Fungal pathogens pose a growing threat to the health of humans, animals, ecosystems, food security, and the global economy, making their effective control necessary (Fisher et al., 2012). Azoles fungicides, with more than 30 compounds available (Fisher et al., 2018), are widely used in agriculture to protect cereal, vegetables, and vines from phytopathogenic fungi, as well as in the cultivation of ornamental plants and to preserve materials such as timber. These fungicides are used worldwide, particularly in Europe and Asia, where they represent one of the most commonly used classes of pesticides (Chowdhary et al., 2015; Chen et al., 2016). Due to their broad-spectrum action across the fungal kingdom, the azoles are also widely used as first-line drugs in medicine (human and animals) for the treatment of superficial and invasive fungal infections. Within this context of widespread use of antifungals across different compartments, the occurrence of multi-resistant pathogenic fungi represents an emerging threat to the control of plant, animal, and human diseases (Fisher et al., 2018). Recently reviewed (Verweij et al., 2020), substantial progress has been made towards building the argument that ecological “hotspots” occur where biotic and abiotic conditions converge to allow the growth of the thermophilic Aspergillus fumigatus in contact with sub-MIC concentrations of agricultural triazoles, generating conditions that are suitable for adaptation to drug pressure. Specifically, highly related genotypes of azole-resistant A. fumigatus have now been described in clinical and environmental samples, suggesting that humans are increasingly exposed to drug-resistant aerosolized A. fumigatus spores with broad public health consequences (Sewell et al., 2019; Rhodes et al., 2021).

Studying the population genetics and evolution of pathogens can help further our understanding of the evolution and spread of antimicrobial resistance, including tracing potential sources of inocula using molecular epidemiology. Specifically, for azole-resistant A fumigatus (ARAf), it is important to understand whether genotypes are randomly distributed or cluster geographically, and whether a common source of colonization may exist (van der Torre et al., 2021). In 2012, we described a first case of invasive aspergillosis caused by TR₃₄/L98H ARAf in a farmer (Rocchi et al., 2014). In 2013, we described a second case, in a woodworker, who developed sinus aspergillosis due to TR₃₄/L98H A. fumigatus (Jeanvoine et al., 2016). Motivated by these findings, the mycology team of Besançon (Eastern France) undertook a systematic screening of A. fumigatus to assess the burden of azole resistance across both clinical and environmental samples. In 2017, we observed an increase in the number of cystic fibrosis (CF) patients colonized with ARAf, with 16% of patients with A. fumigatus-positive cultures observed carrying the TR₃₄/L98H resistance allele (Godeau et al., 2019) whereas previously, only one patient had this type of isolate in 2015. This percentage of resistance mirrors that reported by a British team (Abdolrasouli et al., 2018) and is among the highest occurrence in CF patients observed in European studies (Amorim et al., 2010; Mortensen et al., 2011; Burgel et al., 2012; Morio et al., 2012; Fischer et al., 2014; Prigitano et al., 2017; Guegan et al., 2018; Seufert et al., 2018; Lavergne et al., 2019).

Since 2017, a number of ARAf baring the TR₃₄/L98H allele were detected in the air of Besançon hospital following fungal aerocontamination monitoring. Undertaken weekly using seven air sample locations in general hospital corridors, our surveillance has detected ARAf periodically between 2017 and 2019 (Rocchi et al., 2021). In parallel, two environmental studies were conducted in agricultural fields where azoles had been sprayed, in sawmills (Jeanvoine et al., 2017) and market gardens (Rocchi et al., 2018).

Across our team, we have collected 1,100 strains of the Aspergilli complex (A. fumigatus and cryptic species) isolated in our region (rural landscape in Eastern France). Here, we assess the genetic relatedness among a retrospective sample of 225 susceptible and resistant A. fumigatus from our collection for both clinical and environmental samples by comparing them to data (worldwide scale) held within pre-existing genotype databases.

