Effects Of D-amino Acids On The Biofilm Formation And Dispersion Against Enterococcus Faecalis

ObjectiveThe aim of this study was to establish monospecies Enterococcus faecalis infection model in vitro,to investigate the inhibition by D-leucine,D-Methionine at different concentration of the formation and dispersion of mature biofilm and screening of the optimal application concentration of D-amino acid in practical application.MethodThe suspension of E.faecalis was inoculated in 96-well plate with addition of Dleucine and D-Methionine at a final concentration of 2.5mM、10 mM 、25 mM 、50 mM,respectively;taking 0.2%CHX as the positive control group,and BHI group as the negative control group.All the groups were incubated under anaerobic condition at 37 °C for 24 h.Biofilm inhibition of different concentration of D-amino acid was assessed using crystal violet assay and the OD values were measured.Screening of the optimal concentration which inhibit biofilm formation and biofilm inhibition rate was determined.Single-species Enterococcus faecalis infection model in vitro was established,D-leucine and D-Methionine at a final concentration of 2.5mM、10 mM 、25 mM 、50 mM were added respectively;regarding 0.2%CHX as the positive control group,and BHI group as the negative control group.After co-cultured for 2h,biofilm dispersion of different concentration of D-amino acid was estimated using crystal violet assay.The OD values were detected and filtering the ideal concentration that induced detachment of biofilm,then biofilm eliminating rates were calculated.The data were subjected to statistical analysis using one-way analysis of variance(ANOVA),Student Newman Keuls Test to examine differences.A P-value of less than 0.05 was considered to be statistically significant.Result1.Compared to D-Leucine and D-Methionine group,0.2% CHX group has a stronger effect on the inhibition of biofilm formation and dispersion of mature biofilm which has a significant difference(P<0.05)between the groups.2.There was no statistical significance(P>0.05)between the effect on inhibition of Enterococcus faecalis biofilm formation(24h)of D-Methionine at the concentration of 10mM、25 mM and 50 mM,whereas 2.5 mM group has a weaker effect on compared with the other three concentration groups(P< 0.05).3.Among these groups at different concentrations,D-leucine at the concentration of 25mM presented the most obvious inhibitory effect on 24 h biofilm,followed by 10mM,50 mM groups respectively,whereas the 2.5 mM group showed the lowest inhibition effect,but no differences were observed between these 2 groups(10mM,50 mM)(P> 0.05).The addition of the inoculum with 2.5mM(0.25 mM D-leucine)produced no significant(P> 0.05)reduction in biofilm optical density when incubated for 24 h when compared with BHI group,while biofilm inhibition at these different D-leucine concentrations and control group was significantly different from each other(P < 0.05).4.No significant difference in biofilm dispersion were observed in the D-Methionine groups at the concentration of 10,25 or 50 mM when incubated for 24h biofilm(P> 0.05),and has stronger effect on biofilm dispersion than the 2.5 mM group which has significantly difference(P<0.05).5.Similarly,D-leucine at the concentration of 50 mM presented the most obvious dispersion effect on 24 h biofilm,followed by 25 mM group,whereas the10mM and 2.5 mM group showed the lowest dispersal and all groups were significantly different from each other(P < 0.05)except for 10 mM and 2.5 mM group(P>0.05).Conclusion 1.Both D-leucine and D-Methionine can inhibit formation and promoted disassembly of Enterococcus faecalis biofilm.2.D-leucine at the concentration of 10mM、25 mM and 50 mM has equivalent effect on inhibition of biofilm formation and dispersion in the range of 0-50 mM,the restraint rate ranges from38% to 46%.The eliminating rate ranges from 38% to 45%.3.In the range of 0-50 mM,the optimal concentration of D-leucine that inhibit the formation of Enterococcus faecalis biofilm and discrete the 24 h Enterococcus faecalis biofilm is 25 mM,50 mM respectively.The restraint rate is 74.79% whereas the eliminating rate is 56.02%.4.D-leucine exhibited stronger inhibitive and dispersive effects on Enterococcus faecalis biofilms than D-Methionine.5.D-leucine,D-Methionine presented a weaker inhibitive effect and promoted dispersal effect on 24 h Enterococcus faecalis biofilms than0.2%CHX.