DOI: 10.5061/dryad.zs7h44jfd
title: Azole resistance mechanisms and population structure of Aspergillus fumigatus on retail plant products

description: Aspergillus fumigatus is a ubiquitous saprotroph and human-pathogenic
 fungus that is life-threatening to the immunocompromised.
 Triazole-resistant A. fumigatus was found in patients without prior
 treatment with azoles, leading researchers to conclude that resistance had
 developed in agricultural environments where azoles are used against plant
 pathogens. Previous studies have documented azole-resistant A. fumigatus
 across agricultural environments, but few have looked at retail plant
 products. Our objectives were to determine if azole-resistant A. fumigatus
 is prevalent in retail plant products produced in the United States
 (U.S.), as well as to identify the resistance mechanism(s) and population
 genetic structure of these isolates. Five hundred twenty-five isolates
 were collected from retail plant products and screened for azole
 resistance. Twenty-four isolates collected from compost, soil, flower
 bulbs, and raw peanuts were pan-azole resistant. Resistant isolates had
 the TR34/L98H, TR46/Y121F/T289A, G448S, and H147Y cyp51A alleles, all
 known to underly pan-azole resistance, as well as  WT alleles,
 suggesting that non-cyp51A-mechanisms contribute to pan-azole resistance
 in some isolates. Minimum spanning networks showed two lineages containing
 isolates with TR alleles or the F46Y/M172V/E427K allele, and discriminant
 analysis of principle components (DAPC) identified three primary clusters.
 This is consistent with previous studies detecting three clades of A.
 fumigatus and identifying pan-azole-resistant isolates with TR alleles in
 a single clade. We found pan-azole resistance in U.S. retail plant
 products, particularly compost and flower bulbs, which indicates the risk
 of exposure to these products for susceptible populations and that highly
 resistant isolates are likely distributed worldwide on these products.
description: <strong>Collection and isolation of <em>A.
 fumigatus</em> from retail plant products</strong>
 Eight product types were collected from retail stores from
 September 2019 through April 2021 and surveyed for azole-resistant
 <em>A. fumigatus</em>. Grapes, apples, almonds, pecans,
 peanuts, compost, soil, and flower bulbs (tulip, daffodil,
 <em>Gladiolus</em>, daylily, <em>Dahlia</em>,
 <em>Canna</em>, <em>Liatris</em>,
 <em>Caladium</em>, lily of the valley,
 <em>Clematis</em>, <em>Iris ensata</em>, and
 magnum elephant ears) were collected from eight retail grocery stores and
 nine garden centers in the Athens, Georgia, area. The date of collection
 for fruits and nuts depended on their fresh market season in the U.S. Each
 product was collected by purchasing a pre-packaged bag. A wide variety of
 brands, product sizes, and retail stores were sampled. An effort was made
 to collect products that originated in the U.S; however, some packages of
 compost and flower bulbs originated from Canada, Costa Rica, and the
 Netherlands, respectively. Grapes were first sampled
 using amended sampling methods (39, 42) where parts of individual grapes
 including the skin and stems were swabbed or plated directly onto
 Sabouraud dextrose agar (SDA) amended with 50 µg/ml rose bengal dye, 50
 µg/ml chloramphenicol, and 5 µg/ml gentamicin (aSDA) (5). However, this
 resulted in low yields of <em>A. fumigatus </em>and prevented
 the entire sample from being tested efficiently, therefore, the following
 method was devised that was able to be used on grapes, peanuts, pecans,
 almonds, apples, and flower bulbs not packed in soil. The contents of the
 entire bag of the purchased product ranging from approximately 0.5 to 2 kg
 were placed into a sterile 30  38 cm polypropylene bag and
 rinsed thoroughly with 50 ml sterile 0.05% Tween-20 by massaging the bag
 to distribute the liquid over the plant products. All grapes contained
 stems and some peanuts and pecans were unshelled. All almonds were
 shelled. The liquid was collected into a 50 ml conical tube by cutting one
