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United States Department of Agriculture

Agricultural Research Service

Title: Specific Detection of Pathogens in Environmental Samples: Agricultural Research Meets Bioterrorism

Author
item Karns, Jeffrey

Submitted to: Annual Meeting Canadian Society of Microbiologists
Publication Type: Abstract Only
Publication Acceptance Date: May 1, 2003
Publication Date: May 23, 2003
Citation: Karns, J.S. 2003. Specific detection of pathogens in environmental samples: Agricultural research meets bioterrorism [abstract]. 53rd Annual Meeting Canadian Society of Microbiologists, May 25-28, 2003, Ville de Laval, Montreal, Canada. p. 1.

Technical Abstract: Detection of pathogenic forms of microorganisms in the environment presents interesting challenges. For instance, testing the quality of water usually involves determining the number of fecal microorganisms (fecal coliforms, E. coli, or fecal streptococci) by culturing in or on various microbiological media. While these tests are useful for determining the likelihood that water has been contaminated by fecal material they do not give a true indication of actual pathogenic organisms in the sample. To further characterize pathogenic forms of E. coli or Listeria through traditional methods can take several days or weeks. Traditional characterization of Bacillus anthracis in air and surface samples also takes many days for final confirmation. Immunological and molecular detection techniques can significantly shorten the time periods needed for accurate assessment of pathogenic microorganisms in environmental samples. A method developed by this laboratory uses immunomagnetic bead separation (IM) combined with electrochemiluminescent detection (ECL) to quantitatively detect E. coli O157 in water in less than 24 hours. We have also developed quantitative real-time PCR assays for the quantitative assessment of potentially enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli as well as total E.coli in water. Since the population of E. coli in most water samples is too low for direct detection, the real-time PCR can be performed on DNA isolated from enrichment cultures. We have used this method to quantify low levels of EPEC and EHEC strains in water samples from urban and agricultural watersheds. We have also used real-time PCR techniques to detect Salmonella and Listeria in milk after enrichment culture, eliminating 48 to 96 h of traditional confirmation through culture. A novel test of real-time PCR techniques for the detection of pathogenic microbes came during the anthrax attack of October 2001 in Washington, DC. A mobile laboratory was set up at a downtown location where assays using a portable real-time PCR device were used to screen environmental samples (surface swipes, air samples, powders from articles of mail) for the presence of Bacillus anthracis spores. From October 2001 to September 2002 over 4,635 pieces of mail were examined and 405 real-time PCR assays were performed covering 4,639 samples. No B. anthracis spores were directly detected by real-time PCR and subsequent research indicated that the detection limits for B. anthracis spores in these samples were rather high (5,000 spores/swab or 3,700 spores/m3 air). The real utility of the real-time PCR was the quick identification of suspect colonies after overnight incubation on agar which can eliminate 96 h of confirmatory culture techniques. It is clear that new immunological and molecular detection methods offer faster and more specific identification and quantitation of pathogenic organisms in environmental samples.

Last Modified: 11/28/2014
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