Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling

[This corrects the article on p. 226 in vol. 59.].     

Methods for integrated air sampling and dna analysis for detection of airborne fungal spores

Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.     

Cytotoxic fungal spores in the indoor atmosphere of the damp domestic environment

Eight-three fungal isolates collected and cultured from the air spora of damp public sector Scottish housing have been screened for cytotoxic properties. A bioassay procedure has been utilised for this purpose involving cytotoxic effects on human embryonic diploid fibroblast lung cells grown as monolayer cultures within the wells of microtitre plates. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] biochemical assay system employed has revealed that 39 (47%) of the moulds investigated possessed spore-associated cytotoxic properties; a value which emphasises the potential health hazards of the continual inhalation of toxigenic fungal propagules by occupants of mouldy houses. Penicillia, the most prevalent fungi found in the internal atmosphere of the domestic environment, accounted for the great majority of cytotoxic strains identified.     

Airborne bacteria and fungal spores in the indoor environment. A case study in Singapore

The concentration of airborne fungal spores and bacteria as related to room temperature, humidity and occupancy levels within a library building in Singapore was determined. Measurement of indoor air quality with respect to microorganisms is of particular importance in tropical environments due to the extensive use of air‐conditioning systems and the potential implications for human health. This study has revealed a number of interesting relationships between the concentrations of fungal spores and bacteria in relation to both environmental and human factors. The levels of fungal spores measured in the indoor environment were approximately fifty times lower than those measured outside, probably because of the lowered humidity caused by air‐conditioning in the indoor environment. The variation in fungal spore concentration in the outdoor environment is likely to be due to the diurnal periodicity of spore release and the response to environmental factors such as light temperature and humidity. The indoor concentration of fungal spores in air was not clearly correlated to concentrations measured in air outside of the library building and remained relatively constant, unaffected by the difference in the numbers of occupants in the library. In contrast, the indoor concentrations of bacteria in air were approximately ten times higher than those measured outdoors, indicating a signficant internal source of bacteria. The elevated levels of indoor bacteria were primarily attributed to the number of library occupants. Increased human shedding of skin cells, ejection of microorganisms and particulates from the respiratory tract, and the transport of bacteria on suspended dust particles from floor surfaces probably accounts for the strong positive correlation between occupancy levels and the concentration of bacteria in internal air.     

Fate of bioterrorism-relevant viruses and bacteria, including spores, aerosolized into an indoor air environment

An aerosol physics test facility was used in a series of eight experiments to gather an integrated comprehensive broad base of data on the fate of surrogates of microorganisms that cause smallpox, plague, glanders, anthrax, and viral hemorrhagic fevers. The results are directly relevant to the public health issue of how to protect the occupants of buildings against bioterrorism. The test conditions were directly relevant to the indoor air environment situation, and the results can be generalized to buildings that are now occupied. The reductions in concentrations of relevant viruses and bacteria--including gram-negative fermenters and nonfermenters, gram-positive cocci and bacilli, and spores--were substantial and statistically robust. The data show that the bioterrorism-relevant aerosolized viruses and bacteria, including spores, respond like small particulates to the primary (electrical) forces that control the distribution of small particulates in a room. Further, these relevant microorganisms respond like small particulates to means designed to control airborne particulates. The results could be used to anticipate the effects of a bioterrorist attack on the public health, provide information on means that can be used to minimize such effects, and used to make decisions on how best to protect occupants of specific buildings at minimal cost and with assurance of success.     

Monitoring airborne biotic contaminants in the indoor environment of pig and poultry confinement buildings

Given the growing concerns over human and animal health issues related to confined animal feeding operations, an in‐depth examination is required to monitor for airborne bacteria and associated antibiotic resistance genes. Our 16S rRNA‐based pyrosequencing revealed that the airborne microbial community skewed towards a higher abundance of Firmicutes (> 59.2%) and Bacteroidetes (4.2–31.4%) within the confinement buildings, while the office environment was predominated by Proteobacteria (55.2%). Furthermore, bioaerosols in the confinement buildings were sporadically associated with genera of potential pathogens, and these genera were more frequently observed in the bioaerosols of pig and layer hen confinement than the turkey confinement buildings and office environment. High abundances of tetracycline resistance genes (9.55 × 10² to 1.69 × 10⁶ copies ng^?1 DNA) were also detected in the bioaerosols sampled from confinement buildings. Bacterial lineages present in the poultry bioaerosols clustered apart from those present in the pig bioaerosols and among the different phases of pig production, suggesting that different livestock as well as production phase were associated with a distinct airborne microbial community. By understanding the diversity of biotic contaminants associated with the different confinement buildings, this study facilitates the implementation of better management strategies to minimize potential health impacts on both livestock and humans working in this environment.     

