Citizen Science Surveillance of Triazole-Resistant Aspergillus fumigatus in United Kingdom Residential Garden Soils
ABSTRACT
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 TR34/L98H or TR46/Y121F/T289A, as well as the less common resistance mechanisms TR34, TR53, TR46/Y121F/T289A/S363P/I364V/G448S, and (TR46)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.
INTRODUCTION
The fungus Aspergillus fumigatus plays an important role in the environment as a decomposer, recycling nutrients from decaying plant matter into the soil. This highly sporulating mold is commonly found in woodchip piles and compost from household waste, sewage, sludge, and moldy hay (1), where its thermotolerance enables it to proliferate during the thermogenic phase of composting when temperatures reach 40 to 60°C (2). The small size of A. fumigatus spores (2 to 3 μm), and their hydrophobicity means they are easily aerosolized and transported on air currents, making A. fumigatus a globally ubiquitous fungus (3). Exposure to this mold is medically important, and it is estimated that humans inhale several hundred A. fumigatus spores per day (4), which can trigger an immunoinflammatory response resulting in severe asthma with fungal sensitization (SAFS) or allergic bronchopulmonary aspergillosis (ABPA) (5). The size of the spores allows them to bypass mucociliary clearance in the lung (6), whereupon they must then evade clearance by the host innate and adaptive immune responses (7). If they survive, germinated spores establish in lung cavities, where they can eventually cause chronic pulmonary aspergillosis (CPA). CPA affects apparently immunocompetent individuals with an existing lung condition, such as ABPA, chronic obstructive pulmonary disease, tuberculosis, or lung cancer, or an underlying immune dysfunction due to diabetes, rheumatoid arthritis, or alcoholism (8). If the host immune system is unable to prevent spores from entering the bloodstream, then invasive aspergillosis (IA) develops, which is a life-threatening infection associated with ∼58% survival (9). Individuals who are immunocompromised due to treatment with immunosuppressants, chemotherapy, or HIV/AIDS infection are at greatest risk of IA (9). Furthermore, individuals admitted to intensive care units with severe influenza infection are at risk of developing influenza-associated pulmonary aspergillosis, which is associated with increased mortality (10). A similar disease is now being observed for COVID-19-associated pulmonary aspergillosis in individuals with severe COVID-19 infection (11). It was estimated that in the UK in 2011 there were ∼178,000 individuals living with ABPA, 3,600 with CPA, and 2,900 with IA, plus an additional 377 to 1,345 cases of IA in critical care patients (12). The number of patients in the UK presenting with infections that are resistant to one or more of itraconazole (ICZ), voriconazole (VCZ), and posaconazole (PCZ)—the frontline triazole drugs for treating aspergillosis—increased from 3 to 7% between 1999 and 2001 to 14 to 20% between 2007 and 2009 (13). Triazole-resistant infections are associated with treatment failure, salvage therapy with more toxic antifungals and increased case fatality rates (CFRs), with CFRs up to 88% reported for triazole-resistant IA (14).
Triazole resistance is most commonly caused by polymorphisms in the cyp51A gene, which results in increased production of, or configurational changes in, lanosterol-14a-demethylase, an enzyme involved in ergosterol biosynthesis and the binding target of triazole drugs. An environmental route for the acquisition of triazole-resistant infections has been proposed due to the increase of infections caused by A. fumigatus isolates with a tandem repeat (TR) in the promoter region of cyp51A coupled with single-nucleotide polymorphisms in the coding region leading to amino acid substitutions in the protein, which are frequently recovered from air and soil samples globally (15). This is likely due to the use of the fungicides epoxiconazole, tebuconazole, propiconazole, difenoconazole, and bromuconazole, which have similar molecular structures to the medical triazoles and show cross-resistance (16). In 2008, these were the second, third, sixth, ninth, and seventeenth most sprayed triazoles in agriculture in the UK, respectively (17). In agriculture, triazoles are applied to wheat, beans, carrots, oilseed rape, soft fruits and vines; in horticulture, they are used to sterilize bulbs and to control fungal diseases in lawns and ornamentals; and in industry they are used as wood preservatives and antifouling agents in leather, paper, textiles, paints, and adhesives (17).
The UK government is committed to reducing carbon dioxide emissions by diverting waste from landfill and incineration to composting (18), and compost features in the government’s Food 2030 strategy for improving the productive capacity of soil (19). Compost producers accept input material from agriculture, horticulture, forestry, wood, and paper processing, leather and textile industries, and household and garden waste, which are highly likely to contain triazole residues. In 2007, 90% of composting facilities in the UK produced compost in open windrows (20); where organic waste is shredded, mixed, and 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 (20–27), with resulting negative health impacts on compost handlers (28–36), 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 (43–47) 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.
