Toluidine blue O-mediated antimicrobial photodynamic inactivation reinforced with naringenin against Streptococcus...

Combining antimicrobial photodynamic therapy (aPDT) with phytochemicals may enhance antibiofilm activity while reducing reliance on higher photosensitizer concentrations. This study evaluated toluidine blue O (TBO)-mediated aPDT and the adjunctive effect of naringenin (NAR) against Streptococcus mutans biofilms.

Minimum inhibitory concentrations (MICs) of TBO and NAR against S. mutans were determined. Checkerboard assays assessed drug interaction using the fractional inhibitory concentration index (FICI). Mature S. mutans biofilms were allocated to eight groups: control, TBO (100 µg/mL), NAR (156.25 µg/mL), red LED (630 nm, 60 J/cm²), TBO (100 µg/mL) + LED, TBO (6.25 µg/mL) + LED, NAR + TBO (6.25 µg/mL) + LED, and 0.2 % chlorhexidine (CHX). Colony-forming unit (CFU) and crystal violet staining assays were performed.

There was a synergistic interaction at NAR 156.25 µg/mL plus TBO 6.25 µg/mL (FICI = 0.375). Compared with the control, viable counts were significantly lower only in photoactivated TBO (all p < 0.0001). NAR plus TBO (6.25 µg/mL)-aPDT resulted in lower viable counts than TBO (6.25 µg/mL)-aPDT (p < 0.0001) and was not significantly different from TBO (100 µg/mL) + LED (p = 0.69). Crystal violet staining showed reduced biofilm compared to control with TBO (100 µg/mL)-aPDT (p < 0.0001), NAR + TBO (6.25 µg/mL)-aPDT (p < 0.0001), TBO (6.25 µg/mL)-aPDT (p = 0.002), and CHX (p = 0.001). Adding NAR was not significant versus TBO (6.25 µg/mL)-aPDT (p = 0.09) and was comparable to TBO (100 µg/mL)-aPDT (p = 0.51).

NAR enhanced TBO-aPDT killing of S. mutans biofilms, enabling low-concentration TBO-aPDT to match high-concentration efficacy TBO. Further studies are warranted to confirm efficacy and applicability.