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Article

Distinct Phenotype and Secondary Metabolite Profile Mark a Dominant Aspergillus flavus Outbreak Strain

by
Alexander Gewecke
1,
Noam Aviman
2,
Jens C. Frisvad
2,
Maiken Cavling Arendrup
1,3,4,* and
Jakob Blæsbjerg Hoof
2,*
1
Mycology Unit, Department for Bacteria, Parasites and Fungi, Statens Serum Institut, 2300 Copenhagen S, Denmark
2
DTU Bioengineering, Technical University of Denmark, 2800 Kongens Lyngby, Denmark
3
Department for Clinical Microbiology, Copenhagen University Hospital, Rigshospitalet, 2100 Copenhagen Ø, Denmark
4
Department for Clinical Medicine, University of Copenhagen, 2100 Copenhagen Ø, Denmark
*
Authors to whom correspondence should be addressed.
J. Fungi 2026, 12(6), 454; https://doi.org/10.3390/jof12060454 (registering DOI)
Submission received: 1 April 2026 / Revised: 2 May 2026 / Accepted: 15 June 2026 / Published: 22 June 2026
(This article belongs to the Section Fungal Cell Biology, Metabolism and Physiology)
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Abstract

An Aspergillus flavus outbreak strain dominated the indoor environment in a Danish hospital ward for eight years and subsequently multiple isogenic infections occurred. We investigated whether strain-specific traits were present to understand its prevalence and persistence. The outbreak strain was studied alongside comparator A. flavus isolates with respect to altered virulence that could enhance its pathogenic potential and secondary metabolism that could influence environmental persistence. Twenty-four isolates were examined for growth patterns on ten media and by secondary metabolite profiling using high-performance liquid chromatography with diode-array detection. Strain-specific virulence and other phenotypic traits were studied in vivo using Galleria mellonella and in vitro by culturing on specialised media. No indication of virulence alterations was observed in larvae. However, the outbreak strain exhibited a reproducible fingerprint phenotype with distinct morphological features and secondary metabolites. These included mycotoxins known to be harmful to humans and animals. Although this study found no evidence of increased virulence, identification of a distinct phenotypic profile could indicate adaptation or an intrinsic ecological background of the outbreak strain and possibly competitive traits via potentially bioactive secondary metabolites. Moreover, the production of several mycotoxins by this A. flavus strain raises concerns for both patients and staff. Further analyses of the strain’s ecology, toxic potential, virulence, and phylogeny in a global context could be studied in future experiments.

1. Introduction

Aspergillus flavus is the second most common cause of invasive aspergillosis and of nosocomial Aspergillus outbreaks [1,2]. Beyond the clinical implications, identifying sources of contamination, whether from the external environment or within the hospital, and understanding how and why the fungus settles and persists in hospital environments remain challenging. These processes are difficult, expensive, and time-consuming to examine. From 2017 to 2025, a Danish tertiary hospital experienced an A. flavus outbreak mainly affecting children in a haematology ward, but also sporadic cases in non-haematologic patients and one case in the Department of Clinical Microbiology (DCM) following incubator contamination [3,4,5]. Retrospective analysis revealed a monophyletic cluster (Cluster-10) [5] with independent subbranches concurrently inhabiting and dominating each site. Genomics proposed the outbreak strain had been present in the hospital since 2008 [3,5], and that it was closely related to A. flavus isolates from commercially available building materials (wood-based) from an unrelated study [6]. That study had tested different materials for potential dormant mould spores and their germination after water damage [6]. Source investigation in the haematologic ward led to the discovery of extensive spore reservoirs on hard-to-reach surfaces, and indeed, direct growth of the outbreak strain on water-damaged plywood in a kitchen cabinet [5]. Clonal dissemination was ultimately linked to in-hospital construction work, a well-recognised route of dissemination for Aspergillus spores [2].
Following the outbreak, questions arose regarding virulence and why this particular type of strain was successful in persisting and outcompeting, e.g., other A. flavus strains in the outbreak ward. The answers may reflect its biological background. In a broader context, marked ecological versatility defines A. flavus as a saprotroph mould that thrives on decaying biological material, infects plants and animals, and contaminates crops such as maize and wheat, extending to annual crop losses worth billions of dollars [7]. This is largely due to the production of aflatoxins, insecticidal mycotoxins synthesised as secondary metabolites that enhance fungal survival in the field [8]. The toxin became notorious after the Turkey-X outbreak in the UK during the 1960s, in which approximately 100,000 turkeys died from severe hepatic damage following ingestion of the toxins [8]. Subsequent studies have shown both acute, chronic, and fatal cases of aflatoxicosis in humans following ingestion [7,9], with prolonged low-level exposure associated with immunosuppression and hepatocellular carcinoma [7]. Other common A. flavus secondary metabolites are potentially toxic to humans. For instance, cyclopiazonic acid inhibits the sarcoplasmic reticulum Ca2+ ATPase and shows adverse toxic effects in animals [10], and even small doses of aflatrem, found in sclerotia-producing strains, is neurotoxic in rats [11]. Furthermore, some strains of A. flavus can produce the highly neurotoxic 3-nitropropionic acid [12]. Little is known about the potential chronic mycotoxin exposure among hospitalised patients and healthcare staff when Aspergillus persists in hospital wards. However, a plausible hypothesis is that mycotoxins could worsen patient outcomes if produced during infection or tissue colonisation, or if present in the patient environment as a result of surface contamination or aerosolisation [13].
In addition to their toxic effects on mammals and insects, some secondary metabolites serve as chemical defences against other microbes. A famous example is penicillin production by the mould Penicillium rubens. Secondary metabolites also help A. flavus antagonise competing microorganisms via antimicrobials like aspergillic acid, aspirochlorin, and astellolides [14,15,16], and enable fungal persistence following oxidative stress via production of kojic acid, a potent antioxidant [17]. In addition, biological resilience and preservation mechanisms, through the formation of DNA and mycotoxin-packed sclerotia and conidia [18,19], enable A. flavus to survive elevated temperatures, low water activity, and nutrient deprivation [18]. It is not unreasonable to suggest that one or more of these listed factors enabled the outbreak strain to survive in the hospital’s interior.
Whether it has altered virulence in vivo, displays distinct morphological characters in vitro, or indeed produces harmful or specialised mycotoxins that may facilitate persistence and potentially endanger patients and staff is the focus of this study. The aims are to examine whether the outbreak strain possibly displays a specific phenotype profile and whether there is potential for ecological implications to any such features. As comparators, a panel of clinical and environmental A. flavus strains were selected based on phylogeny in our previous study [5]. We also added additional isolates from internal libraries to increase the volume of comparators for some of the analyses performed.

