Experimental parameters defining ultra-low biomass bioaerosol analysis

Abstract

Investigation of the microbial ecology of terrestrial, aquatic and atmospheric ecosystems requires specific sampling and analytical technologies, owing to vastly different biomass densities typically encountered. In particular, the ultra-low biomass nature of air presents an inherent analytical challenge that is confounded by temporal fluctuations in community structure. Our ultra-low biomass pipeline advances the field of bioaerosol research by significantly reducing sampling times from days/weeks/months to minutes/hours, while maintaining the ability to perform species-level identification through direct metagenomic sequencing. The study further addresses all experimental factors contributing to analysis outcome, such as amassment, storage and extraction, as well as factors that impact on nucleic acid analysis. Quantity and quality of nucleic acid extracts from each optimisation step are evaluated using fluorometry, qPCR and sequencing. Both metagenomics and marker gene amplification-based (16S and ITS) sequencing are assessed with regard to their taxonomic resolution and inter-comparability. The pipeline is robust across a wide range of climatic settings, ranging from arctic to desert to tropical environments. Ultimately, the pipeline can be adapted to environmental settings, such as dust and surfaces, which also require ultra-low biomass analytics.

Airborne fungal and bacterial microbiome in classrooms of elementary schools during the COVID ‐19 pandemic period: Effects of school disinfection and other environmental factors

“…Our study used different flow rates by sample types (i.e., 2.5 L/min for each of the three pumps for indoor samples and 4.0 L/min for each of the two pumps for outdoor samples) to ensure that outdoor air sampling collects similar total volume of air to indoor air sampling with fewer pumps. A study reported that using different sampling flow rates (100, 200, or 300 L/min) caused different particle retention efficiency; 51 however, our study used about 2 orders of magnitude lower flow rates, which would not likely to cause a significant difference in collection efficiency between the indoor and outdoor samples, as found in a study using low flow rates (1 to 4 L/min) 52 . Lastly, the controlled laboratory studies to further examine the effect of disinfection on indoor microbial community composition and how long the effect would last may improve our understanding of effectiveness of disinfection in school environments and its implication for occupants' health.…”

Neisseria species as pathobionts in bronchiectasis

“…Washed solutions from SASS air filters were further processed by filtering through 0.02 mm Anodisc filters (Whatman) using a vacuum manifold (DHI). DNA was then extracted from the Anodisc with the DNeasy PowerWater kit (Qiagen) according to the manufacturer's protocol with modifications to increase DNA yield (Luhung et al, 2021). Sterile swabs, filters and sputum DNA extraction reagents were processed simultaneously as extraction controls to assess the levels of experimental background contamination (Figure S3).…”

Airborne SARS‐CoV‐2 surveillance in hospital environment using high‐flowrate air samplers and its comparison to surface sampling

“…As per the manufacturer's specification, the small poresize filter has an efficiency of 90% for particles <0.5 µm with 50 L/ min sampling flowrate, whereas the large pore-size filter has an efficiency of 50% for particles <0.5 µm with 150 L/min sampling flowrate. The air sampler, along with the selected filter media, has been well studied for their use in sampling and analyzing environmental biological [20][21][22] and chemical aerosols. 23 All air samples were duplicated for environmental SARS-CoV-2 test and for culturing (only for positive samples).…”