Sampling efficiency and nucleic acid stability during long-term sampling with different bioaerosol samplers.

Aerosol microbiome studies have received increased attention as technological advancements have made it possible to dive deeper into the microbial diversity. To enhance biomass collection for metagenomic sequencing, long-term sampling is a common strategy. While the impact of prolonged sampling times on microorganisms' culturability and viability is well-established, its effect on nucleic acid stability remains less understood but is essential to ensure representative sample collection. This study evaluated four air samplers (SKC BioSampler, SASS3100, Coriolis µ, BioSpot-VIVAS 300-P) against a reference sampler (isopore membrane filters) to identify nucleic acid stability during long-term sampling. Physical sampling efficiencies determined with a fluorescent tracer for three particle sizes (0.8, 1, and 3 µm), revealed high efficiencies (> 80% relative to reference) for BioSampler, SASS3100, and BioSpot-VIVAS for all particle sizes, and for Coriolis with 3 µm particles. Coriolis exhibited lower efficiency for 0.8 µm (7%) and 1 µm (50%) particles. During 2-h sampling with MS2 and Pantoea agglomerans, liquid-based collection with Coriolis and BioSampler showed a decrease in nucleic acid yields for all test conditions. BioSpot-VIVAS displayed reduced sampling efficiency for P. agglomerans compared to MS2 and the other air samplers, while filter-based collection with SASS3100 and isopore membrane filters, showed indications of DNA degradation for 1 µm particles of P. agglomerans after long-term sampling. These findings show that long-term air sampling affects nucleic acid stability in both liquid- and filter-based collection methods. These results highlight bias produced by bioaerosol collection and should be considered when selecting an air sampler and interpreting aerosol microbiome data.

Reference genome assembly and annotation of two Bacillus cereus sensu lato strains from Etosha National Park, Namibia.

Bacillus cereus sensu lato (s.l.) poses health and security issues. Here, we report the reference genome assembly of two Bacillus cereus s.l. strains, isolated from Etosha National Park, Namibia (FFI_BCgr36 and FFI_BCgr46). These unique genomes open for better understanding of environmental diversity and improvements in detection of threatening species.

Complete reference genome assemblies and annotations of three Escherichia coli MRE162 clones.

Escherichia coli MRE162 was originally isolated from a toilet pan in 1949 and since been utilized in numerous studies. Here, we sequence, assemble, and annotate clones held at three laboratories providing reference-level assemblies. We show the uniqueness of MRE162 to strains in open databases and make the UK clone publically available.

SARS-CoV-2 in the Air Surrounding Patients during Nebulizer Therapy.

Nebulizer therapy is commonly used for patients with obstructive pulmonary disease or acute pulmonary infections with signs of obstruction. It is considered a "potential aerosol-generating procedure," and the risk of disease transmission to health care workers is uncertain. The aim of this pilot study was to assess whether nebulizer therapy in hospitalized COVID-19 patients is associated with increased dispersion of SARS-CoV-2. Air samples collected prior to and during nebulizer therapy were analyzed by RT-PCR and cell culture. Total aerosol particle concentrations were also quantified. Of 13 patients, seven had quantifiable virus in oropharynx samples, and only two had RT-PCR positive air samples. For both these patients, air samples collected during nebulizer therapy had higher SARS-CoV-2 RNA concentrations compared to control air samples. Also, for particle sizes 0.3-5 µm, particle concentrations were significantly higher during nebulizer therapy than in controls. We were unable to cultivate virus from any of the RT-PCR positive air samples, and it is therefore unknown if the detected virus were replication-competent; however, the significant increase in smaller particles, which can remain airborne for extended periods of time, and increased viral RNA concentrations during treatment may indicate that nebulizer therapy is associated with increased risk of SARS-CoV-2 transmission.

Dispersion of SARS-CoV-2 in air surrounding COVID-19-infected individuals with mild symptoms.