2. Materials and Methods

2.1. Strains and Isolates

A total of 24 isolates were included as summarised in Table 1.
Four main comparators were selected based on known genomic differences [5] to assess inter-strain virulence and phenotype differences to the outbreak strain. These differed from 16,825 to 50,462 SNPs from the outbreak strain “SS18” (Cluster-10). Isolate 8981 was previously identified tentatively as A. flavus by single medium morphology (yeast glucose chloramphenicol) [3], but could not be reliably placed in following phylogenetic analysis due to large divergence from other A. flavus genomes. It was nonetheless retained here as a known genetic outsider for phenotypic comparison. Strain NRRL 3357, previously used as genomic mapping reference [5], was included as a control (sourced from the Statens Serum Institut (SSI)). Aside from SS18, four additional Cluster-10 isolates from our previous study were selected to assess Cluster-10 phenotype variability. These differed by 82-86 SNPs (Wood1 to 3) and 76 SNPs (F16180, DCM patient’s isolate) compared to SS18. Additional outbreak ward surface isolates were included. Those were collected more recently from surfaces (N = 6) in the outbreak ward but had not been sequenced prior to this study. Lastly, external isolates with diverse origin (N = 9) were included from the Institut for Bioteknologi (IBT) Culture Collection at the Technical University of Denmark (DTU). These also included an IBT-version of the NRRL 3357 isolate (NRRL 3357 DTU). The two NRRL 3357 isolates (SSI/DTU) had been genotyped (see Table 1) prior to this study via short tandem repeats as described elsewhere [3]. All isolates were stored at −80 °C prior to use.

2.2. Galleria mellonella Virulence Assay

G. mellonella was used for virulence testing according to previous descriptions [20] with the following modifications. SS18 and the main comparators were incubated for three days at 37 °C prior to conidial harvest and suspension in PBS and concentration-adjustment to approximately 1 to 5 × 106 using an optical density (OD) of 1 with verification of inoculum concentration by quantitative plating of serial dilutions. Three groups of 20 larvae for each strain were injected with 10 µL PBS solution of 1.5 × 106 CFU/mL in a 5-fold dilution series hereof (5−1, 5−2, 5−3) using Hamilton syringes (Hamilton Company, Reno, NV, USA). PBS-injected and uninjected controls were included. Larvae were incubated at 37 °C for five days and monitored every 24 h for mortality. Dead larvae were removed upon recognition. Survival analysis was done in R.4.2.3 (package survival) and visualised in a Kaplan–Meier plot. Group survival was quantified via restricted mean survival time (RMST) [21]. A 2000 replicate bootstrap analysis of RMST estimates was employed to compare isolate-specific virulence. Benjamini–Hochberg (BH) adjustment was used to curb type I error.
Growth kinetic assays on solid agar at 37 °C had previously been performed for a Cluster-10 isolate and most of the main comparators (10822, F7734, 8981) with no difference observed.

2.3. Morphological and HPLC-DAD Profiling

Twenty-four isolates were cultured on different media made according to internal protocols (https://dtu.bio-aware.com/page/Cultivation%20media, URL (accessed on 14 June 2026) including A. flavus and parasiticus agar (AFPA), Creatine sucrose agar (CREA), Czapek Yeast Autolysate agar (CYA), Malt Extract agar (MEAox), Oatmeal agar (OAT), Sabouraud agar (SABO), Yeast extract sucrose agar (YES), Nakamura’s medium (MAN) [22], and Wickerham’s antibiotic test medium (WATM). Agar plates were three-point inoculated and grown in the dark for seven days at 25 °C (and at 37 °C for CYA specifically). For the morphological analysis, differences in colony diameter, degree of sporulation, pigment formation and acid production on CREA were assessed. Additionally, SS18 and the main comparators were inoculated on plywood-fibre agar (15 g fibre and 15 g agar per litre of water) at 37 °C for six days to test whether isolates could grow on that material or not. Growth on plywood was stained with Lactoethanol Cotton Blue (SSI Diagnostica, Hillerød, Denmark) prior to microscopy. Dichloran Glycerol agar (DG18) was also used to test xerotolerance among them.
For HPLC-DAD, plugs were extracted in pairs into a shared vial for CYA + YES, AFPA + MAN, MEAox + WATM, while single for SABO, from agar colonies following standardised procedures [23]. Secondary metabolites were separated via the Agilent 1100 system (Agilent Technologies, Waldbronn, Germany) with a Luna C18 column (100 × 2 mm, 3 µm) (Phenomenex, Torrance, California, USA). The mobile phase comprised water with 50 ppm trifluoroacetic acid and acetonitrile. Alkylphenone standards were included. Samples (3 µL) were analysed using the “Luna2ins.M” method on the Trubadurix platform. Detection by diode-array at 210 and 280 nm. Identification (IDs) were given to secondary metabolites via an internal DTU library based on retention indices (RI) of authentic standards by UV-visible (UV-VIS) spectra [23,24] and unique UV-VIS appearances. Additionally, a previous comparative study by Rank et al. 2012 [25] was used as reference for well characterised A. flavus and A. oryzae secondary metabolites. That study included metabolite identification by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) of NRRL 3357 and RIB40 and was used to compare HPLC-DAD identified secondary metabolites found here. Results were reported as presence or absence of identified metabolites across media, and signal intensities of specific metabolites.

