Evaluation of extracellular DNA present in the extracellular matrix of mixed-species cariogenic biofilms

Objectives: Dental caries is a public health problem worldwide, caused by a cariogenic biofilm, which is a community of microbial cells that are covered and immersed in a three-dimensional extracellular matrix (ECM). Streptococcus mutans coordinates the construction of the ECM in the presence of dietary sucrose and starch, beginning the pathogenesis of dental caries. The objectives were to evaluate the presence of extracellular DNA (eDNA) in the extracellular matrix (ECM) of mixed-species cariogenic biofilms.
Methods: Biofilms were formed with three strains: S. mutans UA159 (parental and knockout mutant strains), Actinomyces naeslundii ATCC12104 and Streptococcus gordonii DL-1 on hydroxyapatite discs coated with pellicle. The culture media contained saliva, sucrose and starch, simulating a cariogenic challenge (37°C and 5%CO₂). Mutant strains of the two component system lytTS (ΔSMu0525 e ΔSMu0526), the operon dltABCD (ΔSMu1538 e ΔSMu1541), and the gene gtfB were respectively used to modulate the presence of eDNA, lipotheicoic acids (LTA), and insoluble exopolysaccharides of S. mutans in the ECM of 67h-old biofilms. Biofilms were processed to assess colony-forming units (UFC/ml), total dry weight (biomass) and eDNA.
Results: S. mutans, S. gordonii, and A. naeslundii were present in all biofilms formed by S. mutans parental and mutant strains. The proportion of the three species was similar for biofilms formed by S. mutans parental strain. However, S. gordonii was detected in higher proportion in biofilms grown with ΔgtfB, while the proportion of S. gordonii and A. naeslundii was similar in the other biofilms. In addition, there was no difference in the amount of biomass and eDNA content of biofilms analyzed (p>0.05, one way ANOVA).
Conclusions: At the biofilm age tested, all strains were detected, while there were no differences on biofilm biomass and on eDNA content. Therefore, further analyses of exopolysaccharides and LTA composition of the ECM at distinct biofilm developmental stages may reveal the extent of eDNA and LTA role on cariogenic biofilms.