Aerosolization of Mycotoxins after Growth of Toxinogenic Fungi on Wallpaper.

Many fungi can develop on building material in indoor environments if the moisture level is high enough. Among species that are frequently observed, some are known to be potent mycotoxin producers. This presence of toxinogenic fungi in indoor environments raises the question of the possible exposure of occupants to these toxic compounds by inhalation after aerosolization. This study investigated mycotoxin production by Penicillium brevicompactum, Aspergillus versicolor, and Stachybotrys chartarum during their growth on wallpaper and the possible subsequent aerosolization of produced mycotoxins from contaminated substrates. We demonstrated that mycophenolic acid, sterigmatocystin, and macrocyclic trichothecenes (sum of 4 major compounds) could be produced at levels of 1.8, 112.1, and 27.8 mg/m2, respectively, on wallpaper. Moreover, part of the produced toxins could be aerosolized from the substrate. The propensity for aerosolization differed according to the fungal species. Thus, particles were aerosolized from wallpaper contaminated with P. brevicompactum when an air velocity of just 0.3 m/s was applied, whereas S. chartarum required an air velocity of 5.9 m/s. A. versicolor was intermediate, since aerosolization occurred under an air velocity of 2 m/s. Quantification of the toxic content revealed that toxic load was mostly associated with particles of size ≥3 µm, which may correspond to spores. However, some macrocyclic trichothecenes (especially satratoxin H and verrucarin J) can also be found on smaller particles that can deeply penetrate the respiratory tract upon inhalation. These elements are important for risk assessment related to moldy environments.IMPORTANCE The possible colonization of building material by toxinogenic fungi in cases of moistening raises the question of the subsequent exposure of occupants to aerosolized mycotoxins. In this study, we demonstrated that three different toxinogenic species produce mycotoxins during their development on wallpaper. These toxins can subsequently be aerosolized, at least partly, from moldy material. This transfer to air requires air velocities that can be encountered under real-life conditions in buildings. Most of the aerosolized toxic load is found in particles whose size corresponds to spores or mycelium fragments. However, some toxins were also found on particles smaller than spores that are easily respirable and can deeply penetrate the human respiratory tract. All of these data are important for risk assessment related to fungal contamination of indoor environments.

Resistance of Aerosolized Bacterial Viruses to Four Germicidal Products.

Viral diseases can spread through a variety of routes including aerosols. Yet, limited data are available on the efficacy of aerosolized chemicals to reduce viral loads in the air. Bacteriophages (phages) are often used as surrogates for hazardous viruses in aerosol studies because they are inexpensive, easy to handle, and safe for laboratory workers. Moreover, several of these bacterial viruses display physical characteristics similar to pathogenic human and animal viruses, like morphological size, type of nucleic acids, capsid morphology, and the presence of an envelope. In this study, the efficacy of four chemicals was evaluated on four airborne phages at two different relative humidity levels. Non-tailed bacteriophages MS2 (single-stranded RNA), ϕ6 (double-stranded RNA, enveloped), PR772 (double-stranded DNA), and ϕX174 (single-stranded DNA) were first aerosolized in a 55L rotative environmental chamber at 19°C with 25% and 50% relative humidity. Then, hydrogen peroxide, Eugenol (phenylpropene used in commercial perfumes and flavorings), Mist® (automobile disinfectant containing Triethylene glycol), and Pledge® (multisurface disinfectant containing Isopropanol, n-Alkyl Dimethyl Benzyl Amonium Chlorides, and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride) were nebulized with the phages using a separate nebulizer. Aerosols were maintained in suspension during 10 minutes, 1 hour, and 2 hours. Viral aerosols were sampled using an SKC BioSampler and samples were analyzed using qPCR and plaque assays. The resistance levels of the four phages varied depending on the relative humidity (RH) and germicidal products tested. Phage MS2 was the most stable airborne virus under the environmental conditions tested while phage PR772 was the least stable. Pledge® and Eugenol reduced the infectivity of all airborne phages tested. At 25% RH, Pledge® and Eugenol were more effective at reducing infectivity of RNA phages ϕ6 and MS2. At 50% RH, Pledge® was the most effective agent against phage MS2. These findings illustrate that various airborne viruses should be tested to demonstrate the effectiveness of germicidal treatments. This research also provides a set of parameters for testing germicidal products in large-scale settings to reduce the risk of virus transmission.

A new approach to detect early or hidden fungal development in indoor environments.

