This study investigated the dynamics of Aspergillus fumigatus azole-resistant phenotypes in two compost heaps with contrasting azole exposures: azole free and azole exposed. After heat shock, to which sexual but not asexual spores are highly resistant, the azole-free compost yielded 98% (49/50) wild-type and 2% (1/50) azole-resistant isolates, whereas the azole-containing compost yielded 9% (4/45) wild-type and 91% (41/45) resistant isolates. From the latter compost, 80% (36/45) of the isolates contained the TR ₄₆ /Y121F/T289A genotype, 2% (1/45) harbored the TR ₄₆ /Y121F/M172I/T289A/G448S genotype, and 9% (4/45) had a novel pan-triazole-resistant mutation (TR ₄₆ ³ /Y121F/M172I/T289A/G448S) with a triple 46-bp promoter repeat. Subsequent screening of a representative set of clinical A. fumigatus isolates showed that the novel TR ₄₆ ³ mutant was already present in samples from three Dutch medical centers collected since 2012. Furthermore, a second new resistance mutation was found in this set that harbored four TR ₄₆ repeats. Importantly, in the laboratory, we recovered the TR ₄₆ ³ mutation from a sexual cross between two TR ₄₆ isolates from the same azole-containing compost, possibly through unequal crossing over between the double tandem repeats (TRs) during meiosis. This possible role of sexual reproduction in the emergence of the mutation was further implicated by the high level of genetic diversity of STR genotypes in the azole-containing compost. Our study confirms that azole resistance mutations continue to emerge in the environment and indicates compost containing azole residues as a possible hot spot. Better insight into the biology of environmental resistance selection is needed to retain the azole class for use in food production and treatment of Aspergillus diseases. IMPORTANCE Composting of organic matter containing azole residues might be important for resistance development and subsequent spread of resistance mutations in Aspergillus fumigatus . In this article, we show the dominance of azole-resistant A. fumigatus in azole-exposed compost and the discovery of a new resistance mutation with clinical relevance. Furthermore, our study indicates that current fungicide application is not sustainable as new resistance mutations continue to emerge, thereby threatening the use of triazoles in medicine. We provide evidence that the sexual part of the fungal life cycle may play a role in the emergence of resistance mutations because under laboratory conditions, we reconstructed the resistance mutation through sexual crossing of two azole-resistant A. fumigatus isolates derived from the same compost heap. Understanding the mechanisms of resistance selection in the environment is needed to design strategies against the accumulation of resistance mutations in order to retain the azole class for crop protection and treatment of Aspergillus diseases.

Resistance to medical triazoles inAspergillus fumigatusis an emerging problem for patients at risk of aspergillus diseases. There are currently two presumed routes for medical triazole-resistance selection: (i) through selection pressure of medical triazoles when treating patients and (ii) through selection pressure from non-medical sterol-biosynthesis-inhibiting (SI) triazole fungicides which are used in the environment. Previous studies have suggested that SI fungicides can induce cross-resistance to medical triazoles. Therefore, to assess the potential of selection of resistance to medical triazoles in the environment, we assessed cross-resistance to three medical triazoles in lineages ofA. fumigatusfrom previous work where we applied an experimental evolution approach with one of five different SI fungicides to select for resistance. In our evolved lines we found widespread cross-resistance indicating that resistance to medical triazoles rapidly arises through selection pressure of SI fungicides. All evolved lineages showed similar evolutionary dynamics to SI fungicides and medical triazoles, which suggests that the mutations inducing resistance to both SI fungicides and medical triazoles are likely to be the same. Whole-genome sequencing revealed that a variety of mutations were putatively involved in the resistance mechanism, some of which are in known target genes.

The treatment of patients suffering from Aspergillus diseases is hampered due to infections with Aspergillus fumigatus that are already resistant to medical azoles. Previous work has suggested that A. fumigatus likely gains resistance through environmental azole exposure in so-called hot spots. Here, we investigated A. fumigatus resistance dynamics over time at three sites at which farmers used azole fungicides for crop protection. Over 16 months, 114 samples were taken from stockpiles of decaying plant waste. A. fumigatus and azole fungicide residues were ubiquitously present in the plant waste. On average, 10⁵A. fumigatus CFU/g was recovered, of which roughly half were itraconazole and tebuconazole resistant. Similar tandem repeat-mediated resistance mechanisms were found in colonies cultured from plant waste as reported in clinical azole-resistant isolates. Our results show a consistent high burden of azole-resistant A. fumigatus in azole-containing plant waste and underscores the need to further investigate resistance-reducing interventions and transmission routes. IMPORTANCEAspergillus fumigatus is consistently present independently on season at a high abundance in plant waste material throughout the sampling period. Our study confirmed that long-term storage sites of azole-containing decaying plant material can indeed be considered hot spots, which can sustain resistance development and maintenance in A. fumigatus. Roughly half of individual isolates were azole resistant and carried genetic mutations that are highly similar to those found in patients with azole-resistant invasive aspergillosis. Our work suggests that environmental sources of azole resistance in A. fumigatus may be important, underscoring the need for further studies on environment-to-patient transmission routes.

Aspergillus fumigatusis a saprobic fungus that causes a range of pulmonary diseases, some of which are characterised by fungal persistence such as is observed in cystic fibrosis (CF) patients. Creation of genetic variation is critical forA. fumigatusto adapt to the lung environment, but biofilm formation, especially in CF patients, may preclude mutational supply inA. fumigatusdue to its confinement to the hyphal morphotype. We tested our hypothesis that genetic variation is created through parasexual recombination in chronic biofilms by phenotypic and genetic analysis ofA. fumigatusisolates cultured from different origins.As diploids are the hallmark of parasex, we screened 799A. fumigatusisolates obtained from patients with CF, chronic pulmonary lung disease and acute invasive aspergillosis, and from the environment for spore size. Benomyl sensitivity, nuclear content measurements through fluorescence-activated cell sorting and scanning electron microscopy were used to confirm the diploid state of large size spores. Whole genome sequencing was used to characterise diploid-associated genetic variation.We identified 11 diploids in isolates recovered from six of 11 (55%) CF patients and from one of 24 (4%) chronic aspergillosis patients, but not in 368 isolates from patients with acuteAspergillusinfection and the environment. Diploid formation was associated with accumulation of mutations and variable haploid offspring including a voriconazole-resistant isolate.Parasexual recombination allowsA. fumigatusto adapt and persist in CF patients, and plays a role in azole resistance development. Our findings are highly significant for understanding the genetics and biology ofA. fumigatusin the human lung.

With population genetic evidence of recombination ongoing in the natural Aspergillus fumigatus population and a sexual cycle demonstrated in the laboratory the question remained what the natural niche for A. fumigatus sex is. Composting plant-waste material is a known substrate of A. fumigatus to thrive and withstand temperatures even up to 70°C. Previous studies have shown indirect evidence for sexual reproduction in these heaps but never directly demonstrated the sexual structures due to technical limitations. Here, we show that flower bulb waste material from stockpiles undergoing composting can provide the conditions for sexual reproduction. Direct detection of ascospore structures was shown in agricultural flower bulb waste material by using a grid-based detection assay. Furthermore, we demonstrate that ascospores can germinate after exposure to 70°C for up to several days in contrast to asexual conidia that are unable to survive a two-hour heat shock. This indicates a sufficient time frame for ascospores to survive and escape composting stockpiles. Finally, sexual crosses with cleistothecium and viable ascospore formation could successfully be performed on flower bulb waste material. Recombination of A. fumigatus can now be explained by active sexual reproduction in nature as we show in this study that flower bulb waste material provides an environmental niche for sex.