Since the beginning of the pandemic, the transmission modes of SARS-CoV-2-particularly the role of aerosol transmission-have been much debated. Accumulating evidence suggests that SARS-CoV-2 can be transmitted by aerosols, and not only via larger respiratory droplets. In this study, we quantified SARS-CoV-2 in air surrounding 14 test subjects in a controlled setting. All subjects had SARS-CoV-2 infection confirmed by a recent positive PCR test and had mild symptoms when included in the study. RT-PCR and cell culture analyses were performed on air samples collected at distances of one, two, and four meters from test subjects. Oronasopharyngeal samples were taken from consenting test subjects and analyzed by RT-PCR. Additionally, total aerosol particles were quantified during air sampling trials. Air viral concentrations at one-meter distance were significantly correlated with both viral loads in the upper airways, mild coughing, and fever. One sample collected at four-meter distance was RT-PCR positive. No samples were successfully cultured. The results reported here have potential application for SARS-CoV-2 detection and monitoring schemes, and for increasing our understanding of SARS-CoV-2 transmission dynamics. Practical implications. In this study, quantification of SARS-CoV-2 in air was performed around infected persons with mild symptoms. Such persons may go longer before they are diagnosed and may thus be a disproportionately important epidemiological group. By correlating viral concentrations in air with behavior and symptoms, we identify potential risk factors for viral dissemination in indoor environments. We also show that quantification of total aerosol particles is not a useful strategy for monitoring SARS-CoV-2 in indoor environments.

Quantification of aerosol dispersal from suspected aerosol-generating procedures.

BACKGROUND: Oxygen-delivering modalities like humidified high-flow nasal cannula (HFNC) and noninvasive positive-pressure ventilation (NIV) are suspected of generating aerosols that may contribute to transmission of disease such as coronavirus disease 2019. We sought to assess if these modalities lead to increased aerosol dispersal compared to the use of non-humidified low-flow nasal cannula oxygen treatment (LFNC). METHODS: Aerosol dispersal from 20 healthy volunteers using HFNC, LFNC and NIV oxygen treatment was measured in a controlled chamber. We investigated effects related to coughing and using a surgical face mask in combination with the oxygen delivering modalities. An aerodynamic particle sizer measured aerosol particles (APS3321, 0.3-20 µm) directly in front of the subjects, while a mesh of smaller particle sensors (SPS30, 0.3-10 µm) was distributed in the test chamber. RESULTS: Non-productive coughing led to significant increases in particle dispersal close to the face when using LFNC and HFNC but not when using NIV. HFNC or NIV did not lead to a statistically significant increase in aerosol dispersal compared to LFNC. With non-productive cough in a room without air changes, there was a significant drop in particle levels between 100 cm and 180 cm from the subjects. CONCLUSIONS: Our results indicate that using HFNC and NIV does not lead to increased aerosol dispersal compared to low-flow oxygen treatment, except in rare cases. For a subject with non-productive cough, NIV with double-limb circuit and non-vented mask may be a favourable choice to reduce the risk for aerosol spread.

A global metagenomic map of urban microbiomes and antimicrobial resistance.

We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities.

Performance evaluation of high-volume electret filter air samplers in aerosol microbiome research.

BACKGROUND: Reliable identification and quantification of bioaerosols is fundamental in aerosol microbiome research, highlighting the importance of using sampling equipment with well-defined performance characteristics. Following advances in sequencing technology, shotgun metagenomic sequencing (SMS) of environmental samples is now possible. However, SMS of air samples is challenging due to low biomass, but with the use of high-volume air samplers sufficient DNA yields can be obtained. Here we investigate the sampling performance and comparability of two hand-portable, battery-operated, high-volume electret filter air samplers, SASS 3100 and ACD-200 Bobcat, previously used in SMS-based aerosol microbiome research. RESULTS: SASS and Bobcat consistently delivered end-to-end sampling efficiencies > 80% during the aerosol chamber evaluation, demonstrating both as effective high-volume air samplers capable of retaining quantitative associations. Filter recovery efficiencies were investigated with manual and sampler-specific semi-automated extraction procedures. Bobcat semi-automated extraction showed reduced efficiency compared to manual extraction. Bobcat tended towards higher sampling efficiencies compared to SASS when combined with manual extraction. To evaluate real-world sampling performance, side-by-side SASS and Bobcat sampling was done in a semi-suburban outdoor environment and subway stations. SMS-based microbiome profiles revealed that highly abundant bacterial species had similar representation across samplers. While alpha diversity did not vary for the two samplers, beta diversity analyses showed significant within-pair variation in subway samples. Certain species were found to be captured only by one of the two samplers, particularly in subway samples. CONCLUSIONS: SASS and Bobcat were both found capable of collecting sufficient aerosol biomass amounts for SMS, even at sampling times down to 30 min. Bobcat semi-automated filter extraction was shown to be less effective than manual filter extraction. For the most abundant species the samplers were comparable, but systematic sampler-specific differences were observed at species level. This suggests that studies conducted with these highly similar air samplers can be compared in a meaningful way, but it would not be recommended to combine samples from the two samplers in joint analyses. The outcome of this work contributes to improved selection of sampling equipment for use in SMS-based aerosol microbiome research and highlights the importance of acknowledging bias introduced by sampling equipment and sample recovery procedures.

Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research.

BACKGROUND: Aerosol microbiome research advances our understanding of bioaerosols, including how airborne microorganisms affect our health and surrounding environment. Traditional microbiological/molecular methods are commonly used to study bioaerosols, but do not allow for generic, unbiased microbiome profiling. Recent studies have adopted shotgun metagenomic sequencing (SMS) to address this issue. However, SMS requires relatively large DNA inputs, which are challenging when studying low biomass air environments, and puts high requirements on air sampling, sample processing and DNA isolation protocols. Previous SMS studies have consequently adopted various mitigation strategies, including long-duration sampling, sample pooling, and whole genome amplification, each associated with some inherent drawbacks/limitations. RESULTS: Here, we demonstrate a new custom, multi-component DNA isolation method optimized for SMS-based aerosol microbiome research. The method achieves improved DNA yields from filter-collected air samples by isolating DNA from the entire filter extract, and ensures a more comprehensive microbiome representation by combining chemical, enzymatic and mechanical lysis. Benchmarking against two state-of-the-art DNA isolation methods was performed with a mock microbial community and real-world air samples. All methods demonstrated similar performance regarding DNA yield and community representation with the mock community. However, with subway samples, the new method obtained drastically improved DNA yields, while SMS revealed that the new method reported higher diversity. The new method involves intermediate filter extract separation into a pellet and supernatant fraction. Using subway samples, we demonstrate that supernatant inclusion results in improved DNA yields. Furthermore, SMS of pellet and supernatant fractions revealed overall similar taxonomic composition but also identified differences that could bias the microbiome profile, emphasizing the importance of processing the entire filter extract. CONCLUSIONS: By demonstrating and benchmarking a new DNA isolation method optimized for SMS-based aerosol microbiome research with both a mock microbial community and real-world air samples, this study contributes to improved selection, harmonization, and standardization of DNA isolation methods. Our findings highlight the importance of ensuring end-to-end sample integrity and using methods with well-defined performance characteristics. Taken together, the demonstrated performance characteristics suggest the new method could be used to improve the quality of SMS-based aerosol microbiome research in low biomass air environments.

The subway microbiome: seasonal dynamics and direct comparison of air and surface bacterial communities.

BACKGROUND: Mass transit environments, such as subways, are uniquely important for transmission of microbes among humans and built environments, and for their ability to spread pathogens and impact large numbers of people. In order to gain a deeper understanding of microbiome dynamics in subways, we must identify variables that affect microbial composition and those microorganisms that are unique to specific habitats. METHODS: We performed high-throughput 16S rRNA gene sequencing of air and surface samples from 16 subway stations in Oslo, Norway, across all four seasons. Distinguishing features across seasons and between air and surface were identified using random forest classification analyses, followed by in-depth diversity analyses. RESULTS: There were significant differences between the air and surface bacterial communities, and across seasons. Highly abundant groups were generally ubiquitous; however, a large number of taxa with low prevalence and abundance were exclusively present in only one sample matrix or one season. Among the highly abundant families and genera, we found that some were uniquely so in air samples. In surface samples, all highly abundant groups were also well represented in air samples. This is congruent with a pattern observed for the entire dataset, namely that air samples had significantly higher within-sample diversity. We also observed a seasonal pattern: diversity was higher during spring and summer. Temperature had a strong effect on diversity in air but not on surface diversity. Among-sample diversity was also significantly associated with air/surface, season, and temperature. CONCLUSIONS: The results presented here provide the first direct comparison of air and surface bacterial microbiomes, and the first assessment of seasonal variation in subways using culture-independent methods. While there were strong similarities between air and surface and across seasons, we found both diversity and the abundances of certain taxa to differ. This constitutes a significant step towards understanding the composition and dynamics of bacterial communities in subways, a highly important environment in our increasingly urbanized and interconnect world. Video abstract.

Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions.

