Flow cytometric analyses on the damage pattern of phenalen-1-one-mediated antimicrobial photodynamic therapy towards biofilms in vitro

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In view of increasing numbers of resistant microorganisms, it should be major goals in dentistry, where usually no life-threatening diseases need to be treated, to restrict the use of conventional antibiotics and antiseptics and to promote research on alternative antimicrobial approaches.In this light, the antimicrobial photodynamic therapy (aPDT) may be an alternative antimicrobial approach. The principle of aPDT is based on incubation of bacteria with a per se non-toxic dye (photosensitizer; PS) and subsequent irradiation by light of an appropriate wavelength in the presence of molecular oxygen. Due to the emergence of reactive oxygen species or singlet oxygen during irradiation bacterial structures are destroyed non-selectively by an oxidative burst. PS based on phenalen-1-one structure seem to be particularly advantageous due to their high singlet oxygen quantum yield and consequential pronounced antimicrobial efficacy. However, the underlying mechanisms and damage patterns still are virtually unknown. Since treatment periods in the seconds range are sufficient for inactivation of planktonic bacteria, it was proposed that bacterial cytoplasmic membranes may be the target structure, which, however, has been disproved within the applicant’s preceding DFG-project. Nevertheless, precise understanding of the mechanism of action and the target structures of a given antimicrobial approach are of vital importance for optimization of this approach and evaluation of the risk of resistance induction.Therefore, the aim of this proposal is to investigate the damage pattern and the target structures of aPDT with phenalen-1-one PS towards bacteria organized in biofilms by means of multi-parameter flow cytometry. First, appropriate conditions shall be evaluated for aPDT with two phenalen-1-one derivates towards monospecies biofilms of Actinomyces naeslundii and Escherichia coli. Then, the method of flow cytometry combined with distinct fluorescent stains shall be established in order to assess the following vital parameters on the single-cell level following aPDT-treatment: membrane integrity, membrane potential, metabolic activity and the level of intracellular ROS. Furthermore, the damage mediated by aPDT shall be visualized by means of scanning electron microscopy and transmission electron microscopy. Furthermore, cytotoxic effects of aPDT with both PS on periodontal ligament cells shall be evaluated.These findings provide the basis for a future clinical application and further development of this class of PS. The flow cytometric method developed here can further be applied in order to investigate the damage pattern of other antimicrobial approaches and, thus, is interesting for other questions in and beyond the field of medicine.