Screening Mold Colonies by Using Two Toxicity Assays Revealed Indoor Strains of Aspergillus calidoustus Producing Ophiobolins G and K

Abstract

The occurrence and toxin production of the opportunistic pathogen Aspergillus calidoustus in Finnish buildings is not well documented in the literature. We tracked and identified four A. calidoustus colonies cultivated from indoor settled dusts and revealed the biological activities of crude biomass extracts. The toxic substances were identified as 6-epi-ophiobolin K, ophiobolin K, and ophiobolin G by high-performance liquid chromatography–mass spectrometry (HPLC-MS) based on chromatographic and mass spectrometry data (MS and MS/MS) on the crude extract of A. calidoustus strain MH34. A total of 29 fungal colonies collected from settled dust in an office room reported for indoor-air-related illnesses were screened for toxins that inhibited boar sperm motility in the BSMI (boar sperm motility inhibiting) assay and cell proliferation in the ICP (inhibition of cell proliferation) assays with PK-15 cells. Out of the 27 colonies tested as toxic, 12 colonies exhibiting conidiophores representative of the genera Chaetomium, Penicillium, and Paecilomyces were excluded from the study, while 13 colonies exhibited Aspergillus-like conidiophores. Biomass suspensions of these colonies were divided into two categories: Category 1 colonies (n = 4), toxic in the BSMI assay and the ICP assays, emitted blue fluorescence and grew at 37 °C; Category 2 colonies (n = 9), only toxic in the ICP assay, emitted orange fluorescence and exhibited limited or no growth at 37 °C. Colonies in Category 1 were pure-cultured, and the strains were named as MH4, MH21, MH34, MH36. Strain MH34 was identified as A. calidoustus by the internal transcribed spacer (ITS) sequences. Ethanol-soluble dry substances extracted from the biomass of the pure cultures exhibited a toxicological profile in the BSMI assay, SMID (sperm membrane integrity damage) assay, and ICP assay similar to that exhibited by pure ophiobolin A. Overall, the viable conidia of A. calidoustus in indoor settled dusts deserve attention when potentially hazardous mold species are monitored.

Detection of Chaetomium globosum, Ch. cochliodes and Ch. rectangulare during the Diversity Tracking of Mycotoxin-Producing Chaetomium-like Isolates Obtained in Buildings in Finland

“…Lethal toxicity was measured as sperm membrane integrity disruption assay with motile spermatozoa (SMID M ), was assessed by staining with PI as described [ 18 ] with modifications. Aliquots of 50 μL PBS were pipetted into a microtiter plate.…”

“…The methods used for cultivating the mold colonies on malt extract agar (15 g malt extract from Sharlab, Spain, and 12 g of agar from Amresco, Solon OH, USA, in 500 mL of H 2 O) were described previously [17,18]. After three weeks of incubation, the colonies on the primary isolation plates (not yet single spored) were numbered and screened for toxicity [17,18].…”

“…The toxicity assays, namely BSMI assay after exposure for 30 min (rapid inhibition) and for one day (slow inhibition), SMID assay and ICP assay, were all described previously [15,16]. The toxicity assays involving the ethanol extracts obtained from pure fungal cultures were performed using porcine cells (sperms and somatic cell lines) as indicators according to methods described before [13,18,41,50]. The calculation of EC 50 -the half maximal effective concentration for the ethanol-dry substances and pure mycotoxins in the BSMI, SMID and ICP assays-were described previously [15,18].…”

“…They regulate metabolic and ecological processes essential for a producer [ 10 ]. In mammalian cells, they may interfere with cellular structures (e.g., biomembranes) and with important cellular processes (e.g., protein, RNA and DNA syntheses; ion homeostasis; energy metabolism; and mitochondrial functions) [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. Most mycotoxins exert immunosuppressive effects, and many of them are cytotoxic/cytostatic and thus could potentially damage the skin and lungs and could affect the gut microbiota [ 19 , 20 , 21 , 22 ].…”

Bioreactivity, Guttation and Agents Influencing Surface Tension of Water Emitted by Actively Growing Indoor Mould Isolates

“…Characterization of the indoor microbial isolates proceeded as follows: biomass lysates of fungal colonies were initially categorized based on toxic responses in two toxicity assays: BSMI (boar sperm motility inhibition assay) and ICP (inhibition of cell proliferation assay) and autofluorescence as described earlier [38,40]. The obtained categories were further identified as morphotypes down to the genus level based on colony morphology on MEA, ability to grow at 37 • C, conidiophore morphology and conidial size as seen under light microscope (Olympus CKX41, Tokyo, Japan; magnification 400×, image recording software Cellsense ® standard version 11.0.06) [40][41][42]. Selected representatives of the morphotypes were identified during previous studies by ITS and tef1αsequencing with the primer pairs ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 )/ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) and EF595F (5 -CGTGACTTCATCAAGAAGATG-3 )/EF1160R (5 -CCGATCTTGTAGACGTCCTG-3 ), respectively (see Table 1 for GenBank accession numbers of the resulted sequences), followed by sequence analysis performed with Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov), and/or identified by DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) or WI (Westerdijk Institute, Wageningen, The Netherlands).…”