Mechanistic Insight into the Anti-Bacterial/Anti-Biofilm Effects of Low Chlorhexidine Concentrations on Enterococcus...

Endodontic treatment failures are often linked to the persistence of Enterococcus faecalis in the root canal system. This study aimed to investigate the antibacterial/antibiofilm mechanism of chlorhexidine (CHX), particularly at low concentrations, against E. faecalis, to improve endodontic treatment protocols.

The antibacterial activity of CHX (0.125-20 μg/mL) was evaluated against E. faecalis ATCC 29212 using various assays, including planktonic growth inhibition, colony-forming units (CFUs), membrane permeability and potential assays, high-resolution scanning electron microscopy (HR-SEM), confocal laser scanning microscopy of biofilms, biomass and metabolic activity assays on matured biofilm, and quantitative real-time PCR for gene expression. Statistical analysis was performed using Student's t-test and ANOVA.

CHX demonstrated concentration-dependent inhibition of E. faecalis, significantly reducing planktonic growth and CFUs. Membrane assays showed increased permeability and depolarization, indicating damage. HR-SEM revealed morphological changes, such as pore formation, while confocal microscopy showed a reduction in biofilm mass and extracellular substances. Gene expression analysis indicated the downregulation of virulence genes and upregulation of stress response genes.

CHX at low concentrations disrupts E. faecalis at multiple levels, from membrane disruption to gene expression modulation, affecting mature biofilm. These findings support the refinement of endodontic disinfection protocols to reduce microbial persistence.