Treatment of Candida infections is often difficult due to, between othersfactors, the ability of Candida speciesto form biofilms.  These highly resistantstructures exhibit resistance to a variety of antifungal agents with clinicaluse. Therefore, combining them with compounds obtained from natural sourcesseems to be one of the strategies in order to restore the sensitivity of themicroorganism to conventional antifungals such as azole drugs. In previousworks, we reported inhibitory activity of usnic acid (UA), a natural compoundobtained from lichens, against azole-resistant Candida albicans biofilm. The biofilms inhibitory concentration(BIC) was 4 μg/ml compared to fluconazole (FLZ, BIC, 2 μg/ml) with inhibitionspercentages of about 70%.

The present study investigated thesensitization (restoring the sensitivity of the microorganism to azole drugsused in the clinic) of azole-resistant C.albicans biofilms to FLZ by combining it with an active compound (UA)obtained from Argentinean native flora (Usneaamblyclada).

UA was purified from the benzeneextract of lichen U. amblyoclada.
An azole-resistant strain of C. albicansisolated from the oral cavity (RCa) that overexpresses efflux transportersgenes of type CDR1, CDR2 and MDR1 was used.

Biofilm formation was measured byadhesion to a 96-well plate and quantified by Crystal Violet (CV) staining andspectrophotometric reading of Optical Density (OD) at 595 nm. The biofilmbiomass unit (BBU) was defined as 0.1DO_595nm = 1UBB.

For antifungal activitydetermination different concentrations of UA (1 to 4 μg/ml) dissolved indimethyl sulfoxide (DMSO), FLZ (0.5 to 2 μg/ml) or their combinations wereadded to each well containing the mature biofilm and incubated at 37 °C for48h. The counts of Colony Forming Units (CFU/ml) were performed for BBUcorrelation studies.

ForScanningConfocal Laser Microscopy (SCLM), the samples were stained with Calcofluor White (0.05%v/v).

UA and FLZ combined atconcentrations four-fold lower than their BICs had a greater inhibitory effecton biofilms. In fact, the combination of UA (1 μg/ml) and FLZ (0.5 μg/ml)achieved an inhibition of 79, 82% while UA and FLZ combined at 1 μg/ml and 0.5μg/ml respectively, almost eradicated de mature biofilm with an inhibitionof   97.69% (^∗p < 0.01).

Analysis by SCLM showed aconsiderable decrease in biomass of biofilms treated with the combination of UA(1 μg/ml) and FLZ (0.5 μg/ml), compared to untreated RCa biofilms (^∗p < 0.01).

These results suggest that thecombination of UA with FLZ effect enhanced the activity of FLZ in the treatmentof azole-resistant of C. albicansbiofilms. This promising action would imply an improvement of the therapeuticsdue to the decrease of the concentrations used of antifungal drugs.