Artificial vs natural Stachybotrys infestation-Comparison of mycotoxin production on various building materials.

The genus Stachybotrys belongs to filamentous fungi found in indoor environment, mostly on cellulose-rich substrates after water-damage. The major purpose of this study was to investigate the influence of different building materials in case of mold infestation on the mycotoxin production of Stachybotrys species. Fifteen Stachybotrys mycotoxins including satratoxins, phenylspirodrimanes, and recently discovered stachybotrychromenes were in the focus of the investigations. Artificial and natural infestations were compared to determine whether environmental factors, for example, time of growth, temperature, humidity, and material additives have an influence on the observed mycotoxin profiles. It turned out that mycotoxin profiles from Stachybotrys spp. on building materials can be influenced by cellulose, paints, and paste of the materials. The total toxin levels of artificially and naturally contaminated gypsum board samples ranged up to 30 µg/cm2 , whereas wallpaper samples showed total toxin levels in the range of 20-66 µg/cm2 . A naturally infested sample disclosed the conversion of the dialdehyde components to the corresponding lactone isomers under the influence of light.

Cultivation and aerosolization of Stachybotrys chartarum for modeling pulmonary inhalation exposure.

Objective:Stachybotrys chartarum is a hydrophilic fungal species commonly found as a contaminant in water-damaged building materials. Although several studies have suggested that S. chartarum exposure elicits a variety of adverse health effects, the ability to characterize the pulmonary immune responses to exposure is limited by delivery methods that do not replicate environmental exposure. This study aimed to develop a method of S. chartarum aerosolization to better model inhalation exposures. Materials and methods: An acoustical generator system (AGS) was previously developed and utilized to aerosolize and deliver fungal spores to mice housed in a multi-animal nose-only exposure chamber. In this study, methods for cultivating, heat-inactivating, and aerosolizing two macrocyclic trichothecene-producing strains of S. chartartum using the AGS are described. Results and discussion: In addition to conidia, acoustical generation of one strain of S. chartarum resulted in the aerosolization of fungal fragments (<2 µm aerodynamic diameter) derived from conidia, phialides, and hyphae that initially comprised 50% of the total fungal particle count but was reduced to less than 10% over the duration of aerosolization. Acoustical generation of heat-inactivated S. chartarum did not result in a similar level of fragmentation. Delivery of dry, unextracted S. chartarum using these aerosolization methods resulted in pulmonary inflammation and immune cell infiltration in mice inhaling viable, but not heat-inactivated S. chartarum. Conclusions: These methods of S. chartarum growth and aerosolization allow for the delivery of fungal bioaerosols to rodents that may better simulate natural exposure within water-damaged indoor environments.

Nonsteroidal Anti-inflammatory Drugs (NSAIDS) Inhibit the Growth and Reproduction of Chaetomium globosum and Other Fungi Associated with Water-Damaged Buildings.

Indoor mold due to water damage causes serious human respiratory disorders, and the remediation to homes, schools, and businesses is a major expense. Prevention of mold infestation of building materials would reduce health problems and building remediation costs. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit yeasts and a limited number of filamentous fungi. The purpose of this research was to determine the possible inhibitory activity of nonsteroidal anti-inflammatory drugs (NSAIDs) on germination, fungal growth, and reproduction of Chaetomium globosum and other important filamentous fungi that occur in water-damaged buildings. Several NSAIDs were found to inhibit C. globosum germination, growth, and reproduction. The most effective NSAIDs inhibiting C. globosum were ibuprofen, diflunisal, and diclofenac. Fusarium oxysporum, Fusarium solani, Aspergillus niger, and Stachybotrys atra were also tested on the various media with similar results obtained. However, F. oxysporum and A. niger exhibited a higher level of resistance to aspirin and NaSAL when compared to the C. globosum isolates. The inhibition exhibited by NSAIDs was variable depending on growth media and stage of fungal development. These compounds have a great potential of inhibiting fungal growth on building materials such as gypsum board. Formulations of sprays or building materials with NSAID-like chemical treatments may hold promise in reducing mold in homes and buildings.

