Actinomyces oris-Derived Membrane Vesicles Promote Insoluble Glucan-Independent Streptococcus mutans Early Biofilms

Actinomyces oris (A. oris), an early colonizer in oral microbiota, possesses two antigenically distinct fimbriae types. Oral bacteria-derived membrane vesicles (MVs) contain diverse bacterial components and are associated with dental caries; however, their role in A. oris-mediated biofilm interactions remains unclear. This study investigated the effects of A. oris-derived MVs on early biofilm formation by Streptococcus mutans (S. mutans), a notable cariogenic bacterium. Biofilm formation was evaluated in salivacoated 96-well plates using MVs from A. oris MG-1 (wild type, WT) and fimbriae A mutant (ΔfimA) in Tryptic Soy Broth (TSB) without dextrose, supplemented with 0.25% sucrose. S. mutans growth was quantified by absorbance at 595 nm. Biofilm formation was measured by safranin staining at 492 nm; representative images were obtained by phase-contrast microscopy. A. oris-derived MVs exhibited no statistically significant effect on the early growth of S. mutans WT or the glucosyltransferases B and C (GtfB and GtfC) mutants (ΔgtfBC). Both MVs also showed no statistically significant effect on early S. mutans WT biofilms. A. oris WT-derived MVs significantly and dose-dependently promoted early biofilm formation of S. mutans ΔgtfBC, whereas A. oris ΔfimA-derived MVs showed no statistically significant effect. Visual observation revealed biofilm formation at 7.81–125 μg/mL in the presence of only A. oris WT-derived MVs. This difference proposed structural variation in MVs associated with fimbriae. Scanning electron microscopy (SEM) analysis revealed that WT-derived MVs were significantly larger than ΔfimA-derived MVs. These findings indicate that FimA-containing A. oris MVs promote insoluble glucan-independent S. mutans biofilm formation.