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Role of autochthonous filamentous fungi in bioremediation of a soil historically contaminated with aromatic hydrocarbons.

Nine fungal strains isolated from an aged and heavily contaminated soil were identified and screened to assess their degradative potential. Among them, Allescheriella sp. strain DABAC 1, Stachybotrys sp. strain DABAC 3, and Phlebia sp. strain DABAC 9 were selected for remediation trials on the basis of Poly R-478 decolorization associated with lignin-modifying enzyme (LME) production. These autochthonous fungi were tested for the abilities to grow under nonsterile conditions and to degrade various aromatic hydrocarbons in the same contaminated soil. After 30 days, fungal colonization was clearly visible and was confirmed by ergosterol determination. In spite of subalkaline pH conditions and the presence of heavy metals, the autochthonous fungi produced laccase and Mn and lignin peroxidases. No LME activities were detected in control microcosms. All of the isolates led to a marked removal of naphthalene, dichloroaniline isomers, o-hydroxybiphenyl, and 1,1'-binaphthalene. Stachybotrys sp. strain DABAC 3 was the most effective isolate due to its ability to partially deplete the predominant contaminants 9,10-anthracenedione and 7H-benz[DE]anthracen-7-one. A release of chloride ions was observed in soil treated with either Allescheriella sp. strain DABAC 1 or Stachybotrys sp. strain DABAC 3, suggesting the occurrence of oxidative dehalogenation. The autochthonous fungi led to a significant decrease in soil toxicity, as assessed by both the Lepidium sativum L. germination test and the Collembola mortality test.

Anthraquinones↗

Production of reactive oxygen metabolites by opsonized fungi and bacteria isolated from indoor air, and their interactions with soluble stimuli, fMLP or PMA.

Changes in the levels of free intracellular calcium ([Ca2+]i) and the production of reactive oxygen metabolites (ROM) induced by opsonized indoor air fungi and bacteria in human polymorphonuclear leukocytes (PMNL) were measured. Moreover, modification of a chemotactic peptide (fMLP)-and a tumor promoter (PMA)-induced production of ROM by opsonized fungi and bacteria were studied. The cells were exposed to graded doses of opsonized Candida sp., Aspergillus sp., Cladosporium sp., Stachybotrys sp., Penicillium sp., Paecilomyces sp., or A4 or A91 Streptomyces sp. alone, or together with fMLP or PMA. All the organisms were isolated from air samples of mold-problem buildings. None of the fungi or bacteria induced changes in [Ca2+]i or the production of ROM without opsonization with human serum. Of all opsonized fungi and bacteria, only Candida sp. elevated [Ca2+]i. All fungi and bacteria, except Paecilomyces sp. and Stachybotrys sp., markedly increased the production of ROM in PMNL. Furthermore, A91 Streptomyces sp. and Aspergillus sp. amplified fMLP-induced production of ROM. Only Candida sp. increased PMA-induced phenomen that normally occurs in the lung, was required for biological activity of the fungi and bacteria. Amplification by opsonization of fungi- or bacteria-induced leukocyte activation revealed remarkable changes between these biologically active particles. The present results suggest that many indoor air fungi and bacteria may activate leukocytes to produce oxidative stress, perhaps associated with harmful effects in exposed individuals.

Adult↗

Mycotoxin production by indoor molds.

Fungal growth in buildings starts at a water activity (a(w)) near 0.8, but significant quantities of mycotoxins are not produced unless a(w) reaches 0.95. Stachybotrys generates particularly high quantities of many chemically distinct metabolites in water-damaged buildings. These metabolites are carried by spores, and can be detected in air samples at high spore concentrations. Very little attention has been paid to major metabolites of Stachybotrys called spirocyclic drimanes, and the precise structures of the most abundant of these compounds are unknown. Species of Aspergillus and Penicillium prevalent in the indoor environment produce relatively low concentrations of mycotoxins, with the exception of sterigmatocystins that can represent up to 1% of the biomass of A. versicolor at a(w)'s close to 1. The worst-case scenario for homeowners is produced by consecutive episodes of water damage that promote fungal growth and mycotoxin synthesis, followed by drier conditions that facilitate the liberation of spores and hyphal fragments.

