Study On Drug Resistance And Virulence Of Klebsiella Pneumoniae In Bloodstream Infection

Objective:1.To study the clinical characteristics,risk factors,drug resistance and virulent gene of carbapenem-resistant Klebsiella pneumoniae in bloodstream infection.2.To study the clinical characteristics,drug resistance,virulence and molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae co-harbouring blaKPC-2-carrying plasmid and pLVPK-like virulence plasmid in bloodstream infection.3.To study the relationship between virulence plasmid and the hypervirulent phenotype and its transferability in hypervirulent Klebsiella pneumonia.Method:1.A total of 248 strains non-repeated Klebsiella pneumoniae from patients with bloodstream infection were collected during June 2016 to June 2019 from the First Affiliated Hospital of Nanchang University.Bacterial identification and antimicrobial susceptibility testing was done for all isolates using Vitek 2 automated systems.Patient information were queried from the medical records.PCR and sequencing were used to detect the bacterial resistance genes,capsular serotype genes and virulence genes.The strains were classified into carbapenem-resistant Klebsiella pneumoniae?CRKP?and carbapenem-sensitive Klebsiella pneumoniae?CSKP?based on drug sensitivity data.By analyzing the clinical data,drug sensitivity data,drug resistance gene,capsule serotype gene and virulence gene of CRKP group and CSKP group with spss21,the clinical characteristics,risk factors,drug resistance characteristics and virulence characteristics of CRKP in bloodstream infection were obtained.2.From the first part of the strains,through the preliminary screening of KPC-2gene and related genes of pLVKP-like virulence plasmid,and then through S1-PFGE and Southern blotting,24 strains CRKP co-harbouring blaKPC-2-carrying plasmid and pLVPK-like virulence plasmid were selected.The clinical data were inquired in the hospital medical record system.The resistance gene,capsule serotype gene and virulence gene of bacteria were detected by PCR and sequencing.blaKPC-2-carrying plasmid and pLVPK-like virulence plasmid were located by S1-PFGE and Southern blotting.The affinity of strains was analyzed by PFGE,MLST and WZI typing.The virulence of strains was analyzed by Galleria mellonella infection model.3.The retrospective genomic study of the first part of the strains screened out the hypervirulent Klebsiella pneumoniae AP8555,and sequenced it whole genome.We analyzed its chromosome and virulence plasmid genomics,and compared it with the sequenced strain genome and analyzed its affinity.Study the transferability of virulence plasmids through conjugation experiments,and determine their biofilm formation ability by crystal violet method.The results of serum resistance test,the wax moth larvae infection model and the lethality experiment of mice infected in vitro reflected the virulence of the strain.Results:1.Multivariate logistic regression analysis showed that admission to ICU,kidney disease,burn,mechanical ventilation,indwelling urinary catheter,indwelling drainage tube,use of carbapenem antibiotics and use of tegacyclin were independent risk factors of CRKP?P<0.05?.The mortality rate of CRKP group?53.42%?was significantly higher than that of CSKP group?10.86%?,and there was a significant difference?P<0.05?.In addition to the natural resistance to ampicillin,the resistance rate of CRKP group to other 15 antibiotics was higher than that of CSKP group and there was a significant difference?P<0.001?.Among 73 CRKPstrains,69 strains were detected with KPC gene,with a positive rate of 94.5%;4 strains with NDM gene,with a positive rate of 5.5%;one strain carried both KPC and NDM genes;another strain did not detect these five carbapenem genes.3 strains K1-KP,1 strains K2-KP,1 strains K20-KP and 2 strains K54-KP were detected in CRKP BSI.2.24 strains CRKP co-harbouring blaKPC-2-carrying plasmid and pLVPK-like virulence plasmid were screened from 248 bloodstream infected patients in the first part.Most of the patients were hospitalized for a long time,using multiple antibiotics.and the high mortality rate?66.7%?.All strains were multi-drug resistant and carried multiple virulence genes.Pulsed-field gel electrophoresis?PFGE?and multilocus sequence typing?MLST?results indicated four clusters,of which cluster A?n=21,87.5%?belonged to ST11 and the three remaining isolates?ST412,ST65,ST23?had different pulsotypes?cluster B,C,D?.The size of the bla_KPC-2-carrying plasmids ranged from¹00kb to³90 kb.Nineteen strains?79.2%?had a 219kb virulence plasmid possessed high similarity to pLVPK from CG43 with serotype K2.Two strains had a 224kb virulence plasmid resembled to plasmid pK2044 from K.pneumoniae NTUH-K2044?ST23?.Moreover,three strains carried 3 different hybrid resistance-and virulence-encoding plasmids.Infection assays in the Galleria mellonella model demonstrated the virulence level of these isolates was found to be consistently higher than that of classic Klebsiella pneumoniae.Among the 24 strains,15 were similar to NUTH-K2044?P>0.05?,but 9 were lower than NUTH-K2044?P<0.05?.3.The retrospective genomic study of the first part of the strains screened out the hypervirulent Klebsiella pneumoniae AP8555.Ap8555 is a ST23 hvKP strain with a new virulence plasmid,which caused a metastatic infection and fatal septic shock in a critical patient.Further experiments demonstrated that this strain killed 100%of Galleria mellonella inoculated with 1×10⁶ cfu of the specimen within 36 hours.The AP8555 strain exhibited hypervirulence with a mouse lethality LD50 of 2.5×10² cfu.The conjugation experiment showed that the virulence plasmid could be transferred to the recipient with a frequency of transfer of³.2×10^-4 per recipient cell,and further enhancing their virulence potential.The whole genome sequencing revealed that AP8555 genome consists of a single 5.46Mb chromosome and a large virulence plasmid.The plasmid pAP8555-vir harboring the virulence genes was 357.8kb in size and was almost structurally similar to various known pLVPK-like plasmids reported previously,except for an extra 130kb region encoding homologs of the type IV secretion systems?T4SS?components and toxin-antitoxin?TA?systems.Conclusion:1.CR-KP strains mainly produced KPC carbapenemase,but accompanied with the prevalence of NDM carbapenemase in this area.CR-KP strains had high mortality,multiple drug resistance.There were the emergence hypervirulent carbapenem-resistant Klebsiella pneumonia among CR-KP strains.Clinicians should intervene and control the independent risk factors of CRKP BSI.2.CRKP co-harbouring blaKPC-2-carrying plasmid and pLVPK-like virulence plasmid had high mortality,multiple drug resistance and enhanced virulence.Three strains carried 3 different hybrid resistance-and virulence-encoding plasmids,which maybe co-transmit virulence and resistance gene.It would be better to track the virulence and resistance plasmids?and genes?rather than just specific host strain and we should implement control policy and infection prevention to control its spread in the hospital.3.AP8555 strain is a ST23 type hvKP strain carrying a new virulence plasmid,showing a hypervirulent phenotype.Its virulence plasmid contains toxin antitoxin?TA?system and type IV secretion system.The virulence plasmid is metastatic.Strengthening the acquisition of virulence plasmids is essential for understanding the potential spread of virulence plasmids in Klebsiella pneumoniae.