Erythrosine B의 Exopolysaccharide 결합을 통한 Streptococcus mutans 생물막 형성 억제 및 항균 효과 증대 = Erythrosine B Inhibits Streptococcus mutans Biofilm Formation and Enhances Bactericidal Efficacy via Exopolysaccharide Binding

다국어 초록 (Multilingual Abstract)

Oral biofilms are highly organized microbial communities encased within an exopolysaccharide (EPS), which strengthens bacterial adhesion, stabilizes the structural architecture, and protects resident microorganisms from antimicrobial agents. Streptococcus mutans, the primary etiological agent of dental caries, metabolizes dietary sucrose to generate acids that demineralize enamel while producing substantial amounts of EPS, thereby supporting the development, persistence, and increased tolerance of cariogenic biofilms. Although disclosing agents such as erythrosine B (EB) have been widely used in clinical settings to visualize dental plaque, their potential ability to modulate biofilm formation and affect bacterial survival remains insufficiently explored; therefore, this study aimed to investigate the inhibitory and bactericidal effects of EB on S. mutans biofilms, elucidate its EPS-associated mechanism of action, and determine its safety on human oral epithelial cells. An initial 96-well plate model was used to screen and identify the effective concentration of EB capable of inhibiting S. mutans growth and suppressing biofilm formation. Afterward, human saliva-coated hydroxyapatite (sHA) discs were employed to mimic the clinical tooth-surface environment and provide a physiologically relevant model for plaque biofilm formation. EB was applied for 10 min during biofilm development to evaluate its inhibitory activity, and it was also applied once for 10 min to 24-h preformed biofilms to assess its bactericidal effects, with chlorhexidine (CHX 0.12%) serving as a positive control for comparison. Biofilm inhibition and killing were evaluated through viable cell counts, glycolytic pH-drop measurements, and confocal analysis of biofilm spatial architecture, while glucanohydrolases were used to determine whether the effects of EB depended on the integrity of the EPS matrix. Additionally, proliferation assays on human oral epithelial cells were conducted to verify the biocompatibility of EB. The results demonstrated that EB at 0.1 mg/mL inhibited S. mutans growth and biofilm formation by more than 50% in the 96-well plate model. In the sHA disc model, repeated short-term exposure to EB substantially reduced biofilm formation by decreasing bacterial adhesion, limiting initial colonization, and suppressing EPS accumulation. In preformed biofilms, EB showed a potent bactericidal effect, resulting in an almost complete reduction of viable cells and surpassing the efficacy of CHX. Furthermore, both EB and CHX significantly reduced the glycolytic pH drop compared with the untreated control, indicating effective suppression of acidogenic activity within the biofilm. Confocal analysis revealed that EB binds directly to the EPS matrix, enhancing its antimicrobial performance. When the EPS layer was enzymatically disrupted, this enhanced killing effect was abolished, indicating that the bactericidal activity of EB is strongly dependent on EPS integrity. Importantly, the concentrations of EB that effectively inhibited and eliminated biofilms did not exhibit cytotoxicity toward human oral epithelial cells, leading to the conclusion that EB inhibits S. mutans biofilm development and enhances bactericidal activity through an EPS-dependent mechanism while exhibiting no cytotoxicity toward human oral epithelial cells.

목차 (Table of Contents)

• Abstract 1
• Introduction 4
• Materials and methods 8
• Results and discussion 23
• Conclusion and future direction 53
• References 55
• 국문초록 63
• Acknowledgements 65