Antimicrobial and antibiofilm properties of Malaysian stingless bee propolis against Enterococcus faecalis: an in vitro study for endodontic intracanal application

Enterococcus faecalis is a persistent pathogen associated with endodontic treatment failures due to its resistance to most common intracanal medicament, non-setting calcium hydroxide [NS-Ca(OH)₂]. The limitations of current treatment have led to interest in researching alternative compounds, with stingless bee propolis emerging as a possible option. Hence, this study aimed to evaluate the antimicrobial efficacy of its ethanolic extracts from three Malaysian stingless bee species (Heterotrigona itama, Geniotrigona thoracica, Tetrigona apicalis) against both E. faecalis ATCC 29212 and ATCC 4082 via agar-well diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), antibiofilm, anti-adherence assays, time-kill study (TKS), and scanning electron microscope (SEM) analysis. Further evaluation was made on potential synergistic effects compared to positive controls, 45 mg/mL NS-Ca(OH)₂ and 2% (20 mg/mL) chlorhexidine (CHX) gel. Statistical analyses were conducted using two-way ANOVA (for agar-well diffusion, antibiofilm, and anti-adherence) and repeated-measure ANOVA (for TKS). For non-normally distributed data, the Kruskal-Wallis test (for MIC and MBC) was used. CHX gel demonstrated the strongest antimicrobial effect for most of the microbiological tests (agar-well diffusion; ATCC 29212 = 16 ± 0.6 mm, antibiofilm effect; ATCC 29212 = 92.40 ± 1.70% and ATCC 4082 = 91.31 ± 1.18%, anti-adherence effect; ATCC 29212 = 85.54 ± 0.65% and ATCC 4082 = 90.07 ± 0.70%). Notably, propolis obtained from the same varied in its antimicrobial properties as H. itama propolis demonstrated the highest efficacy among the propolis extracts, with low MIC values (ATCC 29212 = 1.56 mg/mL (IQR: 1.56), ATCC 4082 = 0.78 mg/mL (IQR: 0.78)) and complete bactericidal activity (0 CFU/mL) within 24 hours. Its anti-adherence activity (ATCC 29212 = 68.50 ± 4.11% and ATCC 4082 = 76.54 ± 2.03%) was also substantially higher than NS-Ca(OH)₂. In contrast, other propolis extracts showed comparatively weaker and inconsistent effects. Synergistic effect was observed in extract mixture against ATCC 4082 (ΣFIC = 0.364), while additive effect for ATCC 29212 (ΣFIC = 0.752). SEM revealed cellular disruption in both treated E. faecalis strains. Regarding bacterial strains, E. faecalis ATCC 4082 was observed to be more susceptible to NS-Ca(OH)2, CHX gel, and all three propolis extracts compared to ATCC 29212 based on all microbiological test. H. itama propolis exhibits strong antimicrobial potential comparable to conventional intracanal medicaments, supporting its potential application as an alternative agent in endodontic medicament.