Sucrose is an important dietary factor in cariogenic biofilm formation and subsequent initiation of dental caries. This study investigated the functional relationships between sucrose concentration and Streptococcus mutans adherence and biofilm formation. Changes in morphological characteristics of the biofilms with increasing sucrose concentration were also evaluated. S. mutans biofilms were formed on saliva-coated hydroxyapatite discs in culture medium containing 0, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, or 40% (w/v) sucrose. The adherence (in 4-hour biofilms) and biofilm composition (in 46-hour biofilms) of the biofilms were analyzed using microbiological, biochemical, laser scanning confocal fluorescence microscopic, and scanning electron microscopic methods. To determine the relationships, 2nd order polynomial curve fitting was performed. In this study, the influence of sucrose on bacterial adhesion, biofilm composition (dry weight, bacterial counts, and water-insoluble extracellular polysaccharide (EPS) content), and acidogenicity followed a 2nd order polynomial curve with concentration dependence, and the maximum effective concentrations (MECs) of sucrose ranged from 0.45 to 2.4%. The bacterial and EPS bio-volume and thickness in the biofilms also gradually increased and then decreased as sucrose concentration increased. Furthermore, the size and shape of the micro-colonies of the biofilms depended on the sucrose concentration. Around the MECs, the micro-colonies were bigger and more homogeneous than those at 0 and 40%, and were surrounded by enough EPSs to support their structure. These results suggest that the relationship between sucrose concentration and cariogenic biofilm formation in the oral cavity could be described by a functional relationship.

Fluoride is commonly used as an ingredient of topical oral hygiene measures. Despite the anti-acidogenic activities of fluoride against cariogenic biofilms, the recovery of the biofilms from fluoride damage is unclear. Herein, we investigated the recovery of acid production in Streptococcus mutans biofilms after short-term or during periodic 1-min fluoride treatments. For this study, 46-hour-old S. mutans biofilms were treated with fluoride (0-2,000 ppm F^-) for 1-8 min and then incubated in saliva for 0-100 min. The 74-hour-old biofilms were also periodically treated with the fluoride concentration during biofilm formation (1 min/treatment). Changes in acidogenicity and viability were determined via pH drop and colony-forming unit assays, respectively. In this study, acid production after a 1-min fluoride treatment was recovered as saliva incubation time increased, which followed a linear pattern of concentration dependence (R = 0.99, R² = 0.98). The recovery pattern was in a biphasic pattern, with an initial rapid rate followed by a second slow recovery. Furthermore, recovery from fluoride damage was retarded in a concentration-dependent manner as treatment time increased. In periodic 1-min fluoride treatments, acid production in the biofilms was not diminished during the non-fluoride treatment period; however, it was reduced in a concentration-dependent manner during the fluoride treatment period. The viability of the biofilm cells did not change, even at high fluoride concentrations. Collectively, our results suggest that brief fluoride treatment does not sustain anti-acidogenic activity against S. mutans in biofilms since the damage is recoverable with time.

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The aim of this study was to investigate the difference in composition and virulence of Streptococcus mutans biofilms according to substratum direction. S. mutans biofilms (46-h-old) were formed on three different saliva-coated hydroxyapatite (sHA) disc direction groups: downward (discs placed in the direction of gravity), vertical (discs placed parallel to gravity direction), and upward (discs placed opposite to gravity). The 46-h-old biofilms on sHA discs in the upward direction showed the highest biofilm accumulation, colony forming unit (CFU) count, and extracellular polysaccharide (EPS) amount, followed by those in the vertical and downward directions. In the confocal laser scanning microscopy (CLSM) study, the biofilms in the upward direction also showed the highest bacterial count (live or dead cells) and EPS biovolume. Scanning electron microscopy (SEM) analysis confirmed the microbiological and biochemical results. In addition, biofilm density and acid production were higher in the upward direction than those in the other directions. Our findings suggest that substratum direction, which might be related to gravity, strongly influences the formation and virulence of cariogenic biofilms and subsequent initiation of dental caries. Collectively, the differences in the formation and virulence of cariogenic biofilms are related to the direction of tooth surface (occlusal surfaces of mandibular teeth > proximal surfaces > occlusal surfaces of maxillary teeth).