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Published online 16 October 2007
Published in J Environ Qual 36:1695-1703 (2007)
DOI: 10.2134/jeq2007.0006
© 2007 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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TECHNICAL REPORTS

Waste Management

Response of Antibiotics and Resistance Genes to High-Intensity and Low-Intensity Manure Management

Heather N. Stortebooma, Sung-Chul Kima, Kathy C. Doeskenb, Kenneth H. Carlsona, Jessica G. Davisb and Amy Prudena,*

a Dep. of Civil and Environmental Engineering, Colorado State Univ., Fort Collins, CO 80523
b Dep. of Soil and Crop Sciences, Colorado State Univ., Fort Collins, CO 80523

* Corresponding author (Amy.Pruden{at}ColoState.edu).

Received for publication January 3, 2007.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
The purpose of this study was to determine the response of antibiotics and antibiotic resistance genes (ARG) to manure management. A pilot field study was conducted using horse manure containing no antibiotics, into which chlortetracycline (CTC), tylosin (TYL), and monensin (MON) were spiked and compared to unspiked controls. Subsequently, a large-scale field study was conducted comparing manure from a dairy with minimal use of antibiotics and a feedlot with regular subtherapeutic use of antibiotics. The manures were subjected to high-intensity management (HIM) (amending, watering, and turning) and low-intensity management (LIM) (no amending, watering, or turning) and were monitored for antibiotic concentrations and levels of tetracycline ARG [tet(W) and tet(O)] using quantitative real-time polymerase chain reaction. All three antibiotics in the pilot study dissipated more rapidly in HIM manure, with half-lives ranging from 4 to 15 d, compared to LIM manure, with half-lives ranging from 8 to 30 d. Levels of tet(W) were significantly higher after 141 d of treatment, but levels of tet(O) were significantly lower in all treatments. In the large-scale study, the feedlot manure had higher initial concentrations than the dairy manure of tetracycline (TC), oxytetracycline (OTC), and CTC as well as tet(W) and tet(O). Tetracycline and OTC dissipated more rapidly in HIM manure, with half-lives ranging from 6 to 15 d, compared to LIM manure, with half-lives ranging from 7 to 31 d. After 6 mo of treatment, tet(W) and tet(O) decreased significantly in feedlot manure, whereas dairy manure required only 4 mo of treatment for similar results.

Abbreviations: ARG, antibiotic-resistant genes • CTC, chlortetracycline • HIM, high-intensity management • HPLC/MS/MS, high performance liquid chromatography tandem mass spectrometry • LIM, low-intensity management • MON, monensin • OTC, oxytetracycline • RMRCCS, Rocky Mountain Region Compost Classification System • STZ, sulfathiazole • TC, tetracycline • TMECC, test methods for examination of composting and compost • TYL, tylosin


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
VARIOUS studies have confirmed the presence of antibiotics in surface waters and sediments, municipal wastewater, animal waste lagoons, and groundwater underlying lagoons (Chee-Sanford et al., 2001; Kolpin et al., 2002; Kummerer and Henninger, 2003; Yang and Carlson, 2003; Pei et al., 2006). The presence of low levels of antibiotics in these environments can lead to selection of antibiotic resistant organisms, whose ability to resist antibiotics is typically conferred by antibiotic-resistant genes (ARG). Antibiotic-resistant genes are often carried on mobile genetic elements that can be passed between diverse species of microorganisms (Roberts, 2005a). The greater environment may serve as a reservoir or transfer pathway for ARG (Rysz and Alvarez, 2004; Yu et al., 2005; Pei et al., 2006), contributing to the ineffectiveness of antibiotics in human medicine. For these reasons, there is growing acceptance for the consideration of ARG as environmental contaminants (Pruden et al., 2006).

