Stachybotrys chartarum

Stachybotrys chartarum on gypsum boardStachybotrys chartarum on woodStachybotrys chartarum on a ceiling tileStachybotrys chartarum on EM agarStachybotrys chartarum on RB agarStachybotrys chartarum - Microscopy (EM Culture)

Basics

The International Mycological Association recognises, in its fungal database, 87 named species within the genusStachybotrys {3971}, whereas there are 15 named species of Stachybotrys registered with the international data bank of the Universal Protein Resource agency (UniProt) along with over 10 unnamed strains {3318}. S. chartarum is the more known species of this genus and has been the object of multiple scientific studies and lay articles because of its potential role in the occurrence of health problems associated with poor air quality of mould- contaminated buildings as well as in sick building syndrome (SBS).

Taxonomy

Kingdom Fungi Order Hypocreales
Phylum Ascomycota Family Dematiaceae
Class Sordariomycetes Genus Stachybotrys

The type species is Stachybotrys chartarum (Stachybotrys atra Corda). S. chartarum was formerly known as S. atra orS. alternans.

Strains of Stachybotrys spp. have been reported to have teleomorph stages within three different genera: Chaetosphaeria pomiformis, Cordyceps sinensis and Melanopsamma pomiformis {3842}.

Two chemotypes of Stachybotrys chartarum have been identified in water-damaged buildings {4271}. The S. chartarum(sensu lato) can be segregated in three: two chemotypes and one new species, S. chlorohalonata with its smooth spores. The two chemotypes of S. chartarum, chemotype S and chemotype A, have similar morphology but differ in their production of metabolites. Chemotype S produces macrocyclic trichothecenes, satratoxins and roridins, while chemotype A produces atranones and dolabellanes {4271}.

Habitat/Ecology

Stachybotrys chartarum is a dematiaceous fungus. Having a worldwide distribution, it is generally found in soil and strata rich in cellulose (hay, straw, grain, plant debris, dead roots, wood pulp, fabrics and paper) {102}. It can contaminate grains, tobacco, insulator foams, paper, textiles, indoor air and water-damaged buildings {725; 816; 724}.

Spores of S. chartarum do not readily disseminate in the air, primarily because they usually occur in a cluster covered with dried slime; the spores become airborne only when dry and disturbed or when they become attached to dust particles {102}. 

Once it has utilised the substrate’s free sugars to initiate growth, the strong cellulolytic activity of Stachybotrys allows it to continue growing rapidly {3729}. The fungus proliferates slowly, facilitating overgrowth by other moulds {102}.

In the indoor environment, the frequency of isolation in air samples of Stachybotrys is approximately 13% of dwellings and 5% of samples.

Growth requirements

S. chartarum does not compete well with other rapidly growing fungi. It grows from 2 to 40 °C (optimum 23-27 °C) {724}. The optimum pH range is 5.6-6.0, although the fungus can also grow at pH 3.0-9.8 {995}.

S. chartarum requires high water activity and its development is enhanced by a high cellulose content in the substratum. In fact, Stachybotrys species require a very high water activity (Aw) in order to grow, i.e. at least 0.94 (optimum >0.98) {817}. On building materials, a water activity of 0.96 yields poorer growth of S. chartarum as compared to an Aw of 0.98 {605}.

Water Activity :         minimal Aw = 0.94  
optimal  Aw > 0.98

Growth on building materials or indoor environment

Stachybotrys is a greenish black mould that grows on substrates with a high cellulose content and a low nitrogen content such as hay, straw, wicker and wood chips, as well as building materials such as ceiling tile, drywall, paper vapour barriers, wallpaper, insulation backing, cardboard boxes, paper files, fiberboard, the paper covering of gypsum wallboard, particleboard, jute, dust and wood when these items become water damaged {3729; 725; 817}.

This mould requires very wet or high humid conditions lasting days or weeks in order to grow. Most mould spores are able to begin growth after just 24 hours of wetness, whereas Stachybotrys spores require at least 48 hours of sustained wetness to germinate and commence growing.