 corner of the bag. Almonds and pecans contained large quantities of broken
 shells, skin, and dust so the liquid was strained through sterile
 cheesecloth during collection. The tube was centrifuged at 3000
 <em>g</em> for 5 min (49) and the supernatant
 poured off. After observing that pellets from some products contained less
 <em>A. fumigatus</em> on the surface (grapes, pecans, and
 almonds), these samples were resuspended in 1 ml of 0.05% Tween-20, and
 100 µl aliquots of the entire solution were plated and spread onto 5-15
 Petri plates of aSDA so that we could obtain multiple isolates of
 <em>A. fumigatus</em>. Pellets from products observed with
 more abundant <em>A. fumigatus</em> (peanuts and flower bulbs
 not packed in soil), were resuspended in 2.5 ml 0.05% Tween-20 solution
 and distributed in 100 µl aliquots among 10 plates of aSDA, 10 plates of
 aSDA amended with 3 µg/ml itraconazole (ITC, Thermo Sci Acros Organics,
 New Jersey, USA), and 10 plates of aSDA amended with 3 µg/ml tebuconazole
 (TEB, TCI America, Oregon, USA). Flower bulbs packed in
 soil contained very abundant amounts of <em>A. fumigatus</em>
 so they were processed differently. Bulbs were removed from the soil and
 placed into a separate bag, shaking off as much soil as possible first.
 The bulbs were rinsed with 0.05% Tween-20 as described above. The liquid
 collected into a 50 ml conical tube was immediately vortexed at maximum
 speed, and 3 ml were transferred to another tube, avoiding any soil that
 may have been collected in the liquid. The liquid was not centrifuged,
 unlike with other samples, because the amount of <em>A. fumigatus
 </em>present was so great that the sample did not need to be
 concentrated. One and a half milliliters of the suspension were spread
 plated in 100 µl aliquots onto 5 plates of aSDA, 5 plates of aSDA amended
 with 3 µg/ml ITC, and 5 plates of aSDA amended with 3 µg/ml TEB. The
 remaining liquid in the tube was diluted 1:2 with fresh 0.05% Tween-20,
 and 1.5 ml in 100 µl aliquots was spread plated amongst 5 plates of aSDA,
 5 plates of aSDA amended with 3 µg/ml ITC, and 5 plates of aSDA amended
 with 3 µg/ml TEB. The additional dilution was plated in case the initial
 sample plating was not dilute enough to visualize individual colonies of
 <em>A. fumigatus.</em> Compost and soil
 were sampled using an amended sampling method (5, 32). Briefly, 2 or 4 g
 of the material was suspended in 0.1 M sodium pyrophosphate. Only 2 g of
 soil and compost with lower densities were collected in order to
 accommodate the size limit of the 50 mL conical tubes, otherwise, 4 g were
 collected. The suspensions were vortexed for 30 s and allowed to settle
 for 1 min. Two and a half milliliters of the supernatant was plated as
 described above for peanuts and flower bulbs not packed in soil.
 All agar plates were incubated at 45°C for 2 to 4 days.
 Individual colonies that were confirmed as <em>A. fumigatus
 </em>based on morphology were quadrant-streaked onto SDA to obtain a
 single-spore culture. All colonies were preliminarily screened for azole
 resistance by quadrant-streaking onto SDA with 3 µg/ml of TEB and SDA with
 3 µg/ml of ITC alongside known resistant and susceptible control isolates
 (5). Plates were incubated at 37°C for 2 days, after which resistance to
 TEB and ITC was preliminarily scored as resistant, intermediate, or
 susceptible based on a visual assessment of growth. Isolates scored as
 resistant had proficient growth in at least two quadrants of an aSDA
 plate; intermediate had spotty growth in one quadrant of an aSDA plate;
 and susceptible did not grow at all. Azole sensitivity was then quantified
 as described in the section below. For long-term storage, a single-spore
 colony was selected from SDA and streaked onto complete media (50) using a
 sterile cotton swab. For each isolate, conidia from complete media slants
 incubated for 2 days at 37°C were harvested, suspended in 15% glycerol in
 cryotubes, frozen in liquid nitrogen, and stored at -80°C.
