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. 2020 Nov 24;11(6):e02213-20.
doi: 10.1128/mBio.02213-20.

Effects of Agricultural Fungicide Use on Aspergillus fumigatus Abundance, Antifungal Susceptibility, and Population Structure

Amelia E Barber #  1   2 , Jennifer Riedel #  3 , Tongta Sae-Ong  4 , Kang Kang  4 , Werner Brabetz  5 , Gianni Panagiotou  4   6 , Holger B Deising  3 , Oliver Kurzai  1   2
Affiliations

Effects of Agricultural Fungicide Use on Aspergillus fumigatus Abundance, Antifungal Susceptibility, and Population Structure

Amelia E Barber et al. mBio. .

Abstract

Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is both an environmental saprobe and an opportunistic human fungal pathogen, resistance is suggested to arise from fungicide use in agriculture, as the azoles used for plant protection share the same molecular target as the frontline antifungals used clinically. However, limiting azole fungicide use on crop fields to preserve their activity for clinical use could threaten the global food supply via a reduction in yield. In this study, we clarify the link between azole fungicide use on crop fields and resistance in a prototypical human pathogen through systematic soil sampling on farms in Germany and surveying fields before and after fungicide application. We observed a reduction in the abundance of A. fumigatus on fields following fungicide treatment in 2017, a finding that was not observed on an organic control field with only natural plant protection agents applied. However, this finding was less pronounced during our 2018 sampling, indicating that the impact of fungicides on A. fumigatus population size is variable and influenced by additional factors. The overall resistance frequency among agricultural isolates is low, with only 1 to 3% of isolates from 2016 to 2018 displaying resistance to medical azoles. Isolates collected after the growing season and azole exposure show a subtle but consistent decrease in susceptibility to medical and agricultural azoles. Whole-genome sequencing indicates that, despite the alterations in antifungal susceptibility, fungicide application does not significantly affect the population structure and genetic diversity of A. fumigatus in fields. Given the low observed resistance rate among agricultural isolates as well the lack of genomic impact following azole application, we do not find evidence that azole use on crops is significantly driving resistance in A. fumigatus in this context.IMPORTANCE Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is an environmental fungus that also causes life-threatening infections in humans, antimicrobial resistance is suggested to arise from fungicide use in agriculture, as the chemicals used for plant protection are almost identical to the antifungals used clinically. However, removing azole fungicides from crop fields threatens the global food supply via a reduction in yield. In this study, we survey crop fields before and after fungicide application. We find a low overall azole resistance rate among agricultural isolates, as well as a lack of genomic and population impact following fungicide application, leading us to conclude azole use on crops does not significantly contribute to resistance in A. fumigatus.

Keywords: Aspergillus; Aspergillus fumigatus; antibiotic resistance; antifungal resistance; azole; fungicide; population genomics.

