An atypical Bacillus anthracis infection in a bull-A potential occupational health hazard.

Bacillus anthracis infecting cattle is usually identified based on the typical symptom: sudden death. Bacillus anthracis causing atypical symptoms may remain undiagnosed and represent a potential occupational health hazard for, that is veterinarians and producers, butchers and tanners. In the year 2004, one case of sudden death in a dairy farm in southern Finland was diagnosed as bovine anthrax. Four years later 2008, an atypical case of anthrax was diagnosed in the same holding. The bull was taken to the Production Animal Hospital of the Faculty of Veterinary Medicine, University of Helsinki because of fever, loss of appetite and a symmetrically swollen scrotal sac. Penicillin treatment cured the fever but not the swollen scrotum. Before the intended therapeutic castration, a punctuate consisting of 10 ml fluid collected into a syringe from the scrotal sac was cultivated on blood agar at 37°C. After 24 hr, an almost pure culture of a completely non-hemolytic Bacillus cereus-like bacteria was obtained. The strain was identified as B. anthracis using Ba-specific primers by the Finnish Food Safety Authority (RUOKAVIRASTO). After the diagnosis, the bull was euthanized and destroyed, the personnel were treated with prophylactic antibiotics and the clinic was disinfected. In this particular case, treatment with water, Virkon S and lime seemed to be effective to eliminate endospores and vegetative cells since no relapses of anthrax have occurred in 10 years. This case is the last reported anthrax case in Finland.

The peptide toxin amylosin of Bacillus amyloliquefaciens from moisture-damaged buildings is immunotoxic, induces potassium efflux from mammalian cells, and has antimicrobial activity.

Amylosin, a heat-stable channel-forming non-ribosomally synthesized peptide toxin produced by strains of Bacillus amyloliquefaciens isolated from moisture-damaged buildings, is shown in this paper to have immunotoxic and cytotoxic effects on human cells as well as antagonistic effects on microbes. Human macrophages exposed to 50 ng of amylosin ml(-1) secreted high levels of cytokines interleukin-1ß (IL-1ß) and IL-18 within 2 h, indicating activation of the NLRP3 inflammasome, an integral part of the innate immune system. At the same exposure level, expression of IL-1ß and IL-18 mRNA increased. Amylosin caused dose-dependent potassium ion efflux from all tested mammalian cells (human monocytes and keratinocytes and porcine sperm cells) at 1 to 2 µM exposure. Amylosin also inhibited the motility of porcine sperm cells and depolarized the mitochondria of human keratinocytes. Amylosin may thus trigger the activation of the NLRP3 inflammasome and subsequently cytokine release by causing potassium efflux from exposed cells. The results of this study indicate that exposure to amylosin activates the innate immune system, which could offer an explanation for the inflammatory symptoms experienced by occupants of moisture-damaged buildings. In addition, the amylosin-producing B. amyloliquefaciens inhibited the growth of both prokaryotic and eukaryotic indoor microbes, and purified amylosin also had an antimicrobial effect. These antimicrobial effects could make amylosin producers dominant and therefore significant causal agents of health problems in some moisture-damaged sites.

Toxicity Screening of Fungal Extracts and Metabolites, Xenobiotic Chemicals, and Indoor Dusts with In Vitro and Ex Vivo Bioassay Methods.

It is controversial how useful bioassays are for identifying the in vivo toxicity of hazardous environmental exposures. In this study, fruiting bodies of forest mushrooms (n = 46), indoor mold colonies (n = 412), fungal secondary metabolites (n = 18), xenobiotic chemicals such as biocides and detergents (n = 6), and methanol extracts of indoor dusts from urban buildings (n = 26) were screened with two different bioactivity assays: boar sperm motility inhibition (BSMI) and inhibition of cell proliferation (ICP) tests. For the forest mushrooms, the toxicity testing result was positive for 100% of poisonous-classified species, 69% of non-edible-classified species, and 18% of edible-classified species. Colonies of 21 isolates of Ascomycota mold fungal species previously isolated from water-damaged buildings proved to be toxic in the tests. Out of the fungal metabolites and xenobiotic chemicals, 94% and 100% were toxic, respectively. Out of the indoor dusts from moldy-classified houses (n = 12) and from dry, mold-free houses (n = 14), 50% and 57% were toxic, respectively. The bioassay tests, however, could not differentiate the samples from indoor dusts of moldy-classified buildings from those from the mold-free buildings. Xenobiotic chemicals and indoor dusts were more toxic in the BSMI assay than in the ICP assay, whereas the opposite results were obtained with the Ascomycota mold colonies and fungal secondary metabolites. The tests recognized unknown methanol-soluble thermoresistant substances in indoor settled dusts. Toxic indoor dusts may indicate a harmful exposure, regardless of whether the toxicity is due to xenobiotic chemicals or microbial metabolites.

