Inhibition of S. mutans Biofilms

Biofilms of S. mutans UA159 were formed on saliva-coated hydroxyapatite (sHA) discs (surface area of 2.93 ± 0.2 cm², Clarkson Chromatography Products Inc., South Williamsport, PA, USA) in batch cultures for 5 days, as detailed elsewhere [21 (link)]. The biofilms were grown in ultrafiltered (10 kDa molecular-weight cut-off) buffered tryptone yeast-extract broth containing 1% (w/v) sucrose [21 (link)]. The culture medium was replaced daily; the organisms were grown undisturbed for 22 h to allow initial biofilm formation. At this point (22 h old), the biofilms were then treated twice-daily (at 10 a.m. and 4 p.m.) until the end of the experimental period (118-h-old biofilm) with one of the following: (i) 1.0 mM myricetin + 2.5 mM tt-farnesol + 125 ppm fluoride (MFar125F); (ii) 1.0 mM myricetin + 2.5 mM tt-farnesol + 250 ppm fluoride (MFar250F); (iii) 250 ppm fluoride (250F); (iv) vehicle control (20% ethanol containing 2.5% DMSO in water); fluoride at 125 ppm F was not included because it is devoid of any significant anti-biofilm effects [12 (link),13 (link)]. The biofilms were exposed to the treatments for 1 min., dip-rinsed three times in sterile saline solution (to remove excess of agents or vehicle-control) and transferred to culture medium. The treatments and rinsing procedures were repeated 6 h later. The pH of culture medium surrounding the biofilms was also determined during the experimental period (until 118 hour biofilms, at 8 a.m., 12 a.m., 4 p.m., 6 p.m.). Our previous studies have shown that the vehicle control (1 min exposure, twice daily) allowed the continued formation of biofilm, and did not affect the biochemical composition and cell viability when compared to biofilms treated with saline solution [20 (link),21 (link)]. Each biofilm was exposed to the respective treatment a total of 8 times. Biofilm assays were performed in duplicate in at least six different experiments.