Azole resistance mechanisms and population structure of Aspergillus fumigatus on retail plant products

Collection and isolation of A. fumigatus from retail plant products Eight product types were collected from retail stores from September 2019 through April 2021 and surveyed for azole-resistant A. fumigatus. Grapes, apples, almonds, pecans, peanuts, compost, soil, and flower bulbs (tulip, daffodil, Gladiolus, daylily, Dahlia, Canna, Liatris, Caladium, lily of the valley, Clematis, Iris ensata, 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 A. fumigatus 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 g for 5 min (49) and the supernatant poured off. After observing that pellets from some products contained less A. fumigatus 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 A. fumigatus. Pellets from products observed with more abundant A. fumigatus (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 A. fumigatus 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 A. fumigatus 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 A. fumigatus. 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 A. fumigatus 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. Azole-resistance phenotyping 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 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 A. fumigatus 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 > R, VOR S ≤ 1 µg/ml > R, and POS S ≤ 0.125 µg/ml and 0.25 µg/ml > 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 > 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. DNA extraction 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 g 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 g to pellet the remaining solids. The supernatant was pipetted into a QIAshredder mini spin column and centrifuged for 2 min at 18,407 g. 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 g. 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 g. 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 g 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). cyp51A sequencing 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 cyp51A. 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 <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 A. fumigatus A1163 Cyp51A protein (GenBank accession EDP50065). STRAf genotyping Single tandem repeats of A. fumigatus (STRAf) 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 STRAf markers (STRAf2A, 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 cyp51A 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. Population genetic analyses Multilocus genotypes used in the analyses were based on the STRAf 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 A. fumigatus which is genotypically diverse and known to sexually reproduce (64). Moreover, each STRAf 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, cyp51A genotype, geographic sampling location, or another factor. Clusters were determined using K-means clustering of principal components.