Identification of Stachybotrys spp. by MALDI-TOF mass spectrometry.

Stachybotrys (S.) spp. are omnipresent cellulolytic molds. Some species are highly toxic owing to their ability to synthesize various secondary metabolites such as macrocyclic trichothecenes or hemolysins. The reliable identification of Stachybotrys at species level is currently limited to genome-based identification. This study aimed to establish a fast and reliable MALDI-TOF MS identification method by optimizing the pre-analytical steps for protein extraction for subsequent generation of high-quality fingerprint mass spectra. Eight reference strains of the American Type Culture Collection and the Technical University of Denmark were cultivated in triplicate (biological repetitions) for 2 days in malt extract broth. The mycelia (1.5 ml) were first washed with 75 % ethanol and an additional washing step with dimethyl sulfoxide (10 %) was added to remove unspecific low weight masses. Furthermore, mycelia were broken with roughened glass beads in formic acid (70 %) and acetonitrile. The method was successfully applied to a total of 45 isolates of Stachybotrys originating from three different habitats (indoor, feed, and food samples; n = 15 each): Twenty-seven isolates of S. chartarum and 18 isolates of S. chlorohalonata could be identified by MALDI-TOF MS. The data obtained exactly matched those obtained by genome-based identification. The mean score values for S. chartarum ranged from 2.509 to 2.739 and from 2.148 to 2.622 for S. chlorohalonata with a very good reproducibility: the relative standard deviations were between 0.3 % and 6.8 %. Thus, MALDI-TOF MS proved to be a fast and reliable alternative to identification of Stachybotrys spp. by nucleotide amplification and sequencing.

Microbial volatile organic compound emissions from Stachybotrys chartarum growing on gypsum wallboard and ceiling tile.

BACKGROUND: Stachybotrys chartarum is a filamentous mold frequently identified among the mycobiota of water-damaged building materials. Growth of S. chartarum on suitable substrates and under favorable environmental conditions leads to the production of secondary metabolites such as mycotoxins and microbial volatile organic compounds (MVOCs). The aim of this study was to characterize MVOC emission profiles of seven toxigenic strains of S. chartarum, isolated from water-damaged buildings, in order to identify unique MVOCs generated during growth on gypsum wallboard and ceiling tile coupons. Inoculated coupons were incubated and monitored for emissions and growth using a closed glass environmental growth chamber maintained at a constant room temperature. Gas samples were collected from the headspace for three to four weeks using Tenax TA tubes. RESULTS: Most of the MVOCs identified were alcohols, ketones, ethers and esters. The data showed that anisole (methoxybenzene) was emitted from all of the S. chartarum strains tested on both types of substrates. Maximum anisole concentration was detected after seven days of incubation. CONCLUSIONS: MVOCs are suitable markers for fungal identification because they easily diffuse through weak barriers like wallpaper, and could be used for early detection of mold growth in hidden cavities. This study identifies the production of anisole by seven toxigenic strains of Stachybotrys chartarum within a period of one week of growth on gypsum wallboard and ceiling tiles. These data could provide useful information for the future construction of a robust MVOC library for the early detection of this mold.

Fungal and microbial volatile organic compounds exposure assessment in a waste sorting plant.

In the management of solid waste, pollutants over a wide range are released with different routes of exposure for workers. The potential for synergism among the pollutants raises concerns about potential adverse health effects, and there are still many uncertainties involved in exposure assessment. In this study, conventional (culture-based) and molecular real-time polymerase chain reaction (RTPCR) methodologies were used to assess fungal air contamination in a waste-sorting plant which focused on the presence of three potential pathogenic/toxigenic fungal species: Aspergillus flavus, A. fumigatus, and Stachybotrys chartarum. In addition, microbial volatile organic compounds (MVOC) were measured by photoionization detection. For all analysis, samplings were performed at five different workstations inside the facilities and also outdoors as a reference. Penicillium sp. were the most common species found at all plant locations. Pathogenic/toxigenic species (A. fumigatus and S. chartarum) were detected at two different workstations by RTPCR but not by culture-based techniques. MVOC concentration indoors ranged between 0 and 8.9 ppm (average 5.3 ± 3.16 ppm). Our results illustrated the advantage of combining both conventional and molecular methodologies in fungal exposure assessment. Together with MVOC analyses in indoor air, data obtained allow for a more precise evaluation of potential health risks associated with bioaerosol exposure. Consequently, with this knowledge, strategies may be developed for effective protection of the workers.