The sources of antibiotics and ARG in the environment are both human and agricultural (Chee-Sanford et al., 2001; Kolpin et al., 2002; Kummerer and Henninger, 2003; Yang and Carlson, 2003; Pei et al., 2006). However, little is known about the transport and fate of antibiotics and ARG in agricultural pathways and treatment systems. Antibiotics are used in animal agriculture for treatment of infections, prevention of disease (prophylaxis), or growth promotion by amending feed with low (subtherapeutic) levels for sustained periods of time (McEwen and Fedorka-Cray, 2002). Tetracyclines are broad-spectrum antibiotics used for both treatment and growth promotion in cattle, swine, and poultry. Chlortetracycline (CTC) is used most commonly, though oxytetracycline (OTC) is also used for growth promotion and prophylaxis in cattle including the treatment of mastitis in dairy cows (National Academy of Sciences Committee on Drug Use in Food Animals, 1999; McEwen and Fedorka-Cray, 2002). The macrolides TYL and erythromycin are administered at therapeutic and subtherapeutic levels to cattle, swine, and poultry, while monensin (MON) is administered to cattle at subtherapeutic levels for growth promotion (National Academy of Sciences Committee on Drug Use in Food Animals, 1999). Previous studies have monitored the degradation of antibiotics in manure and manure-amended soils (Ingerslev and Halling-Sørensen, 2001; De Liguoro et al., 2003; Soeborg et al., 2004; Arikan et al., 2006; Carlson and Mabury, 2006). In general these studies have found that antibiotic dissipation/degradation kinetics depend on characteristics of the particular antibiotic such as water solubility and soil sorption capacity and on environmental conditions such as temperature, light, pH, and oxygen levels (Thiele-Bruhn, 2003; Soeborg et al., 2004).

When animals regularly consume antibiotics, it can lead to an increase in the levels of ARG in the animals' commensal bacteria (Chopra and Roberts, 2001; Wegener, 2003), which can serve as a reservoir for ARG (Aminov et al., 2001). Low levels of antibiotics present in the waste (Elmund et al., 1971; Feinman and Matheson, 1978) as well as the passage of commensal bacteria out of the animal's gut make the manure an ideal habitat for ARG to persist. Animal manure is often managed with varying levels of intensity using methods such as stockpiling, composting, or lagoon systems. The treated product is typically land applied as natural fertilizer for edible crops. Therefore, there is a need to understand the fate of ARG during treatment of manure before land application.

The objective of this study was to understand the response of antibiotics and the ARG tet(W) and tet(O) to different intensities of manure management. Tet(W) and tet(O) are two ribosomal protection tetracycline ARG commonly found in the gut and rumen of animals (Scott et al., 1997; Barbosa et al., 1999; Billington et al., 2002; Stanton and Humphrey, 2003). These genes have also been found in diverse environmental samples such as swine feces, swine lagoons, dairy lagoons, irrigation ditches, surface water sediments, and municipal wastewater (Aminov et al., 2001; Chee-Sanford et al., 2001; Smith et al., 2004; Yu et al., 2005; Pei et al., 2006), which makes them ideal for monitoring. It was hypothesized that high-intensity management of animal manures may stimulate the degradation of antibiotics and minimize the spread of ARG into the environment. Two management strategies, high-intensity management (HIM) and low-intensity management (LIM), were studied to determine which factors promote the degradation of antibiotics and the attenuation of ARG. In this study, HIM was defined as amending with alfalfa (a source of nitrogen) and dried leaves (a bulking agent), and regularly watering and turning manure to enhance degradation. Low-intensity management was defined as piling or windrowing manure, which was not amended, watered, or turned once constructed. Currently, only a few studies have quantified the levels of ARG in animal manure or manure management systems (Smith et al., 2004; Yu et al., 2005; Pruden et al., 2006; Peak et al., 2007), and none have simultaneously monitored the effect of treatment on both ARG, using Q-PCR, and antibiotic concentrations, using HPLC/MS/MS.


   Materials and Methods
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results and Discussion
 Conclusions
 REFERENCES
 
Pilot Field Study Design and Construction
The purpose of the pilot study was to determine the response of ARG to manure that had not been acclimated to antibiotics, and to provide a controlled study environment to test the feasibility of methods and management strategies to be applied in the large-scale study. Horse manure was chosen for this baseline study because horses are not given antibiotics as growth promoters. Horse manure was collected from the Colorado State University Equine Center and subjected to HIM or LIM strategies in three replicate piles of 5.35 m3. The experiment was set up on 24 Sept. 2004 and maintained for 141 d. The antibiotics CTC (Sigma-Aldrich, St. Louis, MO), TYL (Sigma-Aldrich), and MON (added as pulverized animal feed) were spiked into each pile at 0.328 mg kg–1. Chlortetracycline and TYL were dissolved in water and sprayed onto the piles while mixing, and MON was sprinkled onto the piles while mixing and watering. High-intensity management manure was amended with 1.5 m3 of leaves and alfalfa. Two control piles, one for each treatment, were constructed without antibiotic addition. All treatments heated to a temperature of 55°C within the first 14 d of the experiment; however, the temperature of LIM manure declined steadily thereafter, whereas the HIM manure maintained this temperature up until Day 28 before finally declining. Because the piles were small, watering and turning was required infrequently for the HIM manure (Day 4, Day 17, and Day 38).

