The evolutionary dynamics of multiazole resistance in pathogenic Aspergillus fungi

Lead Research Organisation: Rothamsted Research
Department Name: Biointeractions and Crop Protection

Abstract

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

Planned Impact

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.
 
Description A much better understanding is obtained about 'hot spots' of azole resistance development in environmental populations of Aspergillus fumigatus and how resistance is spreading using molecular tools. Follow-up projects will focus on measures to slow down the evolution and spread of azole resistance.
Exploitation Route Several papers are being prepared and outcomes of this funding will be used for follow-up proposals
Sectors Agriculture, Food and Drink,Chemicals,Healthcare,Government, Democracy and Justice,Pharmaceuticals and Medical Biotechnology

 
Description "One health consequences of circularity. What lessons to learn from the saprophytic and human pathogenic fungus Aspergillus fumigatus?" 
Organisation University of Wageningen
Country Netherlands 
Sector Academic/University 
PI Contribution Consortium partner and advisor on this NWO funded project in the Netherlands
Collaborator Contribution Advisory role, project just started
Impact No outcomes yet, project just started
Start Year 2020
 
Description FRAG-UK meeting presentation 'Update on understanding the sources and spread of Azole resistance in environmental Aspergillus fumigatus populations' 20 March 2019, Cambridge, UK 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Provided update on the understanding the sources and spread of Azole resistance in environmental Aspergillus fumigatus populations. Knowledge transfer to AgroChem Industry and the Chemicals Regulation Division.
Year(s) Of Engagement Activity 2019
 
Description Invited speaker 'Azole fungicides in agricultural systems: cereal disease control and resistance development in Aspergillus fumigatus' at session 'Impacts of agricultural fungicides on clinical anti-fungal resistance', Panel Discussion APS Conference, Cleveland, USA, 6 August 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Important international debate on how fungicide resistance can spread in environmental populations of Aspergillus fumigatus, despite being a non-target pathogen
Year(s) Of Engagement Activity 2019
URL https://www.apsnet.org/meetings/annual/meetingarchives/planthealth2019/Documents/Abstracts/aps2019ab...
 
Description Invited speaker 'Fungicide resistance in different habitats and produce' at workshop 'Azole resistance in Aspergillus fumigatus: the next step' Trippenhuis Royal Netherlands Society of Arts and Science, Amsterdam, The Netherlands (31 January 2019). 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Azole resistance is a concern for the management of diseases caused by Aspergillus fumigatus in humans. Azole fungicide use in the environment has been identified as possible cause for resistance development, and thus increases the complexity and number of stakeholders involved in this emerging problem. We aim to bring together relevant stakeholders including medical and agricultural researchers, representatives from the government, fungicide producers and users, and public health to review the current evidence supporting environmental resistance selection and to discuss which research and measures are needed to retain the azole class for environmental and medical applications.
Year(s) Of Engagement Activity 2019
 
Description Invited speaker 'Use of azoles in agriculture and resistance risk' at the Scientific meeting 'Azole Resistance in a One Health Perspective', Oslo, Norway 22-23 January 2019. 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact WHY THIS MEETING?

Fungal infections caused by opportunistic fungi such as Aspergillus fumigatus kill more than 1 million people annually worldwide (equivalent to deaths caused by tuberculosis or malaria). The number will likely increase considerably because medical treatment becomes less effective as the fungus develops resistance to the commonly used antifungal compounds containing azoles. The resistance against azoles is dispersed globally at a disturbing speed. Fungal infections that are resistant to antifungal treatment are an emerging public health problem, requiring immediate action. The global community has a strong focus on antimicrobial resistance (AMR), but almost solely on antibacterial resistance.

Azoles are efficient fungicides, commonly used both to treat and prevent fungal infections (mycoses) in humans and animals, to manage fungal diseases in food production and horticulture, and to protect materials against mould growth. Azole-based antifungal treatment has made it possible to cure Aspergillus fumigatus infections. However, several international studies have shown that the widespread use of azoles in agriculture, horticulture and forestry have caused a selection pressure driving the fungal population to develop resistance to azoles, threatening effectivity of the whole azole class. Furthermore, residues of azoles in nature are regarded as environmental toxins and are suggested to have general endocrine disrupting properties. The case fatality rates in patients with culture-positive azole-resistant invasive aspergillosis (IA) varies between 50% and 100%. Reports of clinical and environmental azole resistant A. fumigatus strains world-wide indicate that azole resistance is increasing. Surveillance studies have shown that in areas where Aspergillus is endemic, the environmental route of resistance selection contributes to > 90 % of resistance mechanisms in azole-resistant Aspergillus diseases.

The scope of the meeting is to get an overview of the current situation and discuss how we can prevent the rapid spread of azole resistance globally. Where are the hot spots or resistance development? What are our priorities? What is the situation in Norway? The impact of climate changes. Biodiversity. One Health. Sustainability.

The ResAzoleNet project focuses on a neglected part of the One Health AMR resistance perspective, and addresses both mechanisms of AMR and strategies to prevent AMR. The project consortium includes research groups in Norway, Denmark and the Netherland
Year(s) Of Engagement Activity 2019
 
Description Invited speaker at the 20th Congress of the International Society for Human and Animal Mycology (ISHAM) in Amsterdam, The Netherlands (1 July 2018) 'Role of agricultural pesticide use in the evolution of triazole resistance in fungi' 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 20th Congress of the International Society for Human and Animal Mycology, 30 June-4 July 2018, Amsterdam, the Netherlands

Congresses of the International Society for Human and Animal Mycology (ISHAM) are held every three years and provide clinicians, scientists, and students an opportunity to meet at an international forum dedicated to the study of medical mycology. The 20th ISHAM is co-hosted by the Netherland Society of Medical Mycology (NVMy).
Year(s) Of Engagement Activity 2018
 
Description PhD late stage review 'Environmental drivers of azole-resistance in Aspergillus fumigatus and its relevance to human health' Imperial College London', 4 October 2019 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Advise on progress of PhD study 'Environmental drivers of azole-resistance in Aspergillus fumigatus and its relevance to human health'
Year(s) Of Engagement Activity 2019