Cellobiose dehydrogenase influences the production of S. microspora ß-glucosidase.

BglG, a Stachybotrys microspora ß-glucosidase produced in the presence of glucose and cellobiose, was used individually as sole carbon source. The time course synthesis of BglG showed two aspects: (1) an exponential curve, observed in glucose Mandels medium, and (2) a cloche curve, observed in cellobiose containing cultures. A decrease was observed in bglG production at the 6th, 8th and 10th days during mycelium growth in cellobiose Mandels medium, which allowed for the assumption that the anabolism of a bglG inhibitor factor was produced with cellobiose but not with glucose. Cellobiose dehydrogenases (CDH) activity was, on another hand detected in cellobiose grown cultures but not in glucose containing ones. The aliquots, withdrawn at the time course of bglG production in the presence of cellobiose, gave rise to an inhibitory effect on bglG activity. This result was obtained with and without the heat treatment (5 min at 100°C) of the aliquots, which supported the non-proteinaceous nature of the inhibitor factor. Furthermore, sugar chromatographic analyses revealed the appearance of a secondary metabolite in the cellobiose Mandels medium and indicated that the factor behind the bglG activity cloche curve was a δ-gluconolactone. Seeing that the latter follows a strong inhibitory effect on bglG activity, it is speculated that the decrease in bglG activity during the time course of bglG synthesis in cellobiose Mandels medium is assigned to the release of δ-gluconolactone. This paper presents and validates an explanatory model for this hypothesis.

The influence of water activity and temperature on germination, growth and sporulation of Stachybotrys chartarum strains.

The objectives were to determine the influence of water activity (a(w), 0.997-0.92) and temperature (10-37°C) and their interactions on conidial germination, mycelial growth and sporulation of two strains of Stachybotrys chartarum in vitro on a potato dextrose medium. Studies were carried out by modifying the medium with glycerol and either spread plating with conidia to evaluate germination and germ tube extension or centrally inoculating treatment media for measuring mycelial growth rates and harvesting whole colonies for determining sporulation. Overall, germination of conidia was significantly influenced by a(w) and temperature and was fastest at 0.997-0.98 a(w) between 15 and 30°C with complete germination within 24 h. Germ tube extension was found to be most rapid at similar a(w) levels and 25-30°C. Mycelial growth rates of both strains were optimal at 0.997 a(w) between 25 and 30°C, with very little growth at 37°C. Sporulation was optimum at 30°C at 0.997 a(w). However, under drier conditions, this was optimum at 25°C. This shows that there are differences in the ranges of a(w) x temperature for germination and growth and for sporulation. This may help in understanding the role of this fungal species in damp buildings and conditions under which immune-compromised patients may be at risk when exposed to such contaminants in the indoor air environment.

Molecular and bioinformatics analyses reveal two differentially expressed intracellular GH1 ß-glucosidases from the rare alkalophilic fungus Stachybotrys microspora.

