| Klebsiella pneumoniae(K.pneumoniae)normally exists in the human respiratory tract and intestine,which can induce infection when the body is in destroyed resistance or dysbacteriosis causing by long-term use of lots of antibiotics.K.pneumoniae,belonging to the enterobacteriaceae Klebsiella spp.,is opportunistic pathogenic bacteria and iatrogenic pathogenic bacteria,which has pathogenicity to human,threatening human health seriously.K.pneumoniae can cause many infectious diseases,such as pneumonia,urinary tract infections,liver abscess,endophthalmitis,and meningitis.Dendritic cells(DCs),acting as a bridge between innate and adaptive immunity,exert immune surveillance when pathogens invade the body.After entering the body from the respiratory tract,K.pneumoniae adhere to and colonize respiratory epithelial cells,DCs recruit under the respiratory mucosa and ingest bacteria on the surface of the respiratory tract,and then antigen-stimulated DCs are activated and matured,migrate to lymph nodes,induce T cell proliferation and differentiation,and initiate adaptive immunity.Biofilm is the survival state of many bacteria in nature,and the formation of biofilm greatly enhances the resistance of bacteria to external pressure.So what is the effect of biofilm formation on K.pneumoniae escaping DCs immune surveillance?There are no specific studies yet.This study was divided into three main parts.First,the biological characteristics of K.pneumoniae clinical strains were tested.Then,one of the clinical strain was selected to study the effect of biofilm on K.pneumoniae infecting mice.Finally,the effect of biofilm on the maturation of DCs induced by K.pneumoniae was studied in vitro and in vitro.This study aims to provide theoretical basis for the prevention and control of K.pneumoniae related diseases by detecting the biological characteristics of K.pneumoniae clinical strains and studying the influence of biofilm on the pathogenicity in mice,and revealing its mechanism from the perspective of immune escape.1.Detection of biological characteristics of K.pneumoniae clinical strains32 K.pneumoniae clinical strains were obtained from two tertiary A hospitals in China,and the biological characteristics of these strains were tested,including viscosity(string test and mucoviscosity assay),virulence gene,drug resistance,biofilm formation,adhesion and invasion to epithelial cells,and LD50.The results showed that 5 of the 32 K.pneumoniae clinical strains had positive string test,accounting for 15.6%,and their mucoviscosity was high,which belong to K.pneumoniae with hyper-mucoviscosity.The mucoviscosity of K.pneumoniae clinical strains has great difference.Nine virulence genes(ytbS,mrkD,entB,rmpA,K2,kfu,allS,iutA,magA)were detected from 32 K.pneumoniae clinical strains by PCR,among them,mrkD and entB had the highest detection rate,both of which were 81.25%,other was below 60%.Antimicrobial susceptibility testing conducted by MIC assay plate find that the K.pneumoniae clinical strains have strong drug resistance and multidrug resistance,and the resistance rate to ampicillin is highest,as high as 96.9%.53.1%of the isolates were resistant to three or more types of drugs.The formation of biofilm was detected by crystal violet staining,and the results showed that 84.4%of the K pneumoniae clinical strains could form biofilm,among which,strain numbered 20 had the strongest ability to form biofilms.In addition,the adhesion and invasion of the K.pneumoniae clinical strains to epithelial cells was tested in this study,as well as the survival rate and LD50 of the K.pneumoniae clinical strains to Galleria mellonella larvae.The results showed that the adhesion and invasion to epithelial cells and the virulence to Galleria mellonella larvae of the K.pneumoniae clinical strains have great difference.2.Effect of K.pneumoniae biofilm on pathogenicity in miceIn this study,numbered 20 K.pneumoniae clinical strain was selected to explore the effect of biofilm on K.pneumoniae infecting mice.Mice infected with K.pneumoniae by endotracheal intubation injection.The results showed that compared with the planktonic bacteria group,the body weight,survival rate of mice and LD50 in the biofilm bacteria group decreased significantly;and the enlargement,hyperemic and thickening of alveolar wall of the lungs in the biofilm bacteria group were more obvious;the lung wet weight/mice body weight and the bacteria-load in the lungs were significantly increased.In addition,the adhesion and invasion of K.pneumoniae to epithelial cells was detected.The results showed that compared with the planktonic bacteria group,the adhesion rate and invasion rate of Calu-3 cells in the biofilm bacteria group were significantly increased.It was shown that the formation of biofilm enhanced the infectious capacity of K.pneumoniae to mice.Moreover,transcriptome analysis was also performed on two states of K.pneumoniae,and the results showed that compared with planktonic bacteria,the biofilm bacteria expressed 151 differential genes,of which 116 were upregulated and 35 were downregulated.The up-regulated genes were mainly concentrated in the ribosome synthesis pathway and RNA polymerase pathway,while the down-regulated genes were mainly concentrated in the riboflavin metabolism pathway,C5-branched-chain dibasic acid metabolism pathway and sulfur relay system pathway.3.Effect of K.pneumoniae biofilm on DCs innate immune responseIn order to explore the effect of biofilm on the maturation of DCs induced by K.pneumoniae,the two states of K.pneumoniae were incubated with mouse bone marrow derived DCs for 24 h,cells and supernatant were collected to detect DCs phenotypic markers and the capacity of stimulating CD4+T cell proliferation.The results showed that compared with the planktonic bacteria group,the expression of DCs surface markers(CD80,CD86,CD40 and MHC II)in the biofilm bacteria group decreased significantly,and the expression of early activation marker CD69 and migration marker CCR7 decreased significantly.In the mixed lymphocyte reaction,the ability of DCs in the biofilm bacteria group to stimulate CD4+T cell proliferation was significantly downregulated.And the DCs in the biofilm bacteria group had a stronger phagocytic ability to FITC-Dextran.In addition,the detection of DCs in the lung showed that the expression of MHC Ⅱ in the biofilm bacteria group decreased significantly compared with the planktonic bacteria group.The above results showed that the formation of biofilm weakened the ability of K.pneumoniae to induce the maturation of DCs.Moreover,transcriptome analysis was performed on the DCs stimulated by two states of K.pneumoniae,and the results showed that compared with the planktonic bacteria group,the biofilm bacteria group expressed 44 differential genes,of which 23 were upregulated and 21 were downregulated.The up-regulated genes were mainly concentrated in the starch sucrose metabolism pathway and cytokine receptor interaction pathway,and the down-regulated genes were mainly concentrated in the amino sugar and nucleoside sugar metabolism pathway,Fc ε RI signaling pathway and synaptic vesicle cycle pathway. |
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