Dual-light photodynamic therapy administered daily provides a sustained antibacterial effect on biofilm and prevents Streptococcus mutans adaptation

Abstract

Antibacterial photodynamic therapy (aPDT) and antibacterial blue light (aBL) are emerging treatment methods auxiliary to mechanical debridement for periodontitis. APDT provided with near-infrared (NIR) light in conjunction with an indocyanine green (ICG) photosensitizer has shown efficacy in several dental in-office-treatment protocols. In this study, we tested Streptococcus mutans biofilm sensitivity to either aPDT, aBL or their combination dual-light aPDT (simultaneous aPDT and aBL) exposure. Biofilm was cultured by pipetting diluted Streptococcus mutans suspension with growth medium on the bottom of well plates. Either aPDT (810 nm) or aBL (405 nm) or a dual-light aPDT (simultaneous 810 nm aPDT and 405 nm aBL) was applied with an ICG photosensitizer in cases of aPDT or dual-light, while keeping the total given radiant exposure constant at 100 J/cm2. Single-dose light exposures were given after one-day or four-day biofilm incubations. Also, a model of daily treatment was provided by repeating the same light dose daily on four-day and fourteen-day biofilm incubations. Finally, the antibacterial action of the dual-light aPDT with different energy ratios of 810 nm and 405 nm of light were examined on the single-day and four-day biofilm protocols. At the end of each experiment the bacterial viability was assessed by colony-forming unit method. Separate samples were prepared for confocal 3D biofilm imaging. On a one-day biofilm, the dual-light aPDT was significantly more efficient than aBL or aPDT, although all modalities were bactericidal. On a four-day biofilm, a single exposure of aPDT or dual-light aPDT was more efficient than aBL, resulting in a four logarithmic scale reduction in bacterial counts. Surprisingly, when the same amount of aPDT was repeated daily on a four-day or a fourteen-day biofilm, bacterial viability improved significantly. A similar improvement in bacterial viability was observed after repetitive aBL application. This viability improvement was eliminated when dual-light aPDT was applied. By changing the 405 nm to 810 nm radiant exposure ratio in dual-light aPDT, the increase in aBL improved the antibacterial action when the biofilm was older. In conclusion, when aPDT is administered repeatedly to S. mutans biofilm, a single wavelength-based aBL or aPDT leads to a significant biofilm adaptation and increased S. mutans viability. The combined use of aBL light in synchrony with aPDT arrests the adaptation and provides significantly improved and sustained antibacterial efficacy.

Fig 1. Effect of single-dose application of aBL, aPDT or dual-light aPDT on one-day S. mutans biofilms.

A one-day S. mutans biofilm was treated with a single-dose application of aBL, aPDT, or dual-light aPDT. The total amount of light irradiance was the same at 100 mW/cm² for all three modalities (aBL vs. control, p = 0.045; aPDT vs. control, p = 0.0025; dual-light aPDT vs. control, p = p = 0.0043; aBL vs. dual-light aPDT, p = 0.0022; aPDT vs. dual-light aPDT, p = 0.0064; aBL vs. aPDT, p = 0.0012; Mann-Whitney U Test). The columns display medians. Each marking on the columns represents an independent assay. The T-bars show 95% confidence interval (CI). A detailed protocol and number of assays are shown at Table 1.

Fig 2. Effect of single-dose or daily-dose application of aBL, aPDT or dual-light aPDT on four-day S. mutans.

A four-day biofilm was exposed to aBL, aPDT or dual-light aPDT as a single-dose exposure at the end of the biofilm maturation period or as a repetitive, daily-dose exposure repeating the same treatment dose. The columns display medians. Single-dose aBL vs. control, p = 0.025; single-dose aPDT vs. control, p = 0.0001; dual-light, single-dose aPDT vs. control, p = 0.0001; single- dose aBL vs. dual-light, single-dose aPDT, p = 0.0022; single-dose aPDT vs. dual-light, single-dose aPDT, p = 0.74; single-dose aBL vs. single-dose aPDT, p = 0.0022; daily-dose aBL vs. control, p = 0.35; daily-dose aPDT vs. control, p = 0.0001; dual-light, daily-dose aPDT vs. control, p = 0.0001; daily-dose aBL vs. dual-light, daily-dose aPDT, p = 0.00022; daily-dose aPDT vs. dual-light, daily-dose aPDT, p = 0.026; daily-dose aBL vs. daily-dose aPDT, p = 00022; single-dose aBL vs. daily-dose aBL, p = 0.0087; single-dose aPDT vs. daily-dose aPDT, p = 0.048; dual-light single dose vs. dual-light daily dose, p = 0.04; Mann-Whitney U Test.). The columns display medians. Each marking on the columns represents an independent assay. The T-bars show 95% confidence interval (CI). A detailed protocol and number of assays are shown at Table 1.

