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Applied and Environmental Microbiology, June 2006, p. 4054-4060, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.00023-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Competitive Metagenomic DNA Hybridization Identifies Host-Specific Microbial Genetic Markers in Cow Fecal Samples{dagger}

Orin C. Shanks,1 Jorge W. Santo Domingo,1* Regina Lamendella,1 Catherine A. Kelty,1 and James E. Graham2

U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, Cincinnati, Ohio 45268,1 Department of Microbiology and Immunology and Department of Biology, University of Louisville, Louisville, Kentucky 402022

Received 4 January 2006/ Accepted 18 March 2006

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host-specific markers. Here we describe the application of a genome fragment enrichment (GFE) method to identify host-specific genetic markers from fecal microbial community DNA. As a proof of concept, bovine fecal DNA was challenged against a porcine fecal DNA background to select for bovine-specific DNA sequences. Bioinformatic analyses of 380 bovine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode membrane-associated and secreted proteins. Oligonucleotide primers capable of annealing to select Bacteroidales-like bovine GFE sequences exhibited extremely high specificity (>99%) in PCR assays with total fecal DNAs from 279 different animal sources. These primers also demonstrated a broad distribution of corresponding genetic markers (81% positive) among 148 different bovine sources. These data demonstrate that direct metagenomic DNA analysis by the competitive solution hybridization approach described is an efficient method for identifying potentially useful fecal genetic markers and for characterizing differences between environmental microbial communities.

* Corresponding author. Mailing address: U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 West Martin Luther King Drive, MS-387, Cincinnati, OH 45268. Phone: (513) 569-7085. Fax: (513) 569-7328. E-mail: santodomingo.jorge{at}epa.gov.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, June 2006, p. 4054-4060, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.00023-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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