| Title |
Investigators | Department | Objectives | Approach Keywords | Progress Reports | Impact Statements | Publications | |
Project * COL00700 | |
| Title | Best Management Practices for Antibiotic Resistance Gene (ARG) Treatment in Livestock Lagoons |
| Investigator(s) | Pruden, A; |
| Department | Civil and Environmental Engineering |
| Objectives | Recently there has been growing local, national, and international concern about the potential impacts of antibiotic use in agriculture. In particular, a better understanding about the effects of farm practices on the spread of antibiotic resistance genes (ARG) is needed. ARG impart the ability of microbes to survive in the presence of antibiotics, and thus can diminish the effectiveness of antibiotics for treating human disease. However, our recent AES-supported research demonstrated promising results, indicating that appropriate management of animal wastes can help to reduce the spread of ARG in the environment. Therefore, the main goal of this proposed research will be to take our findings a step further by determining the response of ARG to treatment in full-scale on-farm lagoons. By investigating a cross-section of animal operations as well as different lagoon management strategies, best management practices for reducing the spread of ARG will be formulated and disseminated to the farming community. This research will be driven by the following three synergistic objectives: Objective 1 is to monitor concentrations of tetracycline, sulfonamide, and macrolide ARG in on-farm animal waste lagoons representing a cross-section of animal operations. Objective 2 is to explore the relationship of temporal ARG concentrations to lagoon operating and water quality characteristics. Objective 3 is to characterize ARG signatures in lagoons to aid source-tracking of ARG in the environment. |
| Approach | A cross-section of existing full-scale animal waste lagoons, including two organic dairies, two traditional dairies, two beef feedlots, one pork confined animal feeding operation (CAFO), and two poultry farms will be monitored over a two year period. Where possible, sampling will be targeted at the lagoon inlet, outlet, and the middle, including both water column samples and settled solids. This will help provide an idea of the effectiveness of treatment in each lagoon. An inflatable raft will be used to access the samples and a portable probe will be used to measure the dissolved oxygen, pH, and temperature in the field. Samples will be transported to the lab for chemical oxygen demand (COD), nitrate, ammonia, and total phosphorus measurements in order to determine the relationship between these water quality characteristics and ARG. Additionally, the metals present in the samples will be profiled using inductively coupled plasma atomic emission spectroscopy (ICP-AES) using the facilities available at the Colorado State University Soil and Water Testing Laboratory. Based on recent reports in the literature and on our recent work (Pei et al., 2007), the presence of heavy metals is a strong correlate with ARG, perhaps because the metal resistance genes are linked to the ARG. DNA will be extracted from the samples and ARG will be quantified implementing real-time quantitative polymerase chain reaction (Q-PCR) techniques developed by the PIs research group. Six ARG from three different classes will be quantified: tetracycline ARG (tetO and tetW), sulfonamide ARG (sulI and sulII), and macrolide ARG (ereA, and msrA). A Cepheid Smart Cycler and an Applied Biosystems 3100 real-time PCR cyclers are available in the PIs laboratory to facilitate high-throughput analysis of these ARG. Bacterial 16S rRNA genes will also be quantified by Q-PCR in order to provide a reference for normalizing the number of ARG quantified. In parallel work, ARG signatures will be characterized using a capillary electrophoresis single strand conformation polymorphism (CE-SSCP) procedure currently under development in the PIs lab. This will help support larger goals of identifying human versus agricultural sources of ARG in the environment. ARG concentrations with time will be analyzed with respect to lagoon operating conditions. In particular, we will examine the effect of the kind of manure and the estimated lagoon retention time. Correlation analyses will be conducted in order to determine if there is a relationship between the levels of ARG and the concentrations or characteristics of the various water quality characteristics being monitored. This will provide a means to identify: 1.) Which practices are associated with attenuating initially high levels of ARGs and 2.) Which practices are associated with maintaining low levels of ARG. Statistical analyses will be implemented as appropriate in order to make sound judgments in this regard. The final results will be synthesized into practical operating guidelines in terms of best management practices. |
| Keywords | antibiotics, antibiotic resistance genes, tetracycline, sulfonamide, macrolide, lagoons |
| Progress Reports | |
