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.

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.

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.

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.

Standoff detection of biological agents using laser induced fluorescence-a comparison of 294 nm and 355 nm excitation wavelengths.

Standoff detection measuring the fluorescence spectra of seven different biological agents excited by 294 nm as well as 355 nm wavelength laser pulses has been undertaken. The biological warfare agent simulants were released in a semi-closed aerosol chamber at 210 m standoff distance and excited by light at either of the two wavelengths using the same instrument. Significant differences in several of the agents' fluorescence response were seen at the two wavelengths. The anthrax simulants' fluorescence responses were almost an order of magnitude stronger at the shorter wavelength excitation. However, most importantly, the fluorescence spectra were significantly more dissimilar at 294 nm than at 355 nm excitation with ~7 nm spectral resolution. This indicates that classification of the substances should be possible with a lower error rate for standoff detection using 294 nm rather than 355 nm excitation wavelength, or even better, utilizing both.

Dispersion of Legionella-containing aerosols from a biological treatment plant, Norway.

Legionella was detected in aeration ponds (biological treatment plant) at Borregaard Ind. Ltd., Norway, and in air samples harvested directly above these ponds. Since 2005, three outbreaks of legionellosis occurred within a 10 km radius from this plant. This work addresses the dispersion patterns of Legionella-containing particles by characterizing the aerosol plume emitted from these ponds (outbreak source) less than 500 meters using wind-tunnel measurements, CFD simulations, and real-life measurements. The most abundant particles directly over the ponds were less than 6 and more than 15 microm. The results showed that the aerosol plume remained narrow; 180 meters wide at 350 meters downwind of the ponds, and that 2 and 18 microm aerosols were mainly deposited in the vicinity of the ponds ( 150 - 200 meters). Furthermore, the maximum aerosol concentration level appeared 5-10 meters above ground level and the maximum concentration 500 meters downwind was approximately 2 per cent of the concentration level directly above the ponds. Our study demonstrates the strength of combining modeling with real-life aerosol analyses increasing the understanding of dispersion of airborne (pathogenic) microorganisms.

Evaluation of a highly discriminating multiplex multi-locus variable-number of tandem-repeats (MLVA) analysis for Vibrio cholerae.

Vibrio cholerae is the etiological agent of cholera and may be used in bioterror actions due to the easiness of its dissemination, and the public fear for acquiring the cholera disease. A simple and highly discriminating method for connecting clinical and environmental isolates of V. cholerae is needed in microbial forensics. Twelve different loci containing variable numbers of tandem-repeats (VNTRs) were evaluated in which six loci were polymorphic. Two multiplex reactions containing PCR primers targeting these six VNTRs resulted in successful DNA amplification of 142 various environmental and clinical V. cholerae isolates. The genetic distribution inside the V. cholerae strain collection was used to evaluate the discriminating power (Simpsons Diversity Index=0.99) of this new MLVA analysis, showing that the assay have a potential to differentiate between various strains, but also to identify those isolates which are collected from a common V. cholerae outbreak. This work has established a rapid and highly discriminating MLVA assay useful for track back analyses and/or forensic studies of V. cholerae infections.

Tracking airborne Legionella and Legionella pneumophila at a biological treatment plant.

Biological treatment plants are frequently used to degrade organic substances in wastewater from wood refinement processes. Aeration ponds in such plants provide an optimal growth environment for many microorganisms, including Legionella species. To investigate whether legionellae could be dispersed as aerosols from the ponds and transported by the wind, the wetted-wall cyclone SASS 2000(PLUS) and the impactors MAS-100 and STA-204 were used to collect air samples directly above, upwind, and downwind of aeration ponds during a 4-month period. Computational fluid dynamics was used a priori to estimate the aerosol paths and to determine suitable air-sampling locations. Several Legionella species, including Legionella pneumophila, were identified in air samples at the biological treatment plant using microbiological and molecular methods. L. pneumophila was identified up to distances of 200 m downwind from the ponds, but, in general, not upwind nor outside the predicted aerosol paths. The highest concentration level of viable legionellae was identified directly above the aeration ponds (3300 CFU/m3). This level decreased as the distance from the aeration ponds increased. Molecular typing indicated that a single clone of L. pneumophila was dispersed from the ponds during the period of the study. Thus, our study demonstrated that aerosols generated at aeration ponds of biological treatment facilities may contain L. pneumophila, which then can be transported by the wind to the surroundings. The methods used in this study may be generically applied to trace biological aerosols that may pose a challenge to environmental occupational health.

Genetic distribution of 295 Bacillus cereus group members based on adk-screening in combination with MLST (Multilocus Sequence Typing) used for validating a primer targeting a chromosomal locus in B. anthracis.

The genetic distribution of 295 Bacillus cereus group members has been investigated by using a modified Multilocus Sequence Typing method (MLST). By comparing the nucleic acid sequence of the adk gene fragment, isolates of B. cereus group members most related to B. anthracis may be easily identified. The genetic distribution, with focus on the B. anthracis close neighbours, was used to evaluate a new primer set for specific identification of B. anthracis. This primer set, BA5510-1/2, targeted the putative B. anthracis specific gene BA5510. Real-time PCR using BA5510-1/2 amplified the target fragment from all B. anthracis strains tested and only two (of 289) non-B. anthracis strains analysed. This is one of the most thoroughly validated chromosomal B. anthracis markers for real-time PCR identification, in which the screened collection contained several very closely related B. anthracis strains.

Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification.

A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.

Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR.

A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.