Synergistic growth, metabolic products, and matrix quantification in dual-species biofilms of oral pathogens

Biofilms exhibit virulence traits that promote microbial protection, such as an extracellular matrix composed of exopolysaccharides (EPS), and contribute to host tissue damage via acid production. This study evaluated bacterial viability, biovolume, matrix (β-polysaccharides), and EPS (α-polysaccharides) formation in monospecies and mixed biofilms of Streptococcus mutans and Enterococcus faecalis, as well as pH and lactic acid production.

Biofilms were grown on dentin blocks for 7 days, and aliquots were collected for colony-forming unit (CFU/mL) counts. Subsequently, biofilms were stained with LIVE/DEAD®, Calcofluor White®, and Alexa Fluor 647-dextran conjugate® to assess bacterial viability, matrix formation, and EPS formation, respectively, using confocal laser scanning microscopy (CLSM). Lactic acid production was determined by enzymatic spectrophotometry, and the pH of the culture medium was measured using a pH meter. Kruskal-Wallis and Dunn's tests were used for pH, viability, biovolume, matrix, and EPS analyses, while one-way ANOVA followed by Tukey's test was applied for CFU/mL counts and lactic acid production (α = 0.05).

Dual-species biofilms showed higher viability and biovolume than S. mutans monospecies biofilms (p < 0.05), whereas the other parameters did not differ among groups (p > 0.05).

Dual-species biofilms of S. mutans and E. faecalis may represent a suitable model for investigating antimicrobial strategies, as their association increased viability and biovolume, which are relevant features in cariology and endodontics.