Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2015 Exploring the relationship between attachment and antibiotic resistance of Escherichia coli from swine manure Martha Reye Zwonitzer Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Agriculture Commons, and the Microbiology Commons Recommended Citation Zwonitzer, Martha Reye, "Exploring the relationship between attachment and antibiotic resistance of Escherichia coli from swine manure" (2015). Graduate Theses and Dissertations. 14704. https://lib.dr.iastate.edu/etd/14704 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Exploring the relationship between attachment and antibiotic resistance of Escherichia coli from swine manure by Martha Reye Zwonitzer A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE i Major: Environmental Science Program of Study Committee: Michelle L. Soupir, Co-major Professor Laura R. Jarboe, Co-major Professor Steve Mickelson Iowa State University Ames, Iowa 2015 Copyright © Martha Reye Zwonitzer, 2015. All rights reserved ii TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................. iv LIST OF TABLES .................................................................................................................... v ACKNOWLEGEMENTS ........................................................................................................ vi ABSTRACT ............................................................................................................................ vii CHAPTER 1: GENERAL INTRODUCTION ......................................................................... 1 1.1 Introduction ................................................................................................................ 1 1.2 Goals and Objectives .................................................................................................. 3 1.3 Hypothesis .................................................................................................................. 4 1.4 Thesis Organization.................................................................................................... 5 CHAPTER 2: LITERATURE REVIEW .................................................................................. 6 2.1 Antibiotic Resistance.................................................................................................. 6 2.2 Antibiotic use by the swine industry .......................................................................... 8 2.3 Antibiotic Classifications ......................................................................................... 11 2.4 Mechanisms of Resistance ....................................................................................... 16 2.5 Bacterial Attachment and Implications for Transport .............................................. 24 2.6 Summary .................................................................................................................. 30 2.7 Literature Cited ........................................................................................................ 31 iii CHAPTER 3. QUANTIFYING ATTACHMENT AND ANTIBIOTIC RESISTANCE OF ESCHERICHIA COLI FROM CONVENTIONAL AND ORGANIC SWINE MANURE ... 43 Abstract ............................................................................................................................... 44 3.1 Introduction .............................................................................................................. 45 3.2 Materials and Methods ............................................................................................. 48 3.3 Results and Discussion ............................................................................................. 53 3.4 Conclusions .............................................................................................................. 59 3.5 Acknowledgements .................................................................................................. 60 3.6 References ................................................................................................................ 60 CHAPTER 4: GENERAL CONCLUSIONS.................................................................... 73 4.1 General Discussion and Conclusions ....................................................................... 73 4.2 Implications and Recommendation for Future Research ......................................... 75 APPENDIX A: RAW DATA ................................................................................................. 77 iv LIST OF FIGURES Page Figure 1 Major targets for antibacterial action.....………………………………………………. 12 Figure 2 Bacterial antibiotic resistance mechanisms .............................................. 20 v LIST OF TABLES Page Table 1 Description of research farms for manure collection used in selecting and enumeration of bacterial isolates.....………………………………………………. ...... 67 Table 2 Minimum inhibitory concentrations (MICs) for antimicrobial agents tested 556 Escherichia coli isolates and three control isolates ............................ 68 Table 3 Chi-square tests of independence were performed to investigate differences in number of isolates for each of the three levels of resistance (Susceptible (S), Intermediate (I), and Resistance (R)) to the thirteen antibiotics for isolates, by management system (Conventional vs. Organic) ................... 69 Table 4 Percentage of total (n = 557) isolates displaying resistance to each of the 13 antibiotics tested, as well as the percentage of those isolates which are resistant to multiple antibiotics……….... .................................................... 70 Table 5a Crosstabulation of isolate attachment vs. resistance level for isolates collected under the conventional management system ........................... 71 Table 5b Crosstabulation of isolate attachment vs. resistance level for isolates collected under the organic management system .................................... 72 vi ACKNOWLEGEMENTS I would like to thank my co-committee chairs, Michelle Soupir and Laura Jarboe and my committee members, Steve Mickelson and Ramesh Kanwar, for their guidance and support throughout the course of this research. I would also like to thank my friends, colleagues, the department faculty and staff for making my time at Iowa State University a wonderful experience. In addition, I would like to thank my family for standing by me throughout this tedious process and for their willingness to help as needed. vii ABSTRACT Antibiotics are widely utilized in swine production for treatment and prevention of disease, growth promotion, and to improve the efficiency of feed. Antibiotic resistance has been present since the invention and subsequent use of antibiotics and bacteria have used antibiotic resistant mechanisms to overcome attacks in the environment. Under antibiotic pressure resistant mutants can spontaneously form and resistance genes can be passed between bacteria. Use of antibiotics at subtherapeutic levels is thought to add selective pressure for the development of antibiotic resistant bacteria both enteric and in the environment. However, there is still a large knowledge gap in understanding why some bacteria develop resistance rapidly and others remain susceptible. The ability to attach to surfaces has been shown to result in bacterial persistence in the environment. Presence of attachment factors, such as pili, could be linked to the persistence of uropathogenic E. coli in humans. Few studies have been completed investigating the mechanisms responsible for adhesion of bacteria in the environment, and even fewer still have been performed examining relationships between attachment and antibiotic resistance. Understanding these relationships might have an impact on the timing and methodology of manure application or the use of antibiotics utilized in both human and animal prophylaxis at subtherapuetic levels in feeding operations. The objectives of this study were to: 1) detect and quantify the fraction of bacteria isolated from manure collected from conventional and organic swine production facilities in Iowa showing preferential attachment to quartz; 2) quantify the levels of resistance (susceptible, viii intermediate, or resistant) of isolates collected from conventional and organic swine production facilities to amoxicillin (AMX), ampicillin (AMP), chloramphenicol (CMP), chlortetracycline (CTC), erythromycin (ERY), gentamycin (GEN), kanamycin (KAN), nalidixic acid (NAL), neomycin (NEO), tetracycline (TET), tylosin (TYL), streptomycin (STP), and sulfamethazine (SMZ); and, 3) statistically quantify any relationships between antibiotic resistance and attachment under different management practices (conventional and organic). E. coli isolates were enumerated from manure collected from six swine production facilities under two management systems—organic, with no antibiotics given and conventional, with antibiotics fed sub-therapeutically. Isolates were subjected to