Project Number: BC-TAC-M100

Project Number: BC-TAC-M100 PHAGE BIOCONTROL OF E. COLI O157:H7 ON PROCESSED MEAT A Major Qualifying Project Report: submitted to the Faculty of the WORCESTER POLYTECHNIC INSTITUTE in partial fulfillment of the requirements for the Degree of Bachelor of Science by _________________________ Nathalia Arenas _________________________ Katelyn M. Ryan Date: April 22, 2008 Approved: _____________________________ Professor Terri Camesano, Advisor _____________________________ Professor William Hobey, Co-Advisor ABSTRACT Foodborne Escherichia coli (E. coli) outbreaks have been a continual public health concern for decades, costing the U.S. $405.2 million annually, in premature deaths, medical care, and lost productivity [3]. Antibiotics have been a common defense against bacteria; however, increase findings of antibiotic-resistant bacteria have lead to research of bacteriophage as a method to reduce foodborne illnesses. This research project investigated the effectiveness of phage for controlling the propagation of artificially contaminated E. coli O157:H7 meat samples. The samples were inoculated by dipping with an E. coli concentration of ~108CFU/ml of to achieve a bacterial coverage of ~103CFU/cm2. Following the bacterial application, the meat samples were inoculated with pp01 via dipping using a phage concentration of ~2- 6X108PFU/ml. The samples were evaluated after various incubation conditions: 0h, 0.5h, 2h, 24h, 48h, and 72h. Two replicate trials were performed; the only difference being that in Trial 1 a non-filtered phage stock was used, while in Trial 2 a phage stock that was filtered with 0.45μm filter was used. Of the two trials performed, the results displayed that the highest reduction of E. coli after phage application occurred at 24h with ~2.7X105CFU/cm2. ii EXECUTIVE SUMMARY E. coli is a gram-negative bacterium found in the intestinal tract of warm blooded animals. Besides causing 60 deaths per year in the U.S., these resistant organisms can survive on counter tops for several weeks and require an extremely small dose of bacterial colonies for infection. In an effort to reduce foodborne illnesses, antibiotics and antiseptic agents are applied directly to foods and to machinery in contact with foods at packaging plants to allow for control of bacterial growth. These methods have posed problems in that mutant bacteria have acquired resistance to such treatments and in that some of these treatments change the taste, smell, and appearance of the foods. Unlike antibiotics and antiseptic agents, the introduction and distribution of phage within these foods can be viewed as a natural process. Phages are observed as natural enemies of bacteria and are commonly found in natural settings where bacteria live. Phage are the most abundant self-replicating units in our environment and are present in significant numbers in water and foods of various origins, and nonfood sources such as: feces, soil, sewage, farms and processing facility effluents. Previous research has shown promising results in the intervention of phage to control E. coli. Phage strategies for food preservation have the advantages of being self-perpetuating, highly discriminatory, natural, and cost- effective. Common negative critiques of phage use are its limited host range, the requirement for threshold numbers of bacterial targets, and phage resistant mutant development. This project focuses on determining the effectiveness of phage application as a method to control propagation of artificially contaminated meat samples with E. coli iii O157:H7. In doing this, an experimental protocol was designed to test for the CFU/cm2 (Colony Forming Units) reduction of E. coli O157:H7 strain EDL933 on processed meat samples after 0h, 0.5h, 2h, 24h, 48h, and 72h treatments with phage pp01. A multiplicity of infection (MOI) of 100 was established as a constant parameter for the phage-to-bacteria ratio. Two replicate trials were performed; the only difference being that in Trial 1 a non-filtered phage stock was used, while in Trial 2 a phage stock that was filtered with 0.45μm filter was used The stock phage concentration obtained for Trial 1 was of 6.4X108PFU/ml (Phage Forming Units) and of 1.96X108PFU/ml for Trial 2. With respect to the phage concentration used, the bacterial concentration was adjusted to ~108CFU/ml in order to achieve a bacterial coverage of ~103CFU/cm2 in each trial. This bacterial coverage corresponded to an MOI of 100 on the meat samples. The meat samples were cut, inoculated with bacteria by dipping, inoculated with either phage or control media by dipping, incubated at 22oC for each time condition, stomached, and plated to obtain the number of CFU. From the two trials performed, the highest bacterial reduction on meat samples occurred after 24h of treatment with pp01. For the 24h condition, Trial 1 showed a bacterial reduction of ~3X105CFU/cm2 and Trial 2 showed a bacterial reduction of ~1X105CFU/cm2 (p0.0036 for both trials). In addition, there was no statistical difference in the reduction of E. coli at 0h from 0.5h, suggesting the exclusion of the 0.5h condition for future research. No data was obtained for incubating the meat samples with bacteria and phage for 48h and 72h as contamination arose from the meat samples themselves. For future research we suggest conduction of this experimental protocol with an MOI>10 or <100, to test for incubation times between 2h-24h and 24h-48h, and to raise the initial bacterial coverage from ~103CFU/cm2 to iv ~105CFU/cm2, in order to determine the conditions at which the most bacterial reduction can be obtained. v ACKNOWLEDGEMENTS We thank Dr. Terri Camesano (Chemical Engineering, WPI), Dr. William Hobey (Chemistry and Biochemistry, WPI), Dr. Nathalie Tufenkji (Chemical Engineering, McGill University), Michael Coussa (Chemical Engineering, McGill University), Charles Poitras (Chemical Engineering, McGill University), and Joshua Strauss (Chemical Engineering, WPI) for their consistent support and guidance. vi AUTHORSHIP The completion of this project was done equally by Nathalia Arenas and Katelyn M. Ryan. vii TABLE OF CONTENTS ABSTRACT ...................................................................................................................ii EXECUTIVE SUMMARY ......................................................................................... iii ACKNOWLEDGEMENTS .......................................................................................... vi AUTHORSHIP ............................................................................................................vii TABLE OF CONTENTS ........................................................................................... viii TABLE OF FIGURES .................................................................................................. ix LIST OF TABLES ......................................................................................................... x CHAPTER 1: INTRODUCTION AND BACKGROUND ........................................... 1 1.1 Bacterial Food Poisoning ..................................................................................... 1 1.2 Escherichia coli O157:H7 .................................................................................... 2 1.3 Bacteriophage Control of Foodborne Bacteria .................................................... 4 1.4 Bacteriophage Control of E. coli O157:H7 .......................................................... 9 CHAPTER 2: METHODOLOGY ............................................................................... 10 2.1 Bacteria Strains and Inoculums ......................................................................... 10 2.2 Bacteriophage Propagation and Isolation .......................................................... 12 2.3 Experimental Protocol ....................................................................................... 13 2.5 Experimental Parameters ................................................................................... 16 CHAPTER 3: RESULTS ............................................................................................. 19 3.1 Trial 1: pp01#1 ................................................................................................... 19 3.2 Trial 2: pp01#2 ................................................................................................... 21 3.3 Trial 3: Baseline with pp01#1 and pp01#2 ........................................................ 23 CHAPTER 4: DISCUSSION ....................................................................................... 24 CHAPTER 5: RECOMMENDATIONS AND FUTURE RESEARCH ..................... 29 BIBLIOGRAPHY ........................................................................................................ 31 APPENDIX I: PROTOCOLS ...................................................................................... 32 1.1 Media Preparation .............................................................................................. 32 1.2 Agar Preparation ................................................................................................ 32 1.3 Peptone Water Preparation ................................................................................ 33 1.4 E. coli O157:H7 Inoculum Preparation ............................................................. 33 1.6 Phage Propagation ............................................................................................. 34 1.6 Phage Titer for PFU/ml Calculation .................................................................

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