Molecular Identification of Fungal Isolates

Resistant isolates detected by screening and EUCAST microdilution broth reference methods were identified by sequencing a portion of the β-tubulin gene [31 (link)]. Complete genomic DNA was extracted from a Malt-extract agar fungal culture using QIAamp DNA blood minikit (Qiagen Sciences Ing, Courtaboeuf, France). Briefly, conidia and hyphae were disrupted with glass beads (VWR, ref: 432-0064) and lysis buffer on MagNA Lyser instrument (Roche Diagnostics, Meylan, France). The resulting suspension was then treated according to the manufacturer’s instructions. PCRs were performed in a 50 µL-final volume containing 1× HF buffer (ThermoFisher, Les Ulis France), 200 µM of deoxynucleoside triphosphates (dNTPs), 1 µM of each primer, 3% of DMSO, 1 unit of Phusion™ High-Fidelity DNA Polymerase (ThermoFisher), and 100 ng of genomic DNA. The primers used for β-tubulin gene amplification were Bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3′) and Bt2b (5′-ACCCTC AGTGTAGTGACCCTTGGC-3′) as previously described [32 (link)]. Sanger sequencing was performed at the Genomic platform of Henri Mondor Hospital Biomedical Research Institute (IMRB). Sequences were analyzed using DNA Baser Assemble v5.15.0 and compared to GenBank and MycoBank databases sequences. Nucleotide identification was achieved at >99% match.