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Monitoring and Quantifying Tetracycline Resistance Genes in a Swine Waste Anaerobic Digester Over a 100-Day Period

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Monitoring and Quantifying Tetracycline Resistance Genes in a Swine Waste Anaerobic Digester Over a 100-Day Period Western Kentucky University TopSCHOLAR® Masters Theses & Specialist Projects Graduate School Spring 2018 Monitoring and Quantifying Tetracycline Resistance Genes in a Swine Waste Anaerobic Digester over a 100-Day Period Melanie Couch Western Kentucky University, [email protected] Follow this and additional works at: https://digitalcommons.wku.edu/theses Part of the Molecular Biology Commons Recommended Citation Couch, Melanie, "Monitoring and Quantifying Tetracycline Resistance Genes in a Swine Waste Anaerobic Digester over a 100-Day Period" (2018). Masters Theses & Specialist Projects. Paper 2449. https://digitalcommons.wku.edu/theses/2449 This Thesis is brought to you for free and open access by TopSCHOLAR®. It has been accepted for inclusion in Masters Theses & Specialist Projects by an authorized administrator of TopSCHOLAR®. For more information, please contact [email protected]. MONITORING AND QUANTIFYING TETRACYCLINE RESISTANCE GENES IN A SWINE WASTE ANAEROBIC DIGESTER OVER A 100-DAY PERIOD A Thesis Presented to The Faculty of the Department of Chemistry Western Kentucky University Bowling Green, Kentucky In Partial Fulfillment Of the Requirements for the Degree Master of Science By Melanie Couch May 2018 I dedicate this thesis to my sister whom we lost too soon, Mary Magdalene Eversole, who is a great inspiration to me. ACKNOWLEDGEMENTS “Alone we can do so little; together we can do so much.” --- Helen Keller This work would not have been possible without the help of many others. I would first like to thank my advisor Dr. Eric Conte of the chemistry department at Western Kentucky University. Dr. Conte was always an open door or an email away whenever I ran into difficulties or had questions. He has allowed this thesis to be my own work but provided much needed guidance along the way. I would like to show my gratitude to my additional committee members, Dr. Kevin Williams, and Dr. John Loughrin. Thank you for your input and support. It is truly appreciated. I am grateful to all of those with whom I had the pleasure to work alongside during this research. Each person has given me extensive professional and personal guidance that taught me about research and life in general. I would especially like to thank Biological Science Technician, Rohan Parekh, at the USDA Agricultural Research Service Food Animal Environmental Systems Research Unit. As a mentor he has taught me more than I could ever express here. He has shown me by example, what a good scientist (and person) should be. I would also like to show my warm thanks to Dr. Agga, Dr. Loughrin, and Stacy Antle at the USDA for their guidance and allowing me this opportunity. I am eternally grateful to Western Kentucky University’s Department of Chemistry for this amazing opportunity as well as funding. All the faculty and staff have provided tremendous support. Dr. Sarah Edwards, my lab coordinator, helped me grow into the teacher I am today. Alicia Pesterfield, Laboratory Safety and Chemistry Supply iv Coordinator, made teaching labs and getting research done a smooth process. A special thanks to Haley Smith, the first person I met at WKU, who was not only my lifeline but a dear friend. Thank you for listening to me and understanding everything. I could not have made it without her help and support. Each instructor never failed to provide a friendly greeting and an encouraging word. WKU will always be a home to me. This research would not be possible without the support of friends and family. To my friends, Drake, Konnor, Jesús and Ali, thank you for your encouragement and laughs. Your different perspectives allowed me to approach my research in many different directions and your humor kept me sane during long study/writing/practice sessions. I am forever grateful to each of you. I must also thank my friend Dr. Michael Guy at NKU for all the support, encouragement, and my strong foundation in biochemistry. I am indebted to my friends at Hazard Community and Technical College, Dr. Paul Currie, Jeremy Wood, and Amy Wernette, for the encouragement and belief that I could do this. I would have given up before I even got started if they were not there to say, “you can do this”. I am humbled by the gentle encouragement from LeeAnn Helton at the University of Kentucky. You are an inspiration and role model. I hear your encouragement every step of the way. To my family, thank you for your emotional support and your belief in me. Without my family I would be nothing. v TABLE OF CONTENTS 1. Introduction……………………………………………………………………………..1 1.1 Background……………………………………………………………………1 1.2 Tetracycline Class……………………………………………………………..3 1.3 Tetracycline Use in Agriculture…………………………………………….....4 1.4 Waste Management……………………………………………………………5 1.5 Antibiotic Resistance Genes…..