2.4. Genomic Basis of Secondary Metabolites

Illumina short read sequences of SS18 and the main comparators generated in our previous study [5] were compared using BLAST (National Library of Medicine) against query sequences of some commonly identified secondary metabolites from the NRRL 3357 reference (GenBank ac no.: GCA_009017415.1), RIB40 (GCF_000184455.2), and E1404 (GCA_013146025.1) [26,27] to further identify length and percentage similarity of gene clusters in the isolate-group. That was done via BLAST-assembly in CLC Main Workbench 25.0.3, sequence alignments, and pairwise gene cluster comparisons. The aim was to verify presence of the biosynthetic gene clusters (BGCs) for HPLC-DAD identified metabolites and compare the isolates in a broader context of A. flavus strains [26].
The internal transcribed spacer (ITS) and calmodulin (CaM) were compared to sequences of type cultures of the other members of the section Flavi by the multiple sequence alignment tool in CLC Main Workbench 25.0.3 to assign species identification for 8981 [28].

3. Results

3.1. Virulence Studies in the Galleria mellonella Virulence Assay

We used a larvae-based virulence assay to assess whether SS18 showed higher virulence than the main comparators Figure 1.
Concentration dependent (5−1, 5−2, 5−3) decrease in mortality of NRRL 3357-infected larvae was observed in a previous pilot study. The 5−1 injections remained potent for all strains, while mortality differences observed from 5−2 to 5−3 were minor. Therefore, only the 5−1 assay is presented here (Figure 1).
RMST survival comparison showed mean survival times of 2.30–3.60 days but the RMST difference in the bootstrap analysis among SS18 (Cluster-10) infected larvae was only significantly different (worse) when compared to the F7734 non-outbreak clinical isolate, but that difference was no longer significant after BH-adjustment. The differences in survival are presented as (days) deviation from the SS18 in Table 2.

3.2. Morphological Profiling

All isolates presented in this study were subjected to comprehensive One Strain Many Compounds analysis (OSMAC). This assay would reveal whether Cluster-10 isolates had an alternative morphotype and metabolic repertoire compared to an array of clinical and environmental isolates when exposed to a wide selection of conditions.
Firstly, we established a common morphotype for Cluster-10, presented in a merged collection of the best images of Cluster-10 isolates that will serve as an overall ID of the lineage Figure 2.
Among Cluster-10 isolates, the uniform morphotype was shared by all subbranches, although some variation was observed on MAN and WATM media; see Figure 3.
For SS18 and the main comparators, the most distinctive media were reported for comparison, while an overview of all media is provided in the Supplementary Files for all isolates in Figure S1.
The isolates showed substantial differences across the media, but most prominently on MAN, CYA, CYA37, YES, WATM, and CREA; see Figure 4.
Growth was comparable for SS18, 10822, and F7734 on DG18 media, but NRRL 3357 showed less growth and 8982 showed a vegetative cotton-like appearance.
Among isolates outside Cluster-10, eight formed sclerotia on WATM, seven on CYA, six on CYA37 and MEAox, and two on YES and OAT, see Figure 4 and Figure S1.
SS18 and the main comparators were further analysed for growth differences on plywood, which was a surface Cluster-10 isolates had previously shown active growth on inside the hospital [5]. All strains had comparable growth on plywood-fibre agar and on the plywood-fibres directly when wetted. Only slight colour variation and overall macro and micromorphology was observed among isolates except for 8981, which displayed darker, more patchy growth; see Figure 5.

3.3. Secondary Metabolite Profiling via HPLC-DAD

A. flavus is a rich producer of bioactive secondary metabolites, where some may play a role in virulence, adaptation with specific advantages, or as competitive traits for strain persistence. At DTU, HPLC-DAD is frequently used for routine analyses to examine metabolic responses of A. flavus in various conditions. The method can detect a number of bioactive molecules, and we hypothesised that the Cluster-10 isolates could display an altered profile given the specific monophyletic nature and unique morphotype. In this analysis, IDs of secondary metabolites were given via standardised retention indices from the internal DTU-library. UV-VIS chromatograms were compared to authentic standards and well-characterised chromatograms. Also, identified metabolites were compared to a previous study that characterised secondary metabolites from A. flavus (NRRL 3357) and A. oryzae (RIB40) via HPLC-DAD and LC-MS/MS [25]. Typical UV-VIS of the secondary metabolites presented here, retention indices distribution, retention times, and logA estimates are available in the Supplementary Files. An overview of metabolites detected in the isolates and their respective retention indices are provided in Table 3.
Secondary metabolite profiles were compared for combined plug-extracts from carbon rich CYA + YES media due to their promotion of a diverse secondary metabolism [28]. This showed a distinct chemical fingerprint for Cluster-10 isolates, different from the main comparators and the IBT isolates Figure 6.
Although the Cluster-10 isolates did not produce aflatoxin B1 on the CYA + YES media, signals for aflatoxin B1 were detected on the SABO medium for most of them. Generally, the other aflatoxin producers showed enhanced aflatoxin signals on SABO. One additional compound was only detected in the AFPA + MAN plugs, aspergillic acid, and was produced by all isolates but one (Wood3). A heatmap displays metabolite profiles across different media; see Figure 7.

3.4. Genomic Basis of Secondary Metabolites

Representative BGCs from reference sequences available at NCBI for aflatoxin, cyclopiazonic acid, ditryptophenaline, and dideacetylparasiticolide were identified and compared for SS18 and the main comparators from whom short-read genomes were available from our previous study (ENA Acc. No. PRJEB105462) [5]. Prior to the analysis, sequences of ITS and CaM for 8981 were blasted to NCBI and both showed a 100% similarity to A. parasiticus. Consequently, this isolate was excluded from further gene cluster comparison, as it was probably a different species within Aspergillus section Flavi. Remaining isolates harboured BGCs with a high level of isogeneity to the BGCs of NRRL 3357 for aflatoxin, cyclopiazonic acid, and ditryptophenaline. The dideacetylparasiticolide gene cluster is defined in [27] and was examined due to this specific compound’s prominence in outbreak-related isolates. An intact BGC was found in SS18, whereas it was indeed partially absent in comparators, which did not produce the metabolite Table 4.
A. flavus strain E1404 [26] was included to verify the dideacetylparasiticolide clusters’ similarity to either E1404 or the query sequence from RIB40 (A. oryzae). Closest similarity was observed between SS18 and E1404 versus SS18 and RIB40, with 99.55% and 98.64% respectively.

