Introduction: Endodontic treatment in the presence of resistant Enterococcus faecalis biofilms remains challenging due to the organism’s ability to form resilient biofilms, leading to persistent infections and treatment failure. Calcium hydroxide (CH) is widely used as an intracanal medicament, but biofilm formation may reduce its effectiveness. Combining CH with antibacterial agents such as chitosan, pantoprazole, and nisin may improve antimicrobial efficacy. This study evaluated the combined effects of CH with these agents on E. faecalis biofilm eradication.
Methods: E. faecalis (ATCC 29212) was cultured in heart brain infusion broth at 37 °C. CH, nisin, pantoprazole, and chitosan were prepared, and minimum inhibitory concentrations (MICs) were determined by broth microdilution. Biofilm formation was quantified using crystal violet staining, and changes in biofilm-related gene expression (gelE and efaA) were assessed by quantitative PCR. Statistical analyses were performed using STATA and ANOVA.
Results: Nisin exhibited the strongest growth inhibition (MIC: 125 μg/mL). Chitosan caused the greatest reduction in biofilm biomass (mean OD: 0.262 ± 0.078, *P* < 0.001). The combination of chitosan with CH and EDTA reduced OD to 0.235 ± 0.065, significantly more than other combinations (*P* < 0.001). Chitosan and nisin significantly downregulated gelE and efaA expression (*P* < 0.05).
Conclusion: The combination of CH with chitosan significantly reduced E. faecalis biofilm biomass and suppressed biofilm-related gene expression, suggesting its potential as an adjunctive agent in endodontic therapy. Nisin also demonstrated significant antimicrobial activity, while pantoprazole had relatively limited effects. These findings support the inclusion of chitosan in intracanal medicament strategies to improve treatment outcomes.