# Comparative Evaluation of the Antibacterial Efficacy of Metronidazole, Chlorhexidine, and Normal Saline as Laser- and Sonic-Activated Intracanal Irrigants Against Enterococcus faecalis: In Vitro Study Protocol

## TL;DR

This study compares how well metronidazole, chlorhexidine, and saline work as root canal cleaners when activated by laser or sonic methods against a stubborn bacteria called Enterococcus faecalis.

## Contribution

The study introduces a novel in vitro protocol to evaluate laser- and sonic-activated irrigants against E. faecalis biofilms in human teeth.

## Key findings

- The antibacterial efficacy of MTR, CHX, and saline will be compared under laser and sonic activation.
- Results will provide insights into optimizing root canal disinfection protocols.
- Findings may improve endodontic treatment outcomes by reducing persistent infections.

## Abstract

The persistence of Enterococcus faecalis is a significant challenge in endodontic therapy, due to its ability to form biofilms and penetrate the dentinal tubules, frequently leading to treatment failures. Therefore, despite the proven antimicrobial activity of metronidazole (MTR) and chlorhexidine (CHX), the resilience of E. faecalis necessitates integrating activation techniques. Laser and sonic activation methods show promise in enhancing the antibacterial performance of irrigants. However, studies on the activation-enhanced efficacy of MTR, CHX, and saline remain limited.

This study aimed to compare the antibacterial efficacy of MTR, CHX, and normal saline as intracanal irrigants when activated by laser and sonic techniques against E. faecalis in extracted human mandibular premolars.

It is a laboratory-based in vitro experimental study involving extracted human teeth. Ninety freshly extracted single-rooted mandibular premolars will be obtained, decoronated, and biomechanically prepared. Canals will be inoculated with E. faecalis (ATCC 29212) and incubated for 7 days to allow biofilm formation. The teeth will then be randomly divided into 3 main groups ([MTR], [CHX], and saline), each subdivided based on the activation method used (laser or sonic). Irrigation protocols will be standardized, and pre- and postirrigation bacterial samples will be collected on paper points and cultured on brain heart infusion agar. Colony-forming units will be counted to evaluate antibacterial efficacy.

As of June 2025, ethical approval for the study has been obtained. Sample collection of extracted human teeth commenced in July 2025, and 90 extracted teeth have been collected as of January 2026, corresponding to the planned sample size. Laboratory procedures and data analysis have not yet commenced.

This study aims to provide evidence-based insights into optimizing root canal disinfection protocols by evaluating and comparing the synergistic effects of antimicrobial agents and advanced activation techniques. The findings could contribute significantly to improving endodontic treatment outcomes and reducing the incidence of persistent infections caused by E. faecalis.