4. Discussion

Intrigued by the notable dominance in the hospital environment and among patients in the outbreak ward, this study explored virulence in Galleria mellonella, morphological characteristics, secondary metabolite profiles and gene clusters for an A. flavus strain from a previously reported clonal outbreak [3,4,5]. No indication of increased virulence was observed among tested isolates, although increased mortality was indeed observed in larvae receiving a fungal strain versus pure PBS. Morphology on several culture agars had defined A. flavus features [29], but with a distinct and reproducible fingerprint morphotype for all isolates included in Cluster-10 (the monophyletic cluster with all outbreak isolates [5]). That was relative to genetically distinct isolates in the main comparators group and isolates from the IBT collection, which had not been sequenced. Similarly, the Cluster-10 isolates shared a uniform secondary metabolite pattern. The HPLC-DAD method yielded distinctive UV-VIS chromatograms that matched spectra and retention indices for LC-MS/MS characterised metabolites [25] identified here via authentic standards. For some metabolites, their detection was supported by the presence of BGCs. The Cluster-10 unique secondary metabolite profile comprised the combination of dideacetylparasiticolide [14,27] and ustilaginoidin C both produced by them all, and remarkably the lack of aflatoxin B1 production on all media except SABO. We speculate whether Cluster-10 dominance in the outbreak ward could be coupled to an overall unique phenotype defined for this monophyletic lineage.
The morphological development of A. flavus undergoes complex genetic regulation [18]. The overall macromorphology of Cluster-10 isolates was preserved across isolates. Prominent conidiation, lack of sclerotia, and low levels of acid production were some of their distinct features. The morphotype was reproducible, and that was also the case for the two inter-laboratory controls (NRRL 3357) on most media. Differences in NRRL 3357 morphology possibly reflected isolate fitness upon inoculation since their genotype and secondary metabolite profile were identical.
Conidia and sclerotia formation enable reproduction and perseverance in A. flavus. Common for both structures are their genetic regulation machinery [18,19]. Lack of sclerotial formation by Cluster-10 isolates could inversely reflect their ecological background indicating an evolutionary or regulatory trait that distances them from agricultural strains like NRRL 3357 that use sclerotia packed with aflatoxins to avoid predation by insects and preserve DNA during unfavourable environmental conditions [30]. Further investigation into regulatory genes could possibly determine which is more likely.
SS18 and the main comparators all grew on plywood substrates and had similar morphology except 8981 with patchy growth and notably dark coloration on several media. Of note, although 8981 was previously morphologically identified as A. flavus upon patient isolation, it showed ITS and CaM sequence homology with A. parasiticus here. This was supported by typical A. parasiticus morphology and a secondary metabolite profile with aflatoxin G1 and absence of cyclopiazonic acid [29]. Nonetheless, all STR loci in the STRAfla assay were previously amplified for isolate 8981 [3], implying homologous loci may be distributed across section Flavi. We previously verified STRAfla for reliable clonality detection with high discriminatory power and consistent identification of Cluster-10 isolates [5]. Thus, STRAfla can be used for clonality assessment in confirmed A. flavus outbreaks, provided species identification within section Flavi is independently verified by robust methods and not only determined by morphology. Interestingly, this A. parasiticus showed virulence comparable to both SS18 and the other main comparators in this study, although it seems rarely associated with human infection in the literature.
Secondary metabolite profiles of Cluster-10 isolates were mapped to authentic standards and showed reproducibility and narrow retention indices for prominent chromatograms of well-characterised A. flavus metabolites [25]. Similar reproducibility of secondary metabolite profile was observed for the two interlaboratory NRRL 3357 isolates compared to one another and to previous reports [25].
Contested microbiological environments like soil, fields, and human or animal tissues warrant antagonistic substances at disposal. Not surprisingly, some secondary metabolites have antimicrobial properties [31]. Application of non-aflatoxigenic A. flavus strains for crop biocontrol is a well-established method to curb contamination in agriculture [32]. Alshannaq et al. demonstrated that co-inoculation even at a 1:10 ratio effectively inhibits growth of aflatoxigenic A. flavus (NRRL 3357) strains [32]. Also, the filtrate alone from non-aflatoxigenic strains effectively inhibited growth of NRRL 3357 supporting a chemical aspect. However, these studies focused on aflatoxin mitigation via non-aflatoxigenic strains. In our study, aflatoxin B1 production by Cluster-10 isolates was only detected on Sabouraud medium, indicating a medium-dependent phenotype. Sabouraud medium is relatively simple, comprising a rich carbon source (glucose or dextrose) and a nitrogen source (peptone), while its pH is kept low at 5.6. The mechanism behind this Sabouraud-specificity for aflatoxin B1 in Cluster-10 isolates remains unknown, but other medium-dependent variations in aflatoxin biosynthesis have been described previously [33,34]. The nitrogen source, as an example, influences toxin production and gene-expression within the aflatoxin cluster in a strain-dependent manner [33]. Medium acidity is another modulator for some A. flavus strains [34].
Cluster-10 isolates dominated the affected ward [5], and hypothetically, a potential reason could include antagonism of other A. flavus strains. As an example, the chemical class of astellolides, that includes dideacetylparasiticolide, was stereotypic for Cluster-10 isolates, and other members of that class have previously shown potent antifungal effects with MICs reaching 1.56 mg/L for Penicillium italicum [14]. Hypothetically, the secondary metabolite profile of the Cluster-10 isolates could reflect adaptations to their combatant needs when they settle in specific environments. But that remains speculative. Future studies are needed for proper experimental testing of that hypothesis via MIC determination of potential antifungal compounds produced by Cluster-10 isolates and other inhibitory traits when co-cultured, or indeed by their filtrate directly added to colonies of other A. flavus strains [32].
A different aspect to mycotoxin presence in hospitals is the potential dangers of human exposure. Aflatoxins pose a well-known threat to humans when ingested, but there is limited evidence regarding other toxins like cyclopiazonic acid, which is known to be toxic to animals [10]. There is an ongoing debate as to whether mycotoxins contaminate indoor environments and whether that poses a realistic threat to human health. For mycotoxins to constitute a clinical challenge, patients must be exposed via ingestion or inhalation. However, Al Hallak et al. recently emphasised that the presence and aerosolization of mycotoxins are multifactorial. The level of fungal contamination on surfaces matters and varies greatly [13]. Studies show that a wide range of mycotoxins are indeed produced when moulds grow on interior building materials [35,36,37,38], and that surfaces can be contaminated with mycotoxins in moisture-damaged homes at quantities of 1.3 ppb in dust and 21–110 ppb on interior building materials [36]. Furthermore, Aleksic et al. demonstrated that the toxic aflatoxin-precursor, sterigmatocystin, is produced in quantities of 82.02–142.18 mg/m2 by Aspergillus versicolor when growing on wallpaper, but with aerosolisation of only 0.2% at an air velocity of 2 m/s [35]. Note that regular movement typically generates air velocities of approximately 0.3 m/s, whereas slamming doors can produce velocities of approximately 6 m/s [35]. Moularat et al. reported slightly higher sterigmatocystin aerosolisation of 1–4% depending on substrate (incl. wallpaper) [37]. Sterigmatocystin may not be a surrogate marker to infer aerosolisation of mycotoxins in general, though it supports that surface contamination and some aerosolisation of the mycotoxins reported here are possible. The outbreak ward surfaces initially contained a median of 800 (0–25,200) CFU/m2 (A. flavus) and 2400 (0–28,000) CFU/m2 (Aspergillus spp.) in total [5]. For context, NRRL 3357 can produce up to 200 ppb aflatoxin from 50 spores per mL cultured at 30 °C for 12 days in potato dextrose broth [32], whereas the WHO sets the limit for aflatoxins in food commodities at 0.5–15 ppb [39].
Examination of BGC similarities (length in base pairs and sequence similarities) supported the detected secondary metabolites in a manner of presence or absence and SS18 (Cluster-10) had intact aflatoxin, cyclopiazonic acid, ditryptophenaline, and dideacetylparasiticolide gene clusters. We previously speculated that the Cluster-10 isolates might be a subspecies different from other A. flavus [3], although subspecies and varieties are not accepted in Aspergillus taxonomic practice. That theory reflected the emphasis placed in literature on distinguishing A. flavus from A. oryzae [26,40,41], a domesticated, non-aflatoxigenic derivative of A. flavus used in the food industry. Specificity of current separation methods are debated [41]. Kim et al. proposed a phylogenetic approach combined with designation of functionally intact aflatoxin and cyclopiazonic acid gene clusters to determine A. flavus [26], whereas Han et al. were sceptical given the apparent non-systematic dissemination of accessory genes (secondary metabolite genes) among A. flavus and A. oryzae [41]. Cluster-10 isolates cannot decisively be designated as either at this point, but morphology and secondary metabolism point to A. flavus in this study, although the cluster may have emerged from a niche wild type background.
This study has several limitations. Galleria mellonella is a recognised model to study fungal virulence and the injection of approximately 3000 CFU per larva gave a sufficient response within a five-day scope in this study. However, only mortality was reported as standardisation of movement and melanisation assessment was too difficult [42] whereby potential valuable information was lost. Additionally, right-wing censoring was chosen due to cocoon formation by day six. Assay credibility could be verified by replications and additional approaches to virulence-testing could also be applied. Compound identification was based on comparison of retention indices and UV-VIS spectra with authentic standards of well-characterised A. flavus metabolites specified previously via LC-MS/MS by Rank et al., 2012 [25]. Minor deviations in retention index were within expected analytical variability. While this supports the proposed identifications, confirmation by an orthogonal technique such as LC-MS/MS was not performed in the present study and would provide additional structural specificity. BGC-comparisons were limited to sequence length and percentage identity relative to query sequences, however without assessment of gene integrity. Finally, none of the identified compounds were evaluated for bioactive properties, and any proposed ecological role in the persistence of the Cluster-10 isolates remains speculative.
In conclusion, this study did not identify a single factor explaining the prolonged dominance of the Cluster-10 isolates in the affected ward or its success in causing infection. Rather, two working hypotheses seem reasonable. (i) Introduction to the hospital via pre-contaminated wood material followed by reseeding of water damaged sites in the ward and prolonged dust-dwelling. Those scenarios were previously supported [5]. (ii) Furthermore, no evidence of increased virulence in G. mellonella was observed here, although that may not be reflective of human virulence. However, dominance in the outbreak ward may not be virulence driven. The Cluster-10 isolates exhibited a distinct and reproducible phenotype. This included production of metabolites that could be further studied to determine whether the phenotype of Cluster-10 isolates confers fitness advantages over competing A. flavus strains. In addition, Cluster-10 isolates produced several poisonous mycotoxins with potential implications for human health, which also calls for further investigation in future studies.
Determinants of ward-dominance, which likely explain the A. flavus outbreak among patients, remain unclear, but this study highlighted potential features to study the success of Cluster-10 isolates that could prove useful in future A. flavus hospital outbreaks.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof12060454/s1, Figure S1: Morphology of all strains; Figure S2: UV-VIS chromatograms; Table S1: HPLC-DAD data.

