Environ. Sci. Technol. 2010, 44, 6102–6109
tet and sul Antibiotic Resistance Genes in Livestock Lagoons of Various Operation Type, Configuration, and Antibiotic Occurrence CHAD W. MCKINNEY,† KEITH A. LOFTIN,‡ MICHAEL T. MEYER,‡ JESSICA G. DAVIS,§ AND A M Y P R U D E N * ,†,| Via Department of Civil and Environmental Engineering, 418 Durham Hall, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, U.S. Geological Survey, 4821 Quail Crest Place, Lawrence, Kansas 66049, Department of Soil & Crop Sciences, Colorado State University, Fort Collins, Colorado 80523, and Department of Civil and Environmental Engineering, Colorado State University, Fort Collins, Colorado 80523
Received December 18, 2009. Revised manuscript received May 26, 2010. Accepted June 25, 2010.
Although livestock operations are known to harbor elevated levels of antibiotic resistant bacteria, few studies have examined the potential of livestock waste lagoons to reduce antibiotic resistance genes (ARGs). The purpose of this study was to determine the prevalence and examine the behavior of tetracycline [tet(O) and tet(W)] and sulfonamide [sul(I) and sul(II)] ARGs in a broad cross-section of livestock lagoons within the same semiarid western watershed. ARGs were monitored for one year in the water and the settled solids of eight lagoon systems by quantitative polymerase chain reaction. In addition, antibiotic residues and various bulk water quality constituents were analyzed. It was found that the lagoons of the chicken layer operation had the lowest concentrations of both tet and sul ARGs and low total antibiotic concentrations, whereas sul ARGs were highest in the swine lagoons, which generally corresponded to the highest total antibiotic concentrations. A marginal benefit of organic and small dairy operations also was observed compared to conventional and large dairies, respectively. In all lagoons, sul ARGs were observed to be generally more recalcitrant than tet ARGs. Also, positive correlations of various bulk water quality constituents were identified with tet ARGs but not sul ARGs. Significant positive correlations were identified between several metals and tet ARGs, but Pearson’s correlation coefficients were mostly lower than those determined between antibiotic residues and ARGs. This study represents a quantitative characterization * Corresponding author phone: (540)231-3980; fax: (540)231-7916; e-mail:
[email protected]. Current address: Via Department of Civil and Environmental Engineering, 418 Durham Hall, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. † Virginia Polytechnic Institute and State University. ‡ U.S. Geological Survey. § Department of Soil & Crop Sciences, Colorado State University. | Department of Civil and Environmental Engineering, Colorado State University. 6102
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of ARGs in lagoons across a variety of livestock operations and provides insight into potential options for managing antibiotic resistance emanating from agricultural activities.
Introduction Considering that no clear solutions to the problem of antibiotic resistance have yet been found (1, 2), new strategies beyond the hospital setting are needed to attenuate antibiotic resistance and prolong the useful lifespan of antibiotics. For this reason, environmental reservoirs and pathways of antibiotic resistance are gaining increasing attention (3-5). One promising approach in conceptualizing the fate and transport of antibiotic resistance in the environment is the consideration of antibiotic resistance genes (ARGs or the DNA encoding resistance) as the primary contaminants of interest rather than their microorganismal hosts (5). Because bacteria are capable of various horizontal gene transfer mechanisms of ARGs (6), an ideal strategy should seek to move beyond disinfection of the host cell and contain/destroy the DNA as well (5, 7). Considering that the precise risks associated with exposure to ARGs are extremely difficult to quantify, this represents a conservative, yet potentially simple means of minimizing the proliferation of resistant pathogens. Livestock operations have been established as harboring elevated concentrations of antibiotics and ARGs compared to surrounding environmental matrices (e.g. refs 5 and 8-10). Generally it is estimated that 30% to 70% of antibiotics used in the U.S. are administered to livestock, but accuracy is questionable due to lack of reporting requirements (11, 12). The concentrations of various antibiotics in land-applied manures and biosolids are commonly in the milligram-perkilogram concentration range (10). With respect to tetracyclines specifically, it has been suggested that about 25% of the oral dose is excreted in feces and another 50 to 60% is excreted unchanged or as an active metabolite in urine (13). Although the main concern is that antibiotics in manure may persist and stimulate resistance, even at low concentrations (4, 14, 15), other constituents, such as heavy metals, may also select for antibiotic resistance (16, 17). Few studies have examined the fate of ARGs in lagoons used to treat animal waste (8, 18) and in particular fieldscale studies considering a variety of livestock operations, lagoon configurations, and ARG classes are lacking. The objective of this study therefore was to examine the behavior of tetracycline [tet(O) and tet(W)] and sulfonamide [sul(I) and sul(II)] ARGs in lagoons on a cross section of field-scale livestock operations within the same semiarid western watershed. This research specifically targeted tetracycline and sulfonamide ARGs because of the widespread use of the corresponding antibiotics in U.S. agriculture and their persistence relative to other commonly used antibiotics, such as beta lactams (19). A survey of the occurrence of the 11 tet and the 2 sul ARGs in the cattle lagoons investigated in this study was recently reported (20), indicating that tet(O), tet(W), sul(I), and sul(II) were particularly abundant and thus suitable for monitoring by quantitative polymerase chain reaction (Q-PCR). Monitoring these four ARGs also provided a point of comparison with prior research on ARG fate in benchscale dairy lagoons (18). To assist the overall objective, ARGs were monitored during a period of 1 year within a range of animal livestock operations including one chicken layer farm, one swine producer, two conventional dairies (large and small), two organic dairies (large and small), and two beef feedlots. Corresponding measures of antibiotics, trace elements, and 10.1021/es9038165
2010 American Chemical Society
Published on Web 07/21/2010
water quality constituents were obtained to assess their relationship to ARGs in animal waste lagoons. The results of the study are discussed with reference to the influence of livestock type, antibiotic use and occurrence, season/climate, and lagoon treatment efficacy on the concentration of ARGs.
