Effects of photodynamic and sonodynamic therapies on the extracellular matrix of Candida albicans biofilm

Abstract

Infections caused by Candida spp. biofilms are of great importance, especially in immunocompromised patients. The resistance of Candida spp. biofilms to medications may be associated with the protective effect of the extracellular matrix (ECM) components. Antimicrobial Photodynamic Therapy (PDT) and Sonodynamic Therapy (SDT) have been suggested as alternative treatments for the inactivation of microorganisms. SDT follows the basis of PDT, combining a sensitizer agent with ultrasound instead of light. However, there is no research that evaluated the effect of PDT and SDT on the ECM of Candida albicans biofilm. Therefore, the aim of this study is to evaluate the effect of PDT and SDT, both mediated by the sensitizers Rose Bengal (RB) or Photodithazine® (PDZ), on the ECM of C. albicans biofilm and the ability of these therapies to inactivate this fungus. This project will be conducted under the supervision of Professor John Callan, at University of Ulster. Standardized suspensions (107 CFU/mL) of C. albicans (ATCC 90028) will be grown onto polystyrene surfaces in RPMI medium at 37ºC for 48 h, for biofilm formation. For treatment with SDT, biofilms will be incubated with RB or PDZ in the dark, for a period of 10 or 20 minutes, respectively. Then, biofilms will be sonicated for 60 min (1 MHz of frequency, 3 W/cm² of ultrasound power density, 50% of duty cycle and pulse frequency of 100 Hz). Additional samples will be treated only with sensitizer (RB or PDZ), only with ultrasound or will receive no treatment (Control group). For PDT treatment, biofilms will also be incubated with RB or PDZ for the same periods, and then, biofilms will be irradiated with a fiber optic-delivered light (560 nm, 20 J/cm²) or a light emitting diode (660 nm, 50 J/cm²), respectively. As the same way as SDT, additional samples will be treated only with sensitizer, only with light or will receive no treatment. The combination of SDT+PDT will be also assessed. In order to do this, additional samples will be incubated with the sensitizers (PDZ or RB), then, the biofilms will simultaneously irradiated by light and sonicated, under the same conditions. After treatments, biofilms will be processed for evaluation of ECM components: water-soluble polysaccharides -WSP, alkali soluble polysaccharide -ASP, proteins and extracellular DNA- eDNA, evaluation of colony forming units - CFU and total biomass. Effects of the treatment on protein expression will also be determined using gel electrophoresis and mass spectrometry. Structural changes will be examined using confocal microscopy. The data will be analyzed by appropriate statistical tests. (AU)