Inhibitory effect of surface pre-reacted glass-ionomer (S-PRG) eluate against adhesion and colonization by Streptococcus mutans

Abstract

Surface Pre-reacted Glass-ionomer (S-PRG) filler is a bioactive filler produced by PRG technology, which has been applied to various dental materials. A S-PRG filler can release multiple ions from a glass-ionomer phase formed in the filler. In the present study, detailed inhibitory effects induced by S-PRG eluate (prepared with S-PRG filler) against Streptococcus mutans, a major pathogen of dental caries, were investigated. S-PRG eluate effectively inhibited S. mutans growth especially in the bacterium before the logarithmic growth phase. Microarray analysis was performed to identify changes in S. mutans gene expression in the presence of the S-PRG eluate. The S-PRG eluate prominently downregulated operons related to S. mutans sugar metabolism, such as the pdh operon encoding the pyruvate dehydrogenase complex and the glg operon encoding a putative glycogen synthase. The S-PRG eluate inhibited several in vitro properties of S. mutans relative to the development of dental caries especially prior to active growth. These results suggest that the S-PRG eluate may effectively inhibit the bacterial growth of S. mutans following downregulation of operons involved in sugar metabolism resulting in attenuation of the cariogenicity of S. mutans, especially before the active growth phase.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Inhibition on S. mutans MT8148 grown by the S-PRG eluate. (A,B) Bacterial growth by adding varying concentration of the eluate followed by 18 h incubation. Growth was determined by OD550 values in BHI broth (A) and recovered bacterial numbers on MSB plates (B). (C) Bacterial growth by adding 1 × 107 CFU/ml of S. mutans at multiple time points, which was determined by OD550 values in BHI broth. (D) Bacterial survival by adding 1 × 107 CFU/ml of S. mutans at multiple time points, which was determined by adding serial dilutions of the bacterial suspensions to MSB plates.

Figure 2

Altered expression of key genes in both S. mutans MT8148 and UA159 under three different conditions using DNA microarray analysis. (A) Detection of altered genes in the presence of S-PRG eluate. (B) Changes in the expression of genes in the pdh operon of MT8148 and UA159 in the presence of S-PRG eluate.

Figure 3

Altered expression of key genes in both S. mutans MT8148 and UA159 under two different conditions detected by DNA microarray analysis. (A) Detection of altered genes in the presence of S-PRG eluate. (B) Changes in the expression of genes in the glg operon of MT8148 and UA159 in the presence of S-PRG eluate.

Figure 4

Altered expression of key genes in either S. mutans MT8148 or UA159 under three different conditions using DNA microarray analysis. (A) Detection of altered genes in the presence of S-PRG eluate. (B) Changes in the expression of genes in the lac operon of MT8148 and UA159 in the presence of S-PRG eluate.

Figure 5

Sucrose-dependent adhesion of S. mutans MT8148 in the presence of various concentrations of S-PRG eluate. Bacterial growth for cells used in the sucrose-dependent adhesion assay was determined by OD550 values (A) and recovered bacterial numbers on MSB plates (B). (C) Sucrose-dependent adhesion rates. Significant differences were determined using ANOVA with Bonferroni correction. *P < 0.05, **P < 0.01, and ***P < 0.001 versus no S-PRG eluate.

Figure 6

Biofilm formation by S. mutans MT8148 grown in BHI with 0.25% sucrose in the presence of various concentrations of S-PRG eluate. (A) Quantity of biofilm formation. (B) Representative images of formed biofilms using confocal scanning laser microscopy. (C) Biofilm thickness. (D) Representative images of biofilm thickness using confocal scanning laser microscopy. Significant differences were determined using ANOVA with Bonferroni correction. ***P < 0.001 versus no S-PRG eluate.

Figure 7

Cellular hydrophobicity of S. mutans MT8148 in the presence of various concentrations of S-PRG eluate. (A) Cellular hydrophobicity in the presence of S-PRG eluate. (B) Bacterial numbers used in the cellular hydrophobicity assay (C) Cellular hydrophobicity following the addition of S-PRG eluate 18 h before the assay. Significant differences were determined using ANOVA with Bonferroni correction. *P < 0.05 and **P < 0.01 versus no S-PRG eluate.

Figure 8

In vitro properties of S. mutans MT8148 in the late logarithmic phase in the presence of various concentrations of S-PRG eluate. (A, B) Bacterial growth by adding 1 × 109 CFU/ml of S. mutans with 18-h incubation, which was determined by OD550 values in BHI broth (A) and recovered bacterial numbers on MSB plates (B). (C) Bacterial growth by adding 1 × 109 CFU/ml of S. mutans at multiple time points, which was determined by OD550 values in BHI broth. (D) Sucrose-dependent adhesion rates. (E) Quantitation of biofilm formation. Significant differences were determined using ANOVA with Bonferroni correction. *P < 0.05, **P < 0.01, and ***P < 0.001 versus no S-PRG eluate.

Figure 9

The in vitro properties of Streptococcus sobrinus B13 and Streptococcus gordonii ATCC10558 in the presence of various concentrations of S-PRG eluate. Bacterial growth using S. sobrinus before reaching the logarithmic phase (adjusted to 1 × 105 CFU/ml) (A) and in the post-logarithmic phase (adjusted to 1 × 109 CFU/ml) (B) following 18 h incubation, which was determined by recovered bacterial numbers on MSB plates. Bacterial growth using S. gordonii reaching the logarithmic phase (adjusted to 1 × 105 CFU/ml) (C) and in the post-logarithmic phase (adjusted to 1 × 109 CFU/ml) (D) with 18 h incubations, which was determined by recovered bacterial numbers on MSB plates. Sucrose-dependent adhesion rates using bacterial suspensions of S. sobrinus B13 adjusted before reaching the logarithmic phase (adjusted to 1 × 107 CFU/ml) (E) and in the post-logarithmic phase (adjusted to 1 × 109 CFU/ml) (F), respectively. Quantitation of biofilm formation using bacterial suspensions of S. sobrinus B13 adjusted to 1 × 107 CFU/ml (G) and 1 × 109 CFU/ml (H), respectively. Significant differences were determined using ANOVA with Bonferroni correction. *P < 0.05, **P < 0.01, and ***P < 0.001 versus no S-PRG eluate.