NAGase activity in airborne biomass dust and relationship between NAGase concentrations and fungal spores

Inhalation of airborne fungal spores or fungalenzymes may cause adverse pulmonary healtheffects. The enzyme NAGase(N-acetyl--D-glucosaminidase) is achitinase presumed to be secreted by all fungi. In this study, NAGase activities andconcentrations of fungi are estimated inairborne biomass dust to acquire knowledgeabout the level of NAGase activity and therelationship between NAGase activity andconcentrations of airborne fungal spores.NAGase was sampled on both teflon andpolycarbonate filters, and polycarbonatefilters proved to be better for extraction ofNAGase than teflon filters. NAGase was foundin airborne dust at a biofuel plant and in dustgenerated from biomass. At a biofuel plant, themedian level of exposure to NAGase was 21 pmols^–1 m^–3. Significant correlationswere found between NAGase activities, totalnumber of fungal spores and CFU of fungi, withthe highest degree of correlation being betweenthe total number of fungal spores and theNAGase activity (r = 0.802, n = 76). Furthermore,when dust was stored for different periods, theculturability of fungal spores was stronglyreduced and the NAGase activity was not or onlyslightly reduced after up to 40 days ofstorage. Accordingly, NAGase activity may beused as a rapid method to get an estimate ofthe exposure level to airborne fungal spores.Whether pure NAGase or the NAGaseconcentrations observed here cause any healtheffects is not known, although it has beenshown that other fungal enzymes can causerespiratory disorders and a chitinase isdescribed as an allergen.     

Molecular characterization of airborne fungal spores in boreal forests of contrasting human disturbance

In this study we present a new approach to characterize fungal diversity with DNA sequencing of mycelium grown from trapped airborne spores. Fungal spores were extracted systematically from air in three boreal forest sites (clear-cut, young and old-growth forests) using an air sampling device. Internal transcribed spacer (ITS) sequences from the nuclear ribosomal DNA (nrDNA) were generated, and the sequences most likely taxon affinities were established through DNA homology searches. Phylogenetic analyses were used to classify similar sequences into operational taxonomic units (OTUs). The analyses indicated that a total of 84 different OTUs had been sampled, 24 basidiomycetes and 60 ascomycetes. OTUs belonging to the ascomycete orders Helotiales and Pleosporales were most frequent (31 and 18 respectively). A total of 54, 29 and 33 OTUs were sampled, respectively, in the old-growth, young and clear-cut forest sites. Although heavy generalization should be avoided due to few replicates, the results could indicate that old-growth boreal forests have significantly higher airborne fungal species richness than recently managed forests. The study shows that the spore-trapping approach has a great potential for targeting and studying anonymous fungi.     

An assessment of the air quality in indoor and outdoor air with reference to fungal spores and pollen grains in four working environments in Kerala, India

Qualitative and quantitative analyses of airborne fungal spores and pollen grains in four working environments (market, saw mill, poultry and cow sheds) in Thiruvananthapuram, the capital city of Kerala, India, were carried out for 2 years using Burkard Personal Slide Sampler and Andersen Two-Stage Sampler. Total spore concentration in these sites was always higher in indoor environments than in outdoor environments. Difference in concentration was not statistically significant in any of these work places except in saw mill (t test, p < 0.05). The highest spore concentration was recorded here followed by market, poultry and cow sheds. A total of 32 fungal spore types from indoor environments and 33 spore types from outdoor environments were recorded. Of them, 16 spore types were common to all the sites. Ameropsores, Cladosporium, other basidiospores, Ganoderma and Nigrospora were the dominant spore types in both indoor and outdoor environments. A total of 27 species of viable fungi from indoor and 24 species from outdoor environments were identified. Penicillium citrinum, Aspergillus flavus and Aspergillus niger were the most dominant viable fungi isolated. In contrast, total pollen concentration was always higher in outdoor environments than in indoor environments. Twenty-nine pollen types from indoor and 32 pollen types from outdoor were captured during the sampling. Poaceae, Cocos, Artocarpus, Amaranthus/Chenopodium and Tridax were the common and dominant pollen types observed in all the sites. Peak spore and pollen incidence were recorded during the late rainy and dry seasons (October–February) in both indoor and outdoor environments. The study revealed high prevalence of predominantly allergenic fungal spores and pollen grains in all the four work places. Workers/visitors are at potential risk of susceptibility to respiratory/allergic disorders.     