Potato crop as a source of emetic Bacillus cereus and cereulide-induced mammalian cell toxicity.

Bacillus cereus, aseptically isolated from potato tubers, were screened for cereulide production and for toxicity on human and other mammalian cells. The cereulide-producing isolates grew slowly, the colonies remained small (~1 mm), tested negative for starch hydrolysis, and varied in productivity from 1 to 100 ng of cereulide mg (wet weight)(-1) (~0.01 to 1 ng per 10(5) CFU). By DNA-fingerprint analysis, the isolates matched B. cereus F5881/94, connected to human food-borne illness, but were distinct from cereulide-producing endophytes of spruce tree (Picea abies). Exposure to cell extracts (1 to 10 µg of bacterial biomass ml(-1)) and to purified cereulide (0.4 to 7 ng ml(-1)) from the potato isolates caused mitochondrial depolarization (loss of ΔΨm) in human peripheral blood mononuclear cells (PBMC) and keratinocytes (HaCaT), porcine spermatozoa and kidney tubular epithelial cells (PK-15), murine fibroblasts (L-929), and pancreatic insulin-producing cells (MIN-6). Cereulide (10 to 20 ng ml(-1)) exposed pancreatic islets (MIN-6) disintegrated into small pyknotic cells, followed by necrotic death. Necrotic death in other test cells was observed only after a 2-log-higher exposure. Exposure to 30 to 60 ng of cereulide ml(-1) induced K(+) translocation in intact, live PBMC, keratinocytes, and sperm cells within seconds of exposure, depleting 2 to 10% of the cellular K(+) stores within 10 min. The ability of cereulide to transfer K(+) ions across biological membranes may benefit the producer bacterium in K(+)-deficient environments such as extracellular spaces inside plant tissue but is a pathogenic trait when in contact with mammalian cells.

In Search of Clinical Markers: Indicators of Exposure in Dampness and Mold Hypersensitivity Syndrome (DMHS).

Potential markers were sought to diagnose mold hypersensitivity. Indoor air condensed water and human macrophage THP-1 test were applied to evaluate the buildings. Basophil activation tests (BAT) were conducted and mold-specific immunoglobulins (IgE, IgG, IgA, and IgD) were measured in study subjects' serum and feces. Exposed subjects reported markedly more symptoms from occupational air than controls. Basophils from exposed subjects died/lost activity at 225 times lower concentrations of toxic extracts from the target building than recommended in the common BAT protocol. Fecal IgG and IgD levels against Acrostalagmus luteoalbus and Aspergillus versicolor produced receiver operating curves (ROC) of 0.928 and 0.916, respectively, when plotted against the inflammation marker MRP8/14. Assaying serum immunoglobulin concentrations against the toxic Chaetomium globosum (MTAV35) from another building, a test control, did not differentiate study individuals. However, if liver metabolism produced the same core molecule from other Chaetomium globosum strains, this would explain the increased response in fecal immunoglobulins in the exposed. The altered immunoglobulin values in the samples of exposed when compared to controls revealed the route of mold exposure. The toxicity of indoor air condensed water samples, BAT and serology confirmed the severity of symptoms in the target building's employees, supporting earlier findings of toxicity in this building.

Biomonitoring of Indoor Air Fungal or Chemical Toxins with Caenorhabditis elegans nematodes.

