key: cord-0788660-wm345kzv authors: Shelton, Jennifer M. G.; Collins, Roseanna; Uzzell, Christopher B.; Alghamdi, Asmaa; Dyer, Paul S.; Singer, Andrew C.; Fisher, Matthew C. title: Citizen Science Surveillance of Triazole-Resistant Aspergillus fumigatus in United Kingdom Residential Garden Soils date: 2022-02-22 journal: Applied and environmental microbiology DOI: 10.1128/aem.02061-21 sha: f97c81bc4a2134f7eec78d72bf41a6ee64f13641 doc_id: 788660 cord_uid: wm345kzv Compost is an ecological niche for Aspergillus fumigatus due to its role as a decomposer of organic matter and its ability to survive the high temperatures associated with the composting process. Subsequently, composting facilities are associated with high levels of A. fumigatus spores that are aerosolized from compost and cause respiratory illness in workers. In the UK, gardening is an activity enjoyed by individuals of all ages, and it is likely that they are being exposed to A. fumigatus spores when handling commercial compost or compost they have produced themselves. In the present study, 246 citizen scientists collected 509 soil samples from locations in their gardens in the UK, from which were cultured 5,174 A. fumigatus isolates. Of these isolates, 736 (14%) were resistant to tebuconazole: the third most-sprayed triazole fungicide in the UK, which confers cross-resistance to the medical triazoles used to treat A. fumigatus lung infections in humans. These isolates were found to contain the common resistance mechanisms in the A. fumigatus cyp51A gene TR(34)/L98H or TR(46)/Y121F/T289A, as well as the less common resistance mechanisms TR(34), TR(53), TR(46)/Y121F/T289A/S363P/I364V/G448S, and (TR(46))(2)/Y121F/M172I/T289A/G448S. Regression analyses found that soil samples containing compost were significantly more likely to grow tebuconazole-susceptible and tebuconazole-resistant A. fumigatus strains than those that did not and that compost samples grew significantly higher numbers of A. fumigatus than other samples. IMPORTANCE The findings presented here highlight compost as a potential health hazard to individuals with predisposing factors to A. fumigatus lung infections and as a potential health hazard to immunocompetent individuals who could be exposed to sufficiently high numbers of spores to develop infection. Furthermore, we found that 14% of A. fumigatus isolates in garden soils were resistant to an agricultural triazole, which confers cross-resistance to medical triazoles used to treat A. fumigatus lung infections. This raises the question of whether compost bags should carry additional health warnings regarding inhalation of A. fumigatus spores, whether individuals should be advised to wear facemasks while handling compost, or whether commercial producers should be responsible for sterilizing compost before shipping. The findings support increasing public awareness of the hazard posed by compost and investigating measures that can be taken to reduce the exposure risk. placed in uncovered rows that are turned regularly during the composting process to improve oxygenation of the waste and to distribute heat and moisture. Composting facilities are known to produce large numbers of A. fumigatus spores (20) (21) (22) (23) (24) (25) (26) (27) , with resulting negative health impacts on compost handlers (28) (29) (30) (31) (32) (33) (34) (35) (36) , and there is evidence from the Netherlands that composting material also produces large numbers of triazole-resistant spores (37, 38) . In 2017, UK households spent approximately £450 million on compost (39) and apply it more liberally to their gardens at 300 tonnes per hectare (t/ha) than the 50 t/ ha applied to agricultural land (40) . Furthermore, more than a third of households with access to a garden report composting their garden and/or kitchen waste (41) . This means that a substantial proportion of the UK population is handling compost on a regular basis, with potential exposure to high levels of A. fumigatus spores that may have developed triazole resistance from composts that contain triazole residues. Indeed, there have been reports of hypersensitivity pneumonitis (42) and IA (43) (44) (45) (46) (47) in apparently immunocompetent individuals following gardening activities; however, no clinical links following exposure to triazole