University of Nevada, Reno Bacteriophage solutions as processing aids in beef production: An essential hurdle to reduce E. coli O157:H7 and the “Big Six” serotypes. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Animal and Rangeland Science by Erica L. Shebs Maurine Dr. Amilton de Mello/Thesis Advisor May 2020 Copyright by Erica L. Shebs Maurine 2020 All Rights Reserved THE GRADUATE• SCHOOL We recommend that the dissertation prepared under our supervision by entitled be accepted in partial fulfillment of the requirements for the degree of Advisor Committee Member Committee Member Committee Member Graduate School Representative David W. Zeh, Ph.D., Dean Graduate School i Abstract The research presented herein demonstrates the applications of bacteriophage solutions against pathogenic E. coli on fresh beef. The first chapter demonstrates the effects of a novel seven-bacteriophage (Mello-Shebs 1 phages) cocktail application against the seven USDA adulterant Shiga Toxin-producing E. coli (STEC) O157:H7 and the “Big Six” in vitro and on beef trim before grinding. The in vitro killing efficiency of each phage against the strain used for isolation ranged from 96.2% to 99.9%. On beef, inoculated and treated samples were held for 30 min or 6 h and at 7°C or 25°C before grinding to simulate refrigeration and ambient air temperatures. Both phage application (P≤0.0001) and temperature (P= 0.0451) had significant effects on STEC reduction. After 30 minutes of lysing time, phage application led to a significant 0.76 Log at 7°C and 0.81 Log reduction at 25°C. This study shows that the application of a seven-bacteriophage cocktail that target STEC O157:H7 and ‘Big Six’ serotypes are effective in reducing STEC on ground beef. The research presented in chapter two demonstrates the effects of a commercially available bacteriophage cocktail compared to lactic acid and peroxyacetic acid in reducing E. coli O157:H7 on fresh beef surface when kept under vacuum or aerobic conditions simulating a High Event Period, a period of high E. coli rates in beef trim. During which, beef subprimals are typically removed from vacuum and treated with antimicrobials, then re-packaged and later tested to verify the absence of STEC. Bacterial loads were evaluated 30 min and 6 h after antimicrobial applications. Under ii aerobic conditions for 30 min and 6 h, phage reduced STEC in beef by approximately 1.4 Log CFU/cm2 whereas organic acids resulted in approximately 0.5 Log CFU/cm2 reduction. Under vacuum conditions for 30 min, bacteriophage significantly reduced STEC by 1 Log CFU/cm2, while no significant effects were observed from organic acid treatment. Under vacuum conditions for 6 h, bacteriophage reduced STEC loads by 1.4 Log CFU/cm2, lactic acid reduced by 0.6 Log CFU/cm2, and no significant effects were observed with peroxyacetic acid application. The research presented in chapter three demonstrates the effects of multiple antimicrobial treatments against STEC O157:H7 and the “Big Six” on fresh beef surface: a novel seven-bacteriophage cocktail (P), peroxyacetic acid (PAA), ultraviolet light (UV), and acidified chlorite (ASC) each alone and in combinations of two under simulated High Event Period scenarios. All treatment combinations that included phage significantly decreased STEC populations (P<0.0001). Under vacuum conditions, Phage + PAA resulted in the lowest STEC load (1.49 Log CFU/cm2 reduction), followed by Phage, Phage + UV, Phage + ASC, Phage + PAA and UV + ASC, respectively. Under aerobic conditions, P + UV provided the greatest reduction (1.46 Log CFU/cm2), followed by Phage + PAA, P, Phage + ASC, UV+ASC, and PAA, respectively. When analyzing contrasts, treatments with phage significantly decreased STEC loads when compared to other treatments (P<0.0001) and control (P<0.0001). Overall, bacteriophage application has been demonstrated to have a high efficacy in reducing STEC loads on beef alone or with other antimicrobial treatments during a simulated industry application. iii Overall, bacteriophage applications targeting STEC provided an efficient control against all adulterant serotypes. It is clear that organic acids commonly used during meat processing are not efficient in controlling STEC. Bacteriophages emerged as an effective antimicrobial that can be applied in different processing steps and during different production scenarios. Currently, according to the United States Department of Agriculture (USDA), bacteriophage solutions allowed to be used in meat processing have Generally Recognized as Safe (GRAS) status only against O157:H7. This research showed that phages can also lower the “Big Six” serotypes. Therefore, when producing phage cocktails that successfully target the “Big Six”, biotechnology companies must request to the Food and Drug Administration (FDA) GRAS status for solutions not only targeting STEC O157:H7, but also the O145, O121, O111, O103, O45, and O26 serotypes. iv Acknowledgements I would like to express my deepest gratitude to those who have supported me through my educational and professional journey. First, thank you to my patient husband Drew for being by my side through this time. Thank you to my lab mates, Francine, Skye, Madison, and Edgar who have spent countless hours in the lab with me during the long experiment days. A special thank you to my advisor, Dr. Amilton de Mello. Thank you for your continuous guidance, you have truly helped me become a better professional and a better person! Also, thank you to my family and friends for all of your love and support. v Table of Contents Abstract ............................................................................................................................................ i Acknowledgements ........................................................................................................................ iv Table of Contents ............................................................................................................................. v List of Tables .................................................................................................................................. vii List of Figures ................................................................................................................................ viii List of Appendices .......................................................................................................................... ix Introduction ..................................................................................................................................... 1 Review of Literature ......................................................................................................................... 3 Foodborne diseases..................................................................................................................... 3 Escherichia coli ............................................................................................................................ 4 Routes of contamination ............................................................................................................ 7 Food Safety interventions ........................................................................................................... 9 Organic Acids ............................................................................................................................. 11 Acid Resistance .......................................................................................................................... 12 Ultraviolet Light ......................................................................................................................... 14 Bacteriophage (phage) .............................................................................................................. 14 Mechanism of Infection ............................................................................................................ 16 Phage Therapy as Biocontrol in Food ....................................................................................... 18 Conclusion ................................................................................................................................. 19 References ................................................................................................................................. 21 CHAPTER ONE ................................................................................................................................ 36 Abstract ..................................................................................................................................... 37 Introduction ............................................................................................................................... 38 Materials and methods ............................................................................................................. 40 Results ....................................................................................................................................... 43 Discussion .................................................................................................................................. 44 Conclusion ................................................................................................................................. 49 References ................................................................................................................................