Air Pollution, Indoor↗

Evaluation of fungal growth on cellulose-containing and inorganic ceiling tile.

Buildings with poor indoor air quality (IAQ) frequently have many areas with surface fungal contamination. Studies have demonstrated that certain fungal genera (e.g., Cladosporium, Penicillium, and Stachybotrys) are able to grow on building materials such as wallpaper, drywall, and ceiling tiles, particularly after water damage has occurred. Due to the increasing awareness of sick building syndrome (SBS), it has become essential to identify building materials that prevent the interior growth of fungi. The objective of this study was to identify building materials that would not support the growth of certain fungal genera, regardless of whether an external food source was made available. The growth of three fungal genera (Cladosporium, Penicillium, and Stachybotrys) was evaluated on cellulose-containing ceiling tile (CCT) and inorganic ceiling tile (ICT). Both types of ceiling tile were exposed to environmental conditions which can occur inside a building. Our results show that ICT did not support the growth of these three fungal genera while CCT did. Our data demonstrate that ICT could serve as an ideal replacement for CCT.

Cellulose↗

Effects of satratoxins and other macrocyclic trichothecenes on IL-2 production and viability of EL-4 thymoma cells.

The macrocyclic trichothecenes are a group of potent protein synthesis inhibitors that have been encountered in indoor air and food as a result of infestation by the fungus Stachybotrys. To evaluate the capacity of these mycotoxins to alter immune functions, the effects of satratoxin G, H, F, roridin A, and verrucarin A on interleukin 2 (IL-2) production and viability were evaluated in a murine T-cell model. EL-4 thymoma cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin and concurrently exposed to various concentrations of the trichothecenes. Enzyme-linked immunosorbent assay (ELISA) of supernatants revealed that IL-2 concentrations at 24 and 72 h were significantly increased in cultures that were incubated in the presence of 0.5 to 1 ng/ml of satratoxin H, 1 to 5 ng/ml of isosatratoxin F, 0.1 to 0.5 ng/ml of roridin A, and 0.25 to 0.5 ng/ml of verrucarin A. However, IL-2 levels at these time points were significantly depressed when incubated in the presence of higher concentrations of satratoxin G (> or =2.5 ng/ml), satratoxin H and isosatratoxin F (> or =5 ng/ml), and roridin A and verrucarin A (> or =1 ng/ml). Cell viability, as measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, was depressed by each of the trichothecenes in a concentration-dependent manner. MTT responses were significantly decreased by as little as 0.5 ng/ml satratoxin G, roridin A, and verrucarin A and by 2.5 ng/ml of isosatratoxin F and satratoxin H. When these data were compared to those found in EL-4 cells for the 8-ketotrichothecene vomitoxin (deoxynivalenol), a common food contaminant, the macrocyclic trichothecenes were at least 100 times more potent. The results indicate that, at low concentrations, macrocyclic trichothecenes as a group could superinduce IL-2 production even while partially decreasing cell viability, whereas higher concentrations suppressed cytokine production and were markedly cytotoxic. The capacity of these compounds to dysregulate cytokine production in a biphasic fashion may play an etiologic role in outbreaks of human illnesses associated with indoor Stachybotrys contamination.

Air Microbiology↗

Induction of cytotoxicity and production of inflammatory mediators in raw264.7 macrophages by spores grown on six different plasterboards.

