Genetic background influences heterogeneity of adaptive response and genome stability during prolonged antifungal drug exposure in Candida albicans

The roles of genetic background and within-species diversity in determining the evolutionary potential of a strain to acquire drug resistance are not well understood. To analyze evolution in the presence of fluconazole, a common antifungal drug, we evolved replicates of twenty different clinical isolates of the human fungal pathogen Candida albicans from four different clades and with different initial levels of drug resistance in drug for 100 generations. The majority of replicates rapidly increased in fitness in the evolutionary environment, with the degree of improvement inversely correlated with ancestral strain fitness in the drug. Unexpectedly, very few replicates from any strain background acquired bona fide drug resistance. Rather, we found prevalent changes in drug tolerance (slow subpopulation growth at high concentrations of drug) and karyotype (changes in chromosome number and/or deviations from diploidy) in a strain-specific manner. Importantly, poor ancestral strain growth in drug was correlated with high levels of heterogeneity among evolved replicates in growth, tolerance, and karyotype, such that the ancestral strain genotype, along with the ancestral fitness level, were associated with the degree of acquired genotypic and phenotypic diversity during adaptation. Thus, the possible responses of a species to a given stress can be diverse and are not necessarily captured by in-depth study of a single strain background. Furthermore, the acquisition of increased fitness in drug is much more frequently associated with a change in tolerance, with or without accompanying karyotypic variation, rather than increased resistance. Abbreviated abstract We evolved replicates of twenty different clinical isolates of the human fungal pathogen Candida albicans in drug for 100 generations. Ancestral strain fitness was negatively correlated with fitness improvement in the drug, the heterogeneity of growth among replicates, the level of drug tolerance, and large-scale genome changes (ploidy levels). Improved fitness in drug was much more frequently associated with changes in drug tolerance than with changes in drug resistance. Impact Statement The acquisition of drug resistance is a serious threat to global public health. Antimicrobial resistance is inherently an evolutionary phenomenon: in the presence of drug, resistant individuals arise and spread within susceptible populations. The acquisition of resistance is critically understudied in fungi, relative to bacteria, and is usually examined with a single lab or clinical isolate. Unlike bacteria, which frequently acquire plasmid-mediated beneficial genes and alleles from the environment, fungal microbes primarily adapt via vertical transmission (mother to offspring) through a range of both genomic mutations and larger-scale karyotypic changes in ploidy level (the number of chromosome sets) and aneuploidy (the altered copy number of one or several chromosomes). We conducted a large-scale experimental evolution study to probe how different strain backgrounds of Candida albicans, a prevalent human fungal pathogen, evolve to fluconazole, the most commonly-prescribed antifungal drug. The results indicate that strain background, mediated in part by the ancestral growth ability in drug, significantly influenced both the mean response and the variability of responses: replicates from strains that grew most slowly when initially exposed to the drug tended to undergo the biggest improvement in growth in drug, but also exhibited more variability in a range of measured phenotypes than replicates from initially fast-growing strain backgrounds. Importantly, very few replicates acquired resistance, defined as the genetically stable ability to grow well at drug concentrations above those used in the evolution conditions. Nevertheless, we found frequent changes in both genome size (DNA content) and antifungal drug tolerance, a newly defined parameter, distinct from resistance, that involves the slow growth of subpopulations of cells in drug concentrations that inhibit the growth of most cells. These results demonstrate that caution should be exercised when generalizing experimental results from a single strain background, and highlight the large range of variability in drug responses within and between populations of microbial populations adapting to drug.