AAAR 33rd Annual Conference
October 20 - October 24, 2014
Rosen Shingle Creek
Orlando, Florida, USA
Abstract View
Effect of Bioaerosol Sampling Stress on 16S rRNA/rRNA-gene Ratio of Airborne Bacteria
HUAJUN ZHEN, Valdis Krumins, Donna Fennell, Gediminas Mainelis, Rutgers, The State University of New Jersey
Abstract Number: 251 Working Group: Bioaerosols
Abstract Enumeration and characterization of ribosomal RNA (rRNA) is widely used in microbiology when analyzing metabolically active species within microbial communities. A higher cellular ribosome content (often represented as 16S rRNA normalized to 16S rRNA-gene) is interpreted as greater potential for metabolic activity of bacteria. The same methodology could be applied to investigate metabolic activity of bacteria in the airborne phase. However, airborne bacteria experience mechanical and/or desiccation stress during sampling, which may affect rRNA stability, raising questions regarding representativeness of measured rRNA content in bioaerosol samples.
To simulate the stress during sampling by filtration, fresh E. coli cultures were aerosolized and collected on filters. Particle-free air was then passed through the filters for 0, 2, 4 and 6 hours. Our initial results showed high variability in rRNA/rRNA-gene ratio as function of sampling time. We determined that this inconsistency was largely due to the losses of rRNA and rRNA-gene (up to 90%) during sample processing.
To overcome this issue, a dual-internal-reference technique was developed utilizing exogenous Pseudomonas fluorescens 16S rRNA and DNA as internal standards to control for losses of E. coli rRNA and DNA during sample processing. This technique was applied to investigate the change of rRNA/rRNA-gene ratio during growth of E. coli in Tryptic Soy Broth at 37 °C. Results showed that the ratio increased rapidly to ~104 during the initial 3 hours of exponential growth phase, and then gradually decreased and finally leveled off at around 5000 after 16 hours. This confirmed the positive correlation between bacterial growth and cellular ribosome content as demonstrated by previous studies.
Tests with sampling devices that utilize collection mechanisms of filtration, impingement and electrostatic precipitation are currently ongoing. The preliminary data indicate that the choice of sampling technique might bias the amount of rRNA detected in bioaerosol samples.