Potential prebiotic substrates modulate composition, metabolism, virulence and inflammatory potential of an in vitro multi-species oral biofilm

Figures & data

Table 1. Overview of bacterial species and strains used in this study

commensals/beneficials	periodontal pathogens	cariogenic pathogens
A. naeslundii ATCC 51655	A. actinomycetemcomitans ATCC 43718	S. mutans ATCC 25175
A. viscosus ATCC 15987	F. nucleatum ATCC 10953	S. sobrinus ATCC 33478
S. gordonii ATCC 49818 ^#	P. gingivalis ATCC 33277
S. mitis DSM 12643 ^#	P. intermedia ATCC 25611
S. oralis DSM 20627 ^#
S. salivarius TOVE-R
S. sanguinis LMG 14657 ^#
V. parvula DSM 2008

^# Species that are often considered as beneficial commensals due to their production of H₂O₂, which plays an important role in, for instance, the inhibition of other (pathogenic) species and during oral biofilm development.

A. naeslundii: Actinomyces naeslundii, A. viscosus: Actinomyces viscosus, S. gordonii: Streptococcus gordonii, S. mitis: Streptococcus mitis, S. oralis: Streptococcus oralis, S. salivarius: Streptococcus salivarius, S. sanguinis: Streptococcus sanguinis, V. parvula: Veillonella parvula; A. actinomycetemcomitans: Aggregatibacter actino-mycetemcomitans, F. nucleatum: Fusobacterium nucleatum, P. gingivalis: Porphyromonas gingivalis, P. intermedia: Prevotella intermedia, S. mutans: Streptococcus mutans, S. sobrinus: Streptococcus sobrinus.

Figure 1. Effects of repeated rinsing with potential prebiotic substrates at 1 M on multi-species biofilm composition

Panel a: Absolute abundances of pathogenic oral species (periodontal and cariogenic pathogens) (upper graph) and beneficial/commensal oral species (lower graph) are shown as mean ± SD (n = 3) logarithmic values of the genome equivalents per millilitre (log(Geq/mL)). Panel b: Relative abundances of the different groups (beneficial/commensals, periodontal pathogens, cariogenic pathogens) of bacterial species are shown as mean ± SD (n = 3) percentage of the genome equivalents per millilitre (%(Geq/mL)). All substrates were dissolved in PBS at a concentration of 1 M. Statistically significantly different values when compared to the control (PBS) are marked with ‘*’ (P < 0.05, ANOVA + Dunnett’s correction for simultaneous hypothesis testing). Aa: A. actinomycetemcomitans; Fn: F. nucleatum; Pg: P. gingivalis; Pi: P. intermedia; An: A. naeslundii; Av: A. viscosus; S. gord.: S. gordonii; S. sal.: S. salivarius; S. sang.: S. sanguinis; Vp: V. parvula; NADG: N-acetyl-D-glucosamine.

Figure 2. Effects of repeated rinsing with potential prebiotic substrates at 1%_(w/v) on multi-species biofilm composition

Panel a: Absolute abundances of pathogenic oral species (periodontal and cariogenic pathogens) (upper graph) and beneficial/commensal oral species (lower graph) are shown as mean ± SD (n = 3) logarithmic values of the genome equivalents per millilitre (log(Geq/mL)). Panel b: Relative abundances of the different groups (beneficial/commensals, periodontal pathogens, cariogenic pathogens) of bacterial species are shown as mean ± SD (n = 3) percentage of the genome equivalents per millilitre (%(Geq/mL)). All substrates were dissolved in PBS at a concentration of 1%_(w/v) (corresponding molar concentrations: 45 mM (NADG), 29 mM (α-D-lactose), 17 mM (D-(+)-raffinose) and 26 mM (D-(+)-trehalose)). Statistically significantly different values when compared to the control (PBS) are marked with ‘*’ (P < 0.05, ANOVA + Dunnett’s correction for simultaneous hypothesis testing). Aa: A. actinomycetemcomitans; Fn: F. nucleatum; Pg: P. gingivalis; Pi: P. intermedia; An: A. naeslundii; Av: A. viscosus; S. gord.: S. gordonii; S. sal.: S. salivarius; S. sang.: S. sanguinis; Vp: V. parvula; LOD: limit of detection (=2.65 log(Geq/mL)); NADG: N-acetyl-D-glucosamine.