Fig 3. Effect of daily-dose application of aBL, aPDT, or dual-light aPDT on fourteen-day S. mutans biofilm.

An extended daily-dose study protocol of fourteen days was established to test the ability of the biofilm to adapt to a repetitive aBL or aPDT application (aBL vs control, p = 0.02; aPDT vs. control, p = 0.0022; aPDT vs. aBL, p = 0.0022). The dual-light, daily-dose light application showed the most effective antibacterial effect (dual-light vs. aBL, p = 0.0022, dual-light vs. aPDT, p = 0.0087; dual-light vs. control, p = 0.0022, Mann-Whitney U-Test). The columns display medians. Each marking on the columns represents an independent assay. The T-bars show 95% confidence interval (CI). A detailed protocol and number of assays are shown at Table 1.

Fig 4. Effect of change in the radiant exposure ratio of aBL to aPDT light in the antibacterial efficacy of the dual-light aPDT.

To assess the amount of blue light needed to increase the antibacterial efficacy of the aPDT, we established an experiment where the radiant exposure ratio of between aBL to aPDT was varied. Firstly, one-fourth of the total light irradiance was given as aBL (405 nm light) and three-fourths were given as aPDT (810 nm light). The exact amounts were 42 mW/cm² for the aBL and 135 mW/cm² for the aPDT, corresponding to a 1:3 ratio. Secondly, half of the total light irradiance was given as aBL (405 nm light) and half as aPDT (810 nm light), and the exact amounts were at 73 mW/cm² for the aBL and 79 mW/cm² for the aPDT, corresponding to 1:1 ratio. Thirdly, three-fourths of the total light irradiance were given as aBL (405nm light), and one fourth was given as aPDT (810nm light). The exact amounts in this case were at 130 mW/cm² for the aBL and 38 mW/cm² for the aPDT, corresponding to a 3:1 ratio. Single-day, single-dose, dual-light aPDT: 1:3 vs. 1:1, p = 0.003; 1:3 vs. 3:1, p = 0.43; 1:1 vs. 3:1; p<0.0001. Four-day, single-dose, dual-light aPDT: 1:3 vs. 1:1, p = 0.12; 1:3 vs. 3:1, p = 0.0057; 1:1 vs. 3:1; p = 0.067. Four-day, daily-dose aPDT: 1:3 vs. 1:1, p = 0.045; 1:3 vs. 3:1, p = 0.36; 1:1 vs. 3:1; p = 0.27; Mann-Whitney U Test.). The columns display medians. Each marking on the columns represents an independent assay. The T-bars show 95% confidence interval (CI). A detailed protocol and number of assays are shown at Table 1.

Fig 5. Confocal 3D images of the four-day maturated biofilms stained with live/dead bacterial staining.

A. A single-dose application of aPDT. B. A single dose application of dual-light aPDT, C. A daily-dose application of aPDT for four days. D. A daily-dose application of dual-light aPDT for four days. E. A control four-day S. mutans biofilm.

Fig 6. Absorption spectrometry of ICG adherence to S. mutans.

The absorption spectrum of ICG bound to S. mutans and the absorption spectrum of free ICG dissolved in 0.9% NaCl.

Fig 7. Antibacterial activity of S. mutans bound Indocyanine green.

ICG dyed S. mutans was excited with 810 nm light and compared to non-ICG dyed control. The light exposure resulted in significant reduction of CFU levels (aPDT vs control, p = 0.0357, Mann-Whitney U test)