Progress 07/01/07 to 06/30/10

Outputs
OUTPUTS: Collection of antibiotic occurrence data in livestock lagoons was completed, which marked the completion of the experimental phase of the livestock lagoon study. Tetracyclines and sulfonamides, as well as tylosin and quinolones were measured through collaboration with the USGS. Research efforts this year focused on analyzing the data in order to identify trends with respect to livestock lagoon treatment of antibiotic resistance genes (ARGs) and the write-up of a manuscript for publication, which was submitted to Environmental Science and Technology in December, 2009. The data analyzed included: concentrations of four ARG (tetW, tetO, sulI and sulII), antibiotic concentrations, chemical oxygen demand, phosphorus, nitrate, ammonia, total nitrogen, and metals. Graphical and statistical comparisons were made with respect to livestock type (conventional dairy, organic dairy, beef, swine, and laying hens), season, water solids versus settled solids, and number of lagoons in series. The second experimental phase was also complete, in which we are working to track the sources of ARG present in the Poudre River sediments. ARG signatures were analyzed at pristine and impacted zones in the Poudre River and compared to the ARG signatures of putative sources: wastewater treatment plants and livestock lagoons. A GIS database was developed and the data was analyzed using descriptive statistics to identify patterns associated with sources versus the pristine river and to identify sources in the impacted river. A book on the topic of Pharmaceuticals and CAFOS, co-edited by the PI, was published in July, 2009. PARTICIPANTS: The co-PI on this project is Mazdak Arabi. Chad McKinney is an MS student who was supported on this project and focused on the livestock lagoon study. Heather Storteboom is a PhD student who was supported on this project and focused on the ARG source-tracking study. Keith Loftin from the USGS collaborated in this project by providing antibiotic analytical services. He is a co-author on the livestock lagoon manuscript. Jessica Davis from Soil and Crop Sciences at CSU assisted in working with livestock producers and is a co-author on both of the manuscripts produced in this project. TARGET AUDIENCES: The target audience for this research is livestock producers. The results provide guidance on antibiotic use and livestock lagoon design. The scientific community is also an audience for this research. The results will help advance the field of understanding the behavior of antibiotic resistance in the environment and the contributions of livestock operations and lagoon management. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
It was found that ARG concentrations varied with livestock type, with swine being the highest and laying hens being the lowest. Also, organic dairies were marginally lower than conventional dairies in terms of ARG concentrations. Antibiotic occurrence data followed similar trends, suggesting that reduced antibiotic use plays a major role in reducing the reservoir of resistance in livestock lagoons. Several significant correlations were identified between antibiotic and ARG concentrations and also between metals and ARG concentrations. However, the correlations were stronger between ARG and antibiotics than between ARG and metals, indicating that antibiotics likely exert a stronger selective pressure than the metals. In terms of indications for best management practices, it was found that multiple lagoons in series (three or more) provided greater attenuation of ARGs. Sul and tet ARG tended to follow distinct patterns in response to treatment. sul ARG were more recalcitrant and tended to increase in concentration, while tet genes decreased. Tet and sul also responded differently seasonally, so there is not likely an ideal season for land application to reduce the spread of resistance. For the source-tracking aspect of the project, the data support the hypothesis that resistant bacteria are transported from livestock operations and wastewater treatment plants to impacted river sites. This is in contrast to the possibility of elevated levels of antibiotics and other pollutants selecting antibiotic resistant bacteria from the native river populations. A GIS database was developed, as well as a novel approach, implementing multiple lines of evidence, to identify sources of ARGs in river environments.

Publications

• McKinney, C.W., Loftin, K.A, Meyer, M.T., Davis, J.G., and A. Pruden. (2009). tet and sul Antibiotic Resistance Genes in Livestock Lagoons of Various Operation Type, Configuration, and Antibiotic Occurrence. (submitted to Environmental Science and Technology, December 2009).
• Storteboom, H.N., Arabi, M., Davis, J.G., Crimi, B., and A. Pruden. (2009). Identification of antibiotic resistance gene molecular signatures suitable as tracers of pristine river, urban, and agricultural sources. (under revision for Environmental Science and Technology).
• Shore, L.S. and A. Pruden (eds) (2009). Hormones and Pharmaceuticals Generated from Concentrated Animal Feeding Operations: Transport in Water and Soil, Springer, New York. ISBN 978-0-387-92833-3.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The first experimental phase of the project was completed this year. The lagoons of four dairies, two beef feedlots, one poultry farm, and one swine operation were monitored over four time points representative of the seasons. Spatial sampling was also implemented in order to capture possible lagoon treatment impacts on antibiotic resistance. All samples were analyzed by quantitative polymerase chain reaction in order to determine the concentration of four different antibiotic resistance genes: tetO, tetW, sulI, and sulII. Nutrient concentrations were also monitored, as well as heavy metals and chemical oxygen demand (COD). The second experimental phase is currently underway in which we are working to track the sources of ARG present in the Poudre River sediments. We have analyzed gene signatures from various sampling points and are developing a GIS database in order to guide best management practices. The PI also developed and delivered a webcast through the National Livestock, Poultry and the Environment Learning Center (NLPE) in order to explain the findings and discuss the issue of antibiotic resistance with the livestock community. She also worked with the NLPE team members to write text for the website pertaining to this topic. The PI also worked with Co-author Larry Shore to write and edit a book on the topic of Pharmaceuticals and CAFOS. The PI and her PhD student joined the Institute for Livestock and the Environment (ILE) led by Jessica Davis at CSU. PARTICIPANTS: One PhD student, Heather Storteboom, worked on this project. Also, one MS student, Chad McKinney, assisted with this project. Dr. Mazdak Arabi is providing technical assistance to the project and co-advising the Ph.D. student. Dr. Jessica Davis provided assistance in contacting appropriate livestock producers to participate in this project. TARGET AUDIENCES: In addition to sharing results with the academic community, the PI has worked to make results available to the broader agricultural community. The webcast presented through the National Livestock, Poultry, and the Environment Learning Center is one example, and the draft of the book that is currently out for peer review is another. Additionally, the PI and her students have worked closely with the producers who participated in this study to communicate the results with them. We intend to put together a custom report for each of the producers who participated in this project in order to explain the overall results with respect to their operation. PROJECT MODIFICATIONS: None.

