Atcc 24067

ATCC 24067 (American Type Culture Collection, Manassas, VA), C. neoformans 52D strain, was used to infect mice in this study. Cryptococcal cells from frozen stocks (10% glycerol) were plated on Yeast Peptone Dextrose (YPD, BD Difco) agar plates containing 50 mg/ml chloramphenicol (Sigma) at 30°C. Single colonies were subsequently picked and grown overnight in YPD broth (BD Difco) containing 50 mg/ml chloramphenicol at 30°C with 225 rpm shaking. Fungal cells were washed three times in PBS, counted on a hemocytometer with trypan blue, and adjusted to a concentration of 5 × 10⁶ per mL before infection. Mice were infected with 10⁶ yeast (in 200 μl of sterile PBS) via tail-vein intravenous injection. Serial dilutions of the C. neoformans suspension were plated on YPD agar to confirm the number of viable fungi in the inoculum. Weights were monitored three times a week. For analysis of brain fungal burdens, animals were euthanized and organs weighed, brains homogenized in PBS, and serially diluted before plating onto YPD agar supplemented with chloramphenicol (Sigma). Colonies were counted after incubation at 30 °C for 48 hours. For analysis of brain cytokines, brain homogenates were diluted 2-fold in PBS before measurement using the Mouse Cytokine 32-plex Discovery Assay (Mouse Cytokine Array/Chemokine Array 32-Plex Panel; Cat#: MD31; Eve Technologies, Alberta, Canada).

We initially assessed the antifungal activity of EE and its fractions against two strains of C. gattii and two strains of C. neoformans. The ethyl acetate fraction (EAF) presented better antimicrobial activity and yield; therefore, it was chosen for this study.
For the “antifungal drug susceptibility testing” assays, we tested two reference strains of C. gattii [American Type Culture Collection (ATCC) 24065 and ATCC 32608] and four reference strains of C. neoformans (ATCC 24067, ATCC 28957, ATCC 62066, and ATCC H99), which were obtained from the Culture Collection of the University of Georgia (Atlanta, GA, United States). Seven clinical isolates of C. gattii, five clinical isolates of C. neoformans, and one environmental isolate of each species, all from the Culture Collection of the Mycology Laboratory/ICB-UFMG, were also used in this study (Magalhães et al., 2013 (link)). Isolates were maintained on Sabouraud dextrose broth (SDB) at −80°C. Prior to each test, the strains were subcultured on Sabouraud dextrose agar (SDA) for 48 h at 35°C.
The ATCC 32608 and L27/01 of C. gattii and ATCC 2895 and ATCC H99 of C. neoformans strains were randomly chosen for further experiments, except for ITC, in which we used ATCC 32068 and ATCC H99 strains.