In addition to the biodegradation problems encountered in buildings, exposure of their occupants to mold is responsible for numerous diseases such as respiratory infections, immediate or delayed allergies and different types of irritations. However, current techniques are unable to detect mold at an early stage of development or hidden contaminants. Moularat et al., in 2008 has established chemical fingerprints of moldy growth from Volatile Organic Compounds (VOCs) arising specifically from fungal metabolism and developed the Fungal Contamination Index (FCI) (Moularat et al., 2008a,b). This index has the advantage of detecting fungal development both reliably and rapidly before any visible signs of contamination could be detected. However, even though the FCI has been widely tested, VOCs' analysis by GC/MS, which is required for index calculation, is incompatible with real-time monitoring strategy for indoor environments. In this context, researches around FCI exploitation have been followed up in order to provide a monitoring device widely deployable which is the result of the miniaturization of an analytical chain for portable, reliable and low-cost applications. This device is based on one hand the selection and concentration of chemical compounds from the sample of interest and on the other hand the development of an array of different conducting polymer based sensors in order to obtain a specific footprint. This fungal contamination detection device was the subject of patent applications by the CSTB. The modularity of the system (ability to vary both the elements of detection polymers and retention time of interest) allows for expansion of its use to other pollutants.

'Core species' in three sources of indoor air belonging to the human micro-environment to the exclusion of outdoor air.

Although we spend the majority of our lives indoors, the airborne microbial content of enclosed spaces still remains inadequately described. The objective of this study was to characterize the bacterial diversity of indoor air in three different enclosed spaces with three levels of occupancy, and, in particular, to highlight the 'core' species, the opportunistic pathogens and their origins. Our findings provide an overall description of bacterial diversity in these indoor environments. Data gathered from the three enclosed spaces revealed the presence of a common indoor signature (60% of total sequences in common). This work will provide a clearer understanding of the dominant groups of bacteria encountered in enclosed spaces: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. Thus, certain evidence revealed a connection between 'core' species and the human micro-environment (20% of phylotypes and 12% of sequences of human origin). Overall PCA analysis showed that the indoor environment is influenced mainly by the microbial diversity from nose and skin. Among the 'core species' found during this study, a large number (72% of all pathogen-related sequences were concentrated in 'core species') of genera and species are known to be responsible for opportunistic or nosocomial diseases or to include human commensal bacteria such as Mycobacterium sp., Acinetobacter baumanii, Aerococcus viridians, Thermoactinomyces vulgaris or Clostridium perfringens.

Effects of disinfection on Legionella spp., eukarya, and biofilms in a hot water system.

Legionella species are frequently detected in hot water systems, attached to the surface as a biofilm. In this work, the dynamics of Legionella spp. and diverse bacteria and eukarya associated together in the biofilm, coming from a pilot scale 1 system simulating a real hot water system, were investigated throughout 6 months after two successive heat shock treatments followed by three successive chemical treatments. Community structure was assessed by a fingerprint technique, single-strand conformation polymorphism (SSCP). In addition, the diversity and dynamics of Legionella and eukarya were investigated by small-subunit (SSU) ribosomal cloning and sequencing. Our results showed that pathogenic Legionella species remained after the heat shock and chemical treatments (Legionella pneumophila and Legionella anisa, respectively). The biofilm was not removed, and the bacterial community structure was transitorily affected by the treatments. Moreover, several amoebae had been detected in the biofilm before treatments (Thecamoebae sp., Vannella sp., and Hartmanella vermiformis) and after the first heat shock treatment, but only H. vermiformis remained. However, another protozoan affiliated with Alveolata, which is known as a host cell for Legionella, dominated the eukaryal species after the second heat shock and chemical treatment tests. Therefore, effective Legionella disinfection may be dependent on the elimination of these important microbial components. We suggest that eradicating Legionella in hot water networks requires better study of bacterial and eukaryal species associated with Legionella in biofilms.

Chemical disinfection of Legionella in hot water systems biofilm: a pilot-scale 1 study.

Legionella bacteria encounter optimum growing conditions in hot water systems and cooling towers. A pilot-scale 1 unit was built in order to study the biofilm disinfection. It consisted of two identical loops, one used as a control and the other as a 'Test Loop'. A combination of a bio-detergent and a biocide (hydrogen peroxide + peracetic acid) was applied in the Test Loop three times under the same conditions at 100 and 1,000 mg/L with a contact time of 24 and 3-6 hours, respectively. Each treatment test was preceded by a three week period of biofilm re-colonization. Initial concentrations of culturable Legionella into biofilm were close to 10(3) CFU/cm2. Results showed that culturable Legionella spp. in biofilm were no longer detectable three days following each treatment. evertheless, initial Legionella spp. concentrations were recovered 7 days after the treatments (in two cases). Before the tests, Legionella spp. and L. pneumophila PCR counts were both about 10(4) GU/cm2 in biofilm and they both decreased by 1 to 2 log units 72 hours after each treatment. The three tests had a good but transient efficiency on Legionella disinfection in biofilm.

