Figure 7.

Photodynamic properties of the M13_Gram− phage vector

(A) Amplex Red assay for the generation of peroxides using different concentrations of RB (pink) and M13_Gram−-RB (purple).

(B) Determination of ¹O₂ generation following the decrease of ABMDMA absorbance over the irradiation time under white light irradiation for RB (pink line), M13_Gram−-RB (purple line), and PBS (black line).

(C) Flow cytometry analysis of retargeted and RB-conjugated M13_Gram− phages directed to the lipopolysaccharides of Gram-negative bacteria: dark lines correspond to the control cells treated with PBS, whereas colored ones highlight binding of the engineered M13_Gram−-RB to S. aureus (orange), P. aeruginosa (magenta), and A. baumannii (cyan).

(D) Percentage of bacterial fluorescent cells measured by flow cytometry analysis.

(E–I) (E) Percentage of bacteria survived to light irradiation after preincubation with M13_Gram−-RB phages. The activity of phages was tested at two concentrations, 0.1 and 0.25 μM, of equivalent RB. Live/dead assay was performed on P. aeruginosa and A. baumannii preincubated with M13_Gram−-RB and (F–H) kept in dark or (G–I) irradiated.

(J–M) Percentage of live, injured, and dead cells measured in live/dead assay 20 min after irradiation. Metabolic activity of (K) P. aeruginosa and (M) A. baumannii after phage-mediated aPDT. Data are represented as mean ± SD. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001.