An in vitro comparison of cold atmospheric plasma and methylene blue-mediated antimicrobial photodynamic therapy with 660 nm laser on Streptococcus mutans biofilms

This study aimed to comparatively evaluate the antimicrobial and antibiofilm efficacy of helium-based cold atmospheric plasma (CAP) and methylene blue-mediated antimicrobial photodynamic therapy (MB-aPDT) against mature Streptococcus mutans biofilms formed on human enamel surfaces. In this study, standardized enamel slabs prepared from extracted human premolars were inoculated with S. mutans ATCC 35,668 and incubated for 7 days to allow mature biofilm formation in the presence of sucrose and human saliva. Specimens were randomly allocated to nine experimental groups (n = 10): negative control, 0.2% chlorhexidine (CHX), laser only (660 nm, 100 mW, 20 J/cm², 100 s), 0.02% methylene blue (MB) only, MB-aPDT (0.02% MB + 660 nm laser), and CAP applied for 90, 120, 150, and 180 s. Quantitative assessment of biofilm viability was performed by colony-forming unit (CFU/mL) counting. Structural alterations were qualitatively analyzed using scanning electron microscopy (SEM). Data were analyzed using one-way ANOVA and Tukey post hoc tests (α = 0.05). Both MB-aPDT and CAP significantly reduced S. mutans biofilm viability compared with the untreated control (p < 0.001). MB-aPDT achieved a 90.7% reduction in CFU counts. CAP demonstrated a time-dependent antibacterial effect, with progressive reductions from 53.5% at 90 s to 87.0% at 180 s. MB-aPDT was significantly more effective than CAP at 90, 120, and 150 s (p < 0.001), whereas no significant difference was observed between MB-aPDT and CAP at 180 s (p = 0.266). SEM analysis corroborated quantitative findings, revealing marked disruption of biofilm architecture following MB-aPDT and CAP-180 s treatments. Both MB-aPDT and helium-based CAP exhibit significant antibiofilm activity against mature S. mutans biofilms on enamel surfaces. While MB-aPDT demonstrated superior efficacy compared with shorter CAP exposures, prolonged CAP treatment achieved comparable antibacterial effects. These findings support the potential integration of both modalities as adjunctive, minimally invasive strategies for biofilm control in preventive and restorative dentistry.