The present study reports the isolation and analysis of two novel GH1 ß-glucosidases from the alkalophilic fungus Stachybotrys microspora, using PCR and Nested-PCR. Three major gene fragments were obtained by PCR: the first two are very similar and constitute a novel gene, which was named Smbgl1A, and the third PCR fragment is part of a different gene, named Smbgl1B. The truncated gene sequences were completely filled using the recent partial whole genome sequencing data of S. microspora (data not yet published). Moreover, we investigated the relative effects of glucose in comparison to cellulose rather than evaluate their absolute effects. In fact, RT-PCR analysis showed that while Smbgl1A was expressed when the fungus was grown in the presence of cellulose but not when grown with glucose, Smbgl1B was equally expressed under both conditions. The putative catalytic residues and the conserved glycone binding sites were identified. Zymogram analysis showed the intracellular production of ß-glucosidases in S. microspora. The predicted secondary structure exhibited a classical (ß/α)8 barrel fold, showing that both SmBGL1A and SmBGL1B belong to the GH1 family. Phylogenetic studies showed that SmBGL1A and SmBGL1B belong to the same branch as ß-glucosidases from Stachybotrys chlorohalonata and Stachybotrys chartarum. However, SmBGL1A and SmBGL1B form two distinct clades.

Testing antimicrobial paint efficacy on gypsum wallboard contaminated with Stachybotrys chartarum.

The goal of this research was to reduce occupant exposure to indoor mold through the efficacy testing of antimicrobial paints. An accepted method for handling Stachybotrys chartarum-contaminated gypsum wallboard (GWB) is removal and replacement. This practice is also recommended for water-damaged or mold-contaminated GWB but is not always followed completely. The efficacy of antimicrobial paints to eliminate or control mold regrowth on surfaces can be tested easily on nonporous surfaces. The testing of antimicrobial efficacy on porous surfaces found in the indoor environment, such as gypsum wallboard, can be more complicated and prone to incorrect conclusions regarding residual organisms. The mold S. chartarum has been studied for toxin production and its occurrence in water-damaged buildings. Research to control its growth using seven different antimicrobial paints and two commonly used paints on contaminated, common gypsum wallboard was performed in laboratory testing at high relative humidity. The results indicate differences in antimicrobial efficacy for the period of testing, and that proper cleaning and resurfacing of GWB with an antimicrobial paint can be an option in those unique circumstances when removal may not be possible.

Stachybotrychromenes A-C: novel cytotoxic meroterpenoids from Stachybotrys sp.

In the course of gaining new insights into the secondary metabolite profile of various Stachybotrys strains, in particular concerning triprenyl phenol-like compounds, so far, unknown metabolites with analogous structural features were discovered. Three novel meroterpenoids containing a chromene ring moiety, namely stachybotrychromenes A-C, were isolated from solid culture of the filamentous fungus Stachybotrys chartarum DSMZ 12880 (chemotype S). Their structures were elucidated by means of comprehensive spectroscopic analysis (1D and 2D NMR, ESI-HRMS, and CD) as well as by comparison with spectroscopic data of structural analogues described in literature. Stachybotrychromenes A and B exhibited moderate cytotoxic effects on HepG2 cells after 24 h with corresponding IC50 values of 73.7 and 28.2 µM, respectively. Stachybotrychromene C showed no significant cytotoxic activity up to 100 µM. Moreover, it is noteworthy that stachybotrychromenes A-C are produced not only by S. chartarum chemotype S but also S. chartarum chemotype A and Stachybotrys chlorohalonata.

Ketamine intervention limits pathogen expansion in vitro.

Ketamine is one of several clinically important drugs whose therapeutic efficacy is due in part to their ability to act upon ion channels prevalent in nearly all biological systems. In studying eukaryotic and prokaryotic organisms in vitro, we show that ketamine short-circuits the growth and spatial expansion of three microorganisms, Stachybotrys chartarum, Staphylococcus epidermidis and Borrelia burgdorferi, at doses efficient at reducing depression-like behaviors in mouse models of clinical depression. Although our findings do not reveal the mechanism(s) by which ketamine mediates its antifungal and antibacterial effects, we hypothesize that a function of L-glutamate signal transduction is associated with the ability of ketamine to limit pathogen expansion. In general, our findings illustrate the functional similarities between fungal, bacterial and human ion channels, and suggest that ketamine or its metabolites not only act in neurons, as previously thought, but also in microbial communities colonizing human body surfaces.

Enhanced antimicrobial efficacy of thermal-reduced silver nanoparticles supported by titanium dioxide.