……………………………………………,,10 1.6 Tetracycline Resistance Genes..……………………………………………..12 1.7 Polymerase Chain Reaction………………………………………………….13 1.8 Research Proposal……………………………………………………………16 2. Experimental…………………………………………………………………………..16 2.1 Digester Design………………………………………………………………16 2.2 Digester Operation…………………………………………………………...18 2.3 Sample Collection……………………………………………………………19 2.4 Quantitative Polymerase Chain Reaction……………………………………19 2.5 Primer Optimization…………………………………………………………21 2.6 Statistical Analysis…………………………………………………………...32 3. Results…………………………………………………………………………………33 3.1 Distribution of Tetracycline Resistance Genes………………………………33 3.2 Tetracycline Antibiotic Concentration……………………………………….38 3.3 Gas Production……………………………………………………………….39 4. Conclusion…………………………………………………………………………….40 vi References…..……………………………………………………………………………42 Appendix…………………………………………………………………………………52 Effect of anaerobic digestion of swine waste on the concentrations of tetracycline resistance genes and tetracycline antibiotics……………………………………..52 Abstract…………………………………………………………………………..53 Introduction………………………………………………………………………54 Materials and Methods…………………………………………………………...56 Digester Design…………………………………………………………..56 Digester Operation……………………………………………………….57 Sample Collecting and Processing……………………………………….58 Quantification of Tetracycline Resistance Genes………………………..58 Analysis of Tetracycline Antibiotics…………………………………….61 Chemicals………………………………………………………...61 Sample Extraction for Tetracycline Antibiotic Concentration Measurements……………………………………………………61 LC-MS/MS Analysis…………………………………………….62 Gas and Water Quality Measurements…………………………………..63 Statistical Analysis……………………………………………………….63 Results and Discussion………………………………………………………..…64 Tetracycline Resistance Genes……………...…………………………...64 Concentration of Tetracycline Antibiotics……………………………….70 Relationship between Tetracycline Antibiotics and ARGs…..………….72 Gas Production and Water Quality Measurements………………………75 vii Acknowledgments………………………………………………………………..76 Abbreviations Used………………………………………………………………78 References………………………………………………………………………..79 viii MONITORING AND QUANTIFYING TETRACYCLINE RESISTANCE GENES IN A SWINE WASTE ANAEROBIC DIGESTER OVER A 100-DAY PERIOD Melanie Couch May 2018 83 Pages Directed by: Dr. Eric Conte, Dr. Kevin Williams, and Dr. John Loughrin Department of Chemistry Western Kentucky University Unregulated use of growth promoting antibiotics like Tetracyclines in agricultural feeds is becoming an increasing problem in antibiotic resistance. Undigested antibiotics leads to significant concentrations in livestock waste. These concentrations provide continuous selection pressure for the development of antibiotic resistance genes in the environment. Antibiotic resistance related deaths are projected to surpass cancer related deaths by 2050 making antibiotic resistance a pressing public health issue. The purpose of this study is to determine the abundance and persistence of tetracycline (tet) resistance genes in swine waste over a period of 100 days in an anaerobic digester system. Tet(A), tet(B), tet(G), tet(M), tet(O), tet(Q), and tet(W) were quantified by quantitative polymerase chain reaction after DNA extraction. Primers that target ribosomal protection proteins and efflux proteins were used. Antibiotic resistance genes decreased from day one but were found to be present throughout the study. ix 1. Introduction 1.1 Background Paul Ehrlich and Alexander Fleming are the pioneers of modern antibiotics.1 An important quality for antibiotics is the ability to selectively kill or inhibit microbial targets without causing harm to the human or animal host. Ehrlich realized the possibility of microbe selectivity while observing synthetic dyes. Aniline and other synthetic dyes would stain one microbe and not another. This observation led Ehrlich to begin a large- scale screening program in 1904 for a drug to treat syphilis, which was rampant and untreatable at the time. One at time, compounds were treated for specificity and effectiveness until a suitable treatment was found. With the help of fellow scientist, Alfred Bertheim, Salvarsan was synthesized. Salvarsan successfully treated syphilis and was the most frequently prescribed drug until the discovery of penicillin in the 1940s. The effective systematic screening method for drug discovery using diseased live animal models was adapted by many scientists. For example, Bayer scientists used the method to discover sulfa drugs in 1935. On September 3, 1928, Alexander Fleming observed the antibacterial properties of mold that led him to the discovery of penicillin. Alexander’s relentless determination brought awareness to the antibiotic’s potential while trying to get chemists to purify and stabilize the drug. In 1940, an Oxford led team
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