In our study, we describe linked patient sampling, healthcare bioaerosol monitoring, and environmental sampling of A. fumigatus in order to explore potential links using molecular epidemiology. STRAf genotyping that we undertook demonstrated spatial heterogeneity, with some clones occurring far more widely than others. These data highlight the importance of incorporating spatial scale towards understanding of the local population genetic structure of A. fumigatus and the epidemiology ARAf.

Currently, the modes of dissemination, the occurrence of resistant genotypes in the environment, and the likelihood of exposure to at-risk patients remain poorly known. When applied locally, genotyping methods can shed light into the source of infection with various studies showing possible nosocomial acquisition of A. fumigatus and potentially ARAf (van der Torre et al., 2021). Three studies have suggested that dissemination from patient to patient occurs, as well as from patient to the healthcare environment (Pegues et al., 2002; Lemaire et al., 2018; Engel et al., 2019). For animals, the dynamics of resistant animal populations in livestock in relation to the environment (except for poultry) are poorly described and are unknown in wildlife. In both cases, it is likely that exposure to ARAf genotypes is higher for individuals in environment that are frequently exposed to fungicides and/or also to organic matter (leaf litter, compost, and cultivated soils rich in organic matter). Clearly, studies to assess the extent that resistant genotypes are stratified by region and environment will lend nuanced understanding to the ecological processes that are thought to generate ARAf.

In this study, as in others, we observe a reduced genetic diversity of TR₃₄/L98H isolates (the most common resistance mechanism) compared to susceptible strains, suggesting a more recent emergence of this genotype (Camps et al., 2012). This allele is suggested to have emerged around 1997, perhaps in the Netherlands and dispersed across Europe, although other regions of origin are possible (Snelders et al., 2012; Abdolrasouli et al., 2015; Bader et al., 2015; Garcia-Rubio et al., 2018). Currently, TR₃₄/L98H is increasingly described in other global settings and the occurrence of TR₃₄/L98H genotypes does not seem to be limited to Europe. Aerial dispersal is most likely a major contributor to the long-distance migration of airborne A. fumigatus conidia owing to the widespread detection of aerosolized A. fumigatus spores (Shelton et al., 2020), which can also be moved in traded products (Dunne et al., 2017). Indeed, identical genotypes were described in several centers and countries, sometimes separated by thousands of kilometres (Chowdhary et al., 2012; Sewell et al., 2019). In 2012, a study of environmental isolates from Iran reported a TR₃₄/L98H MLG closely related to Dutch and Indian isolates (Badali et al., 2013). A recent Japanese publication described TR₃₄/L98H isolates clustering with strains from the Netherlands and France (Tsuchido et al., 2019).

In our study, one of the two dominant MLGs (MLG B) isolated in different environments in the Besançon region and isolated from different patients over several years was identical to an MLG in Australia and Ireland. Owing to the highly diverse nature of the STRAf barcoding loci, the likelihood by these MLGs being identical by chance rather than by descent is vanishingly unlikely (Sewell et al., 2019). Conversely, we have also found MLGs that have so far not been found in other countries. This is particularly the case for MLG A, which has high frequency in varied environments (sawmills and hospital air) as well as in different patients over several years. In Taiwan, a team has observed a potential global spread of TR₃₄/L98H isolates but also a local genetic clustering of some clinical and environmental TR₃₄/L98H (Wang et al., 2018). Other works have also reported local clusters, as in China with clinical and environmental TR₃₄/L98H/S297T/F495I isolates (Deng et al., 2017; Chen et al., 2018). The two isolates carrying this mutation found in the French sawmill do not have the same STRAf profiles as those described in these works. In Germany, local factors have been evoked as the reason for the increased prevalence of specific genotypes (Bader et al., 2015). Resistance could therefore also result from the appearance of different clones located in different countries or regions when there is sufficient selection pressure. This could explain the greater diversity of genotypes found in vegetable crops in the market gardens, for example.