零售植物源产品中烟曲霉（Aspergillus fumigatus，下文简称A. fumigatus）的采集与分离
2019年9月至2021年4月期间，从零售门店采集8类产品，针对耐唑类A. fumigatus开展调查。本次研究从佐治亚州雅典地区的8家零售杂货店与9家园艺中心采集了葡萄、苹果、扁桃仁、碧根果、花生、堆肥、土壤以及球根花卉（郁金香、黄水仙、唐菖蒲、萱草、大丽花、美人蕉、蛇鞭菊、彩叶芋、铃兰、铁线莲、花菖蒲及巨型象耳芋）。果蔬与坚果的采集日期需契合美国当地的鲜食上市季。所有产品均通过采购预包装袋装样品完成采集，涵盖了多样化的品牌、产品规格及零售门店。研究人员尽量采集原产于美国的产品，但部分堆肥与球根花卉样品分别来自加拿大、哥斯达黎加与荷兰。
研究初期采用改良采样方法（参考文献39、42）对葡萄进行处理：擦拭或直接涂布包含果皮与果梗在内的葡萄组织至添加了50 μg/ml孟加拉红染料、50 μg/ml氯霉素与5 μg/ml庆大霉素的沙氏葡萄糖琼脂（Sabouraud dextrose agar，SDA，下文简称aSDA）培养基（参考文献5）。但该方法的A. fumigatus分离率较低，无法高效完成全部样品检测，因此研究人员开发了适用于非土壤包裹型葡萄、花生、碧根果、扁桃仁、苹果以及非土壤包裹球根花卉的新采样方案：将整袋采购的样品（重量约0.5~2 kg）转移至无菌30×38 cm聚丙烯袋中，加入50 ml无菌0.05%吐温-20（Tween-20），通过揉搓袋体使液体均匀覆盖植物源产品。所有葡萄均带有果梗，部分花生与碧根果未脱壳，扁桃仁均为带壳样品。通过剪下角袋一角将冲洗液收集至50 ml离心管中：由于扁桃仁与碧根果含有大量破碎果壳、种皮与碎屑，收集过程中需通过无菌脱脂纱布过滤冲洗液。将离心管以3000×g离心5分钟（参考文献49），弃去上清液。
前期观察发现，部分样品（葡萄、碧根果与扁桃仁）的表面沉淀中A. fumigatus含量较低，因此将其沉淀重悬于1 ml 0.05% Tween-20中，取全部溶液的100 μl等分试样涂布于5~15个aSDA培养皿上，以获取多株A. fumigatus分离物。对于A. fumigatus含量较高的样品（未脱壳花生与非土壤包裹球根花卉），将沉淀重悬于2.5 ml 0.05% Tween-20溶液中，取100 μl等分试样分别涂布于10个aSDA培养皿、10个添加了3 μg/ml伊曲康唑（itraconazole，ITC，赛默飞世尔科技奥德里奇有机试剂部，美国新泽西州）的aSDA培养皿，以及10个添加了3 μg/ml戊唑醇（tebuconazole，TEB，东京化成工业美国分部，美国俄勒冈州）的aSDA培养皿。
带土包裹的球根花卉中A. fumigatus含量极高，因此采用差异化处理流程：先将球根从土壤中取出并转移至单独袋中，尽量抖落附着土壤后，用0.05% Tween-20冲洗，收集冲洗液至50 ml离心管中，随后全速涡旋混匀，取3 ml上清液转移至新管，避免混入收集到的土壤。与其他样品不同，该冲洗液无需离心，因A. fumigatus载量极高，无需浓缩处理。取1.5 ml悬浮液，以100 μl等分试样涂布于5个aSDA培养皿、5个添加3 μg/ml ITC的aSDA培养皿以及5个添加3 μg/ml TEB的aSDA培养皿。将管中剩余液体用新鲜0.05% Tween-20以1:2比例稀释，取1.5 ml以100 μl等分试样涂布于5个aSDA培养皿、5个添加3 μg/ml ITC的aSDA培养皿以及5个添加3 μg/ml TEB的aSDA培养皿，此举用于应对初始涂布浓度过高导致无法清晰观察单个A. fumigatus菌落的情况。
堆肥与土壤样品采用改良采样方法（参考文献5、32）处理：取2 g或4 g样品悬浮于0.1 M焦磷酸钠溶液中。为适配50 ml离心管的体积限制，低密度的土壤与堆肥样品仅取2 g，其余样品取4 g。将悬浮液涡旋混匀30秒后静置1分钟，取2.5 ml上清液，按照未脱壳花生与非土壤包裹球根花卉的流程进行涂布。
所有琼脂平板均置于45℃培养2~4天。根据菌落形态确认的A. fumigatus单菌落，通过四区划线法接种至SDA培养基以获得单孢子培养物。所有菌落均通过四区划线法分别接种至添加3 μg/ml TEB的SDA培养基与添加3 μg/ml ITC的SDA培养基，同时设置已知耐药与敏感的对照分离物（参考文献5），完成耐唑类菌株的初筛。平板置于37℃培养2天后，通过肉眼观察生长情况，将菌株初步划分为耐药、中介或敏感三类：耐药菌株在aSDA平板至少两个象限中可良好生长；中介菌株仅在一个象限中出现斑点状生长；敏感菌株无任何生长。随后按照下文所述方法定量检测唑类药物敏感性。
对于长期保存，从SDA培养基上挑取单孢子菌落，用无菌棉拭子接种至完全培养基（参考文献50）。将37℃培养2天的完全培养基斜面上的分生孢子收获至15%甘油溶液中，分装至冻存管，经液氮速冻后于-80℃保存。