Because Stachybotrys spores are rarely airborne, it is more often identified by laboratory analysis of direct swabs or lift tape samples than by air sampling.

Although Stachybotrys is only one of the fungal genera isolated in mouldy buildings and, in fact, is less common in the air and in lesser amounts compared to other mould genera {816}, the presence of Stachybotrys and its mycotoxins on building materials have interested health professionals for many reasons. One of these is their possible role in the development of sick building syndrome, especially since this mould is one of the key contaminants infesting buildings shown to have major problems in mechanical system design, construction and/or operational strategies, leading to excess indoor moisture.

In one study, Stachybotrys was cultured from samples of 13% of contaminated buildings {4274}. In another study conducted throughout the United Stated to establish the profile of indoor and outdoor fungi, S. chartarum was found present indoors in 0-27% of buildings while its presence in the outdoor environment was usually below 2%.

When active and growing in a wet environment, Stachybotrys can appear black, shiny and slimy; when it dries out it takes on a powdery or sooty appearance.

More details

Indoors, Stachybotrys grows on papered surfaces moistened by water penetration or severe condensation. Heavily wetted gypsum panels, often used for interior walls and ceilings, can be attacked {725}. At a relative humidity of 84-100%, this fungus reproduces rapidly on wallpaper and plaster and even produces toxins {995}. Because of its ability to degrade cellulose-containing material, and because of its hydrophilic nature, it is an excellent indicator of chronically water-damaged building materials {413}. However, because it is generally a slower-growing species, a surface overgrown byStachybotrys may have been previously dominated by other species at an earlier stage of the water damage, at a lower relative humidity {16}.

Moreover, since spores of S. chartarum are usually covered with dried slime, aerosolisation of the spores in ambient air occur only after the colonies have dried out and become brittle {809}. It is usually difficult to detect Stachybotrys in indoor air samples unless it is physically disturbed. The spores are in a gelatinous mass and die readily after release, although dead spores remain both allergenic and toxigenic {817}.

Many reports have related the prevalence of Stachybotrys in indoor air samples; in general this mould is found in approximately 13% of contaminated dwellings or less and in approximately only 5% of air samples. Furthermore, this fungus proliferates slowly, facilitating overgrowth by other moulds {102}.

A study carried out on more than 12,000 fungal air samples from 1,717 buildings throughout the United States identifiedS. chartarum in the indoor air in 6% of buildings studied {696}.

In a similar study, 200 houses with a history of water incursion were sampled near Houston, Texas {809}; in addition to culturing air samples, investigators used a spore trap for direct microscopic count. Stachybotrys spp. were detected with at least one of the methods in 58.5% of the houses tested. Only 9.6% of the 821 room air samples containedStachybotrys spores, whereas 28.0% of wall-cavity-air samples were positive; outdoor air revealed 2.7% positive samples. Median spore counts were 13 CFU/m³ for outdoor air, 53 CFU for room air and 14,190 CFU for wall cavity air. The authors concluded that the high Stachybotrys spore concentrations present in wall cavities did not contribute significantly to that detected in room air, further suggesting that contamination by this species can be relatively contained if it is well confined within wall cavities.

A study conducted on wet spray-applied cellulose insulation (WSACI) in water-damaged buildings revealed that, of the total airborne mould spore content, Stachybotrys was usually in low concentrations, representing under 5% of overall moulds in 2 out 4 buildings; however, on occasions, concentrations could also be high, reaching 27 million CFU/g of bulk ceiling overspray {670}.

A study performed on 72 samples of mould-infested building materials from 23 buildings in Denmark revealed thatStachybotrys was the second most frequently isolated mould genera after Aspergillus sp., with S. chartarum being the species most often identified on material samples, followed by A. versicolor. Trichothecenes were detected on all sampled types of building materials naturally infested with Stachybotrys; on experimentally infested materials in the laboratory, four of five cultures of S. chartarum were able to produce these mycotoxins {605}.

