10th International Aerosol Conference September 2 - September 7, 2018 America's Center Convention Complex St. Louis, Missouri, USA
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Comparative Assessment of Conventional, Fluorometric and Molecular Biomarkers of Airborne Microbial Activity during Condensation Capture and Direct Filtration
MARINA NIETO-CABALLERO, Patricia Keady, Nicole Savage, Mark T. Hernandez, University of Colorado Boulder
Abstract Number: 220 Working Group: Bioaerosols
Abstract While the field of aerobiology has experienced rapid growth over the last decade, technology is still lacking that enables engineers and scientists to accurately assess the identity, abundance and activity of airborne microorganisms—both indoors and out. Accurate bioaerosol assessment remains limited by conventional filtration and impaction methods that can compromise, or otherwise influence, the physiologic and metabolic state of airborne microbes, as they are collected from the atmospheric environment.
In response, emerging condensation capture technology was assessed for its ability to preserve bioaerosols in the same physiologic state they exist when suspended in the atmosphere. As judged by direct microscopy, culturing and genetic material content (DNA and RNA), the impacts of whole cell bioaerosol recovery using Condensation Growth Tube Capture ((CGTC), Aerosol Devices, Fort Collins, CO) was juxtaposed to conventional particle filtration. Under this scenario, a full-scale environmental chamber (ca. 11 m3) was charged with well-characterized pure cultures of bacteria and fungal spores that have often been used to model the behavior of bioaerosols that are relevant to the built environment and environmental health (Bacillus sp., Mycobacteria sp., Legionella sp., and Aspergillus sp.). With exception to spores that were dry-aerosolized from a desiccated state, vegetative cells, harvested from liquid culture, were introduced to the chamber air and allowed to age under controlled temperature and humidity conditions. Over several hours, airborne microbes were periodically and concomitantly collected using CGTC and filtration, while being counted, sized and characterized by cytometry using a conventional laser coupled with Ultraviolet Induced Fluorescence ((UVIF), InstaScope, DetectionTek, Boulder, CO).
Bacteria, while in their late exponential growth phase, were aerosolized for ten minutes using a 6-jet Collison nebulizer, reaching an initial concentration at or above 105 cells/L in the chamber air. These airborne bacteria were collected by CGTC directly in the following liquids that were continuously chilled to 4oC: phosphate buffered saline and a genetic preservative (DNA/RNA Shield, ZymoBIOMICS, Irvine, CA). Cellular preparation (washing) and extended nebulization time had no effect on culturability, cell size or cell count.
A time-series of recovery experiments was performed at different humidity levels (30% and 80%). Samples were aseptically taken from CGTC wells for 10 minutes after 30, 60, 90, 120, 150 and 180 minutes of aerosol aging in the chamber, following the initial aerosolization. This provided a means to independently isolate and compare cellular deposition rates as judged by the different recovery methods. Direct microscopic and cytometric counts were in agreement with the time-series recovery of airborne DNA gene copy numbers, obtained using quantitative PCR (qPCR) with universal bacterial primers. Quantitative PCR steadily recovered between 3 and 4 fold higher gene copy numbers than its direct microscopic counterpart, consistent with whole cell microorganisms containing multiple copies of the target 16s rDNA gene. As anticipated, a significantly lower and age-dependent response was observed from the standard culturing of colony forming units (CFUs) on rich, broad spectrum agar (R2A). About 0.1% of the microorganisms aerosolized could be recovered as CFUs; however, culturable cells rapidly declined below detection (<60 min) while the optical methods indicated orders of magnitude higher airborne cell numbers, consistent with amplicons of (phylo)genetic DNA and RNA targets. The direct recovery of whole cells into genomic preservative, suggests the persistence of different RNA pools that can be used as a robust activity indicator in this time window. Moreover, the optical diameter of the airborne bacteria collected significantly diminished during aerosol aging as judged by aerosol cytometry and direct microscopy.