Photodynamic inactivation of microbial biofilms: impact of Hsp70 expression and non-invasive optical monitoring of oxygen during photodynamic inactivation

Candida albicans is the main type of fungi able to form biofilms, which cause superficial skin and mucous membrane infections as well as invasive mycoses, particularly in immunocompromised patients. In these patients, invasive infections are often associated with high morbidity and mortality. Furthermore, the increase in antifungal resistance has decreased the efficacy of conventional therapies. The use of aPDT as an antimicrobial topical agent against superficial and cutaneous diseases represents an effective method for eliminating microorganisms. The first chapter describes: a) the complex problem of C. albicans infections, b) what a biofilm is, why they are so resistant to antimicrobial agents and their importance in infections. In addition the first chapter describes the antimicrobial Photodynamic Therapy (aPDT) and suggests it as an effective approach for killing of microbial cells. The second chapter shows for the first time the use of XF-73, a new porphyrine derivative, combined with blue light to inactivate C. albicans biofilms, causing 5 log10 cell killing. The results were compared to TMPyP, an already known PS The efficacy of aPDT was also compared with the efficacy of Amphotericin B (AmB), the gold standard for the treatment of fungal infections. The presence of extracellular polymeric substance (EPS), which is a hallmark characteristic of microbial biofilms, was also detected. Besides, for the first time, singlet oxygen (1O2) was directly detected in biofilms treated by XF-73-aPDT. In chapter 3, a model of duo-species biofilm, formed by C. albicans and S. epidermidis, was developed. The susceptibility of C. albicans, co-aggregated in this duo- species biofilm, was evaluated using XF-73-aPDT. Duo-species biofilms were less susceptible to XF-73-aPDT than monomicrobial C. albicans biofilms. The results showed that the susceptibility to aPDT decreases with the increase of the complexity of the biofilms composition. Since the over expression of heat shock proteins is a mechanism, whereby cells can become resistant to aPDT, in chapter 4 it was examined, whether the heat shock protein 70 (Hsp70) over expression after thermal stress protects C. albicans cells against the oxidative damage caused by aPDT. In addition, it was investigated, whether C. albicans cells, treated by aPDT, might over express Hsp70. For this, the kinetics of Hsp70 expression in C. albicans cells, treated with a sub lethal dose of aPDT, were determined over a period of 24 h. The results, obtained, showed that the over expression of Hsp70 by C. albicans cells is not a mechanism, by which microbial cells might develop resistance to TMPyP-mediated aPDT. Also, the expression of Hsp70 in C. albicans did not increase after sub-lethal TMPyP-mediated aPDT. Chapter 5 reports the use of a luminescence based oxygen-sensitive sensor for monitoring oxygen concentration of C. albicans and S. epidermidis biofilms during aPDT. The knowledge of oxygen concentration during aPDT is important to improve the efficacy of aPDT