High-throughput Bioluminescence Assessment of Argon Plasma Efficacy Against Streptococcus mutans

Objectives: To determine the antibacterial efficacy of cold argon plasma against biofilms of Streptococcus mutans using a real-time and high-throughput (HTS) bioluminescence assay and viable colony counts (VCC).
Methods: Specimens (n=16/group; d= 6.0mm; h=0.5mm) of Point 4Ô resin-composite (Kerr Corp., USA) were fabricated, polymerized (20s/each; VALO, Ultradent Products, Inc.) and wet-polished (180-1200grit SiC; final 0.5mm diamond suspension). Specimens were then immersed in sterile ultrapure water for monomer extraction (37°C, 24h). Bioluminescent Streptococcus mutans (UA159::JM10) biofilms were grown onto UV-sterilized specimens (24h) using 0.65xTHY+0.1% sucrose (w/v; anaerobic, 37°C). Biofilms were individually treated (30s, 90s, 120s or 150s) with cold plasma (Model SAP, Surface Engineering and Plasma Solutions, Brazil). Specimens that were left untreated (positive control), or treated using either chlorhexidine gluconate (CHX; n=18, 2min; negative control) or argon (150s; treatment control) served as control groups. After treatment, cells were replenished using fresh 1xTHY+1.0% glucose and incubated (37°C, 1h) in preparation for bioluminescence (BL) and VCC. Measurements of biofilms’ BL (Relative Luminescence Units [RLU]) were obtained with a Synergy HT (Biotek, USA) after the addition of 100mM D-Luciferin substrate. Inter-group comparisons were statistically analyzed for BL and VCC using General Linear Models and post-hoc Student-Newman-Keuls tests (a=0.05; SAS software).


Results: Mean values of BL indicated that plasma treatment for 90s, 120s or 150s resulted in statistically significantly lower metabolic activity (p<0.001) when compared to those of positive control groups or treated with 30s plasma. Mean VCC values corroborated BL results and have indicated that cells’ viability decreased in a time-dependent manner. Biofilms treated with CHX (2min) displayed the lowest mean values of BL and VCC amongst all groups investigated.
Conclusions: Plasma application was demonstrated to significantly lower the metabolism and viability of S. mutans in a time-dependent manner, and therefore can be used as an adjunct intra-oral surface-decontamination strategy.