耐唑类耐药表型分析
选取初筛判定为耐药的111株分离物，以及初筛判定为敏感或中介的19株分离物，针对杀菌剂TEB以及临床抗真菌药物ITC、伏立康唑（voriconazole，VOR，赛默飞世尔科技奥德里奇有机试剂部，美国新泽西州）、泊沙康唑（posaconazole，POS，艾美捷科技，美国得克萨斯州）开展最低抑菌浓度（minimum inhibitory concentration，MIC）检测，以明确其耐唑类耐药表型。选取覆盖各类采样产品、在唑类修饰aSDA上生长量较高的分离物，采用美国临床实验室标准化协会肉汤微量稀释法（参考文献51）进行检测。由于同一样品（如同一袋堆肥或球根花卉）的多数分离体表型一致，因此并非所有分离物均进行该阶段检测，同时纳入部分敏感菌株作为对照。
具体流程如下：从培养4天的完全培养基斜面上用3 ml无菌0.05% Tween-20收获分生孢子，使用分光光度计将孢子悬液调整至530 nm波长下吸光度为0.09~0.13，取20 μl悬液加入11 ml RPMI 1640液体培养基（赛默飞世尔科技Gibco分部，美国加利福尼亚州）。将该溶液以100 μl等分试样加入96孔微孔板，微孔板中已预先加入两倍系列稀释的抗真菌药物，最终药物浓度范围为0.015625 μg/ml至16 μg/ml。将微孔板置于37℃培养48小时。通过肉眼观察确定每株分离物对每种抗真菌药物的MIC：选取完全无真菌生长的第一孔，该孔对应的药物浓度即为MIC。使用已知TEB、ITC、VOR与POS MIC值的敏感与耐药A. fumigatus对照菌株验证MIC检测的准确性。采用2020年2月公布的欧洲临床微生物和感染病学会（EUCAST）药敏折点标准（参考文献52）将菌株划分为敏感（S）或耐药（R）：ITC S≤1 μg/ml，>1 μg/ml为R；VOR S≤1 μg/ml，>1 μg/ml为R；POS S≤0.125 μg/ml，>0.25 μg/ml为R。ITC与VOR的MIC值为2 μg/ml、POS的MIC值为0.25 μg/ml时，被归类为技术不确定区间（ATU），即此类菌株在特定情况下可采用对应抗真菌药物治疗，但本研究中将其划分为耐药菌株（参考文献52）。参考既往研究（参考文献5），将TEB的耐药折点定义为>2 μg/ml。
将抗真菌耐药表型进一步划分为四类：唑类敏感、TEB耐药、泛唑类耐药以及唑类耐药。无任何唑类耐药性的菌株划分为唑类敏感；仅对TEB耐药、对临床用唑类药物无耐药性的菌株划分为TEB耐药；仅对一种临床用唑类药物耐药，或对一种临床用唑类药物及TEB耐药的菌株划分为唑类耐药；对多种临床用唑类药物耐药的菌株划分为泛唑类耐药。

DNA提取
选取102株分离物进行DNA提取，其中包括80株经MIC检测的菌株以及22株覆盖各类采样产品的敏感菌株。将孢子接种至液体完全培养基中，于30℃、1 g轨道摇床中培养16~20小时以获取菌丝体（参考文献5）。通过40 μm细胞筛过滤收集菌丝体，并用无菌棉拭子挤干残留液体。取约100~200 mg菌丝体置于2 ml离心管中。按照凯杰DNeasy植物迷你试剂盒（QIAGEN，美国马里兰州）的操作流程进行DNA提取，仅做少量调整（参考文献53）：
将AP1缓冲液预热至65℃至少10分钟后使用。向每管菌丝体样品中加入400 μl AP1缓冲液与4 μl核糖核酸酶A（RNase A），涡旋混匀至少2分钟至菌丝体完全悬浮。将样品置于65℃孵育10分钟，孵育期间每10秒涡旋一次，共三次。向每管样品中加入130 μl P3缓冲液，涡旋混匀后置于-20℃孵育5分钟。将样品以18407×g离心5分钟以沉淀固体杂质。将上清液转移至QIAshredder微型离心柱中，以18407×g离心2分钟。取500 μl流穿液至新的无菌2 ml锁盖管中，加入750 μl AW1缓冲液（1.5倍体积），立即通过移液枪混匀。取650 μl该混合液加入DNeasy微型离心柱中，以18407×g离心1分钟，弃去流穿液，剩余混合液重复该步骤。将DNeasy离心柱转移至新的2 ml离心管中，加入500 μl AW2缓冲液，以18407×g离心1分钟，弃去流穿液，重复该步骤但将离心时间延长至2分钟。将DNeasy离心柱转移至无菌1.5 ml离心管中，向膜上加入50 μl AE缓冲液，室温孵育5分钟后以18407×g离心1分钟，重复该步骤一次，最终DNA于4℃保存。使用NanoDrop One超微量分光光度计（赛默飞世尔科技，美国新泽西州）定量DNA浓度。

