In Situ Antibacterial Activity of Essential Oils with and without Alcohol on Oral Biofilm: A Randomized Clinical Trial

Abstract

Currently, there is little evidence on the <i>in situ</i> antibacterial activity of essential oils (EO) without alcohol. This study aimed to evaluate <i>in situ</i> the substantivity and antiplaque effect on the plaque-like biofilm (PL-biofilm) of two solutions, a traditional formulation that contains EO with alcohol (T-EO) and an alcohol-free formulation of EO (Af-EO). Eighteen healthy adults performed a single mouthwash of: T-EO, Af-EO, and sterile water (WATER) after wearing an individualized disk-holding splint for 2 days. The bacterial viability (BV) and thickness of the PL-biofilm were quantified at baseline, 30 s, and 1, 3, 5, and 7 h post-rinsing (Test 1). Subsequently, each volunteer wore the splint for 4 days, applying two daily mouthwashes of: T-EO, Af-EO, and WATER. The BV, thickness, and covering grade (CG) of the PL-biofilm were quantified (Test 2). Samples were analyzed by confocal laser scanning microscopy after staining with the LIVE/DEAD® BacLight™ solution. To conduct the computations of the BV automatically, a Matlab toolbox called Dentius Biofilm was developed. In test 1, both EO antiseptics had a similar antibacterial effect, reducing BV after a single rinse compared to the WATER, and keeping it below baseline levels up to 7 h post-rinse (<i>P</i> < 0.001). The mean thickness of the PL-biofilm after rinsing was not affected by any of the EO formulations and ranged from 18.58 to 20.19 μm. After 4 days, the T-EO and Af-EO solutions were significantly more effective than the WATER, reducing the BV, thickness, and CG of the PL-biofilm (<i>P</i> < 0.001). Although, both EO antiseptics presented a similar bactericidal activity, the Af-EO rinses led to more significant reductions in the thickness and CG of the PL-biofilm than the T-EO rinses (thickness = 7.90 vs. 9.92 μm, <i>P</i> = 0.012; CG = 33.36 vs. 46.61%, <i>P</i> = 0.001). In conclusion, both essential oils antiseptics had very high immediate antibacterial activity and substantivity <i>in situ</i> on the 2-day PL-biofilm after a single mouthwash. In the 4-day PL-biofilm, both essential oils formulations demonstrated a very good antiplaque effect <i>in situ</i>, although the alcohol-free formula performed better at reducing the biofilm thickness and covering grade.