A Finnish survey conducted on children in moisture damaged schools in order to study the relationship between the presence of indoor air microbes and respiratory symptoms, showed that Stachybotrys was found more frequently in samples collected from schools with humidity problems than in control schools, thus confirming its status as an excess moisture indicator {694}.

Mycotoxins have also been identified in several studies relating to mould-contaminated buildings: in an indoor environment study, significantly higher concentrations of trichothecenes were present in contaminated buildings comparatively to control buildings {425}. In another Finnish study, fifteen out of 79 samples (19%) of mouldy water damaged building materials were found to contain trichothecenes at concentrations ranging from 2.5 to 3500 ng/g of sample–fresh weight {16}. It is however important to note that fewer metabolites and organic volatile compounds are produced on building materials (such as gypsum board) than on nutrient media; this difference is more likely linked to the nutritional value of the substrates {596}.

Laboratory section

Normal laboratory precautions should be exercised in handling cultures of this species within Biosafety Level 2 practices and containment facilities.

This saprophyte fungus is easily isolated and cultured on common fungal media. For the correct identification of the fungus, cornmeal agar (CMA) and malt extract agar (MEA) are recommended {413}; to facilitate isolation, highly specific non standard media should be used {876}.

Colony, macroscopic morphology

Colonies are usually quite rapid growing, maturing within 4 days if the media offers the required free water {412}. Colonies at 25 °C attain a diameter of 2.5-3 cm on malt extract agar. Colonies are effuse, cottony or powdery (due to conidial masses) and color of colony is initially white, turning blackish-green with aging; the reverse is at first colourless, then black {412; 724}. A diffuse brown pigment may be produced.

Microscopic morphology

Hyphae are septate and will grow superficially and immersed in the culture media; hyphae may form ropes.

Conidiophores, macronematous, mononematous, are usually 100 µm up to 1 mm long, and 3-6 µm wide; they are simple or branched, hyaline or greyish at first, becoming olivaceous brown to black; the conidiophores bear a crown of phialides at their apices {3971; 724}. Each conidiophore (stipe and branch) is straight or flexuous, either colourless, grey, brown or olivaceous, and at times partly covered with dark granules.

The conidiogenous cells are monophialidic, discrete, in groups and are ellipsoidal or broadly fusiform. These phialides, in clusters of 3 to 10, are hyaline or pigmented single cells, cylindrical in shape with a swollen upper portion; they are usually with a very small opening and no collarette.

The conidia aggregate in large, slimy, often black and glistening heads; each conidium is acrogenous, simple, cylindrical or oblong, rounded at one end or both ends, ellipsoidal, reniform or subspherical, either grey, greenish, dark brown, blackish brown or black, smooth or verrucose, sometimes covered with dark granules, sometimes with longitudinal striations. The conidia of S. chartarum in particular are mostly dark, oval (average 4.5 X 9 µm), hyaline and smooth-walled at first, becoming dark olivaceous-grey and verrucose {725; 412; 816; 724}.

Specific metabolites

Several secondary metabolites, volatile organic compounds and mycotoxins, have been isolated and characterized fromS. chartarum.

Organics compounds (including VOCs)

The specific microbial volatile organic compound (mVOC) profile of S. chartarum is partially known. In a study of mVOCs produced by this species when grown on rice, a team of researchers mainly identified butanol derivates and thujopsene (a sesquiterpene) {596}. Growth of S. chartarum on gypsum board led to only one of these compounds, 1-butanol, whereas about 20 minor compounds (alcohols, ketones and terpenes) were also identified. Wilkins et al.  reported the synthesis of trichodiene, a sesquiterpene involved in the biosynthesis of antibiotics and some mycotoxins {707}. Other MVOCs have also been reported, such as sesquiterpene hydrocarbons {995}.

Other mVOCs common to many moulds have also been identified in Stachybotrys cultures.