Introducing DNA-based methods to compare fungal microbiota and concentrations in indoor,outdoor, and personal air

Inhalation of airborne fungi is known to cause respiratory illnesses such as allergies. However, the association between exposure and health outcomes remains largely unclear, in part due to lack of knowledge about fungal exposure in daily life. This study aims to introduce DNA-based methods such as high-throughput sequencing (HTS) and quantitative polymerase chain reaction (qPCR) to compare fungal microbiota and concentrations in indoor, outdoor, and personal air. Five sets of concurrent indoor, outdoor, and personal air samples were collected, each with duration of 4 days. Sequencing analysis revealed greater species richness in personal than indoor air for four out of the five sets, indicating that people are exposed to outdoor species that are not in indoor air. The personal–indoor (P/I) and personal–outdoor (P/O) ratios of total fungi were 1.2 and 0.15, respectively, suggesting that personal exposure to total fungi is better represented by indoor than outdoor concentrations. However, the ratios were taxon dependent, highlighting the complexity of generalizing personal exposure to the diverse kingdom Fungi. These results demonstrate that the HTS/qPCR method is useful for assessing taxon-specific fungal exposure, which might be difficult to achieve effectively using conventional, non-DNA-based techniques.     

The distribution of bacteria and fungal spores in Blelham Tarn with particular reference to an experimental overturn

Summary During an experimental overturn at Blelham Tarn, surface water bacteria and fungal spore contents (determined by plating) ranged from 600–6,000 and from 0.3–30 per ml respectively. Following heavy rainfall however, bacterial numbers rose to over 10,000 and fungal spore numbers to over 100 per ml. These high concentrations normally extended down to the thermocline but not below it. The evidence was that particulate matter washed in by rain, and presumably carrying an attached bacterial and fungal flora, was largely lost to the lake through the outflow stream and did not sediment through to the hypolimnion.     

Influence of various dust sampling and extraction methods on the measurement of airborne endotoxin.

The influence of various filter types and extraction conditions on the quantitation of airborne endotoxin with the Limulus amebocyte lysate test was studied by using airborne dusts sampled in a potato processing plant. Samples were collected with an apparatus designed to provide parallel samples. Data from the parallel-sampling experiment were statistically evaluated by using analysis of variance. In addition, the influence of storage conditions on the detectable endotoxin concentration was investigated by using commercially available lipopolysaccharides (LPS) and endotoxin-containing house dust extracts. The endotoxin extraction efficiency of 0.05% Tween 20 in pyrogen-free water was seven times higher than that of pyrogen-free water only. Two-times-greater amounts of endotoxin were extracted from glass fiber, Teflon, and polycarbonate filters than from cellulose ester filters. The temperature and shaking intensity during extraction were not related to the extraction efficiency. Repeated freeze (-20 degrees C)-and-thaw cycles with commercial LPS reconstituted in pyrogen-free water had a dramatic effect on the detectable endotoxin level. A 25% loss in endotoxin activity per freeze-thaw cycle was observed. Storage of LPS samples for a period of 1 year at 7 degrees C had no effect on the endotoxin level. House dust extracts showed a decrease of about 20% in the endotoxin level after they had been frozen and thawed for a second time. The use of different container materials (borosilicate glass, "soft" glass, and polypropylene) did not result in different endotoxin levels. This study indicates that the assessment of endotoxin exposure may differ considerably between groups when different sampling, extraction, and storage procedures are employed.     

Detection of airborne Stachybotrys chartarum macrocyclic trichothecene mycotoxins in the indoor environment

The existence of airborne mycotoxins in mold-contaminated buildings has long been hypothesized to be a potential occupant health risk. However, little work has been done to demonstrate the presence of these compounds in such environments. The presence of airborne macrocyclic trichothecene mycotoxins in indoor environments with known Stachybotrys chartarum contamination was therefore investigated. In seven buildings, air was collected using a high-volume liquid impaction bioaerosol sampler (SpinCon PAS 450-10) under static or disturbed conditions. An additional building was sampled using an Andersen GPS-1 PUF sampler modified to separate and collect particulates smaller than conidia. Four control buildings (i.e., no detectable S. chartarum growth or history of water damage) and outdoor air were also tested. Samples were analyzed using a macrocyclic trichothecene-specific enzyme-linked immunosorbent assay (ELISA). ELISA specificity was tested using phosphate-buffered saline extracts of the fungal genera Aspergillus, Chaetomium, Cladosporium, Fusarium, Memnoniella, Penicillium, Rhizopus, and Trichoderma, five Stachybotrys strains, and the indoor air allergens Can f 1, Der p 1, and Fel d 1. For test buildings, the results showed that detectable toxin concentrations increased with the sampling time and short periods of air disturbance. Trichothecene values ranged from <10 to >1,300 pg/m3 of sampled air. The control environments demonstrated statistically significantly (P < 0.001) lower levels of airborne trichothecenes. ELISA specificity experiments demonstrated a high specificity for the trichothecene-producing strain of S. chartarum. Our data indicate that airborne macrocyclic trichothecenes can exist in Stachybotrys-contaminated buildings, and this should be taken into consideration in future indoor air quality investigations.     

