Antifungal Tolerance and Resistance Emerge at Distinct Drug Concentrations and Rely upon Different Aneuploid Chromosomes.

Antifungal drug tolerance is a response distinct from resistance, in which cells grow slowly above the MIC. Here, we found that the majority (69.2%) of 133 Candida albicans clinical isolates, including standard lab strain SC5314, exhibited temperature-enhanced tolerance at 37°C and 39°C, and were not tolerant at 30°C. Other isolates were either always tolerant (23.3%) or never tolerant (7.5%) at these three temperatures, suggesting that tolerance requires different physiological processes in different isolates. At supra-MIC fluconazole concentrations (8 to 128 µg/mL), tolerant colonies emerged rapidly at a frequency of ~10-3. In liquid passages over a broader range of fluconazole concentrations (0.25 to 128 µg/mL), tolerance emerged rapidly (within one passage) at supra-MICs. In contrast, resistance appeared at sub-MICs after 5 or more passages. Of 155 adaptors that evolved higher tolerance, all carried one of several recurrent aneuploid chromosomes, often including chromosome R, alone or in combination with other chromosomes. Furthermore, loss of these recurrent aneuploidies was associated with a loss of acquired tolerance, indicating that specific aneuploidies confer fluconazole tolerance. Thus, genetic background and physiology and the degree of drug stress (above or below the MIC) influence the evolutionary trajectories and dynamics with which antifungal drug resistance or tolerance emerges. IMPORTANCE Antifungal drug tolerance differs from drug resistance: tolerant cells grow slowly in drug, while resistant cells usually grow well, due to mutations in a few known genes. More than half of Candida albicans clinical isolates have higher tolerance at body temperature than they do at the lower temperatures used for most lab experiments. This implies that different isolates achieve drug tolerance via several cellular processes. When we evolved different strains at a range of high drug concentrations above inhibitory levels, tolerance emerged rapidly and at high frequency (one in 1,000 cells) while resistance appeared only later at very low drug concentrations. An extra copy of all or part of chromosome R was associated with tolerance, while point mutations or different aneuploidies were seen with resistance. Thus, genetic background and physiology, temperature, and drug concentration all influence how drug tolerance or resistance evolves.

Genetic variation in aneuploidy prevalence and tolerance across Saccharomyces cerevisiae lineages.

Individuals carrying an aberrant number of chromosomes can vary widely in their expression of aneuploidy phenotypes. A major unanswered question is the degree to which an individual's genetic makeup influences its tolerance of karyotypic imbalance. Here we investigated within-species variation in aneuploidy prevalence and tolerance, using Saccharomyces cerevisiae as a model for eukaryotic biology. We analyzed genotypic and phenotypic variation recently published for over 1,000 S. cerevisiae strains spanning dozens of genetically defined clades and ecological associations. Our results show that the prevalence of chromosome gain and loss varies by clade and can be better explained by differences in genetic background than ecology. The relationships between lineages with high aneuploidy frequencies suggest that increased aneuploidy prevalence emerged multiple times in S. cerevisiae evolution. Separate from aneuploidy prevalence, analyzing growth phenotypes revealed that some genetic backgrounds-such as the European Wine lineage-show fitness costs in aneuploids compared to euploids, whereas other clades with high aneuploidy frequencies show little evidence of major deleterious effects. Our analysis confirms that chromosome gain can produce phenotypic benefits, which could influence evolutionary trajectories. These results have important implications for understanding genetic variation in aneuploidy prevalence in health, disease, and evolution.