resistant spores have been documented. The aims of this study were to (i) determine the numbers of triazole-susceptible and -resistant A. fumigatus spores in soil samples collected from residential gardens in the UK, (ii) characterize the cyp51A polymorphisms responsible for resistance, and (iii) find environmental variables associated with the presence/numbers of A. fumigatus spores in soil samples. In order to simultaneously sample a wide range of UK gardens, we were assisted by a network of citizen-scientists trained in the collection of samples that may contain A. fumigatus. Our aim was to ascertain whether gardening activities may lead to exposure to triazole-resistant genotypes of this mold that could present a risk to susceptible individuals. Based on our findings, we present thoughts on how these exposure risks in susceptible individuals might be mitigated. Tebuconazole-susceptible and tebuconazole-resistant A. fumigatus in soil samples. Of the 509 soil samples collected, 327 (64%) samples between them grew 5,174 A. fumigatus isolates and 101 (20%) samples grew 736 tebuconazole-resistant isolates ( Table 1) . Most of the samples (n = 451; 89%) were assigned a singlelocation in the garden where they were collected, whereas the remainder were assigned multiple locations. These multiple locations occurred when a border or pot/planter had recently been topped up with manure or compost. The concentration of spores and mycelial fragments averaged across the samples that grew A. fumigatus was 316 CFU/g, which ranged from 0 CFU/g in the sample collected from a border plus manure bag to 600 CFU/g in the sample collected from a manure bag. The concentration of spores and mycelial fragments averaged across the samples that grew tebuconazole-resistant A. fumigatus was 146 CFU/g, which ranged from 0 CFU/g in samples collected from several garden locations to 214 CFU/g in samples collected from compost heaps. Figure 1 shows the geographical locations in the UK where soil samples were collected. Cyp51A polymorphisms in tebuconazole-resistant A. fumigatus isolates. Of the 736 tebuconazole-resistant A. fumigatus isolates, 93 (13%) failed to regrow from refrigerated storage for cryopreservation and DNA extraction. In the 643 isolates that regrew, TR 34 /L98H was detected in 542 (85%), TR 46 /Y121F/T289A was detected in 16 (3%), TR 53 was detected in 2, and (TR 48 ) 2 /Y121F/M172I/T289A/G448S was detected in 1 sample, and no cyp51A polymorphisms were detected in 27 (4%) isolates. A total of 14 isolates failed to sequence with the cyp51A promoter and coding region primers, and beta-tubulin sequencing confirmed their identities as A. fischeri (n = 8), A. fumigatus (n = 2), A. oerlinghausenensis (n = 3), and unknown (n = 1). Uncommon polymorphisms detected were TR 34 without accompanying amino acid substitutions in three isolates, (TR 34 ) 2 /L98H in one isolate, and (TR 130 ) 3 /D430G in four isolates. The remaining isolates contained one or more amino acid substitutions in cyp51A, with or without accompanying TRs ( Table 2 ). Further details regarding the tebuconazole-resistant A. fumigatus isolates can be found in Table S1 in the supplemental material. Environmental variables influencing growth and numbers of A. fumigatus colonies. (i) Growth of A. fumigatus from soil samples. Eight samples were excluded from the logistic regression with growth of A. fumigatus as the outcome, which left 501 samples in the analysis. These samples were excluded because the Sabouraud dextrose agar (SDA) plates were too contaminated to determine the presence of A. fumigatus. The location in the garden where the soil sample was collected was the only variable that significantly affected whether a sample grew A. fumigatus (x 2 = 67.3, df = 12, P , 0.01). The odds ratios and P values from the logistic regression model are shown in Table 3 . Samples collected from a compost bag, compost heap, pot/planter, and pot/planter plus compost bag had significantly increased odds of growing A. fumigatus (P , 0.01) compared to samples collected from a border. There were no significant changes in odds of growing A. fumigatus from other sampling locations. (ii) Number of A. fumigatus colonies grown from soil samples. The first negative binomial regression was run on the 335 samples that grew A. fumigatus. The