In fact, in the indoor environment of damp buildings, many organic compounds, including microbial volatile organic compounds (mVOCs), have been identified associated with several fungal species. Some of these compounds are common to most fungal species and probably contribute to a number of health problems associated with indoor air quality. However many of the fungal metabolites identified are non reactive and are in low concentrations in indoor air {594}.

Mycotoxins

Stachybotrys are known to produce numerous mycotoxins. S. chartarum produces mycotoxins belonging to the following families: satratoxins, roridins, verrucarins and stachybocins {4272}. The majority of available toxicological studies focus on macrocyclic trichothecenes.

Stachybotrys toxins are produced in phialides, conidia and conidiophores. It should be emphasised that only about one-third of S. chartarum isolates actually produce toxic macrocyclic trichothecenes, the remaining producing simple, less toxic trichothecenes {996}.

A recent review of the toxicological literatureidentifiedover 30 mycotoxins produced by Stachybotrysthat could be deleterious to humans or animals, at least active in vitro in cell lines {995}:  Atranones  (Atranone  A, Atranone B, Atranone C, Atranone D, Atranone E), Dechlorogriseofulvin, Epidechlorogriseofulvin, Epiroridin E, Isororidins (Isororidin A, Isororidin E),  Isosatratoxins (sosatratoxin F, Isosatratoxin G, S-Isosatratoxin H), Neosolaniol, Phenylspirodimane(s), Roridins (Roridin A, Roridin C, Roridin E), Roridinic acid, Roridin L 2, (+)-Roridin L-2, Satratoxins (Satratoxin F, Satratoxin G, Satratoxin H), Solaniol, Stachybocins (Stachybocin A, Stachybocin B, Stachybocin C, Stachybocin D), Stachybotramide, Stachybotrin, Stachybotrydial (Mer-NF 5003F, NF 5003F, F 1839M), Stachybotrylactam, Stachybotrylactone, Stachybotrolide, Stachybotryotoxin, Stachybotriotoxin, Trichoverrins (Trichoverrin A, Trichoverrin B), Trichoverrols (Trichoverrol A, Trichoverrol B), Verrucarins (Verrucarin A, Verrucarin B, Verrucarin C).

All macrocyclic trichothecene compounds are chemically stable, which explains why certain contaminated materials such as hay maintain their toxic properties for many years {725}. Subjects may be exposed to these toxins by ingestion of food products contaminated with the fungus, or experimentally via direct inhalation of the spores {817}.

More details

Trichothecenes resist sunlight, ultraviolet light, X-rays, heat up to 120 °C as well as acids. They are however readily destroyed by alkalis, which allows for detoxification, a key characteristic that has important ramifications for building remediation {102}.

The roridins A and E, verrucarin B and J, satratoxins F, G and H as well as trichoverrols A and B are toxins purported to be associated with adverse human health effects {425; 102}. Less toxic simple trichothecenes (nonmacrocyclic) have also been reported, including trichodermol and trichodermin {995}.

It should be emphasised that S. chartarum is the species most closely associated with trichothecene mycotoxin production {130}. In addition to its mycotoxins, Stachybotrys produces stachylysin, a proteinaceous haemolytic agent known to lyse sheep erythrocytes {995; 816}.

Adverse health reactions

Interest in Stachybotrys is mostly related to its toxic potential resulting in varying mycotoxicosis; its role in cases of hemosiderosis and sick building syndrome are controversial although case control studies, case reports and experimental studies have suggested a possible association with environmental exposure to Stachybotrys mycotoxins  {1128; 2649; 893}. Some unsubstantiated reports in the 1970s mentioned that aerosolized trichothecenes could have been used as biological weapons in Southeast Asia.

The pathogenicity of Stachybotrys was first observed in cattle and horses in the Soviet Union in the 1920’s. Development of stomatitis, rhinitis, conjunctivitis and neurological disorders were observed in the animals following ingestion of hay contaminated with the mould. The syndrome was later called stachybotrytoxicosis. Animal studies have since been undertaken to demonstrate the pathogenic effects of trichothecenes.