Airborne fungal volatile organic compounds in rural and urban dwellings: detection of mould contamination in 94 homes determined by visual inspection and airborne fungal volatile organic compounds method.

Moulds can both degrade the materials and structures they colonise and contribute to the appearance of symptoms and diseases in the inhabitants of contaminated dwellings. Only few data have compared the levels of contamination in urban and rural environments and the results are not consistent. The aim of this study was to use a fungal contamination index, based on the detection of specific Microbial Volatile Organic Compounds (MVOC), to determine the exposure to moulds of individuals living in urban and rural dwellings. For this purpose, 94 dwellings (47 in an urban setting in Clermont-Ferrand and 47 in rural areas of the Auvergne region, France) were studied. By demonstrating marked disparities between the proportion of visible contamination (19%) and that of active, visible and/or hidden contamination (59%) and the fact that almost all visible contamination was identified by MVOC, we were able to show that use of the index seemed relevant to confirm the actual presence of fungal contamination in a dwelling. Furthermore, it was possible to demonstrate a relationship between moulds and the presence of water on surfaces (condensation, infiltrations, water damage, etc.). A higher proportion of positive fungal contamination index in rural homes was observed compared to the proportion in urban ones (68% versus 49%; p<0.05).

Detection of fungal development in a closed environment through the identification of specific VOC: demonstration of a specific VOC fingerprint for fungal development.

The occurrence of disease amongst the occupants of "mouldy" environments has been widely described in the literature. However, the detection of such moulds in closed environments remains difficult, particularly in the event of recent (before the first deterioration) or masked contamination (behind a material). In this context, the present study aimed to determine a specific chemical fingerprint for fungal development detectable in closed environments (dwellings, office, museum...). To achieve this, chemical emissions from sterile and artificially contaminated by moulds materials were analyzed and compared using a descriptive statistical method. Principal Component Analysis is thus chosen to analyze the results. PCA generated optimum and similar graphical representations of the scatterplot representing the data matrix. This statistical approach made it possible to identify an emission fingerprint without applying any preconception as to the type of emitted compound. Statistical analysis of the data then enabled confirmation of the impact of moulds on total VOC emissions. This emission of specific compounds resulted in obtaining a signature for the presence of fungal development in an environment, defined by specific ions. This analysis, and use of these ions applied to dwellings, made it possible to distinguish those with proven fungal development from those with no sign of mould or with a context favorable to fungal development, thus demonstrating that a chemical fingerprint specific to fungal development could be detected in indoor environments.

Detection of fungal development in closed spaces through the determination of specific chemical targets.

In addition to the biodegradation problems encountered in buildings, exposure of their occupants to moulds is responsible for numerous diseases: infections (invasive nosocomial aspergillosis), immediate or delayed allergies, food-borne infections and different types of irritation. In this context, the aim of our work has been to determine specific chemical tracers for fungal development on construction materials. More generally, by detecting a specific chemical fingerprint of fungal development, our objective was to propose a microbiological alert system which could control systems and/or procedures for the microbiological treatment of indoor areas. We therefore characterized the chemical emissions from six types of construction material contaminated artificially by moulds. Chemical fingerprints were established for 19 compounds arising specifically from fungal metabolism: 2-ethylhexanoic acid methyl ester, 1-octen-3-ol, 3-heptanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 1,3-octadiene, 2-(5H)-furanone, 2-heptene, alpha-pinene, 2-methylisoborneol, 4-heptanone, 2-methylfuran, 3-methylfuran, dimethyldisulfide, methoxybenzene, a terpenoid and three sesquiterpenes. Determining the origin of these compounds and their specific links with a growth substrate or fungal species made it possible to judge the pertinence of choosing these compounds as tracers. Thus the detecting specific volatile organic compounds emitted as from the second day of fungal growth demonstrated that this approach had the advantage of detecting fungal development both reliably and rapidly before any visible signs of contamination could be detected.

Impact of Health on Particle Size of Exhaled Respiratory Aerosols: Case-control Study.

Individuals with viral infection could possibly emit an infectious aerosol. The distinction between exhaled breaths of infected and healthy individuals should facilitate an understanding of the airborne transmission of infections. In this context, the present study is aimed at distinguishing healthy individuals from symptomatic ones by the study of their exhaled breath. A setup composed of a modified hood connected to an electrical low pressure impactor, which allows for the study of a wide range of particle sizes (from 7 nm to 10 µm), has been developed in order to collect exhaled breaths. This setup has been used with seventy eight volunteers. The results obtained using Principal Component Analysis (PCA) showed that exhaled breaths of individuals without symptoms have statistical similarities and are different from those of individuals with symptoms. This separation was made by the greater proportional emission by individuals with symptoms of particles collected on stages 3 (D 50 = 0.09 µm), 6 (D 50 = 0.38 µm), 8 (D 50 = 0.95 µm), 10 (D 50 = 2.40 µm), and 12 (D 50 = 4.02 µm) of the impactor. There was not a specific size distribution obtained for the individuals with symptoms. As a consequence, further research on the exhaled breath should be undertaken with symptomatic volunteers and would require the analysis of this wide range of particle sizes.