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Figures

FIG 1
FIG 1
Abundance of A. fumigatus in the soil of conventional and organic farms. (A) A. fumigatus (CFU/g) at various soil depths. n = 10 samples per depth. (B) Estimated fungicide treatment rates and areas in Germany. The fraction of each district that is theoretically treated with fungicides was calculated using land use and organic agriculture share data reported by the Statistical Office of Germany in December 2016. Districts where no data on land use were available are shaded gray. (C and D) Abundance of A. fumigatus in the spring as measured by number of CFU/g soil in the spring of 2017 (C) and 2018 (D). For 2017, boxplots represent n = 50 soil samples per field for farms A, B, C, E, F, H, and K and n = 25 for farms D, G, and L. For 2018, n = 50 soil samples per field for farms A, B, C, D, E, G, H, and K and n = 25 for farms F and L. (E) Comparison of the mean number of CFU/g soil on farms between 2017 and 2018.
FIG 2
FIG 2
Abundance of A. fumigatus in the soil of conventional farms before and after the vegetative period and fungicide application. (A and D) A. fumigatus (CFU/g) on conventional fields sampled in April, prior to the vegetative period and fungicide application (orange), and in July, after the vegetative period and 3 months of fungicide application, including azole fungicides (gray) in 2017 (A) and 2018 (D). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; NS, not significant; determined by Mann-Whitney U test. (A) Boxplots represent n = 50 samples per field and time point for farms A, B, C, E, and H and n = 25 samples for farms D and L. (D) n = 50 samples per field and time point for farms A, B, C, D, E, and H and n = 25 samples for farm L. (B and C) A. fumigatus (CFU/g) during the months of April, May, June, July, October, and November of a conventional field applying fungicides from May to July (C) and an organic field not applying nonnatural fungicides (B). Bars represent means ± standard errors of the means from 50 soil samples per month. No significant difference was found in abundance between the months of April, May, June, and July for the organic field using a Kruskal-Wallis test. In contrast, we found a significant difference (P = 0.004) for the abundances in this time period on the conventional field. P values from subsequent pairwise comparisons between months are indicated. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; determined by Wilcoxon signed rank.
FIG 3
FIG 3
Azole resistance among agricultural A. fumigatus. (A and B) Fraction of the isolates per field that grow at 1 mg/liter difenoconazole (A) and 2 mg/liter tebuconazole (B). For 2017, n = 340 isolates from 9 conventional and 8 organic fields, ≈20 isolates per field, were used. For 2018, n = 213 isolates from 8 conventional and 7 organic fields, ≈20 isolates per field, were used (full summary in Tables S2 and S3). (C to F) Comparison of the proportion of isolates that grow at 1 mg/liter difenoconazole (C and D) and 2 mg/liter tebuconazole (E and F) before and after the vegetative period and fungicide application. For 2017 (C and E), n = 275 isolates from 7 fields were used, and for 2018, n = 261 isolates from 8 fields were used (full summary in Tables S4 and S5). (Left) Overall summary of all isolates tested that year. (Right) Within-field changes (n ≈ 40 isolates per field; 20 before, 20 after). P values were calculated by Wilcoxon signed-rank test between before and after values. (G) Temporal changes in antifungal susceptibility of A. fumigatus on apple fields sampled before and after fungicide exposure over a 2-year period. Shown is the fraction of isolates that can grow at 1 mg/liter difenoconazole (top) and 2 mg/liter tebuconazole (bottom). n = 12 to 20 isolates/field and time point. (H) cyp51a genotypes of isolates resistant to one or more medical azole. (I) MICs of agricultural A. fumigatus isolated before and after azole exposure. n = 159 randomly selected isolates from 2017 and 2018; n = 80 before and n = 79 after. P values were calculated by Wilcoxon signed rank test between before and after values.
FIG 3
FIG 3
Azole resistance among agricultural A. fumigatus. (A and B) Fraction of the isolates per field that grow at 1 mg/liter difenoconazole (A) and 2 mg/liter tebuconazole (B). For 2017, n = 340 isolates from 9 conventional and 8 organic fields, ≈20 isolates per field, were used. For 2018, n = 213 isolates from 8 conventional and 7 organic fields, ≈20 isolates per field, were used (full summary in Tables S2 and S3). (C to F) Comparison of the proportion of isolates that grow at 1 mg/liter difenoconazole (C and D) and 2 mg/liter tebuconazole (E and F) before and after the vegetative period and fungicide application. For 2017 (C and E), n = 275 isolates from 7 fields were used, and for 2018, n = 261 isolates from 8 fields were used (full summary in Tables S4 and S5). (Left) Overall summary of all isolates tested that year. (Right) Within-field changes (n ≈ 40 isolates per field; 20 before, 20 after). P values were calculated by Wilcoxon signed-rank test between before and after values. (G) Temporal changes in antifungal susceptibility of A. fumigatus on apple fields sampled before and after fungicide exposure over a 2-year period. Shown is the fraction of isolates that can grow at 1 mg/liter difenoconazole (top) and 2 mg/liter tebuconazole (bottom). n = 12 to 20 isolates/field and time point. (H) cyp51a genotypes of isolates resistant to one or more medical azole. (I) MICs of agricultural A. fumigatus isolated before and after azole exposure. n = 159 randomly selected isolates from 2017 and 2018; n = 80 before and n = 79 after. P values were calculated by Wilcoxon signed rank test between before and after values.
FIG 4
FIG 4
Phylogeny of agricultural A. fumigatus isolates from before and after the vegetative period and azole application. From top to bottom, the colored bars indicate the farm where the isolate was collected, the collection period, voriconazole resistance (susceptible, intermediate, or resistant, according to EUCAST definitions), posaconazole resistance, itraconazole resistance, and the presence of the TR34/L98H allele in cyp51a. A. fischeri is indicated as an outgroup, and the two A. fumigatus reference strains, Af293 and A1163 (CEA10), are also marked. Branches with support values of less than 0.9 are marked in red.
FIG 5
FIG 5
Genetic diversity among isolates from before and after the vegetative period and azole exposure. (A to C) Nucleotide diversity (π) (A), nucleotide polymorphism (Watterson estimator, or θ) (B), and Tajima’s D (C) along 5-kb windows with a 500-bp step size before and after the vegetative period and azole exposure. n = 8 isolates per farm and time point, 64 in total.
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References

    1. Leading International Fungal Education . Invasive aspergillosis. http://www.life-worldwide.org/fungal-diseases/invasive-aspergillosis. Accessed 1 November 2019.
    1. Patterson TF, Thompson GR, Denning DW, Fishman JA, Hadley S, Herbrecht R, Kontoyiannis DP, Marr KA, Morrison VA, Nguyen MH, Segal BH, Steinbach WJ, Stevens DA, Walsh TJ, Wingard JR, Young J-AH, Bennett JE. 2016. Practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the Infectious Diseases Society of America. Clin Infect Dis 63:433–442. doi:10.1093/cid/ciw444. - DOI - PMC - PubMed
    1. Buil JB, Snelders E, Denardi LB, Melchers WJG, Verweij PE. 2019. Trends in azole resistance in Aspergillus fumigatus, the Netherlands, 1994–2016. Emerg Infect Dis 25:176–178. doi:10.3201/eid2501.171925. - DOI - PMC - PubMed
    1. Steinmann J, Hamprecht A, Vehreschild MJ, Cornely OA, Buchheidt D, Spiess B, Koldehoff M, Buer J, Meis JF, Rath PM. 2015. Emergence of azole-resistant invasive aspergillosis in HSCT recipients in Germany. J Antimicrob Chemother 70:1522–1526. doi:10.1093/jac/dku566. - DOI - PubMed
    1. Chowdhary A, Kathuria S, Xu J, Meis JF. 2013. Emergence of azole-resistant Aspergillus fumigatus strains due to agricultural azole use creates an increasing threat to human health. PLoS Pathog 9:3–7. doi:10.1371/annotation/4ffcf1da-b180-4149-834c-9c723c5dbf9b. - DOI - PMC - PubMed

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