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 in situ antibacterial activity of essential oils (EO) without alcohol. This study aimed to evaluate in situ 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 (P < 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 (P < 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, P = 0.012; CG = 33.36 vs. 46.61%, P = 0.001). In conclusion, both essential oils antiseptics had very high immediate antibacterial activity and substantivity in situ 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 in situ, although the alcohol-free formula performed better at reducing the biofilm thickness and covering grade.

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“…5 Bacterial ability to form biofilms on the tooth surface, i.e., dental plaque is a factor that increases possibility for the development of both caries and periodontitis. 6 Bacterial plaque can be removed with tooth brushing and dental floss, but usage of mouth rinse could be one more step towards complete control of this problem.…”

In situ substrate-formed biofilms using IDODS mimic supragingival tooth-formed biofilms

“…This procedure defines the inclusion and exclusion criteria applied [15]. The inclusion criteria were as follows: systemically healthy adult volunteers aged between 20 and 45 with a good periodontal health status (a minimum of 24 permanent teeth with no evidence of gingivitis or periodontitis – community periodontal index score = 0-) [16] and an absence of untreated caries at the start of the study).…”

“…The protocol described by Quintas et al [15] was followed to evaluate the different fields within the discs, with the thickness and bacterial viability analysed. In each field, the maximum biofilm thickness was divided into three layers: the outer layer (layer 1), the middle layer (layer 2), and the inner layer (layer 3).…”

“…Data were captured using the same settings in all cases, and certain parameters were established following previous investigations by our group [15]. The quantification of bacterial viability in the series of XY images was determined using a cytofluorographic analysis (Leica confocal software).…”

“…In this analysis, the images of each fluorochrome were defined as ‘channels’ (SYTO 9 occupies the green channel and propidium iodide the red channel). For automatic computations, a Matlab toolbox called the Dentius Biofilm was developed, and its usability characteristics have already been described [15]. Determination of the mean viability percentage in each field required sections with a minimum biofilm area of 250 µm 2 (approximately 4,750 pixels).…”