| Aims To analyze the detection status,antibiotic resistance,mucoviscous phenotypes,virulence genotypes,capsular serotypesand Multilocus Sequence Typing(MLST)of hypervirulent Klebsiella pneumoniae(hvKP)in order to provide evidence for the prevention and control of hvKP infection as well as accurate diagnosis and treatment of hvKP in our hospital.To explore the biomarkers that can distinguish hvKP from classical Klebsiella pneumoniae(c KP)by proteomics.To establish the methodology for the identification of hvKP and c KP by terE gene then evaluate its performance.To provide reference for the methodology selection of hvKP identification in clinical microbiology laboratory.Methods 1.411 non-repetitive strains of Klebsiella pneumoniae isolated from the first affiliated Hospital of Fujian Medical University during March 2019 to August 2019 were collected.Strain identification and antimicrobial susceptibility analysis were performed by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry(MALDI-TOF MS)and Vitek 2 Compact system,respectively.Four common virulence genes(iuc A,iro B,rmp A,rmp A2)and six capsule genes(K1,K2,K5,K20,K54,K57)were amplified by PCR.The mucoviscous phenotype of Klebsiella pneumoniae was detected by sting test.The homology among 60 hvKP strains was analyzed by MLST.The clinical data of 121 patients with hvKP infection were collected for retrospective analysis.2.Six strains of hvKP,three strains of c KP and one strain of Klebsiella pneumoniae quality control strain 700603 were selected for label-free proteomic detection.Bioinformatics analyses such as GO and KEGG enrichment were carried out to find out differential proteins and related functional information.The selected Ter E protein was verified by Quantitative Real-time PCR(Real-time PCR).The Ter E protein was induced and purified by constructing p ET28 a(+)-terE overexpression plasmid.The Ter E protein peak was detected by MALDI-TOF MS.3.According to the proteomics results,the PCR method was used to detect the Ter E protein encoding gene terE.The clinically isolated 411 Klebsiella pneumoniae strains were used to evaluate the diagnostic performance of the sting test.Chi-square test and Cohen’s Kappa were used for statistical analysis.Results 1.Based on the PCR results of four virulence genes(iuc A,iro B,rmp A and rmp A2),all positive was considered as the criteria of hvKP identification.121 strains of hvKP(29.4%)and 290 strains of c KP(70.6%)were screened out from 411 strains of Klebsiella pneumoniae.The detection rates of virulence genes were iuc A 39.2%(161/411),iro B 38.7%(159/411),rmp A 37.7%(155/411),rmp A2 30.9%(127/411),respectively.The positive rates of K1,K2,K5,K20,K54 and K57 were 35.5%(43/121),28.1%(34/121),5.0%(6/121),3.3%(4/121),5.8%(7/121)and 8.3 %(10/121),respectively.In addition,14.0%(17/121)of hvKP capsular serum were undetectable.The positive rate of sting test in 411 strains of Klebsiella pneumoniae was 32.4%(133/411).Among them,the positive rates of hvKP and c KP were 77.7%(94/121)and 13.4%(39/290),respectively.The results of MLST typing showed that 60 hvKP strains could be divided into ST23、ST65、ST268,ST592、ST36、ST1049、ST202、ST218、ST29、ST374、ST1265、ST1764、ST355、ST412、ST86 and ST889.The detection rates were 35.0 %(21/60),16.7 %(10/60),8.3 %(5/60),8.3 %(5/60),5.0 %(3/60),3.3 %(2/60),3.3 %(2/60),3.3 %(2/60),3.3 %(2/60),3.3 %(2/60),1.7 %(1/60),1.7 %(1/60),1.7 %(1/60),1.7 %(1/60),1.7 %(1/60)and 1.7 %(1/60),respectively.Among them,ST23 was mainly K1 serotype.The results of antimicrobial sensitivity showed that hvKP strain was sensitive to most commonly used clinical antibiotics.Its resistance rates of piperacillin 、 ampicillin / sulbactam 、piperacillin / tazobactam、cefazolin、cefuroxime、ceftriaxone、ceftazidime、cefepime、cefuroxime axetil、aztreonam、imipenem、meropenem、tobramycin、gentamicin、amikacin、levofloxacin and ciprofloxacin were lower than c KP(P< 0.001).2.Proteomic analysis showed that compared with c KP,there were 59 differentially expressed proteins in hvKP,of which 21 proteins such as Ter E,Ter Z,Ter F and outer membrane proteins were up-regulated,and 38 proteins were down-regulated,including DNA-damage-inducible protein D,2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase,Ethanolamine utilization protein eut Kand small heat shock protein,etc.GO enrichment analysis results of differentially expressed proteins of hvKP and c KP showed that the differentially expressed proteins were mainly related to cell components,molecular functions and biological processes.While KEGG annotations showed that differential proteins were mainly involved in fructose and mannose metabolism,amino sugarand nucleotide sugar metabolism,synthesis and degradation of ketone bodies.The screened Ter E protein was used as a potential biomarker.Real-time PCR verification results showed that the relative m RNA expression of terE was significantly higher in hvKP group than c KP group(11293.93±11522.90 VS 0.80±0.67,P <0.0001).Through the construction of p ET28 a(+)-terE overexpression plasmid and MALDI-TOF MS detection,it was found that there was a corresponding protein peak at 11 k Da of Ter E protein.3.Clinically isolated 411 strains of Klebsiella pneumoniae were used to evaluate the performance of the sting test and the terE gene detection method,it was found that the sensitivity,specificity,accuracy,positive predictive value and negative predictive value of the sting test identified hvKP were 77.7%,86.6%,83.9%,70.7% and 90.3%,respectively.The consistency check of the sting test and the reference method showed that the Kappa value was 0.624,which is generally consistent.PCR analysis showed that the positive rate of terE gene in 411 strains of Klebsiella pneumoniae was 35.0%(144/411),among which the carrier rate of terE gene in hvKP was 95.9%(116/121),and the carrier rate of terE gene in c KP was 9.7%(28/290).Ter E gene detection had a sensitivity of 95.9%,a specificity of 90.3%,an accuracy of 92.0%,a positive predictive value of 80.6% and a negative predictive value of 98.1% when it was used as an index to judge hypervirulent Klebsiella pneumoniae.The consistency check of the terE gene detection method and the reference method showed that the Kappa value was 0.817.These two methods had strong consistency in the detection of hvKP.Conclusions 1.The detection rate of hvKP strains in our hospital was 29.4%.It was highly sensitive to most clinical antimicrobials.The serotypes of hvKP isolated clinically were mainly K1 and K2 serotypes,and the ST typing was mainly ST23,in which ST23 hvKP was dominated by K1 serotype.This study reveals the epidemic characteristics of hvKP in our hospital,laying a theoretical foundation for its source tracing,prevention and control as well as clinical diagnosis and treatment.2.Ter E protein is expected to become a virulence marker for hvKP identification.The detection of Ter E protein specific mass spectrometry peaks by MALDI-TOF MS provides the possibility for KP detection and hvk P identification at the same time.But the subsequent performance verification of clinical hvKP strains is still needed.3.The clinical isolated hvKP strain was used to evaluate the performance of terE gene detection.It was found that the established methodology had high sensitivity,specificity and accuracy,and could be used as a method for identification of hvKP. |
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