The ability to perform controlled experiments with bioaerosols is a fundamental enabler of many bioaerosol research disciplines. A practical alternative to using hazardous biothreat agents, e.g., for detection equipment development and testing, involves using appropriate model organisms (simulants). Several species of Gram-negative bacteria have been used or proposed as biothreat simulants. However, the appropriateness of different bacterial genera, species, and strains as simulants is still debated. Here, we report aerobiological stability characteristics of four species of Gram-negative bacteria (Pantoea agglomerans, Serratia marcescens, Escherichia coli, and Xanthomonas arboricola) in single-cell particles and cell clusters produced using four spray liquids (H2O, phosphate-buffered saline[PBS], spent culture medium[SCM], and a SCM-PBS mixture). E. coli showed higher stability in cell clusters from all spray liquids than the other species, but it showed similar or lower stability in single-cell particles. The overall stability was higher in cell clusters than in single-cell particles. The highest overall stability was observed for bioaerosols produced using SCM-containing spray liquids. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research.IMPORTANCE The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns.

Complete Genome Sequences of Six Legionella pneumophila Isolates from Two Collocated Outbreaks of Legionnaires' Disease in 2005 and 2008 in Sarpsborg/Fredrikstad, Norway.

Here, we report the complete genome sequences of Legionella pneumophila isolates from two collocated outbreaks of Legionnaires' disease in 2005 and 2008 in Sarpsborg/Fredrikstad, Norway. One clinical and two environmental isolates were sequenced from each outbreak. The genome of all six isolates consisted of a 3.36 Mb-chromosome, while the 2005 genomes featured an additional 68 kb-episome sharing high sequence similarity with the L. pneumophila Lens plasmid. All six genomes contained multiple mobile genetic elements including novel combinations of type-IVA secretion systems. A comparative genomics study will be launched to resolve the genetic relationship between the L. pneumophila isolates.

Microbial community structure in a full-scale anaerobic treatment plant during start-up and first year of operation revealed by high-throughput 16S rRNA gene amplicon sequencing.

High-throughput amplicon sequencing of six biomass samples from a full-scale anaerobic reactor at a Norwegian wood and pulp factory using Biothane Biobed Expanded Granular Sludge Bed (EGSB) technology during start-up and first year of operation was performed. A total of 106,166 16S rRNA gene sequences (V3-V5 region) were obtained. The number of operational taxonomic units (OTUs) ranged from 595 to 2472, and a total of 38 different phyla and 143 families were observed. The predominant phyla were Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria, and Spirochaetes. A more diverse microbial community was observed in the inoculum biomass coming from an Upflow Anaerobic Sludge Blanket (USAB) reactor, reflecting an adaptation of the inoculum diversity to the specific conditions of the new reactor. In addition, no taxa classified as obligate pathogens were identified and potentially opportunistic pathogens were absent or observed in low abundances. No Legionella bacteria were identified by traditional culture-based and molecular methods.

Identification of airborne bacteria by 16S rDNA sequencing, MALDI-TOF MS and the MIDI microbial identification system.

The aim of this study was to collect and identify airborne bacteria in Norway, Sweden and Finland and to compare three different technologies for identifying collected airborne bacterial isolates: the "gold standard" method 16S rDNA sequencing, MALDI-TOF MS using the MALDI Biotyper 2.0 and the MIDI Sherlock® Microbial Identification System (MIDI MIS system). Airborne bacteria were collected during three different periods from May to October 2009 using air sampling directly on agar plates. A total of 140 isolates were collected during three sampling campaigns, and 74 % (103) of these isolates were analyzed by all three methods. The dominant genera in Norway and Finland were the gram-positive bacteria Bacillus and Staphylococcus, whereas the gram-negative bacterium Acinetobacter was the dominant genus in Sweden. Using 16S rDNA sequencing, MALDI-TOF MS and MIDI MIS analysis, 83, 79 and 75 %, respectively, of the isolates were identified and assigned to order or higher taxonomic levels. In this study, the MALDI-TOF MS combining with the MALDI Biotyper 2.0 classification tool was demonstrated to be a fast and reliable alternative for identifying the airborne bacterial isolates. These studies have increased knowledge about the airborne bacterial background in outdoor air, which can be useful for evaluating and improving the operational performance of biological detectors in various environments. To our knowledge, this is the first time that 16S rDNA sequencing, MALDI-TOF MS and MIDI MIS analysis technologies have been compared for their efficiency in identifying airborne bacteria.

Temporal variability of the bioaerosol background at a subway station: concentration level, size distribution, and diversity of airborne bacteria.