Toxic-metabolite-producing bacteria and fungus in an indoor environment.

Toxic-metabolite-emitting microbes were isolated from the indoor environment of a building where the occupant was suffering serious building-related ill-health symptoms. Toxic substances soluble in methanol and inhibitory to spermatozoa at <10 microg (dry weight) ml(-1) were found from six bacterial isolates and one fungus. The substances from isolates of Bacillus simplex and from isolates belonging to the actinobacterial genera Streptomyces and Nocardiopsis were mitochondriotoxic. These substances dissipated the mitochondrial membrane potential (Deltapsi) of boar spermatozoa. The substances from the Streptomyces isolates also swelled the mitochondria. The substances from isolates of Trichoderma harzianum Rifai and Bacillus pumilus damaged the cell membrane barrier function of sperm cells.     

Detection of arbuscular mycorrhizal fungal spores in the air across different biomes and ecoregions

Aerial dispersal of fungal spores is common, but the role of wind and air movement in dispersal of spores of arbuscular mycorrhizal (AM) fungi is largely unknown. Several studies have examined the possibility of AM fungal spores being moved by wind vectors without observing spores taken from the air environment. For the first time this study observed the presence of AM fungal spores in the air. The frequency of AM fungal spores in the air was determined in six North American biomes composed of 18 ecoregions. Multiple samples were taken from both the air and the soil at each location. AM fungal spores were found in high abundance in the soil (hundreds of spores per gram of soil), however, they were rarely found in the air (most samples contained no AM fungal spores). Furthermore, only the Glomus morphotype was found in the air, whereas spores in the soil were taxomomically more diverse (Glomus, Acaulospora, Gigaspora, Scutellospora morphotypes were observed). The proportion of Glomus spores in the air relative to Glomus spores in the soil was highest in more arid systems, indicating that AM fungi may be more likely to be dispersed in the air in such systems. Nonetheless, the results indicate that the air is not likely a dominant mode of dispersal for AM fungi.     

A survey of airborne algae and cyanobacteria within the indoor environment of an office building in Kuala Lumpur,Malaysia

This study investigates the occurrence of airborne algae and cyanobacteria (AAC) within the indoor environment of an office building in Kuala Lumpur, Malaysia. Samples of air, wall scrapings and soils of potted plants were collected from various sites within the building and surrounding areas. In addition, AAC were collected by exposing a culture medium to the indoor air. Based on the cultured material, 14 taxa of AAC consisting of cyanobacteria such as Phormidium angustissima and Chroococcus minor and chlorophytes such as Chlorella vulgaris and Chlorococcum humicola were recorded. The surrounding areas of the building recorded the highest occurrence (75%) of AAC. Within the building, the highest occurrence of AAC (45%) was recorded on the lower ground floor, an area exposed to the outdoor environment. Some of the AAC recorded were also detected in the wall scraping and soil samples. Areas with heavy human movement appeared to have high occurrence of AAC. Human movement appeared to be an important factor in affecting the dispersal of the AAC.     

Analysis of the antimicrobial effects of nonthermal plasma on fungal spores in ionic solutions

The antimicrobial efficiency of reactive species-based control strategies is significantly affected by the dynamics of reactive species in the biological environment. Atmospheric-pressure nonthermal plasma is an ionized gas in which various reactive species are produced. The various levels of antimicrobial activity may result from the dynamic interaction of the plasma-generated reactive species with the environment. However, the nature of the interaction between plasma and environments is poorly understood. In this study, we analyzed the influence of the ionic strength of surrounding solutions (environment) on the antimicrobial activity of plasma in relation to the plasma-generated reactive species using a model filamentous fungus, Neurospora crassa. Our data revealed that the presence of sodium chloride (NaCl) in the background solution attenuated the deleterious effects of plasma on germination, internal structure, and genomic DNA of fungal spores. The protective effects of NaCl were not explained exclusively by pH, osmotic stability, or the level of reactive species in the solution. These were strongly associated with the ionic strength of the background solution. The presence of ions reduced plasma toxicity, which might be due to a reduced access of reactive species to fungal spores, and fungal spores were inactivated by plasma in a background fluid of nonionic osmolytes despite the low level of reactive species. Our results suggest that the surrounding environment may affect the behavior of reactive species, which leads to different biological consequences regardless of their quantity. Moreover, the microbicidal effect of plasma can be synergistically regulated through control of the microenvironment.