Outer Membrane Vesicles From Hypervirulent Klebsiella Pneumoniae Mediate Virulence Factor Transfer To Antimicrobial-resistant Klebsiella Pneumoniae And Host Cells

Background and objectiveHypervirulent Klebsiella pneumoniae(hvKp)is a notorious clinical pathogen that is more likely to cause severe primary and metastatic abscesses.The dissemina-tion of antimicrobial-resistant hvKp isolates have been reported worldwide,posing a great challenge to clinical anti-infection treatment.However,the mechanisms of antimicrobial-resistant hvKp isolates prevalent in the world are not well precise.Outer membrane vesicles(OMVs)secreted from gram-negative bacteria are an important vehicle for delivery of effector molecules inter-and intra-species,and a unique way of horizontal gene transfer.The aim of this study is to explore OMVs as the vector of virulence genes horizontal transfer in bacteria-bacteria and bacteria-host interactions,elucidating a new mechanism for the emergence of antimicrobialresistant hvKp isolates,and laying a foundation for the application of OMVs in the diagnosis and treatment of hypervirulent Klebsiella pneumoniae.Contents and methods1.Isolation,identification and characterization of hvKp-OMVshvKp-OMVs were isolated and then characterized by transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA).2.Extraction and identification of key virulence factors of hvKp-OMVs.The genomic DNA,plasmid and protein of hvKp-OMVs were extracted to detect virulence genes and functional proteins.3.To explore the role of hvKp-OMVs mediate virulence factors disseminate among bacteria in vitrohvKp-OMVs was incubated with antimicrobial-resistant Klebsiella pneumoniae for 48 hours.After the transformants were identified,the successful transformation were detected by the mucoviscosity assay,uronic acid quantification,string test,virulence genes detection(Polymerase chain reaction,PCR)and drug susceptibility test.4.To determine the virulence level of transformants in mouse infection modelsWe conducted an animal model using wild-type C57BL/6(WT)mice.Mice were infected through tail vein injection,and the virulence level of transformants were determined.5.Analysis of the role of hvKp-OMVs elicite an immune response in vitroTo decipher the effect of hvKp-OMVs on host cells(RAW264.7 cells):(1)the production of reactive oxygen species(ROS)and nitric oxide(NO);(2)the production of interleukin(IL)-6,interleukin(IL)-8 and tumour necrosis factor(TNF)-α;(3)cell apoptosis.6.Analysis of the role of hvKp-OMVs induce an immune response in vivoWe conducted an animal model using wild-type C57BL/6(WT)mice.Different doses of hvKp-OMVs intravenously injected into C57BL/6 mice,and the sections of target organs were stained with hematoxylin-eosin(HE)staining to observe the infiltration of inflammatory cells.Results1.hypervirulent Klebsiella pneumoniae can secrete outer membrane vesicles(hvKp-OMVs),which are characterized by typical structure and particle size through TEM and NTA.2.hvKp-OMVs contain key virulence genes prmpA and iroB consistent with parent bacterium.3.The protein profile of the vesicles shows that hvKp-OMVs are enrich with functional proteins.4.hvKp-OMVs mediate virulence-encoding genes transfer to antimicrobial-resistant Klebsiella pneumoniae,enhancing the virulence level of latter.5.hvKp-OMVs mediate functional proteins transfer to host cells(RAW264.7 macrophages),inducing inflammatory responses and macrophage apoptosis.ConclusionsHypervirulent Klebsiella pneumoniae(hvKp)naturally secrete outer membrane vesicles(OMVs),which contain virulence-encoding genes and functional proteins.hvKp-OMVs carried virulence genes that could be transferred from hvKp horizon-tally to antimicrobial-resistant Klebsiella pneumoniae,allowing increase the virulence level of latter and elucidating a new mechanism for the emergence of antimicrobial-resistant hvKp isolates.hvKp-OMVs delivered functional proteins to RAW264.7 macrophages,inducing inflammatory response and macrophage apoptosis.