Impacts
There are two outcomes/Impacts to highlight at this point in the project. The first is the webcast that was delivered via the National Livestock Poultry and Environment Learning Center (NLPE). This provided the opportunity to share the results of the research with the greater farming community in an interactive manner. The second is the compilation of a book on the topic of pharmaceuticals in CAFOS. This book is currently out for peer-review. With respect to the research, we are still interpreting the results thus far and have put together a draft manuscript of the results of the lagoon monitoring study. Thus far the results indicate that there is a modest benefit of organic dairy operation in terms of reduction of antibiotic resistance, however, antibiotic resistance is not eliminated. There are also benefits in terms of antibiotic resistance for small dairy operation. The lagoons at the pork operation had the highest concentrations of antibiotic resistance, and the lagoons at the poultry operation had the lowest concentrations of antibiotic resistance. These results provide some preliminary guidance regarding the end use of these respective lagoon residuals. Finally, a seasonal effect was found, suggesting that ARG concentrations are higher during cold seasons, suggesting that land application should be avoided in late fall, winter. These results are preliminary, but clearly indicate that the information gained will be able to advance the development of BMPs for better controlling antibiotic resistance emanating from livestock operations.

Publications

• Pruden, A. Webcast entitled, Human and Environmental Impacts of Antibiotics. Broadcast March 28, 2008 through the National Livestock, Poultry, and the Environment Learning Center. Archive available at: University of Nebraska, Lincoln. http://www.extension.org/pages/Livestock_and_Poultry_Environmental_Le arning_Center_Webcast_Series

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: 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). There is concern that the antibiotics may increase the concentration of bacteria that are resistant to these antibiotics. The purpose of this study was to investigate the effect of livestock lagoon treatment of animal waste on antibiotic resistance genes (ARG). ARG are the pieces of DNA that make bacteria resistant to medicines. Past AES work demonstrated that ARG do occur at higher concentrations where antibiotics are also present at high concentrations. An encouraging find was that even though ARG are present in the environment at high concentrations when impacted by human activity, ARG do appear to respond to treatment. In a study of manure management by composting, ARG were observed to decline in concentration. Similarly, in a study comparing aerobic and anaerobic treatment of dairy lagoon water, it was found that some ARG responded well to treatment. The focus of the current ARG project is treatment in on-farm lagoons. This study is important because it is providing information about how ARG behave in actual field-scale systems. We have made rapid progress in the first 6 months of this project and have collected samples from Colorado lagoons over 5 time points. The farms that have agreed to participate in this study include: 1 large organic dairy, 1 small organic dairy, 1 large conventional dairy, 1 small conventional dairy, 1 large beef feedlot, 1 small beef feedlot, 1 poultry farm (eggs), and 1 hog farm. This provides a good cross section of the behavior of ARG in different kinds of animal waste and different lagoon arrangements. Of particular interest is the effect of organic farming and innovative treatment, such as the use of algae. At this stage of the project we are collecting data on the samples, including: measurement of four different ARG, pH, total nitrogen, total phosphorus, and heavy metals. The ARG that are the target of this study include two sulfonamide ARG (sulI and sulII) and two tetracycline ARG (tetO and tetW). Initial results suggest that there are clear differences between the farms, but it is too soon to identify which approaches to lagoon operation best control ARG. PARTICIPANTS: Amy Pruden Chad McKinney Heather Storteboom Kathy Doesken Jessica Davis TARGET AUDIENCES: Colorado Farmers Colorado Livestock Association Colorado Water Resources Research Institute Livestock Poultry and Environmental Learning Center PROJECT MODIFICATIONS: None.

Impacts
Although in its infancy, this project has had significant impact. On November 1st, 2006, the PI received an award from the President of the United States in part for recognition of her work on antibiotic resistance, including current work on this AES-supported project. An article in Discover magazine is anticipated in February, 2008. The PI is also making efforts to share results with the agricultural community. For example, she is part of the LPE Pharmaceutical Team organized by Jessica Davis in Soil and Crop Sciences at Colorado State University. As a member of this team, she is helping to prepare a website that will help educate the public, and particularly farmers, about what is known about ARG in animal waste. She will be giving a live webcast on the subject on March 28th. This project is expected to have further direct impact because of its applied nature and current outreach efforts with the agricultural community. Lagoon management works 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 Center for Disease Control, and other national and international agencies.

Publications

• No publications reported this period

Progress 01/01/06 to 12/31/06

Outputs
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.

Impacts
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

• 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).
• 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).
• 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).
• Choi, C.Q. (2007) Pollution in Solution: Drug-Resistance DNA as the Latest Freshwater Threat. Scientific American. 296 (1): 22-23.

Progress 01/01/05 to 12/31/05

Outputs
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.

Impacts
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.

Publications

• 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.

Progress 01/01/04 to 12/31/04

Outputs
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.

Impacts
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.

Publications

• No publications reported this period