Large-Scale Field Study Design and Construction
The purpose of the large-scale study was to determine the response of tetracyclines and corresponding tet ARG to treatment in two manures acclimated to regular exposure to antibiotics. Manure was obtained from a feedlot facility that used antibiotics regularly for therapeutic and non-therapeutic purposes. For comparison, manure was obtained from a dairy using antibiotics for only therapeutic purposes to determine the response to treatment of manure with low expected initial levels of ARG and antibiotics. Neither monensin nor tylosin were found at high enough concentrations that they could be monitored with time (data not shown). The windrows were constructed on 6 July 2005 and maintained for 182 d. Four windrows were constructed: one HIM windrow and one LIM windrow of each type of manure. The HIM windrows were amended with equal amounts of a mixture of dried leaves and freshly harvested alfalfa. The average size of the HIM windrows was 20.5 by 1.5 by 1 m (average volume = 15.75 m3). Unamended LIM windrows were smaller with an average size of 19.8 by 1.5 by 0.75 m (average volume = 11.1 m3). Windrows were divided into three sampling regions (~5 m length) separated by two internal non-sampling regions (2–3 m length). After initial construction, the LIM windrows were neither watered nor turned.

High-intensity management windrows were watered as necessary and turned weekly with a compost turner during the first 10 wk of the study. Temperatures in the HIM windrows were recorded weekly. The feedlot compost windrow reached its highest temperature (47°C) by Day 5 and remained in the thermophilic stage until Day 27. The dairy compost remained in the mesophilic stage throughout the composting process, reaching its highest temperature (34°C) on Day 20.

Sampling Technique and Storage
A coring device was used to sample the piles in ten locations during each event for the pilot study. To sample the large windrows, eight cores were extracted from each sampling region. For each event, one composite sample was collected from each pile/sampling region. Samples were stored at –20°C for short-term storage (<3 mo) or at –80°C for long-term storage (3–6 mo). Samples were slurried in sterile distilled water to form a homogenous mixture before analysis. Samples from eight dates were chosen for downstream analysis for the pilot-scale study (Day 1, 4, 7, 19, 28, 54, 61, and 141) and nine dates for the large-scale study (Day 0, 14, 28, 42, 56, 70, 112, 161, and 182).

Chemical Analysis of High- and Low-Intensity Management Manures
High-intensity management and LIM piles/windrows were sampled on Day 0 in the large-scale study and on the final day in both studies for moisture content, ammonia-N/nitrate N ratio, carbon to nitrogen ratio (C/N ratio), pH, and soluble salts analyses. Samples were analyzed using standard test methods for the examination of composting and compost (TMECC). Pilot study samples were analyzed by Colorado State University's Soil, Water, and Plant Testing Laboratory and large-scale study samples were analyzed by Colorado Analytical Laboratories. The results of these analyses are presented in Table 1 .


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  		Table 1. Manure and compost characteristics before and after treatment

 
The similar temperature profiles of HIM and LIM manure from the pilot study suggest that microbial degradation of the manure occurred in all treatments. When compared to the standards for compost/soil amendments set by the Rocky Mountain Region Compost Classification System (RMRCCS), the manure managed by both strategies had ammonia-N/nitrate N ratio, C/N ratio, pH, and soluble salts concentration representative of fully composted, stable, mature product (Class I soil amendment) by the final day of the pilot field study (A1 Organics, 2006). Although the dairy manure used in the construction of the large-scale study had the appearance of moist, raw manure, chemical analysis revealed that it could be initially classified as a fully composted, stable, mature product (Class I or Class II soil amendment) on the basis of the manure's initial ammonia-N/nitrate N ratio, C/N ratio, and soluble salts concentration. The low moisture content of the feedlot manure indicated that it may have been slightly aged before its use in the study, though its texture resembled raw manure. The feedlot manure had a similar C/N ratio and soluble salts concentration as the dairy manure, but had ammonia-N/nitrate N ratio typical of raw manure. While the National Organics Standards Board suggests that adequate composting can occur with starting C/N ratios as low as 15 (National Organic Standards Board, 2002), the inability of the LIM and HIM manures to reach temperatures above 40°C suggest that carbon may have been a limiting factor to microbial activity. Suboptimal moisture in the feedlot manure and low nitrogen content in the dairy manure may have also contributed to low microbial activity (Zibilske, 2005).

