Antifungal Drug Concentration Impacts the Spectrum of Adaptive Mutations in Candida albicans

Invasive fungal infections are a leading global cause of human mortality. Only three major classes of antifungal drugs are widely used, and resistance to all three classes can arise rapidly. The most widely prescribed antifungal drug, fluconazole, disseminates rapidly and reaches a wide range of concentrations throughout the body. The impact of drug concentration on the spectrum and effect of mutations acquired during adaptation is not known for any fungal pathogen, and how the specific level of a given stress influences the distribution of beneficial mutations has been poorly explored in general. We evolved 144 lineages from three genetically distinct clinical isolates of Candida albicans to four concentrations of fluconazole (0, 1, 8, and 64 μg/ml) and performed comprehensive phenotypic and genomic comparisons of ancestral and evolved populations. Adaptation to different fluconazole concentrations resulted in distinct adaptive trajectories. In general, lineages evolved to drug concentrations close to their MIC(50) (the level of drug that reduces growth by 50% in the ancestor) tended to rapidly evolve an increased MIC(50) and acquired distinct segmental aneuploidies and copy number variations. By contrast, lineages evolved to drug concentrations above their ancestral MIC(50) tended to acquire a different suite of mutational changes and increased in drug tolerance (the ability of a subpopulation of cells to grow slowly above their MIC(50)). This is the first evidence that different concentrations of drug can select for different genotypic and phenotypic outcomes in vitro and may explain observed in vivo drug response variation.