| Objective:1.To know the clinical distribution and antibiotics resistance of Klebsiella pneumoniae in our hospital from 2017 to 2018.2.To analyze the risk factors,homology and the expression level of biofilm isolated from invasive infection caused by Hypervirulence Klebsiella pneumoniae3.To investigate the epidemiological characteristics of virulence genes of Klebsiella pneumoniae in our hospital.Methods1.WHONET5.6 software was used to analyze the clinical data and resistance situation of nonrepetitive Klebsiella pneumoniae isolated from the First Affiliated Hospital of Jinan University from 2017 to 2018.2.String Test(ST)was used to identify Klebsiella pneumoniae(KP)as hypermucoviscous Klebsiella pneumoniae(hmKP).PCR was used to amplify the virulence genes of the regulator of mucoid phenotype A and aerobactin in all hmKPs.The two of virulence genes were simultaneously expressed as Hypervirulence Klebsiella pneumonia.All HvKPs were re-identified by using the VITEK2 compact microbial identification system and antimicrobial susceptibility tests3.Multivariate logistic regression was used to analyze the risk factors for the HvKP invasive infection,and to analyze and compare the clinical data and antimicrobial susceptibility results from the isolation of non-HvKP and HvKP from the invasive infection.4.HvKPs isolated from invasive infection were used PCR amplified seven house-keeping genes which were provided on Pasteur website and analyzed by multilocus sequence typing(MLST).And the genetic relationship of HvKP isolates were analyzed by Mega 7.0 software.5.PCR was used to amplify capsular serotypes and some virulence genes in hmKP.6.Crystal violet(CV)and XTT assay were used to quantitative determination of biofilm content and activity of HvKPs from invasive infection.Results:1.Between 2017 and 2018,the number of infected Klebsiella pneumoniae patients was 296 and 256 repectively.And the total number of infected patients was 552.Among them,there were 552 strains of Klebsiella pneumoniae that were not repeatedly isolated.2.Most of the 552 strains of non-repetitive Klebsiella pneumoniae were isolated from the sputum(46.92%,259/552),urine(20.47%,113/552),Blood(13.59%,75/552).Among them,102 strains isolated from sterile body fluid(blood,cerebrospinal fluid,liver puncture,pleural effusion,peritoneal effusion,pathological tissues,bile and catheter),were accounted for(18.7%,102/552).ICU,breath ward,neurosurgery and neurology ward have the isolating rates of 17.39%(96/552),11.41%(63/552),8.51%(47/552)and 7.78%(43/552)respectively.3.552 non-repetitive strains of Klebsiella pneumoniae resistance rate to clinical commonly used antibiotics were as follows: piperacillin 59.8%,cefuroxime sodium 56.8%,cotrimoxazole 56%,gentamicin 55.2%,nitrofurantoin 53.9%,ciprofloxacin 46%,ceftazidime 41.2%,levofloxacin 40.6%,cefepime 26.5%,cefoperazone/sulbactam 28.1%,piperacillin tazobactam 25.7%,imipenem 16.4%,meropenem 16.9%.4.From 2017 to 2018,51 non-repetitive strains were identified as hmKP by string test,accounted for 9.24%(51/552)of the total isolation rate.26 strains of Klebsiella pneumoniae were isolated from invasive klebsiella pneumoniae infection and 25 strains of Klebsiella pneumoniae were isolated from non-invasive klebsiella pneumoniae infection.5.Virulence genes(RmpA,aerobactin)were amplified specifically for 51 strains of hmKP.25 of the 26 strains of hmKP isolated from invasive Klebsiella pneumoniae infection were determined to be HvKP and 18 of the 26 strains isolated from non-invasive Klebsiella pneumoniae infection were determined to be HvKP.The specificity of the ST assay was 84.31%(43/51).6.Multivariate logistic regression analyzed that males,poor in control blood glucose,presence of invasive procedures during hospitalization and occurrence of liver abscess are the independent risk factors of invasive HvKP infection.The OR values were 7.05,1.16,12.97,17.65,respectively.7.HvKP isolated from invasive infections was susceptible to clinical commonly used antibiotics,with sensitive rate of 100.00% to ceftriaxone sodium,amtraxone,gentamicin,ciprofloxacin,levofloxacin,tobramycin,piperacillin tazobactam,imipenem,meropenem,and no ESBL and CRHvKP strains were found in the resistance phenotype.8.MLST revealed the 25 HvKPs isolated from invasive infection were divided into 9 ST types including ST23(40.00%,10/25);ST86(12.00%,3/25);ST65,ST714,ST 1764 and ST828 for every type(8.00%,2/25),ST375,ST660 and ST218 for every type(4.00%,1/25).Another ST type was unknown due to the presence of mutations in allele gene tonB.9.Major capsular serotypes of 43 non-repetitive isolated HvKP strains were K1(41.86%,18/43)and K2(23.26%,10/43),other capsular serotypes were K20(13.95%,6/43),K57(6.98%,3/43),K54(4.65%,2/43),K5(2.32%,1/43)respectively.10.Among 43 strains of non-repetitive isolated HvKP,top four virulence genes of carrying rates were ycfM 100.00%(43/43),entB100.00%(49/51),ureA93.02%(40/43)and wabG 93.02%(40/43).11.The biofilm of 25 strains of HvKP was quantified by CV and XTT method.36.00%(9/25),32.00%(9/25)and 32.00%(8/25)of the isolates could form small,moderate and large amount of biofilm respectively,while 36.00%(9/25),8.00%(2/25)and 56.00%(14/25)of the strains could form moderate active and strongly active biofilm respectively.Conclusion1.Most of Klebsiella pneumoniae isolated from the sputum,urine,blood and mainly from ICU,breath ward,neurosurgery and neurology ward between 2017 to 2018.The isolates of Klebsiella pneumoniae showed seriously to resistance of clinical commonly used antibiotics,which suggests that it is necessary to strengthen the clinical use of antibiotics.2.Male,poor in control blood glucose,presence of invasive procedures during hospitalization and occurrence of liver abscess are the independent risk factors of invasive HvKP infection.The dominant sequence type is ST23,major capsular serotypes are K1 and K2,carrying rates of ycfM,entB,ureA and wabG virulence genes are the highest in the First Affiliated Hospital of Jinan University.3.HvKPs isolated from invasive infection in the First Affiliated Hospital of Jinan University have strongly biofilm activity. |
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