1. Introduction
Persistent endodontic infections pose a significant challenge in clinical practice (1). Persistent endodontic infections are polymicrobial in nature; however, E. faecalis has been detected in 24–77% of failed root canal treatments, making it one of the most clinically relevant pathogens associated with retreatment cases (2). Its survival is attributed to resistance to nutrient deprivation, tolerance to alkaline stress, and the ability to penetrate dentinal tubules and form structured biofilms. The increasing emergence of vancomycin-resistant E. faecalis (VRE) strains further highlights the need for alternative antimicrobial strategies in endodontics (3). Totarol has demonstrated reported MIC values ranging from 0.25–4 µg/mL against Gram-positive bacteria in previous microbiological studies, suggesting potent membrane-targeting and electron-transport inhibitory activity (3). Despite these promising antimicrobial characteristics, its application as an intracanal medicament against mature endodontic biofilms remains insufficiently investigated. These traits enable E. faecalis to persist in root canals despite conventional chemomechanical preparation and irrigation (4).
Calcium hydroxide has long been the standard intracanal medicament because of its high pH and bactericidal activity mediated by hydroxyl ions (5). However, its effectiveness against E. faecalis is often limited due to dentin buffering and the bacterium's intrinsic alkaline tolerance (6-7).
Totarol is a naturally occurring phenolic diterpene derived from Podocarpus species (3). It has demonstrated potent activity against several Gram‑positive bacteria through mechanisms involving disruption of bacterial membranes, interference with metabolic pathways, and inhibition of electron transport (3). Unlike Ca (OH)₂, Totarol is lipophilic, which may allow enhanced penetration into biofilm matrices and dentinal tubules. This property suggests a potential advantage in eradicating bacteria protected within biofilms. The present study was designed to compare the antibacterial and antibiofilm effects of Totarol (Bioactive Totarol Ltd., New Zealand) and Ca (OH)₂ (Metapaste, Meta Biomed. Korea) using clinical E. faecalis isolates in both in vitro assays and an ex vivo 21‑day root canal biofilm model. The null hypothesis was that no significant difference exists between Totarol and Ca (OH)₂ in their antimicrobial effectiveness against E. faecalis.
2. Materials and Methods
2.1 Bacterial Identification
Patients requiring endodontic treatment for pulpal necrosis associated with periapical lesions were recruited from the Karbala Dentistry Center, Iraq. The diagnosis was established based on clinical examination and radiographic findings obtained in the Diagnostic Unit.
Inclusion criteria were: (i) patients aged 25–45 years; (ii) single-rooted teeth diagnosed with pulpal necrosis and periapical pathology; and (iii) the presence of clinical symptoms indicative of endodontic infection. Exclusion criteria included: (i) current or recent use of systemic antibiotics; (ii) teeth with extensive coronal destruction that precluded proper rubber dam isolation; and (iii) teeth presenting with root or crown cracks. Under aseptic conditions, the tooth was isolated with a rubber dam, and the tooth and surrounding area were disinfected with 10% povidone–iodine. Access cavity preparation was done in two steps: first, a sterile carbide fissure bur was used to remove all caries and coronal restorations, followed by disinfection; second, a low-speed round carbide bur was used to remove the roof of the pulp chamber and complete cleaning. A new sterile #20 file was then placed in the root canal to the working length (confirmed radiographically), rotated twice clockwise, and immediately transferred into a tube containing sterile Amies transport medium (8). Because Enterococcus faecalis is a facultative anaerobe, samples were transported under both aerobic and anaerobic conditions to maximize bacterial recovery and then isolated within two hours.
Experiments were performed using a single confirmed clinical isolate of Enterococcus faecalis. All assays were repeated to ensure experimental reproducibility.
2.2 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for Totarol
MIC values for Totarol were determined using a broth dilution method with decreasing concentrations according to the Clinical and Laboratory Standards Institute guidelines (9). Tubes containing Mueller–Hinton broth were inoculated with standardized bacterial suspensions and incubated at 37°C for 24 hours under aerobic conditions. MIC was defined as the lowest concentration of Totarol that prevented visible turbidity. For MBC determination, aliquots from tubes without visible growth were sub‑cultured into agar plates (10). The lowest concentration yielding no bacterial growth on agar was considered the MBC. MIC/MBC determination for Ca (OH)₂ was not performed because its paste formulation and alkaline antimicrobial mechanism are not compatible with standardized broth dilution methods.
2.3 Agar Diffusion Assay
Muller‑Hinton agar plates were inoculated with standardized 0.5 McFarland suspensions of E. faecalis to create a uniform bacterial lawn (11). Sterile wells were prepared in the agar each with a diameter of 6 mm and a depth of 4 mm, were created in each agar plate using agar punch and filled with Totarol at the MBC concentration determined in broth dilution testing and Ca (OH)₂ paste. The plates were incubated aerobically at 37 °C for 24 hours, and the diameter of the inhibition zones around each well was measured in millimeters using an electronic Vernier caliper (12).
