IADR Abstract Archives
Understanding the Mechanism of Action of SnF2-Containing Toothpastes With Metagenomics
Objectives: Stannous fluoride (SnF2) containing toothpastes have been shown to provide clinical anti-gingivitis benefits through both direct antimicrobial properties and inhibition of plaque regrowth. This work aimed to understand the mechanism of action of SnF2 containing toothpaste on oral microorganisms using a combination of cell based, microscopic and metagenomic analyses.
Methods: Streptococcus mutans, as a representative oral microorganism, was treated with either SnF2 raw material or in a full toothpaste formulation for 30s and 2min. Cell viability or growth inhibition were assayed for using viable cell counts or optical density, respectively. Additionally, mixed-species, saliva-derived biofilms were grown for 4 days on hydroxyapatite coupons in an in vitro CDC bioreactor in the presence of twice-daily, 30s treatments with either a stannous or control toothpaste. Assays were conducted assessing cell viability and confocal scanning laser microscopy (CSLM) in combination with BacLight Live/Dead and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining. Additionally, biofilms were harvested for shotgun metagenomics (Illumina).
Results: Concentrations as low as 0.015625% w/v of SnF2 were able to delay exponential growth of S. mutans. At higher concentrations of 0.25% w/v, 30 s and 2 min contact times delayed growth S. mutans growth by 3.75h and 7.875h, respectively. The stannous containing toothpaste resulted in significantly greater log reduction in viable cells (3.40 +/- 0.139 at 30s and 5.07 +/- 0.001 at 2min) compared to control toothpastes (1.29 +/- 0.057 at 30s and 2.41 +/- 0.008 at 2min) at both time points (p<0.0001). Imaging via CSLM using CTC and Live/Dead staining showed a significant reduction in biofilm metabolic activity in response to stannous toothpaste treatment with metagenomic analysis showing a reduction in several pathways involved in bacterial growth, biofilm formation and survival.
Conclusions: This study has provided significant observations into the effectiveness of SnF2-containing toothpastes in terms of their underpinning mechanisms of action in controlling oral microbial growth and survival.
Methods: Streptococcus mutans, as a representative oral microorganism, was treated with either SnF2 raw material or in a full toothpaste formulation for 30s and 2min. Cell viability or growth inhibition were assayed for using viable cell counts or optical density, respectively. Additionally, mixed-species, saliva-derived biofilms were grown for 4 days on hydroxyapatite coupons in an in vitro CDC bioreactor in the presence of twice-daily, 30s treatments with either a stannous or control toothpaste. Assays were conducted assessing cell viability and confocal scanning laser microscopy (CSLM) in combination with BacLight Live/Dead and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining. Additionally, biofilms were harvested for shotgun metagenomics (Illumina).
Results: Concentrations as low as 0.015625% w/v of SnF2 were able to delay exponential growth of S. mutans. At higher concentrations of 0.25% w/v, 30 s and 2 min contact times delayed growth S. mutans growth by 3.75h and 7.875h, respectively. The stannous containing toothpaste resulted in significantly greater log reduction in viable cells (3.40 +/- 0.139 at 30s and 5.07 +/- 0.001 at 2min) compared to control toothpastes (1.29 +/- 0.057 at 30s and 2.41 +/- 0.008 at 2min) at both time points (p<0.0001). Imaging via CSLM using CTC and Live/Dead staining showed a significant reduction in biofilm metabolic activity in response to stannous toothpaste treatment with metagenomic analysis showing a reduction in several pathways involved in bacterial growth, biofilm formation and survival.
Conclusions: This study has provided significant observations into the effectiveness of SnF2-containing toothpastes in terms of their underpinning mechanisms of action in controlling oral microbial growth and survival.