 <strong>Azole-resistance phenotyping
 </strong> One-hundred-eleven of the isolates
 preliminarily scored as resistant and 19 of the isolates preliminarily
 scored as susceptible or intermediate in the screening were selected for
 azole-resistance phenotyping via minimum inhibitory concentration (MIC)
 assays to the fungicide TEB, and clinical antifungals ITC, voriconazole
 (VOR; Thermo Sci Acros Organics, New Jersey, USA), and posaconazole (POS;
 Apexbio Technology, Texas, USA). Isolates representing a variety of
 products and samples with elevated growth on azole-amended
 SDA<strong> </strong>were selected and assessed using the
 Clinical Laboratory Standard Institute broth microdilution method (51).
 Not all isolates were assayed at this stage since many had similar
 phenotypes from the same sample, such as the same bag of compost or flower
 bulbs. Some susceptible isolates were included for comparison. Briefly,
 conidia were harvested from 4-day-old complete media slants using 3 ml
 sterile 0.05% Tween-20. The spore suspensions were adjusted to 0.09 to
 0.13 OD at 530 nm using a spectrophotometer, and 20 µl of suspension was
 added to 11 ml RPMI 1640 liquid medium (Thermo Sci Gibco, California,
 USA). The solution was distributed in 100 µl aliquots among 96-wells in
 microtiter plates containing two-fold serial dilutions of antifungals with
 the final concentrations ranging from 0.015625 µg/ml to 16 µg/ml. The
 plates were incubated at 37°C for 48 hr. The MIC (minimum inhibitory
 concentration) of each isolate to each antifungal was determined visually
 by selecting the first well that had no fungal growth; the corresponding
 concentration of the antifungal in that well was the MIC. The accuracy of
 the MIC assays was checked using both susceptible and resistant
 <em>A. fumigatus</em> control isolates with known MIC values
 for TEB, ITC, VOR, and POS. The EUCAST breakpoints defined in February
 2020 (52) were used to classify isolates as sensitive (S) or resistant
 (R): ITC S ≤ 1 µg/ml &gt; R, VOR S ≤ 1 µg/ml &gt; R, and POS S ≤
 0.125 µg/ml and 0.25 µg/ml &gt; R. MIC values of 2 µg/ml for ITC and
 VOR and 0.25 µg/ml for POS are classified as areas of technical
 uncertainty (ATU) meaning that treatment with these antifungals may be
 used for isolates with this resistance breakpoint under certain
 situations, but for this study they were considered resistant (52). A
 breakpoint cutoff for TEB was defined as &gt; 2 µg/ml according to
 previous studies (5). Antifungal resistance phenotypes were further
 classified into four categories: azole-susceptible, TEB-resistant,
 pan-azole-resistant, and azole-resistant. Isolates with no azole
 resistance were classified azole-susceptible. Isolates with a
 TEB-resistant phenotype but no resistance to any medical azole were
 classified TEB-resistant. Isolates that were either resistant to only one
 medical azole or one medical azole and TEB were classified as
 azole-resistant. Finally, isolates with resistance to more than one
 medical azole were classified as pan-azole-resistant.
 <strong>DNA extraction</strong>
 Hyphae of 102 isolates, including 80 isolates screened by MIC
 assays and 22 susceptible isolates representing a variety of the sampled
 products, were grown from spores in liquid complete medium for 16 to 20 hr
 at 30°C in a 1 <em>g</em> orbital shaker (5).
 Tissue was gathered by filtering through a 40 µm cell strainer and
 squeezing the residual liquid from the tissue using a sterile cotton swab.
 Approximately 100 to 200 mg of tissue was collected and set aside in 2-ml
 tubes. DNA extractions were performed according to the QIAGEN DNeasy Plant
 Mini Kit protocol (QIAGEN, Maryland, USA), with a few amendments (53).