Because many of the initial characteristics of the manures were typical of stable, mature soil amendments, the management of the manures resulted in slight reductions in the ammonia-N/nitrate N ratio, C/N ratio, pH, and soluble salts of most treatments. However, both management strategies resulted in a noticeable reduction in the ammonia-N/nitrate N ratio of the feedlot manure by the end of the study period, though the manure subjected to LIM still had an ammonia-N/nitrate N ratio exceeding the range suggested for a Class I or Class II stable, mature soil amendment.

Quantification of Antibiotics
General sample preparation was adapted from United States Department of Agriculture (USDA) guideline (USDA, 2003) and modified as necessary. Briefly, a 1 g (wet weight) subsample from each manure slurry was placed in 40 ml Teflon tube, and 20 ml of McIlvaine buffer solution was used to extract residual from solid phase to liquid phase followed by adding 200 µL of 5% Na2EDTA (w/v, 1 mmol in solution) to complex any metals. Each sample was then vigorously mixed in a parallel shaker (Model No-4626, Lab-line instrument) for 20 min at 400 excursions min–1 followed by centrifuging at 1434 g (IEC Clinical Centrifuge, International Equipment Co., Needham Heights, MA) for 15 min. After separating solid and liquid phase, the supernatant was filtered using 0.2 µm glass fiber filters. Filtered samples were decanted into another 40 mL vial and kept at 4°C. The extraction procedure was repeated with the remaining solid in the Teflon tube in the same manner as described above, and supernatants were combined to make total volume of 40 ml. Prepared samples were then cleaned up with solid phase extraction (SPE) method, and high performance liquid chromatography (HPLC) tandem mass spectrometry (HPLC/MS/MS) was used for separation and detection. Detailed information of the SPE procedure and HPLC/MS/MS is described elsewhere (Yang et al., 2004; Zhu et al., 2001). Average recovery was 98 and 95% of the known concentrations of 0.03 and 0.09 mg kg–1, respectively. The limit of quantification was determined to be 0.0007 mg kg–1.

DNA Extraction and Purification
DNA was extracted in duplicate from pilot manure samples from ~1 g of the slurried sample using the UltraClean Soil DNA Kit according to the manufacturer's instructions (MO Bio Laboratories, Inc., Carlsbad, CA). Extracted DNA was purified using the GENECLEAN SPIN Kit according to the manufacturer's instructions (Q·BIOgene, Morgan Irvine, CA). Extraction yield and DNA quality were verified by agarose gel electrophoresis. After purification, duplicate samples were combined for subsequent DNA analysis. In the large-scale study, the PowerMax Soil DNA Isolation Kit (MO Bio) was used to reduce inhibitors and eliminate the need for downstream purification. DNA was extracted from ~5 g of the slurried sample per manufacturer's instructions with the following exceptions: the beadbeating step was extended to 30 min, and the final elution volume was reduced from 5 to 4 ml to concentrate the DNA. DNA extracts were stored at –20°C for short-term storage (<3 mo) and at –80°C for long-term storage (>3 mo).

Quantitative Real-Time Polymerase Chain Reaction (Q-PCR)
Quantitative real-time polymerase chain reaction was performed on DNA extracts in triplicate in independent runs for each ARG. Tet(W) and tet(O) were quantified with a SYBR Green assay using previously published primers (Aminov et al., 2001) and protocols (Pei et al., 2006). To normalize the quantities of ARG to the total bacterial community, the bacterial 16S rRNA gene was quantified using the universal primers 1369F and 1492R and a TaqMan Q-PCR assay as described in Suzuki et al. (2000). This also provided a means to account for variations in overall extraction efficiency. The conditions were the same except that Qiagen SYBR Green PCR Master Mix (Valencia, CA) was applied in the SmartCycler (Cepheid, Sunnyvale, CA) and SYBR Green PCR Master Mix (Applied Biosystems) was applied in the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA), with a reduced denaturing step of 10 min. Calibration curves, negative controls, and spiked matrix controls were included in every run on the 7300 cycler.

