.String test was conducted to high mucous phenotype of K.pneumoniae.Six common kinds of K.pneumoniae capsule serotypes?K1,K2,K5,K20,K54,K57?and 11 virulence genes?prmpA?wcaG?aerobaction?mrkD?entB?ybtS?fimH?iutA?terB?prmpA₂?iucA?were tested by PCR.CAS double-layer agar blue medium was prepared to detect the siderophore of K.pneumoniae.The homogeneity of the strains was analyzed by ERIC-PCR.K.pneumoniae Kpn472,Kpn1031 and Kpn1050 were choosed to perform whole genome sequencing,and then the original data was assembled to obtain the genome sequence.Glimmer software was used for gene prediction.Gene function annotation was carried out in GO and KEGG databases,and the differences of virulence,drug resistance and secretory system among the three strains were analyzed.Results:According to the clinical data and the result of virulence test of the Galleria mellonella infection model,151 K.pneumoniae strains consist with65 hypervirulent K.pneumoniae strains and 86 classic K.pneumoniae strains.In antimicrobial susceptibility testing,hvKP showed lower common antimicrobactial drug resistance than cKP except piperacillin,cefoxitin,tobramycin,piperacillin/tazobactam,imipenem,andmeropenem.The aerobaction,prmpA,iucA and prmpA₂ genes had accuracy greater than 95%for the identification of hypervirulent K.pneumoniae.The results of string test showed that 50 of 65 hvKP strains were positive and 7 of 86 cKP strains were positive,with an accuracy of 85.43%.The combined detection of six capsular serotypes k1/k2/k5/k20/k54/k57 increased the accuracy to 91.39%.The siderophore was detected by CAS double-layer agar blue medium,and its accuracy was 90.72%in the identification of hvKP.The results of ERIC-PCR DNA fingerprinting indicated that hvKP and cKP showed diversity.The results of genome sequencing showed that the size of Kpn472 genome sequence was5640618bp,and the GC content was 57.20%.The size of Kpn1031 was5817945bp,and the GC content was 57.04%.The size of Kpn1050 was5687489bp,and the GC content was 57.19%.The results of Gene prediction found that Kpn472 contained 5180 genes,83 tRNA and 2 5S rRNA;Kpn1031contained 5429 genes,83 tRNA and 5 5S rRNA;and Kpn1050 contained 5840coding genes,80 tRNA,1 23S rRNA,5 5S rRNA.GO and KEGG annotation results showed that the three strains had more genes involved in coding molecular function and metabolism.The hyperviruent K.pneumoniae Kpn472and Kpn1031 strains carried more iron uptake system-associated virulence gene than the classical K.pneumoniae Kpn1050.The carbapenem-resistant strains Kpn1031 and Kpn1050 both carried the bla_KPC-2 resistance gene.The numbers of secretory system related genes in Kpn472,Kpn1031 and Kpn1050 were 48,46 and 47,respectively.These genes were mainly related to type II,type VI and Sec-SRP secretion systems.Conclusion:1.aerobaction,prmpA,iucA,prmpA₂ genes had potential to be biomarkers for the identification of hypervirulent K.pneumoniae.2.CAS double-layer blue medium assay was a powerful method to detect the siderophores,and it could be used by primary hospital without PCR laboratory to identify hvKP.3.Aerobaction played an important role in the virulence of hypervirulent K.pneumoniae.4.The two carbapenem-resistant strains carried the bla_KPC-2 resistance gene.