Effect of cell-photosensitizer binding and cell density on microbial photoinactivation

Abstract

Photodynamic therapy involves the use of nontoxic dyes called photosensitizers and visible light to produce reactive oxygen species and cell killing. It is being studied as an alternative method of killing pathogens in localized infections due to the increasing problem of multiantibiotic resistance. Although much has been learned about the mechanisms of microbial killing, there is still uncertainty about whether dyes must bind to and penetrate various classes of microbe in order to produce effective killing after illumination. In this report, we compare the interactions of three antimicrobial photosensitizers: rose bengal (RB), toluidine blue O (TBO), and a poly-L-lysine chlorin(e6) conjugate (pL-ce6) with representative members of three classes of pathogens; Escherichia coli (gram-negative bacteria), Staphylococcus aureus (gram-positive bacteria), Candida albicans (yeast). We compared fluence-dependent cell survival after illumination with the appropriate wavelengths of light before and after extracellular dye had been washed out and used three 10-fold dilutions of cell concentration. pL-ce6 was overall the most powerful photosensitizer, was equally effective with and without washing, and showed a strong dependence on cell concentration. TBO was less effective in all cases after washing, and the dependence on cell concentration was less pronounced. RB was ineffective after washing (except for S. aureus) but still showed a dependence on cell concentration. The overall order of susceptibility was S. aureus>E. coli>C. albicans, but C. albicans cells were 10 to 50 times bigger than the bacteria. We conclude that the number and mass of the cells compete both for available dye binding and for extracellularly generated reactive oxygen species.

FIG. 1.

Chemical structures of the PS used in this study.

FIG. 2.

Photoinactivation of S. aureus. Cells were incubated with the specified concentration of PS for 20 min followed by illumination with the appropriate wavelength of light (see text). • and ○, 10⁹ cells/ml; ▪ and □, 10⁸ cells/ml; ▴ and ▵, 10⁷ cells/ml; •, ▪, and ▴, PS washed from cells before illumination; ○, □, and ▵, PS left in cell suspension during illumination. The values shown are means of three independent experiments, and bars are the standard error of the mean. ***, P < 0.001; **, P < 0.01; *, P < 0.05; compared with next highest cell concentration. §, P < 0.05, between no wash and corresponding wash.

FIG. 3.

Photoinactivation of E. coli. Cells were incubated with the specified concentration of PS for 20 min followed by illumination with the appropriate wavelength of light (see text). • and ○, 10⁹ cells/ml; ▪ and □, 10⁸ cells/ml; ▴ and ▵, 10⁷ cells/ml; •, ▪, and ▴, PS washed from cells before illumination; ○, □, and ▵, PS left in cell suspension during illumination. The values shown are means of three independent experiments, and bars are the standard error of the mean. ***, P < 0.001; **, P < 0.01; *, P < 0.05, compared with next highest cell concentration. §§§, P < 0.001; and §, P < 0.05, between no wash and corresponding wash.

FIG. 4.

Photoinactivation of C. albicans. Cells were incubated with the specified concentration of PS for 20 min followed by illumination with the appropriate wavelength of light (see text). • and ○, 10⁸ cells/ml; ▪ and □, 10⁷ cells/ml; ▴ and ▵, 10⁶ cells/ml; •, ▪, and ▴, PS washed from cells before illumination; ○, □, and ▵, PS left in cell suspension during illumination. Values shown are means of three independent experiments, and bars are the standard error of the mean. ***, P < 0.001; *, P < 0.05, compared with next highest cell concentration. §§§, P < 0.001; §, P < 0.05, between no wash and corresponding wash.