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. 2005 Mar;49(3):895-902.
doi: 10.1128/AAC.49.3.895-902.2005.

Bactericidal effects of toluidine blue-mediated photodynamic action on Vibrio vulnificus

Tak-Wah Wong  1 , Yin-Yi Wang, Hamm-Ming Sheu, Yin-Ching Chuang
Affiliations

Bactericidal effects of toluidine blue-mediated photodynamic action on Vibrio vulnificus

Tak-Wah Wong et al. Antimicrob Agents Chemother. 2005 Mar.

Abstract

Vibrio vulnificus is a gram-negative, highly invasive bacterium responsible for human opportunistic infections. We studied the antibacterial effects of toluidine blue O (TBO)-mediated photodynamic therapy (PDT) for V. vulnificus wound infections in mice. Fifty-three percent (10 of 19) of mice treated with 100 microg of TBO per ml and exposed to broad-spectrum red light (150 J/cm(2) at 80 mW/cm(2)) survived, even though systemic septicemia had been established with a bacterial inoculum 100 times the 50% lethal dose. In vitro, the bacteria were killed after exposure to a lower light dose (100 J/cm(2) at 80 mW/cm(2)) in the presence of low-dose TBO (0.1 microg/ml). PDT severely damaged the cell wall and reduced cell motility and virulence. Cell-killing effects were dependent on the TBO concentration and light doses and were mediated partly through the reactive oxygen species generated during the photodynamic reaction. Our study has demonstrated that PDT can cure mice with otherwise fatal V. vulnificus wound infections. These promising results suggest the potential of this regimen as a possible alternative to antibiotics in future clinical applications.

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Figures

FIG. 1.
FIG. 1.
Inhibition of V. vulnificus growth (inoculum size, 5 × 105 CFU/ml) after the bacteria were incubated with different concentrations of TBO and kept in the dark (A) or exposed to 100 J of red light/cm2 at 80 mW/cm2 (B). Datum points are the means of triplicate determinations and two separate experiments, and bars indicate standard deviations. In panel B, the curves for 0.1, 1, and 10 μg/ml overlap completely.
FIG. 2.
FIG. 2.
TBO-PDT inhibits V. vulnificus motility. V. vulnificus (108 CFU/ml) was incubated with TBO at 0.1 μg/ml before irradiation with red light. The V. vulnificus cells moved, rotated, and spun in all directions freely and at a high speed (A). Thirty minutes after irradiation, cell motility was markedly reduced and could barely be seen on the video at the same speed. The bacteria also became less reflective (B). No movable bacteria could be observed at 1 h post-PDT. Almost all cells lost their rod shape and became round (C). Bars, 25 μm.
FIG. 3.
FIG. 3.
Effect of photodynamic actions on V. vulnificus proteolytic activities as revealed by azocasein assay after exposure to different TBO concentrations and light doses. The protease-containing supernatants were kept in the dark or exposed to 100 J of red light/cm2 (A). Proteolytic activity was significantly reduced in the presence of TBO concentrations greater than 0.1 μg/ml with light exposure (P < 0.001; Bonferroni t method). The inhibition was TBO dose dependent (P < 0.05; linear regression). Irradiation of the supernatants in the presence of 1 or 10 μg of TBO per ml resulted in a substantial, light-dose-dependent decrease in proteolytic activity (B) (P = 0.002 for TBO at both 1 and 10 μg/ml; linear regression). Protease secretion and proteolytic activities were determined by the well assay (C). Samples were kept in the dark (left panel) or exposed to 100 J of red light/cm2 (right panel). The protease activity was totally inhibited by TBO at concentrations greater than or equal to 0.1 μg/ml when the samples were exposed to light, as shown by the absence of a clear halo in the agar. There was a trend for a very low TBO concentration (0.01 μg/ml) to result in protease inhibition when the sample was exposed to light, although this difference was not significant (P = 0.636). Datum points are the means of triplicate determinations and three separate experiments, and bars indicate standard deviations.
FIG. 4.
FIG. 4.
Free radicals and singlet oxygen scavengers reduced the antibacterial effect of TBO-PDT, while stabilizers enhanced the antibacterial effect of TBO-PDT. Increases in the numbers of viable V. vulnificus cells of 2.2 and 3.7 log10 were found with the addition of the scavengers proline and l-tryptophan, respectively, compared to the numbers obtained by treatment with TBO alone and the same light dose (P < 0.001; Bonferroni t method) (A). Effect of D2O on lethal photosensitization (B). The presence of saline (S) or D2O (D) did not affect bacterial growth when the samples were either kept in the dark or exposed to 100 J of red light/cm2 alone (left two sets of bars). The viable counts were reduced 3.9 log10 in the D2O-treated groups and 1.5 log10 in the saline-treated groups exposed to light compared to the counts for the control kept in the dark (P < 0.001; Bonferroni t method).
FIG. 5.
FIG. 5.
TBO-PDT disrupts the flagellum and causes cell wall pebbling of V. vulnificus. TEM studies showed the typical long single flagellum (arrow), fine hairlike pili (arrowhead), and the intact cell wall of V. vulnificus before PDT treatment (A). Immediately after PDT treatment, the flagellum was fragmented and difficult to find. The cell wall structure was severely damaged, as revealed by the formation of many bubbles on the cell wall (arrow). The pili of the cells remained intact (arrowhead) (B). Bars, 0.5 μm.
FIG. 6.
FIG. 6.
Kaplan-Meier survival plots for TBO-PDT-treated mice. Mice were kept in the dark (D) or were exposed to 150 J of red light/cm2 (L) with increasing TBO concentrations (0 to 100 μg/ml [as indicated by the numbers after L and D]). TBO at 100 μg/ml and light treatment (•), n = 19 animals; TBO at 100 μg/ml with the animals kept in the dark (○), n = 24 animals; TBO at 10 μg/ml and light treatment (▿), n = 20 animals; TBO at 10 μg/ml with the animals kept in the dark (□), n = 20 animals; TBO at 1 μg/ml and light treatment (▪), n = 4 animals; TBO at 1 μg/ml with the animals kept in the dark (□), n = 4 animals; TBO at 0 μg/ml and light treatment (♦), n = 4 animals; TBO at 0 μg/ml with the animals kept in the dark (⋄), n = 4 animals.
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