In our study, we have shown that ARAf occurs in the air of hospital and, in this case, could be acquired by patients during episodes of immunosuppression. In our hospital, resistant isolates were found in the general corridors, not in the hematology departments, thus reducing the risk of acquiring invasive aspergillosis caused by a resistant genotype. However, we described MLGs found in CF patients and in environments such as hospital air, and also with hospital visits coinciding with the isolation of certain resistant genotypes; this is the case, for example, for MLGs A and B. However, the wide distribution of these MLGs, both locally for MLG A (different environments) and globally for MLG B (different environments and in other countries), prevents us from conclusively asserting nosocomial acquisition of these isolates in CF patients. This is because, on a probabilistic basis, there is a non-negligible likelihood that these dominant genotypes may have been acquired through exposures outside of the hospital. However, a single MLG found only in Besançon was isolated from hospital air (1E023) and from sputum of a CF patient (1E017) 1.5 months apart. Even if we do not have the analytical power to definitively prove this, the potential for nosocomial contamination of the healthcare environment cannot currently be excluded.

Microsatellite genotyping is widely used in eukaryotes. In A. fumigatus, it has been used to identify the potential origins of resistant genotypes, and how genotypes are shared between patients, geographical regions, countries, and continents (Korfanty et al., 2019). Genotyping analyses with the nine-microsatellite-based method in A. fumigatus (de Valk et al., 2005) provide high discriminatory power and reproducibility (Klaassen and Osherov, 2007). For our local isolate comparisons, we can be sure that MLG A and B of the Besançon region isolates are different, as they were analyzed with the same methodology (PCR, fragment analysis, and copy number transformation). However, when we compare the profiles of isolates analyzed in different laboratories, how can we be sure that two isolates with only one microsatellite difference, for example, are indeed different or that the difference is due to interlaboratory variations? MLG A (only local isolates) and MLG B (local and found in Australia and Ireland) only differ by one repeat on a microsatellite, STRAf 3A, which is the loci showing the greatest diversity, as has already been shown for agricultural strains (Ashu et al., 2018). It is therefore important to secure or find ways to harmonize methods of analysis on a large scale so that results can be reliably compared, and ensure in our case that it is indeed the MLG A that is local and that the MLG B has a global distribution. Interlaboratory controls should also be considered with, in particular, the implementation of standardization of sizing data by using allelic ladders (de Valk et al., 2009). Moreover, microsatellite genotyping does not provide sufficiently fine-grained information on the degree of proximity of two strains with two MLG, for example, whereas whole genome sequencing (WGS) can (Hagiwara et al., 2014; Rhodes et al., 2021). High-throughput WGS techniques are now poised to surpass other typing methods because of the high resolution of the data generated and the decreasing costs as performance increases. Better genetic knowledge of resistant strains through genomic approaches will help define the population structure of A. fumigatus and inform the future evolutionary trajectory of resistant phenotypes.

While WGS techniques have a higher analysis resolution than other genotyping techniques, the STRAf method, coupled with mutation screening and even mating type characterization, appears to be a good compromise to rapidly and simply screen relatively large numbers of isolates that have resulted from surveillance programs (van der Torre et al., 2021). Then, as in our case, where a “super clone” emerges from the analysis, it would be judicious to use WGS analysis to refine analyses in order to further explore patient–environmental links. Going forward, there is a need to continue to study the environmental conditions (both in host and in the environment) that facilitate the development, selection, and spread of resistance genotypes with large-scale multidisciplinary collaborations.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Conceptualization: SR and MF. Molecular analysis: SR, CG, and AL. Data analysis, SR, TS, and BV. Writing—original draft preparation: SR. Writing, review, and editing: SR, MF, TS, CG, BV, and LM. Funding acquisition: SR and LM. All authors contributed to the article and approved the submitted version.

The routine analyses were carried out within the framework of hospital activity, without external funding. The in-depth analysis of resistant strains was financially supported by LTSER “Zone Atelier Arc Jurassien”, “Observatoire des Sciences de l’Univers Terre Homme Environnement Temps Astronomie”, Bourgogne Franche-Comté-University, and RECOTOX network supported by AllEnvi and co-carried by INRAE and CNRS-INEE. MF and TS were supported by the UK Medical Research Council and the UK Natural Environmental Research Council.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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