The abundance of six tetracycline resistance genes tet(O), tet(Q), tet(W), tet(M), tet(B) and tet(L), were quantified over time in wastewater lagoons at concentrated animal feeding operations (CAFO) to assess how feedlot operation affects resistance genes in downstream surface waters. Eight lagoons at five cattle feedlots in the Midwestern United States were monitored for 6 months. Resistance and 16S-rRNA gene abundances were quantified using real-time PCR, and physicochemical lagoon conditions, tetracycline levels, and other factors (e.g. feedlot size and weather conditions) were monitored over time. Lagoons were sorted according to antibiotic use practice at each site, and designated as 'no-use', 'mixed-use' or 'high-use' for comparison. High-use lagoons had significantly higher detected resistance gene levels (tet(R); 2.8 x 10(6) copies ml(-1)) relative to no-use lagoons (5.1 x 10(3) copies ml(-1); P < 0.01) and mixed-use lagoons (7.3 x 10(5) copies ml(-1); P = 0.076). Bivariate correlation analysis on pooled data (n = 54) confirmed that tet(R) level strongly correlated with feedlot area (r = 0.67, P < 0.01) and 'total' bacterial 16S-rRNA gene level in each lagoon (r = 0.51, P < 0.01), which are both characteristic of large CAFOs. tet(M) was the most commonly detected gene, both in absolute number and normalized to 16S-rRNA gene level, although tet(O), tet(Q) and tet(W) levels were also high in the mixed and high-use lagoons. Finally, resistance gene levels were highly seasonal with abundances being 10-100 times greater in the autumn versus the summer. Results show that antibiotic use strategy strongly affects both the abundance and seasonal distribution of resistance genes in associated lagoons, which has implications on water quality and feedlot management practices.

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 log 10 of the sum of the three resistance gene concentrations was correlated with free-tetracycline levels (r 2 ‫؍‬ 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 g/liter by enzymelinked 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.The tetracycline class of antibiotics is used frequently for the treatment and/or prevention of bacterial disease and for growth promotion in the cattle and swine industries (24, 25). For example, since the mid-1990s, more than 10 6 kg of tetracycline has been used per year in the U.S. livestock industry alone (9). There is growing evidence that such use of antibiotics for therapy, prophylaxis, and animal growth promotion may be resulting in the selection of resistant animal pathogens and commensals in the environment (reviewed by Chopra and Roberts [9] and Wegener [26]) because of the selection of tetracycline-resistant (Tc r ) strains in the rumina and intestines of exposed animals. A recent study on integrated livestock-fish farming indicated a significant increase in bacterial resistance to six antimicrobials in water-sediment samples from fish ponds exposed to manure from antibiotic-fed chickens (18). However, little quantitative information is available on the numbers of resistance genes in the environment.Phenotypic resistance testing of tetracycline and other antibiotics has been performed for years, although these methods tend to detect only resistance in culturable bacterial species and do not directly detect specific genes that confer the resistance. Previous studies have used nonquantitative PCR with Tc r gene-specific primers for the detection of tetracycline efflux genes (2, 5); however, such systems are not optimal for sensitive detection or for precise quantification. Therefore, a quantitative method for tracking resistance genes in environmental samples is needed to help determine whether antibacterial resistance in the environment is truly increasing because of anthropogenic practices.In this study, a new quantitat...