Quantification of tetracycline resistance genes in feedlot lagoons by real-time PCR

A new real-time PCR method is presented that detects and quantifies three tetracyctine 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.