Figure 3. Effects of repeated rinsing with potential prebiotic substrates on multi-species biofilm organic acid balances

Organic acid levels detected in the supernatants of substrate-treated multi-species biofilms are shown as mean ± SD (n = 3) values (mg/L). Values >0 mg/L represent net organic acid production, values <0 mg/L represent net organic acid consumption. Substrates were dissolved in PBS at a concentration of 1 M (panel a) or 1%_(w/v) (panel b) (corresponding molar concentrations: 45 mM (NADG), 29 mM (α-D-lactose), 17 mM (D-(+)-raffinose) and 26 mM (D-(+)-trehalose)). Statistically significantly different values when compared to the control (PBS) are marked with ‘*’ (P < 0.05, ANOVA + Dunnett’s correction for simultaneous hypothesis testing). OA: organic acid; NADG: N-acetyl-D-glucosamine.

Table 2. Effects of repeated rinsing of multi-species biofilms with potential prebiotic substrates on virulence gene expression from A. actinomycetemcomitans.

Fold changes in virulence gene expression were determined relative to the control condition through the 2^−ΔΔCt method and are shown as the geometric mean (C.I.) (n = 3) of the 2^−ΔΔCt values. All substrates were dissolved in PBS at a concentration of 1 M (upper part) or 1%_(w/v) (lower part) (corresponding molar concentrations: 45 mM (NADG), 29 mM (α-D-lactose), 17 mM (D-(+)-raffinose) and 26 mM (D-(+)-trehalose)). Values between 0 and 1 represent relative downregulation, values >1 represent relative upregulation. Statistically, significantly different fold changes relative to the control (PBS) that are <0.5 (more than 2-fold downregulated) or >1.5 (more than 1.5-fold upregulated) are considered biologically relevant and are shown in bold and marked with ‘*’ (P < 0.05, ANOVA + Dunnett’s correction for simultaneous hypothesis testing). The color code represents the magnitude of the fold change in virulence gene expression relative to the control. NADG: N-acetyl-D-glucosamine; C.I.: 95% confidence interval.

Table 3. Effects of repeated rinsing of multi-species biofilms with potential prebiotic substrates on virulence gene expression from P. gingivalis.

Fold changes in virulence gene expression were determined relative to the control condition through the 2^−ΔΔCt method and are shown as the geometric mean (C.I.) (n = 3) of the 2^−ΔΔCt values. All substrates were dissolved in PBS at a concentration of 1 M (upper part) or 1%_(w/v) (lower part) (corresponding molar concentrations: 45 mM (NADG), 29 mM (α-D-lactose), 17 mM (D-(+)-raffinose) and 26 mM (D-(+)-trehalose)). Values between 0 and 1 represent relative downregulation, values >1 represent relative upregulation. Statistically, significantly different fold changes relative to the control (PBS) that are <0.5 (more than 2-fold downregulated) or >1.5 (more than 1.5-fold upregulated) are considered biologically relevant and are shown in bold and marked with ‘*’ (P < 0.05, ANOVA + Dunnett’s correction for simultaneous hypothesis testing). The color code represents the magnitude of the fold change in virulence gene expression relative to the control. NADG: N-acetyl-D-glucosamine; C.I.: 95% confidence interval.

Table 4. Effects of repeated rinsing of multi-species biofilms with potential prebiotic substrates on virulence gene expression from F. nucleatum.