Irritation and inflammation

Specific irritation or inflammation properties associated with Stachybotrys have been attributed to trichothecenes. Satratoxins are known to modulate inflammatory reactions and alter alveolar surfactant concentrations. Satratoxin H is present on fungal spores and is toxic by inhalation {817}.

Experimental animal studies have shown the presence of severe intra-alveolar, bronchiolar and interstitial inflammation following intranasal exposure to Stachybotrys spores {811; 4273}.

Allergic reactions

Allergic reactions and infection are considered to be minor mechanisms involved in Stachybotrys-related health problems. However, some authors have associated rhinitis and asthma symptoms to the presence of Stachybotrys indoors.

More details

According to Vesper et al., Stachybotrys chartarum is a mould significantly found at higher concentrations in the homes of asthmatic subjects {810}. Multiple respiratory exposures to S. chartarum crude allergen extracts cause responses typical of allergic airway disease, as demonstrated by elevated serum and bronchoalveolar lavage IgE {102}. However IgE antibodies directed against S. chartarum were also found in approximately 10% of asymptomatic subjects.

The presence of IgE specific to Aspergillus spp., Cladosporium and Stachybotrys chartarum was found to be related to sick building syndrome in one study {917}. 
In a similar study, subjects exposed to mouldy environments where S. chartarum was identified, presented symptoms consistent with potential asthma and interstitial lung disease {240}.

Allergic components and mechanism

No purified allergenic fraction has yet been reported.

Hypersensitivity pneumonitis

No confirmed human hypersensitivity pneumonitis (HP) associated with Stachybotrys has been reported. In some cases, subjects exposed to mouldy environments where S. chartarum was found have displayed respiratory problems, some compatible with HP.

More details

Hodgson et al. described a building-associated pulmonary disease from exposure to Stachybotrys chartarum andAspergillus versicolor originating from a newly-constructed courthouse and two associated office buildings with moisture problems (window and roof leaks and envelope seepage) {240}; occupants complained of general discomfort, some with potential asthma and interstitial lung disease. Several ceiling tiles were visually covered by a black mould subsequently identified as S. chartarum; presence of the mould was also found in all air samples where books were handled. Bulk samples revealed S. chartarum at high concentrations from 104 CFU to 107 CFU/m².

Toxic effects (mycotoxicosis)

Stachybotrys produces many toxins including satratoxins which are trichothecene mycotoxins.  In general, trichothecenes are potent inhibitors of DNA, RNA and protein synthesis. They modulate inflammatory reactions and subsequently alter alveolar surfactant phospholipid concentrations.

It has been shown that exposure to these toxins may occur through ingestion of contaminated food or experimentally, via direct inhalation of the spores {460; 423; 1363; 896; 890; 4273; 1404; 899; 196}; localised absorption through the skin is also possible {4305}.

Exposure to trichothecene toxins may produce a variety of clinical conditions, including skin irritation, anorexia, vomiting, diarrhoea, haemorrhage, convulsion and death.

In addition to its mycotoxins, Stachybotrys produces an hemolysin, stachylysin, which lyses sheep erythrocytes {883; 882; 885; 884}. The existence of stachylysin has been demonstrated in spores of certain strains.

Inhalation of S. chartarum toxic spores may result in trichothecene absorption {425}.  Respiratory symptoms following inhalation can be observed, ranging from benign, such as congestion, cough and rhinitis, to reactive airways disease as well as more serious syndromes, including pulmonary fibrosis {102}.

Individuals with chronic exposure to the toxins produced by this fungus report cold and flu-like symptoms, sore throats, diarrhoea, headaches, fatigue, dermatitis, intermittent local hair loss and generalized malaise. Inhalation of conidia may also induce respiratory pathological changes (pneumomycotoxicosis) {725}.

Exposure to S. chartarum has been reported to cause human neurotoxicity, although conclusive evidence is still lacking {130}. Animals injected with the toxins exhibited the following symptoms: necrosis and haemorrhage within the brain, thymus, spleen, intestine, lung, heart, lymph node, liver and kidney {817}.  Additional studies are needed to document whether prolonged exposure to these mycotoxins in humans leads to compatible clinical and pathologic pictures as demonstrated in animal models.