Materials and Methods Lagoon Sampling. Lagoons managing waste from a chicken layer facility (1.1 million hens), a swine producer (3600 head), a large conventional dairy (15,000 cows), a large organic dairy (4000 cows), a small conventional dairy (800 cows), a small organic dairy (150 cows), and two conventional beef feedlots (4000-12,000 head and ∼18,000 head) within the same western watershed were selected for study. Details on lagoon configuration and reported antibiotic use are provided in the Supporting Information. Sampling of livestock operations was confined to approximately one week intervals over 1 year: October 13th-November 9th, 2006 (fall); February 28thMarch 9th, 2007 (winter); May 17th-May 24th, 2007 (spring); and September 14th-September 19th, 2007 (summer). All samples were collected from the approximate midpoint of each lagoon via an inflatable raft. Water samples were collected at a depth of approximately 10 cm in autoclaved 1-L Nalgene bottles. Settled solids were subsequently collected by dragging a metal bucket along the bottom of the lagoon and transferring subsamples to 50-mL sterilized conical tubes. Water temperature and pH were measured using field probes. Samples were transported on ice to the laboratory within 6 h of collection. Settled solids and centrifuged (5 min at 3823 × g) pellets of water samples (water solids) were stored at -20 °C until processed. Prior to centrifugation, subsamples were removed for trace element and nutrient analysis and stored at 4 °C. Water-Quality Analysis. Water-quality analysis was performed on unfiltered water samples. Hach Test ‘N Tube kits were used for all analyses using a Hach DR4000 (Hach, Loveland, CO) spectrophotometer using the following methods: chemical oxygen demand (COD) - digestion method 8000 (20- to 1500-mg/L) (USEPA approved); total nitrogen (TN) - persulfate digestion method 10072 (10 to 150 mg/L N); ammonia - salicylate method 10031 (0 to 50.0 mg/L NH3-N); nitrate - chromotropic acid method 10020 (0 to 30.0 mg/L NO3--N); phosphate - acid persulfate digestion method 10127 (0 to 100.0 mg/L PO43-). Manufacturer’s protocol was followed for all methods, except that DI water blanks were utilized instead of background sample water to provide improved consistency. Each sample was analyzed in duplicate over a range of dilutions. Trace Element Analysis. Samples were digested using a CEM MDS-2000 microwave digester (CEM Corporation, Matthews, NC) following USEPA method 3015 for the water samples and USEPA method 3051 for the settled solids. Following digestion, the samples were filtered through a 0.45µm mixed cellulose ester filter. Sample extracts were analyzed by the Soil, Water, and Plant Testing Laboratory at Colorado State University (Fort Collins, CO) by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) (IRIS Advantage, TJA, Franklin, MA). The elements quantified were as follows: Al, B, Ba, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Mo, Ni, P, Pb, Na, S, Si, Sr, Ti, and Zn. DNA Extraction. DNA extraction was carried out on 0.25 g of centrifuged water solids or settled solids using a Mo Bio PowerSoil DNA Isolation Kit (Mo Bio Laboratories Inc., Carlsbad, CA). The manufacturer’s protocol was followed except that a bead beater was used for 3 min instead of vortexing for 10 min in step 5 of the protocol. The extracted DNA was stored at -80 °C for long-term and -20 °C for short-term storage (