 Briefly, buffer AP1 was warmed to 65°C for at least 10 min prior to use.
 Four hundred microliters buffer AP1 and 4 µl RNase A were added to each
 tube with fungal tissue and vortexed for at least 2 min until all tissue
 was suspended. Each sample was incubated at 65°C for 10 min, vortexing for
 10 seconds three times throughout the incubation. One hundred thirty
 microliters of buffer P3 were added to each sample, vortexed, and then
 incubated at -20°C for 5 min. The samples were centrifuged for 5 min at
 18,407 <em>g</em> to pellet the remaining solids.
 The supernatant was pipetted into a QIAshredder mini spin column and
 centrifuged for 2 min at 18,407 <em>g</em>. Five
 hundred microliters of the flow-through fraction were transferred to a new
 sterile 2-ml lock-lid tube and 750 µl buffer AW1 (1.5 volume)
 was added to the flow-through and immediately mixed by pipetting. Six
 hundred fifty microliters of this solution were pipetted into a DNeasy
 mini spin column and centrifuged for 1 min at 18,407
 <em>g</em>. The flow-through was discarded, and the
 previous step was repeated with the remaining solution. The DNeasy spin
 column was transferred into a fresh 2 ml tube, 500 µl buffer AW2 was
 added, and the column was centrifuged for 1 min at 18,407
 <em>g</em>. The flow-through was discarded, and the
 previous step was repeated except the centrifugation lasted for 2 min
 rather than 1 min. The DNeasy spin column was transferred to a sterile 1.5
 ml tube, and 50 µl buffer AE was added to the membrane. The columns were
 incubated at room temperature for 5 min, then centrifuged at 18,407
 <em>g</em> for 1 min. This step was repeated once,
 and the DNA was stored at 4°C. The DNA concentration was quantified using
 NanoDrop One (Thermo Sci, New Jersey, USA).
 <strong><em>cyp51A</em>
 sequencing</strong> Seventy-nine isolates,
 including 37 sensitive and 42 resistant isolates based on MIC assays, were
 selected for Sanger sequencing of the promoter and coding regions of
 <em>cyp51A</em>. These isolates were selected in order to
 obtain representatives of isolates with varying phenotypes from all
 sampled plant-based retail products. The selection of isolates was based
 primarily on elevated MIC values, but some susceptible isolates were
 included as well for comparison. Isolates with low TEB MIC values and
 isolates with similar MIC values from the same sample were not always
 included. PCR was performed using a mix of 12.5 µl OneTaq 2
 Master Mix, 6.5 µl RNA-free sterile ddH2O, 2 µl each of previously
 designed forward primer 5´-CGGGCTGGAGATACTATGGCT-3´ and reverse primer
 5´-GTATAATACACCTATTCCGATCACACC-3´ (5, 54). PCR cycling conditions were as
 follows: 98°C for 2 min followed by 30 cycles of 98°C for 15 sec, 62°C for
 15 sec, and 72°C for 2.5 min, followed by a final extension at 72°C for 5
 min (5). Sanger sequencing was performed by Genewiz (Genewiz by Azenta
 Life Sciences, Massachusetts, USA) using 4 primers:
 5´-GCATTCTGAAACACGTGCGTAG-3´, 5´-GTCTCCTCGAAATGGTGCCG-3´,
 5´-CGTTCCAAACTCACGGCTGA-3´, and 5´-GCGACGAACACTTCCCCAAT-3´ (5). Sequence
 alignment was performed using Geneious v2019.2 (Biomatters, Auckland, NZ).