To determine the effect of PCR inhibitors present in the DNA matrix on quantification, positive control standards were spiked into the DNA matrix at concentrations 1000 times the average concentration of the DNA matrices. A suppression factor, S, was calculated from Eq. [1]:

Formula 1[1]
where c is the number of templates quantified in the positive control, t is the number of templates quantified in the DNA extract, and m is the number of templates quantified in the DNA matrix spiked with the control (Pei et al., 2006). In the pilot study, suppression tests were performed on three samples for each study to determine if inhibitors were present. Suppression tests were performed for all samples from the large-scale field study and calculated suppression factors were used to determine the total number of copies in the DNA extract.

Considering the dilution factor (D) and the suppression factor (S), the concentration of genes in the DNA extract (C) was determined by Eq. [2] where t is the number of copies in the diluted DNA extract:

Formula 2[2]

Statistical Tests and Analysis
All statistical tests were performed using SAS 9.1 (SAS Institute, 2003). Transformations were made when necessary to achieve homogeneity of variance. Significance for all tests was defined as P values ≤ 0.05.

Antibiotic-resistant genes data were analyzed as gene copy numbers normalized to the bacterial 16S rRNA gene (paired by pile or sampling region). Normalized data were transformed and compared in log scale for the pilot study, and using a square-root transformation for the large-scale study. Dixon's extreme value test was used to test for statistical outliers at the treatment level (<5% of all data points were rejected). To analyze differences in the response patterns of ARG with time, mixed linear regressions were fit to ratio data sets using PROC MIXED. Degrees of freedom were calculated using the Kenward-Roger method with repeated time measurements. Least squared means were calculated for all data.

Antibiotic data was fit to the exponential decay function C = C0xe–{lambda}t using PROC NLIN to estimate model parameters and half-lives. The PROC REG function was used to test for significant correlations between average normalized ARG levels and average levels of antibiotics (CTC in pilot study and total tetracyclines in large-scale study), when paired by treatment/windrow. In the large-scale study, initial levels of total tetracyclines and ARG (paired by sampling region) were tested for correlation. The PROC GLM function was used to determine least significant differences between initial antibiotic concentrations in treatments in the large-scale field study.


   Results and Discussion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
Conclusions
 REFERENCES
 
Pilot Field Study
Response of Antimicrobials to Treatment
The quantities of CTC, TYL, and MON normalized to the dry weight of the manure are shown in Fig. 1 as a function of treatment time. The control piles showed no detectable quantities of these antimicrobials during the 141 d study period (data not shown). Estimates and 95% confidence intervals of antibiotic half-lives are presented in Table 2 . For all antibiotics, HIM manures had significantly lower half-lives than LIM manures. Half-lives of TYL were 4.2 d in HIM manure and 13.1 d in LIM manure, compared to reported values of 3.3 to 8.1 d in soil-manure slurries (Ingerslev and Halling-Sørensen, 2001). Estimates of half-lives for CTC in environmental matrices found in the literature vary from 24 to >84 d (Thiele-Bruhn, 2003; Carlson and Mabury, 2006), much higher than the half-lives of 5.11 d in HIM manure and 8.43 d in LIM estimated in this study (Thiele-Bruhn, 2003). However, Soeborg et al. (2004) found CTC half-lives to be significantly lower in soil interstitial water with pH of 8.5 compared to water with pH < 5.0, which corresponds to the pH of manures in the pilot study. In HIM manure, the half-life of MON was 14.7 d compared to 30.1 d in LIM manure. These half-lives fall within reported values ranging from 3.8 d in manure-amended soil (Carlson and Mabury, 2006) to >70 d in anaerobic manure (Thiele-Bruhn, 2003).


Figure 1
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  		Fig. 1. Concentrations of antibiotics quantified in the pilot study with respect to time. Concentrations of antibiotics in control piles were below detection (data not shown). Data points represent average values of all measurements (performed in triplicate for each pile). Error bars represent standard deviation.

 

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  		Table 2. Estimated half-lives of tylosin (TYL), monensin (MON), chlortetracycline (CTC), tetracycline (TC), and oxytetracycline (OTC) in manures subjected to high- and low-intensity management (HIM and LIM, respectively).