| 2004 | Recently concerns have arisen about the problems that could be associated with pharmaceuticals such as antibiotics entering the water supply. One large source of antibiotics is run-off from agricultural fields, feedlots, or waste lagoons. While antibiotics have benefits in terms of increased animal weight gain, many antibiotics are poorly absorbed into an animal's digestive system and thus 25 to 75 percent of the antibiotics are passed unaltered in their waste. Several recent studies, including a study done here at CSU, have confirmed the presence of low levels of antibiotics in river sediments and have linked the source of some of these antibiotics to concentrated animal feed operations (CAFOs). Even though most studies confirmed that the antibiotics were present at low concentrations, antibiotics are still active at low doses and thus pose a concern to public health. Low concentrations of antibiotics allow microorganisms to develop resistance, or may themselves act as endocrine disrupting agents, posing a direct threat to humans consuming the water. Considering our recent awareness that antibiotics are present in the environment and their potential threat, the purpose of this research project is to determine if proper waste management of feedlots will prevent the input of antibiotics and/or antibiotic resistant organisms into our waterways. Immediately after funding became available in August, 2004, a graduate student (Heather Storteboom, Civil Engineering) and a research assistant (Kathy Doeskin, Soil and Crop Sciences) were identified for conducting the experiments, and a preliminary field study was carried out. In this study, horse manure spiked with three antibiotics (monensin, chlortetracycline, and tylosin) and untreated horse manure were subjected to two different management regimes: composting and stockpiling. This preliminary study has been of value in several ways. First, a reliable sampling method has been developed and the machinery necessary to properly manage the compost has been acquired and tested. Also, new methods of sample preparation have been developed to ensure consistency between parallel studies being conducted using microbiological, analytical, and molecular methods. The approach is to sample multiple points throughout the compost and to homogenize them as a slurry in sterile water to ensure thorough mixing and increased consistency between samples. Current work is focused on homogenizing the samples that were collected over the Fall and preparing them for antibiotic and antibiotic gene analysis, which will provide valuable information regarding which management regime best reduces antibiotic impacts. Tests are also underway to compare the level at which each compost treatment is 'finished' and analytical techniques appropriate to compost testing standards are being established. This Spring will be devoted to completing these analysis and preparing for the next series of experiments this Summer and Fall. These follow-up field experiments will be conducted to compare management of cow manure from feedlots using medicated feed with cow manure from operations not using medicated feed. |
| 2005 | The focus of this investigation deals with a topic of growing concern: the spread of antibiotic resistant microorganisms in the environment. The spread of antibiotic resistant pathogens is a growing human and animal health concern. Approaches are therefore needed to help mitigate this problem. This project investigates a practical approach to help reduce the spread of antibiotic resistance by examining directly the fate of antibiotic resistance genes in compost. Since the time of the last report, the year one composting study of horse manure spiked with antibiotics has been completed. All compost samples have been analyzed with respect to antibiotic content and the quantities of two representative tetracycline resistance genes, tetO and tetW. The results showed that tetO and tetW responded differently to composting. tetO decreased continually during composting, while tetW increased before finally decreasing. This provides important guidance for farmers and suggests that longer composting times will provide sufficient time for all resistance genes to attenuate before the compost is land-applied. It was also found that chlortetracycline reduced continually during composting, suggesting that composting can also help minimize the spread of antibiotic pharmaceuticals. A follow-up experiment was conducted this summer and fall, 2005, to see if the effects are the same in dairy manure already acclimated to antibiotics (rather than spiked). As a control, dairy manure from a farm not using antibiotics was used. The compost was sampled regularly and is now in the curing phase. Current efforts are underway to quantify antibiotics and resistance genes in the compost samples. In addition to tetW and tetO, sulfonamide resistance genes will also be monitored. The results of this research are expected to have a positive economic impact by potentially avoiding a ban on sub-therapeutic antibiotic use, while at the same time protecting the environment. The results have also attracted broader scientific attention. For example, NSF funding was recently granted to the PI to look into more fundamental aspects of how antibiotic resistance genes are spread in the environment and how they respond to biological treatment. Topics such as these are of increasing interest as the need becomes apparent to understand how pathogens and the genetic elements that they carry are spread. |
| 2006 | There is growing concern about pharmaceuticals such as antibiotics entering the water supply, especially in Colorado. One large source of antibiotics is run-off from agricultural fields, feedlots, or waste lagoons. Several recent studies, including a study done here at CSU, have confirmed the presence of low levels of antibiotics in river sediments and have linked the source of some of these antibiotics to concentrated animal feed operations (CAFOs). A major goal of this project was to determine if there is a corresponding increase in antibiotic resistance genes (ARG) in these environments that also have high levels of antibiotics. At the same time, lagoon treatment and composting were investigated as a practical approach to ARG treatment. As of December 31, 2006, two major publications have been accepted (listed below) that demonstrate that ARG do occur at higher concentrations where antibiotics are also present at high concentration. Also, a trend was demonstrated in which ARG concentrations are highest near antibiotic sources on farms, lowest in river sediments, and intermediate in irrigation ditches. This suggests a possible pathway for the spread of ARG, which merits future investigation. Some of the same ARG that