Author Contributions

A.G., N.A. and J.C.F. conducted the laboratory work and acquisition of data. A.G., M.C.A., J.B.H. and J.C.F. planned analyses and conceptualised hypotheses. All authors contributed to analysing the data. A.G. wrote the initial draft, which M.C.A. revised. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Genomic data used are publicly available from the following ENA genomic sequence accession number: PRJEB105462.

Conflicts of Interest

The authors declare no conflicts of interest related to this work. Outside this study, the authors declare the following: M.C.A. has, over the past 5 years, received research grants/contract work (paid to the SSI) from Cidara/Mundipharma, F2G/Shionogi, Pfizer, Pulmocide and Scynexis, and speaker honoraria (personal fee) from Gilead and F2G/Shionogi. She is the current immediate past-chair of the EUCAST-AFST. ChatGPT5.2 has been used for grammatical improvement in parts of the text and for code-editing in RStudio version 4.2.3.

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Figure 1. Kaplan–Meier survival. The plot displays comparable G. mellonella survival probability in percent over time (five days) after injection with SS18 or the main comparators. Most strains had killed 50% of the larvae after two days, except F7734, whereas 50% were dead after four days.
Figure 1. Kaplan–Meier survival. The plot displays comparable G. mellonella survival probability in percent over time (five days) after injection with SS18 or the main comparators. Most strains had killed 50% of the larvae after two days, except F7734, whereas 50% were dead after four days.
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Figure 2. Common morphotype of Cluster-10 isolates. Growth on eleven media at 25 °C, 7 d (CYA37 at 37 °C, 7 d): AFPA: Colonies moderately deep, sulcate; mycelium white/cream; texture floccose; conidia absent; reverse bright orange. MAN: Colonies plane; mycelium white; texture floccose/powdery; conidia yellow abundant. CYA: Colonies moderately deep, sulcate; mycelium white; texture floccose centre, granular margin; conidia zonation from centre, brownish, olive-green, greyish-green, abundant; exudate droplets. CYA37 (37 °C, 7 d): Colonies plane, shallow sulci, good growth; mycelium white; texture floccose centre, powdery margin; conidia zonation from centre, brown, green, yellowish, abundant. YES: Colonies shallow/moderately deep, sulcate, lawn growth; texture granular/powdery; Heavy conidiation olive-green, darker-green; exudate large droplets. MEAox: Colonies thick; mycelium white, aerial hyphae; texture fluffy, moderate density; conidia olive-green, light-green, abundant. WATM: Colonies plane; mycelium white; texture floccose centre, powdery margin; conidia light-green, abundant towards margin. SABO: Colonies thick; mycelium white; texture fluffy, moderate/low density; conidia zonation from centre grey/greyish-green, moderate abundance. CREA: Poor growth; acid production absent. OAT: Colonies plane; mycelium white; texture floccose centre, powdery margin; conidia pale-green, abundant towards margin. DG18: Colonies plane, good growth; mycelium white; texture granular; conidia olive-green, abundant. Sclerotia were absent on all media.
Figure 2. Common morphotype of Cluster-10 isolates. Growth on eleven media at 25 °C, 7 d (CYA37 at 37 °C, 7 d): AFPA: Colonies moderately deep, sulcate; mycelium white/cream; texture floccose; conidia absent; reverse bright orange. MAN: Colonies plane; mycelium white; texture floccose/powdery; conidia yellow abundant. CYA: Colonies moderately deep, sulcate; mycelium white; texture floccose centre, granular margin; conidia zonation from centre, brownish, olive-green, greyish-green, abundant; exudate droplets. CYA37 (37 °C, 7 d): Colonies plane, shallow sulci, good growth; mycelium white; texture floccose centre, powdery margin; conidia zonation from centre, brown, green, yellowish, abundant. YES: Colonies shallow/moderately deep, sulcate, lawn growth; texture granular/powdery; Heavy conidiation olive-green, darker-green; exudate large droplets. MEAox: Colonies thick; mycelium white, aerial hyphae; texture fluffy, moderate density; conidia olive-green, light-green, abundant. WATM: Colonies plane; mycelium white; texture floccose centre, powdery margin; conidia light-green, abundant towards margin. SABO: Colonies thick; mycelium white; texture fluffy, moderate/low density; conidia zonation from centre grey/greyish-green, moderate abundance. CREA: Poor growth; acid production absent. OAT: Colonies plane; mycelium white; texture floccose centre, powdery margin; conidia pale-green, abundant towards margin. DG18: Colonies plane, good growth; mycelium white; texture granular; conidia olive-green, abundant. Sclerotia were absent on all media.
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Figure 3. Morphological reproducibility among Cluster-10 and outbreak ward surface isolates. Cluster-10 showed a uniform morphology on most media including the clinical isolate F16180 from an infected DCM patient. A unique variation was shared among building material isolates (Wood1 to 3), and one isolate from an outbreak ward surface (Surface6) on the MAN-medium (sulcate, fluffy, greyish-green) and WATM (yellow with green margin). But the Surface6 isolate lacked exudate droplets on YES, although exudate was abundant for the building material isolates. That implies that isolates with both morphotype variations can be found inside the outbreak ward. Isolates not shown here can be found in Figure S1 in the Supplementary Files.
Figure 3. Morphological reproducibility among Cluster-10 and outbreak ward surface isolates. Cluster-10 showed a uniform morphology on most media including the clinical isolate F16180 from an infected DCM patient. A unique variation was shared among building material isolates (Wood1 to 3), and one isolate from an outbreak ward surface (Surface6) on the MAN-medium (sulcate, fluffy, greyish-green) and WATM (yellow with green margin). But the Surface6 isolate lacked exudate droplets on YES, although exudate was abundant for the building material isolates. That implies that isolates with both morphotype variations can be found inside the outbreak ward. Isolates not shown here can be found in Figure S1 in the Supplementary Files.
