Long-Term Monitoring of the Occurrence of Antibiotic Residues and Antibiotic Resistance Genes in Groundwater near Swine Confinement Facilities

To assess the impact of manure management on groundwater quality, groundwater samples have been collected since 1996 near two swine confinement facilities in Illinois (site A and site C) that use lagoons for manure storage and treatment. Several key contaminant indicators, including inorganic ions, antibiotics, and antibiotic resistance genes and bacteria, were analyzed in groundwater collected from 23 monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on analysis of these constituents, shallow groundwater has been impacted by lagoon seepage at both sites. The extent of migration of contaminants down gradient from the lagoons and the magnitude of contaminant concentrations in groundwater were significantly greater at site A than at site C. Migration of contaminants as much as 30 m down-gradient of the lagoon at site C and 150 m at site A can be attributed to the difference in the local geologic conditions at the sites. Operators at site A used a limited number of antibiotics including bacitracin, chlortetracycline, and tylosin. At site C, chlortetracycline was administered continuously in the feed, and penicillin and tylosin were used occasionally for disease prevention and treatment. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Fewer than 5% of the groundwater samples collected at either of the facilities contained detectable levels of these antibiotics even in samples from wells that had been significantly impacted by manure seepage as evidenced by elevated chloride, ammonium, and potassium concentrations. Only three groundwater samples contained detectable levels of tetracyclines at either site. Degradates of the tetracyclines were also detected in some groundwater samples at site A even when the parent compound was not detected. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 μg/L. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. To help explain the occurrence of the antibiotics, sorption studies were conducted. The tetracyclines sorbed significantly to the site soils, and based on their Freundlich sorption coefficients (Kf ranged from 196 to 4371 L/kg) are considered immobile. The relatively few detections and small concentrations in our manure and groundwater samples were likely a result of the high affinity and irreversible sorption of these antibiotics to soil minerals and organic matter. Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes, tet(M), tet(O), tet(Q), tet(W) were detected. The frequency of detection of the four tet genes in groundwater samples from site A indicated fluctuating resistance gene patterns over time and across the groundwater flow path. In contrast to site A where tet genes occurred persistently in most well samples, there were significant periodic differences in tet gene occurrence in groundwater from site C. In 2001, tet genes were below detection in nearly all groundwater samples from site C, while in 2003, tet genes were detected in nearly all samples. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. Also, in 2003, higher frequencies of tet genes occurred in background wells located up-gradient from the lagoons at both sites. Further analysis using quantitative PCR methods demonstrated that in lagoon samples tet(Q), relative to the total bacteria present, was much greater than tet(M). These results suggested that in the groundwater underlying both facilities, tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period. Using denaturing gradient gel electrophoresis as a DNA fingerprinting method to characterize bacterial community structure, we found that dominant bacterial species differed between the lagoon and groundwater samples. Lagoon samples contained communities that were more complex and stable than those found in the groundwater samples. In several wells, fecal bacteria were detected by cultivation methods. Although the