| Objectives The purpose of this study was to investigate the effects of different D-amino acids(D-AAs)and D-amino acids Mixtures(D-Mixs)on single and multi-strain mixed biofilms in vitro,and to evaluate its inhibitory effect on biofilm and its dispersing effect on the formed biofilm,and to select the optimum concentration.In order to provide thebasis for select new root canal irrigating drugs,we further explored the effect of D-AAs combined with 2% chlorhexidine on the removal of Enterococcus faecalis biofilm.Methods 1.Effects of D-AAs and D-Mixs on Oral Pathogens Bacterial Biofilm1.1 The Effect of D-AAs and D-Mixs on Single Strain Biofilm1)strains of Enterococcus faecalis(E.faecalis),Streptococcus mutans(S.mutans)and Fusobacterium nucleatum(F.nucleatum)biofilms were established on the polystyrene,different concentrations of D-Tryptophan(D-Trp),D-Methionnine(D-Met),D-Leucine(D-Leu)and D-Tyrosine(D-Tyr)were co-cultured with the above-cultured bacterial solutions for 24 h In vitro.Using BHI medium without drug as negative control group and 0.2% chlorhexidine as positive control group.Crystal violet staining was used to test the absorbance value of each pore at 570 nm wavelength by enzyme labeling instrument.The total biomass of bacterial biofilm was calculated.The optimum concentration of D-AAs was screened out.The inhibition rate were calculated.2)In the experiment of biofilm dispersion:Firstly,a 24 h biofilm model of the three strains was established on the surface of polystyrene,and then the drug to be tested was added to it.After co-culture for 2 hours,the total biomass of bacterial biofilm was determined by crystal violet staining,the clearance rate of biofilm was calculated,and the effect of D-AAs on biofilm dispersion was evaluated.3)According to 1)results,choose the most suitable inhibited biofilm concentration comprised of D-Mix1(100m M D-Met,50 m M D-Leu,10 m M D-Trp and 0.25 m M D-Tyr).According to 2)results,selected the most suitable dispersed biofilm concentration consisted of D-Mix 2(100m M D-Met,100 m M D-Leu,10 m M D-Trp and 0.25 m M D-Tyr),to detect the total biomass of biofilm,calculated the inhibitionand clearance rate of biofilm,and evaluated the inhibition and dispersion of D-Mixs on biofilms.1.2 The effect of D-AAs and D-Mixs on multi-bacterial biofilm in vitro1)E.faecalis,S.mutans and F.nucleatum were co-cultured in BHI solid medium containing vitamins and heme chloride for 24 hours to observe whether the growth of the three bacteria could inhibit each other.2)On the surfaceof polystyrene plate,a multi-bacterial biofilm model was established,which consisted of E.faecalis,S.mutans and F.nucleatum.The experimental group and the drugs added were the same as experiment 1.The total biomass of biofilm was calculated by crystal violet staining.The effects of D-AAs and D-Mixs on the formation and dispersion of multi-bacterial biofilm were measured.Additionally,the optimum concentration of D-AAs was selected.The inhibition and clearance rate of biofilms were calculated.1.3 Effect of D-AAs and D-Mixs on multi-bacterial biofilm formed by infected root canals1)Approved by Ethics Committee of Stomatological Hospital of Tianjin Medical University,the tooth with chronic periapical periodontitis were collected according to inclusion criteria.Mixed microorganisms were extracted from root canals by paper-tip method.After bacterial enrichment,a bacterial biofilm model of infected root canals was established on polystyrene plate.2)The effects of D-AAs and D-Mixs on the formation and dispersion of microorganisms biofilm were determined.The experiment group,the added drugs and the detection methods as to experiment 2.Additionally,the optimum concentration of D-AAs was screened out.The inhibition and clearance rate of biofilm were calculated.2.Eliminating effect of D-AAs combined with 2% chlorhexidine on E.faecalis biofilm in vitroA 24 h biofilm model of E.faecalis was established on dentin slices,the species and concentration of D-AAs obtained in above experiment were selected to study.The experimental groups were as follows:Group A:5m M D-Trp + 2% CHX,Group B: 10 m M D-Leu + 2% CHX,Group C: 10 m M D-Met + 2% CHX,Group D: Deionized water + 2% CHX,Group E: Deionized water.After the above drugs and 2% chlorhexidine were continuously co-cultured with the established E.faecalis biofilm for 10 and 1 min,the total biomass of the microorganisms biofilm was determined