More details

Toxic effects were first studied following an outbreak in horses in the mid-thirties, in the Ukraine; the epidemiological data suggested the hypothesis that the outbreak was linked to mouldy fodder. Subsequent identification of Stachybotrys in the straw as well as experimental exposure leading to the same disease confirmed the identity of a disease now known as "stachybotryotoxicosis".  Soon afterwards, an outbreak among husbandry workers was determined to be human stachybotryotoxicosis linked to inhalation exposure to mouldy barley straw.

Farm workers handling Stachybotrys contaminated straw or hay also constitute a risk group for human toxicosis. Following Stachybotrys exposure, the main symptoms described are mainly respiratory and cutaneous irritation at the site of contact with the toxic material {811}. Following direct contact with this mould, primary disease manifestations may appear on skin, with dermatitis. Lesions progress to crusting exudates and ultimately necrosis.

In one study of a contaminated building, satratoxins G and H were identified at concentrations of 2 to 5 ppm in mouldy ceiling tiles {240}. However, in this instance, the authors remained unsure as to whether toxins from different species of moulds represented the primary cause of health problems exhibited by the occupants.

Trichothecenes are among the most potent small-molecule inhibitors of protein synthesis and have been associated with decreased resistance to infectious organisms.

Stachybotrys species can produce a cyclosporine-like immunosuppressive agent capable of increasing skin graft survival in rats {102}.  Current data on the toxicology of mycotoxins produced by Stachybotrys demonstrate that this group of mycotoxins is capable of producing immunosuppression and inflammatory insults to gastrointestinal and pulmonary systems {2382}. Case control studies and case reports have suggested a possible association with environmental exposure to Stachybotrys mycotoxins, although an unequivocal causal relationship has yet to be firmly established.

Although some trichothecenes are potentially carcinogenic, there have been only conflicting reports to date in animal studies. There is no evidence to support claims that individuals exposed to Stachybotrys are at long-term risk of cancer {102}.

Infections and colonisations

No human or animal infections due to Stachybotrys chartarum or to any Stachybotrys species have been reported.

Virulence factors

No virulence factor has been reported for Stachybotrys spp.   

Specific settings

Nosocomial infections

No nosocomial infections due to Stachybotrys have been reported.

No nosocomial outbreaks due to exposure to Stachybotrys spp. have been reported in the hospital setting.

Occupational diseases

No particular outbreaks due to exposure to Stachybotrys spp.have been reported in the workplace in recent years. The only outbreak published was that of the historical outbreak of stachybotryotoxicosis in farmers handling contaminated hay in the Soviet Union.

More details

Because of this particular risk, farm workers handling Stachybotrys contaminated straw or hay constitute a risk group for human toxicosis. Following this type of Stachybotrys exposure, the main symptoms described are mainly respiratory and cutaneous irritation at the site of contact with the toxic material {811}. Following direct contact with this mould, primary disease manifestations may appear on skin, with dermatitis. Lesions progress to crusting exudates and ultimately necrosis.

Diagnostic tools

Cultures

Cultures of clinical specimens are not warranted as there are no reported infections associated with Stachybotrys spp.

Histopathology

Histological examination of clinical specimens is not warranted as there are no reported infections associated withStachybotrys spp.

Immunodiagnosis

While some studies have shown the presence of IgE or IgG in exposed symptomatic subjects, serological testing forStachybotrys is not readily available and is not part of the basic skin test panel. 

Only one allergen, the GL10 - Stachybotrys atra, is registered with the American «Biological Products Deviation Reporting» surveillance program of the Federal Drug Administration (FDA) {3285}.

Test IgE IgG Antigens Other
Skin Tests        
RAST-IgE Experimental      
RAST-IgG   Experimental    
ELISA-ELIFA        
Immunodiffusion        
Immunofluorescence        
Complement fixation        
PCR        
Other        

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