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Applied and Environmental Microbiology, December 2004, p. 7372-7377, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7372-7377.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Quantification of Tetracycline Resistance Genes in Feedlot Lagoons by Real-Time PCR

Marilyn S. Smith,1* Richard K. Yang,1,2 Charles W. Knapp,2 Yafen Niu,1 Nicholas Peak,2 Margery M. Hanfelt,3 John C. Galland,4 and David W. Graham2

Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City,1 Department of Civil, Environmental and Architectural Engineering, University of Kansas, Lawrence,2 Food Science Institute, Kansas State University, Manhattan, Kansas,3 Western Institute for Food Safety and Security, University of California, Davis, Davis, California4

Received 18 February 2004/ Accepted 6 August 2004

A new real-time PCR method is presented that detects and quantifies three tetracycline resistance (Tcr) genes [tet(O), tet(W), and tet(Q)] in mixed microbial communities resident in feedlot lagoon wastewater. Tcr gene real-time TaqMan primer-probe sets were developed and optimized to quantify the Tcr genes present in seven different cattle feedlot lagoons, to validate the method, and to assess whether resistance gene concentrations correlate with free-tetracycline levels in lagoon waters. The method proved to be sensitive across a wide range of gene concentrations and provided consistent and reproducible results from complex lagoon water samples. The log10 of the sum of the three resistance gene concentrations was correlated with free-tetracycline levels (r2 = 0.50, P < 0.001; n = 18), with the geometric means of individual resistance concentrations ranging from 4- to 8.3-fold greater in lagoon samples with above-median tetracycline levels (>1.95 µg/liter by enzyme-linked immunosorbent assay techniques) than in below-median lagoon samples. Of the three Tcr genes tested, tet(W) and tet(Q) were more commonly found in lagoon water samples. Successful development of this real-time PCR assay will permit other studies quantifying Tcr gene numbers in environmental and other samples.

* Corresponding author. Mailing address: Mail Stop 3029, Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160. Phone: (913) 588-7085. Fax: (913) 588-7295. E-mail: msmith6{at}kumc.edu.

Applied and Environmental Microbiology, December 2004, p. 7372-7377, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7372-7377.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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