Combined treatment with bacteriophages and sodium hypochlorite against E. faecalis biofilm in an in vitro root canal model with evaluation of bacterial resistance

Aim To assess the time-dependent antimicrobial activity of a newly isolated bacteriophage against Enterococcus faecalis biofilms in two in vitro models, aloneand combinedwith sodium hypochlorite, followed by the evaluation of potential resistance development.Methodology Bacteriophage CUB_EF02 was isolated from sewage water using E. faecalis ATCC 29212 as host, visualized by TEM, and sequenced. Its antimicrobial effect against biofilms grown on porous glass beads was assessed in a time-killing assay. Additionally, 10-day-old biofilms were established in 36 extracted single-rooted human teeth and treated with 0.9% NaCl, 1% NaOCl, phages, or their combination. Dentine-adherent biofilm was quantified by plate counts at baseline, after treatment, and after 48 h re-incubation. Logarithmic reduction factors were analyzed using one-way ANOVA and Tukey HSD. Phage resistance was evaluated by Top-Agar overlay spot assay.Results Phage treatment reduced biofilm by 1.35 ± 0.2 SD-log after 6 h compared to control. Irrigation of the root canals using NaOCl achieved a significantly higher reduction (LRFred. = 2.98 ± 0.77 SD) than phages alone(LRFred. = 1.50 ± 0.23 SD). Combined treatment showed the strongest antimicrobial effect (LRFred. = 4.05 ± 1.17 SD). Bacterial regrowth occurred in all groups. Resistance developed after phage incubationfor ≥6 h.Conclusion Bacteriophages represent a promising adjunctive in root canal disinfection, but remain less effective than NaOCl and may inducebacterial resistance.