Role of Biofilm in The Pathogenic Mechanism of Klebsiella pneumoniae
克雷白氏肺炎桿菌 ； Klebsiella pneumoniae
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Klebsiella pneumoniae (KP) is a Gram negative facultative anaerobic bacilli with capsule, and normally in oral cavity, skin and intestinal flora of human. It is one of the community-acquired and nosocomial infections, and mainly causes pneumonia, acute cholangitis, urinary tract infection and liver abscess. The major virulence factors of KP include surface antigen (capsular lipopolysaccharide), adhesion factor (fimbriae) and iron acquisition system etc. In recent years, it has been found that replapses of patients with Klebsiella pneumoniae-induced liver abscess often occurr after an operation for removing of abscess. It is proposed causing by other virulence factors such as biofilm. The biofilm will be built by bacteria those secrete the liquid polysaccharide matrix after bacteria attached on the surface of the aqueous environment or tissues, and then parcel bacteria form a loose and easy to wandering state. Because of rendering dormant state in biofilms, bacteria are more tolerant to antibiotics. Furthermore, biofilm protects bacteria from being detected and attecked by immune system, and passes for the main cause of chronic infection in vivo. In the thesis, we hypothesize that biofilm could increase the pathogenicity of KP too. We compared the biofilm-forming abilities of KP strains obtained from patients with liver abscess with strains from patients with abscess not in liver. We found that there was no correlation between bacterial virulence and biofilm-forming ability. We also found that requirements of nutrients and oxygen of different strains were very diverse. So we could not conclude what environmental factor(s) will be helpful for biofilm biognenesis. We selected the KLA1242 strain with the most ability of biofilm formation from those clinical isolates for screening what gene(s) may be involved in biofilm formation by transposome mutagenesis method, and our results demonstrated that the type 3 pili is important for biofilm formation. In further studies, the planktonic of and biofilm from KP standard strain CG43 were cocultured with murine splenocytes respectively for studying the interacting outcome of biofilm with immune cells by quantitating cell activation and cytokine-secreting levels. For more precisely quantifying the biofilm and its bioactivity, the biofilm polysaccharide extracted by KP was be used and the capsule polysaccharide from planktonic bacteria was as a control. Our results demonstraed that immune cells incoulding T cells, B cells and macrophages could be activated by polysaccharide and especially the percentages of activating stages of B cells and macrophages were high at 30%~50%. The polysaccharide treatment could also induce secretions of IL-1β, IL-2, IL-6, IL-10, TNF-α and IFN-γ and especially the levels of IL-6 and IL-10 were polysaccharide dose-dependent. We suggest that IL-6 may help inflammatory responses in the beginning of infection, and the following IL-10 secretion may sustain antibody production. For understanding whether TLR2 will be the receptor of biofilm polysaccharide, the splenocytes obtained from TLR2 mice were treated with biofilm polysaccharide, and our results indicated that the cell activations and levels of cytokines produced by splenocytes from TLR2 mice were significantly less than biofilm polysaccharide-treated splenocytes from wild-type mice. Our results proved that biofilm polysaccharide activate immune cells via TLR2 receptor signaling. In Taiwan, patients with liver abscess caused by KP usually also suffer with diabetes. For understanding the immune responses of individuals with diabetes to biofilm polysaccharide, splenocytes from STZ-induced diabetic mice were treated with polysaccharide under a high glucose condition. Our results demonstrated that levels of L-6, IL-10, TNF-α,TGF-β and IFN-γ secreated by polysaccharide-treated splenocytes from diabetic mice were significantly lower than from wild-type mice. Although our results could not demonstrate the role of biofilm in KP infection in vivo, but we prove the capsular polysaccharide and biofilm polysaccharide could activate immune cells, especially B cells and macrophages, mainly through TLR2 and partial through other receptor. Furthermore, the large-scale productions of IL-6 and IL-10 by biofilm polysaccharide-stimulated immune cells may contribute to regulation of immune responses.
醫學院 > 生化微生物免疫研究所