Advancing Near-Real-Time Metabolic Profiling of Airborne Bacteria
EMILY KRAUS, Bharath Prithiviraj, Jin Seo, Mark Hernandez, University of Colorado Boulder
Abstract Number: 206
Working Group: Bioaerosols
Abstract
Deciphering mechanistic responses underpinning microbial survival in air is indispensable toward understanding their persistence in the atmospheric environment. High-throughput microbial mRNA sequencing can generate a transcriptome to ascertain metabolic activity within an aerosol as it is collected. Transcriptomic studies of bioaerosols must carefully preserve mRNA to overcome significant challenges associated with the low levels of bacterial biomass typically found in air, as well as the rapid turnover of mRNA within cells. We developed methods to collect and extract RNA from live airborne microorganisms for transcriptomic studies while minimizing experimental artifacts during aerosolization, collection, and subsequent sample handling. Mycobacteria (gram +) and Pseudomonas (gram -) spp. harvested during log phase growth (ca. 109 CFU/mL), were aerosolized under temperature-controlled conditions into respirable particle sizes within a 10m3 chamber under environmentally controlled conditions, and subsequently collected via condensation in 5 minute sampling intervals directly into an RNA preservative. Bacterial strains differed in their airborne persistence over the course of an hour both between and within genera tested. Total RNA quantities recovered for Mycobacterium crocinum ranged from 300 – 460 ng over 5, 15, and 25 minutes of suspension at 60% RH, while Pseudomonas syringae yielded 365 ng of total RNA after 5 and 30 minutes airborne under otherwise identical conditions. The developed methodology can be easily adapted for any liquid nucleic acid preservative and desired atmospheric conditions, and, if needed, to include concomitant viability studies (either by cultivability or propidium monoazide qPCR). With these pilot studies and methodological developments, we present recommendations for advancing bioaerosol metabolic profiling while minimizing pervasive experimental artifacts and challenges inherent to both high-fidelity mRNA collection and bioaerosol experiments with low biomass samples. These results may assist in better mechanistic assessments of engineering interventions that can selectively inactivate live bioaerosols in relevant scenarios.