Functional changes in the oral microbiome after use of fluoride and arginine containing dentifrices: a metagenomic and metatranscriptomic study

Abstract

Background: Tooth decay is one of the most prevalent diseases worldwide, and efficient tooth brushing with a fluoride-containing dentifrice is considered fundamental to caries prevention. Fluoride-containing dentifrices have been extensively studied in relation to enamel resistance to demineralization. Arginine (Arg) has also been proposed as a promising prebiotic to promote pH buffering through ammonia production. Here, we present the first metagenomic (DNA sequencing of the whole microbial community) and metatranscriptomic (RNAseq of the same community) analyses of human dental plaque to evaluate the effect of brushing with fluoride (Fl) and a Fl+Arg containing dentifrices on oral microbial composition and activity. Fifty-three patients were enrolled in a longitudinal clinical intervention study with two arms, including 26 caries-active and 27 caries-free adults. After a minimum 1-week washout period, dental plaque samples were collected at this post-washout baseline, 3 months after the use of a 1450-ppm fluoride dentifrice, and after 6 months of using a 1450-ppm fluoride with 1.5% arginine dentifrice.

Results: There was a shift in both the composition and activity of the plaque microbiome after 3 months of brushing with the fluoride-containing toothpaste compared to the samples collected at the 1-week post-washout period, both for caries-active and caries-free sites. Although several caries-associated bacteria were reduced, there was also an increase in several health- and periodontitis-associated bacteria. Over 400 genes changed proportion in the metagenome, and between 180 and 300 genes changed their expression level depending on whether caries-free or caries-active sites were analyzed. The metagenome and metatranscriptome also changed after the subjects brushed with the Fl+Arg dentifrice. There was a further decrease of both caries- and periodontitis-associated organisms. In both caries-free and caries-active sites, a decrease of genes from the arginine biosynthesis pathway was also observed, in addition to an increase in the expression of genes associated with the arginine deiminase pathway, which catabolizes arginine into ammonia, thereby buffering acidic pH. Bacterial richness and diversity were not affected by either of the two treatments in the two arms of the study.

Conclusions: Our data demonstrate that long-term use of both assayed dentifrices changes the bacterial composition and functional profiles of human dental plaque towards a healthier microbial community, both in caries-free and caries-active sites. This observation was especially apparent for the Fl+Arg dentifrice. Thus, we conclude that the preventive benefits of tooth brushing go beyond the physical removal of dental plaque and that the active ingredients formulated within dentifrices have a positive effect not only on enamel chemistry but also on the metabolism of oral microbial populations. Video Abstract.

Keywords: 16S rRNA; Arginine; Arginolytic route; Caries; Fluoride; Metagenome; Metatranscriptome; Oral microbiota.

Conflict of interest statement

The authors declare that they have no competing interests.

© 2022. The Author(s).

Figures

Fig. 1

Structure of the longitudinal clinical intervention study. A After a 1-week washout period, individuals brushed their teeth twice per day for a period of 3 months with a 1450-ppm fluoride dentifrice and six additional months with a 1.5% arginine + 1450 ppm fluoride dentifrice. Dental plaque samples were collected longitudinally at post-washout (marked as “Baseline” in the figure) and after the two treatment periods (arrows). DNA and RNA were extracted from the plaque samples and separately sequenced on an Illumina platform. The corresponding metagenome (MTG) and metatranscriptome (MTT) were analyzed to determine the bacterial composition, gene content, and gene expression of the overall dental plaque community. In addition, the 16S rRNA gene was amplified in the DNA samples and sequenced separately through Illumina Miseq sequencing. B Number of dental plaque samples analyzed in the current manuscript for the two arms of the study, i.e., caries-active (CA) and caries-free (CF) individuals at the three time points. Numbers in the table represent the final number of samples included in the analysis after excluding patients that abandoned the study and samples with low number of reads. MTG, metagenomic (i.e., DNA) dataset; MTT, metatranscriptomic (i.e., RNA) dataset; 16S (i.e., amplified DNA) dataset

Fig. 2

Genus composition of microbial communities associated with caries-active (CA) and caries-free (CF) individuals. Top 20 most abundant genera at baseline (post-washout period), after 3 months of treatment with fluoride dentifrice (Fluoride), and after 6 further months of treatment with fluoride + arginine (Fl+Arg) dentifrice are represented based on data from the metagenome (MTG) or metatranscriptome (MTT). Bacteria that could not be unequivocally assigned to a given genus are indicated with the name of the corresponding family followed by “uc” (unclassified)

Fig. 3

Effect of dentifrice treatment on taxonomic composition. Differences in abundance (MTG) and activity (MTT) of bacterial communities (species) between baseline (post-washout period), fluoride treatment, and fluoride + arginine treatment were studied using canonical correspondence analyses (CCA). The effect on caries-active (CA) and caries-free (CF) samples was studied separately. 95% confidence intervals were included

Fig. 4

Changes in the levels of total and transcriptionally active bacterial species after fluoride + arginine treatments. Each dot represents a species that was significantly under- or over-represented in the 16S rRNA sequencing, metagenomic (MTG), or metatranscriptomic (MTT) dataset after a 6-month use of a fluoride + arginine dentifrice. The abundance (y-axis) and fold change (x-axis, %before/%after) are shown for each bacterium. Dots corresponding to species over-represented after treatment with a fluoride-containing dentifrice are shown in orange, and those in green represent bacteria over-represented after brushing with a Fl+Arg dentifrice. Species differentially represented in caries-active sites (CA) are shown on the left panels while those for caries-free individuals (CF) are on the right panels. The names of the species at > 1% abundance before or after the treatment are indicated

Fig. 5

Effect of toothpaste treatment at the functional level. Differences in gene proportions (as inferred from the MTG dataset) and gene expression (as inferred from the MTT dataset) between baseline (post-washout period), fluoride treatment, and fluoride + arginine treatment were studied using canonical correspondence analyses (CCA). The effect on caries-active sites (CA) and caries-free (CF) patients was studied separately. 95% confidence intervals were included

Fig. 6

Changes in the arginolytic pathway gene content and expression after fluoride + arginine treatment. The degradation of arginine to NH3 and CO2 via the ADS pathway and the genes involved are represented at the top of the figure. The abundance of the genes (as inferred from the MTG dataset) in CA and CF patients before (orange) and after (green) the treatment is represented on the middle panels, while changes in the gene expression (as inferred from the MTT dataset) are shown on the bottom panels. *Statistically significant difference, p < 0.05. st, statistical trend, p < 0.1

Fig. 7

Changes in the arginine biosynthetic route after fluoride + arginine treatment. The abundance (logRLM) of each gene involved in the biosynthetic route is shown before and after treatment with fluoride + arginine. The pathway is shown on the right panel. *p < 0.05

Fig. 8

Effects of fluoride and fluoride + arginine on arginine metabolism. The enzymatic reactions and genes responsible for arginine biosynthesis, arginine degradation through citrulline (arginolytic pathway, ADS), or arginine conversion into agmatine and its further degradation (agmatine deiminase system, AgDS) are plotted. Those genes over-represented or over-expressed after fluoride treatment are colored in orange whereas those that were over-represented or over-expressed after fluoride + arginine treatment are colored in green