Difference in virulence and composition of a cariogenic biofilm according to substratum direction

Abstract

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).

Figure 1

Dry weight (A), CFUs (B), and water-insoluble EPSs (C) of 46-h-old S. mutans biofilms formed on sHA discs placed in downward, vertical, and upward directions. Values followed by the same superscript are not significantly different from each other (p > 0.05).

Figure 2

Acid production in 46-h-old S. mutans biofilms formed on sHA discs placed in downward, vertical, and upward directions. Changes in initial rate of H⁺ production (0–20 min) and total produced concentration of H⁺ (180 min) in 46-h-old S. mutans biofilms, calculated from biofilm pH drop assay data. Values followed by the same superscript are not significantly different from each other (p > 0.05).

Figure 3

Effect of substratum direction on live and dead cells. (A) Bacterial biovolume, (B) bacterial thickness of live and dead cells in 46-h-old S. mutans biofilms, and (C) representative 3-D images of live (green) and dead (red) cells in the biofilms: (C-1) downward substratum surfaces, (C-2) vertical substratum surfaces, and (C-3) upward substratum surfaces. Values followed by the same superscript are not significantly different from each other (p > 0.05). *p < 0.05: significantly different from each other.

Figure 4

Effect of substratum direction on EPS. (A) EPS biovolume, (B) EPS thickness of 46-h-old S. mutans biofilm, and (C) representative 3-D images of EPSs (red) in the biofilms: (C-1) downward substratum surfaces, (C-2) vertical substratum surfaces, and (C-3) upward substratum surfaces. Values followed by the same superscript are not significantly different from each other (p > 0.05).

Figure 5

Biofilm density of 46-h-old S. mutans biofilms formed on sHA discs placed in downward, vertical, and upward directions. Values followed by the same superscripts are not significantly different from each other (p > 0.05).

Figure 6

Representative SEM images (×100, ×5000) of 46-h-old S. mutans biofilms formed on sHA discs placed in different directions: (A) downward substratum surfaces, (B) vertical substratum surfaces, and (C) upward substratum surfaces.

Figure 7

S. mutans biofilm formation on saliva-coated hydroxyapatite (sHA) discs placed in different directions and the experimental plan.