The anti-biofilm effect of ozonated water and super-oxidized water applied with different activation procedures against Enterococcus faecalis biofilm

The objective of this in vitro study was to assess the anti-biofilm efficacy of super-oxidized water and ozonated water, activated through various methods, against Enterococcus faecalis (E. faecalis) biofilms within the root canal system. Seventy-four single-root mandibular premolars were prepared, inoculated with E. faecalis, and incubated for 4 weeks to allow mature biofilm formation. Infected root canals were divided into 7 experimental groups (n = 10), including 3 main experimental groups consisting of activation methods (Standard needle irrigation-SNI, EndoActivator-EA, Erbium: yttrium-aluminum-garnet-Er: YAG laser), 2 sub-experimental groups consisting of irrigation solutions (Super-oxidized water, ozonated water) and a positive control group consisting of sodium hypochlorite (NaOCl). Biofilm was assessed using colony-forming units (CFU) on blood agar plates. The data were statistically evaluated using the Mann-Whitney U and Kruskal-Wallis tests, with the significance threshold set at 0.05. Super-oxidized water showed significantly higher anti-biofilm efficacy than ozonated water across all activation methods (p < 0.05). EA combined with super-oxidized water was more effective than Er: YAG laser (p < 0.05). Ozonated water groups showed no significant differences between activation methods (p > 0.05). NaOCl exhibited significantly higher anti-biofilm activity compared to all other groups (p < 0.05). The results demonstrated that neither super-oxidized water nor ozonated water was capable of achieving complete bacterial elimination within the root canal system following the application of SNI, EA, or Er: YAG laser techniques. In addition, it can be concluded that the activation of super-oxidized water with EA has a remarkable anti-biofilm effect. It was found that super-oxidized water was more effective in bacterial elimination than ozonated water in all activation methods.