Ex vivo vs. in vivo antibacterial activity of two antiseptics on oral biofilm

Isabel Prada-López, Víctor Quintas, Maria A Casares-De-Cal, Juan A Suárez-Quintanilla, David Suárez-Quintanilla, Inmaculada Tomás, Isabel Prada-López, Víctor Quintas, Maria A Casares-De-Cal, Juan A Suárez-Quintanilla, David Suárez-Quintanilla, Inmaculada Tomás

Abstract

Aim: To compare the immediate antibacterial effect of two application methods (passive immersion and active mouthwash) of two antiseptic solutions on the in situ oral biofilm.

Material and methods: A randomized observer-masked crossover study was conducted. Fifteen healthy volunteers wore a specific intraoral device for 48 h to form a biofilm in three glass disks. One of these disks was used as a baseline; another one was immersed in a solution of 0.2% Chlorhexidine (0.2% CHX), remaining the third in the device, placed in the oral cavity, during the 0.2% CHX mouthwash application. After a 2-weeks washout period, the protocol was repeated using a solution of Essential Oils (EO). Samples were analyzed for bacterial viability with the confocal laser scanning microscope after previous staining with LIVE/DEAD® BacLight™.

Results: The EO showed a better antibacterial effect compared to the 0.2% CHX after the mouthwash application (% of bacterial viability = 1.16 ± 1.00% vs. 5.08 ± 5.79%, respectively), and was more effective in all layers (p < 0.05). In the immersion, both antiseptics were significantly less effective (% of bacterial viability = 26.93 ± 13.11%, EO vs. 15.17 ± 6.14%, 0.2% CHX); in the case of EO immersion, there were no significant changes in the bacterial viability of the deepest layer in comparison with the baseline.

Conclusions: The method of application conditioned the antibacterial activity of the 0.2% CHX and EO solutions on the in situ oral biofilm. The in vivo active mouthwash was more effective than the ex vivo passive immersion in both antiseptic solutions. There was more penetration of the antiseptic inside the biofilm with an active mouthwash, especially with the EO. Trial registered in clinicaltrials.gov with the number NCT02267239. URL: https://ichgcp.net/clinical-trials-registry/NCT02267239.

Keywords: PL-biofilm; antiseptic; chlorhexidine; essential oils; immersion; mouthwash.

Figures

Figure 1
Figure 1
Protocol of the study.
Figure 2
Figure 2
Intraoral view of the Intraoral Device Overlaid Disk-holding Splint (IDODS).
Figure 3
Figure 3
Representative images of the PL-biofilm (“stacked projection” of images in the “Z” axis) bacterial viability under basal conditions, after immersion and after mouthwash with 0.2% Chlorhexidine and Essential Oils. (They are images of representative fields of the PL-biofilm. It is a maximum projection of all obtained images in the plane XY in the Z axis for a same field. That is commonly called “stacked projection.” These images do not represent nor the outer, the middle or the inner layers, they represent all of them projected in the same axis).
Figure 4
Figure 4
Total bacterial viability and by PL-biofilm layers in the 0.2% Chlorhexidine series. (PL-biofilm, plaque like-biofilm; 0.2% CHX, 0.2% of Chlorhexidine; CLSM, confocal laser scanning microscope).
Figure 5
Figure 5
Total bacterial viability and by PL-biofilm layers in the Essential Oils series. (PL-biofilm, plaque like-biofilm; EO, Essential Oils; CLSM, confocal laser scanning microscope).

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