by using the plate colony counting method,and the biofilm clearance rate was calculated.3.SPSS 23.0 was used forstatistical analysis: one-way ANOVA was used to compare the overall means of each group,SNKand L-SD method were used for comparison between groups.P<0.05 was considered statistically significant.Results 1.The Effect of D-AAs and D-Mixs on Single Strain Biofilm in vitro1)Inhibition of biofilm formation experiments showed that D-AAs had different effects on the same single bacterial biofilm.The total biomass of 2.5m M D-Leu group of S.mutans and E.faecalis biofilm had no significant difference compared with that of BHI group(P>0.05),the total biomass of biofilm of other experimental groups was significantly lower than that of BHI group(P<0.05).2)Similarly,the discrete effects of D-AAs and D-Mixs on single strain of biofilm are also different.For the S.mutans and E.faecalis biofilms,the total biomass of the biofilm in the experimental group was significantly lower than that in the BHI group.However,for the F.nucleatum biofilm,2.5m M of D-Leu and D-Trp group,the total biofilm biomass was not significantly different from the BHI group(P>0.05).The total biomass of other groups was significantly reduced(P<0.05).The D-Mix2 group only significantly reduced the biofilm biomass of S.mutans,and had no significant effect on other biofilms.2.The effect of D-AAs and D-Mixs on multi-bacterial biofilm in vitro1)When co-culture of E.faecalis,S.mutans and F.nucleatum,the growth of the three strains did not inhibit each other.2)Inhibition of biofilm formation experiments showed that except for D-Mix2 group,the total biomass of biofilm of other experimental groups was significantly lower than that of BHI group(P<0.05).3)Biofilm discreted experiment showed that except for 50 m M D-Leu group and D-Tyr groupthe biofilm biomass of other groups was lower than that of BHI group,the difference was statistically significant(P<0.05).the total biofilm biomass of D-Leu group were lower than D-Met and D-Tyr group,and the difference was statistically significant.3.Effect of D-AAs and D-Mixs on multi-bacterial biofilm formed by infected root canals1)Inhibition experiment showed that 10 m M D-Leu group and D-Tyr groupin total biofilm biomasshave no significant difference between BHI group(P>0.05),while the total biofilm biomass of other groups was lower than that of BHI group(P<0.05).The 50 m M D-Met group had the lowest biofilm biomass.2)In all the experimental groups,the biofilm biomassof 10 m M D-Leu and D-Tyr groupswas no significantly different from that of the BHI group(P>0.05),and the biofilm biomass of other groups was lower than that of the BHI group.The difference was statistically significant(P<0.05).4.Eliminating effect of D-AAs combined with 2% chlorhexidine on E.faecalis biofilm in vitroWhen 2% chlorhexidine was combined with D-Trp,the number of remaining viable bacteria in E.faecalisbiofilm was less than that of 2% chlorhexidine alone(P< 0.05);when 2% chlorhexidine was combined with D-Met and D-Leu respectively,the number of remaining viable bacteria in E.faecalis biofilm had no significant difference compared with the control group(P> 0.05).Conclusion 1.D-Met,D-Leu,D-Trp,D-Tyr and D-Mix1 all have the effect of inhibiting the formation of biofilms of S.mutans,E.faecalis and F.nucleatum.D-AAs had no dose-dependent effect on single strain biofilm.The effect of 50 m M D-Met inhibited the formation of biofilm of S.mutans and F.nucleatumis the highest.25 m M D-Leu inhibited the formation of F.nucleatum biofilm is the highest.2.D-Met,D-Leu,D-Trp and D-Tyr all have the functions of discrete single strain biofilm.D-Mix 2 has the function of discrete of S.mutans biofilm.50 mM of D-Met,50m M of D-Leu and 5m M of D-Trp have the highest effect on S.mutans,F.nucleatum and E.faecalis biofilm,respectively.3.D-AAs(D-Met,D-Leu,D-Trp,D-Tyr)can inhibit and discrete multi-bacterial biofilm.D-Mix1 can inhibit multi-bacterial biofilm.4.D-Met,D-Leu,D-Trp can inhibit and discrete multi-bacterial biofilm that extract from infected root canals.D-Met of 50 m M had the strongest inhibit effect.5.D-Mixs did not show a stronger inhibitory and dispersion effect on the mixed biofilms than single D-AAs.6.2% chlorhexidine combined with D-Trp can improve the biofilm clearance effect of E.faecalis on dentin tablets,but D-Met and D-Leu can not. |
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