A total of 19 xerophilic/xerotolerant identified Aspergillus species were obtained from the IBT fungal culture collection. These isolates were originally isolated in different countries and from different environments and materials such as air, house dust, mouldy paper, wallpaper and paintings (Table 1). These species were all the Aspergillus sect. Restricti and contained representatives of the Aspergillus restrictus/Aspergillus conicus clade, the Aspergillus vitricola clade, the Aspergillus penicillioides clade and the Aspergillus halophilicus clade [16].

The fungi were cultivated on Dichloran Glycerol Agar (DG18, Thermo Fisher Scientific Oxoid, Basingstoke, UK) [29] supplemented with 100 mg/L chloramphenicol. The species A. halophilicus was grown on Malt Yeast 50% Glucose Agar (MY50G) [30] as it does not grow well on DG18. All fungi were incubated at 25 °C for 14–21 days. Of the 19 Aspergillus spp. included in this study, A. penicillioides was already present in the Bruker BDAL Filamentous Fungi Library version 4 [MaldiBiotyperDBUpdate_V4_Fungi-856(RUO)] (Bruker, Bremen, Gemany), and a strain of A. penicillioides was included in the supplementary library as a positive control.

Protein extraction was performed using a modified version of the manufacture’s ethanol-formic acid extraction protocol. In brief, the fungal isolates were inoculated in 1.5 mL Eppendorf tubes containing a broth and incubated at room temperature for 1–14 days until visible growth was observed. Samples were inactivated by centrifugation, discarding the supernatant and resuspending the pellet in 70% ethanol. The ethanol was removed, and the sample was dried at room temperature, after which 5–20 μL (depending on pellet size) 70% formic acid was added. After 2 min, the same volume (5–20 μL, depending on pellet size) of acetonitrile was added. The suspension were then centrifuged and 1 μL of the supernatant was spotted onto a MALDI 96 main spectral profile (MSP) polished steel target plate (Bruker, Bremen, Germany) along with 1 μL (α-cyano-4-hydroxycinnamic acid (HCCA) matrix solution (#8255344, Merck, Darmstadt, Germany).

Spectra were acquired on a Microflex LT mass spectrometer (Bruker Daltonics Inc.), using the Bruker Biotyper software (v. 3.1) with the Filamentous Fungi Library version 4, MaldiBiotyperDBUpdate_V4_Fungi-856(RUO).

Each species in the supplementary database was represented by a series of MSPs, constructed by combining raw mass spectra obtained from the species isolates inoculated in four different liquid broth media [Sabouraud (SAB) agar [31], filtered MY50 [32], Czapek yeast 20% sucrose broth (CY20) [31] or filtered Malt Yeast 70% Glucose Broth (MY70)], at room temperature until growth was visible (which was for 3–5 days for all species except for A. halophilicus, which had to be incubated for 14 days, and A. halophilicus should have a double layer of matrix in contrast to other species).

For fungal identification, the standard procedure in this laboratory was to inoculate isolates in SAB broth, therefore, the majority of the MSPs were created based on raw mass spectra obtained from isolates inoculated in SAB broth. Our previous studies and other studies show that SAB supports growth of many different Aspergillus species [31,33]. MSPs were later created based on mass spectra obtained from isolates inoculated in MY50, MY70 or CY20 broth. Protein extraction was as described above.

Prior to MSP creation, all the raw spectra were visually examined using the flexAnalysis software (Bruker Daltonics Inc.), where spectra with outlier peaks and low mass to charge ratio (m/z) were excluded. Then, using the MALDI Biotyper Compass Explorer module, raw mass spectra were ‘preprocessed’ by default settings, which includes Mass Adjustment, Smoothing, Baseline Subtraction, Normalisation and Peak picking. This was followed by creating a principal component analysis plot of the spectra, enabling further examination and removal of outlying spectra. Finally, the MSPs obtained for all species were presented as an MSP dendrogram using the MALDI Biotyper Compass Explorer module (Bruker, Bremen, Germany). This analysis enables visualisation of how close the MSPs representing the different species are related to one another, reflected by an arbitrary distance level, normalised to a maximum of 1000. In summary, for each species, up to four reference MSPs were created based on the broth medium used for inoculation (SAB, MY50, CY20 and MY70).

