Streptococcus mutans Extracellular Vesicles Regulate Early Streptococcus sanguinis Nanoadhesion

Dental caries remains a globally prevalent disease, driven by microbial dysbiosis and competitive ecological shifts within the oral biofilm. A key interaction in this context is the antagonism between Streptococcus mutans and Streptococcus sanguinis , two early colonizers of the dentin surfaces. While bacterial extracellular vesicles (bEVs) have emerged as important mediators of microbial communication, their role in interspecies adhesion and early biofilm dynamics remains poorly understood. This study explored how S. mutans –derived bEVs, isolated from planktonic cultures and biofilms grown on native or glycated type I collagen substrates, modulate S. sanguinis nanoadhesion and initial biofilm formation. Bacterial EVs were characterized using nanoparticle tracking analysis and transmission electron microscopy, and their functional effects were assessed via atomic force microscopy–based single-cell force spectroscopy. This approach enabled direct quantification of bacterial adhesion forces and single-molecule unbinding events under different vesicle-exposure conditions. Our results demonstrated that planktonic-derived bEVs enhanced S. sanguinis adhesion at 5-s contact times, while bEVs from collagen-bound S. mutans biofilms, particularly those grown on glycated matrices, consistently reduced adhesion forces, rupture lengths, and unbinding events. Optical coherence tomography imaging confirmed that these nanoscale effects translated into altered early biofilm architecture, with planktonic bEVs promoting thicker, denser biofilms and biofilm-derived bEVs leading to sparser colonization. Our findings suggest that S. mutans bEVs exhibit a context-dependent modulatory effect on S. sanguinis , enhancing adhesion under planktonic conditions and suppressing it upon biofilm establishment. This biphasic behavior may represent a strategic mechanism for niche domination during caries initiation. Moreover, collagen glycation—mimicking aged or hyperglycemic dentin—further influenced bEV function, underscoring the importance of the host matrix state in microbial interactions. Overall, this study highlights a previously unrecognized role for bEVs in shaping early oral dysbiosis at the single-cell level.