Effect of arginine-based synbiotics on multispecies biofilm.

Objective: To examine the effect of arginine (Arg)-based synbiotics on multispecies biofilm.

Methods: In vitro biofilms (Streptococcus mutans UA159, S. gordonii DL1, S. sanguinis DSS-10) were grown on HA discs under anaerobic conditions (37°C, 5% CO₂, 24 h) and treated with: (1) Arg (0.25%, 0.5% w/v.), (2) Lacticaseibacillus rhamnosus GG (LRG) at 10⁷ CFU/mL, or (3) their combinations, 2× daily for 3 days. At 96 h, biofilm matrix components (exoproteins, eDNA, and carbohydrates) and microbial viability (confocal microscopy and PMA-qPCR) were quantified. Relative gene expression analysis was also conducted with species-specific genes (gtfB, sagP, arcA, argG, argH).

Results: For carbohydrates, no significant difference was identified among treatment groups (p>0.05). Protein content for 0.5% Arg+LRG was significantly lower than LRG (p<0.05). The lowest eDNA content was observed in LRG (p<0.05); while eDNA content of 0.5% Arg+LRG, was similar to 0.5% Arg and control (p>0.05). Using confocal imaging, the highest proportion of live cell was found in 0.5% Arg+LRG (p<0.05), followed by 0.25% Arg+LRG, 3-D biofilm imaging demonstrated increased biomass with smoother architecture in biofilms treated with 0.5% Arg+LRG (p<0.05). 0.5% Arg+LRG significantly enhanced growth of commensal streptococci (S. gordonii, S. sanguinis) compared to monotherapies and control, while also reducing viability of S. mutans compared to control (p<0.05). Gene expression analysis revealed downregulation of S. mutans virulence (gtfB) and upregulation of commensal metabolism (sagP, arcA) for 0.5% Arg+LRG (p<0.05).

Conclusion: The 0.5% Arg+LRG synbiotics uniquely integrates ecological modulation by regulating biochemical matrix components, promoting commensal enrichment and suppressing cariogenic pathogens.

Clinical significance: A deliverable Arg-LRG synbiotics for caries prevention addresses a global public health priority. This strategy aligns with microbial homeostasis principles, presenting a novel paradigm for caries prevention. The Arg-LRG synbiotics can counter the limitations of fluorides to establish a diverse oral microbiome, imparting an ecologically driven approach to caries prevention.