Bad indoor air quality due to toxins and other impurities can have a negative impact on human well-being, working capacity and health. Therefore, reliable methods to monitor the health risks associated with exposure to hazardous indoor air agents are needed. Here, we have used transgenic Caenorhabditis elegans nematode strains carrying stress-responsive fluorescent reporters and evaluated their ability to sense fungal or chemical toxins, especially those that are present in moisture-damaged buildings. Liquid-based or airborne exposure of nematodes to mycotoxins, chemical agents or damaged building materials reproducibly resulted in time- and dose-dependent fluorescent responses, which could be quantitated by either microscopy or spectrometry. Thus, the C. elegans nematodes present an easy, ethically acceptable and comprehensive in vivo model system to monitor the response of multicellular organisms to indoor air toxicity.

Cereulide produced by Bacillus cereus increases the fitness of the producer organism in low-potassium environments.

Cereulide, produced by certain Bacillus cereus strains, is a lipophilic cyclic peptide of 1152 Da that binds K(+) ions with high specificity and affinity. It is toxic to humans, but its role for the producer organism is not known. We report here that cereulide operates for B. cereus to scavenge potassium when the environment is growth limiting for this ion. Cereulide-producing B. cereus showed higher maximal growth rates (µ(max)) than cereulide non-producing B. cereus in K(+)-deficient medium (K(+) concentration ~1 mM). The cereulide-producing strains grew faster in K(+)-deficient than in K(+)-rich medium with or without added cereulide. Cereulide non-producing B. cereus neither increased µ(max) in K(+)-deficient medium compared with K(+)-rich medium, nor benefited from added cereulide. Cereulide-producing strains outcompeted GFP-labelled Bacillus thuringiensis in potassium-deficient (K(+) concentration ~1 mM) but not in potassium-rich (K(+) concentration ~30 mM) medium. Exposure to 2 µM cereulide in potassium-free medium lacking an energy source caused, within seconds, a major efflux of cellular K(+) from B. cereus not producing cereulide as well as from Bacillus subtilis. Cereulide depleted the cereulide non-producing B. cereus and B. subtilis cells of a major part of their K(+) stores, but did not affect cereulide-producing B. cereus strains. Externally added 6-10 µM cereulide triggered the generation of biofilms and pellicles by B. cereus. The results indicate that both endogenous and externally accessible cereulide supports the fitness of cereulide-producing B. cereus in environments where the potassium concentration is low.

Psychrotolerant Paenibacillus tundrae isolates from barley grains produce new cereulide-like depsipeptides (paenilide and homopaenilide) that are highly toxic to mammalian cells.

Paenilide is a novel, heat-stable peptide toxin from Paenibacillus tundrae, which colonizes barley. P. tundrae produced 20 to 50 ng of the toxin mg(-1) of cells (wet weight) throughout a range of growth temperatures from +5°C to +28°C. Paenilide consisted of two substances of 1,152 Da and 1,166 Da, with masses and tandem mass spectra identical to those of cereulide and a cereulide homolog, respectively, produced by Bacillus cereus NS-58. The two components of paenilide were separated from those of cereulide by high-performance liquid chromatography (HPLC), showing a structural difference suggesting the replacement of O-Leu (cereulide) by O-Ile (paenilide). The exposure of porcine spermatozoa and kidney tubular epithelial (PK-15) cells to subnanomolar concentrations of paenilide resulted in inhibited motility, the depolarization of mitochondria, excessive glucose consumption, and metabolic acidosis. Paenilide was similar to cereulide in eight different toxicity endpoints with porcine and murine cells. In isolated rat liver mitochondria, nanomolar concentrations of paenilide collapsed respiratory control, zeroed the mitochondrial membrane potential, and induced swelling. The toxic effect of paenilide depended on its high lipophilicity and activity as a high-affinity potassium ion carrier. Similar to cereulide, paenilide formed lipocations, i.e., lipophilic cationic compounds, with K(+) ions already at 4 mM [K(+)], rendering lipid membranes electroconductive. Paenilide-producing P. tundrae was negative in a PCR assay with primers specific for the cesB gene, indicating that paenilide was not a product of plasmid pCER270, encoding the biosynthesis of cereulide in B. cereus. Paenilide represents the first potassium ionophoric compound described for Paenibacillus. The findings in this paper indicate that paenilide from P. tundrae is a potential food-poisoning agent.

