Sabouraud agar plates

A. fumigatus isolates were grown on Sabouraud agar plates (Oxoid, Wesel, Germany) for 2–5 days at 35 °C. Isolates were resuspended in a solution of water plus 0.1% tween 20. The solution was filtered using a filter membrane of 11 µm pore size (Merck, Milano, Italy) for the recovery of the conidia. The suspension was diluted to obtain a final solution at a concentration of 2–5 × 10⁵.

Twelve C. glabrata strains, isolated from different patients and of different origins, seven from hemoculture (12, 20, 30, 42, 45, 52 and 62), one vaginal (64), one from sputum (63), two exudates (60, 61), and one from sample preservation liquid (65) at a tertiary care hospital (Hospital Universitario de Badajoz, Spain) were included in this study. The isolated C. glabrata strains were sent to us anonymized, in accordance with the protocol approved by the Ethical Committee of the Hospital, (Comité de Ética de la Investigación Clínica del CHUB), in order to maintain the confidentiality of the patients, and were included in our collection (MicromedBA). The clinical isolates were identified by sowing in chromogenic medium CHROMagar Candida® and with biochemical methods using the API Candida ID32C system (bioMèrieux, Marcy L’Étoile, France). Sensitivity to antifungals was assessed by means of the automatic system Sensititre YeastOneR (Trek Diagnostic Systems, United Kingdom). Yeasts were conserved in vials at − 80 °C (Microbanc, Prolab, Ontario, Canada). Later, the strains were cultivated on Sabouraud agar plates (OXOID LTD., Basingstoke, Hampshire, UK) and incubated at 37 °C for 24 h; two subcultures were made in RPMI-1640 (Sigma, St. Louis, MO, USA.) medium supplemented with 2% glucose, and at pH 7 in MOPS buffer (Sigma) for the different experiments.

Isolates were taken from storage at −80 °C and grown on Sabouraud agar plates (Oxoid, Hampshire, UK) at 30 °C. For STR genotyping and WGS, a single colony of each isolate was resuspended in 400 µL MagNA Pure bacteria lysis buffer and MagNA Lyser green beads. These were mechanically lysed for 30 s at 6500 rpm using the MagNA Lyser system (all Roche Diagnostics GmbH, Mannheim, Germany). DNA was extracted and purified with the MagNA Pure 96 instrument and the MagNA Pure DNA and Viral NA Small Volume Kit (Roche Diagnostics), following the manufacturer’s instruction. For WGS, all samples were subsequently treated with RNase (Sigma-Aldrich, Burlington, MA, USA) at a final concentration of 5 µg/µL for one hour at room temperature, after which DNA was extracted and purified as described above. Purified DNA was measured with a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) using the double-stranded DNA (dsDNA) high sensitivity option.