Another advantage of PDT is that, unlike the vast majority of antibiotics, it can also inactivate microbial virulence factors in addition to its microbicidal effect. Hence, the biological activities of the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis and the lipopolysaccharide of Escherichia coli have all been AG-120 shown to be reduced by irradiation in the presence of a LAAA [29, 30]. The future
of LAAAs for the prevention and/or treatment of infectious diseases looks promising following the recent report of the use of methylene blue to successfully treat periodontitis – one of the most prevalent infectious diseases of humans. Conclusion In this study we have shown that PDT using the light-activated antimicrobial agent, methylene blue, kills MRSA in superficial and deep excisional wounds in mice. However, killing is less effective than when performed in-vitro. This bactericidal effect was not due to the heat generated as a consequence of the treatment. Histological examination of the wounds showed neither collateral tissue necrosis nor architectural disturbance. Methods Bacteria
The organism used in this KPT-8602 in vitro investigation was the prototypic UK epidemic MRSA: EMRSA-16 (NCTC 13143). EMRSA-16 was maintained by weekly sub-culture on blood agar (BA, Oxoid Ltd, Basingstoke, UK) supplemented with 5% (v/v) horse blood. For experimental purposes, a few colonies were inoculated into brain heart infusion broth before (BA, Oxoid Ltd, Basingstoke, UK) and grown aerobically with shaking for 16 hours at 37°C. Cells were then harvested by centrifugation, washed and resuspended in sterile phosphate buffered saline (PBS) to a concentration of 4 × 109 bacteria per ml. Twenty five μl of the bacterial suspension (108 CFU of EMRSA-16) was then added to the wound. Photosensitiser and laser Methylene blue (MB, Sigma, UK) solution was prepared fresh for each experiment in sterile PBS to a final concentration of 100 μg/ml. The light source used was a 665
nm diode laser (PerioWave system, Ondine Biopharma, Vancouver, Canada) with a measured output of 200 mW distributed by a fibreoptic cable and a diffusing head. The source was held at a Selleckchem CB-839 constant distance from the wound to produce a 1 cm2 circle of illumination. Animals All animal experiments were carried out in accordance with the Animals (Scientific Procedures) Act 1986 and with approval of the local Ethics Committee. Eight-week old female C57 Black mice (Charles River, Margate, Kent, UK), of 14–18 g body weight were housed in the local animal unit for 7 days prior to experimentation, with free access to food and water. Excisional wound model Mice were anaesthetised with an intramuscular injection of ketamine-xylazine mixture (90 mg/kg ketamine, 9 mg/kg xylazine), and their backs shaved and depilated with a commercial cream (Veet®, Reckitt Benckiser, UK). Intramuscular Carpofen (5 mg/kg) was used to provide analgesia.