Light source
A Periowave™ laser (Ondine Biopharma Inc., Canada), which emits light with a wavelength of 665 nm was used for all irradiation experiments. For experimental purposes, the laser system was set up to give a power density of 32 mW/cm2. The power output of the laser was measured using a thermopile power meter (TPM-300CE, Genetic, Canada) and was found to be 73 mW at the plate surface.
Photosensitiser
Methylene blue (C16H18ClN3S.3H2O) was purchased from Sigma-Aldrich (UK). Stock solutions of 0.1 mg/ml were prepared in phosphate buffered saline (PBS) and kept in the dark at room temperature.
Bacterial strains
EMRSA-16 was maintained by weekly subculture on Blood Agar (Oxoid Ltd, UK), supplemented with 5% horse blood (E & O Laboratories Ltd). For experimental purposes, bacteria were grown aerobically in Brain Heart Infusion broth (Oxoid Ltd, UK) at 37°C for 16 hours in a shaking incubator at 200 rpm. Cultures were centrifuged and resuspended in an equal volume of PBS and the optical density was adjusted to 0.05 at 600 nm, corresponding to approximately 1 × 107 colony forming units (CFU) per mL.
The effect of photosensitiser dose on the lethal photosensitisation of EMRSA-16
Methylene blue was diluted in PBS to give final concentrations of 1, 5, 10 and 20 μM. 50 μL of methylene blue was added to an equal volume of the inoculum in triplicate wells of a sterile, flat-bottomed, untreated 96-well plate and irradiated with 665 nm laser light with an energy density of 1.93 J/cm2 (L+S+), with stirring. Three additional wells containing 50 μL methylene blue and 50 μL of the bacterial suspension were kept in the dark to assess the toxicity of the photosensitiser alone (L-S+). 50 μL PBS was also added to 50 μL of the inoculum in a further six wells, three of which were irradiated with laser light as above (L+S-) and the remaining three were kept in the dark (L-S-). Following irradiation/dark incubation, each sample was serially diluted 10-fold in PBS. 10 μL of each dilution was spotted onto 5% horse blood agar plates in triplicate and the plates incubated aerobically overnight at 37°C. The surviving CFU/mL were enumerated by viable counting. Experiments were performed three times in triplicate.
The effect of laser light dose on the lethal photosensitisation of EMRSA-16
Methylene blue was diluted in PBS to give a final concentration of 20 μM. 50 μL of methylene blue was added to an equal volume of the inoculum in triplicate wells of a sterile, flat-bottomed, untreated 96-well plate and irradiated with 665 nm laser light with energy densities of 1.93 J/cm2, 3.86 J/cm2 or 9.65 J/cm2, corresponding to 1, 2 or 5 minutes irradiation respectively, with stirring (L+S+). Three additional wells containing 50 μL methylene blue and 50 μL of the bacterial suspension were kept in the dark (L-S+) and 50 μL PBS was also added to 50 μL of the inoculum in a further six wells, three of which were irradiated with laser light (L+S-) and the remaining three were kept in the dark (L-S-). Following irradiation/dark incubation, each sample was serially diluted 10-fold in PBS. 10 μL of each dilution was spotted onto 5% horse blood agar plates in triplicate and the plates incubated aerobically overnight at 37°C. The surviving CFU/mL were enumerated by viable counting. Experiments were performed three times in triplicate.
Azocasein hydrolysis assay
Endoproteinase Glu-C (also known as V8 protease) from S. aureus V8 was purchased from Sigma-Aldrich (UK) and stored at -20°C at a concentration of 1 mg/mL in dH2O. A final concentration of 5 μg/mL was obtained by diluting the enzyme in PBS after preliminary experiments to determine the appropriate concentration for the assay conditions. 50 μL of V8 protease was added to an equal volume of either methylene blue (S+) or PBS (S-) in triplicate wells of a 96-well plate and samples were irradiated with laser light (L+) or incubated in the dark (L-). For photosensitiser dose experiments, final concentrations of 1, 5, 10 and 20 μM methylene blue were used and samples were irradiated with 665 nm laser light with an energy density of 1.93 J/cm2. For laser light dose experiments, a final concentration of 20 μM methylene blue was used and samples were irradiated with 665 nm laser light for either 1, 2 or 5 minutes, corresponding to energy densities of 1.93 J/cm2, 3.86 J/cm2 or 9.65 J/cm2.
