Influence Of Klebsiella Pneumoniae Biofilm On Host Immunological Function

PART1 ESTABLISHMENT THE MODEL OF KLEBSIELLA PNEUMONIAE BIOFILM IN VITROObjective: To build Klebsiella pneumoniae biofilm formed on silicone elastomer (SE) as the vector in vitro.Methods: The in vitro model of Klebsiella pneumoniae bacterial biofilm was form by modified plating method.:SE discs (1cm diameter) were immersed in 200ul of a standardized Klebsiella pneumoniae suspension (1×107 cells/ml) , then placed in fresh Luri-bertain medium in a 24-well tissue culture plate and allowed to form mature biofilms. Biofilm was identified through light microscope and scanning elecrton microscope.Results: After 5 days culture, 20 Klebsiella pneumoniae bioflims had formed on the SE discs. They were an assemblage of microbial cells that was irreversibly associated with a surface and enclosed in a matrix of primarily polysaccharide material. Conclusion: The method could be used to established the BF models in vitro.PART2 INFLUENCE OF KLEBSIELLA PNEUMONIAE BIOFILM ON EXPRESSION OF TOLL LIKE RECEPTOR 2 IN MONOCYTIC LINE THP-1Objective: To study influence of bacterial biofilm on expression of Toll like receptor2 in monocytic line THP-1.Methods: Biofilm of Klebsiella pneumoniae was prepared in vitro. The supernatants of bacterial biofilm and bacterioplankton culture were incubated with THP-1. The mRNA expression level of TLR2 was detected by reverse transcription-polymerase chain reaction and protein expression level was measured by FACS Calibur.Results: The TLR2 expression levels of THP-1 in experiment group decreased significantly in the levels of both mRNA and protein compared to those of control group.(P<0.05) Conclusion: Our results showed that bacterial biofilm reduced the TLR2 expression level. This may be one mechanism for bacterial biofilm to escape from host immune defense.PART3 INFLUENCE OF KLEBSIELLA PNEUMONIAE BIOFILM ON IMMUNOLOGICAL FUNCTION IN MOUSE MACROPHAGEObjective: To study influence of Klebsiella pneumoniae biofilm on expression of Toll like receptors mRNA and Cytokines and to explore the mechanism of anti-bacterial biofilm in mouse peritoneal macrophage.Methods: Forty male Kunming (KM) mice were randomly divided into the two groups (n=20 each). Mice in the experiment group were peritoneal implanted silicone elastomer (SE) which Klebsiella pneumoniae biofilm formed in vitro to establish BF infection model of indwelling medical devices.Mice in the control group were embedded in planktonic bacteria with the same amount of the experiment group.The mRNA expression level of TLR2 was detected by rea-time polymerase chain reaction and protein expression level was measured by FACS Calibur. Plasma Cytokines levels were measured by double antibody enzyme linked-immunosorbent assay.Results: The TLR2 and TLR4 mRNA expression of macrophage in experiment group were 0.23 and 0.24 fold compared to the control group, whereas TLR5,TLR9 were no difference.The protein expression of experiment group decreased significantly compared to the control group(P<0.05). After the stimulation,the levels of IL-1 and IL-2 in experiment group decreased significantly compared to the control group, but the level of IL-4 was opposite. (P<0.01).Conclusion: Our results showed that bacterial biofilm reduced the TLR2 and TLR4. expression levels and induce Th2 immune response compared to bacterioplankton. This may be one mechanism for bacterial biofilm to escape from host immune defense.