Objectives: Aspergillus fumigatus is an environmentally ubiquitous saprophyte capable of causing life-threatening invasive infections and allergic bronchopulmonary disease and current management strategies rely on the azoles. Unfortunately, over the last decade there has been a global emergence in resistance to the azoles in A. fumigatus and the dominant resistance mechanism is of environmental origin, suggesting that resistance is emerging through a selective pressure applied by the widespread usage of azoles in agriculture.
Methods: To examine the link between the use of azoles in agriculture and the emergence of clinical resistance, systematic soil sampling was performed over a three year period on seven conventional agricultural sites in Germany utilizing azole fungicides and on six organic sites withholding these compounds. Conventional sites were evaluated both before and after azole application. We also performed WGS on 250 environmental and 50 clinical isolates.
Results: In total, 2875 soil samples were analyzed between 2016 and 2018. We observed a high degree of variation in the abundance of A. fumigatus across sites, however, fields with high A. fumigatus density tended to be consistently so from year to year. Strikingly, we observed a significant reduction in the abundance of A. fumigatus on conventional fields following azole treatment – a finding that was not repeated on an organic agriculture control field – indicating that the application of azoles is imposing a bottleneck on A. fumigatus. We detected a low overall resistance frequency amongst agricultural isolates, with only 1-5% of isolates from 2016-2018 showing medical azole resistance – a rate lower than the 15-20% resistance frequency observed by the German National Reference Center for Invasive Fungal Infections during the same time period. Susceptibility to commonly-applied agricultural azoles was also assessed and isolates resistant to medical azoles almost always had elevated MICs to these agricultural azoles, suggesting cross resistance. Importantly, we observed an increased tolerance to both agricultural and medical azoles in the samples taken after the growing season and application of azoles. At the genome level, isolates from different regions and types of agriculture did not cluster separately, indicating a lack of population structure in the fungus. Comparison of environmental isolates with clinical isolates revealed several subgroups present in the environment that were not represented among clinical samples. We also observed differences in the distribution of resistance mutations between environmental and clinical isolates. Resistant environmental isolates were exclusively either wild type at the cyp51a loci or carried the environmentally-derived TR34/L98H mutation. Clinical isolates, in contrast, showed a much wider range mutations, including substitutions at positions F219, M220, S297, F495, and G448.Ongoing work is focused on defining fungal determinants critical for human infection as well as genetic mechanisms associated with cyp51a-independent azole resistance.
Conclusion: We observed a marked reduction in A. fumigatus fields following azole application and increased azole tolerance after the growing season and azole exposure. No population structure was observed among environmental isolates, but whole genome phylogeny identified genetic backgrounds enriched for among clinical isolates.
Full conference title:
- TIMM (2019)