 
In the HIM manure, TYL was no longer detectable after 28 d. All other treatments had detectable levels of antibiotics on the final day of the study. After 141 d of treatment, CTC and TYL were present at statistically higher concentrations in the LIM manure than in the HIM manure (P < 0.0001). Final concentrations of MON were not statistically different between treatments. The dissipation of the antibiotics was likely due to a combination of photolysis, heat, and microbial degradation. There are only three known genes that confer resistance to tetracycline via tetracycline degradation, and based on current research, these have limited ecological importance (Roberts, 2005b). However, one enzymatic alteration gene, tet(X) was present (data not shown), which may indicate microbial degradation. In both treatments, the half-life of MON was nearly triple the half-lives of CTC and TYL. This may be related to the fact that MON was added as a solid pulverized feed, which could have made it more recalcitrant. Monensin is also the least water-soluble, and therefore may have had limited bioavailability (Thiele-Bruhn, 2003).

Response of Tetracycline Antibiotic-Resistant Genes to Treatment
Results from Q-PCR analysis of tetracycline ARG, tet(W), and tet(O), are shown in Fig. 2 . Tet(W) increased in all treatments, reaching the highest level on Day 28. Levels of tet(W) decreased from Day 61 to Day 141 in all the treatments, but were still significantly higher on the final day than the initial day of the study (P < 0.03) in the treatments spiked with antibiotics. This suggests that it may be crucial to carry out treatment to completion, as increased tetracycline resistance could result from stopping treatment prematurely. It is possible that the diverse community promoted by manure management could actually enhance the transfer and persistence of ARG because the natural process of succession favors populations that carry ARG. However, allowing the treated manure to sit after antibiotics have fully degraded may allow these ARG to attenuate over time.


Figure 2
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  		Fig. 2. Detected levels of tetracycline antibiotic-resistant genes (ARG) normalized to copies of bacterial 16S rRNA genes present in the pilot field samples with respect to time. Spiked treatments had 0.328 mg each of chlortetracycline, tylosin, and monensin added per kg manure. Data points represent average values of all measurements (performed in triplicate for each pile) for the specified treatment.

 
The levels of tet(O) decreased by an order of magnitude during the first 4 wk of the study, and continued to decrease nearly another order of magnitude during the final sixteen weeks of the study (Fig. 2). Final levels of tet(O) were significantly lower than initial levels in the spiked treatments (P < 0.0001) and in the control treatments (P < 0.01).

Although tet(O) started out at higher copy numbers than tet(W), the subsequent rapid decline of tet(O) reversed this trend so that by the midpoint and endpoint of the study, levels of tet(W) were two orders of magnitude greater than the levels of tet(O) in all treatments. The overall response of tet(W) and tet(O) in the controls was not significantly different from the response to the spiked treatments. This indicates that the spiked antibiotics did not influence the microbial community, suggesting that the concentrations were too low or not bioavailable, or there was insufficient time to select for ARG. There was also no significant difference in the response of ARG between the two different levels of manure management. Overall, time appeared to be the most significant factor in reducing ARG.

The wide host range (19 genera) (Roberts, 2005b) of tet(W) may have been a factor in its observed increase in response to treatment. Its wide host range may be due to its association with the conjugative transposon TnB1230 (Melville et al., 2004). In contrast, the tet(O) gene has a narrower host range (11 genera) (Roberts, 2005b), and was only recently found to be associated with a conjugative transposon (Giovanetti et al., 2003). Antibiotic-resistant genes carried on transposons often have increased frequency of transfer and greater stability in the host once transferred when compared to other genetic elements (Levy, 2002; Roberts, 2005b). If the source of tet(O) in this study was a plasmid as opposed to a conjugative transposon, the ability of tet(O) to transfer to diverse hosts would limit its ability to survive the microbial succession that takes place during manure management.

Correlation of Antibiotic-Resistant Genes Levels to Concentrations of Total Tetracyclines
The levels of tet(W) were not correlated to the concentration of CTC (r2 < 0.33) in either treatment, but levels of tet(O) were positively correlated to the concentration of CTC in the spiked HIM manure (r2 = 0.53, P < 0.04) and LIM manure (r2 = 0.73, P < 0.01). However, because the response of ARG levels was found to be independent of the addition of antibiotics, it is unlikely that this correlation is indicative of a true relationship.

