In vitro inhibition of Streptococcus mutans biofilm formation on hydroxyapatite by subinhibitory concentrations of anthraquinones

Abstract

We report that certain anthraquinones (AQs) reduce Streptococcus mutans biofilm formation on hydroxyapatite at concentrations below the MIC. Although AQs are known to generate reactive oxygen species, the latter do not underlie the observed effect. Our results suggest that AQs inhibit S. mutans biofilm formation by causing membrane perturbation.

FIG. 1.

Susceptibility of S. mutans LMG 14558^T to increasing emodin concentrations in various media. MICs were determined using a modified microdilution assay in 96-well microtiter plates, as previously described (8), in the presence and absence of 1.5 mM GSH (Sigma), 0.025% (wt/vol) cysteine (Sigma), and 10 mM mannitol (Sigma). These compounds are ROS scavengers, protect cells from oxidative damage (18, 23), and will increase the MIC of emodin if its antibacterial effect is due to ROS production. M1 is the chemically defined medium described in reference .

FIG. 2.

Fluorescence emission spectra of DPH-labeled S. mutans LMG 14558^T cells grown in BHI in the presence or absence of 5 μg/ml emodin. DPH is allowed to insert into the membrane, the sample is subsequently excited with polarized light, and the extent of polarization of the emitted light (which depends on the rotational Brownian motion) is measured. The magnitude of the rotational diffusion of DPH depends on the temperature and the microviscosity (fluidity) of the surrounding membrane. Cell suspensions were incubated with 5 × 10⁻⁶ M DPH for 1 h at 37°C, and subsequently steady-state fluorescence anisotropy was measured with a Photon Technology International spectrofluorimeter (excitation with vertically polarized light of 360 nm; emission at 430 nm).