Fold changes in virulence gene expression were determined relative to the control condition through the 2^−ΔΔCt method and are shown as the geometric mean (C.I.) (n = 3) of the 2^−ΔΔCt values. All substrates were dissolved in PBS at a concentration of 1 M (upper part) or 1%_(w/v) (lower part) (corresponding molar concentrations: 45 mM (NADG), 29 mM (α-D-lactose), 17 mM (D-(+)-raffinose) and 26 mM (D-(+)-trehalose)). Values between 0 and 1 represent relative downregulation, values >1 represent relative upregulation. Statistically significantly different fold changes relative to the control (PBS) that are <0.5 (more than 2-fold downregulated) or >1.5 (more than 1.5-fold upregulated) are considered biologically relevant and are shown in bold and marked with ‘*’ (P < 0.05, ANOVA + Dunnett’s correction for simultaneous hypothesis testing). The color code represents the magnitude of the fold change in virulence gene expression relative to the control. NADG: N-acetyl-D-glucosamine; C.I.: 95% confidence interval; but.-coA transf.: butyrate-acetoacetate CoA-transferase; ABC transp. perm.: ABC transporter permease.

Table 5. Effects of repeated rinsing of multi-species biofilms with potential prebiotic substrates on virulence gene expression from P. intermedia.

Fold changes in virulence gene expression were determined relative to the control condition through the 2^−ΔΔCt method and are shown as the geometric mean (C.I.) (n = 3) of the 2^−ΔΔCt values. All substrates were dissolved in PBS at a concentration of 1 M (upper part) or 1%_(w/v) (lower part) (corresponding molar concentrations: 45 mM (NADG), 29 mM (α-D-lactose), 17 mM (D-(+)-raffinose) and 26 mM (D-(+)-trehalose)). Values between 0 and 1 represent relative downregulation, values >1 represent relative upregulation. Statistically significantly different fold changes relative to the control (PBS) that are <0.5 (more than 2-fold downregulated) or >1.5 (more than 1.5-fold upregulated) are considered biologically relevant and are shown in bold and marked with ‘*’ (P < 0.05, ANOVA + Dunnett’s correction for simultaneous hypothesis testing). The color code represents the magnitude of the fold change in virulence gene expression relative to the control. NADG: N-acetyl-D-glucosamine; C.I.: 95% confidence interval.

Table 6. Effects of repeated rinsing with potential prebiotic substrates on multi-species biofilm inflammatory potential towards human oral keratinocytes

Fold changes in inflammatory mediator gene expression from human oral keratinocytes (HOK-18A) exposed to substrate-treated multi-species biofilms were determined relatively to the control through the 2^−ΔΔCt method and are shown as the geometric mean (C.I.) (n = 3) of the 2^−ΔΔCt values. All substrates were dissolved in PBS at a concentration of 1 M (upper part) or 1%_(w/v) (lower part) (corresponding molar concentrations: 45 mM (NADG), 29 mM (α-D-lactose), 17 mM (D-(+)-raffinose) and 26 mM (D-(+)-trehalose)). Values between 0 and 1 represent relative downregulation, values >1 represent relative upregulation. Statistically significantly different fold changes relatively to the control (PBS) that are <0.5 (more than 2-fold downregulated) or >1.5 (more than 1.5-fold upregulated) are considered biologically relevant and are shown in bold and marked with ‘*’ (P < 0.05). The color code represents the magnitude of the fold change in virulence gene expression relatively to the control. NADG: N-acetyl-D-glucosamine; C.I.: 95% confidence interval.

Figure 4. Effects of repeated rinsing with potential prebiotic substrates on multi-species biofilm inflammatory potential

IL-8 levels detected in the supernatants of human oral keratinocytes (HOK-18A) cultures exposed to substrate-treated multi-species biofilms are shown as mean ± SD (n = 3) values (pg/mL). Substrates were dissolved in PBS at a concentration of 1 M (panel a) or 1%_(w/v) (panel b) (corresponding molar concentrations: 45 mM (NADG), 29 mM (α-D-lactose), 17 mM (D-(+)-raffinose) and 26 mM (D-(+)-trehalose)). Statistically significantly different values when compared to the control (PBS) are marked with ‘*’ (P < 0.05, ANOVA + Dunnett’s correction for simultaneous hypothesis testing). IL-8: interleukin-8; NADG: N-acetyl-D-glucosamine.