Characterization of antifungal effect of natural compounds

MARIANA A PERALTA, MARÍA A DA SILVA, MARÍA G ORTEGA, JOSÉ L CABRERA MARÍA G PARAJE

Rosario

Congreso; X Congreso Argentino de Microbiología General (SAMIGE); 2013

SAMIGE

The continued emergence of infections with antifungal resistant Candida strains has been tried to solve with higher doses or with combination antifungal therapies. This health problem requires constant search and researching of new antifungal drugs; and the plant kingdom represents a rich source of new chemical structures. The aim of this study was to investigate if two flavonoid, 6PP (new structure) and robinetin (reference structure) have effect against biofilms formed by two Candida albicans strains, one of them fluconazole sensitive and the other azole-resistant with overexpression mutations of the multidrug efflux transporters. In addition, we studied if the reactive oxygen species (ROS) and antioxidant activity of sessile cells are affected. Materials and methods Compounds: flavonoid 6PP (from Dalea elegans); fluconazole and robinetin (Sigma). Microorganisms: two strains of C. albicans isolated from the oral cavity were used (kind gift from Dr. White University of Washington, Seattle, USA). The azole-resistant strain (RCa) overexpresses the transporter genes CDR1, CDR2 and MDR1, whereas the sensitive strain (SCa) lacks these transporter genes. Biofilm formation: was measured by adhesion to 96-well plates and crystal violet stain. The biofilm biomass unit (BBU) was defined as 0.1 OD595nm=1 BBU. Different conditions (pH, incubation time and culture medium) were assayed. The supernatant was separated by measuring: ROS: by the reduction of the nitro-blue tetrazolium (NBT) reaction. Total superoxide dismutase (SOD) activity: was assayed photochemically based on the inhibition of NBT reduction. Total antioxidant capacity of biofilms: by measuring the ferrous reducing activity (FRAP method) Results: For RCa and SCa strains the optimal biofilm formation (BBU=2.2 and 2.5, respectively) was obtained with precoating the plates with fetal bovine serum in Sabouraud broth at pH 5.5 and 48 h of incubation. Fluconazole inhibited the mature biofilm at 0.25 and 5 µg/mL, whereas, the greatest inhibition for 6PP and robinetin was observed at 50 µM; being more significant in SCa strain. In both strains it was found that all compounds, at the inhibitory concentration, stimulated ROS production. For RCa the SOD increase was significant in presence of fluconazole or flavonoids. The total antioxidant capacity of biofilm showed higher basal levels in RCa than SCa. However, this strain evidenced a considerable increase in FRAP in presence of fluconazole or flavonoids, respectably. Conclusions: The new natural compound 6PP showed antifungal activity on biofilms similar to the reference (robinetin) and fluconazole in C. albicans strains, even in RCa, with an increase of ROS and the antioxidant system. However, with none of the three compounds could be eradicate completely the biofilms. These results encourage the study of mechanisms of action and combination assays with other antifungals agents used in medicine.