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. 2023 Jan 31:13:1130255.
doi: 10.3389/fcimb.2023.1130255. eCollection 2023.

In vitro versus in situ biofilms for evaluating the antimicrobial effectiveness of herbal mouthrinses

Nicole Schönbächler  1 , Thomas Thurnheer  1 , Pune Nina Paqué  1   2 , Thomas Attin  1 , Lamprini Karygianni  1
Affiliations

In vitro versus in situ biofilms for evaluating the antimicrobial effectiveness of herbal mouthrinses

Nicole Schönbächler et al. Front Cell Infect Microbiol. .

Abstract

For centuries, diverse mouthrinses have been applied for medicinal purposes in the oral cavity. In view of the growing resistance of oral microorganisms against conventional antimicrobial agents e.g. chlorhexidine, the implementation of alternative treatments inspired by nature has lately gained increasing interest. The aim of the present study was to compare in vitro biofilm models with in situ biofilms in order to evaluate the antimicrobial potential of different natural mouthrinses. For the in vitro study a six-species supragingival biofilm model containing A. oris, V. dispar, C. albicans, F. nucleatum, S. mutans and S. oralis was used. Biofilms were grown anaerobically on hydroxyapatite discs and treated with natural mouthrinses Ratanhia, Trybol and Tebodont. 0.9% NaCl and 10% ethanol served as negative controls, while 0.2% CHX served as positive control. After 64h hours, biofilms were harvested and quantified by cultural analysis CFU. For the in situ study, individual test splints were manufactured for the participants. After 2h and 72h the biofilm-covered samples were removed and treated with the mouthrinses and controls mentioned above. The biofilms were quantified by CFU and stained for vitality under the confocal laser scanning microscope. In the in vitro study, 0.2% CHX yielded the highest antimicrobial effect. Among all mouthrinses, Tebodont (4.708 ± 1.294 log10 CFU, median 5.279, p<0.0001) compared with 0.9% NaCl showed the highest antimicrobial potential. After 72h there was no significant reduction in CFU after 0.2% CHX treatment. Only Trybol showed a statistically significant reduction of aerobic growth of microorganisms in situ (5.331 ± 0.7350 log10 CFU, median 5.579, p<0.0209). After treatment with the positive control 0.2% CHX, a significant percentage of non-vital bacteria (42.006 ± 12.173 log10 CFU, median 42.150) was detected. To sum up, a less pronounced effect of all mouthrinses was shown for the in situ biofilms compared to the in vitro biofilms.

Keywords: chlorhexidine (CHX); confocal laser scanning microscopy (CLSM); herbal mouthrinses; in situ; multispecies oral biofilm.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Individual acrylic splints with six enamel slabs attached to different locations. The slabs were placed in the front, in the middle and in the back on both sides, right and left. All sides of the slabs, except the enamel surface were covered with silicone and embedded in the appliance. Due to the fact that biofilm formation can differ from molar to premolar, the treatment groups were shifted one position further at each subject. C/E: Back; B/F: Middle; A/D: Front.
Figure 2
Figure 2
Box plots illustrating the colony-forming units (CFUs) of six-species oral biofilms after simultaneous exposure to the natural mouthrinses Ratanhia, Trybol, Tebodont. 0.2% CHX (positive control), 0.9% NaCl and 10% EtOH (negative control) were also used. The CFU values were shown on a log10 scale per mm2 (log10/mm2). The line dividing the box shows the median. The p-values of the significantly different data are plotted: ***= P-value <0.001, **** = P-value < 0.0001.
Figure 3
Figure 3
Box plots show the number of CFUs that demonstrate the antimicrobial effect of the tested substances on aerobic (A) and anaerobic (B) bacteria after an oral exposure time of 2 hours. A positive control (0.2% CHX), two negative controls (0.9% NaCl, 10% EtOH) and the natural mouthrinses Ratanhia, Trybol and Tebodont were also tested. The CFU values were shown on a log10 scale per mm2 (log10/mm2). The horizontal line within the box shows the median values. The p-values of the significantly different data are plotted: *= P-value <0.05.
Figure 4
Figure 4
The graphs show the number of CFUs that demonstrate the antimicrobial effect of the tested substances on aerobic (A) and anaerobic (B) bacteria after an oral exposure time of 72 hours. A positive control (0.2% CHX), two negative controls (0.9% NaCl, 10% EtOH) and three different natural mouthrinses Ratanhia, Trybol and Tebodont were also used. The CFU values were shown on a log10 scale per mm2 (log10/mm2). The horizontal line within the box shows the median values. The p-values of the significantly different data are plotted: *= P-value <0.05.
Figure 5
Figure 5
Box plots illustrating the comparison between the two treatment methods in vitro and in situ. It was measured by colony-forming units (CFUs) on a log10 scale per mm2 (log10/mm2). In vitro and in situ biofilms were treated with Ratanhia, Trybol, Tebodont. 0.2% CHX (positive control), 0.9% NaCl and 10% EtOH (negative control) were also used. The line dividing the box shows the median. The p-values of the significantly different data are plotted: *= P-value <0.05, **** = P-value < 0.0001.
Figure 6
Figure 6
Boxplots illustrating the outcomes of image analysis. The in situ biofilm was grown 72 hours on HA discs and afterwards treated with different mouthrinses: 0.2% CHX, 10% EtOH, 0.9% NaCl, Ratanhia, Tebodont and Trybol. The visualization of nucleic acids was aided by SYTO 40, a cell-permeant nucleic acid stain, as well as DRAQ7 and TOTO-1, which cell impermeable DNA dyes. The percentage of stained nucleic acids was measured. The line dividing the box shows the median. The p-values of the significantly different data are plotted: ** = P-value < 0.01., **** = P-value < 0.0001.
Figure 7
Figure 7
Confocal laser scanning microscopy (CLSM). 3D reconstructions and images sections of biofilm grown 3 days on HA discs and afterwards treated with different mouthrinse (A) 0.2% CHX, (B) 10% EtOH, (C) 0.9% NaCl, (D) Ratanhia, (E) Tebodont, (F) Trybol. The biofilms were used for viability analysis and visualization of extracellular DNA (eDNA): SYTO 40 a cell-permeant nucleic acid stain in green, DRAQ7 in red and TOTO-1 cell impermeable DNA dyes in blue.
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This study was supported by Institutional funds of the University of Zurich.

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