After irradiation, the azocasein hydrolysis assay (modified from [15]) was performed. 100 μL was removed from each well and added to 50 μL of 6% azocasein (w/v) in 0.5 M Tris buffer, pH 7 (Sigma-Aldrich, UK) in 0.5 mL Eppendorf tubes. Samples were incubated in the dark for one hour at 37°C. The reaction was stopped with an equal volume of 20% acetic acid and the samples centrifuged for 10 minutes at 5590 × g. 75 μL of the supernatant was removed in duplicate and the optical density read at 450 nm using a Dynex plate reader. The enzyme activity at one hour was calculated for each sample; one unit of activity was determined as that which caused a change in absorbance of 0.001 in one hour at 450 nm. Photosensitiser and light dose experiments were performed three times in triplicate.
Haemolytic titration
α-haemolysin from S. aureus was purchased from Sigma-Aldrich (UK) and stored at 2-8°C at a concentration of 0.5 mg/mL in sterile, deionised water plus sodium citrate buffer. For experimental purposes, α-haemolysin was diluted in sterile PBS to a final concentration of 100 μg/mL after preliminary experiments to determine the appropriate concentration for the assay conditions and according to Bhakdi et al. [30]. For photosensitiser dose experiments, the stock solution of methylene blue was diluted in PBS to give final concentrations of 1, 5, 10 and 20 μM. 50 μL of methylene blue was added to an equal volume of α-haemolysin in duplicate wells of a sterile, flat-bottomed, untreated 96-well plate and irradiated with laser light for 1 minute, corresponding to an energy dose of 1.93 J/cm2 (L+S+). Two additional wells containing 50 μL methylene blue and 50 μL of the α-haemolysin were kept in the dark to assess the effect of the photosensitiser alone (L-S+). 50 μL PBS was also added to 50 μL of the α-haemolysin in a further four wells, two of which were irradiated with laser light (L+S-) and the remaining two kept in the dark (L-S-). For laser light dose experiments, a final concentration of 20 μM methylene blue was used and samples were irradiated with 665 nm laser light for either 1, 2 or 5 minutes, corresponding to energy densities of 1.93 J/cm2, 3.86 J/cm2 or 9.65 J/cm2. Following irradiation/dark incubation, samples were removed and aliquoted into round-bottomed 96-well plates for the haemolytic titration assay.
For the haemolytic titration assay, samples were serially diluted using doubling dilutions in PBS. Sterile, deionised water was used as a positive control and sterile PBS as a negative control. Defibrinated rabbit blood (E & O Laboratories, UK) was centrifuged at 503 × g for 10 minutes and the supernatant discarded. The cells were washed and resuspended in sterile PBS to a final concentration of 2%. 50 μL was added to the serially diluted toxin and control wells and incubated in the dark at 37°C for 1 hour. After incubation, the haemolytic titre for each sample was determined as the highest dilution giving rise to lysis. Photosensitiser dose experiments were performed twice in duplicate and light dose experiments were performed twice in triplicate
The effect of human serum on the photosensitisation of S. aureusα-haemolysin
α-haemolysin was diluted to a final concentration of 100 μg/mL in either PBS or PBS + 12.5% human serum (Sigma Aldrich, UK) in order to determine the effect of serum on the photoinactivation of the toxin. 12.5% serum was added to model in vivo conditions as this concentration provided a protein concentration similar to that found in an acute wound [29]. α-haemolysin in either the presence or absence of human serum was exposed to 20 μM methylene blue and laser light with energy densities of 1.93 J/cm2, 3.86 J/cm2 or 9.65 J/cm2 and the haemolytic titration assay was performed as previously described. Experiments were performed twice in triplicate.