Previously obtained and stored mass spectra, originating from an unrelated study on drilling waste treatment plants, were used to perform a test of the supplementary database, using the Bruker Biotyper 3 software and the BDAL Filamentous Fungi Library version 4 [MaldiBiotyperDBUpdate_V4_Fungi-856(RUO)]. Exposure samples had previously been plated on DG18 and SAB agar. The number of mass spectra obtained from isolates grown on DG18 agar (n_DG18) and SAB agar (n_SAB) was 197 and 116, respectively. Further information on the study has been published elsewhere [34].

The supplementary database was evaluated on environmental samples from three different environments, utilising different sampling techniques. The environments included in this study were domestic homes (n_samples = 27), a museum (n_samples = 15) and a warehouse (n_samples = 9). Environmental samples can be collected using different methods, depending on the environment and the aim of the research. Therefore, for each environment, a different sampling method was utilised. Air samples were taken in the warehouse using Gesamtstaubprobenahme (GSP, CIS by BGI Inc.) sampling actively at 3.5 L/min. Electrostatic dust collectors (EDC) were used in homes as passive samplers. In an open-air museum environmental surface swabs (eSwab collection and transport system designed for microbiological sampling) were taken from the surfaces of artefacts.

The warehouse was used for storage of gardening and roadwork equipment, as well as tools and machinery used by workers for the maintenance of public spaces. The warehouse was selected for this study, because fungi seemed to grow on the walls, and it is important to protect the workers from exposure. The warehouse is made of heat-treated pine wood (Thermowood), and fungal growth was suggested to be caused by fluctuations in the humidity. Personal GSPs (n_samples = 6) were carried by workers when they were present in the warehouse in the morning and sampled for 11–21 min and the stationary GSPs (n_samples = 3) sampled for 240 min at specified locations in the warehouse. The GSP samplers were mounted with 37 mm polycarbonate filters (pore size of 0.8/1 μm; Merck, Damstadt Germany).

The museum is an open-air facility consisting of historical houses, buildings and indoor exhibitions representing various eras throughout history. Here, surface samples were collected using eSwab (Copan’s Liquid Amies Elution Swab, eSwab; Copan, Brescia, Italy) from artefacts in various locations. The museum was selected because the staff expressed concerns that the furniture and artefacts were colonised by fungi. Samples were collected from surfaces that appeared to be colonised by fungi, surfaces that appeared clean and surfaces covered in dust.

The homes were randomly selected and with no described water damage. The home environments utilised long-term passive sampling (11–19 days) of dust using an EDC cloth (ZEEMAN, Alphen, The Netherlands, surface exposure area: 209 m²).

Sampled material was extracted using an extraction solution [MilliQ water, 0.85% sodium chloride (NaCl) and 0.05% Tween80] while shaking (GSP and eSwab – 15 min at 500 rpm; EDC – 60 min at 300 rpm). All samples were stored at −80 °C in 30% glycerol until plated.

The GSP samples were plated on DG18 and MY50 agar. The eSwab samples were plated on SAB, DG18 and MY50 agar. The EDC samples were plated on DG18, MY50 and malt extract agar (MEA). In the case of agar plates being overgrown, making colony counting and isolation impossible, the samples were plated in 10 × dilutions (MilliQ water, 0.85% NaCl, 0.001% Bac. peptone, Merck KGaA, Darmstadt, Germany). All agar plates were incubated at 25°C; DG18, SAB, MEA for 1 week and MY50 for 3 weeks.

Following incubation, fungal colonies were counted. Fungal concentrations from EDC and GSP samples were calculated, taking into account the number of colonies, how much samples were diluted, sampling time and volume extracted, and for GSP samples also the flow rate.

The fungal colonies were prepared for MALDI-TOF MS as described above and identified using the Bruker database as well as the extended database. Identification of isolates were analysed as technical duplicates, with the cut-offs: identification (ID) scores <1.75 were unidentified, ID scores 1.75–1.85 were identified at genus level and ID scores >1.58 identified to species level.

Data analysis was conducted using R v.4.2.3 [35] using the packages ‘tidyverse’ [36], ampvis2 [37] and ggplot2 [38] for data handling and visualisation. Data on fungal species (xerophilic/xerotolerant out of total) on different agar media were compared using Fisher’s Exact Test.

