Potentiation of photoinactivation of Gram-positive and Gram-negative bacteria mediated by six phenothiazinium dyes by addition of azide ion

Abstract

Antimicrobial photodynamic inactivation (APDI) using phenothiazinium dyes is mediated by reactive oxygen species consisting of a combination of singlet oxygen (quenched by azide), hydroxyl radicals and other reactive oxygen species. We recently showed that addition of sodium azide paradoxically potentiated APDI of Gram-positive and Gram-negative bacteria using methylene blue as the photosensitizer, and this was due to electron transfer to the dye triplet state from azide anion, producing azidyl radical. Here we compare this effect using six different homologous phenothiazinium dyes: methylene blue, toluidine blue O, new methylene blue, dimethylmethylene blue, azure A, and azure B. We found both significant potentiation (up to 2 logs) and also significant inhibition (>3 logs) of killing by adding 10 mM azide depending on Gram classification, washing the dye from the cells, and dye structure. Killing of E. coli was potentiated with all 6 dyes after a wash, while S. aureus killing was only potentiated by MB and TBO with a wash and DMMB with no wash. More lipophilic dyes (higher log P value, such as DMMB) were more likely to show potentiation. We conclude that the Type I photochemical mechanism (potentiation with azide) likely depends on the microenvironment, i.e. higher binding of dye to bacteria. Bacterial dye-binding is thought to be higher with Gram-negative compared to Gram-positive bacteria, when unbound dye has been washed away, and with more lipophilic dyes.

Antibacterial photodynamic therapy: overview of a promising approach to fight antibiotic-resistant bacterial infections

“…However, the exact mechanism of how the excited PS removes the electron from the azide anion requires further investigation. Similar results were obtained with other phenothiazinium-based PSs, including methylene blue, dimethyl methylene blue, new methylene blue, toluidine blue O, azure A, and azure B [31].…”

Evaluation of Photodynamic Inactivation Efficiency Using Conventional and Decorative Light-Emitting Diode Lamps

“…35 showed that PDI on methicillin-resistant S. aureus did not affect cell proteins; membrane proteins on the surface first exhibited a crosslink phenomenon, and DNA damage appeared only after long-term irradiation, indicating that the hematoporphyrin action occurred on the outside of the cell and that membrane proteins were the primary target; DNA was targeted subsequently. (40,41) This is consistent with the damage mechanism in our study, which is displayed in the SEM image of Fig. 4, indicating the type I reaction damage mechanism.…”

Pigments influence the tolerance of Pseudomonas aeruginosa PAO1 to photodynamically induced oxidative stress

“…In PAO1 photoinactivation experiments with TMPyP confirmed its ability to generate mainly singlet oxygen, as demonstrated by the protection effect exerted by sodium azide, which, by contrast, enhanced the photosensitization effects induced by TBO, as expected for radical inducing PSs (Fig. 4) (Tavares et al, 2011;Kasimova et al, 2014).…”