3,3'-Diindolylmethane (DIM): A Potential Therapeutic Agent against Cariogenic Streptococcus mutans Biofilm

Abstract

Indole, a metabolite of the amino acid tryptophan, has been proven to act as a signal molecule in bacteria, acting in different aspects of biofilm formation. The oral biofilm is a type of biofilm that has consequences for human health. It is a complex, three-dimensional structure that develops on the surface of teeth via the attachment of primary microbial colonizers. Many oral infections are caused by an imbalance occurring in the microorganisms naturally found in oral biofilms and are considered major public health concerns. In this study, we test the effect of a natural bis-indole, 3,3'-Diindolylmethane (DIM), in mitigating the pathogenicity of the oral biofilm inhabiting bacterium Streptococcus mutans, a bacterium that is considered to be a principal etiological agent in dental caries. Our study found that DIM was able to attenuate S. mutans biofilm formation by 92%. Additionally, treatment with DIM lowered extracellular polymeric substance (EPS) production and decreased its durability significantly under acidic conditions. Therefore, the anti-biofilm and anti-virulence properties of DIM against S. mutans bacteria in an "oral setting" provides evidence for its usefulness in reducing biofilm formation and potentially for caries attenuation.

Keywords: 3,3′-Diindolylmethane; S. mutans; biofilm; caries; resistance.

Figure 1

Dose response of 3,3′-Diindolylmethane on biofilm formation. CLSM images of biofilm formed by S. mutans after 24 h of treatment by different concentrations of (B–E) DIM or (A) untreated (DMSO control). Biofilm formed on a glass bottom 96-well plate after static incubation coated with clarified saliva. CLSM images visualize viable cells stained green and dead cells stained red with the BacLight^® LIVE/DEAD Kit, scanned areas of ~318 μm × 318 μm. (F) Statistical analysis showing the means ± SDs of biofilm volume generated from three independent sets of experiments (t-test; *** p < 0.001). Differences were also analyzed for their significance by using one-way ANOVA with Tukey’s test.

Figure 2

Surface morphology of biofilm formation. SEM images of the biofilm formed by S. mutans after 24 h (A,B) untreated (DMSO control) or (C,D) treatment with 0.5 µM of DIM. Biofilm formed on a glass coated with clarified saliva after static incubation. White arrows indicate the presence of mature microcolonies in the control group. Images are shown at 250× and 5000× magnification.

Figure 3

Biofilm dispersal in dynamic culture. S. mutans biofilm (A) control (equivalent concentration of DMSO) or (B) 0.5µM DIM continuously supplemented through the inlet media. Biofilms were stained with the LIVE/DEAD bacterial viability kit and subsequently imaged by CSLM 48 h post inoculation. (C) Bio-volume (μm³/μm²) quantification is based on image analysis by IMARIS software. Images were acquired from three different areas for each treatment (means ± SDs), with at least three independent repetitions. Asterisks indicate significant differences compared to control (t-test; * p < 0.05).

Figure 4

Quantitative EPS production and aciduricity determination. (A) Biofilm’s EPS of S. mutans assessment after 24 h treated with 0.5 µM of DIM or an equivalent amount of DMSO as the control using Anthrone method [24]. Standard curve was prepared with dextran standard with various concentrations (data not shown). (B) Acid tolerance was determined by measuring the survival rate of S. mutans at pH 5.0. in the presence of 0.5 µM DIM compared with the untreated control (DMSO). N0 and N represent CFU counts before and after 2 h and 4 h treatments in pH 5.0 culture, respectively. Asterisks indicate significant differences compared to control (t-test; *** p < 0.001).