Anti-biofilm Chemotherapy Modulates Expression of Streptococcus mutans Virulence in vivo

Fluoride is the mainstay of dental caries prevention, yet current applications offer incomplete protection and may not effectively address the infectious character of the disease.

Objectives: We sought to evaluate the impact of a novel therapeutic approach (combining naturally-occurring anti-biofilm agents with fluoride) on development of carious lesions in vivo, and to identify mechanisms by which topical treatments alter the transcriptional and biochemical properties of S. mutans within biofilms.

Methods: S. mutans UA159 was used to form biofilms on saliva-coated hydroxyapatite discs and to infect Sprague-Dawley rats in a caries model. Treatment regimens, simulating those experienced clinically, were as follows:  twice-daily exposure for 60 s with (1) combination therapy (CT; 2 mM myricetin, 4 mM tt-farnesol, 250 ppm fluoride), (2) 250 ppm fluoride or (3) vehicle control.  Microarrays, RT-qPCR, fluorescence imaging, and biochemical assays identified potential therapeutic targets.  In vivo effectiveness was evaluated from lesion incidence/severity, while plaque was processed for microbiological/transcriptional assessment.

 Results: Genes involved in stress tolerance and EPS synthesis (sloA, sodA, copY, and gtfB) and a unique gene (patB) were severely repressed by CT.  patB, encoding a putative hemolysin, was identified as a potentially novel therapeutic target, as deletion disrupted S. mutans biofilm formation/3D-architecture.  Repression of these targets (sustained hours after treatment) correlated with decreased EPS-matrix production and acid-stress fitness, all without biocidal effect.   CT treatment decreased the incidence and severity of smooth and sulcal surface caries compared to the vehicle, and CT effectiveness was superior to fluoride alone.  S. mutans sloA and gtfB expression was repressed in plaque, while viability of the oral flora was unaffected.  This provides evidence that CT specifically targets the expression of S. mutans virulence in vivo.

Conclusion: CT may represent an innovative, highly-effective, and selective approach to disrupt virulent plaque-biofilm formation, reducing development of dental caries, a costly and ubiquitous disease.