The Toxicity of Wiped Dust and Airborne Microbes in Individual Classrooms Increase the Risk of Teachers' Work-Related Symptoms: A Cross-Sectional Study.

BACKGROUND: The causes and pathophysiological mechanisms of building-related symptoms (BRS) remain open. OBJECTIVE: We aimed to investigate the association between teachers' individual work-related symptoms and intrinsic in vitro toxicity in classrooms. This is a further analysis of a previously published dataset. METHODS: Teachers from 15 Finnish schools in Helsinki responded to the symptom survey. The boar sperm motility inhibition assay, a sensitive indicator of mitochondrial dysfunction, was used to measure the toxicity of wiped dust and cultured microbial fallout samples collected from the teachers' classrooms. RESULTS: 231 teachers whose classroom toxicity data had been collected responded to the questionnaire. Logistic regression analysis adjusted for age, gender, smoking, and atopy showed that classroom dust intrinsic toxicity was statistically significantly associated with the following 12 symptoms reported by teachers (adjusted ORs in parentheses): nose stuffiness (4.1), runny nose (6.9), hoarseness (6.4), globus sensation (9.0), throat mucus (7.6), throat itching (4.4), shortness of breath (12.2), dry cough (4.7), wet eyes (12.7), hypersensitivity to sound (7.9), difficulty falling asleep (7.6), and increased need for sleep (7.7). Toxicity of cultured microbes was found to be associated with nine symptoms (adjusted ORs in parentheses): headache (2.3), nose stuffiness (2.2), nose dryness (2.2), mouth dryness (2.8), hoarseness (2.2), sore throat (2.8), throat mucus (2.3), eye discharge (10.2), and increased need for sleep (3.5). CONCLUSIONS: The toxicity of classroom dust and airborne microbes in boar sperm motility inhibition assay significantly increased teachers' risk of work-related respiratory and ocular symptoms. Potential pathophysiological mechanisms of BRS are discussed.

Chaetomium and Chaetomium-like Species from European Indoor Environments Include Dichotomopilus finlandicus sp. nov.

The genus Chaetomium is a frequently occurring fungal taxon world-wide. Chaetomium and Chaetomium-like species occur in indoor environments, where they can degrade cellulose-based building materials, thereby causing structural damage. Furthermore, several species of this genus may also cause adverse effects on human health. The aims of this research were to identify Chaetomium and Chaetomium-like strains isolated from indoor environments in Hungary and Finland, two geographically distant regions of Europe with drier and wetter continental climates, respectively, and to study their morphological and physiological properties, as well as their extracellular enzyme activities, thereby comparing the Chaetomium and Chaetomium-like species isolated from these two different regions of Europe and their properties. Chaetomium and Chaetomium-like strains were isolated from flats and offices in Hungary, as well as from schools, flats, and offices in Finland. Fragments of the translation elongation factor 1α (tef1α), the second largest subunit of RNA polymerase II (rpb2) and ß-tubulin (tub2) genes, as well as the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster were sequenced, and phylogenetic analysis of the sequences performed. Morphological examinations were performed by stereomicroscopy and scanning electron microscopy. Thirty-one Chaetomium sp. strains (15 from Hungary and 16 from Finland) were examined during the study. The most abundant species was Ch. globosum in both countries. In Hungary, 13 strains were identified as Ch. globosum, 1 as Ch. cochliodes, and 1 as Ch. interruptum. In Finland, 10 strains were Ch. globosum, 2 strains were Ch. cochliodes, 2 were Ch. rectangulare, and 2 isolates (SZMC 26527, SZMC 26529) proved to be representatives of a yet undescribed phylogenetic species from the closely related genus Dichotomopilus, which we formally describe here as the new species Dichotomopilus finlandicus. Growth of the isolates was examined at different temperatures (4, 15, 20, 25, 30, 37, 35, 40, and 45 °C), while their extracellular enzyme production was determined spectrophotometrically.