are found on farms have been found in tap-water, which is raising public interest. Two composting experiments have been completed. The first compared high level management (amendment, watering, and turning) with low level management (no amendment, watering, or turning) of horse manure on a pilot-scale. The second compared high-level and low-level management of dairy (low antibiotic level) and beef (high antibiotic level) manure. It was found that ARG could be reduced below their initial levels in all manures and both scales, but that time was a significant factor. If composting was terminated too early, then it could lead to the spread of ARG. Also, with sufficient time, even manures with high antibiotic concentrations could be reduced to the same levels as manures with low antibiotic concentrations. Antibiotics were also diminished during composting. Similarly, in a study comparing aerobic and anaerobic treatment of dairy lagoon water, it was found that some ARG increased initially before decreasing. This again emphasizes the importance of time in treatment in order to reduce the spread of ARG. At this stage of the project the emphasis is on further analysis of the data and write-up of the results. We are expanding the study to examine several different farms in order to determine the behavior of ARG during actual farm treatment. At the same time, we are developing source-tracking methods in order to distinguish human from agricultural ARG sources in the greater environment. |
| Impact | |
| 2004 | If composting is found to reduce the amount of antibiotics and antibiotic resistant organisms found in waste from feedlots using medicated animals, then it would allow antibiotic contamination to be mitigated before it enters waterways. Also, if composting is found to be a reasonable solution, it could prevent an outright ban of antibiotic use in livestock production, as was done in many European countries. This will not only benefit livestock producers and cost to consumers, but it will also aid in reducing conflict between environmental policy makers and livestock producers. Reducing this conflict will play an important role in future cooperation of livestock producers with environmental regulations. To our knowledge, the present study is the first to directly examine composting as a means to decrease antibiotics and antibiotic resistance genes. Looking at plausible solutions to the problem of antibiotic contamination before regulations or policies are put into place will benefit livestock producers by preparing them to deal with regulations and or policies that may be passed in the future. |
| 2005 | A new treatment is being developed based on this research in that new guidelines for composting will be published that help to prevent the spread of antibiotic resistance genes. This work suggests that time is a critical factor in composting, if it is terminated too early , the antibiotic resistance genes may actually be HIGHER than their initial concentrations. Because the livestock industry across the U.S. depends on the use of agricultural antibiotics, these results have widespread implications for Colorado and beyond. Investigating treatment approaches, such as composting, will help to make the practice safe and help reduce the spread of antibiotic resistant pathogens. Currently the spread of antibiotic resistant pathogens is considered to be on of the most critical concerns of the world health organization (WHO). At the same time, the rate at which new antibiotics are being discovered is decreasing. This emphasizes the need to find ways to stop the spread of resistance as antibiotics become less reliable. |
| 2006 | This project is already having significant impact in Colorado and beyond. On October 5th, the PI presented the results of this study to the CAFO Roundtable, which met in Denver, CO and received favorable reviews from the state and federal attendees. The issue of ARG was clearly viewed as meriting further investigation. At the same time, this project is receiving significant local, national, and international attention, including: Articles in the Denver Post, Rocky Mountain News, Loveland Daily Reporter-Herald, and Fort Collins Coloradoan; Online publications by MS NBC News and EMax Health; A radio interview by Carol Off of the CBC; and a recent publication in Scientific American (listed below). This project is expected to have further direct impact because of its applied nature and current outreach efforts with the agricultural community. Composting and lagoon management work with the existing infrastructure on farms and present a practical means of addressing the problem of the spread of antibiotic resistance. Currently antibiotic resistance is viewed as one of the most serious growing threats to human health by the World Health Organization, the CDC, and other national and international agencies. |
| Publications | |
| 2005 |
Pei, R., Huxford, K. and Pruden, A. 2005. Quantifying Antibiotic Resistance in the Sediments of a Mixed-Landscape River. Conference Proceedings of the Water Environment Federation Technical Conference and Exhibition, October 29, Washington D.C. |
| 2006 |
Choi, C.Q. 2007. Pollution in Solution: Drug-Resistance DNA as the Latest Freshwater Threat. Scientific American. 296 (1): 22-23. Pei, R., Cha, J-M., Carlson, K.H., and A. Pruden. 2007. Biological Treatment of Antibiotic Resistance Genes (ARG) in Dairy Lagoon Water. Environ. Sci. Technol. (Submitted). Pruden, A., Pei, R., Storteboom, H., and K.H. Carlson. 2007. Antibiotic Resistance Genes (ARG) as Emerging Contaminants: Studies in Northeastern Colorado. Environ. Sci. Technol. (in press for Special Issue on Emerging Contaminants). Storteboom, H., Kim, S.C., Doesken, K., Davis, J., Carlson, K.H., and A. Pruden. 2007. Response of Tetracycline and Resistance Genes tet(W) and tet(O) to High-Level and Low-Level Managed Manures. J. Environ. Qual. (Submitted). |