Dampness and microbial growth in buildings are associated with respiratory symptoms in the occupants, but details of the phenomenon are not sufficiently understood. The current study examined the effects of growth conditions provided by six plasterboards on cytotoxicity and inflammatory potential of the spores of Streptomyces californicus, Penicillium spinulosum, Aspergillus versicolor, and Stachybotrys chartarum. The microbes were isolated from mold problem buildings and thereafter grown on six different plasterboards. The spores were harvested, applied to RAW264.7 macrophages (10(4), 10(5), 10(6) spores/10(6) cells), and evaluated 24 h after exposure for the ability to cause cytotoxicity and to stimulate production of nitric oxide (NO), interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6). The data indicate clear differences between spores of different microbes in their ability to induce the production of these inflammatory mediators and to cause cell death in macrophages. Also, for each microbe, the induction ability specifically depended on the brand of plasterboard. The spores of Streptomyces californicus collected from all plasterboards were the most potent at inducing NO and cytokine production. Cytotoxicity caused by P. spinulosum and Streptomyces californicus spores was consistent with NO, IL-1beta and IL-6 production induced by those microbes. However, the production of these inflammatory mediators by the spores of Stachybotrys chartarum was not parallel to their ability to cause cell death. The low productions of NO and cytokines were associated with high cytotoxicity caused by the spores of the A. versicolor. These data suggest that growth condition of microbes on different plasterboards affect the ability of microbial spores to induce inflammatory responses and cytotoxicity in macrophages.

Aspergillus↗

Modulation of lipopolysaccharide-induced proinflammatory cytokine production by satratoxins and other macrocyclic trichothecenes in the murine macrophage.

The satratoxins and other macrocyclic trichothecene mycotoxins are produced by Stachybotrys, a mold that is often found in water-damaged dwellings and office buildings. To test the potential immunomodulatory effects of these mycotoxins, RAW 264.7 murine macrophage cells were treated with various concentrations of satratoxin G (SG), isosatratoxin F (iSF), satratoxin H (SH), roridin A (RA), and verrucarin A (VA) for 48 h in the presence or absence of suboptimal concentra-tion of lipopolysaccharide (LPS, 50 ng/ml), and tumor necrosis factor-alpha (TNF-alpha ) and interleukin-6 (IL-6) production were assayed by enzyme-linked immunosorbent assay (ELISA). In LPS-stimulated cultures, TNF-alpha supernatant concentrations were significantly increased in the presence of 2.5, 2.5, and 1 ng/ml of SG, SH, and RA, respectively, whereas IL-6 concentrations were not affected by the same concentrations these macrocyclic trichothecenes. When cells that were treated with LPS and SG (2.5 ng/ml) were evaluated by real-time polymerase chain reaction (PCR),TNF-alpha mRNA was found to increase at 24, 36, and 48 h compared to control cells. At higher concentrations, cytokine production and cell viability were markedly impaired in LPS-stimulated cells. Without LPS stimulation, neither TNF-alpha, nor IL-6 was induced. These results indicate that low concentrations of macrocyclic trichothecenes superinduce expression of TNF-alpha, whereas higher concentrations of these toxins are cytotoxic and concurrently reduce cytokine production. The capacity of satratoxins and other macrocyclic trichothecenes to alter cytokine production may play an etiologic role in outbreaks of Stachybotrys-associated human illnesses.

Analysis of Variance↗

Correlation between the prevalence of certain fungi and sick building syndrome.

OBJECTIVE: To examine the role of fungi in the production of sick building syndrome. METHODS: A 22 month study in the United States of 48 schools (in which there had been concerns about health and indoor air quality (IAQ). Building indoor air and surface samples, as well as outdoor air samples were taken at all sites to look for the presence of fungi or their viable propagules. RESULTS: Five fungal genera were consistently found in the outdoor air and comprised over 95% of the outdoor fungi. These genera were Cladosporium (81.5%), Penicillium (5.2%), Chrysosporium (4.9%), Alternaria (2.8%), and Aspergillus (1.1%). At 20 schools, there were significantly more colony forming units per cubic metre (CFU/m3) (p < 0.0001) of propagules of Penicillium species in the air samples from complaint areas when compared with the outdoor air samples and the indoor air samples from noncomplaint areas. At five schools, there were more, although not significant (p = 0.10), Penicillium propagules in the air samples from complaint areas when compared with the outdoor air samples and the indoor air samples from noncomplaint areas. In 11 schools, the indoor air (complaint areas) fungal ratios were similar to that in the outdoor air. In these 11 schools Stachybotrys atra was isolated from swab samples of visible growth under wetted carpets, on wetted walls, or behind vinyl wall coverings. In the remaining 11 schools, the fungal ratios and CFU/m3 of air were not significantly different in different areas. Many of the schools took remedial action that resulted in an indoor air fungal profile that was similar to that outdoors. CONCLUSIONS: Propagules of Penicillium and Stachybotrys species may be associated with sick building syndrome.

