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Mitigation of Antimicrobial Drug Resistance and Changes in The Mitigation of Antimicrobial Drug Resistance and Changes in the Microbiome of Feedlot Cattle Supplemented with Lactobacillus salivarius L28 as an Alternative to Sub-Therapeutic Antibiotics by Andrea R. English, M.S. A Dissertation In Animal Science Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Approved Dr. Marcos X. Sánchez-Plata Co-Chair of Committee Dr. Mindy M. Brashears Co-Chair of Committee Dr. Kendra K. Nightingale Dr. Alejandro Echeverry Dr. Jessie Vipham Dr. Mark F. Miller Mark Sheridan Dean of the Graduate School December, 2019 Copyright 2019, Andrea English Texas Tech University, Andrea English, December 2019 ACKNOWLEDGMENTS I would like to first thank my advisor Dr. Mindy Brashears you took a chance on me and I am forever grateful. I am so thankful that I was able to learn and grow as a scientist under your guidance. You have provided me with experiences I will always remember. Thank you for everything you have done for me while at Texas Tech. Thank you to my committee, Dr. Sanchez you have been an incredible mentor and I have truly valued absorbing as much knowledge as I possibly could from you. Dr. Nightingale thank you for investing in me and this project. You pushed me to be a better scientist which I very much appreciate. Dr. Echeverry and Dr. Vipham I cannot say thank you enough for the support you have provided me. You each have always made time for me and that is invaluable. Dr. Miller thank you for continuously providing me with opportunities to grow, learn and dabble in meat science while here at Texas Tech. A very special thank you is warranted to Dr. Handley from Washington University School of Medicine your support and assistance with data analysis and interpretation of the microbiome work is greatly appreciated. Thank you to the graduate students that helped with the completion of this project specifically Keelyn Hanlon, Hector Garnica you both were there for nearly every fecal collection, streaked plate, sensititre run and swab collection. I could not have done it without you both. Diana Ayala your guidance, friendship and assistance during my time in Lubbock is unmeasurable (the cytokine analysis would not have been done without you). I am extremely thankful for you each. ii Texas Tech University, Andrea English, December 2019 A huge thank you to my family. Mom and Dad you both have been my main support system. You have never questioned my goals and have only encouraged me to reach even further. Most importantly thank you for your unconditional love and always reminding me that “I can do all things through Christ who strengthens me” Philippians 4:13. iii Texas Tech University, Andrea English, December 2019 TABLE OF CONTENTS ACKNOWLEDGMENTS .......................................................................................... ii ABSTRACT ................................................................................................................. vi LIST OF TABLES ................................................................................................... viii LIST OF FIGURES ..................................................................................................... x 1. REVIEW OF LITERATURE ................................................................................. 1 Introduction .............................................................................................................. 1 Antimicrobial Drugs Mode of Action ...................................................................... 3 Cell Wall synthesis ............................................................................................. 3 Protein synthesis ................................................................................................ 4 Nucleic Acid metabolism ................................................................................... 5 Membrane structure ........................................................................................... 6 Antimicrobial Resistant Bacteria ............................................................................. 6 Enterococcus spp. ............................................................................................ 10 Salmonella and Escherichia coli ..................................................................... 19 Antibiotic Use and Resistance in Beef Production ................................................ 26 Background ...................................................................................................... 26 Antimicrobial Resistance in Beef Production .................................................. 29 Microbial Diversity in Feedlot Cattle Feces ................................................... 37 Alternatives to Antimicrobial Growth Promoters ............................................ 39 Direct-fed Microbials ............................................................................................. 42 Background ...................................................................................................... 42 Lactic Acid Bacteria ........................................................................................ 44 Direct-fed Microbials in Cattle and Controlling Pathogen Shedding ............. 45 Direct-fed Microbials and Cattle Health ......................................................... 48 Conclusion and Objectives .................................................................................... 52 2. DETECTION OF ESCHERICHIA COLI O157 AND SALMONELLA FROM FEEDLOT CATTLE FECES AND FROM CARCASSES SUPPLEMENTED WITH LACTOBACILLUS SALIVARIUS L28 AS A DIRECT-FED MICROBIAL ................................................................................... 54 Abstract .................................................................................................................. 54 Highlights .............................................................................................................. 56 Introduction ............................................................................................................ 57 Material and Methods ............................................................................................ 59 iv Texas Tech University, Andrea English, December 2019 Results and Discussion .......................................................................................... 65 E. coli O157 Detection. ................................................................................... 65 Salmonella Detection. ...................................................................................... 67 Postharvest. ..................................................................................................... 70 Conclusion ............................................................................................................. 71 3. ANTIMICROBIAL RESISTANCE PATTERNS AND MICROBIOME CHANGES IN FEEDLOT CATTLE SUPPLEMENTED WITH LACTOBACILLUS SALIVARIUS L28 ...................... 77 Abstract .................................................................................................................. 77 Highlights .............................................................................................................. 79 Introduction ............................................................................................................ 80 Material and Methods ............................................................................................ 84 Results and Discussion .......................................................................................... 92 Generic E. coli ................................................................................................. 92 Enterococcus .................................................................................................... 96 E. coli O157 ................................................................................................... 100 Salmonella ..................................................................................................... 102 Microbiome .................................................................................................... 104 Conclusion ........................................................................................................... 107 REFERENCES ........................................................................................................ 135 APPENDIX A ........................................................................................................... 154 Sensitire™ Plate schematics ................................................................................. 154 v Texas Tech University, Andrea English, December 2019 ABSTRACT The objectives of this study were to evaluate the efficacy of utilizing L. salivarius L28 as a direct-fed microbial in feedlot cattle to 1) reduce the frequency of pathogen shedding, 2) determine the AMR in commensal and pathogenic bacteria and investigate the microbiome and cytokine changes. A total of three dietary treatments (control, base and monpro) based on conventional high concentrate diets were fed to finish cattle (n=144) for harvest. Fecal samples were collected every 28 days until harvest at 140 days. Traditional culture methods were used to isolate bacteria. Antibiotic susceptibility testing was done using the micro-broth dilution (Sensititre™) susceptibility minimum inhibitory concentration plates, following the National Antimicrobial Resistance
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