Potential of Prebiotic D-Tagatose for Prevention of Oral Disease

Abstract

Recent studies have shown phenotypic and metabolic heterogeneity in related species including Streptococcus oralis, a typical oral commensal bacterium, Streptococcus mutans, a cariogenic bacterium, and Streptococcus gordonii, which functions as an accessory pathogen in periodontopathic biofilm. In this study, metabolites characteristically contained in the saliva of individuals with good oral hygiene were determined, after which the effects of an identified prebiotic candidate, D-tagatose, on phenotype, gene expression, and metabolic profiles of those three key bacterial species were investigated. Examinations of the saliva metabolome of 18 systemically healthy volunteers identified salivary D-tagatose as associated with lower dental biofilm abundance in the oral cavity (Spearman's correlation coefficient; r = -0.603, p = 0.008), then the effects of D-tagatose on oral streptococci were analyzed in vitro. In chemically defined medium (CDM) containing D-tagatose as the sole carbohydrate source, S. mutans and S. gordonii each showed negligible biofilm formation, whereas significant biofilms were formed in cultures of S. oralis. Furthermore, even in the presence of glucose, S. mutans and S. gordonii showed growth suppression and decreases in the final viable cell count in a D-tagatose concentration-dependent manner. In contrast, no inhibitory effects of D-tagatose on the growth of S. oralis were observed. To investigate species-specific inhibition by D-tagatose, the metabolomic profiles of D-tagatose-treated S. mutans, S. gordonii, and S. oralis cells were examined. The intracellular amounts of pyruvate-derived amino acids in S. mutans and S. gordonii, but not in S. oralis, such as branched-chain amino acids and alanine, tended to decrease in the presence of D-tagatose. This phenomenon indicates that D-tagatose inhibits growth of those bacteria by affecting glycolysis and its downstream metabolism. In conclusion, the present study provides evidence that D-tagatose is abundant in saliva of individuals with good oral health. Additionally, experimental results demonstrated that D-tagatose selectively inhibits growth of the oral pathogens S. mutans and S. gordonii. In contrast, the oral commensal S. oralis seemed to be negligibly affected, thus highlighting the potential of administration of D-tagatose as an oral prebiotic for its ability to manipulate the metabolism of those targeted oral streptococci.

Keywords: Streptococcus gordonii; Streptococcus mutans (S. mutans); Streptococcus oralis; biofilm; d-tagatose; metabolomics (OMICS); transcriptomics.

Figure 1

Association of salivary tagatose with modified plaque index (mPlI). The correlation between D-tagatose and mPlI, considered to be an indicator of oral hygiene status, was evaluated in 18 subjects for whom mPlI data were available. D-tagatose was found to be significantly negatively correlated with mPlI (Spearman’s correlation coefficient; r = -0.603, p = 0.008; ). It is suggested that tagatose may play a role in maintaining a low level of plaque and thus good hygiene in the oral cavity.

Figure 2

Bacterial growth of S. mutans. All data are shown as the mean ± S.D. of triplicate experiments. (A) Growth of S. mutans OMZ175 in BHI containing various concentrations of D-tagatose. (B, D) Growth of S. mutans OMZ175 and UA159 in CDM containing no sugar, 0.8% D-glucose, or 0.8% D-Tagatose. (C, E) Growth of S. mutans OMZ175 and UA159 in CDM containing 0.2% D-glucose with 0%, 1%, or 5% D-tagatose.

Figure 3

Bacterial growth of S. gordonii. All data are shown as the mean ± S.D. of triplicate experiments. (A) Growth of S. gordonii in BHI containing various concentrations of D-tagatose. (B) Growth of S. gordonii in CDM containing no sugar, 0.8% D-glucose, or 0.8% D-Tagatose. (C) Growth of S. gordonii in CDM containing 0.2% D-glucose with 0%, 1%, or 5% D-tagatose.

Figure 4

Bacterial growth of S. oralis. All data are shown as the mean ± S.D. of triplicate experiments. (A) Growth of S. oralis in BHI containing various concentrations of D-tagatose. (B) Growth of S. oralis in CDM containing no sugar, 0.8% D-glucose, or 0.8% D-Tagatose. (C) Growth of S. oralis in CDM containing 0.2% D-glucose with 0%, 1%, or 5% D-tagatose.

Figure 5

Effects on viable cells of S. mutans, S. gordonii, and S. oralis. Cells were incubated in CDM containing 0.2% glucose with 0%, 1%, or 5% tagatose for 48 hours. Statistical differences were calculated using Dunnett’s test. *p < 0.05, **p < 0.01.

Figure 6

Effects of D-tagatose on S. mutans EPS production. (A) Representative CLSM images showing typical architecture of planktonic cells (red) and EPS (green) following reconstruction with Imaris software. (B) Relative amounts of EPS to cells. Ten fields per sample were randomly recorded with CLSM and the relative amount of EPS was calculated using IMARIS software. Control, without tagatose. **P < 0.01.

Figure 7

Effects of D-Tagatose on S. mutans biofilm formation. (A, C) Representative CLSM images showing typical architecture of biofilms following reconstruction with Imaris software. S. mutans cells were stained with hexidium iodide (red), and incubated with saccharides or nothing was added. (B, D) Biovolume analysis of S. mutans. Ten fields per sample were randomly recorded with CLSM and S. mutans biovolume was quantified using IMARIS software. Control, containing no sugar. **P < 0.01.

Figure 8

Effects of D-Tagatose on S. gordonii biofilm formation. (A) Representative CLSM images showing typical architecture of biofilms following reconstruction with Imaris software. S. gordonii cells were stained with hexidium iodide (red), and incubated with saccharides or nothing was added. (B) Biovolume analysis of S. gordonii. Ten fields per sample were randomly recorded with CLSM and S. gordonii biovolume was quantified using IMARIS software. Control, containing no sugar. **P < 0.01.

Figure 9

Effects of D-Tagatose on S. oralis biofilm formation. (A) Representative CLSM images showing typical architecture of biofilms following reconstruction with Imaris software. S. oralis cells were stained with hexidium iodide (red), and incubated with saccharides or nothing was added. (B) Biovolume analysis of S. oralis. Ten fields per sample were randomly recorded with CLSM and S. oralis biovolume was quantified using IMARIS software. Control, containing no sugar. **P < 0.01.

Figure 10

Expressions of transporter genes. Gene expression levels of fructose-specific PTS and amino acid ABC transporters are shown. The three squares show Log₂-transformed fold change values for tagatose-incubated cells calculated as compared to the control S. mutans (left square), S. gordonii (center square), and S. oralis (right square), according to a reported color scale.

Figure 11

Principal component analysis of intracellular metabolome of S. gordonii, S. mutans, and S. oralis. Score plots for PC1 and PC2 are shown.

Figure 12

Contents of tagatose and downstream metabolites from pyruvate. Metabolites were extracted from S. mutans, S. gordonii, and S. oralis cells after three hours of incubation in CDM with or without tagatose, followed by determination of those levels using GC-MS. To minimize the effects of different growth stages, the OD₆₀₀ value was set to 0.8 before starting the culture. Bars represent the mean ± S.D. (n = 4). P-values were determined with Student’s t-test.