Naturally occurring bioaerosol environments may present a challenge to biological detection-identification-monitoring (BIODIM) systems aiming at rapid and reliable warning of bioterrorism incidents. One way to improve the operational performance of BIODIM systems is to increase our understanding of relevant bioaerosol backgrounds. Subway stations are enclosed public environments which may be regarded as potential bioterrorism targets. This study provides novel information concerning the temporal variability of the concentration level, size distribution, and diversity of airborne bacteria in a Norwegian subway station. Three different air samplers were used during a 72-h sampling campaign in February 2011. The results suggested that the airborne bacterial environment was stable between days and seasons, while the intraday variability was found to be substantial, although often following a consistent diurnal pattern. The bacterial levels ranged from not detected to 10(3) CFU m(-3) and generally showed increased levels during the daytime compared to the nighttime levels, as well as during rush hours compared to non-rush hours. The airborne bacterial levels showed rapid temporal variation (up to 270-fold) on some occasions, both consistent and inconsistent with the diurnal profile. Airborne bacterium-containing particles were distributed between different sizes for particles of >1.1 µm, although ∼50% were between 1.1 and 3.3 µm. Anthropogenic activities (mainly passengers) were demonstrated as major sources of airborne bacteria and predominantly contributed 1.1- to 3.3-µm bacterium-containing particles. Our findings contribute to the development of realistic testing and evaluation schemes for BIODIM equipment by providing information that may be used to simulate operational bioaerosol backgrounds during controlled aerosol chamber-based challenge tests with biological threat agents.

Rapid identification of Bacillus anthracis spores in suspicious powder samples by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 10(6) spores, equivalent to a 55-µg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min.

Large scale MALDI-TOF MS based taxa identification to identify novel pigment producers in a marine bacterial culture collection.

A challenge in the rational exploitation of microbial culture collections is to avoid superfluous testing of replicas. MALDI-TOF MS has been shown to be an efficient dereplication tool as it can be used to discriminate between bacterial isolates at the species level. A bacterial culture collection of more than 10,000 heterotrophic marine bacterial isolates from sea-water surface layers of the Norwegian Trondheimsfjord and neighbouring coastal areas has been established. A sub-collection of pigmented isolates was earlier screened for novel carotenoids with UVA-Blue light absorbing properties. This was a comprehensive analytical task and it was observed that a significant number of extracts with identical pigment profile were recovered. Hence, this study was undertaken to explore the use of MALDI-TOF MS as a dereplication tool to quickly characterize the bacterial collection. Furthermore, LC-DAD-MS analysis of pigment profiles was performed to check if pigment profile diversity was maintained among isolates kept after the potential MALDI-TOF MS selection step. Four hundred isolates comprising both pigmented and non-pigmented isolates were used for this study. The resulting MALDI-TOF MS dendrogram clearly identified a diversity of different taxa and these were supported by the pigment profile clustering, thus linking the pigment production as species-specific properties. Although one exception was found, it can be concluded that MALDI-TOF MS dereplication is a promising pre-screening tool for more efficient screening of microbial culture collection containing pigments with potential novel properties.

Characterization of airborne bacteria at an underground subway station.

The reliable detection of airborne biological threat agents depends on several factors, including the performance criteria of the detector and its operational environment. One step in improving the detector's performance is to increase our knowledge of the biological aerosol background in potential operational environments. Subway stations are enclosed public environments, which may be regarded as potential targets for incidents involving biological threat agents. In this study, the airborne bacterial community at a subway station in Norway was characterized (concentration level, diversity, and virulence- and survival-associated properties). In addition, a SASS 3100 high-volume air sampler and a matrix-assisted laser desorption ionization-time of flight mass spectrometry-based isolate screening procedure was used for these studies. The daytime level of airborne bacteria at the station was higher than the nighttime and outdoor levels, and the relative bacterial spore number was higher in outdoor air than at the station. The bacterial content, particle concentration, and size distribution were stable within each environment throughout the study (May to September 2010). The majority of the airborne bacteria belonged to the genera Bacillus, Micrococcus, and Staphylococcus, but a total of 37 different genera were identified in the air. These results suggest that anthropogenic sources are major contributors to airborne bacteria at subway stations and that such airborne communities could harbor virulence- and survival-associated properties of potential relevance for biological detection and surveillance, as well as for public health. Our findings also contribute to the development of realistic testing and evaluation schemes for biological detection/surveillance systems by providing information that can be used to mimic real-life operational airborne environments in controlled aerosol test chambers.