RESULTS
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.
FIG 1
TABLE 1
| Location in garden where the soil sample was collectedb | No. of soil samples | No. of samples that grew: | No. of A. fumigatus isolates grown | Avg no. of A. fumigatus isolates (CFU/g) | No. of tebuconazole-resistant A. fumigatus isolates grown (% A. fumigatus isolates) | Avg no. of tebuconazole-resistant A. fumigatus isolates (CFU/g) | |
|---|---|---|---|---|---|---|---|
| A. fumigatus (%) | Tebuconazole-resistant A. fumigatus (%) | ||||||
| B | 206 | 99 (48) | 19 (9) | 1,009 | 204 | 121 (12) | 127 |
| + CB | 7 | 4 (57) | 1 (14) | 56 | 280 | 8 (14) | 160 |
| + CH | 5 | 3 (60) | 0 (0) | 44 | 293 | 0 (0) | 0 |
| + MB | 1 | 0 (0) | 0 (0) | 0 | 0 | 0 (0) | 0 |
| + MB + CH | 1 | 1 (100) | 0 (0) | 1 | 20 | 0 (0) | 0 |
| CB | 49 | 44 (90) | 20 (41) | 993 | 451 | 137 (14) | 137 |
| CH | 80 | 58 (73) | 27 (34) | 1,464 | 505 | 289 (20) | 214 |
| MB | 1 | 1 (100) | 0 (0) | 30 | 600 | 0 (0) | 0 |
| PP | 115 | 79 (69) | 26 (23) | 1,005 | 254 | 130 (13) | 98 |
| + CB | 38 | 33 (87) | 8 (21) | 529 | 321 | 51 (15) | 128 |
| + CB + CH | 3 | 2 (67) | 0 (0) | 21 | 210 | 0 (0) | 0 |
| + CB + MB | 2 | 2 (100) | 0 (0) | 4 | 40 | 0 (0) | 0 |
| + CH | 1 | 1 (100) | 0 (0) | 18 | 360 | 0 (0) | 0 |
| Total | 509 | 327 (64) | 101 (20) | 5,174 | 316 | 736 (14) | 145 |
a
The table shows a breakdown of the number of soil samples collected, the number and percentage of soil samples that grew tebuconazole-susceptible and tebuconazole-resistant Aspergillus fumigatus, the numbers of tebuconazole-susceptible and tebuconazole-resistant A. fumigatus isolates grown, and the average CFU/g across samples that grew tebuconazole-susceptible and tebuconazole-resistant A. fumigatus by the location(s) in the garden where the soil sample was collected.
b
B, border; CB, compost bag; CH, compost heap, MB, manure bag; PP, pot/planter.
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, TR34/L98H was detected in 542 (85%), TR46/Y121F/T289A was detected in 16 (3%), TR53 was detected in 2, and (TR48)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 TR34 without accompanying amino acid substitutions in three isolates, (TR34)2/L98H in one isolate, and (TR130)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.
TABLE 2
| Tandem repeat in cyp51A promoter region | Amino acid substitution(s) in cyp51A | No. of samples based on the location where the garden soil sample was collecteda | Total | Medical triazole susceptibility (reference) | |||||
|---|---|---|---|---|---|---|---|---|---|
| B | B+CB | CB | CH | PP | PP+CB | ||||
| – | – | 3 | 1 | 23 | 27 | ||||
| – | C270R | 1 | 1 | ||||||
| – | I242V | 5 | 5 | 63 | |||||
| TR34 | – | 1 | 1 | 1 | 3 | 57 | |||
| TR34 | L98H | 82 | 7 | 117 | 237 | 65 | 34 | 542 | 71 |
| TR34 | L98H/Q191E | 1 | 1 | ||||||
| TR34 | L98H/R196L | 1 | 1 | ||||||
| TR34 | L98H/K240R | 1 | 1 | 2 | |||||
| TR34 | L98H/T289A/I364V/G448S | 6 | 6 | 55 | |||||
| TR34 | L98H/K372R | 1 | 1 | ||||||
| TR34 | L98H/P394R | 1 | 1 | ||||||
| TR34 | L98H/F404C/F459S/A460S | 1 | 1 | ||||||
| TR34 | L98H/F404V | 1 | 1 | ||||||
| TR34 | L98H/N406D | 1 | 1 | ||||||
| TR34 | L98H/N406M | 1 | 1 | ||||||
| TR34 | L98H/K421R | 1 | 1 | ||||||
| TR34 | L98H/P443L | 2 | 2 | ||||||
| TR34 | L98H/A460S | 1 | 1 | ||||||
| TR34 | L98H/D481N | 1 | 1 | 2 | |||||
| (TR34)2 | L98H | 1 | 1 | ||||||
| TR46 | Y121F/M178W/T289A/S363P/I364V/G448S | 1 | 1 | ||||||
| TR46 | Y121F/T289A | 15 | 1 | 16 | |||||
| TR46 | Y121F/T289A/S363P/I364V/G448S | 4 | 4 | 52 | |||||
| (TR46)2 | Y121F/M172I/T289A/G448S | 1 | 1 | 38 | |||||
| TR53 | – | 1 | 1 | 2 | 59 | ||||
| (TR130)3 | D430G | 4 | 4 | 72 | |||||
| Failed to sequence | Failed to sequenceb | 3 | 3 | 8 | 14 | ||||
| Total | 91 | 8 | 124 | 276 | 107 | 37 | 643 | ||
a
B, border; CB, compost bag; CH, compost heap, MB, manure bag; PP, pot/planter.
