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

Abstract

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...

Microbial community functional structure in response to antibiotics in pharmaceutical wastewater treatment systems

“…tet(M)-like subfamily, tet(Y) and tet(A) genes were detected, which was in accordance with our previous result that 12.2%, 22.2% and 69.3% of bacterial isolates from oxytetracycline wastewater effluent contained tet(M), tet(Y) and tet(A) (Li et al, 2010). The levels of three gene families involved in antibiotic resistance, including b-lactamase-C, MFS, and SMR were significantly correlated with antibiotic concentration (P < 0.05) (Table S7 In Supporting Information), which was consistent with previous reports that environmental antibiotic residues might impose selective pressure on bacterial communities to acquire antibiotic resistance (Smith et al, 2004;Pei et al, 2007). On the other hand, nine tet genes including tet(M) and tet(A) were successfully quantified using quantitative PCR for the same samples (Table S8 in Supporting Information).…”

Abundance and Distribution of Tetracycline Resistance Genes and Mobile Elements in an Oxytetracycline Production Wastewater Treatment System

“…Tet genes in the environment have been extensively studied in sewage treatment plants (STPs), 11 animal lagoons, 12 and fish farming ponds, 13 where tetracyclines have been frequently detected. Among the 44 known tet genes, more than 29 have been found in the environment.…”

Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics

“…vanC, ampC, mecA (Schwartz et al, 2003;Volkmann et al, 2004), or on genes conferring resistance to a specific class of antimicrobial compounds, e.g. b-lastams (Henriques et al, 2006a, b), chloramphenicol (Dang et al, 2008), or tetracyclines (Akinbowale et al, 2007;Chee-Sanford et al, 2001;Guillaume et al, 2000;Smith et al, 2004).…”