Membrane Vesicles Improve Streptococcus mutans Early Biofilm Formation

Streptococcus mutans (S. mutans), one of the main etiological pathogens of dental caries, forms dental plaque biofilms that drive tooth decay. Although bacterial membrane vesicles (MVs) are increasingly recognized as modulators of biofilm biology, little is known about MVs generated by S. mutans. The objective of this study is to investigate the role of S. mutans-derived MVs in the development of S. mutans biofilms formed under static conditions in plates or confocal dishes. Transmission electron microscopy and nanoparticle tracking analysis revealed that the MVs were cup-shaped with bilayered membranes and averaged 80.49 ± 32.24 nm in diameter. The addition of ≥5 µg/mL MVs enhanced biofilm formation during the initial adhesion stage (0 to 6 h), as demonstrated by crystal violet staining and XTT assays. Confocal laser scanning microscopy and scanning electron microscopy confirmed the incorporation of PKH26-labeled MVs into S. mutans biofilms and showed that supplemental MVs increased bacterial viability and extracellular polysaccharide biomass. Furthermore, RT-qPCR analysis revealed upregulated expression of genes related to adhesion and quorum-sensing systems in MV-treated biofilms. In conclusion, these findings indicate that S. muants MVs are integral biofilm components that promote biofilm establishment at the early stage of biofilm formation.