Use of merocyanine 540 for photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells

Abstract

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 mug of MC 540 per ml and a light dose of 600 J/cm2 or to 20 mug of MC 540 per ml and a light dose of 450 J/cm2. A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.

FIG. 1.

MC 540 bound to S. aureus planktonic cells in response to treatment with 5 μg of MC 540 per ml (•), 10 μg of MC 540 per ml (□), 15 μg of MC 540 per ml (▴), and 20 μg of MC 540 per ml (▵) in the absence of light. The error bars indicate standard errors of the means from three separate experiments.

FIG. 2.

Antimicrobial activity of MC 540-mediated PDI of S. aureus planktonic cells (a) and excitation level of MC 540 (b) in response to treatment with no MC 540 (⋄), 5 μg of MC 540 per ml (•), 10 μg of MC 540 per ml (□), 15 μg of MC 540 per ml (▴), and 20 μg of MC 540 per ml (▵) for 10 min in the dark, followed by green light irradiation at various levels. N/N₀ is the count measurement normalized by the initial count.

FIG. 3.

Antimicrobial activity of MC 540-mediated PDI of S. aureus (a) and excitation level of MC 540 (b) in response to treatment with 15 μg of MC 540 per ml for 10 min in the dark and 1-fold dilution (•), 2-fold dilution (○), 5-fold dilution (▪), and 10-fold dilution (□) with PBS, followed by green light irradiation at various intensities. N/N₀ is the count measurement normalized by the initial count.

FIG. 4.

Antimicrobial activity of MC 540-mediated PDI of S. aureus biofilms. Biofilms were treated with no MC 540 (⋄), 5 μg of MC 540 per ml (•), 10 μg of MC 540 per ml (□), 15 μg of MC 540 per ml (▴), and 20 μg of MC 540 per ml (▵) for 40 min in the dark, followed by green light irradiation at various doses. The error bars indicate standard errors of the means from three separate experiments. N/N₀ is the count measurement normalized by the initial count.

FIG. 5.

Excitation level of MC 540 bound to biofilms. Biofilms were treated with 5 μg of MC 540 per ml (•), 10 μg of MC 540 per ml (□), 15 μg of MC 540 per ml (▴), and 20 μg of MC 540 per ml (▵) for 40 min in the dark, followed by green light irradiation at various doses.