Hyperbaric oxygen therapy augments the photodynamic action of methylene blue against bacteria in vitro

Abstract

Photodynamic therapy (PDT) entails the combination of photosensitizer and light to generate cytotoxic molecules that derive from molecular oxygen (O₂). The presence of sufficient O₂ within the target tissues is critical to the efficiency of PDT. This study investigates the use of hyperbaric oxygen therapy in combination with PDT (HOTPDT) to augment the photodynamic action of methylene blue (MB) or 5-aminolevulinic acid (ALA) against gram positive and gram negative bacterial strains <i>in vitro</i>. <i>Staphylococcus aureus </i>or <i>Pseudomonas aeruginosa </i>were grown in trypticase soy broth as planktonic cultures (~10⁸/mL) or as established biofilms in 48 well plates (3 days old) at 32°C. Dark toxicity and PDT response in the presence or absence of HOT (2 atmospheres, 100% O₂ for 30, 60 or 120 min) was established for both MB (0-0.1 mM) and ALA (0- 1 mM) for a range of incubation times. The number of surviving colonies (CFU/mL) was plotted for each treatment groups. Light treatments (5, 10, 20 or 30 J/cm²) were conducted using an array of halogen bulbs with a red filter providing 90% transmittance over 600-800 nm at 21 mW/cm². HOT increased the dark toxicity of MB (30 min, 0.1 mM) from < 0.2 log cell kill to 0.5 log cell kill. Dark toxicity of ALA (4 hr, 1 mM) was negligible and did not increase with HOT. For non-dark toxic concentrations of MB or ALA, (0.05 mM and 1 mM respectively) HOT-PDT enhanced the antimicrobial effect of MB against <i>Staphylococcus aureus</i> in culture by >1 and >2 logs of cell kill (CFU/mL) at 5 and 10 J/cm² light dose respectively as compared to PDT alone. HOT-PDT also increased the anti-microbial effects of MB against <i>Staphylococcus aureus</i> biofilms compared to PDT, albeit less so (> 2 logs) following 10 J/cm² light dose. Anti-microbial effects of PDT using ALA were not significant for either strain with or without HOT. These data suggest that HOTPDT may be useful for improving the PDT treatment of bacterial infections.