cyp51A基因测序
选取79株分离物进行cyp51A启动子区与编码区的桑格测序（Sanger sequencing），其中包括37株MIC检测为敏感的菌株与42株MIC检测为耐药的菌株。本次选株旨在覆盖所有采样植物源产品中具有不同表型的分离物，选株标准主要为MIC值升高的菌株，同时纳入部分敏感菌株作为对照。部分TEB MIC值较低的菌株以及同一样本中MIC值相近的菌株未被纳入测序。
PCR反应体系配置如下：12.5 μl OneTaq 2预混PCR体系、6.5 μl无RNA酶无菌双蒸水、2 μl上游引物（5´-CGGGCTGGAGATACTATGGCT-3´）与2 μl下游引物（5´-GTATAATACACCTATTCCGATCACACC-3´，参考文献5、54）。PCR循环参数为：98℃预变性2分钟，随后30个循环：98℃变性15秒、62℃退火15秒、72℃延伸2.5分钟，最后72℃终延伸5分钟（参考文献5）。
由Genewiz（现隶属于Azenta生命科学，美国马萨诸塞州）完成桑格测序，使用4条引物：5´-GCATTCTGAAACACGTGCGTAG-3´、5´-GTCTCCTCGAAATGGTGCCG-3´、5´-CGTTCCAAACTCACGGCTGA-3´与5´-GCGACGAACACTTCCCCAAT-3´（参考文献5）。使用Geneious v2019.2软件（Biomatters，新西兰奥克兰）完成序列比对：首先对所有序列进行修剪，去除首尾端碱基质量得分<20的低质量碱基；随后对每株分离物的序列进行比对，目视评估共识序列。将启动子区序列与Ensembl数据库中A. fumigatus A1163基因组版本v43进行比对；将编码区序列翻译为蛋白序列，与A. fumigatus A1163 Cyp51A蛋白（GenBank登录号EDP50065）进行比对。

烟曲霉单串联重复序列（Single tandem repeats of A. fumigatus，STRAf）基因分型
烟曲霉单串联重复序列（STRAf）是一类微卫星标记，常用于评估不同环境来源（如临床或农业环境）分离物的遗传多样性与群体遗传结构（参考文献56~59）。本次研究使用9个已开发的STRAf标记（STRAf2A、2B、2C、3A、3B、3C、4A、4B及4C）对95株分离物进行基因分型：其中72株已完成cyp51A测序，另外23株用于覆盖所有采样的植物源零售产品（参考文献57）。
采用改良的Type-it微卫星PCR试剂盒（凯杰）流程进行多重PCR：每个多重PCR反应（每反应覆盖3个位点）包含5 μl 2×Type-it预混体系、1 μl 10×引物混合液（每条引物终浓度2 μM，共6条多重PCR引物）、1 μl DNA模板与无RNA酶水。热循环参数为：95℃预变性5分钟，随后28个循环：95℃变性30秒、57℃退火90秒、72℃延伸30秒，最后60℃终延伸30分钟。通过1%琼脂糖凝胶（1×TBE缓冲液）电泳验证每个多重PCR产物的扩增情况。将PCR产物以1:15比例稀释后，送至康奈尔生物信息学研究所（美国纽约州伊萨卡），加入Genescan-500 Liz内标与HiDi甲酰胺，随后在应用生物系统3730x1型96毛细管DNA分析仪上进行片段分析。使用Geneious v.6软件（Biomatters，新西兰奥克兰）中的微卫星插件分析数据，以识别9个位点及每个样品的扩增子长度（即等位基因）。

群体遗传分析
本次群体遗传分析使用的多位点基因型数据来自本研究的95株分离物的STRAf数据，以及既往研究中80株来自美国的临床与环境分离物（参考文献5）：其中28株来自临床环境，环境分离物来自农业堆肥（4株）、带有植物残体的土壤（种植苹果2株、西瓜7株、草莓4株、碧根果13株、花生16株、葡萄5株）。既往研究中的实验室参考菌株Af293作为对照纳入分析。研究人员选取覆盖多种基质的分离物以保证样本代表性，但优先选取与本次研究采样的零售产品相关的基质（如葡萄、苹果、花生土壤及植物残体）。
为评估分离物间的遗传相关性，使用R语言的Poppr软件包（参考文献63），基于Bruvo遗传距离模型与Nei 1978年遗传距离构建最小生成网络。Bruvo遗传距离常用于微卫星标记的遗传分析（参考文献60），其假设逐步突变模型，但对于基因型多样且存在有性生殖的A. fumigatus而言，该模型并不完全准确（参考文献64）。此外，每个STRAf位点包含11~37个不同重复数的等位基因，其演化可能并非遵循逐步突变模式（参考文献57），因此研究同时采用Nei遗传距离以纳入无限等位基因模型。
使用R语言中的主成分判别分析（discriminate analysis of principal components，DAPC）分析群体遗传结构，以确定群体是否根据环境类型、来源基质、cyp51A基因型、地理采样地点或其他因素聚类。聚类结果通过主成分的K均值聚类确定。