Mechanistic Insight into the Anti-Bacterial/Anti-Biofilm Effects of Low Chlorhexidine Concentrations on <i>Enterococcus faecalis</i>—In Vitro Study

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

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Bibliographic Details
Main Authors: Nathanyel Sebbane, Itzhak Abramovitz, Nurit Kot-Limon, Doron Steinberg
Format: Article
Language:English
Published: MDPI AG 2024-11-01
Series:Microorganisms
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Online Access:https://www.mdpi.com/2076-2607/12/11/2297
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Summary:Background: Endodontic treatment failures are often linked to the persistence of <i>Enterococcus faecalis</i> in the root canal system. This study aimed to investigate the antibacterial/antibiofilm mechanism of chlorhexidine (CHX), particularly at low concentrations, against <i>E. faecalis</i>, to improve endodontic treatment protocols. Methods: The antibacterial activity of CHX (0.125–20 μg/mL) was evaluated against <i>E. faecalis</i> 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 <i>t</i>-test and ANOVA. Results: CHX demonstrated concentration-dependent inhibition of <i>E. faecalis</i>, 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. Conclusions: CHX at low concentrations disrupts <i>E. faecalis</i> 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.
ISSN:2076-2607