Large-Scale Field Study
Response of Tetracyclines to Treatment
The response of the tetracycline compounds (TC, OTC, and CTC) to treatment are shown in Fig. 3 . The total treatment time was 182 d. Observed half-lives for dairy and feedlot manures are given in Table 2. For TC and OTC, HIM manures had significantly lower half-lives than LIM manures. The half-life of CTC was not significantly different between treatments. All antibiotics had significantly lower half-lives in dairy manure than feedlot manures. Therefore, dairy manure treated with HIM had the highest rates of antibiotic dissipation.


Figure 3
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  		Fig. 3. Concentrations of total tetracyclines, and the individual tetracycline compounds (oxytetracycline, chlortetracycline, and tetracycline) quantified in the large-scale study treatment windrows with respect to time. Data points represent average values of all measurements (performed in triplicate for each sampling region) for the specified windrow. Error bars represent standard deviation.

 
In feedlot manure, the observed half-life of TC was 6.5 d for HIM and 17.2 d for LIM and fell within the range of 4.5 to 105 d reported in the literature for pig manure (Thiele-Bruhn, 2003). The average half-life of CTC was 6.3 d in dairy manure and 13.4 d in feedlot manure, similar to values calculated in the pilot-scale study. The half-lives of OTC ranged from 9.8 d in dairy manure treated by HIM to 31.1 d in feedlot manure treated by LIM. Reported half-lives of OTC in beef manure range from 30 d (De Liguoro et al., 2003) to 56 d (Arikan et al., 2006).

In all samples, the trend observed with respect to tetracycline concentrations was OTC > CTC > TC. It is not surprising that OTC and CTC were found in high quantities, because they are the most commonly used tetracyclines in animal agriculture (McEwen and Fedorka-Cray, 2002). Initial concentrations of OTC, CTC, and TC were significantly higher in the feedlot manures than the dairy manures (P < 0.0001, P < 0.0001, P < 0.01, respectively). For comparison, sulfathiazole (STZ) was quantified, and concentrations similar to the levels of TC seen in dairy manure were detected, with no significant difference between the two types of manures (data not shown). It is likely that the presence of higher concentrations of tetracyclines in the feedlot manure is a result of the use of tetracyclines as growth promoters in the beef cattle. This is supported by the presence of STZ in similar quantities in both manures, because STZ is not used as a growth promoter in cattle.

Response of Tetracycline Antibiotic-Resistant Genes to Treatment
Results from Q-PCR analysis of tetracycline ARG in the large-scale field study are shown in Fig. 4 . Initial levels of tet(W) in dairy manure were significantly lower than in the feedlot manure (P < 0.01). The dairy manure treatments maintained significantly lower levels of tet(W) compared to the feedlot manure treatments through Day 28 for HIM and Day 14 for LIM. After Day 28, the levels of tet(W) were consistent between all treatments. On the final day of the study, the HIM feedlot manure had significantly lower levels of tet(W) than the other three treatments (P < 0.005). Overall response of tet(W) was statistically independent of the level of treatment applied to the manure (HIM or LIM). This similarity may be due to the fact that microbial degradation of the manure was occurring at similar rates in HIM and LIM.


Figure 4
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  		Fig. 4. Copies of tetracycline antibiotic-resistant genes (ARG) normalized to copies 16S bacterial rRNA gene present in the large-scale field samples with respect to time. Data points represent average values of all measurements (performed in triplicate for each sampling region) for the specified windrow. Error bars represent standard deviation.

 
In the large-scale study, tet(O) responded similarly to tet(W) to treatment (Fig. 4). The initial levels of tet(O) were significantly higher in the feedlot manure treatments than in the dairy manure (P < 0.02). Through Day 28, the HIM dairy manure maintained significantly lower levels of tet(O) than either of the feedlot manure treatments (P < 0.02). After Day 28, levels of tet(O) remained similar between all treatments. On the final day of the study, tet(O) in the HIM dairy manure was significantly higher than in the LIM dairy manure and the HIM feedlot manure (P < 0.004). The LIM feedlot manure also had significantly higher levels of tet(O) than the LIM dairy manure on the final day of the study (P < 0.05).

