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 a range of 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 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 directly feed into NERCs new strategic direction 'The Business of the Environment'.

Our research project will benefit a broad range of stakeholders;

Health practitioners and clinicians:
As argued recently in the Lancet by Barnes et al (2014, Vol. 384, p1427), triazole drugs are the only effective oral treatment for invasive aspergillosis and pan-azole resistance in the UK is on the increase. The results of our reasearch are urgently needed by health-care practitioners as there is currently uncertainty as to i) the extent to which triazole resistance is aquired in vivo or is originating in the environment and ii) whether characteristic resistance mutations need to be screened at the bedside in immunocompromised patients / susceptible patients (such as transplant or cystic fibrosis patients) in order to optimise timely treatment. The project will address these questions and will constitute a valuable resource for the health-care community not only in the UK but also elsewhere.

Public Health Authorities:
Our findings are of immediate interest to Public Health England and the Environment Agency, whom are both Project Partners on our application. Our results will feed directly into policy questions surrounding the risk of generating and amplifying antifungal resistance in the environment as a by-product of intensive agribusiness.

Agribusiness:
Our findings are of immediate interest to current debates surrounding the use of various classes of antifungal compounds in agriculture as well as industrial composting factories, and will inform future methodologies to minimise exposure of non-target fungal species to high levels of triazole compounds.

The pharmaceutical industry:
The type and frequency of antifungal mutations that we characterise will be of high interest to those in the pharmaceutical industry whom are developing and optimising antifungal therapies; these pharamaceutical companies include Gilead and Astrazeneca.

The lay public:
Individuals have a right to know the risks that are associated with the intensive use of antifungal compounds in the environment, and how this may effect their health. This project will directly address the publics right to be informed.

How will they benefit?

Methodological contribution:
Scientists interested in the biology and epidemiology of A. fumigatus STRAf genotyping will benefit from our new R-based toolkit for the analysis of A. fumigatus STR profiles. This free software will implement standard population genetics approaches for quantifying and exploring genetic diversity, as well as cutting edge methodology for assessing and describing population structure. It will also implement statistical prediction of antifungal resistance based on STR profiles of the isolates considered. This development will be undertaken in close collaboration with members of the different collaborating laboratories to ensure relevance and address potential practical issues and optimize data analysis pipelines.

To maximize the impact of our methodological contribution, the tools developed during the project will be presented during the workshop we will organise in the third year of the project (see 'workshop' section below).

Workshop:
In the third year of the project we will hold a two-day workshop at St Mary's hospital, London, on the outputs of the project to-date and to integrate our findings with other centres of activity in the UK, such as the Manchester National Aspergillosis Centre and the BBSRC Rothamsted Research Station. This will enable us to write a White Paper that is aimed at policy makers, politicians and the media that will summarise the state of our understanding around the genomics of Aspergillosis antifungal resistance in the UK, and will also focus future research and policy directions. This will also be the occasion to introduce the tools developed for the characterisation of A. fumigatus isolates based on STR data, through lectures and hands-on practicals.