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

Abstract

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.

Copyright 2012 American Association for Aerosol Research

Acknowledgments

This work was supported by the Edgewood Chemical Biological Center of the U.S. Army Research, Development, and Engineering command under contract DAAD13–03-C-0050. The authors wish to express their appreciation to Edward W. Stuebing, Jerold R. Bottiger, and Jana Kasavan of the Army and Dr. Carlos F. Gonzalez of the Department of Plant Pathology at the Texas A&M University for their suggestions and help.

^aCurrent addresses: AR McFarland, PhD, PE, Inc., Houston, TX.

Notes

A.k.a. log phase, which is a time period characterized by the increase in the number of new bacteria appearing per unit time being proportional to the existing population.

Optical density at 600 nm light wave length.

Guanine (G) and cytosine (C) are two of the five main nucleobases found in the nucleic acids DNA and RNA. In DNA, guanine is paired with cytosine via hydrogen bonds.