Air Microbiology↗

Effects of amoebae on the growth of microbes isolated from moisture-damaged buildings.

Dampness, moisture, and mold in buildings are associated with adverse health outcomes. In addition to fungi and bacteria, amoebae have been found in moisture-damaged building materials. Amoebae and a growing list of bacteria have been shown to have mutual effects on each other's growth, but the interactions between amoebae and microbes common in moisture-damaged buildings have not been reported. We co-cultivated the amoeba Acanthamoeba polyphaga with bacteria and fungi isolated from moisture-damaged buildings in laboratory conditions for up to 28 days. The microbes selected were the bacteria Streptomyces californicus, Bacillus cereus, and Pseudomonas fluorescens, and the fungi Stachybotrys chartarum, Aspergillus versicolor, and Penicillium spinulosum. Fungi and bacteria generally benefited from the presence of the amoebae, whereas the growth of amoebae was hindered by Streptomyces californicus, Stachybotrys chartarum, and Bacillus cereus. Pseudomonas fluorescens slightly enhanced amoebae viability. Amoebae were indifferent to the presence of Aspergillus versicolor and Penicillium spinulosum. Thus, our results show that amoebae can alter the survival and growth of some microbes in moisture-damaged buildings.

Acanthamoeba↗

Overview of investigations into pulmonary hemorrhage among infants in Cleveland, Ohio.

Idiopathic pulmonary hemorrhage was diagnosed in 37 infants in the Cleveland, Ohio, area between 1993 and 1998. This rare disorder has been related to 12 deaths, including 7 originally thought to be sudden infant death syndrome. Thirty of the infants were African American, all of whom lived in a limited geographic area of eastern metropolitan Cleveland, an area of older housing stock. An investigation led by the Centers for Disease Control and Prevention has found an association with household exposure to a toxigenic mold, Stachybotrys chartarum, and other fungi. The rapidly growing lungs of young infants appear to be especially vulnerable to the toxins made by toxigenic molds. Environmental tobacco smoke was frequently present in the infants' homes and may be a trigger precipitating the acute bleeding. Stachybotrys, although not thought to be a common mold, is known to have a wide geographic distribution. An additional 101 cases of acute, idiopathic pulmonary hemorrhage have been reported in infants in the United States over the past 5 years. In this overview, the investigations are summarized, the clinical profile is described, the toxicity of S. chartarum is discussed, and pathophysiologic concepts are presented.

Animals↗

Bisabosquals, novel squalene synthase inhibitors. I. Taxonomy, fermentation, isolation and biological activities.

In the course of screening for yeast squalene synthase inhibitors, bisabosqual A was isolated from the culture broth of Stachybotrys sp. RF-7260. The related compounds bisabosquals B, C and D were also isolated from Stachybotrys ruwenzoriensis RF-6853. Bisabosquals inhibited squalene synthases. IC50 values of bisabosqual A against the microsomal squalene synthases from Saccharomyces cerevisiae, Candida albicans, HepG2 cell and rat liver were 0.43, 0.25, 0.95 and 2.5 microg/ml, respectively. Bisabosqual C exhibited inhibitory activities similar to bisabosqual A. Bisabosqual A showed broad spectrum antifungal activity in vitro.

Animals↗

Bisabosquals, novel squalene synthase inhibitors. II. Physico-chemical properties and structure elucidation.

