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ROLE OF POINT SOURCES ON THE DISSEMINATION OF ANTIBIOTIC RESISTANCE IN THE ENVIRONMENT A DISSERTATION SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY IRENE BUENO PADILLA IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Randall S. Singer, Dominic A. Travis (Advisors) October 2018 Irene Bueno Padilla, October 2018© Acknowledgements I would like to first of all acknowledge my advisors, Drs. Randall S. Singer and Dominic A. Travis. Dominic, you have always been so supportive, even before the PhD when you guided me through my MPH and encouraged me to continue with a PhD. Thank you for all the valuable time you have spent with me and for all I have learned with you. Randy, thank you so much for believing and trusting in me to conduct an international project in Chile. Thank you both for so many learning opportunities, for allowing me to be creative while guiding me when I got off track. I would also like to thank my committee members (Drs. Claudia Muñoz-Zanzi, Tim J. Johnson, and Nick Phelps) for their patience and support throughout my PhD. I would also like to give special thanks to Jessica Williams-Nguyen, Haejin Hwang, André Nault, and Jan Sargeant for their contribution to the development of the two systematic reviews. Furthermore, I would like to thank Drs. Satoshi Ishii, Qian Zhang and especially Britta Wass and the rest of the Singer lab staff for their help with the molecular laboratory methods. I would really like to thank all the people in Chile that have made this work possible: Dr. Cristián García, for his enthusiasm and willingness to cooperate with the trout farms work; Drs. Paulina Fry, Guillermo Ramirez and Keith Hood (SEREMI de Salud de la Región de los Ríos, Chile) for their help with the wastewater treatment plant work, as well as the staff from the wastewater treatment plant company, especially to Mr. Jorge Arel. I would also like to thank the field assistants and staff from the E3 lab at the Facultad de Veterinaria de la Universidad Austral de Chile, i especially Dr. Claudio Verdugo for his willingness to collaborate with me and for providing all the field equipment for the bird study. I would also like to acknowledge the investigators from Universidad de Concepcion, especially Drs. Gerardo González- Rocha and Celia Lima for their support and collaboration. In addition, I would like to thank the faculty, other graduate students, and program coordinators (Lisa Hubinger and Kate Barry) at the College of Veterinary Medicine (UMN) for their involvement and for creating such a great working environment, and with especial mention to my great UMN friends Cata, Whitney, and Fernanda who have always been there for me. I could not have got where I am if it was not for Dr. Patrick Redig, the champion I followed to Minnesota to learn from, and the staff and volunteers from The Raptor Center, UMN. On a final personal note, I have to thank all my other numerous great friends, and very especially my best friends, Ana, Paola, and Mari. Also my boyfriend, Omar for his patience and love. Finally, my family, “La Piña”, especially my brothers Luis Miguel and Jose María whom I so admire, for their unconditional support and love, as well as Raluca and Ana, and my adorable and smart nephew, Robert, and my niece, Maria. ii Dedication Para mis padres Luis y Carmen. To my dad for teaching me that with perseverance, discipline, and hard work I can do anything, and to my mom, la “roca”, for giving me the wings to always follow my dreams. Gracias a los dos por vuestro apoyo incondicional, os admiro y quiero mucho. iii Research Abstracts Chapter 2. Systematic Review: Impact of Point Sources on Antibiotic Resistant Bacteria in the Natural Environment Point sources such as wastewater treatment plants and agricultural facilities may have a role in the dissemination of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG). To analyze the evidence for increases of ARB in the natural environment associated with these point sources of ARB and ARG, we conducted a systematic review. We evaluated 5,247 records retrieved through database searches, including both studies that ascertained ARG and ARB outcomes. All studies were subjected to a screening process to assess relevance to the question and methodology to address our review question. A risk of bias assessment was conducted upon the final pool of studies included in the review. This paper summarizes the evidence only for those studies with ARB outcomes (n=47). Thirty five studies were at high (n=11) or at unclear (n=24) risk of bias in the estimation of source effects due to lack of information and/or failure to control for confounders. Statistical analysis was used in ten studies, of which one assessed the effect of multiple sources