 Briefly, all sequences were trimmed to remove low-quality base pairs with
 a base quality score &lt;20 from the beginning and end of each
 sequence. The sequences were aligned for each isolate and the consensus
 sequence was visually assessed. The promoter regions were aligned and
 compared with A1163 genomic sequence v43 from Ensembl (55). The coding
 sequences were translated and aligned to the <em>A.
 fumigatus</em> A1163 Cyp51A protein (GenBank accession
 EDP50065). <strong>STR<em>Af</em>
 genotyping</strong> Single tandem repeats of
 <em>A. fumigatus</em> (STR<em>Af</em>) are
 microsatellite markers commonly used to assess genetic diversity and
 population genetic structure among isolates from different environmental
 origins, such as clinical or agricultural environments (56-59). Nine
 previously-developed STR<em>Af</em> markers
 (STR<em>Af</em>2A, 2B, 2C, 3A, 3B, 3C, 4A, 4B, and 4C) were
 used to genotype 95 isolates collected in this study: 72 of which we had
 collected <em>cyp51A </em>sequence data and 23 others that
 were included to investigate isolates from all of the sampled plant-based
 retail products (57). Multiplex PCR was performed using a modified
 protocol for the Type-it Microsatellite PCR kit (Qiagen). Briefly, each of
 the three multiplex reactions (3 loci per multiplex) contained 5 µl
 2 Type-it Master Mix, 1 µl 10 primer mix (2 µM of
 each of the 6 multiplex primers), 1 µl DNA template, and RNAse-free water.
 Thermal cycling conditions were as follows: 95°C for 5 min followed by 28
 cycles of 95°C for 30 sec, 57°C for 90 sec, 72°C for 30 sec, and a final
 elongation of 60°C for 30 min. Amplification of several PCR products from
 each multiplex was confirmed by electrophoresis on a 1% agarose gel with
 1× TBE buffer. The PCR products were diluted 1:15 and then sent to the
 Cornell Institute of Bioinformatics (Ithaca, New York, USA) for addition
 of the internal size standard Genescan-500 Liz and HiDi-formamide,
 followed by fragment analysis on an Applied Biosystems 3730x1 96-capillary
 DNA analyzer. The data were analyzed using the Microsatellite plugin in
 Geneious v.6 (Biomatters, Auckland, NZ) to identify the nine loci and the
 amplicon length (or allele) in each sample.
 <strong>Population genetic
 analyses</strong> Multilocus genotypes used in
 the analyses were based on the STR<em>Af</em> data for the 95
 isolates from this study and 80 clinical and environmental isolates from
 the U.S. from a previous study (5). There were 28 isolates from clinical
 settings and environmental isolates came from agricultural compost (4) and
 soil with plant debris where apple (2), watermelon (7), strawberry (4),
 pecan (13), peanut (16), and grape (5) were growing. The laboratory
 reference used in the previous study (5), Af293, was included as a control
 for comparison. Isolates from a variety of substrates were chosen to
 obtain a representative sample, but substrates associated with the retail
 products sampled in this study were prioritized (e.g., grape, apple,
 peanut soil, and debris). To estimate the genetic
 relatedness among isolates, minimum spanning networks using Bruvo’s
 genetic distance model (60) and Nei’s 1978 distance (61, 62) were
 constructed with the Poppr package in R (63). Bruvo’s genetic distance is
 often useful for analyses based on microsatellite markers (60) and assumes
 a stepwise mutation model that may not be entirely accurate for an
 organism like <em>A. fumigatus </em>which is genotypically
 diverse and known to sexually reproduce (64). Moreover, each
 STR<em>Af</em> locus contains 11 to 37 alleles that vary in
 repeat number, so they may not be evolving in a stepwise manner (57).
 Therefore, we used Nei’s genetic distance, as well, to incorporate an
 infinite alleles model. Population genetic structure was analyzed using
 discriminate analysis of principal components (DAPC) in R (65) to identify
 if populations clustered based on environmental setting, substrate of
 origin, <em>cyp51A </em>genotype, geographic sampling
 location, or another factor. Clusters were determined using K-means
 clustering of principal components.
