Photodynamic therapy as alternative treatment for disinfection of bacteria in oral biofilms
Tayal, Dharam P.
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A laboratory investigation was undertaken to determine whether disinfection of biofilms of representative oral microorganisms, by application of Photodynamic Therapy (PDT), might favor PDT as an effective alternative to current antiseptic or antibiotic treatments of dental plaques. PDT does not risk development of bacterial resistance to anti-microbial drugs. Two incubator-contained parallel-plate flow cells were connected in series, each containing 12 Radio Frequency Glow Discharge treated (RFGDT) polystyrene test plates (2 x 0.83 x 0.1cm) suitable for readings of both absorbance and fluorescence in 12-well microtiter plates as well as direct inspection by transmitted light microscopy. Reproducible biofilms were formed by Streptococcus mutans strain ATCC 27351 growing in log phase at 37°C in Brain Heart Infusion medium, circulating through the flow cells at 3ml/minute for 36–48 hours, followed by flow of distilled water at 7 ml/minute for 15 minutes before exposure to PDT or control conditions. Supplementary data characterizing the biofilms before and after exposure to PDT were acquired by Multiple Attenuated Internal Reflection InfraRed (MAIR-IR) spectroscopy of similar biofilms formed on germanium prisms (2 x 5 x 0.1cm). The photo-sensitizer used was Photofrin ® hematoporphyrin at 25–500 microgram/ml, with increasing biofilm-immersion times and increasing energy density of post-immersion laser illumination at 630nm. Resultant decreases in bacterial viability in the biofilms were tracked by monitoring AlamarBlue ® conversion to a red colorimetric and fluorescent indicator of the reducing power of retained living bacteria which internally converted nontoxic, cell-permeable blue-colored, non-fluorescent resazurin molecules to bright red fluorescent resorufin molecules. Biofilm bacterial viability, monitored by fluorescence measurements at hourly intervals after PDT, showed that uniform fluorescence counts (RFU) for starting biofilms of 35,000 RFU +/− 10,000 RFU were reducible to as low as 2500 RFU with Photofrin incubation times as short as 5 minutes in solutions as dilute as 25 microgram/ml concentration, illuminated at 630 nm with as little as 30 Joules/cm 2 . Optimum values were 125 micrograms/ml photo-sensitizer concentration and 60 J/cm 2 energy density. MAIR-IR showed that both the PDT process and control saline immersion led to loss of biofilm extra-cellular carbohydrate components, confirming that PDT reduction of bacterial viability was by internalized photo-sensitizer applied at doses about 1000X lower than approved for PDT treatment of esophageal carcinoma. Application of PDT to control of intra-oral infections thus seems both safe and effective, based on these laboratory trials.