Supplementary Material for: Dietary carbohydrates modulate Streptococcus mutans adherence and bacterial proteome

Introduction: Streptococcus mutans adherence to the tooth surface and subsequent biofilm development is modulated by the carbohydrate source, but the corresponding effect on bacterial proteome has not been previously studied. This study aimed to assess the effect of different carbohydrates on S. mutans viability and bacterial proteome at two-time points, early attachment (8 h) and biofilm maturation (24 h). Methods: Hydroxyapatite (HAp) discs coated with parotid saliva proteins were inoculated with S. mutans UA159 in Tryptone Soy Broth without dextrose supplemented with one of the following carbohydrates (n=12/treatment/time point): 1% Sucrose (S); 0.525% Glucose + 0.525% Fructose (G+F); 10% Xylitol (X); 10% Xylitol + 1% Sucrose (X+S); or culture medium without supplementation as negative control (C). Once inoculated, HAp discs were incubated for 8 h or 24 h at 37 °C and 10% CO2. After each incubation period, adhered bacteria were quantified using the plate-counting method for 6 HAp discs/group, and the remaining 6 HAp discs/group were used to extract bacterial cell wall proteins. Extracted proteins were analyzed using liquid chromatography coupled with mass spectrometry and then classified by their biological process. The study was conducted in three independent assays and the number of bacteria adhered to the HAp discs was determined at each time point and analyzed by two-way ANOVA followed by Bonferroni test (=5%). Results: The results suggest that xylitol significantly repressed bacterial adherence and metabolism at 8 h and 24 h; however, bacterial adherence and metabolism were significantly enhanced when xylitol was combined with sucrose, showing no negative effect on S. mutans at both time points. Bacterial proteome was modulated by the carbohydrate source. Conclusion: The cariogenicity of S. mutans biofilms may be reduced by the alternative sweetener xylitol; however, the combination with fermentable sugars may inhibit such a beneficial effect.