description: # Azole resistance mechanisms and population structure of Aspergillus
 fumigatus on retail plant products This README file was generated on
 2024-04-10 by Caroline Wang. GENERAL INFORMATION 1. Title of dataset:
 Azole resistance mechanisms and population structure of Aspergillus
 fumigatus on retail plant products 2. Author Information 1. Principal
 Investigator: Marin Brewer; University of Georgia Dept. Plant Pathology;
 GA USA; [mtbrewer@uga.edu](mailto:marin.brewer@uga.e) 2. First author:
 Caroline Wang; University of Georgia Dept. Plant Pathology; GA, USA;
 [cburks817@gmail.com](mailto:cburks817@gmail.c) 3. Date of data collection
 (single date, range, approximate date): 2019-2022 4. Geographic location
 of data collection: Georgia, USA 5. Information about funding sources that
 supported the collection of the data: National Institute of Food and
 Agriculture 2019-67017-29113 SHARING/ACCESS INFORMATION 1.
 Licenses/restrictions placed on the data: None 2. Links to publications
 that cite or use the data: 1. Kang SE, Sumabat LG, Melie T, Mangum B,
 Momany M, Brewer MT. 2022. Evidence for the agricultural origin of
 resistance to multiple antimicrobials in *Aspergillus fumigatus*, a fungal
 pathogen of humans. G3 (Bethesda)
 12. [https://doi.org/10.1093/g3journal/jkab427](https://doi.org/10.1093/g3journal/jkab427) 2. Wang C, Miller N, Vines D, Severns P, Momany M, Brewer M. 2024. Azole resistance mechanisms and population structure of the human pathogen *Aspergillus fumigatus* on retail plant products. Accepted to AEM. 3. Links to other publicly accessible locations of the data: None 4. Links/relationships to ancillary data sets: None 5. Was data derived from another source? Yes, agriculture and clinical isolates from Microsatellite data obtained from: Kang SE, Sumabat LG, Melie T, Mangum B, Momany M, Brewer MT. 2022. Evidence for the agricultural origin of resistance to multiple antimicrobials in *Aspergillus fumigatus*, a fungal pathogen of humans. G3 (Bethesda) 12. [https://doi.org/10.1093/g3journal/jkab427](https://doi.org/10.1093/g3journal/jkab427) 6. Recommended citation for this dataset: Wang C, Miller N, Vines D, Severns P, Momany M, Brewer M. 2024. Data from: Azole resistance mechanisms and population structure of the human pathogen *Aspergillus fumigatus* on retail plant products. Dryad Digital Repository. doi: 10.5061/dryad.zs7h44jfd DATA & FILE OVERVIEW 1. File List: 1. Sample_data.xlsx 2. MIC_and_genotype_data.xlsx 3. Microsatellite_data.xlsx 2. Relationship between files, if important: None 3. Additional related data collected that was not included in the current data package: None 4. Are there multiple versions of the dataset? Yes, each file has been updated multiple times to fix formatting issues \######################################################################### DATA-SPECIFIC INFORMATION FOR: **Sample_data.xlsx** 1. Number of variables: 14 per page 2. Number of cases/rows: 16-21 per page, Each page corresponds to a different type of product 3. Variable List: 1. Site: Label given to each sample purchased: ex. A19-G1. A(Aspergillus fumigatus) 19(year of project start: 2019) - G(Letter identifier for each type of sample ex. G for grapes) 1(# sample in series) 2. Store: Store where sample was purchased coded for privacy 3. Brand: Brand of item purchased coded for privacy 4. Variety: Additional info about sample purchased ex. color, plant variety, soil makeup, shelled, salted, etc. Varies for each product type. 5. Size: Size of product if listed on bag/product itself 6. Origin: Location of product origin if listed on bag/product itself 7. Treatment: Any treatments provided to product if listed on bag/product itself 8. Collected: Date item was purchased  9. Processed: Date item was screened for *Aspergillus fumigatus* 10. Plates: Number of plates used when screening, additional information about types of plates used may be provided for each product 11. Method: Method used to screen (Briefly): 1. Dunking: washing product with tween-20 2. Peeling: peeling skin off of product and plating directly 3. Swabbing: clean swabs rubbed outside of product and plated 4. Compost/soil: number of grams screened by suspending in sodium pyrophosphate and plating 5. Bulb: washed via dunking method but diluted before plating 6. Bulb+Soil: bulbs removed from soil, dunked, and diluted 12. CFU (A fum): number of colonies of presumed *A. fumigatus* on aSDA (Sabouraud dextrose agar (SDA) amended with 50 µg/ml rose bengal dye, 50 µg/ml chloramphenicol, and 5 µg/ml gentamicin) 13. Teb CFU: number of colonies of presumed *A. fumigatus* on aSDA+3µg/ml tebuconazole plates if screened 14. Itra CFU: number of colonies of presumed *A. fumigatus* on aSDA+3µg/ml itraconazole plates if screened 4. Missing data codes:  1. Unknown: information not listed on the product/bag so not recorded 2. Not recorded: information was not recorded 3. Not screened: products with lower quantities of *A. fumigatus* were not screened using Teb and Itra plates 5. Specialized formats or other abbreviations used: 1. Each page corresponds to a different product type 2. A fum: *Aspergillus fumigatus* 3. TMTC: Too many colonies to count 4. Teb or T: tebuconazole 5. Itra or I: itraconazole 6. NA: aSDA \######################################################################### DATA-SPECIFIC INFORMATION FOR: **MIC_and_genotype_data.xlsx** 1. Number of variables: 19 2. Number of cases/rows: 526 3. Variable List (some repeated for each rep): 1. Isolate ID: Number given to each isolate according to order collected 2. Isolate name: Unique identifier given to each isolate according to naming convention of each sample described above 3. MIC Rep 1: Values with this in the header correspond to the first minimum inhibitory concentration assay  4. MIC Rep 2: Values with this in the header correspond to the second minimum inhibitory concentration assay 5. Analysis: Values with this in the header were used for the classification of isolates as resistant or susceptible 6. Date: Date data were collected 7. TEB: Minimum concentration of tebuconazole needed to inhibit growth 8. ITR: Minimum concentration of itraconazole needed to inhibit growth 9. VOR: Minimum concentration of voriconazole needed to inhibit growth 10. POS: Minimum concentration of voriconazole needed to inhibit growth 11. MIC phenotype:  1. Sensitive: susceptible to all azoles tested 2. Teb: resistant to tebuconazole only 3. Med: resistant to one medical azole and possibly tebuconazole 4. Pan: resistant to more than one medical azole and possibly tebuconazole 12. *cyp51A* genotype:  1. WT: matches A1163 *cyp51A* sequence 2. T1: F46Y/M172V/E427K 3. T2: I242V 4. T3: F46Y/M172V/N248T/D255E/E427K 5. TR34: TR34/L98H 6. TR46: TR46/Y121F/T289A 7. Other: Genotype unique to just one isolate 13. Full genotype: cyp51A genotype fully listed 4. Missing data codes:  1. Isolates not screened: - 5. Specialized formats or other abbreviations used: 1. MIC: minimum inhibitory concentration assay 2. All others described above \######################################################################### DATA-SPECIFIC INFORMATION FOR: **Microsatellite_data.xlsx** 1. Number of variables: 11 2. Number of cases/rows: 176 3. Variable List: 1. Isolate ID: Number given to each isolate according to order collected 2. Product/Substrate of origin: 1. Retail: isolates from this study 2. Agriculture: isolates collected from agricultural environments, obtained from [https://doi.org/10.1093/g3journal/jkab427](https://doi.org/10.1093/g3journal/jkab427) 3. Clinical: isolates collected from the clinic, obtained from [https://doi.org/10.1093/g3journal/jkab427](https://doi.org/10.1093/g3journal/jkab427) 3. Microsatellite alleles: 9 allele markers and number codes associated with each marker 4. Missing data codes: No data collected: 0 5. Specialized formats or other abbreviations used: 1. Microsatellite allele locations: 2A, 2B, 2C, 3A, 3B, 3C, 4A, 4B, 4C