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Figure 4. SS18 and main comparator morphology including two inter-laboratory NRRL 3357 isolates. The most prominent differences were observed on MAN, CYA, CYA37, YES, WATM, and CREA. For SS18 the lack of sclerotia on any media and lack of acid production on CREA was unique, whereas 10822 did not form sclerotia, but did produce acid while F7734 formed sclerotia (small glistening seeds) on CYA and WATM but had no acid production. 8981 stood out with its dark pigmentation, sulcate, suede-like appearance, especially on CYA37 and YES. No sclerotia were observed. NRRL 3357 isolates sourced from different laboratories showed many similarities on AFPA, CYA, CYA37, YES, MEAox, and SABO (here/Figure S1). Common for the two NRRL 3357 isolates (from the SSI and DTU collections, respectively) was fluffy white centres with pale-green conidiation in the margins and abundant sclerotia on CYA37 (brown/white dotted seeds). But more vegetative growth was observed on MAN, MEAox, and WATM for IBT 28520, and increased acid production on CREA.
Figure 4. SS18 and main comparator morphology including two inter-laboratory NRRL 3357 isolates. The most prominent differences were observed on MAN, CYA, CYA37, YES, WATM, and CREA. For SS18 the lack of sclerotia on any media and lack of acid production on CREA was unique, whereas 10822 did not form sclerotia, but did produce acid while F7734 formed sclerotia (small glistening seeds) on CYA and WATM but had no acid production. 8981 stood out with its dark pigmentation, sulcate, suede-like appearance, especially on CYA37 and YES. No sclerotia were observed. NRRL 3357 isolates sourced from different laboratories showed many similarities on AFPA, CYA, CYA37, YES, MEAox, and SABO (here/Figure S1). Common for the two NRRL 3357 isolates (from the SSI and DTU collections, respectively) was fluffy white centres with pale-green conidiation in the margins and abundant sclerotia on CYA37 (brown/white dotted seeds). But more vegetative growth was observed on MAN, MEAox, and WATM for IBT 28520, and increased acid production on CREA.
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Figure 5. Growth of SS18 and main comparators on plywood-fibre agar and plywood-fibres directly. Despite small variations in colour patterns among strains, only 8981 appeared different with a dark green and patchy growth. Micromorphology: SS18: Conidiophore delicate, smooth, moderately thin walled, collapsed; vesicle clavate/globose; conidia globose, smooth, thick walled. 8981: Conidiophore smooth, moderately thick walled, distal branching; vesicles globose, small; conidia globose, rough with projections, moderately thick walled. 10822: Conidiophore smooth, moderately thick walled, collapsed; vesicles clavate/globose; conidia globose, smooth, thick walled. F7734: Conidiophore smooth, moderately thick walled, collapsed; vesicles clavate/globose; conidia globose, smooth, thick walled. NRRL 3357: Conidiophore smooth, moderately thick walled, collapsed; vesicles clavate/globose; conidia globose, smooth, moderately thick walled.
Figure 5. Growth of SS18 and main comparators on plywood-fibre agar and plywood-fibres directly. Despite small variations in colour patterns among strains, only 8981 appeared different with a dark green and patchy growth. Micromorphology: SS18: Conidiophore delicate, smooth, moderately thin walled, collapsed; vesicle clavate/globose; conidia globose, smooth, thick walled. 8981: Conidiophore smooth, moderately thick walled, distal branching; vesicles globose, small; conidia globose, rough with projections, moderately thick walled. 10822: Conidiophore smooth, moderately thick walled, collapsed; vesicles clavate/globose; conidia globose, smooth, thick walled. F7734: Conidiophore smooth, moderately thick walled, collapsed; vesicles clavate/globose; conidia globose, smooth, thick walled. NRRL 3357: Conidiophore smooth, moderately thick walled, collapsed; vesicles clavate/globose; conidia globose, smooth, moderately thick walled.
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Figure 6. Secondary metabolite comparison from CYA + YES plugs combined for 24 A. flavus isolates. Detected secondary metabolites are displayed as log transformed (LogA) signal intensities expressed as area under the curve. Note that UV-absorption is metabolite specific, whereas intensity can only be compared across isolates for identical metabolites. The Cluster-10 isolates showed a distinct secondary metabolite profile with prominent signals for dideacetylparasiticolide, ustilaginoidin C, and absence of aflatoxin B1. Only one other (IBT 28414) produced dideacetylparasiticolide but aflatoxin B1 and paspaline was also detected in that isolate and the latter is found only in sclerotia which were prominent for IBT 28414 on the CYA medium (see Figure S1), but absent in Cluster-10 isolates. Notably, 8981 had intense signals for linoleic acid, kojic acid, aflatoxin B1, G1, and G2, but was lacking other secondary metabolites—a profile normally associated with A. parasiticus. The inter-laboratory NRRL 3357 controls had identical profiles. All isolates shared comparable signals for kojic acid and for the primary metabolism related fatty acid linoleic acid.
Figure 6. Secondary metabolite comparison from CYA + YES plugs combined for 24 A. flavus isolates. Detected secondary metabolites are displayed as log transformed (LogA) signal intensities expressed as area under the curve. Note that UV-absorption is metabolite specific, whereas intensity can only be compared across isolates for identical metabolites. The Cluster-10 isolates showed a distinct secondary metabolite profile with prominent signals for dideacetylparasiticolide, ustilaginoidin C, and absence of aflatoxin B1. Only one other (IBT 28414) produced dideacetylparasiticolide but aflatoxin B1 and paspaline was also detected in that isolate and the latter is found only in sclerotia which were prominent for IBT 28414 on the CYA medium (see Figure S1), but absent in Cluster-10 isolates. Notably, 8981 had intense signals for linoleic acid, kojic acid, aflatoxin B1, G1, and G2, but was lacking other secondary metabolites—a profile normally associated with A. parasiticus. The inter-laboratory NRRL 3357 controls had identical profiles. All isolates shared comparable signals for kojic acid and for the primary metabolism related fatty acid linoleic acid.