only variable found to significantly affect the number of A. fumigatus colonies grown from a sample was garden location from which the sample was collected (x 2 = 50.8, df = 11, P , 0.01). In the regression model, samples collected from compost bag (P , 0.01), compost heap (P , 0.01), and pot/planter plus compost bag (P = 0.02) grew significantly more A. fumigatus colonies than samples collected from borders. Samples collected from a pot/planter plus compost bag plus manure bag grew fewer A. fumigatus colonies than samples collected from borders, although this reduction was marginally significant (P = 0.05). (iii) Growth of tebuconazole-resistant A. fumigatus from soil samples. All 509 soil samples were included in the logistic regression with growth of tebuconazole-resistant A. fumigatus as the outcome. The only variable found to significantly affect whether a sample grew tebuconazole-resistant A. fumigatus was garden location from which the sample was collected (x 2 = 43.0, df = 12, P , 0.01). The odds ratios and P values from the logistic regression model are shown in Table 4 . Samples collected from a compost Samples that failed to amplify with the cyp51A promoter and coding region primers were sequenced using beta-tubulin primers for fungal identification. bag, compost heap, pot/planter, and pot/planter plus compost bag had significantly increased odds of growing tebuconazole-resistant A. fumigatus (P , 0.01) compared to samples collected from a border. There were no significant changes in the odds of growing tebuconazole-resistant A. fumigatus from other sampling locations. (iv) Number of tebuconazole-resistant A. fumigatus colonies grown from soil samples. The second negative binomial regression was run on the 101 samples that grew tebuconazole-resistant A. fumigatus. None of the environmental variables were found to have a significant effect on the outcome. In this study, 5,174 A. fumigatus isolates were cultured from 509 soil samples collected by 249 citizen scientists from their gardens across the UK (48) . Of these soil samples, 327 (64%) grew A. fumigatus isolates, and 101 (20%) grew isolates that were resistant to tebuconazole at a concentration of 6 mg/L. The percentage of soils that grew A. fumigatus in this study was lower than the 78% of soils collected by Sewell et al. from several sites across South West England, including parks, cemeteries, public gardens, flower beds outside hospitals, a lavender farm, a forest, and farmland (49) . (51). This prevalence of 14% is also greater than the 9% in experimental cropland and 12% in commercial wheat fields in the UK reported by Fraaije et al. (52) ; however, it is less than the 37% prevalence in isolates cultured from flower bulbs bought from a garden center in Dublin reported by Dunne et al. (53) . In this study, the average concentration of A. fumigatus from positive soil samples was 316 CFU/g, which is higher than the 43.5 CFU/g in agricultural soils and 106 CFU/g in urban soils from Greater Manchester reported by Bromley et al. (54) and considerably higher than the 0 to 10 CFU/g reported from woodlands, grass verges, experimental cropland, and commercial wheat fields across the UK reported by Fraaije et al. (52) . Given that A. fumigatus is often considered to be ubiquitous in the environment, it is intriguing that 36% of the soil samples collected in this study did not grow this mold. We speculate that A. fumigatus spores and mycelial fragments in garden soils are killed by triazole residues from dipped bulbs (53), for example, if they have not developed triazole resistance. It is also possible that A. fumigatus is outcompeted by other microbes, especially in soils that have not experienced the high temperatures that are associated with composting. Of the 736 A. fumigatus isolates that grew on tebuconazole at 6 mg/L, 93 (13%) did not regrow from short-term storage in the fridge, which left 643 (87%) isolates for sequencing of the cyp51A promoter and gene coding regions. Similar to existing UK studies (49, 50, 54) , the predominant mutation identified in this study was TR 34 /L98H (n = 535; 73%). Of these isolates, 22 had amino acid substitutions in cyp51A in addition to L98H. Six isolates had T289A, I364V, and G448S amino acid substitutions, in addition to TR 34 /L98H, which has been