b
Samples that failed to amplify with the cyp51A promoter and coding region primers were sequenced using beta-tubulin primers for fungal identification.
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 (χ2 = 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.
TABLE 3
| Garden location sampled | OR (95% CI)b | Pr(>|z|) |
|---|---|---|
| Border (baseline) | ||
| + compost bag | 1.43 (0.31–7.40) | 0.64 |
| + compost heap | 1.61 (0.26–10.24) | 0.61 |
| + manure bag | – | 0.99 |
| + manure bag + compost heap | – | 0.99 |
| Compost bag | 15.70 (5.50–66.19) | <0.01 |
| Compost heap | 3.45 (1.93–6.40) | <0.01 |
| Manure bag | – | 0.99 |
| Pot/planter | 2.42 (1.50–3.95) | <0.01 |
| + compost bag | 7.07 (2.88–21.28) | <0.01 |
| + compost bag + compost heap | 2.14 (0.20–46.50) | 0.53 |
| + compost bag + manure bag | – | 0.98 |
| + compost heap | – | 0.99 |
a
Significant results (P ≤ 0.05) are highlighted in boldface.
b
OR, odds ratio. –, Insufficient data to calculate the odds ratio and confidence intervals (CI).
(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 (χ2 = 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 (χ2 = 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 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.
TABLE 4
| Garden location sampled | OR (95% CI)b | Pr(>|z|) |
|---|---|---|
| Border (baseline) | ||
| + compost bag | 1.64 (0.08–10.32) | 0.65 |
| + compost heap | – | 0.99 |
| + manure bag | – | 0.99 |
| + manure bag + compost heap | – | 0.99 |
| Compost bag | 6.79 (3.25–14.37) | <0.01 |
| Compost heap | 4.74 (2.45–9.32) | <0.01 |
| Manure bag | – | 0.99 |
| Pot/planter | 2.88 (1.52–5.53) | <0.01 |
| + compost bag | 3.05 (1.22–7.26) | <0.01 |
| + compost bag + compost heap | – | 0.99 |
| + compost bag + manure bag | – | 0.99 |
| + compost heap | – | 0.99 |
a
Significant results (P ≤ 0.05) are highlighted in boldface.
b
OR, odds ratio. –, Insufficient data to calculate the odds ratio and confidence intervals (CI).
(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.
DISCUSSION
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). However, the percentage of soils in this study that grew tebuconazole-resistant A. fumigatus isolates was greater than the 6% of soils in Sewell et al. that grew A. fumigatus with increased MICs to ICZ, VCZ, and/or PCZ (49). Of the 5,174 A. fumigatus isolates cultured in this study, 736 (14%) were resistant to tebuconazole, which is greater than the 6% prevalence of triazole-resistant A. fumigatus reported by Tsitsopoulou et al. from urban and rural soils in South Wales (50) and the absence of triazole-resistance detected by van der Torre et al. in isolates cultured from soils adhered to vegetables grown in the UK (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 TR34/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 TR34/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). TR68/L98H was detected in one isolate, which was found to be two repeats of the 34-bp insert, and in three isolates TR34 was detected without any accompanying amino acid substitutions, which was first detected in an environmental isolate collected from Scotland (57). TR46/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 TR53, 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 TR92/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 × 103 CFU/g at the start of the process rising to 605.7 × 103 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 × 103 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 >103 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) × 103 CFU/g in compost heaps in residential gardens, up to 2.3 × 105 CFU/g in flower bulb waste, and up to 8.4 × 104 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 (43–47). Although this study has not undertaken susceptibility testing for the tebuconazole-resistant A. fumigatus isolates against medical triazoles, the most commonly detected cyp51A polymorphisms TR34/L98H and TR46/Y121F/T289A are associated with elevated MICs to ICZ, PCZ, and VCZ (68). Furthermore, Hodiamont et al. reported a clinical isolate containing TR53 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.