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Figure 7. Secondary metabolite detection frequency across media for Cluster-10 and outbreak ward surface isolates (OWSI) in one group versus main comparators and IBT isolates (IBT) in the other. The secondary metabolite profile of Cluster-10 isolates and outbreak ward surface isolates were consistent on CYA + YES and MEAox + WATM comprising the combination of dideacetylparasiticolide, ustilaginoidin C, and absence of aflatoxin B1. However, no ustilaginoidin C was observed in AFPA + MAN, and aflatoxin B1 was present on SABO medium. Detection of aspergillic acid was observed only in AFPA + MAN for all isolates.
Figure 7. Secondary metabolite detection frequency across media for Cluster-10 and outbreak ward surface isolates (OWSI) in one group versus main comparators and IBT isolates (IBT) in the other. The secondary metabolite profile of Cluster-10 isolates and outbreak ward surface isolates were consistent on CYA + YES and MEAox + WATM comprising the combination of dideacetylparasiticolide, ustilaginoidin C, and absence of aflatoxin B1. However, no ustilaginoidin C was observed in AFPA + MAN, and aflatoxin B1 was present on SABO medium. Detection of aspergillic acid was observed only in AFPA + MAN for all isolates.
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Table 1. Overview of comparator isolates and available metadata.
Table 1. Overview of comparator isolates and available metadata.
Isolates (Local Name and Info.)Origin (Original Strain Number)Sourced fromMaterialYearSNPs from SS18
Previously Sequenced [5]
Cluster-10 isolates
SS18DKOutbreak ward surfaceDust (Vacuum cleaner bag)2024(Ref.)
F16180DKDCM patientSputum202376
Wood1UnknownDanish retailerOriented strand board (OSB)202082
Wood2UnknownDanish retailerMedium-density fibreboard (MDF)202086
Wood3UnknownDanish retailerPlywood202082
Main comparators
10822DKNon-outbreak patientBAL201916,825
F7734DKNon-outbreak patientBAL202347,448
8981DKNon-outbreak patientBAL2018Not in tree b
NRRL 3357 SSI aUSA (CBS 128202)Statens Serum Institut (DK)Peanut196850,462
Non-sequenced
Outbreak ward surface isolates
Surface 1 to 6DKOutbreak ward surfacesSurface swabs2025
IBT isolates
IBT 5778USA (NRRL 1957)Danish Technical UniversityCellophane1944
IBT 16760EcuadorDanish Technical UniversitySoil1965
IBT 22301FinlandDanish Technical UniversityBuilding materialUnknown
IBT 27193The NetherlandsDanish Technical UniversityUnknownUnknown
IBT 28414DKDanish Technical UniversityLake water2006
IBT 32099USA—Miami (SRRC 2653)Danish Technical UniversityCorneal ulcerUnknown
IBT 32103USA—Chicago (SRRC 2632)Danish Technical UniversityClinical (unknown site)Unknown
IBT 34246DKDanish Technical UniversityWheatUnknown
NRRL 3357 DTU aUSA (NRRL 3357)Danish Technical UniversityPeanut1968
a NRRL 3357 SSI and NRRL 3357 DTU was the same strain, but sourced from SSI and DTU respectively. Both had identical genotypes in all tested short tandem repeat genotype markers (6/9). STRAfla-markers 2A-3C: 15;16;20;19;16;14. b 8981 was previously sequenced but excluded from phylogeny due to few callable positions [5]. SNPs: Single Nucleotide Polymorphisms, BAL: Bronchoalveolar lavage, DK: Denmark, USA: United States of America.
Table 2. Survival time in days for all larval groups infected by SS18 or the main comparators.
Table 2. Survival time in days for all larval groups infected by SS18 or the main comparators.
StrainRMST (Days)Difference
(Days)		CIp a
SS18 (Cluster-10)2.50(Ref.)--
NRRL 33572.30−0.20[−1.00; 0.55]0.68
108222.35−0.15[−1.05; 0.75]0.78
89812.850.35[−0.65; 1.35]0.50
F77343.601.10[0.10; 2.05]0.03
a After BH-adjustment all p-values were >0.05.
Table 3. Detection of secondary metabolites in 24 isolates, including associated retention index standards and ranges.
Table 3. Detection of secondary metabolites in 24 isolates, including associated retention index standards and ranges.
Metabolite IDsNo. Isolates with DetectionRetention Index (RI)RI Range
Kojic acid *24558556–560
Ustilaginoidin C **17849847–849
Ditryptoalanine ***19952942–959
Ditryptophenaline ***23967958–969
Aflatoxin G1 *2815815–820
Aflatoxin G2 *1712
Aflatoxin B1 *18840839–845
Aspirochlorin ***13857850–860
Dideacetylparasiticolide ***1310651065, 1066
Cyclopiazonic acid ***2210991097–1103
Paspaline ***414861485–1486
Linoleic acid * (in all fungi)2416151614–1619
Aspergillic acid *2219861984–1988
Ergosterol * (in all fungi)421352126–2138
* Compared with an in-house authentic standard. ** Based on a very unique UV spectrum. *** Compared with an authentic standard and previously characterised in NRRL 3357 or RIB40 via LC-MS/MS (Rank et al. 2012) [25].
Table 4. Aflatoxin, cyclopiazonic acid, ditryptophenaline, and dideacetylparasiticolide BGC sizes and identity among SS18 and main comparator isolates compared to query sequences from NRRL 3357, RIB40, or E1404.
Table 4. Aflatoxin, cyclopiazonic acid, ditryptophenaline, and dideacetylparasiticolide BGC sizes and identity among SS18 and main comparator isolates compared to query sequences from NRRL 3357, RIB40, or E1404.
Toxin/MetaboliteStrainSeq. Size (bp)Identity (%)
AflatoxinNRRL 3357 (NCBI ref. sequence)70,001(Ref)
SS18 (Cluster-10)69,70697.69
1082269,73297.63
F773469,76398.84
Cyclopiazonic acidNRRL 335724,262(Ref)
SS1820,02978.67
1082221,34483.44
F773424,26999.02
DitryptophenalineNRRL 335714,001(Ref)
SS1813,98299.59
1082213,97599.71
F773413,99299.93
DideacetylparasiticolideRIB40 (A. oryzae NCBI ref. sequence)16,857(Ref)
SS1816,87298.64
1082217259.38
F773417259.40
NRRL 335717259.40
E1404 (A. flavus NCBI ref. sequence)16,87298.99
E1404 vs. SS18-99.55
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MDPI and ACS Style

