Antibiofilm combination of usnic acid with fluconazole on resistant Candida albicans.

PERALTA MARIANA A; PARAJE, MARÍA G; CABRERA JOSE L.

Rosario

Congreso; Congreso Argentino de Microbiología General (SAMIGe) 2017; 2017

Asociación Civil de Microbiología General

IntroductionTreatment of Candida infections is often difficult due to, between others factors, the ability of Candidaspecies to form biofilms. These highly resistant structures exhibit resistance to a variety of antifungalagents with clinical use. Therefore, combining them with compounds obtained from natural sourcesseems to be one of the strategies in order to restore the sensitivity of the microorganism toconventional antifungals such as azole drugs. In previous works, we reported inhibitory activity ofusnic acid (UA), a natural compound obtained from lichens, against azole-resistant Candida albicansbiofilm. The biofilms inhibitory concentration (BIC) was 4 mg/ml compared to fluconazole (FLZ, BIC, 2mg/ml) with inhibitions percentages of about 70%.ObjectiveThe present study investigated the sensitization (restoring the sensitivity of the microorganism to azoledrugs used in the clinic) of azole-resistant C. albicans biofilms to FLZ by combining it with an activecompound (UA) obtained from Argentinean native flora (Usnea amblyclada).Materials and MethodsCompound: UA was purified from the benzene extract of lichen U. amblyoclada.Microorganisms: An azole-resistant strain of C. albicans isolated from the oral cavity (RCa) thatoverexpresses efflux transporters genes of type CDR1, CDR2 and MDR1 was used.Biofilm formation was measured by adhesion to a 96-well plate and quantified by Crystal Violet (CV)staining and spectrophotometric reading of Optical Density (OD) at 595 nm. The biofilm biomass unit(BBU) was defined as 0.1DO595nm = 1UBB.For antifungal activity determination different concentrations of UA (1 to 4 mg/ml) dissolved in dimethylsulfoxide (DMSO), FLZ (0.5 to 2 mg/ml) or their combinations were added to each well containing themature biofilm and incubated at 37 °C for 48h. The counts of Colony Forming Units (CFU/ml) wereperformed for BBU correlation studies.Scanning Confocal Laser Microscopy (SCLM): the samples were stained with Calcofluor White (0.05%v/v).ResultsUA and FLZ combined at concentrations four-fold lower than their BICs had a greater inhibitory effecton biofilms. In fact, the combination of UA (1 mg/ml) and FLZ (0.5 mg/ml) achieved an inhibition of 79,82% while UA and FLZ combined at 1 mg/ml and 0.5 mg/ml respectively, almost eradicated de matureCongreso Argentino de Microbiología General SAMIGE - 2017biofilm with an inhibition of 97.69% (* p < 0.01).Analysis by SCLM showed a considerable decrease in biomass of biofilms treated with thecombination of UA (1 mg/ml) and FLZ (0.5 mg/ml), compared to untreated RCa biofilms (* p < 0.01).ConclusionThese results suggest that the combination of UA with FLZ effect enhanced the activity of FLZ in thetreatment of azole-resistant of C. albicans biofilms. This promising action would imply an improvementof the therapeutics due to the decrease of the concentrations used of antifungal drugs.Congreso Argentino