Biofilm matrix-mediated localization potentiates antimicrobial activity in cariogenic biofilms

The extracellular polymeric substance (EPS) matrix serves as a structural scaffold for biofilm organization, contributing to structural integrity and antimicrobial tolerance by modulating the penetration and distribution of antimicrobial agents. However, whether this EPS matrix can also potentiate antimicrobial localization and activity remains poorly understood. Here, we investigated the role of the EPS matrix in erythrosine B (EB)-mediated killing of Streptococcus mutans within mature biofilms following brief topical exposure. Biofilms formed on saliva-coated hydroxyapatite discs were exposed to EB and evaluated for bactericidal activity, glycolytic acid production, and EB spatial distribution. EB markedly reduced viable bacteria in mature biofilms and suppressed glycolytic acid production. Confocal imaging revealed preferential localization of EB within EPS-rich regions of intact biofilms, whereas selective enzymatic disruption of the glucan-rich matrix, particularly following glucanohydrolases treatment, markedly reduced EB retention. Consistently, EB-mediated bactericidal activity was attenuated in biofilms grown under glucan-deficient conditions, in which sucrose-dependent glucan-rich matrix formation was limited. Together, these findings support a matrix-dependent antimicrobial model in which glucan-rich biofilm matrix promotes EB localization and retention, thereby enhancing bactericidal activity in mature biofilms. This work challenges the conventional view of the biofilm matrix solely as a protective barrier and highlights its broader role as a functional determinant of antimicrobial performance in mature biofilms.