The antimicrobial efficacy of silver nanoparticles (AgNPs) is influenced by many factors, including the particle size, AgNP oxidation state and support materials. In this study, AgNPs are synthesized and supported by two types of TiO2 powders (P25 and Merck TiO2) using two heat-treatment temperatures (120 and 200°C). The formation of well-dispersed AgNPs with diameters ranging from 3.2 to 5.7nm was confirmed using transmission electron microscopy. X-ray photoelectron spectroscopy and X-ray diffraction indicated that the majority of the AgNPs were reduced from Ag+ to Ag0 at 200°C. The AgNP antimicrobial activity was determined by the zone of inhibition against three fungi, A. niger, P. spinulosum and S. chartarum, and two bacteria, E. coli (Gram-negative) and S. epidermidis (Gram-positive). The antimicrobial activity of metallic AgNPs was more pronounced than that of silver nitrate and some antimicrobial drugs. The AgNPs exhibited optimal antimicrobial efficacy when the AgNP dispersion on the surface of TiO2 was in the region between 0.2 and 0.7µg-Ag/m2. The minimum (critical) AgNP concentrations needed to inhibit the growth of bacteria (E. coli) and fungi (A. niger) were 13.48 and 25.4µg/mL, respectively. The results indicate that AgNPs/TiO2 nanocomposites are a promising disinfectant against both bacteria and fungi.

Airborne mold and endotoxin concentrations in New Orleans, Louisiana, after flooding, October through November 2005.

BACKGROUND: The hurricanes and flooding in New Orleans, Louisiana, in October and November 2005 resulted in damp conditions favorable to the dispersion of bioaerosols such as mold spores and endotoxin. OBJECTIVE: Our objective in this study was to assess potential human exposure to bioaerosols in New Orleans after the flooding of the city. METHODS: A team of investigators performed continuous airborne sampling for mold spores and endotoxin outdoors in flooded and nonflooded areas, and inside homes that had undergone various levels of remediation, for periods of 5-24 hr during the 2 months after the flooding. RESULTS: The estimated 24-hr mold concentrations ranged from 21,000 to 102,000 spores/m3 in outdoor air and from 11,000 to 645,000 spores/m3 in indoor air. The mean outdoor spore concentration in flooded areas was roughly double the concentration in nonflooded areas (66,167 vs. 33,179 spores/m3 ; p < 0.05) . The highest concentrations were inside homes. The most common mold species were from the genera of Cladosporium and Aspergillus/Penicillium ; Stachybotrys was detected in some indoor samples. The airborne endotoxin concentrations ranged from 0.6 to 8.3 EU (endotoxin units) /m3 but did not vary with flooded status or between indoor and outdoor environments. CONCLUSIONS: The high concentration of mold measured indoors and outdoors in the New Orleans area is likely to be a significant respiratory hazard that should be monitored over time. Workers and returning residents should use appropriate personal protective equipment and exposure mitigation techniques to prevent respiratory morbidity and long-term health effects.

Hazard identification, dose-response and environmental characteristics of stachybotryotoxins and other health-related products from Stachybotrys.

An extensive growth of Stachybotrys in water-damaged buildings is of great public health concern. It is inconclusive whether Stachybotrys is responsible for the reported health effects on the occupants in these contaminated environments. However, based on the veterinary, occupational and laboratory toxicity studies, it is reasonable to project that Stachybotrys can cause adverse health responses once the toxic level of the corresponding agents reached the target systems. In order to assess the risk to occupants in contaminated buildings, it is essential to outline and collect information for risk assessment. This review paper presents the current information in the format of hazard identification, dose-response and environmental characteristics and aims to discuss existing information with researchers and risk assessors and help to conduct risk characterization under different indoor conditions.

Visualization of the structural changes in plywood and gypsum board during the growth of Chaetomium globosum and Stachybotrys chartarum.