using modeling approaches, and none reported effect measures. Most studies reported higher ARB prevalence or concentration downstream/near the source; however, this evidence was primarily descriptive and it could not be concluded that there is a clear impact of point sources on increases of ARB in the environment. In order to quantify increases of ARB in the environment due to specific point sources, there is a iv need for studies that stress study design, control of biases, and analytical tools to provide effect measure estimates. Chapter 3. Impact of Point Sources on Antibiotic Resistance Genes in the Natural Environment: A Systematic Review of the Evidence There is a growing concern about the role of the environment in the dissemination of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG). In this systematic review we summarized measurable increases of ARG in the natural environment associated with potential sources of ARB and ARG such as agricultural facilities and wastewater treatment plants. A total of 5,247 citations were identified, including both studies that ascertained ARG and ARB outcomes. All studies were screened for relevance to the question and methodology. This chapter summarizes the evidence only for those studies with ARG outcomes (n=24). Sixteen studies were at high (n=3) or at unclear (n=13) risk of bias in the estimation of source effects due to lack of information and/or failure to control for confounders. Statistical methods were used in 9 studies; 3 studies assessed the effect of multiple sources using modeling approaches, and none reported effect measures. Most studies reported higher ARG concentration downstream/near the source, but heterogeneous findings hindered making any sound conclusions. To quantify increases of ARG in the environment due to specific point sources, there is a need for studies that emphasize analytic or design control of confounding, and that provide effect measure estimates. v Chapter 4. Antibiotic Resistance Genes in Freshwater Trout Farms in a Watershed in Chile Point sources such as wastewater treatment plants, terrestrial agriculture, and aquaculture, release antibiotic residues, antibiotic resistant bacteria, and antibiotic resistance genes (ARG) into the aquatic ecosystem. However, increases of ARG in the natural environment associated with specific point sources have not been widely quantified. The goal of this study was to evaluate the role of freshwater trout farms on the release and dissemination of ARG into the environment. Sediment samples upstream and downstream from five trout farms were collected over time in southern Chile. A microfluidic qPCR approach was used to quantify an ARG array covering different mechanisms of resistance. Surveys were conducted to obtain information about management practices including antibiotic use at the farms. Florfenicol and oxytetracycline were used at these farms, although at different rates. A total of 93 samples were analyzed. A statistically significant increase of the abundance of qacG, strB, sul1, and several tet genes, was found downstream from the farms compared to upstream sites. While results from this study indicate an influence of these trout farms on the presence of ARG in the immediate environment, the biological significance of this finding is unknown and deserves further investigation. Chapter 5. Role of Wastewater Treatment Plants on Environmental Abundance of Antibiotic Resistance Genes in Chilean Rivers Point sources such as wastewater treatment plants (WWTPs) commonly discharge their effluent into rivers. Their waste may include antibiotic residues, disinfectants, antibiotic vi resistant bacteria (ARB), and antibiotic resistance genes (ARG). There is evidence that ARG can be found in the natural environment, but attribution to specific point sources is lacking. The goal of this study was to assess the release and dissemination of ARG from three WWTPs in southern Chile via two pathways: through the river systems, and through wild birds. A longitudinal study was conducted in small spatial scales around each WWTP to avoid influence of confounders, collecting river sediment samples at different distances both upstream and downstream from each WWTP. Wild birds were sampled from around one of the WWTPs once a month for 13 months. A microfluidic qPCR approach was used to quantify an ARG array covering different resistance mechanisms. There was a statistically significant increase downstream (p<0.05) of the abundance of strB, sul1, and sul2, but the downstream dissemination through the rivers was not clear. Beta-lactamase genes blaKPC, blaTEM, and blaSHV were the most abundant in birds, with higher abundance in migratory species compared to resident species (p<0.05).

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