Efficient photodynamic therapy against gram-positive and gram-negative bacteria using THPTS, a cationic photosensitizer excited by infrared wavelength

Abstract

The worldwide rise in the rates of antibiotic resistance of bacteria underlines the need for alternative antibacterial agents. A promising approach to kill antibiotic-resistant bacteria uses light in combination with a photosensitizer to induce a phototoxic reaction. Concentrations of 1, 10 and 100microM of tetrahydroporphyrin-tetratosylat (THPTS) and different incubation times (30, 90 and 180min) were used to measure photodynamic efficiency against two Gram-positive strains of S.aureus (MSSA and MRSA), and two Gram-negative strains of E.coli and P.aeruginosa. We found that phototoxicity of the drug is independent of the antibiotic resistance pattern when incubated in PBS for the investigated strains. Also, an incubation with 100microM THPTS followed by illumination, yielded a 6lg (> or =99.999%) decrease in the viable numbers of all bacteria strains tested, indicating that the THPTS drug has a high degree of photodynamic inactivation. We then modulated incubation time, photosensitizer concentration and monitored the effect of serum on the THPTS activity. In doing so, we established the conditions to obtain the strongest bactericidal effect. Our results suggest that this new and highly pure synthetic compound should improve the efficiency of photodynamic therapy against multiresistant bacteria and has a significant potential for clinical applications in the treatment of nosocomial infections.

Figure 1. Viability of Gram-positive bacteria using a live/dead assay.

Cells were incubate for 90min with THPTS and either kept in the dark (A, B) or irradiated (C, D) at 760nm. Healthy bacteria were seen in green whereas dying bacteria were also stained with propidium iodie and appeared therefore in yellow. Dying bacteria could be observed in control panels (arrows in B) but no healthy bacteria were seen in irradiated panel (C and D). Scale bar 40µm in A and C, 10µm in B and D.

Figure 2. Photoinactivation of MSSA by THPTS.

Photoinactivation of S.aureus (MSSA strain: DSM1104) by THPTS after incubation times of 30, 90 or 180min in PBS (A) or in PBS+10% HS (human serum, B). Viability of MSSA was determined by a CFU assay. Even in presence of serum, 10µM of THPTS for 90min had a dramatic bactericidal effect leading to the total absence of colony. Each point is the mean ± standard deviation of three experiments (P<0.005). CD: control in the dark, bacteria were maintained in the dark after incubation with THPTS. CL: control with light, no THPTS incubation before laser treatment. PI: photoinactivation using THPTS at indicated concentrations before laser treatment.

Figure 3. Photoinactivation of MRSA by THPTS.

Photoinactivation of S.aureus (MRSA strain: DSM11729) by THPTS after incubation times of 30, 90 and 180min in PBS (A) and in PBS+10% HS (B). Viability of MRSA was determined by a CFU assay. 10µM of THPTS for 30min were sufficient to obtain a total absence of colony. However this concentration had to be increased to 100µM in presence of serum. Each bar is the mean ± standard deviation of three experiments (P<0.005). CD: control in the dark, CL: control with light, PI: photoinactivation.

Figure 4. Viability of Gram-negative bacteria using live/dead assay.

Cells were incubate with THPTS for 90min and either kept in the dark (A, B) or irradiated (C, D) at 760nm. Healthy bacteria were seen in green whereas dying bacteria were also stained with propidium iodide and appeared therefore in yellow. Dying bacteria could be observed in controls (arrows in A and B) but no healthy bacteria were seen in irradiated samples (C and D). Scale bar 40µm in A and C, 10µm in B and D.

Figure 5. Photoinactivation of E. Coli by THPTS.

Photoinactivation of E. coli (DSM8698) by THPTS photosensitizer (PS) after incubation times of 30; 90 and 180min in PBS (A) or in PBS+10% HS (B). Viability of bacterial cells was determined by a CFU assay. 100µM of THPTS for 90min, or 10µM for 180min were sufficient to obtain an efficient bactericidal effect. However, serum addition reduced the efficiency of the treatment without abolishing it when THPTS was incubated for 90min. Each bar is the mean ± standard deviation of three experiments (P<0.005). CD: control in the dark, CL: control with light, PI: photoinactivation.

Figure 6. Photoinactivation of P.aeruginosa by THPTS.

Photoinactivation of P.aeruginosa (DSM1117) by THPTS photosensitizer (PS) after incubation times of 30, 90 and 180min in PBS (A) and PBS+10% HS (B). 100µM of THPTS for 30min or 10µM of THPTS for 90min were sufficient to obtain a total bactericidal effect leading to the complete absence of colony. Serum addition completely abolished the bactericidal effect of THPTS for this bacterial strain. Viability of bacterial cells was determined by a CFU assay. Each bar is the mean ± standard deviation of three experiments (P<0.005). CD: control in the dark, CL: control with light, PI: photoinactivation.