previously detected in Korea in a patient with IA (55) and in Japan on tulip bulbs imported from The Netherlands (56) . TR 68 /L98H was detected in one isolate, which was found to be two repeats of the 34-bp insert, and in three isolates TR 34 was detected without any accompanying amino acid substitutions, which was first detected in an environmental isolate collected from Scotland (57) . TR 46 / Y121F/T289A was detected in 16 (2%) isolates and was accompanied by S363P, I364V, and G448S in four additional isolates, a combination reported from The Netherlands in 2018 (52) . Additional polymorphisms detected in this study included TR 53 , which has been previously reported from flower fields in Colombia (58) and from a patient with multiple-azole-resistant A. fumigatus osteomyelitis in The Netherlands (59) , and TR 92 / Y121F/M172I/T289A/G448S, which has been previously detected in flower bulb waste in The Netherlands (38) and is two repeats of the 46-bp insert. There were 33 (4%) isolates in this study that did not contain any TRs: five contained I242V, one contained C270R, and 27 had no amino acid substitutions in cyp51A. I242V is the only single cyp51A amino acid substitution detected in this study to have been reported in studies summarizing cyp51A polymorphisms (60-63), which may suggest these polymorphisms occurred in situ. The 28 isolates that did not contain any cyp51A polymorphisms may well be using non-cyp51A mechanisms for triazole resistance, such as the overexpression of efflux pumps, cyp51B overexpression, cholesterol import, or hapE mutation, which were not explored in this study (64) . The only environmental variable measured in this study that was found to have a significant effect on whether a sample grew A. fumigatus or on the numbers of A. fumigatus grown was the garden location from which the sample was collected. The greatest concentration of A. fumigatus was cultured from a bag of manure at 600 CFU/g, followed by homemade compost heap samples at 505 CFU/g, commercial compost bag samples at 451 CFU/g, and pot/planters containing commercial compost at 321 CFU/g. Soil samples that did not contain compost grew fewer A. fumigatus isolates: 254 CFU/g from pot/planters and 204 CFU/g from borders. Similar observations were made for tebuconazole-resistant A. fumigatus, with concentrations of 128 to 289 CFU/g recorded for samples containing compost and of 98 to 127 CFU/g for samples without compost. As citizen scientists were only asked to indicate one garden location from which the soil sample was collected, it is possible that the concentrations of A. fumigatus spores from borders and pot/planters were inflated by the recent addition of compost that was not indicated on the questionnaire. In the regression models, soils collected from commercial compost bags, homemade compost heaps, pot/planters, and pot/planters plus commercial compost had significantly greater odds of growing A. fumigatus and tebuconazole-resistant A. fumigatus (P , 0.01 for all associations) compared to soil samples collected from borders. Furthermore, samples collected from commercial compost bags, homemade compost heaps, and pot/planter plus commercial compost grew significantly more A. fumigatus colonies compared to samples collected from borders. No association was found for garden locations sampled from and numbers of tebuconazole-resistant A. fumigatus. Several existing studies have looked for triazole-resistant A. fumigatus specifically in compost in the UK and globally, and the findings have been highly variable. Tsitsopoulou et al. collected 11 compost samples from agricultural fields, a horticultural nursery and public areas across South Wales that grew 10 A. fumigatus isolates in all-none of which were triazole resistant (50). Dunne et al. do not report how many samples they collected from commercial compost bought from a garden center in Dublin or how many A. fumigatus were cultured from these samples; only that one isolate was triazole-resistant (53). Sewell et al. collected two samples from a compost heap in London that, combined with three samples collected from a flower bed ;500 m away, gave a 60% prevalence of triazole-resistant A. fumigatus (49) . Pugliese et al. sampled from composting orange peel in Italy and found A. fumigatus concentrations of 8.8 Â 10 