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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

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 (Tc^r) genes [tet(O), tet(W), and tet(Q)] in mixed microbial communities resident in feedlot lagoon wastewater. Tc^r gene real-time TaqMan primer-probe sets were developed and optimized to quantify the Tc^r 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 Tc^r 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 Tc^r gene numbers in environmental and other samples.

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