title: 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.
authors.affiliation: The Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA. hamblin@helix.mgh.harvard.edu.
authors.forename: Kamola R
authors.initials: KR
authors.lastname: Kasimova
authors.forename: Magesh
authors.initials: M
authors.lastname: Sadasivam
authors.forename: Giacomo
authors.initials: G
authors.lastname: Landi
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Sarna
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hamblin
mesh_terms: D001399:Azure Stains / Q000737:chemistry / Q000494:pharmacology; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D008027:Light; D008751:Methylene Blue / Q000737:chemistry / Q000494:pharmacology; D010640:Phenothiazines / Q000737:chemistry* / Q000494:pharmacology; D017319:Photosensitizing Agents / Q000737:chemistry* / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000737:chemistry* / Q000378:metabolism; D019810:Sodium Azide / Q000737:chemistry*; D014048:Tolonium Chloride / Q000737:chemistry / Q000494:pharmacology
chemical_list: D001399:Azure Stains; D010640:Phenothiazines; D017319:Photosensitizing Agents; D017382:Reactive Oxygen Species; D014048:Tolonium Chloride; C025819:Azure B; D019810:Sodium Azide; C025818:Azure A; D008751:Methylene Blue