MATERIALS AND METHODS
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-μL 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 μL 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 5′-GGACTGGCTGATCAAACTATGC-3′ and the reverse primer 5′-GTTCTGTTCGGTTCCAAAGCC-3′ 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 μL: 10 μL of FIREPol DNA polymerase (Solis Biodyne, Estonia), 10 μL of forward primer (1.5 μM; Invitrogen, USA), 10 μL of reverse primer (1.5 μM; Invitrogen, USA), 18 μL of nuclease-free water (Merck, Germany), and 2 μL 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 5′-ATGGTGCCGATGCTATGG-3′ and the reverse primer 5′-CTGTCTCACTTGGATGTG-3′ 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 μL: 0.2 μL of Q5 high-fidelity DNA polymerase (New England Biolabs, UK), 10 μL of Q5 reaction buffer (5×; New England Biolabs, UK), 0.5 μL of deoxynucleotide (dNTP) solution mix (40 μM; New England Biolabs, UK), 1 μL of forward primer (10 μM; Invitrogen, USA), 1 μL of reverse primer (10 μM; Invitrogen, USA), 35.3 μL of nuclease-free water (Merck, Germany), and 2 μL 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 5′-TACGTTGACATCATCAATCAG-3′ and 5′-GATTCACCGAACTTTCAAGGCTCG-3′. 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 5′-AATTGGTGCCGCTTTCTGG-3′ and the reverse primer 5′-AGTTGTCGGGACGGAATAG-3′ 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.
TABLE 5
| Environmental variables ascertained for sampling date and location | Source of information (references) |
|---|---|
| Garden location where soil sample was collected | Citizen scientist |
| Date the sample was collected | Citizen scientist |
| Maximum daily temp at sampling location on sampling date | Met Office HadUK-Grid dataset (73) |
| Land cover classification of sampling location | UKCEH Land Cover Map 2019 (74) |
| Urban or rural classification of sampling location | Calculated from land cover classification |
| Percent arable land in 2-km buffer surrounding sampling location | Calculated from UKCEH Land Cover Map 2019 using QGIS 3.16.4 (75) |
| Distance of sampling location to nearest composter with open windrow or outdoor activity | Composter locations were obtained from Environment Agency, Scottish Environment Agency (SEPA) website (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). |
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.
ACKNOWLEDGMENTS
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.
This study was supported by the Natural Environment Research Council (NERC; NE/L002515/1 and NE/P000916/1) and the UK Medical Research Council (MRC; MR/R015600/1). M.C.F. is a fellow in the CIFAR Fungal Kingdoms program. A.A. was supported by a postgraduate studentship from Al-Baha University, Saudi Arabia.
The authors have no competing interests to declare.
J.M.G.S., A.C.S., and M.C.F. conceptualized the study. A.A. and P.S.D. contributed experimental techniques. J.M.G.S. and R.C. processed samples. J.M.G.S. and C.B.U. analyzed the data. J.M.G.S. drafted the original manuscript, which C.B.U., A.C.S., and M.C.F. reviewed and edited.
Footnote
[This article was published on 22 February 2022 with a CC BY 4.0 copyright line (“Copyright © 2022 Shelton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.”). The authors elected to remove open access for the article after publication, necessitating replacement of the original copyright line, and this change was made on 10 March 2022.]
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Information & Contributors
Information
Published In
Applied and Environmental Microbiology
Volume 88 • Number 4 • 22 February 2022
eLocator: e02061-21
Editor: Irina S. Druzhinina, Nanjing Agricultural University
Copyright
© 2022 American Society for Microbiology. All Rights Reserved.
History
Received: 18 October 2021
Accepted: 20 December 2021
Accepted manuscript posted online: 5 January 2022
Published online: 22 February 2022
Keywords
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Irina S. Druzhinina
Editor
Nanjing Agricultural University
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