The influence of drug dosing regimen on extinction and adaptation during experimental evolution in Candida albicans

The antifungal azole drug fluconazole (FLC) is frequently prescribed for treating Candida albicans infections. Azole drugs are typically fungistatic; they inhibit cell growth rather than kill susceptible cells within a population. Therefore, fungal populations can adapt to fluconazole through increased drug resistance or increased drug tolerance (the ability of a subpopulation of susceptible cells to grow slowly at inhibitory drug concentrations). To our knowledge, no prior experiments have examined how different factors influence extinction propensity, although previous in vitro studies show that both drug concentration and strain background can influence adaptation. To determine how the drug dosing regimen, namely fluconazole concentration and transfer duration (i.e., the amount of time between transfer into fresh medium), influenced extinction and adaptation in Candida albicans, we conducted a fully factorial series of short in vitro evolution experiments. Dosing regimen significantly influenced extinction: all replicates evolved at all drug concentrations survived the 48 h and 72 h transfer experiments, yet the extinction rate increased sharply from low to high drug concentration with 24 h transfers. Increases in drug tolerance were frequent regardless of the dosing regimen. In contrast, drug resistance and growth rate both tended to decrease following evolution at higher drug concentrations. Fluconazole-evolved replicates exhibited frequent increases in cross-tolerance and decreases in cross-resistance in a panel of other azole drugs. Genome size changes were common in some strain backgrounds evolved to higher fluconazole concentrations, regardless of transfer times. Combined, our results indicate that many experimental factors can influence the trajectory of adaptation, and show that extinction in a fungistatic drug occurs readily at high drug concentrations with rapid transfers.