D-leucine Enhances Antibiofilm Activity Of Chlorhexidine Against Caries-causing Streptococcus Mutans Biofilm

Objective: Chlorhexidine(CHX)mouthwash is widely used clinically to remove the established Streptococcus mutans(S.mutans)biofilm and prevent caries.However,the long-term use with high concentration of CHX leads to the staining of tooth and mucous membrane.In this study,D-leucine(D-leu)was used as an antimicrobial enhancer for CHX against mature S.mutans biofilm.This study investigated whether D-leu could enhance the killing effect of chlorhexidine on S.mutans around stainless steel brackets,reduce the occurrence of caries and reduce the clinical application concentration and side effects of chlorhexidine.Methods: The biocidal efficacy was evaluated by means of quantitative antibacterial test and biofilm observation.The bactericidal effect of D-leu on CHX was evaluated by plate counting.The surface morphology of the biofilm around the stainless steel bracket was observed and analyzed by scanning electron microscope.The influence of D-leu on the extracellular polysaccharide content of S.mutans biofilm around the stainless steel bracket was analyzed by detecting the content of nucleic acid,protein and polysaccharide.The content of live dead bacteria of S.mutans around the stainless steel bracket was evaluated by laser confocal microscopy.The effects of D-leu on S.mutans biofilm around the stainless steel bracket were compared by ATP content analysis.The biocompatibility of D-leu was evaluated by CCK8 assay and real-time cell analysis.Results: The results indicated that 100 ppm D-leu alone was ineffective to remove the S.mutans biofilm.Moreover,the biocidal effect of 1 ppm CHX was also inadequate to eradicate the biofilm.When 100 ppm D-leu was mixed with 1 ppm CHX,most of the mature S.mutans biofilm on 316 L stainless steel coupon surfaces were successfully removed,and the removal efficacy was equivalent to that of 10 ppm CHX alone.Conclusion: The results confirmed that D-leu significantly enhanced the antibiofilm effect of CHX,dissembling the tenacious biofilm into easily killed planktonic counterparts.D-leu represents a novel strategy to reduce the dosage of CHX with a guaranteed effect for caries prevention.