Genetic and physiological effects of noncoherent visible light combined with hydrogen peroxide on Streptococcus mutans in biofilm

Abstract

Oral biofilms are associated with the most common infections of the oral cavity. Bacteria embedded in the biofilms are less sensitive to antibacterial agents than planktonic bacteria are. Recently, an antibacterial synergic effect of noncoherent blue light and hydrogen peroxide (H(2)O(2)) on planktonic Streptococcus mutans was demonstrated. In this study, we tested the effect of a combination of light and H(2)O(2) on the vitality and gene expression of S. mutans embedded in biofilm. Biofilms of S. mutans were exposed to visible light (wavelengths, 400 to 500 nm) for 30 or 60 s (equivalent to 34 or 68 J/cm(2)) in the presence of 3 to 300 mM H(2)O(2). The antibacterial effect was assessed by microbial counts of each treated sample compared with that of the control. The effect of light combined with H(2)O(2) on the different layers of the biofilm was evaluated by confocal laser scanning microscopy. Gene expression was determined by real-time reverse transcription-PCR. Our results show that noncoherent light, in combination with H(2)O(2), has a synergistic antibacterial effect through all of the layers of the biofilm. Furthermore, this treatment was more effective against bacteria in biofilm than against planktonic bacteria. The combined light and H(2)O(2) treatment up-regulated the expression of several genes such as gtfB, brp, smu630, and comDE but did not affect relA and ftf. The ability of noncoherent visible light in combination with H(2)O(2) to affect bacteria in deep layers of the biofilm suggests that this treatment may be applied in biofilm-related diseases as a minimally invasive antibacterial procedure.

FIG. 1.

Bacterial growth following exposure of bacterial biofilm to noncoherent visible light (at wavelengths of 400 to 500 nm) in combination with different concentrations of H₂O₂. The growth of nonexposed (control) bacterial samples and samples exposed to light for 30 s (equivalent to ∼34 J/cm²) or 60 s (equivalent to ∼68 J/cm²) in the absence or presence of H₂O₂ at a concentration of 3, 30, or 300 mM is expressed as CFU counts on a logarithmic scale. Asterisks indicate statistically significant differences (P < 0.001).

FIG. 2.

Comparison of the number of CFU under planktonic conditions and in biofilm of nonexposed (control) bacterial samples and samples exposed to light for 30 s (equivalent to ∼34 J/cm²) or 60 s (equivalent to ∼68 J/cm²) in the presence of 30 mM H₂O₂. A statistically significant difference between the biofilm and planktonic groups was found (P < 0.02).

FIG. 3.

CSLM images of different layers, from the biofilm surface (left upper image) to the deepest layer of the biofilm (right lower image). All samples were exposed to noncoherent visible light (at wavelengths of 400 to 500 nm) for 60 s (equivalent to ∼68 J/cm²) in the presence of 30 mM H₂O₂. Panels: a, immediately after exposure; b, 3 h after exposure; c, 6 h after exposure. Green indicates live bacteria, red indicates dead bacteria, and yellow indicates the presence of both live and dead bacteria.

FIG. 4.

Bacterial ATP levels measured before (zero time) and at different time points after exposure to noncoherent visible light (at wavelengths of 400 to 500 nm) for 60 s (equivalent to ∼68 J/cm²) in the presence of 30 mM H₂O₂, H₂O₂ alone, and light exposure alone. There was a statistically significant difference in ATP levels at 0.2, 3, and 6 h after combined irradiation and H₂O₂ treatment (P < 0.001) (no significant difference was found after 1 h).

FIG. 5.

Relative expression of several genes related to biofilm formation 24 h after treatment of bacterial biofilm by exposure to noncoherent visible light (at wavelengths of 400 to 500 nm) for 60 s (equivalent to ∼68 J/cm²) in the absence (horizontal lines) or presence (vertical lines) of 30 mM H₂O₂ or to 30 mM H₂O₂ alone (white columns) and in a nontreated control (black columns). All samples were normalized to the endogenous 16S rRNA. The expression of all genes was significantly enhanced following treatment, except for ftf and relA (P < 0.05).