Abstract Antifungal drug resistance and tolerance poses a serious threat to global public health. In the human fungal pathogen, Candida auris , resistance to triazole, polyene, and echinocandin antifungals is rising, resulting in multidrug resistant isolates. Here, we use genome analysis and in vitro evolution of seventeen new clinical isolates of C. auris from clades I and IV to determine how quickly resistance mutations arise, the stability of resistance in the absence of drug, and the impact of genetic background on evolutionary trajectories. We evolved each isolate in the absence of drug as well as in low and high concentrations of fluconazole. In just three passages, we observed genomic and phenotypic changes including karyotype alterations, aneuploidy, acquisition of point mutations, and increases in MIC values within the populations. Fluconazole resistance was stable in the absence of drug, indicating little to no fitness cost associated with resistance. Importantly, two isolates substantially increased fluconazole resistance to ≥256µg/ml fluconazole. Multiple evolutionary pathways and mechanisms to increase fluconazole resistance occurred simultaneously within the same population. Strikingly, the sub-telomeric regions of C. auris were highly dynamic as deletion of multiple genes near the sub-telomeres occurred during the three passages in several populations. Finally, we discovered a mutator phenotype in a clinical isolate of C. auris . This isolate had elevated mutation rates compared to other isolates and acquired substantial resistance during evolution in vitro and in vivo supporting that the genetic background of clinical isolates can have a significant effect on evolutionary potential. Importance Drug resistant Candida auris infections are recognized by the CDC as an urgent threat. Here, we obtained and characterized a set of clinical isolates of C. auris including multiple isolates from the same patient. To understand how drug resistance arises, we evolved these isolates and found that resistance to fluconazole, the most commonly prescribed antifungal, can occur rapidly and that there are multiple pathways to resistance. During our experiment, resistance was gained, but it was not lost, even in the absence of drug. We also found that some C. auris isolates have higher mutation rates than others and are primed to acquire antifungal resistance mutations. Furthermore, we found that multidrug resistance can evolve within a single patient. Overall, our results highlight the high stability and high rates of acquisition of antifungal resistance of C. auris that allow evolution of pan-resistant, transmissible isolates in the clinic.

Genomic diversity across Candida auris clinical isolates shapes rapid development of antifungal resistance in vitro and in vivo