Elevated mutation rates in multi-azole resistant Aspergillus fumigatus drive rapid evolution of antifungal resistance
The evolution of antifungal resistance is an emerging global threat. Particularly concerning is the widespread occurrence of azole resistance within Aspergillus fumigatus, a globally ubiquitous environmental mould that causes over one million life-threatening invasive infections in humans each year. The environmental use of azole fungicides has led to selective sweeps across multiple loci in the A. fumigatus genome causing the rapid expansion of a genetically distinct cluster of genotypes (clade A) resulting in globally widespread resistance to frontline clinical azoles. Isolates within this cluster are more likely to be resistant to agricultural antifungals with unrelated modes of action suggesting they are adapting rapidly to antifungal challenge. Here we show that this cluster is not only multi-azole resistant but has increased propensity to develop resistance to next generation antifungals because of variants in the DNA mismatch repair system. A variant in msh6 (G233A) is found almost exclusively within clade A, occurs in 88% of multi-azole resistant isolates harbouring the canonical cyp51A azole resistance allelic variant TR34/L98H, and is globally distributed. Naturally occurring isolates with this msh6 variant display up to 5-times higher rate of mutation, leading to an increased propensity to evolve resistance to next generation antifungals. We argue that pervasive environmental use of fungicides has resulted in a selective sweep of resistance alleles upon which a mutator allele has hitchhiked leading to the spread of genotypes of A. fumigatus with increased adaptive capability which thrive in the face of strong directional selection. These results may explain the pronounced clustering of multiple independent resistance mechanisms within the mutable clade A. Furthermore, unlike hypermutator strains that are unable to persist due to the selective disadvantage of high mutation rates, the G233A variant conveys no measurable fitness cost and has become globally distributed.  Our findings further suggest that resistance to next-generation antifungals is more likely to emerge within organisms that are already multi-azole resistant due to the close linkage between TR34/L98H and msh6-G233A, posing a major problem due to the prospect of dual use of novel antifungals in clinical and agricultural settings.