Anthrax surrogate spores are destroyed by PDT mediated by phenothiazinium dyes

Abstract

ABSTRACT Some Gram-positive bacteria (including the causative agent of anthrax - Bacillus anthracis) survive conditions ofstress and starvation by producing dormant stage spores. The spore’s multilayered capsule consists of inner andouter membranes, cortex, proteinaceous spore coat, and in some species an exosporium. These outer layers enclosedehydrated and condensed DNA, saturated with small, acid-soluble proteins. These protective structures makespores highly resistant to damage by heat, radiation, and commonly employed anti-bacterial agents. PreviouslyBacillus spores have been shown to be resistant to photodynamic inactivation (PDI) using dyes and light that easilydestroy the corresponding vegetative bacteria, but recently we have discovered that they are susceptible to PDI.Photoinactivation, however, is only possible if phenothiazinium dyes are used. Dimethylmethylene blue, methyleneblue, new methylene blue and toluidine blue O are all effective photosensitizers. Alternative photosensitizers such asRose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin and benzoporphyrin derivative are ineffectiveagainst spores even though they can easily kill vegetative cells. Spores of B. cereus and B. thuringiensis are mostsusceptible, B. subtilis and B. atrophaeus are also killed, while B. megaterium is resistant. Photoinactivation is mosteffective when excess dye is washed from the spores showing that the dye binds to the spores and that excess dye insolution can quench light delivery. The relatively mild conditions needed for spore killing could have applicationsfor treating wounds contaminated by anthrax spores and for which conventional sporicides would have unacceptabletissue toxicity.Keywords: photosensitizer, anthrax, spore, photodynamic therapy, toluidine blue, methylene blue, Bacillus species