Title: The evolutionary dynamics of multiazole resistance in pathogenic Aspergillus fungi
Funder: Natural Environment Research Council

Abstract: The evolutionary dynamics of multiazole resistance in pathogenic Aspergillus fungi The fungus Aspergillus fumigatus is globally ubiquitous in the environment, being present on decaying vegetation and in soils, where it performs a valuable role in nutrient recycling. The fungus is a minimal health threat to healthy individuals. However, patients that suffer from cystic fibrosis, cancer or have received organ transplants and are undergoing corticosteroid therapy, are at risk from 'invasive aspergillosis'. Current estimates indicate that over 63,000 patients develop this fungal disease annually across Europe. The primary method for controlling infections is by administering azole antifungal drugs. However, we and others have shown a sharp increase in the resistance of A. fumigatus to frontline azole antifungals, with unacceptably high mortality rates in these at-risk patient groups. The mutations that confer resistance of A. fumigatus to these drugs appear to have evolved in the environment, rather than in the patient. Azole compounds are also used as fungicides to control crop diseases. This has led to the hypothesis that the widespread use in agricultural crops of azole antifungal sprays is leading to the environmental selection for resistance in A. fumigatus, which is then resulting in decreased patient survival following infection. Our project aims to examine this hypothesis by determining the relative proportions of azole-resistant and azole-sensitive A. fumigatus in the UK by sampling environmental populations using growth media containing antifungal drugs. This environmental exposure assessment approach will target environments that have had high to low applications of crop-antifungals and will enable us to statistically examine whether there are links between the intensive use of these azole-based compounds in the environment and the occurrence of drug-resistant A. fumigatus. We will then use powerful technologies to sequence the genomes of many hundreds of A. fumigatus that are sensitive, or resistant, to azole antifungals. We already have numerous isolates pre-collected from around the world though a broad network of project partners, and we now know that there are two main azole-resistance mutations that widely occur. Our plan is to use our genome sequences and cutting-edge statistical genetic methods in order to determine when and where these mutations originated globally, use our newly isolated samples to test whether they occur within the UK environment and patient populations, whether they are spreading to invade new environments here and elsewhere, and whether novel undescribed resistance mutations exist. A. fumigatus is capable of sexual, as well as asexual, reproduction. In this case, the rate at which a newly-evolved resistance mutation can be integrated into new genetic backgrounds depends on the fertility of the A. fumigatus populations. In order to directly measure the 'sexiness' of the A. fumigatus populations, we will therefore perform sexual crosses using sequenced isolates that represent not only the range of genetic diversity that we encounter, but also the range of azole-resistance mutations. By measuring the number and fitness of progeny, we will be able to determine the rate at which resistance mutations can recombine into new genetic backgrounds, and also discover unknown drug-resistance mechanisms. By addressing these questions, we will directly measure the risk that the use of antifungal compounds has on evolving resistance in non-target fungal species, and also answer important questions on the distance that these airborne fungi are able to spread and share genes with one another. Our findings will not only be of high relevance to health care professionals, directly informing diagnostic protocols and disease management in intensive-care settings, but will also inform current debates on the costs of widespread use of antimicrobial compounds in the environment. These goals all


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION