Influence of fluconazole drug-dosing regimen on the rate of adaptation and extinction in Candida albicans

Abstract

Invasive infections caused by human fungal pathogens are a global health concern due to high mortality and morbidity rates. Many fungal infections are caused by species from the genus Candida. The most common, Candida albicans, is both a commensal member of the healthy human microbiota and a prevalent opportunistic fungal pathogen. The antifungal azole drug fluconazole (FLC) is frequently prescribed for treating C. albicans infections. Azole drugs are typically fungistatic—they inhibit cell growth rather than kill susceptible cells within a population. Therefore, fungal populations can potentially adapt to fluconazole through increases in drug resistance or increases in drug tolerance (the ability of some cells of a susceptible population to grow slowly at inhibitory drug concentrations). Given the limited arsenal of antifungal drugs and the difficulty in developing novel antifungals due to the close phylogenetic relationship between human and fungal cells, there is a need to design treatment strategies that prolong drug efficacy and delay the evolution of drug tolerance or resistance. To understand the influence of fluconazole drug-dosing regimen on the rate of adaptation and extinction in Candida albicans, I performed nine short-term in vitro evolution experiments. I independently evolved twenty-four replicates from four C. albicans strains to 0.25 μg/mL FLC ("FLC0.25"), 1 μg/mL FLC ("FLC1"), and 16 μg/mL FLC ("FLC16"), with 1:1000 dilutions done at three transfer durations (24 h, 48 h, and 72 h). Transfer duration significantly influenced the extinction rate: 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 very frequent regardless of the dosing regimen. In contrast, large increases in drug susceptibility occurred for higher drug concentrations- FLC1 and FLC16 evolved replicates with 48 h and 72 h transfers. Reduction in drug susceptibility and growth rates of the evolved replicates were common. Genome size changes were common in some strain backgrounds evolved to higher fluconazole concentrations and longer transfer times. Overall, we found a significant effect of the drug-dosing regimen on the phenotype and genome size and was strain background specific.