3 CFU/g at the start of the process rising to 605.7 Â 10 3 CFU/g by the end, and yet none of the 30 isolates selected for susceptibility testing were triazole resistant (65). Santoro et al. sampled from 11 green and brown composts across Spain, Hungary, and Italy and found concentrations of A. fumigatus ranging from 100 to 10.6 Â 10 3 CFU/g; none of the 30 isolates selected for susceptibility testing were triazole resistant (66). Ahangarkani et al. screened isolates cultured from 300 compost samples collected in Iran and detected 57 isolates with elevated MICs to ICZ and VCZ (67). Zhang et al. collected 114 samples from a plant waste stockpile over 16 months in the Netherlands and detected .10 3 A. fumigatus CFU/g in 74% of samples, with the prevalence of triazole-resistant A. fumigatus averaging 50% across all samples (37) . Also in The Netherlands, Schoustra et al. found concentrations of triazole-resistant A. fumigatus of 200 CFU/g in household green waste, (1.5 to 1.8) Â 10 3 CFU/g in compost heaps in residential gardens, up to 2.3 Â 10 5 CFU/g in flower bulb waste, and up to 8.4 Â 10 4 CFU/g in organic waste from landscaping (38) . The key findings of this study are that 64% of soil samples collected from residential gardens in the UK grew A. fumigatus and that 20% of samples grew tebuconazole-resistant A. fumigatus. This means that individuals are very likely to be exposed to both A. fumigatus and triazole-resistant A. fumigatus spores that are aerosolized from soil when they are undertaking gardening activities (43) (44) (45) (46) (47) . Although this study has not undertaken susceptibility testing for the tebuconazole-resistant A. fumigatus isolates against medical triazoles, the most commonly detected cyp51A polymorphisms TR 34 /L98H and TR 46 /Y121F/T289A are associated with elevated MICs to ICZ, PCZ, and VCZ (68) . Furthermore, Hodiamont et al. reported a clinical isolate containing TR 53 as being resistant to ICZ and VCZ, with reduced susceptibility to PCZ (59) . This study also reports that the likelihood of being exposed to A. fumigatus and triazole-resistant A. fumigatus spores is significantly greater when handling commercial or homemade compost compared to soils in borders or pots/ planters. The 14% prevalence of triazole resistance detected in garden soil samples in this study is higher than most existing studies that have sampled from rural and urban locations in the UK, which is likely being driven by the concentrated application of compost in residential settings. The National Aspergillosis Centre advises that people take care when opening bags of compost and recommends wearing a facemask while doing so to avoid dust inhalation. Currently, the only health warning on commercial compost bags is for women to not handle compost without gloves if they are pregnant, presumably to avoid toxoplasmosis infection (69) . The evidence presented here supports the recommendation for users to wear a mask while handling compost and the introduction of health warnings on bags of compost with regard to inhaling A. fumigatus. Measures could also be taken by compost producers to sterilize the composting before packaging, thereby killing viable A. fumigatus spores and eliminating the immediate hazard it poses to the user. Culturing Aspergillus fumigatus from residential garden soil samples. The soil samples from which A fumigatus isolates were cultured for this study were collected as part of a citizen science project undertaken in June 2019, which involved 246 volunteers in the UK collected a total of 509 soil samples from different locations in their gardens (48) . Participants indicated on a questionnaire whether samples were collected from a border, pot or planter, compost heap, bag of manure, or bag of compost. Upon receipt, 2 g of each soil sample was suspended in 8 mL of buffer (0.85% NaCl and 0.01% Tween 20 in distilled water), shaken vigorously, and left to settle for 30 min. No adjustment was made for the moisture content of the soil when weighing it out. One 200-mL aliquot from the surface of the buffer was spread onto a plate containing SDA, penicillin (200 mg/L), and streptomycin (400 mg/L) and a second aliquot of 200 mL was spread onto a plate containing SDA, penicillin (200 mg/L), streptomycin (400 mg/L), and tebuconazole (6 mg/L). The