Detection of airborne Legionella while showering using liquid impingement and fluorescent in situ hybridization (FISH).

Aerosols of water contaminated with Legionella bacteria constitute the only mode of exposure for humans. However, the prevention strategy against this pathogenic bacteria risk is managed through the survey of water contamination. No relationship linked the Legionella bacteria water concentration and their airborne abundance. Therefore, new approaches in the field of the metrological aspects of Legionella bioaerosols are required. This study was aimed at testing the main principles for bioaerosol collection (solid impaction, liquid impingement and filtration) and the in situ hybridization (FISH) method, both in laboratory and field assays, with the intention of applying such methodologies for airborne Legionella bacteria detection while showering. An aerosolization chamber was developed to generate controlled and reproducible L. pneumophila aerosols. This tool allowed the identification of the liquid impingement method as the most appropriate one for collecting airborne Legionella bacteria. The culturable fraction of airborne L. pneumophila recovered with the liquid impingement principle was 4 and 700 times higher compared to the impaction and filtration techniques, respectively. Moreover, the concentrations of airborne L. pneumophila in the impinger fluid were on average 7.0 x 10(5) FISH-cells m(-3) air with the fluorescent in situ hybridization (FISH) method versus 9.0 x 10(4) CFU m(-3) air with the culture method. These results, recorded under well-controlled conditions, were confirmed during the field experiments performed on aerosols generated by hot water showers in health institutions. This new approach may provide a more accurate characterization of aerobiocontamination by Legionella bacteria.

Characterisation of exposure to airborne fungi: measurement of ergosterol.

In order to gain a clearer understanding of the level of fungal air contamination in indoor environments, we have adapted and tested a method to evaluate fungal biomass. Liquid phase chromatography (HPLC) of ergosterol, a component of the cell membrane of microscopic fungi, was employed. This method permits the detection and identification of ergosterol molecules at a concentration of 40 microg/ml (n=33, sigma=5). By combining this assay with a rotating cup collection apparatus, it was possible to measure fungal flora levels with a limit of quantification of 0.4 ng/m3 or a theoretical value of 150 spores per cubic meter (m3). Measurements of ergosterol levels performed on different sites showed that this method reflected the different situations of exposure of occupants to airborne fungal flora.

Fungal fragments as indoor air biocontaminants.

The aerosolization process of fungal propagules of three species (Aspergillus versicolor, Penicillium melinii, and Cladosporium cladosporioides) was studied by using a newly designed and constructed aerosolization chamber. We discovered that fungal fragments are aerosolized simultaneously with spores from contaminated agar and ceiling tile surfaces. Concentration measurements with an optical particle counter showed that the fragments are released in higher numbers (up to 320 times) than the spores. The release of fungal propagules varied depending on the fungal species, the air velocity above the contaminated surface, and the texture and vibration of the contaminated material. In contrast to spores, the release of fragments from smooth surfaces was not affected by air velocity, indicating a different release mechanism. Correlation analysis showed that the number of released fragments cannot be predicted on the basis of the number of spores. Enzyme-linked immunosorbent assays with monoclonal antibodies produced against Aspergillus and Penicillium fungal species showed that fragments and spores share common antigens, which not only confirmed the fungal origin of the fragments but also established their potential biological relevance. The considerable immunological reactivity, the high number, and the small particle size of the fungal fragments may contribute to human health effects that have been detected in buildings with mold problems but had no scientific explanation until now. This study suggests that future fungal spore investigations in buildings with mold problems should include the quantitation of fungal fragments.

Assessing bactericidal properties of materials: the case of metallic surfaces in contact with air.

A new method for assessing bactericidal properties of metallic materials, soiled by aerosol, was developed and applied to stainless steel in conditions close to reality. The airborne bacteria survival on different stainless steel grades and massive copper is presented here. The investigating bacterium was Enterococcus faecalis, which is a well-known contaminant strain in the indoor environments. It was observed that the bacterial aerosol lethality increased proportionally with the relative humidity (RH) of the environment. A significant difference in survival rate was measured depending on the tested supports, the greatest lethality being observed on clean massive copper. Moreover, the addition of nutrients on metallic surfaces, even in small quantities, was enough to ensure the revival of quiescent microorganisms.