Research Of D-methionine Dispersing Porphyromonas Gingivalis Biofilms By Reducing The Concentration Of C-di-GMP

Objectives: In this study,we aimed to investigate the mechanism of D-Methionine dispersing porphyromonas gingivalis biofilm by affecting the formation of c-di-GMP,in order to provide a new strategy for the treatment of periodontitis and peri-implantitis.Methods: D-Met was prepared with different concentrations(0、5、10、15、20、25、30、35、40、45 m M)and respectively dissolved in cell and bacteria culture media.The cell counting kit-8(CCK-8)assay was used to determine the cell viability of D-Met on L929 cell.Time-kill curve,minimum inhibitory concentration test(MIC)and minimum bactericidal concentration test(MBC)were utilized to determine the effects of D-Met on bacterial activities of P.gingivalis.Crystal violet biofilm biomass assay,extracellular polysaccharide measurement assay and scanning Electron Microscope(SEM)were used to evaluate the inhibition and dispersion effects of D-Met on P.gingivalis biofilm.c-diGMP concentration was detected by HPLC to evaluate the mechanism of D-Methionine on P.gingivalis biofilm.Results: 1.D-Methionine ≤40m M had no effect on L929 cell proliferation.2.The MIC of D-Met on P.gingivalis was 35 m M,and the MBC of D-Methionine on P.gingivalis was 40 m M.3.The inhibition of D-Met on the formation of P.gingivalis biofilms: 3.1 Compared with the control group,when D-Met ≥20m M,the amount of biofilm formation of P.gingivalis was inhibited,and the amount of extracellular polysaccharide was reduced.3.2 The results of SEM showed that the number of P.gingivalis layers of control group(0m M)is more than 4,and P.gingivalis sticked to each other as biofilm structure.When D-Met ≥ 20 m M,P.gingivalis was scattered and did not stick to each other,and the biofilm structure became incomplete.When the concentration of D-Met was 35 m M,the integrity of a few P.gingivalis membrane was destroyed and interbacterial matrix was significantly reduced.4.The disassemble effects of D-Met on P.gingivalis biofilms:4.1 Compared with the control group,when D-Met ≥25m M,it had the effect of reducing biofilm biomass and the amount of extracellular polysaccharide.4.2 The results of SEM showed that the number of P.gingivalis layers of control group(0m M)is morethan 4,and P.gingivalis sticked to each other as biofilm structure.When D-Met ≥ 25 m M,the mature biofilm was disassembled,and bacteria were scattered and did not stick to each other.When the concentration of D-Met was 35 m M,the integrity of a few P.gingivalis membrane was destroyed and interbacterial matrix was significantly reduced.5.The effects of D-Met on the content of P.gingivalis c-di-GMP : Compared with control group(0m M),when D-Met ≥20m M,c-di-GMP in P.gingivalis was significantly reduced.Moreover,c-di-GMP was negatively correl ated with D-Met concentration:the higher concentration of D-Met,the lower content of c-di-GMP.Conclusion: 1.D-Met concentration ≤ 40 m M has good biological safety.The D-Met concentration of 40 m M can inhibit the bacterial activity and has bactericidal effect on P.gingivalis.2.When the D-Met concentration ≥20m M,it can inhibit the biofilm formation of P.gingivalis.D-Met≥25m M has the effects of dispersing mature P.gingivalis biofilm.3.D-Met disperses the P.gingivalis biofilm by reducing the content of c-di-GMP.