Gewecke, A.; Aviman, N.; Frisvad, J.C.; Arendrup, M.C.; Hoof, J.B. Distinct Phenotype and Secondary Metabolite Profile Mark a Dominant Aspergillus flavus Outbreak Strain. J. Fungi 2026, 12, 454. https://doi.org/10.3390/jof12060454

AMA Style

Gewecke A, Aviman N, Frisvad JC, Arendrup MC, Hoof JB. Distinct Phenotype and Secondary Metabolite Profile Mark a Dominant Aspergillus flavus Outbreak Strain. Journal of Fungi. 2026; 12(6):454. https://doi.org/10.3390/jof12060454

Chicago/Turabian Style

Gewecke, Alexander, Noam Aviman, Jens C. Frisvad, Maiken Cavling Arendrup, and Jakob Blæsbjerg Hoof. 2026. "Distinct Phenotype and Secondary Metabolite Profile Mark a Dominant Aspergillus flavus Outbreak Strain" Journal of Fungi 12, no. 6: 454. https://doi.org/10.3390/jof12060454

APA Style

Gewecke, A., Aviman, N., Frisvad, J. C., Arendrup, M. C., & Hoof, J. B. (2026). Distinct Phenotype and Secondary Metabolite Profile Mark a Dominant Aspergillus flavus Outbreak Strain. Journal of Fungi, 12(6), 454. https://doi.org/10.3390/jof12060454

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