Fig. 2. BRI+CAS have synergism against A. fumigatus.

a A. fumigatus conidia were incubated for 48 h at 37 °C with different combinations of BRI + CAS and BRI + VOR. After this period, non-germinated conidia were plated on MM and colony forming units (CFUs) were assessed. The results are expressed as the % of viable conidia with respect to initial inoculum (n = 100) and are the average of three repetitions ±SD (t-test; p < 0.0001). b Microscopic images of A. fumigatus after 48 h exposure to BRI 20 µM, CAS 0.5 µg/mL, BRI 20 µM+CAS 0.5 µg/mL, VOR 0.25 µg/mL, and BRI 20 µM+ VOR 0.25 µg/mL. Bars, 20 µm. c, d The Fractional Inhibitory Concentration (FIC) index for BRI + CAS and BRI + VORI, respectively. Results show the average of three independent experiments. e BRI + CAS disrupts the A. fumigatus membrane potential. A. fumigatus was grown 16 h at 30 °C in MM and transferred to MM without glucose (non-carbon source, NCS) or MM for 4 h before adding CAS [0.2 µg/mL], BRI [20 µM], or CAS + BRI and 3 µg/ml DIBAC₄(3) for 10 min. The results are expressed as the % of fluorescent cells and are the average of three independent repetitions of 50 germlings each ± SD (n = 50; t-test; p < 0.0001). f Metabolic activity expressed by XTT of A. fumigatus biolfilm treated with VOR or CAS alone and combinations of BRI + VOR and BRI + CAS. Biofilm was formed for 24 h of incubation at 37 °C. After this period 50 µL of fresh MM containing CAS, VOR or a combination of VOR + CAS or CAS + BRI were added to the biofilm to reach the final concentration as indicated and incubated for further 12 h at 37 °C. Untreated biofilm was used as a positive control. The graphic shows the value of 9 technical replicates from three independent experiments ± SD deviation (n = 3; 2way ANOVA, Tukey´s post-test; *p < 0.05, **p < 0.005, and ****p < 0.0001).