E DELARZE¹, S ZNAIDI², M LEGRAND², M ARENDRUP³, C MUNRO⁴, C D'ENFERT², D SANGLARD<sup>1

1</sup>University of Lausanne and University Hospital Center, Lausanne, SWITZERLAND, ²Pasteur Institute, Paris, FRANCE, ³Statens Serum Institut, Copenhagen, DENMARK, ⁴University of Aberdeen, Aberdeen, UNITED KINGDOM

Antifungal tolerance can be defined as the ability of C. albicans cells to survive high drug concentrations without acquiring mutations associated with resistance. The study of drug tolerant strains may reveal factors contributing to treatment failures and facilitate the design of improved therapies. In this study we aimed to identify mediators of tolerance to fluconazole (FLC), using two different approaches. The first was to assess the tolerance profile of 26 clinical C. albicans isolates against FLC using the EUCAST drug susceptibility assay. Tolerant strains were identified by their ability to sustain residual growth after 24 h at a drug concentration above the FLC resistance breakpoint. This screening revealed different profiles grouped from low to high tolerance. These FLC tolerant strains are candidates for further characterization by genetic and transcriptomic approaches and will be used to test the importance of drug tolerance in animal models. In a second approach, in order to identify genetic mediators of tolerance, we used a collection of 582 tetracycline-inducible overexpression barcoded strains, which were pooled and maintained under FLC pressure for five days of repeated subculture. This strategy was used to enrich the pool in FLC resistant and/or tolerant strains. After amplification and sequencing of all barcodes from the cultures, the fractional index of each strain was calculated. This analysis yielded 53 strains that were at least 2-times more enriched under FLC pressure than in conditions without FLC or ORF overexpression. The FLC tolerance profile of these strains will be tested individually.