Photoinactivation of staphylococcus aureus in vitro by red light (660 nm) in the presence of methylene blue

Abstract

Introduction. Оpen wounds and ulcers treatment involves the use of bandage material, antibiotics and
antiseptic to prevent the development of a pathogenic microflora and to provide the necessary conditions for
tissue regeneration. An emergence of multi-resistant strains of microorganisms reduces the effectiveness of such
technology and requires the new treatment approaches. One of the promising areas is a photodynamic therapy
with the use of external photosensitizers.
The aim of the investigation is to determine the effectiveness of the synergistic action of red light (660 nm)
and different concentrations of methylene blue on the inhibition of Staphylococcus aureus growth.
Materials and methods. We used the hydrogels based on the copolymers dextran-polyacrylamide with
the different concentration of crosslinking agent 0.2 %, 0.4 %, 0.6 % (w/w) for investigation a rate of diffusion
methylene blue into and out from hydrogel. Microbiological research was performed on wild strains of S. aureus
isolated on a Yolk-salt agar. The evaluation of a bactericidal action of methylene blue was carried out on a
Müller-Hinton No. 2 agar similarly to the disc-diffusion method for assessing the resistance of microorganisms
to antibiotics. For irradiation by different wavelengths was used «LIKA-Led» (Photonics Plus) LEDs 390 nm,
460 nm and 660 nm. The radiation power for each wavelength was 100 mW, duration — 20 min, 30 min, 40 min.
According to the duration, the irradiation doses were 21 J/cm2, 31.5 J/cm2, 42.1 J/cm2. Mathematical and
statistical data processing was performed in the OriginLab 8.0 software package.
Results. Increasing the amount of crosslinking agent in the hydrogel based on the copolymer dextran-polyacrylamide
provides a decrease in the diffusion rate of methylene blue from the hydrogel. 390 nm ultraviolet
radiation reduces the number of S. aureus colonies for 80% at 20 min exposure. Further increase in the exposure
did not contribute to significant changes in this indicator. Blue light (460 nm) reduces the presence of this
strain of microorganisms for 66% at 20 min exposure and reaches the effect of UV at 30 min exposure. Red
light (660 nm) has no bactericidal effect. Minimal activity was found for methylene blue at concentrations of
0.001% and 0.0001% which was around 6 mm. The synergistic effect of 0.001% methylene blue and red light
increases the activity for 40% up to 10 mm.
Conclusions. For the photoinactivation of Staphylococcus aureus, it is advisable to use a low energy red
light with a 660 nm wavelength in combination with a saturated methylene blue (0.001%) hydrogels. Perhaps
the synergistic action of red light and dye provides a generation of active radicals that contribute to the growth
retardation of microorganisms.