Role Of Luxs In The Formation And Detachment Of Biofilms Which Be Induced By Glucose On The Surface Of Biomaterials Of Biacteria

Background:Staphylococcus epidermidis which is one of the main pathogenic bacterias of the biomaterial centered infections can be detected with higher rate in iatrogenic infection site such as surgical suture sites, parts of the drainage port, central venous catheter, heart valve replacement surgery, implantable prostheses. The key link of BCI is bacterial biofilm formation. The change of environmental factors such as low nutrition, high permeability, low temperature, ph value, extracellular slime substance can affect the formation of biofilms of staphylococcus. In a previous study we find the bacteria have obvious biological membrane formation on the presence of glucose (11.1mmoL/L). The function of skin barrier has been impaired because of body immune disorders, oxidative stress, atherosclerosis, vaso-motor function in diabetes. In addition, hyperglycemia can lead to decrease of the function of neutrophils and increase of surface adhesion factor so that it will induce microbial gathering to form biofilms easily. Patients with diabetes are vulnerable to infection and antibiotic resistance, so that BCI in diabetes are refractory performance. The detection of staphylococcus epidermidis rate was21%n the tube of subcutaneous insulin infusion in diabetes. To study infection of staphylococcus epidermidis is more important. The purpose of this study is to understand the effect of glucose on the formation of biofilms in bacteria and the change of value of ica,agr, lux gene expression in vivo. By studying BCI animal model with diabetes, we investigated the role of luxS quorum-sensing system in BCI and discussed the specific mechanism of luxS quorum-sensing system in formation and separation of biofilms induced glucose.Part I:Effect of glucose on biofilms formation on the surface of biomaterials in Staphylococcus epidermidisPurpose:To investigate the effect of different concentration of glucose on the biofilms on the surface of biomaterial in Staphylococcus epidermidis.Methods:Staphylococcus epidermidis model of biomaterial infection was cultured in different concentrations of glucose. Biofilms mass was semi-quantified by crystal violet dye and its growth curve was drawn. The thickness of biofilms was measured by laser scanning confocal microscope. The ultrastructure of biofilms was observed by scanning electron microscope.Results:The amount of the adhesion in staphylococcus epidermidis1457strains in different concentration of glucose on PVC surface gradually increases with growth time.The amount of the adhesion is maximum in12to24hours. There are biofilm formation in all groups in Se1457. The amount of the adhesion reaches the peak in24hours and decreases after24hours gradually. The amount of the adhesion of bacteria increase gradually with the increasing concentration of glucose in the culture medium in glucose concentration in11.1mmoL/L and above. Confocal laser microscopy demonstrated that, the biofilm was significantly thicker in=11.1mmoL/L glucose group compared with no glucose group (P <0.05). The maximum value of biofilm formation occurred with30-50mmoL/L glucose, and the peak of it with40mmoL/L glucose. When glucose concentration is=60mmoL/L, the thickness of biofilm decreases, however, it is still higher than that of in the groups with=20mmoL/L glucose concentration. The biofilm formation on the surface of PVC in Se1457can be observed by scanning electron microscopy when the bacteria grow in different glucose concentration after24hours. Biofilms thicken gradually as the glucose concentration increasing. The compact structure and a highly organized structure of cells can de observed by scanning electron microscopy in40mmoL/L glucose. There were no biofilm formed in biofilm negative strains (Staphylococcus epidermidis ATCC12228)Conclusions:The amount of the adhesion in staphylococcus epidermidis1457strains in different concentration of glucose on PVC surface gradually increases with growth time. The thickness of biofilm on PVC surface gradually increases with the concentration of glucose during the time of growth. The thickness of biofilm on PVC surface gradually decreases when the concentration of glucose is higher. However.there were no biofilm formed in biofilm negative strainsPart II:Role of the luxs quorum-sensing system in biofilm formation which is induced by glucose on the surface of biomaterials of Staphylococcus epidermidisPurpose:To study molecular mechanism of biofilm formation which is induced by glucose on the surface of biomaterials of Staphylococcus epidermidis.Methods:To study expression of the key gene during the formation and separation induced by glucose by determination mRNA of icaC, lux, hld gene in staphylococcus epidermidis with40mmol/L glucose concentration. To investigate the role of luxS quorum-sensing system in the formation and separation induced by glucose by determination mRNA of icaC, hld gene in Alux mutations strains with40mmol/L glucose concentrations.Results:The maximum amount of mRNA expression of ica gene induced by glucose is in24hours.The peak of mRNA expression of hld gene induced by glucose will delay. Glucose down regulate the expression of lux gene mRNA in Se1457. Glucose up regulate the expression of icaC gene mRNA in lux-negative mutation strains. The amount of mRNA expression of hld gene induced by glucose increases in lux-negative mutation strains, and the peak of it has been extended from30hours to72hours.Conclusions:Glucose promote the formation of biofilm by inducing the mRNA expression of icaC gene in biofilm positive strains. Glucose promote the separation of bacterial biofilm by inducing the mRNA expression of hld gene during the separation of mature bacterial biofilms.Lux gene have negative regulation on ica gene and inhibit the high level expression of hid gene in the separation of biofilms. When lux gene absent, the negative regulation attenuate.The level of expression in hld gene increase significantly and the peak extend and the thickness become obvious.This kind of phenomenon is obvious in lux-negative mutation strains.Part â…¢:The study of the ability of adhesion and clearing in Alux strains on the surface biofilms of biomaterial in Tupaia belangeri with hyperglycemia.Purpose:To investigate the ability of adhesion and clearing inâ–³lux strains on the surface biofilms of biomaterial in Tupaia belangeri with hyperglycemia.Methods:The model of hyperglycaemia Tupaia belangeri was induced by intraperitoneal injection of STZ. PVC catheters were inserted into their left femoral vein and Staphylococcus epidermidis were inoculated into the catheters. One week after inoculation, the animals were killed and the bacteria recovered from catheters, blood, hearts, kidneys, pancreas and livers were counted. The formation of biofilms was detected by SEM to study the infiltrating of inflammatory cells in the hearts, kidneys, pancreas and livers of animal model by the pathological examination.Results:There were higher rate of infection, bacteria community quantity of tissue and blood in Tupai belangeri infected Alux Staphylococcus epidermidis with11.1mmoL/L or higher than that of with<11.1mmoL/L glucose groups (P<0.05). Detected by SEM, biofilm formation was thicker with11.1mmoL/L or higher than that of with<11.1mmoL/L glucose groups (P<0.05). Pro-inflammatory cytokines increase significantly and the formation of anti-inflammatory cytokines is insufficient in Tupai belangeri infected Alux Staphylococcus epidermidis. The infiltrating of inflammatory cells in the hearts, kidneys, pancreas and livers of the animal models infiltration of inflammatory cells in the tissue in these animal model were obvious.Conclusions:Hyperglycemia induced the decreasing activity of eliminating bacteria and the increasing formation of biofilms on the surface of biomaterial in Staphylococcus epidermidis. The pathogenicity and invasiveness ofâ–³lux Staphylococcus epidermidis improved significantly in animals with hyperglycaemia.