2.4 Agar Diffusion Assay
Extracted human single-rooted teeth with fully formed apices were selected. The teeth selected for this study were first examined carefully, and any teeth showing cracks or defects in the cementum were excluded. Extracted teeth were stored in sterile saline at room temperature until use to prevent dehydration. The crowns were removed to obtain root segments of about 12 mm in length. A size 15 K-file was used to confirm apical patency and create a glide path. The working length was set 1 mm short of the point where the file tip became visible at the apical foramen. Specimens were randomly allocated into experimental groups (n = 10 per group). Root canals were shaped with a crown-down technique using the ProTaper Next files (X1–X4). Final irrigation for 1 minute was done with 17% EDTA and 2.5% NaOCl to remove debris and the smear layer, then the canals were rinsed with sterile distilled water. After instrumentation, the apical foramina of all roots were sealed with a flowable composite resin. The specimens were then placed vertically in silicone impression blocks. Before inoculation, they were sterilized in an autoclave at 121°C and 15 psi for 30 minutes. Resin integrity and apical sealing were verified after sterilization to ensure structural stability.
2.5 Biofilm Development (21‑Day Model)
One milliliter of bacterial isolates grown in Brain–heart infusion broth (BHI) for 24 hours was transferred into 5 mL of fresh Brain Heart Infusion broth to prepare the bacterial suspension. Using sterile 1-mL insulin syringes, the suspension was carefully injected into each root canal, taking care to avoid any overflow. A size 15 sterile K-file was then introduced with a single in-and-out movement to help spread the bacteria along the full length of the canal. After inoculation, the roots were stored in an incubator at 37°C for 21 days under aseptic conditions. All procedures were performed inside a laminar flow cabinet to minimize contamination risk. To maintain bacterial viability, fresh suspension was added every three days. Sterile broth-only controls were incubated in parallel to confirm absence of contamination during the biofilm development period.
2.6 Intracanal Medication
After biofilm maturation, specimens were randomly assigned into two groups: Totarol and Ca (OH)₂. Totarol was applied inside the canals at its MBC concentration using 30-gauge syringe. Ca (OH)₂ paste was placed using a Lentulo spiral according to manufacturer instructions in the canals of the comparator group After that, access cavities were sealed with temporary restorative material. All specimens were incubated at 37°C under aerobic conditions for an additional 3 days to allow interaction between medicaments and the biofilm. Totarol was dissolved in dimethyl sulfoxide (DMSO) to ensure complete solubility for intracanal application. A DMSO vehicle-control was tested separately and produced no measurable antibacterial effect under the experimental conditions. Each medicament was applied in the form consistent with its standard laboratory or commercial preparation to reflect realistic clinical handling conditions. The study aimed to compare functional antimicrobial performance rather than identical formulation viscosity.
A 3-day medication period was intentionally selected to evaluate short-term antimicrobial performance under standardized laboratory conditions.
2.7 Dentin Shavings Collection and CFU Enumeration
At the end of the medication period, dentin shavings were collected from the canal walls using sterile H file instruments (H-file, Dentsply Maillefer, USA) with 10 standardized vertical strokes. The shavings were transferred into tubes containing 1 mL of sterile transport medium and vortexed for 30 seconds at high speed to detach adherent bacteria from dentin. Serial dilutions were prepared and plated on agar. After incubation, CFU were counted and expressed as CFU ×10³. Baseline and post‑medication CFU counts were recorded for each group.
2.8 Statistical Analysis
All experimental data were analyzed using SPSS software (v. 26). Normality was verified prior to evaluation. Paired t-tests were used to evaluate within-group changes (before vs after medication). Independent t-tests were applied for direct comparison between Totarol and Ca (OH)₂ groups. One-way ANOVA followed by Tukey HSD post-hoc testing was used to confirm overall differences among experimental conditions. Data were expressed as mean ± SD, and statistical significance was set at p < 0.05 (highly significant when p < 0.0001).
3. Result
Statistical analysis demonstrated a pronounced reduction in bacterial load following totarol treatment relative to baseline. Raw CFU values were consistent across specimens and fell within a narrow experimental range, supporting measurement reliability. Confidence interval analysis confirmed the statistical robustness of the observed differences between groups. MIC and MBC testing were performed in triplicate to ensure reproducibility. Agar diffusion testing followed standardized well-diameter methodology; identical well dimensions were maintained across all groups to ensure valid comparative interpretation. The proximity between MIC and MBC values suggests strong bactericidal activity, although time-kill dynamics were not evaluated in this study. Data distribution normality was verified prior to parametric testing, and confidence interval analysis supported the statistical reliability of the observed reductions.
3.1 Identification of isolated bacteria
Colonies grew well on blood agar and showed no hemolysis. They were sized 1.0–1.5 mm and had a circular, smooth, raised edge. E. faecalis grows on Pfizer agar, resulting in a black colony radius. The VITEK 2 system confirmed identification with ≥99% confidence (Table 1).