5.03] Photodynamic therapy for the treatment of pathogen bacteria

Abstract

Objective Dental caries is one of the most common diseases in Western countries. Its pathoetiology is multifactorial; however, the Gram-positive, facultatively anaerobic bacterium Streptococcus mutans may be one of the key factors involved in this process. The fact that therapeutic approaches to eradicate S. mutans are still limited prompted us to investigate the potential effects of photodynamic therapy with hypericin and Foslipos in vitro . Material and methods S. mutans (OMZ 176) was cultivated in fluid universal medium (FUM) under standard conditions. In total 100 μl bacteria (OD1 at 550 nm) were incubated with the photosensitizers hypericin (Invitrogen, Basel, Switzerland), the meso-tetrahydroxyphenylchlorin derivative Foslipos (Biolitec, Jena, Germany) or a mixture of both at concentrations ranging between 1.25 and 10 μg/ml for 1–90 min in FUM, followed by a thorough washing step in buffer. Bacteria were then irradiated for 60–120 s with a dental polymerization instrument (450 nm). All samples were subjected to serial dilutions and spiral plating on blood agar plates. Viable colony counts were determined after 48 h in culture. Adhesion and uptake of photosensitizers was analyzed by confocal and TIRF microscopy. Results Using the mix of photosensitizers (10 μg/ml total), a photosensitizer incubation time of 90 min and irradiation for 120 s, 100% of S. mutans could be killed. However, further optimization studies showed that irradiation time could be shortened to 60 s without loss of efficacy. In addition, the incubation time of the photosensitizers could be scaled down to 5 min, still resulting in 99.9999994% bacterial death. Comparisons of the concentration range indicated that a total photosensitizer concentration of as low as 2.5 μg/ml is sufficient for complete mortification of S. mutans . Under the same conditions, use of single photosensitizers instead of the mix appears to be less effective. Microscopic analyses confirmed the adherence of photosensitizers to bacteria. Conclusion Using a short protocol with a novel combination of low-dose photosensitizers, a complete eradication of S. mutans could be achieved in vitro . Future studies will include investigations on the efficacy of our protocol for other oral pathogens in vitro and in biofilms. Our data may set the stage for more effective treatment modalities not only within the oral cavity, but also for other systems prone to bacterial infections, such as the lower urinary tract.