Study Of D-glutamic Acid Against Porphyromonas Gingivalis Biofilm

Background:Biofilm is an ecological environment formed by a microbial community on the surface of a certain medium through its extracellular matrix.More than 80%of bacterial infections,especially chronic diseases,are clinically related to biofilms.Dental plaque is a typical biofilm structure that can accommodate the survival of multiple floras in the oral ecosystem.Bacteria in biofilms are different from their planktonic state because of their naturally resistant to the elimination of host defense and antibiotic agents.In addition,biofilm bacteria have metabolic activities and gene expression differences from their planktonic state.Peri-implantitis and peri-implant mucositis are related to the accumulation of plaque biofilm in the oral cavity.The biofilm destroys the host-microbial homeostasis on the implant and mucosal interface,leading to inflammatory lesions.P.gingivalis is recognized as the most closely bacteria related to the development of peri-implantitis and the failure of implants.Therefore,in order to prevent or control the occurrence and development of peri-implantitis,the key is to destroy or prevent.gingivalis biofilm on the implant surface effectively.However,currently methods used for treating peri-implantitis,such as manual treatment,laser therapy,antibiotic agents,and photodynamic therapy,cannot guarantee effectively removing the biofilm without damaging the implant surface and surrounding tissue.The dispersal effects of D-amino acids on biofilms and their inhibition on biofilm formation have been confirmed by inhibiting biofilm matrix growth and expression of related genes,proteins,or by the way of incorporating into the peptide side chain of peptidoglycan during synthesis to inhibit the formation of bacterial biofilms.Studies have shown that different D-amino acids have different effects on biofilms of different strains.At present,the effect of D-amino acids on biofilms is mostly concentrated on Pseudomonas aeruginosa,Bacillus subtilis and Staphylococcus aureus.P.gingivalis plays important roles in the development of periodontal disease and peri-implant disease.However,there are few studies on P.gingivalis biofilm.Whether D-glutamic acid,an important component of cell wall peptidoglycan,anti-P.gingivalis biofilm has not been reported.Therefore,this experiment will explore the effects of different concentration gradients of D-glutamic acid on P.gingivalis biofilms,in order to provide theoretical support for the clinical application of D-glutamic acid.Methods:Different concentrations(0.05 mM、0.5 mM、5 mM、10 mM、25 mM、35 mM、45 mM)of D-glutamic acid solution were prepared and tested for the effects on P.gingivalis ATCC 33277 planktonic bacteria and biofilm.The cultured P.gingivalis bacterial suspension was inoculated in a 24-well/96-well plate for 72 hours,and the effects of anti-P.gingivalis biofilm after adding D-glutamic acid were detected by crystal violet staining,MTT,extracellular polysaccharide,scanning electron microscopy and live/dead bacteria staining.Results:1.0.05 mM,0.5 mM,5 mM,10 mM,25 mM,35 mM D-glutamic acid had no significant effect on the growth curve of P.gingivalis planktonic bacteria.D-glutamic acid at a concentration of 45 mM reduced the number of planktonic bacteria and prolonged the stagnation period.2.When D-glutamic acid was used to prevent P.gingivalis biofilm formation,the presence of D-glutamic acid at concentrations of 5 mM,10 mM,25 mM,35 mM,45 mM results in biofilm biomass of P.gingivalis significantly reducing.10 mM,25 mM,35 mM,45 mM D-glutamate resulted in a significant decrease in the metabolic activity of P.gingivalis biofilm.0.05 mM,0.5 mM,5 mM,10 mM,25 mM,35 mM,45 mM D-glutamate reduced the production of extracellular polysaccharides.After the addition of 5 mM-45 mM D-glutamic acid,the number of bacteria in the biofilm decreased,while the proportion of live bacteria decreased,and the proportion of dead bacteria increased.3.D-glutamate at concentrations of 0.5 mM,5 mM,10 mM,25 mM,35 mM,45 mM resulted in a significant decrease in P.gingivalis biofilm biomass when being used to reduce the P.gingivalis biofilm that had been formed.35 mM,45 mM D-glutamic acid resulted in a significant decrease in the metabolic activity of P.gingivalis biofilm.D-glutamic acid at 0.05 mM,0.5 mM,5 mM,10 mM,25 mM,35 mM,45 mM decreased the production of extracellular polysaccharides.After the addition of 5 mM-45 mM D-glutamic acid,the number of bacteria in the biofilm decreased,and it was observed that individual bacteria became long rods.The proportion of living bacterna did not change significantly.Conclusions:1.0.05 mM,0.5 mM,5 mM,10 mM,25 mM,35 mM D-glutamic acid had no sigjnificant effect on the growth of P.gingivalis planktonic bacteria.D-glutamic acid at a concentration of 45 mM inhibited the growth of.gingivalis planktonic bacter:ia.2.When used to prevent the formation of P.gingivalis biofilm,D-glutamic acid concentration≥10 mM can reduce the formation of biofilm biomass,change the activity of bacteria in the biofilm and reduce the production of EPS.3.When used to destroy P.gingivalis biofilm that has been formed,D-glutamic acid concentration≥35 mM can reduce the biofilm biomass,change the activity of bacteria in the biofilm and reduce the production of EPS.