Comparing the results of the pilot-scale and the large-scale studies provides some insight into the behavior of ARG in antibiotic-acclimated versus unacclimated manures. In contrast to the pilot-scale study, overall responses of tet(W) and tet(O) in the large-scale study were relatively similar. Final levels of tet(W) were also significantly lower than the initial levels in both the feedlot (P < 0.0001) and dairy manures (P < 0.05). The final levels of tet(O) were significantly lower than initial levels in all of the treatments, except HIM dairy manure, which maintained relatively low levels throughout the study. While tet(W) was higher than tet(O) only in the second half of the pilot study, levels of tet(W) were initially an order of magnitude higher than tet(O) and maintained higher levels throughout the large-scale study. Thus, ARG in acclimated versus unacclimated manures appear to have different behaviors. Also, ARG levels in the unacclimated horse manure were consistently lower than in the acclimated manures. The length of time the manure had cured before being subjected to treatment could also have affected the initial levels of both the antibiotics and the ARG in the large-scale study. The analysis of the physical and chemical properties of the manures on Day 1 of the study demonstrated that both manures may have gone through some curing before treatment (Table 1), which likely affected the initial concentrations of the antibiotics and ARG. However, further reduction was observed in most treatments.

Correlation of Antibiotic-Resistant Genes Levels to Concentrations of Total Tetracyclines
Levels of tet(W) in the dairy manure and feedlot manure (independent of treatment) were positively correlated to the total concentration of tetracyclines with r2 values of 0.63 (P < 0.0002) and 0.68 (P < 0.0001), respectively. Levels of tet(O) were positively correlated to total tetracyclines in the HIM feedlot manure (r2 = 0.83, P < 0.002) and LIM feedlot manure (r2 = 0.50, P < 0.05), but not in either of the dairy manure treatments. Positive correlations were also observed when initial levels of antibiotics and ARG were paired according to sampling region, with an r2 value of 0.68 (P < 0.002) for tet(W) and an r2 value of 0.82 (P < 0.0001) for tet(O). These results suggest that the reduction in antibiotic levels with time may reduce selective pressure, allowing ARG to be lost from some hosts. On the other hand, the correlation between ARG and antibiotics appears to be lost when either is initially present at low concentrations, indicating that ARG may persist even once antibiotics are non-detectable.


   Conclusions
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 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
REFERENCES
 
The overall results suggest that the manure type and treatment time are the main factors in achieving low levels of both antibiotics and ARG in manure before land application. High-intensity management was more effective than LIM for increasing the rate of antibiotic dissipation, but it was not a significant factor in reducing levels of ARG. Future studies comparing more contrasting treatments would be of interest. The formation of recalcitrant antibiotic degradation products may be one reason that ARG persist and would be of interest for further study. For example, some CTC degradation products have half-lives as high as 400 d in soil interstitial water, depending on environmental conditions (Soeborg et al., 2004). These metabolites are often still effective, and may allow the selection pressure to remain after the parent compounds have been degraded (Thiele-Bruhn, 2003). However, developing techniques suitable for quantifying metabolites within the complex manure/compost matrix will be challenging. Future studies would also benefit from monitoring additional genes such as tetracycline efflux ARG.

With respect to manure type, the feedlot manure had significantly higher levels of ARG than the dairy manure, which was higher than the horse manure. This is reasonable given that cattle from the feedlot were routinely fed subtherapeutic concentrations of antibiotics, whereas the dairy cattle were only given therapeutic antibiotics during non-lactating periods, and the horses were given no antibiotics. With respect to time, 6 mo of treatment was necessary for reduction of ARG levels and antibiotic concentrations in feedlot manure to below 0.01 mg antibiotic per kg dry manure. In the dairy manure, antibiotic concentrations fell below the detection limit after only 4 mo. While no comprehensive data is available on the minimum concentration of antibiotics that causes selection in the environment, these studies show that at least 4 mo was necessary for the reduction of total tetracycline concentrations below 0.01 mg kg–1 in a manure with initial concentration of ~0.4 mg kg–1, while a manure with initial concentration ~2 mg kg–1 required treatment times of at least 6 mo. On the final day of the large-scale study, levels of tet(W) and tet(O) were still above the detection limit in both manures. In some manures, levels of tet(W) and tet(O) did not directly correlate with a decrease in antibiotics. This provides evidence that ARG may be maintained for extended time periods following the dissipation of the antibiotics, possibly due to the presence of antibiotic degradation products; therefore, longer treatment times may be necessary to further reduce levels of these metabolites and ARG.


   ACKNOWLEDGMENTS
 
Funding for this study was provided by the USDA Agricultural Experiment Station at Colorado State University and the NSF CAREER program.


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