Bioaerosol Sampling with a Wetted Wall Cyclone: Cell Culturability and DNA Integrity of Escherichia coli Bacteria

摘要

Contemporary near-real-time bioaerosol identifiers that read labeled DNA require a minimum DNA length of about 500,000 base pairs; and for critical applications, instrumental identification results must be verified through the use of classical microbiological culturing techniques. A 300 L/min Wetted Wall Cyclone (WWC) and an 800 L/min inertial impactor were used in a comparative study to collect aerosolized single cells of Escherichia coli (E. coli) at temperatures of 24°C and 46°C. Classical microbiological plating techniques showed that the culturability of E. coli collected with a WWC is a factor of about 100 higher than that of the impactor when the sampled aerosol is at room temperature (RT) and a factor of about 4000 higher when the sampled aerosol is at 46°C. DNA integrity was qualitatively evaluated with pulsed field gel electrophoresis (PFGE) and photographic evidence shows a significant difference in the amount of high molecular weight DNA (molecules larger than 500,000 base pairs) collected with the WWC compared with the impactor. Extracted DNA was also digested by the NotI enzyme, and the qualitative results of the restriction analysis showed there to be high integrity of the WWC-collected DNA, whereas the impactor-collected DNA showed considerable fragmentation. Real-Time polymerase chain reaction (RT-PCR) showed samples required for E. coli identification need to be about 100 times more concentrated if they are collected with the impactor rather than that of the WWC. Also, it appears that only the intact genomic DNA of the culturable cells provides adequate templates for traditional and RT-PCR amplification.