MSPs were successfully created from all four broth media for 15 out of the 19 xerophilic/xerotolerant Aspergillus species. However, for Aspergillus clavatophorus, A. restrictus, A. penicillioides and A. halophilicus, it was not possible to obtain spectra from all media (Table 1). It was only possible to obtain spectra of A. halophilicus if the fungus was inoculated in broth media with low water activity (MY50 and MY70) for 10 to 14 days. In contrast, spectra of A. restrictus, A. clavatophorus and A. penicillioides were of insufficient quality when inoculated in MY50, MY70 or CY20 (Table 1).

Discrimination between the 19 xerophilic/xerotolerant Aspergillus species of the supplementary database was evaluated by an MSP dendrogram (Fig. 1). The majority of the MSPs formed species-specific groups, with the exception of Aspergillus destruens and Aspergillus penicilloides, which were indistinguishable from each other. Generally, the MSP species groups formed clades, with similar patterns to previously published data based on genomic data, albeit with some discrepancies [16]. The Aspergillus restrictus clade, consisting of Aspergillus pachycaulis, Aspergillus caesiellus and A. restrictus were grouped together, forming a distinct cluster. The A. conicus clade (A. villosus, Aspergillus gracilis, Aspergillus domesticus, Aspergillus pseudogracilis, A. conicus, A. destruens), instead of forming one clade, formed two clades of A. villosus, A. gracilis and A. domesticus, A. conicus, A. pseudogracilis, with A. destruens being indistinguishable from A. penicillioides from the A. penicillioides clade. The two species included from the A. vitricola clade (Aspergillus glabripes, A. vitricola) were grouped with species of the A. penicillioides clade and A. conicus clade. The species included from the A. penicillioides clade (Aspergillus magnivesiculatus, Aspergillus hordei, A. penicillioides, A. clavatophorus, Aspergillus infrequens, A. canadensis), were split into two distinct clades.

When queried against the Bruker fungi database, only A. penicillioides, which already was present in library, was correctly identified. The remaining MSPs were not identified, as expected. The MSPs of A. destruens were indistinguishable from A. penicillioides and would be identified as A. penicillioides by the Bruker fungi library. The MSPs were kept in the supplementary database regardless.

The supplementary database was used to identify mass spectra previously obtained from a study on drilling waste treatment plants [34]. Xerophilic/xerotolerant fungi had been reported in this environment, therefore, a total of 313 mass spectra originating from isolates grown on DG18 and/or SAB agar plates (n_DG18 = 197; n_SAB = 116), respectively, were attempted to be re-identified, using the supplementary database. Of these, two previously unidentified isolates (originating from the same DG18 agar plate) were now identified as A. caesiellus, with ID scores >2.00 (Fig. A1).

The two agar media used in this study appeared to affect the total number of xerophilic/xerotolerant species detected (Fig. A1). We observed that the samples inoculated on DG18 agar plates, contained a higher fraction of xerophilic/xerotolerant species (n = 7/21) of the genera Penicillium, Cladosporium and Aspergillus when compared to the samples detected on SAB agar plates (n = 3/14) – although the differences were not significant (p = 0.70).

The supplementary database was further evaluated using environmental samples collected from domestic homes (n = 27, via EDC), a museum (n = 15, via eSwab) and a warehouse (n = 9, via GSP) (Fig. 2). Across all environments, isolates identified to species level by the Bruker fungi library were not misidentified by the new supplementary database.

The fungal concentration of the air exposure samplers (GSP) was between 268 and 1.15 × 10⁴ colony forming units (CFU)/m³. The fungal concentration of the sedimented dust (EDC) was between 205 and 4.45 × 10⁴ CFU/m²/day. The fungal surface concentrations (eSwab) were between 50 and 9.2 × 10⁶ CFU/location range (Fig. A2).

Xerophilic Aspergillus species, not present in the Bruker library, were detected in the home and the warehouse samples when using the supplementary database. The xerophilic Aspergillus species identified using the supplementary database were A. pseudogracilis (nine isolates), A. glabripes (one isolate), A. domesticus (three isolates) and A. conicus (one isolate). Aspergillus domesticus and A. pseudogracilis were identified in two of the environments tested (Figs 3–5). Other xerophilic/xerotolerant fungi were identified from these environments using the Bruker library, including A. glaucus (six isolates), Penicillium brevicompactum/olsonii (64 isolates), Penicillium chrysogenum (15 isolates), Penicillium corylophilium (14 isolates) and Cladosporium herbarum (27 isolates) identified in several samples (Figs 3–5).

