Photodynamic therapy using systemic administration of 5-aminolevulinic acid and a 410-nm wavelength light-emitting diode for methicillin-resistant Staphylococcus aureus-infected ulcers in mice

Abstract

Bacterial resistance to antibiotics has become a worldwide problem. One potential alternative for bacterial control is photodynamic therapy. 5-aminolevulinic acid is a natural precursor of the photosensitizer protoporphyrin IX. Relatively little is known about the antibacterial efficacy of photodynamic therapy using the systemic administration of 5-aminolevulinic acid; a few reports have shown that 5-aminolevulinic acid exerts photodynamic effects on methicillin-resistant Staphylococcus aureus (MRSA) in vitro. In this study, we evaluated the effectiveness of photodynamic therapy using 5-aminolevulinic acid and a 410-nm wavelength light-emitting diode in vitro and in vivo for the treatment of MRSA. We found that 5-aminolevulinic acid photodynamic therapy with the light-emitting diode had an in-vitro bactericidal effect on MRSA. In vivo, protoporphyrin IX successfully accumulated in MRSA on ulcer surfaces after intraperitoneal administration of 5-aminolevulinic acid to mice. Furthermore, 5-aminolevulinic acid photodynamic therapy accelerated wound healing and decreased bacterial counts on ulcer surfaces; in contrast, vancomycin treatment did not accelerate wound healing. Our findings indicate that 5-aminolevulinic acid photodynamic therapy may be a new treatment option for MRSA-infected wounds.

Figure 1. Mouse model of MRSA-infected cutaneous ulcers.

(A) Mouse model of infected cutaneous ulcers after inoculation with MRSA. (B) Light shielding and wound protection with a bandage.

Figure 2. MRSA counts after PDT with 5-ALA in vitro.

(A) MRSA counts (in CFU/mL) 15 min, 4 h, and 16 h after ALA-PDT with 5 mg/mL 5-ALA and LED irradiation at 50 J/cm². (B) MRSA counts after ALA-PDT with various 5-ALA concentrations and LED total fluencies. Data are expressed as mean ± SEM. *P<0.01.

Figure 3. ALA-PDT selectivity of MRSA over 3T3 cells.

In-vitro survival curves of MRSA and 3T3 cells after PDT with 5 mg/mL 5-ALA and irradiation at 0, 5, 20, and 50 J/cm² with a 410-nm LED. Data are expressed as mean ± SEM. N = 3 in each group.

Figure 4. Accumulation of PpIX in MRSA after intraperitoneal injection with 5-ALA.

(A) Cutaneous skin ulcers with (right) or without (left) MRSA. (B) Strong red fluorescence was detected in MRSA-infected ulcers 24 h after intraperitoneal injection of 200 mg/kg 5-ALA and irradiation with a Wood’s lamp (right). (C) Transmission image of a smear on a cutaneous ulcer infected with MRSA after intraperitoneal injection of 5-ALA. (D) Confocal laser scanning microscopy of PpIX fluorescence in MRSA. 405-nm diode, ultraviolet light, emission bandwidth 620–650 nm.

Figure 5. Change in ulcer area over time after MRSA infection.

Wound healing over time in MRSA-infected or non-infected cutaneous ulcers. Each point represents the mean percentage of the area of the original wound. Data are expressed as mean ± SEM. N = 5 in each group. *P<0.01.

Figure 6. Change in ulcer area over time after PDT.

(A) Healing of MRSA-infected cutaneous ulcers treated with 410-nm irradiation at 50 J/cm². (B) Wound healing over time in mice with MRSA-infected cutaneous ulcers treated with 200 mg/kg 5-ALA with or without PDT. Each point represents the mean percentage of the area of the original wound. Data are expressed as mean ± SEM. N = 5 in each group. *P<0.01.

Figure 7. Changes in ulcer area after VCM treatment.

Wound healing over time in PDT-treated and VCM-treated mice. Each point represents the mean percentage of the area of the original wound. Data are expressed as mean ± SEM. N = 5 in each group.

Figure 8. Bacterial counts in wound tissue after PDT.

Bacterial cell viability in wound tissue from PDT-treated mice (200 mg/kg 5-ALA and 50 J/cm² irradiation) or VCM-treated mice. Data are expressed as mean ± SEM. N = 3 in each group. *P<0.01.

Figure 9. Gross morphology of cutaneous ulcers after PDT.

General morphologies of cutaneous ulcers were imaged on days 1, 4, 7, 10, and 13 in (A) C57BL/ksj db/db mice without MRSA, (B) C57BL/ksj db/db mice with MRSA, (C) VCM-treated mice, and (D) PDT-treated mice (200 mg/kg 5-ALA and 50 J/cm² irradiation).