Persistence strategies of Bacillus cereus spores isolated from dairy silo tanks.

Survival of Bacillus cereus spores of dairy silo tank origin was investigated under conditions simulating those in operational dairy silos. Twenty-three strains were selected to represent all B. cereus isolates (n = 457) with genotypes (RAPD-PCR) that frequently colonised the silo tanks of at least two of the sampled eight dairies. The spores were studied for survival when immersed in liquids used for cleaning-in-place (1.0% sodium hydroxide at pH 13.1, 75 degrees C; 0.9% nitric acid at pH 0.8, 65 degrees C), for adhesion onto nonliving surfaces at 4 degrees C and for germination and biofilm formation in milk. Four groups with different strategies for survival were identified. First, high survival (log 15 min kill < or =1.5) in the hot-alkaline wash liquid. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterised by slow germination in rich medium and well preserved viability when exposed to heating at 90 degrees C. Fourth, spores capable of germinating at 8 degrees C and possessing the cspA gene. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot-alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus.

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

The diversity of Chaetomium-like isolates in buildings in Finland is poorly documented. This paper describes a set of methods for rapid diversity tracking of 42 indoor Chaetomium-like isolates. These isolates were categorized based on their fluorescence emission, ascomatal hair morphology, responses in three bioassays and resistance/sensitivity to the wetting agent Genapol X-080. Thirty-nine toxigenic isolates were identified [Ch. globosum (n = 35), Ch. cochliodes (n = 2) and Ch. rectangulare (n = 2)]. These isolates were identified down to the species level by tef1α gene sequencing. The major toxic substances in the ethanol extracts of the Ch. globosum and Ch. cochliodes strains were chaetoglobosin, chaetoviridin A and C, chaetomugilin D and chaetomin, identified based on HPLC-UV and mass spectrometry data (MS and MS/MS). Ethanol extracts from pure Ch. globosum cultures exhibited a toxicological profile in the boar sperm motility inhibition assay (BSMI), sperm membrane integrity damage assay (SMID) and inhibition of cell proliferation (ICP) assay, similar to that exhibited by pure chaetoglobosin A. Overall, differences in fluorescence, morphology, toxicity profile, mycotoxin production and sensitivity to chemicals were consistent with those in tef1α sequencing results for species identification. The results indicate the presence of Ch. cochliodes and Ch. rectangulare in Finnish buildings, representing a new finding.

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

The secretion of metabolites in guttation droplets by indoor moulds is not well documented. This study demonstrates the guttation of metabolites by actively growing common indoor moulds. Old and fresh biomasses of indoor isolates of Aspergillus versicolor, Chaetomium globosum, Penicillium expansum, Trichoderma atroviride, T. trixiae, Rhizopus sp. and Stachybotrys sp. were compared. Metabolic activity indicated by viability staining and guttation of liquid droplets detected in young (<3 weeks old) biomass were absent in old (>6 months old) cultures consisting of dehydrated hyphae and dormant conidia. Fresh (<3 weeks old) biomasses were toxic more than 10 times towards mammalian cell lines (PK-15 and MNA) compared to the old dormant, dry biomasses, when calculated per biomass wet weight and per conidial particle. Surfactant activity was emitted in exudates from fresh biomass of T. atroviride, Rhizopus sp. and Stachybotrys sp. Surfactant activity was also provoked by fresh conidia from T. atroviride and Stachybotrys sp. strains. Water repealing substances were emitted by cultures of P. expansum, T. atroviride and C. globosum strains. The metabolic state of the indoor fungal growth may influence emission of liquid soluble bioreactive metabolites into the indoor air.

Corrigendum: Structural Diversity and Bioactivities of Peptaibol Compounds From the Longibrachiatum Clade of the Filamentous Fungal Genus Trichoderma.

[This corrects the article DOI: 10.3389/fmicb.2019.01434.].

Exposure to indoor air contaminants in school buildings with and without reported indoor air quality problems.

