Fig. 5.

Heat-inactivated OMVs exhibit bactericidal and antibiofilm activities against S. mutans. S. mutans biofilms were grown on chamber-slides in BMGS for 24 h before being treated with (A) PBS, (B) 100 µg/mL heat-inactivated OMVs, or (C) 100 µg/mL native OMVs for another 24 h. After staining with LIVE/DEAD BacLight fluorescent dye (SYTO 9/propidium iodide), biofilms were subjected to optical dissection using confocal microscopy. Green: total biomass. Red: dead cells. Side bars indicate the thickness of the biofilms. Post-acquisition analyses of the fluorescent images were performed using COMSTAT 2.0. Treatments with heat-inactivated OMVs, native OMVs, or PBS were compared for (D) biofilm biomass, (E) dead cell ratio, (F) average thickness, and (G) roughness coefficient. Roughness coefficients of the biofilms were calculated as an indication of biofilm integrity. The results were analyzed using one-factor ANOVA. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, NS = not significant)