Fungal growth in indoor environments is associated with many negative health effects. Many studies focus on brown- and white-rot fungi and their effect on wood, but there is none that reveals the influence of soft-rot fungi, such as Stachybotrys spp. and Chaetomium spp., on the structure of building materials such as plywood and gypsum wallboard. This study focuses on using micro-computed tomography (microCT) to investigate changes of the structure of plywood and gypsum wallboard during fungal degradation by S. chartarum and C. globosum. Changes in the materials as a result of dampness and fungal growth were determined by measuring porosity and pore shape via microCT. The results show that the composition of the building material influenced the level of penetration by fungi as shown by scanning electron microscopy (SEM). Plywood appeared to be the most affected, with the penetration of moisture and fungi throughout the whole thickness of the sample. Conversely, fungi grew only on the top cardboard in the gypsum wallboard and they did not have significant influence on the gypsum wallboard structure. The majority of the observed changes in gypsum wallboard occurred due to moisture. This paper suggests that the mycelium distribution within building materials and the structural changes, caused by dampness and fungal growth, depend on the type of the material.

Rapid detection and identification of Stachybotrys and Chaetomium species using tissue PCR analysis.

Indoor fungi are a worldwide problem causing negative health effects for infected building's occupants and even deterioration of building structures. Different fungal species affect buildings and their inhabitants differently. Therefore, rapid and accurate identification of fungi to the species level is essential for health risk assessment and building remediation. This study focuses on molecular identification of two common indoor fungal genera: Stachybotrys and Chaetomium. This study proposes two new DNA barcode candidates for Stachybotrys and Chaetomium: the gene encoding mitogen activated protein kinase (hogA) and the intergenic region between histone 3 and histone 4 (h3-h4) as well as it introduces a rapid - 3.5h - protocol for direct Stachybotrys and Chaetomium species identification, which bypasses culture cultivation, DNA extraction and DNA sequencing.

The Pathogenicity of Stachybotrys chartarum.

Stachybotrys chartarum is a dematiaceous fungus that is ubiquitous in our living environment. This fungus has long been regarded as non-pathogenic and its inhalation effect on humans has been scarcely studied. Recently, however, epidemiologic studies on acute idiopathic pulmonary hemorrhage in infants suggested that the fungus might be potentially pathogenic to humans. To determine the pathogenicity of this fungus, its interaction with the host defense system was studied using polymorphonuclear leukocytes (PMNs) and macrophages. Histopathological analysis of mice intratracheally injected with this fungus was also performed. The results disclosed that the conidia of S. chartarum were resistant to the antifungal activities of alveolar macrophages in terms of phagocytosis, killing and inhibition of germination. However, the conidia could not survive in the lungs of mice when injected intratracheally. Lavage fluid of mycelia that contained the dark slimy material coating the surface of conidia showed cytotoxic activity against macrophages and PMNs. Intratracheal injection of conidia in mice resulted in intraalveolar infiltration of PMNs. When using multiple injections during a 3-week period, strong eosinophilic infiltration into the proximal alveoli and perivascular tissues was observed. Our results suggest that inhalation of conidia may cause serious damage to the human lung, particularly when repeated.

[Wall relative humidity: a simple and reliable index for predicting Stachybotrys chartarum infestation in dwellings]. / Le taux d'hygrométrie murale, indicateur de la présence de la moisissure Stachybotrys chartarum dans les logements.

As the indoor mold Stachybotrys chartarum (SC) has been linked to serious health disorders, its identification in water-damaged dwellings is of utmost importance. The aim of this work was to compare wall relative humidity (RH) measurements with the results of mold identification studied on 458 samples collected from 100 dwellings. Mold identification was based on direct microscopic examination of wall samples collected by the gummed paper technique. Mean (+/- SD) wall RH (%) was much higher (97.0 +/- 6.1) when SC was identified (30 samples) than when other molds were identified (291 samples, 41.8 +/- 36.9) and when no molds were identified (137 samples, 38.9 +/- 34.8). There was a direct relationship between wall and room-air RH but the scatter of results implies that the latter cannot be used as a surrogate for the former. This study suggests that simple wall RH measurement can serve as a reliable indicator of SC infestation of dwellings.