concentration of 6 mg/liter tebuconazole was chosen after testing the growth of 30 isolates with known CYP51A mutations on SDA supplemented with 0, 4, 6, 8, and 16 mg/L tebuconazole. The only concentration that showed no growth of any isolates without CYP51A mutations and partial or full growth of all isolates with CYP51A mutations was 6 mg/L. Both plates were incubated at 37°C for 48 h, the number of colonies that morphologically resembled A. fumigatus on each plate recorded, and the colonies growing on the plate containing tebuconazole were picked into tubes containing mold preservation solution (0.2% agar and 0.05% Tween 20 in deionized water) and stored at 4°C. These isolates were subsequently cryopreserved in 50% glycerol solution and were DNA extracted as detailed by Boyle et al. (70) . Sequencing of A. fumigatus cyp51A gene. The promoter region of cyp51A was amplified using forward primer 59-GGACTGGCTGATCAAACTATGC-39 and the reverse primer 59-GTTCTGTTCGGTTCCAAAGCC-39 and the following PCR conditions: 95°C for 5 min; 30 cycles of 98°C for 20 s, 65°C for 30 s, and 72°C for 30 s; followed by 72°C for 5 min. The PCR volume used was 50 mL: 10 mL of FIREPol DNA polymerase (Solis Biodyne, Estonia), 10 mL of forward primer (1.5 mM; Invitrogen, USA), 10 mL of reverse primer (1.5 mM; Invitrogen, USA), 18 mL of nuclease-free water (Merck, Germany), and 2 mL of DNA. Amplicons were visualized by gel electrophoresis, and samples with visible bands were sent for sequencing using the forward primer. The coding region of cyp51A was amplified using the forward primer 59-ATGGTGCCGATGCTATGG-39 and the reverse primer 59-CTGTCTCACTTGGATGTG-39 and the following PCR conditions: 94°C for 2 min; 35 cycles of 94°C for 30 s, 60°C for 45 s, and 72°C for 45 s; followed by 72°C for 5 min. The PCR volume used was 50 mL: 0.2 mL of Q5 high-fidelity DNA polymerase (New England Biolabs, UK), 10 mL of Q5 reaction buffer (5Â; New England Biolabs, UK), 0.5 mL of deoxynucleotide (dNTP) solution mix (40 mM; New England Biolabs, UK), 1 mL of forward primer (10 mM; Invitrogen, USA), 1 mL of reverse primer (10 mM; Invitrogen, USA), 35.3 mL of nuclease-free water (Merck, Germany), and 2 mL of DNA. Amplicons were visualized by gel electrophoresis, and samples with visible bands were sent for sequencing using the Sanger chain termination method in two segments using the primers 59-TACGTTGACATCATCAATCAG-39 and 59-GATTCACCGA ACTTTCAAGGCTCG-39. Sequences were aligned using Molecular Evolutionary Genetics Analysis (MEGA) software (Penn State University). Identification of isolates. For isolates that failed to sequence using the primers for the promoter and coding regions of cyp51A, part of the beta-tubulin gene was sequenced using the forward primer 59-AATTGGTGCCGCTTTCTGG-39 and the reverse primer 59-AGTTGTCGGGACGGAATAG-39 and the following PCR conditions: 94°C for 3 min; 30 cycles of 94°C for 15 s, 55°C for 30 s, and 68°C for 30 s; followed by 68°C for 3 min. Amplicons were visualized by gel electrophoresis, and samples with visible bands were sent for sequencing using the forward primer. The Basic Local Alignment Search Tool (BLAST) was used to align the sequences to those in the National Center for Biotechnology Information (NCBI, Bethesda, MD) to identify the isolate. Environmental variables that may influence growth of Aspergillus fumigatus. Table 5 details the environmental variables that were ascertained for the locations in the UK where soil samples were collected, the date the sampling occurred, and the sources from which the data were obtained. Generalized linear models. Generalized linear models (GLMs) were run using R version 4.0.0 to find associations between the environmental variables in Table 5 and (i) the likelihoods of a sample growing susceptible or triazole-resistant A. fumigatus and (ii) the number of susceptible or triazole-resistant A. fumigatus colonies grown from a sample. Growth of triazole-susceptible or triazole-resistant A. fumigatus from a sample was categorized as 0/1 and logistic regressions ("glm" function; family = "binomial") were performed. The numbers of susceptible and triazole-resistant A. fumigatus colonies grown from samples were overdispersed; therefore, negative binomial regressions (library "MASS"; "glm.nb" function) were performed. Environmental variables were included in the regression model based on a significant improvement on the null model, as determined by analysis of variance (ANOVA) using chi-squared test. Results were considered significant when P # 0.05. The regression model with the best fit was chosen based on a reduced Akaike information criterion (AIC) score and a significant improvement on the null model. Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.3 MB. (76), Natural Resources Wales website (77) , and Northern Ireland Environment Agency website (78) . Distances were calculated using package "geosphere" in R version 4.0.0 (79). Occurrence of Aspergillus fumigatus during composting of sewage sludge Mycological control and surveillance of biological waste and compost Aspergillus fumigatus: what makes the species a ubiquitous human fungal pathogen? Aspergillus fumigatus and aspergillosis Treatment options in severe fungal asthma and allergic bronchopulmonary aspergillosis Aspergillus fumigatus: principles of pathogenesis and host defense Preparations for invasion: modulation of host lung immunity during pulmonary aspergillosis by gliotoxin and other fungal secondary metabolites The Management of chronic pulmonary aspergillosis: the UK National Aspergillosis Centre approach Aspergillosis case-fatality rate: systematic review of the literature Invasive aspergillosis in patients admitted to the intensive care unit with severe influenza: a retrospective cohort study Confronting and mitigating the risk of COVID-19 Estimating the burden of invasive and serious fungal disease in the United Kingdom Azole antifungal resistance in Aspergillus fumigatus Clinical implications of azole resistance in Aspergillus fumigatus Clinical implications of globally emerging azole resistance in Aspergillus fumigatus Triazole fungicides can induce cross-resistance to medical triazoles in Aspergillus fumigatus European Centre for Disease Prevention and Control: risk assessment on the impact of environmental usage of triazoles on the development and spread of resistance to medical triazoles in Aspergillus species Government review of waste policy in England. Department for Environment, Food and Rural Affairs Food 2030, Department for Environment, Food and Rural Affairs Managing the potential public health risks from bioaerosol liberation at commercial composting sites in the UK: an analysis of the evidence base Endotoxin emissions from commercial composting activities Preliminary results of monitoring the release of bioaerosols from composting facilities in the UK: interpretation, modelling and appraisal of mitigation measures. In Occupational and environmental exposure to bioaerosols from composts and potential health effects-a critical review of published data. HSE Books, Norwich Generation and dispersion of airborne microorganisms from composting facilities Bioaerosol emissions from waste composting and the potential for workers' exposure. HSE Books Bioaerosol releases from compost facilities: evaluating passive and active source terms at a green waste facility for improved risk assessments Estimating fugitive bioaerosol releases from static compost windrows: feasibility of a portable wind tunnel approach Bioaerosols from composting facilities: a review Prevalence of workrelated rhino-conjunctivitis and respiratory symptoms among domestic waste collectors Health status and health-related quality of life of municipal waste collection workers: a cross-sectional survey Prevalence of and relationship between rhinoconjunctivitis and lower airway diseases in compost workers with current or former exposure to organic dust Symptoms, spirometry, and serum antibody concentrations among compost workers exposed to organic dust Work-related health symptoms among compost facility workers: a cross-sectional study Respiratory health of municipal solid waste workers Two year follow-up of a garbage collector with allergic bronchopulmonary aspergillosis (ABPA) Allergic bronchopulmonary aspergillosis in garden waste (compost) collectors-occupational implications A 5-year followup study on respiratory disorders and lung function in workers exposed to organic dust from composting plants Dynamics of Aspergillus fumigatus in azole fungicide-containing plant waste in the Netherlands Environmental hot spots for azole resistance selection