Effects of Growth Phase and Extracellular Slime on Photodynamic Inactivation of Gram-Positive Pathogenic Bacteria

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Abstract
The emergence of antibiotic resistance among pathogenic bacteria has led to efforts to find alternative antimicrobial therapeutics to which bacteria will not be easily able to develop resistance. One of these may be the combination of nontoxic dyes (photosensitizers [PS]) and visible light, known as photodynamic therapy, and we have reported its use to treat localized infections in animal models. While it is known that gram-positive species are generally susceptible to photodynamic inactivation (PDI), the factors that govern variation in degrees of killing are unknown. We used isogenic pairs of wild-type and transposon mutants deficient in capsular polysaccharide and slime production generated from Staphylococcus epidermidis and Staphylococcus aureus to examine the effects of extracellular slime on susceptibility to PDI mediated by two cationic PS (a polylysine-chlorine6 conjugate, pL-ce6, and methylene blue [MB]) and an anionic molecule, free ce6, and subsequent exposure to 665-nm light at 0 to 40 J/cm2. Free ce6 gave more killing of mutant strains than wild type, despite the latter taking up more PS. Log-phase cultures were killed more than stationary-phase cultures, and this correlated with increased uptake. The cationic pL-ce6 and MB gave similar uptakes and killing despite a 50-fold difference in incubation concentration. Differences in susceptibility between strains and between growth phases observed with free ce6 largely disappeared with the cationic compounds despite significant differences in uptake. These data suggest that slime production and stationary phase can be obstacles against PDI for gram-positive bacteria but that these obstacles can be overcome by using cationic PS.