The worldwide rise in the rates of antibiotic resistance of bacteria underlines the need for alternative antibacterial agents. A promising approach to the killing of gram-positive antibiotic-resistant bacteria of the skin uses light in combination with a photosensitizer to induce a phototoxic reaction. Different concentrations (0 to 100 M) of porphyrin-based photosensitizers (CTP1, XF70, and XF73) and different incubation times (5 min, 1 h, and 4 h) were used to determine phototoxicity against two methicillin-resistant Staphylococcus aureus strains, one methicillin-sensitive S. aureus strain, one methicillin-resistant Staphylococcus epidermidis strain, one Escherichia coli strain, and human keratinocytes and fibroblasts. Incubation with 0.005 M XF70 or XF73, followed by illumination, yielded a 3-log 10 (>99.9%) decrease in the viable cell numbers of all staphylococcal strains, indicating that the XF drugs have high degrees of potency against gram-positive bacteria and also that the activities of these novel drugs are independent of the antibiotic resistance pattern of the staphylococci examined. CTP1 was less potent against the staphylococci under the same conditions. At 0.005 M, XF70 and XF73 demonstrated no toxicity toward fibroblasts or keratinocytes. No inactivation of E. coli was detected at this concentration. XF73 was confirmed to act via a reactive oxygen species from the results of studies with sodium azide (a quencher of singlet oxygen), which reduced the killing of both eukaryotic and prokaryotic cells. When a quencher of superoxide anion and the hydroxyl radical was used, cell killing was not inhibited. These results demonstrate that the porphyrin-based photosensitizers had concentration-dependent differences in their efficacies of killing of methicillin-resistant staphylococcal strains via reactive oxygen species without harming eukaryotic cells at the same concentrations.

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Following extensive in vitro screening of new photosensitizers the purpose of the present study was to examine penetration as well as antibacterial efficacy of a lead photosensitizer against MRSA using an ex vivo porcine skin model. Two different applications were performed: (i) preincubation of bacteria in solution with a porphyrin-based photosensitizer XF73 and subsequent application on the ex vivo porcine skin; (ii) application of pure bacteria on the explants followed by an incubation with XF73 in a water-ethanol formulation for up to 60 min under occlusion. The localisation of XF73 was restricted to the stratum corneum. Different concentrations (0-10 microM) of XF73 and different incubation times (5-60 min) were used to determine phototoxicity against methicillin-resistant and methicillin-sensitive S. aureus, which was applied on the explants. Preincubation of S. aureus with 0.1 microM XF73 in solution prior to the application of these XF73-incubated bacteria on the skin demonstrates a higher efficacy (>3 log10) after irradiation. Antibacterial photodynamic inactivation resulted in a approximately 1 log10 (0.1 microM)-3.64+/-0.035 (10 microM) log10 growth reduction independently of the antibiotic resistance pattern of used S. aureus strains. Irradiation of applied bacteria without photosensitizer incubation did not show any marked decrease (<1 log10) of bacteria cell number, indicating a significant phototoxicity of the XF73. Histological evaluations of untreated and treated skin areas upon irradiation within 24 h showed no significant degree of necrosis or apoptosis determined by TUNEL-assay indicating that the porcine skin is still vital. This study demonstrates that this XF73 porphyrin-based photosensitizer had concentration-dependent differences in killing efficacy of MRSA in comparison to skin cells using an ex vivo porcine skin model. The results described here imply that topical delivery of XF73 may be considered as a possible treatment in patients with superficial infections of the skin.

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