Photobiological study of hypericin for application as photosensitizer in Antimicrobial Photodynamic Therapy on Staphylococcus aureus and Escherichia coli colonies

With the increase of cases of microbial resistance to common treatments (antibiotics and antimicrobials), the antimicrobial photodynamic therapy (aPDT) appears as a light at the end of the tunnel, because the interaction of three components: a photosensitizer, light and oxygen of the mediu], resulting in the formation of reactive species capable of to reach different cellular targets (proteins, enzymes, unsaturated lipids) leading to rupture of the wall and cell membrane. This property of acting on different targets hinders any mechanism of resistance, favouring the aPDT. In this work we verified the efficiency of hypericin as a photosensitizer on Sthapylococcus aureus and Escherichia coli culture. For this, we first analyzed the minimal inhibition concentration -MIC by liquid inhibition tests.After we studied the uptake of hypericin by the bacteria through indirect studies using the ultraviolet-visible spectrophotometry method (Ultravioletvisible photodiode spectrophotometer, model Cary 50, Varian - UV-VIS) and Flow Cytometer ((BD FACSCantoTM II) where the incubation time and compound concentration were varied. To established the best conditions of treatment, we evaluated the photodynamic activity of hypericin on the bacteria, carried out with different concentrations of hypericin and variations in incubation and doses light. It also determined the mechanism of action triggered by aPDT from microbial growth kinetics under the best treatment conditions. For MIC determination tests we obtained a MIC 50 at a concentration of 1 μM hypericin, and the uptake assay showed no significant changes between concentrations and incubation time for both bacterias. When evaluating the incubation time and the light dose, we observed that the incubation time was not a determining factor in the study, being established at 5 minutes for both microorganisms, however, the light dose induced the inhibition of colony growth, This was evidenced by the kinetics test, which demonstrated the antimicrobial potential of hypericin as a bacteriostatic compound. In conclusion we have that hypericin was able to reduce the viability of microbial cells, although we observed growth resumption, which indicates the potential application of aTFD associated with other antimicrobial treatments.