of Aspergillus fumigatus, the Netherlands The economic impact of ornamental horticulture and landscaping in the UK: a report for the Ornamental Horticulture Round Table Group Hazard to man and the environment posed by the use of urban waste compost: a review Household waste prevention evidence review: a report for Defra's Waste and Resources Evidence Programme Hypersensitivity pneumonitis from residential composting: residential composter's lung Acute pulmonary aspergillosis in immunocompetent subjects after exposure to bark chippings Fatal acute granulomatous pulmonary aspergillosis in a healthy subject after inhalation of vegetal dust Gardening can induce pulmonary failure: Aspergillus ARDS in an immunocompetent patient, a case report Gardening can seriously damage your health Locally invasive pulmonary aspergillosis occurring in a gardener: an occupational hazard? Campaign-based citizen science for environmental mycology: the Science Solstice and Summer Soil-Stice projects to assess drug resistance in air-and soil-borne Aspergillus fumigatus Elevated prevalence of azole-resistant Aspergillus fumigatus in urban versus rural environments in the United Kingdom Determination of the prevalence of triazole resistance in environmental Aspergillus fumigatus strains isolated in South Wales Absence of azole antifungal resistance in Aspergillus fumigatus isolated from root vegetables harvested from UK arable and horticultural soils The Multi-fungicide resistance status of Aspergillus fumigatus populations in arable soils and the wider European environment Intercountry transfer of triazole-resistant Aspergillus fumigatus on plant bulbs Occurrence of azole-resistant species of Aspergillus in the UK environment Epidemiology and antifungal susceptibility profile of Aspergillus species: comparison between environmental and clinical isolates from patients with hematologic malignancies Isolation of azole-resistant Aspergillus fumigatus from imported plant bulbs in Japan and the effect of fungicide treatment Tracing patterns of evolution and acquisition of drug resistant Aspergillus fumigatus infection from the environment using population genomics Azole-resistant Aspergillus fumigatus harboring TR 34 /L98H, TR 46 / Y121F/T289A and TR 53 mutations related to flower fields in Colombia Multiple-azole-resistant Aspergillus fumigatus osteomyelitis in a patient with chronic granulomatous disease successfully treated with long-term oral posaconazole and surgery Emerging threat of triazole-resistant Aspergillus fumigatus Frequency and evolution of azole resistance in Aspergillus fumigatus associated with treatment failure Detecting azole-antifungal resistance in Aspergillus fumigatus by pyrosequencing Diagnostics and susceptibility testing in Aspergillus Exploring azole antifungal drug resistance in Aspergillus fumigatus with special reference to resistance mechanisms Molecular characterization and sensitivity to demethylation inhibitor fungicides of Aspergillus fumigatus from orange-based compost Abundance, genetic diversity and sensitivity to demethylation inhibitor fungicides of Aspergillus fumigatus isolates from organic substrates with special emphasis on compost First azole-resistant Aspergillus fumigatus isolates with the environmental TR46/Y121F/T289A mutation in Iran Itraconazole, voriconazole, and posaconazole CLSI MIC distributions for wild-type and azole-resistant Aspergillus fumigatus isolates Toxoplasmosis prevention and testing in pregnancy, survey of obstetrician-gynaecologists. Zoonoses Public Health Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time TaqMan PCR assay Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism 2020. D430G mutation of cyp51A in Aspergillus fumigatus causes azole-resistance HadUK-Grid datasets Land Cover Map QGIS: a free and open source geographic information system Waste sites Natural Resources Wales. 2021. Environmental permitting regulationswaste sites Waste licenses register R: a language and environment for statistical computing. R Foundation for Statistical Computing We thank all the citizen scientists who collected soil samples for this study. We also thank Pippa Douglas for providing the locations of composters in England with open windrow or outdoor activity and Jianhua Zhang for sharing the cyp51A coding region primers.