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Applied and Environmental Microbiology, March 2006, p. 1843-1851, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.1843-1851.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Using DNA Microarrays To Identify Library-Independent Markers for Bacterial Source Tracking

Marilyn Soule,¹ Edward Kuhn,¹ Frank Loge,² John Gay,³ and Douglas R. Call^1*

Department of Microbiology and Pathology, Washington State University, Pullman, Washington,¹ Department of Civil and Environmental Engineering, University of California at Davis, Davis, California,² Department of Veterinary Clinical Sciences, Washington State University, Pullman, Washington³

Received 28 June 2005/ Accepted 27 December 2005

Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50 Enterococcus isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four Enterococcus species (Enterococcus casseliflavus, E. faecalis, E. hirae, or E. mundtii). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of library-independent markers.

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* Corresponding author. Mailing address: Department of Microbiology and Pathology, Washington State University, 402 Bustad Hall, Pullman, WA 99164-7040. Phone: (509) 335-6313. Fax: (509) 335-8529. E-mail: drcall{at}wsu.edu.

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Applied and Environmental Microbiology, March 2006, p. 1843-1851, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.1843-1851.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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