Reported indoor air quality (IAQ) complaints are common even in relatively new or renovated school buildings in Finland. However, detecting the causes for complaints with commonly used indoor air measurements is difficult. This study presents data on perceived and measured IAQ in six comprehensive school buildings in Finland. The aim of this study was to discover the possible differences of perceived and measured IAQ between schools with reported IAQ complaints and schools without reported IAQ complaints. The initial categorisation of schools with ('problematic schools') and without ('comparison schools') complaints was ensured via a validated indoor climate survey and a recently developed online questionnaire, which were completed by 186 teachers and 1268 students from the six schools. IAQ measurements of physical parameters, gaseous pollutants, particulate matter and bioaerosols were conducted in four problematic school buildings (26 classrooms) and two comparison school buildings (12 classrooms). Using air sampling as well as exhaust air filters and classroom settled dust to detect the presence of elevated concentrations of airborne cultivable microbes and pathogenic, toxigenic and mycoparasitic Trichoderma strains were the most indicative methods in distinguishing problematic schools from comparison schools. Other IAQ-related measurements did not detect clear differences between problematic and comparison schools, as the concentration levels were very low. The results indicate that the complaints reported by occupants could have been related to excess moisture or mould problems that had not been found or repaired. Ventilation pressure condition investigations and simultaneous exhaust and supply air filter dust culture should be addressed precisely in future studies.

Contrasting immunological effects of two disparate dusts - preliminary observations.

BACKGROUND: Modern lifestyle and urbanization have been associated with a raised risk for atopic diseases whereas early and long-term exposure to a farm environment confers protection against atopic sensitization. Immunomodulatory potential and microbiological characteristics of settled airborne dust from an urban house and a barn were examined. METHODS: Pulmonary inflammation was induced in mice by repeated intranasal administration of dusts. Monocyte-derived human dendritic cells (moDCs) were exposed to dusts followed by coculture with purified naïve T cells. Cytokine/chemokine mRNA and protein levels were analyzed by real-time polymerase chain reaction, enzyme-linked immunosorbent assay and flow cytometry. The dusts were analyzed by cloning and sequencing of 16S rRNA genes (290 sequences) for DNA, lipids, endotoxin and beta-glucan, by live-dead staining, viable counting, isolation and identification of pure cultures (n = 76). RESULTS: Repeated exposure to house dust elicited pulmonary eosinophilia in mice whereas exposure to barn dust elicited neutrophilic and lymphocytic airway inflammation. Stimulation of moDCs with urban house dust elicited expression of Th2-promoting OX40L and Jagged-1 costimulatory molecules. Dendritic cells (DCs) exposed to house dust directed naïve T cells towards Th2 responses. Exposure of DCs to barn dust elicited the development of Th1-dominated immune responses. Urban house dust contained bacterial debris almost exclusively of human commensal species (corynebacteria, streptococci) whereas barn dust comprised mainly intact, viable bacteria of high diversity and no commensal species. CONCLUSION: Contact to debris originating from human commensal bacteria in urban house dust elicited a Th2-type response whereas barn dust with high bacterial diversity directed the cells towards a Th1 response.

Novel mycotoxin from Acremonium exuviarum is a powerful inhibitor of the mitochondrial respiratory chain complex III.

A novel mycotoxin named acrebol, consisting of two closely similar peptaibols (1726 and 1740 Da), was isolated from an indoor strain of the mitosporic ascomycete fungus Acremonium exuviarum. This paper describes the unique mitochondrial toxicity of acrebol, not earlier described for any peptaibol. Acrebol inhibited complex III of the respiratory chain of isolated rat liver mitochondria (1 mg of protein mL(-1)) with an IC(50) of approximately 80 ng mL(-1) (50 nM) after a short preincubation, and 350 ng mL(-1) caused immediate and complete inhibition. Acrebol thus is a complex III inhibitor almost as potent as antimycin A and myxothiazol but completely different in structure. Similarly to myxothiazol but in contrast to antimycin A, acrebol decreased the level of mitochondrial superoxide anion detectable by chemiluminescent probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one. Unlike other peptaibols, acrebol in toxic concentrations did not increase the ionic and solute permeability of membranes of isolated rat liver mitochondria, did not induce disturbance of the ionic homeostasis or the osmotic balance of mitochondria, and did not release apoptogenic proteins like cytochrome c from the intermembrane space of mitochondria. In boar spermatozoa, acrebol inhibited the respiratory chain and caused ATP depletion by activation of the oligomycin-sensitive F(0)F(1)-ATPase, which resulted in the inhibition of the progressive movement. In mouse insulinoma MIN-6 cells, whose energy supply solely depends on oxidative phosphorylation, acrebol induced necrosis-like death. The pathophysiological relevance of these findings is discussed.

