Interaction of the photobactericides methylene blue and toluidine blue with a fluorophore in Pseudomonas aeruginosa cells

Background and Objectives: The difference in photobactericidal efficacy between methylene blue (MB) and toluidine blue (TB) may be explained by their involvement with proteins, lipopolysaccharides (LPS), and siderophores and siderophore-receptor protein complexes on the bacterial outer membrane. This study aims to determine if this is the case by using the fluorescence given off by a pseudomonal siderophore named pyoverdin. Study Design/Materials and Methods: Confocal laser scanning microscopy was used to observe the fluorescence of Pseudomonas aeruginosa cells excited at 488 nm in the presence of increasing dye concentrations. Results: Cellular fluorescence at 522 nm progressively decreased with increasing dye concentrations. The Stern-Volmer constants for cellular fluorescence quenching with the dyes were compared to the association constants for dyes complexed with LPS. The quenching of cellular fluorescence was associated with the formation of a ground-state complex between the dyes and pyoverdin-FpvA protein system. MB readily complexed with this system, whereas TB complexed more strongly with LPS. Conclusion: The different affinities of the dyes for both pyoverdin-protein and LPS will affect the contributions of the dyes' interactions with these biopolymers to the overall bacterial photodamage.