The home environments were dominated by the genus Cladosporium, with Cladosporium cladosporioides and C. herbarum being present in high concentrations of 7.0 × 10³ CFU/m²/day and 4.4 × 10³ CFU/m²/day, respectively. Species of Penicillium were also present in high concentrations. The three xerophilic Aspergillus species identified from these environments, A. domesticus, A. glabripes and A. pseudogracilis, using the supplementary database were reported in concentrations of 520 CFU/day, 410 CFU/day and 210 CFU/day, respectively (Fig. 3).

The most abundant fungi detected in the museum were Penicillium aurantiogriseum (6.5 × 10⁴ CFU/location), P. chrysogenum (1.1 × 10⁴ CFU/location) and isolates belonging to the Cladosporium genus (2.2 × 10⁴ CFU/location). No additional xerophilic Aspergillus species were identified from the museum samples using the supplementary database (Fig. 4).

The warehouse samples were dominated by Penicillium camemberti commune cyclopium (Penicillium CCC) (5.5 × 10³ CFU/m³), Cladosporium langeronii (2.2 × 10³ CFU/m³) and the yeast Filibasidium magnum (2.8 × 10³ CFU/m³). In this environment, A. pseudogracilis, A. domesticus and A. conicus were found in concentrations of 270 CFU/m³, 39 CFU/m³ and 100 CFU/m³, respectively (Fig. 5).

The various agar media used for fungal growth tended to affect the detection of xerophilic fungi (Figs 3–5). In the home environment, a larger fraction of the detected fungi tended to be xerophilic species when using MY50 (10/21 = 48% of species) and DG18 (8/29 = 27% of species), contra MEA (5/26 = 19% of species); (for MY50 vs MEA, p = 0.059). None of the xerophilic Aspergillus species included in the supplementary database were detected on DG18 or MEA. A similar trend was observed in the warehouse environment with 7/10 (70%) of species on MY50 being xerophilic species compared to 1/5 (20%) of species on DG18 (p = 0.12). This trend, however, was not observed in the museum environment (MY50, 6/22 = 27% of species; DG18, 5/25 = 20% of species; SAB, 7/25 = 28% of species) (p = 0.77).

Culture condition and sample preparation methods can influence the mass spectrum obtained [39,40]. Therefore, the database was constructed using MSPs based on raw mass spectra originating from isolates inoculated in four different broth media. Raw mass spectra of the highly xerophilic fungus, A. halophilicus, were only obtained when the fungus was inoculated in broth media of low water activity for at least 14 days. The inability to obtain raw mass spectra from A. halophilicus might be partly explained by it being obligately xerophilic and not readily cultured in high water-activity media [41–44]. This suggests that, when working with environments where extreme/obligate xerophilic fungi thrive, it is relevant to re-evaluate the protocols (i.e., inoculation media and incubation time) used to inoculate unknown fungal isolates, to obtain mass spectra and hence ensure species identification.

Importantly, none of the MSPs of the supplementary database matched entries in the Bruker fungi library except for the species A. destruens identified as A. penicillioides. The MSP dendrogram showed that the MSPs formed species-specific clusters of the various xerophilic Aspergillus species. An exception of the species-specific clusters was A. penicillioides and A. destruens, for which MSPs were indistinguishable. By the time of submission, we observed that the species designation was updated for A. destruens (strain IBT no. 34818), which is now designated Aspergillus salinarum. This species is a halophilic fungus isolated from a hypersaline environment (Table 1). However, this does not explain why MSPs of these two species are indistinguishable.

Regarding the overall clade structure of the MSP dendrogram, some disagreements in the phylogeny between proteomic-based data and published phylogenetic data based on genomic data were observed [16]. It should be noted that the supplementary database was based on a single strain per fungal species, which is insufficient for taxonomic characterisation of fungal species. Future studies, improving the supplementary database, by including biological replicates, will be relevant to provide better characterisation and clarify discrepancies.

The supplementary database was first tested on previously acquired spectra from a study on drilling waste treatment facilities. Two unidentified isolates were now identified as A. caesiellus, demonstrating how already performed studies can easily be revised with supplementary fungal databases. Isolates identified to species level by the Bruker fungi library were not identified by the supplementary database, and thus it did not lead to misidentification.