The squalene synthase inhibitor bisabosqual A was isolated from the culture broth of Stachybotrys sp. RF-7260, and its structure was determined on the basis of spectroscopic methods including detailed 2D NMR analyses. The structures of bisabosquals B, C and D isolated from Stachybotrys ruwenzoriensis RF-6853 were determined by spectroscopic methods and chemical reactions. The absolute stereochemistry of bisabosquals A, B and D was determined by X-ray crystallographic analysis. They have novel cis-fused tetracyclic structures with a bisabolane-type sesquiterpene and phenol moieties.

Crystallography, X-Ray↗

Update: Pulmonary hemorrhage/hemosiderosis among infants--Cleveland, Ohio, 1993-1996.

A review within CDC and by outside experts of an investigation of acute pulmonary hemorrhage/hemosiderosis in infants has identified shortcomings in the implementation and reporting of the investigation described in MMWR and detailed in other scientific publications authored, in part, by CDC personnel. The reviews led CDC to conclude that a possible association between acute pulmonary hemorrhage/hemosiderosis in infants and exposure to molds, specifically Stachybotrys chartarum, commonly referred to by its synonym Stachybotrys atra, was not proven. This report describes the specific findings of these internal and external reviews.

Disease Outbreaks↗

Cobalt 60 radiation and growth of eleven species of micro-fungi from Evolution Canyon, Lower Nahal Oren, Israel.

Eleven micro-fungal species isolated from both the north facing slope (NFS) and the south facing slope (SFS) of Evolution Canyon, Lower Nahal Oren, Israel, were examined for growth rates before and after exposure to 60Co irradiation. Species of Alternaria, Aspergillus, Humicola, Oidiodendron, and Staphylotrichum from SFS grew faster than the NFS isolates while Fusarium, Sordaria, and Stachybotrys grew at greater rates from the NFS than from the SFS. Mucor and Ulocladium isolates grew at the same rate from both SFS and NFS. The eleven isolates from each slope were next subjected to 60Co irradiation. At 40,000 rads exposure, Alternaria, Fusarium, and Stachybotrys grew more rapidly when isolated from the NFS, while Humicola and Staphylotrichum grew at a faster rate when isolated from the SFS. Aspergillus, Mucor, Sordaria, and Ulocladium from both the NFS and the SFS had relatively the same growth rate at 40,000 rads exposure. At 400,000 rads exposure, growth rates remained much the same for both the N and S exposed isolates as they were at 40,000 rads. Above 10(6) rads, growth ceased but recovery occurred at various times for individual isolates of the same species from opposing canyon slopes.

Alternaria↗

Microfungi in cultivated fields in Eskişehir provience (Turkey).

The soil microfungi flora was investigated in four locations of Eskişehir (Turkey). 56 soil samples were seasonaly collected from 14 stations in the areas of Karacahöyük, Bahçecik, OGU I, and OGU II. A total of 110 species belonging to 32 genera were encountered including Absidia, Acremonium, Alternaria, Aspergillus, Beauveria, Botryoderma, Chaetomium, Chrysosporium, Cladosporium, Eupenicillium, Eurotium, Fusarium, Geotrichum, Gliocladium, Gonytrichum, Metarrhizium, Mucor, Myrothecium, Paecilomyces, Penicillium, Phoma, Plectosphaerella, Rhizoctania, Rhizopus, Scopulariopsis, Septonema, Stachybotrys, Trichocladium, Trichoderma, Ulocladium, Verticillium, and Wardomyces. Twenty five species were more frequent (all locations) while twenty seven species were rare (only one sample). Mainly, Acremonium kiliense, Aspergillus ochraceus, A. terricola var. americanus, A. versicolor, Cladosporium cladosporioides, Fusarium oxysporum, F. solani, Gliocladium roseum, Penicillium chrysogenum, P. corylophum, P. expansum, P. griseofulvum, P. implicatum, P. restrictum, and Stachybotrys chartarum were the most common and abundant microfungi in all locations. Five species Aspergillus subsessilis, A. terreus var. africanus, Eupenicillium egyptiacum, Paecilomyces ramosus, and Penicillium novae-zeelandiae are likely to be newly recorded for Turkey. The microfungi number in Eskişehir soils was between 25,000-234,000 CFU/g (mean value at 126,375 CFU/g).