Spectrophotometric assay for sphingomyelinase activity
Sphingomyelinase (also known as β-haemolysin or β-toxin) from S. aureus was purchased from Sigma-Aldrich (UK) in buffered aqueous glycerol containing 0.25 M phosphate buffer, pH 7.5. For experimental purposes, the enzyme was diluted to a final concentration of 0.5 Units/mL in 250 mM Tris-HCl buffer with 10 mM magnesium chloride, pH 7.4 at 37°C according to the manufacturer's instructions, based on the spectrophotometric assay for sphingomyelinase described by Gatt [31]. 25 μL of sphingomyelinase was added to either 25 μL of 1, 5, 10 or 20 μM methylene blue (S+) or 25 μL PBS (S-) and irradiation of the enzyme suspension was carried out using an energy density of 1.93 J/cm2, with the appropriate controls (L-S-, L-S+, L+S-). Experiments were performed three times in duplicate. For laser light dose experiments, 20 μM methylene blue and energy densities of 1.93 J/cm2, 3.86 J/cm2 or 9.65 J/cm2 were used and experiments were performed three times in triplicate
Following irradiation/dark incubation, the spectrophotometric assay for sphingomyelinase activity (modified from [32]) was performed. 10 μL from each sample was removed and added to 190 μL of incubation buffer containing 0.02 mg Trinitrophenylaminolauroyl-Sphingomyelin (TNPAL-Sphingomyelin; Sigma-Aldrich, UK), 250 mM Tris-HCl, 10 mM MgCl2 and 1% Triton X-100 in 0.5 mL Eppendorf tubes and incubated in the dark at 37°C for 5 minutes, with shaking. 150 μL of Isopropanol:Heptane:H2SO4 (40:10:1) was added to stop the reaction and the tubes were immediately placed on ice. 100 μL of n-heptane (Sigma-Aldrich, UK) and 80 μL deionised water were then added and the samples were centrifuged for ten minutes at 1398 × g. Following centrifugation, the tubes were left to settle at room temperature for 5 minutes, after which 60 μL of the upper layer was removed and the optical density at 330 nm recorded using a UV-VIS spectrophotometer. A blank sample containing 10 μL incubation buffer instead of sphingomyelinase was used as a reference.
The effect of human serum on the photosensitisation of S. aureussphingomyelinase
Sphingomyelinase was diluted to a final concentration of 0.5 Units/mL in either 250 mM Tris-HCl buffer with 10 mM magnesium chloride, pH 7.4 at 37°C or the buffer with the addition of 12.5% human serum (Sigma Aldrich, UK) in order to model acute wound conditions and exposed to 20 μM methylene blue and laser light with energy densities of 1.93 J/cm2 or 9.65 J/cm2. The spectophotometric assay for sphingomyelinase activity was performed as previously described. Experiments were performed twice in triplicate.
SDS PAGE analysis
After photosensitisation or dark incubation as previously described, the V8 protease and α-haemolysin were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). Briefly, 20 μL of each sample was added to 5 μL reducing SDS PAGE sample buffer (Pierce, UK) and boiled for 5 minutes to denature the protein. Samples were then analysed by SDS PAGE using a 5% stacking gel and 15% resolving gel. After electrophoresis, gels were placed in a fixative solution (40% methanol, 15% acetic acid) and then stained with Brilliant Blue G (Sigma, UK). V8 protease samples were incubated on ice with 100 mM phenylmethanesulfonyl fluoride for 30 minutes prior to SDS PAGE in order to minimise self-digestion. The expected molecular masses of the V8 protease and α-haemolysin were given as 29 kDa and 33 kDa respectively, as specified by the manufacturer.
Statistical analysis
Data are expressed as means ± standard error. The results of the azocasein hydrolysis assay and sphingomyelinase assay were analysed using the univariate ANOVA test with Bonferroni analysis. The results from the lethal photosensitisation of EMRSA-16 were analysed using the Mann Whitney U test. For both statistical analyses, a P value of less than 0.05 was considered statistically significant. For photosensitiser dose experiments, the P values refer to samples in the absence of light versus irradiated samples. For light dose experiments, the P values refer to samples in the absence of methylene blue versus samples irradiated in the presence of methylene blue.