Evidence that increased azole persistence and stress resistance precede the in vivo evolution of azole resistance in Aspergillus fumigatus

ABSTRACT Aspergillus fumigatus is the major causative agent of human aspergillosis. Azoles are the first-line therapy, but resistance is increasing. Here, we characterized voriconazole persistence by screening a global collection of 495 clinical and environmental A. fumigatus isolates. We demonstrate two persistence phenotypes: non-growth persisters (NGPs) and slow-growth persisters (SGPs). We focused on nine sequential clinical isolates from a single patient treated with voriconazole for 2 years. Genome sequencing and phylogenomic analyses show that these isolates form a single genetically related population exhibiting within-host diversification. While temporal isolation order does not strictly correlate with genetic relatedness, we observe that increased azole persistence and stress resistance are early adaptive steps in the in vivo evolution of azole resistance, preceding the emergence of voriconazole resistance. Our findings indicate that increased persistence and stress resistance may create a permissive adaptive landscape within the host, highlighting the complex evolutionary dynamics underlying antifungal treatment failure. IMPORTANCE Azole resistance in Aspergillus fumigatus is a major clinical concern; however, treatment failures occur without the presence of classic resistance mutations. This study provides evidence that azole persistence—the ability of susceptible cells to survive drug exposure—and stress resistance precede the in vivo evolution of azole resistance in Aspergillus fumigatus . We identified two persistence types in a global isolate collection and tracked their evolution in a patient over a 2-year period. Our findings reveal that increased voriconazole persistence and stress resistance emerged before the acquisition of the cyp51A resistance mutation. This adaptive phase involved significant transcriptional and metabolic reprogramming, including the upregulation of secondary metabolism. We propose that persistence is not a passive survival state, but an active evolutionary stepping stone that fosters a permissive landscape for the development of resistance. Understanding this “persistence-first” pathway is crucial for developing more effective diagnostics and therapeutic strategies to combat antifungal treatment failure.