Fungi↗

Mycoflora of air-conditioners dust from Riyadh, Saudi Arabia.

Using the hair baiting technique, 6 genera and 14 species were collected on Sabouraud's dextrose agar from 37 dust samples from air-conditioners. The most common fungi were Chrysosporium tropicum, C. indicum, C. keratinophilum, Aspergillus flavus followed by Acremonium strictum and Scopulariopsis brevicaulis. Using the dilution-plate method, 26 genera and 52 species were collected from 37 dust samples on glucose-(23 genera and 45 species) and cellulose-(18 genera and 34 species) Czapek's agar at 28 degrees C. The most prevalent species were Aspergillus niger, A. flavus, Penicillium chrysogenum, Stachybotrys chartarum, Ulocladium atrum, Mucor racemosus and Fusarium solani and A. niger, A. flavus, Trichoderma viride, P. chrysogenum, Ulocladium atrum, Chaetomium globosum, C. spirale, Stachybotrys chartarum and Mucor racemosus on the two media, respectively.

Air Conditioning↗

Fast methods for screening of trichothecenes in fungal cultures using gas chromatography-tandem mass spectrometry.

The paper presents a fast method for trichothecene profiling and chemotaxonomic studies in species of Fusarium, Stachybotrys. Trichoderma and Memnoniella. Micro scale extracted crude Fusarium extracts were derivatised using pentafluoropropionic anhydride and analysed by gas chromatography with simultaneous full scan and tandem mass spectrometric detection. It was possible to monitor for up to four compounds simultaneous, making detection of acetyl T-2 toxin, T-2 toxin, HT-2 toxin, T-2 triol. T-2 tetraol, neosolaniol, iso-neosolaniol, scirpentriol, 4,15-diacetoxyscirpenol, 15-acetoxyscirpenol, 4-acetoxyscirpentriol, nivalenol, fusarenon-X, deoxynivalenol, 15-acetyl-deoxynivalenol and 3-acetyldeoxynivalenol possible during a 23-min GC run. A slightly modified method could detect trichothecenes produced by Stachybotrys, Memnoniella and Trichoderma, by hydrolysing crude extracts prior to derivatisation with heptafluorobuturyl imidazole. All types of derivatised extracts could be reanalysed using negative ion chemical ionisation (NICI) GC-MS for molecular mass determination and verification purposes. A retention time index could be used for correction in retention time drifts between sequences and worked both in EI+ and NICI mode.

Fungi↗

Determination of the anamorph of Cordyceps sinensis inferred from the analysis of the ribosomal DNA internal transcribed spacers and 5.8S rDNA.

The anamorph determination of Cordyceps sinensis remains problematic due to the lack of clear links between the sexual and conidial forms of the fungus. In this study, we applied molecular approaches to analyze the genetic variation of Cordyceps sinensis and its allies to identify the anamorph-teleomorph connection. The sequences of the internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal RNA gene of Cordyceps sinensis (teleomorph) collected from Qingzang plateau (altitude over 4000m), Tibet and several related asexual conidial forms were determined. The sequence comparison showed that Cordyceps sinensis was most closely related to Hirsutella sinensis, and was clearly divergent from Paecilomyces sinensis, Stachybotrys sp. or Tolypocladium sp.; distance values, estimated according to Kimura two-parameter models between Cordyceps sinensis and Hirsutella sinensis, were extremely low (<0.02), whereas distance values between Cordyceps sinensis and Paecilomyces sinensis, Stachybotrys sp. and Tolypocladium sp. were 0.34, 0.21 and 0.25, respectively. Taken together, Hirsutella sinensis and Cordyceps sinensis are the different stages of the life cycle stages of the same organism. Hirsutella sinensis is therefore the anamorph of Cordyceps sinensis, rather than Paecilomyces sinensis or other species. The possible reasons as to why different taxa can be obtained when culturing Cordyceps sinensis are also discussed.

Journal Article↗