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

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.

Structural Diversity and Bioactivities of Peptaibol Compounds From the Longibrachiatum Clade of the Filamentous Fungal Genus Trichoderma.

This study examined the structural diversity and bioactivity of peptaibol compounds produced by species from the phylogenetically separated Longibrachiatum Clade of the filamentous fungal genus Trichoderma, which contains several biotechnologically, agriculturally and clinically important species. HPLC-ESI-MS investigations of crude extracts from 17 species of the Longibrachiatum Clade (T. aethiopicum, T. andinense, T. capillare, T. citrinoviride, T. effusum, T. flagellatum, T. ghanense, T. konilangbra, T. longibrachiatum, T. novae-zelandiae, T. pinnatum, T. parareesei, T. pseudokoningii, T. reesei, T. saturnisporum, T. sinensis, and T. orientale) revealed several new and recurrent 20-residue peptaibols related to trichobrachins, paracelsins, suzukacillins, saturnisporins, trichoaureocins, trichocellins, longibrachins, hyporientalins, trichokonins, trilongins, metanicins, trichosporins, gliodeliquescins, alamethicins and hypophellins, as well as eight 19-residue sequences from a new subfamily of peptaibols named brevicelsins. Non-ribosomal peptide synthetase genes were mined from the available genome sequences of the Longibrachiatum Clade. Their annotation and product prediction were performed in silico and revealed full agreement in 11 out of 20 positions regarding the amino acids predicted based on the signature sequences and the detected amino acids incorporated. Molecular dynamics simulations were performed for structural characterization of four selected peptaibol sequences: paracelsins B, H and their 19-residue counterparts brevicelsins I and IV. Loss of position R6 in brevicelsins resulted in smaller helical structures with higher atomic fluctuation for every residue than the structures formed by paracelsins. We observed the formation of highly bent, almost hairpin-like, helical structures throughout the trajectory, along with linear conformation. Bioactivity tests were performed on the purified peptaibol extract of T. reesei on clinically and phytopathologically important filamentous fungi, mammalian cells, and Arabidopsis thaliana seedlings. Porcine kidney cells and boar spermatozoa proved to be sensitive to the purified peptaibol extract. Peptaibol concentrations ≥0.3 mg ml-1 deterred the growth of A. thaliana. However, negative effects to plants were not detected at concentrations below 0.1 mg ml-1, which could still inhibit plant pathogenic filamentous fungi, suggesting that those peptaibols reported here may have applications for plant protection.

In vitro toxicity of cereulide on porcine pancreatic Langerhans islets.

Cereulide is a K(+) ionophore cytotoxic and mitochondriotoxic to primary cells and cell lines of human and other mammalian origins. It is a heat-stable, highly lipophilic (logK(ow) 5.96) peptide (1152 g mol(-1)) produced by certain strains of Bacillus cereus, a bacterium connected to emetic food poisonings. In this study the pancreatic toxicity of purified cereulide, and cereulide-containing bacterial extracts, was studied using fetal porcine Langerhans islets in culture. Exposure to 1ngml(-1) of purified cereulide caused necrotic cell death of the islet cells impairing their insulin content within 2 days. Cell extracts of cereulide-positive B. cereus strains connected to food poisoning or isolated from foodstuffs were toxic, corresponding to their measured cereulide content. Extracts of B. cereus strains producing or not producing the B. cereus diarrheal toxin, but no cereulide, were tolerated by the porcine islet cultures up to concentrations 1000-fold higher compared to extracts from strains containing cereulide, and up to exposure times of 7d. Cereulide thus was identified as the B. cereus-produced substance toxic towards porcine fetal Langerhans islets and beta cells.