Abstract
The importance of within-species diversity in determining the evolutionary potential of a population to evolve drug resistance or tolerance is not well understood, including in eukaryotic pathogens. To examine the influence of genetic background, we evolved replicates of 20 different clinical isolates of Candida albicans, a human fungal pathogen, in fluconazole, the commonly used antifungal drug. The isolates hailed from the major C. albicans clades and had different initial levels of drug resistance and tolerance to the drug. The majority of replicates rapidly increased in fitness in the evolutionary environment, with the degree of improvement inversely correlated with parental strain fitness in the drug. Improvement was largely restricted to up to the evolutionary level of drug: only 4% of the evolved replicates increased resistance (MIC) above the evolutionary level of drug. Prevalent changes were altered levels of drug tolerance (slow growth of a subpopulation of cells at drug concentrations above the MIC) and increased diversity of genome size. The prevalence and predominant direction of these changes differed in a strain-specific manner, but neither correlated directly with parental fitness or improvement in fitness. Rather, low parental strain fitness was correlated with high levels of heterogeneity in fitness, tolerance, and genome size among evolved replicates. Thus, parental strain background is an important determinant in mean improvement to the evolutionary environment as well as the diversity of evolved phenotypes, and the range of possible responses of a pathogen to an antimicrobial drug cannot be captured by in-depth study of a single strain background.IMPORTANCE Antimicrobial resistance is an evolutionary phenomenon with clinical implications. We tested how replicates from diverse strains of Candida albicans, a prevalent human fungal pathogen, evolve in the commonly prescribed antifungal drug fluconazole. Replicates on average increased in fitness in the level of drug they were evolved to, with the least fit parental strains improving the most. Very few replicates increased resistance above the drug level they were evolved in. Notably, many replicates increased in genome size and changed in drug tolerance (a drug response where a subpopulation of cells grow slowly in high levels of drug), and variability among replicates in fitness, tolerance, and genome size was higher in strains that initially were more sensitive to the drug. Genetic background influenced the average degree of adaptation and the evolved variability of many phenotypes, highlighting that different strains from the same species may respond and adapt very differently during adaptation.
Highlights
The acquisition of drug resistance is an inherently evolutionary process
A small, finite number of cellular mechanisms can lead to drug resistance, defined as the ability of all cells in a population to grow at drug concentrations above a specific, clinically-established breakpoint, measured as the minimal inhibitory concentration (MIC) of the drug
Karyotypic variation, whether via changes in ploidy or aneuploidy, is frequently observed in laboratory populations subjected to stress (Wellington and Rustchenko 2005; Bouchonville et al 2009)or passaged through mice (Forche et al 2009, 2018), as well as in clinical isolates of Candida albicans, Candida glabrata and Saccharomyces cerevisiae(Ford et al 2015; Zhu et al 2016)
Summary
The acquisition of drug resistance is an inherently evolutionary process. In principle, a small, finite number of cellular mechanisms can lead to drug resistance, defined as the ability of all cells in a population to grow at drug concentrations above a specific, clinically-established breakpoint, measured as the minimal inhibitory concentration (MIC) of the drug. A property distinct from drug resistance, is the ability of cells to grow slowly at drug concentrations above the minimum inhibitory concentration (MIC; Fridman et al 2014; Delarze and Sanglard 2015), and correlates well with the size of the subpopulation of cells that can form small colonies on drug as well as the rate at which they do so (Rosenberg et al 2018).We recently developed the first methods to quantify tolerance using either disk diffusion assays or broth microdilution measurements (Gerstein et al 2016; Rosenberg et al 2018)and found that strains that exhibit tolerance wasare more likely to cause clinically persistent infections (i.e., those not resolved by a single course of antifungal therapy) compared to strains with similar MIC levels that are cleared by a single course of the drug (Rosenberg et al 2018). The true degree of karyotypic variation among populations is likely to be even higher than what has been observed, as strain isolation methods (i.e., growth on rich medium) may select against karyotypic variants
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