The supplementary database was used to complement the Bruker database from three different environments. Isolates of xerophilic Aspergillus species were identified in two of these environments (homes and warehouse).

The museum samples did not reveal any xerophilic Aspergillus species using the supplementary database, despite other xerophilic/xerotolerant species being identified by the Bruker database from this environment. It should be mentioned that the museum environment from which these samples originate was not a closed, confined or climate-controlled site, and may not have favoured the growth of the specific xerophilic Aspergillus species included in this study.

The warehouse was selected for this study because the walls were described as being dry and yet had fungal growth on the walls, and therefore the presence of airborne xerophilic fungi could be expected to be present. In line with this, the supplementary database detected two species, A. glabripes and A. pseudogracilis. Other xerophilic/xerotolerant were also identified from this environment by the Bruker database, including A. glaucus, Aspergillus pseudoglaucus, P. brevicompactum and Penicillium corylophilum. The low number of fungal species detected in this environment could be explained by the relatively short sampling time for the GSP samplers [11–21 min for the personal samplers (n = 6) and 240 min for the stationary samplers (n = 3)], resulting in a small sampled volume. The sampling for personal samplers covered the period the workers had tasks in the warehouse in the morning.

We analysed samples from 27 randomly selected homes. The xerophilic Aspergillus species identified by the supplementary database (A. domesticus, A. glabripes and A. pseudogracilis) constituted only a small part of the potential exposure to fungi in the homes. This is in accordance with what could be expected, as fungi in home environments may have several sources and not only sources supporting growth of xerophilic fungi. The relatively low concentration of xerophilic Aspergillus species, on the one hand, suggests that these species do not pose a problem in these homes. On the other hand, their presence, combined with the fact that methods selecting for these xerophilic fungi have not been widely used in previous studies in homes, highlights the need for further studies on their prevalence in homes. Previous studies in homes also using EDCs for sampling have typically used DG18 agar [45] and SAB agar [46]. The most common species observed are in accordance with what has previously been found in homes, such as certain Cladosporium (Cladosporium sphaerospermum, C. herbarum, Cladosporium cladosporides), Penicillium (P. brevicompactum, P. camemberti, P. chrysogenum, Penicillium citrinum, Penicillium commume, Penicillium glabrum, P. olsonii) and Aspergillus (A. glaucus) species [17,47,48].

The type of agar media tended to have an effect on detection of xerophilic/xerotolerant species. With the exception of A. caesiellus (Fig. A1), all xerophilic Aspergillus species from the supplementary database were only detected on MY50. Some xerophilic/tolerant species, such as P. brevicompactum/olsonii, P. chrysogenum, C. cladosporioides, C. herbarum, A. pseudoglaucus and A. glaucus, were detected regardless of the media used. It should be noted that the MY50 plates were incubated longer than DG18, MEA and SAB agar plates as it takes more time for fungi to grow on this water-restricted medium. One explanation as to why the xerophilic Aspergillus species in the supplementary database were not detected on DG18 (with exception of A. caesiellus, Fig. A1), and that fewer xerophilic/xerotolerant fungi were observed on other media, might be that they were outcompeted by faster growing species on less restrictive media.

As described previously in this discussion, the broth medium also has an impact on the creation of MSPs for the new database, and raw mass spectra of A. halophilicus could only be obtained if it was inoculated in MY50 or MY70 while A. clavatophorus only formed raw mass spectra on SAB agar. These results, along with other studies [49], underline the importance of appropriate media (i.e., low a_w media) and incubation time, both for constructing and for using the database.

Xerophilic/xerotolerant Aspergillus species were detected across multiple environments and sample types though they constituted only a small fraction of all fungi. For future studies, we recommend incorporating low activity media (e.g., MY50) into both initial plating and broth cultivation protocols to obtain more knowledge on the presence of xerophilic/xerotolerant fungi in indoor environments. While this may not be necessary for routine surveys, it could be warranted in cases where environmental conditions, such as dry, climate-controlled buildings, suggest a potential niche for xerophilic species. The data from the warehouse, along with prior reports from museum settings [26,50], indicates that xerophilic fungi may persist even under stringent environmental controls. As such, targeted methods may be crucial for accurate assessment of fungal exposure in vulnerable settings.