1 AN ABSTRACT OF THE THESIS OF John Jorgensen for the degree of Master of Science in Food Science and Technology presented on September 13, 2019. Title: Prevalence and Characterization of Listeria spp. Recovered from Pacific Northwest Produce Handling and Processing Facilities. Abstract approved: _________________________________________________________________________________________________________ Joy Waite-Cusic Jovana Kovacevic Foodborne illness in the United States continues to be a complex and recurring issue despite our increased understanding of the pathogenic microorganisms responsible. Foodborne illness outbreaks and product recalls linked to pathogenic bacteria have been more frequent in the produce industry (e.g. fruits and vegetables) in the last ten years. Currently one of the most concerning foodborne bacterial pathogens in the produce industry is Listeria monocytogenes. This foodborne pathogen has been linked to multistate produce-associated outbreaks causing hundreds of illnesses and dozens of deaths. In several of these outbreaks, L. monocytogenes strains isolated from clinical patients were found to be persistent in produce handling and processing (PHP) facilities. This suggests that L. monocytogenes may contaminate product through cross-contamination events in PHP facilities and current food safety interventions in these environments may be inadequate to prevent transfer to food. Since the passage of the Food Safety and Modernization Act (FSMA) in 2011, States in the Pacific Northwest (PNW) that supply the U.S. with hundreds of specialty crops have been more focused on food safety. The produce industry in the PNW needs data and knowledge to effectively control L. monocytogenes in PHP facilities and comply with FSMA. The objective of this study was to investigate the prevalence of Listeria spp. in seven PHP facilities in Oregon and Washington through environmental monitoring on non-food contact surfaces only, with emphasis on the pathogenic species L. monocytogenes. The facility with the highest prevalence would receive additional and more intensive environmental sampling. A secondary objective was to characterize Listeria spp. strains recovered from PHP facilities and group potentially related strains. Characterization of strains was done through speciation, a multiplex PCR serogrouping assay, and antimicrobial resistance (AMR) profiling. A third objective was to track related strains throughout one facility and identify potential contamination sources. Environmental samples were collected from all PHP facilities at least twice (Rounds 1 and 2) from 2018-2019 and tested for Listeria spp. using a modified ISO 11290-1 method. Listeria spp. were not recovered from two PHP facilities (5/7, 70%). The prevalence of Listeria spp. through the first two rounds varied significantly across all PHP facilities and overall prevalence was relatively low (24/350, 6.9%). Additionally, L. monocytogenes was recovered in all PHP facilities positive for Listeria spp. One facility contributed >50% of the positive samples for the entire study. This facility minimally processes and packs raw produce only, does not have an environmental monitoring program and it is not subject to environmental monitoring regulations included in FSMA. Throughout the next rounds of sampling in only this facility (Rounds C and D) L. monocytogenes was more frequently recovered from environmental samples (26/100, 26%). A majority of L. monocytogenes strains were recovered from production room drains, foot traffic and forklift traffic entry points, samples taken outside the facility and in high traffic production floor areas. Characterization and tracking suggested that Listeria spp. are commonly brought into this facility on the bottom of employee shoes or forklifts and subsequently deposited throughout the facility. Serogrouping of L. monocytogenes strains showed that isolates from all facilities may be serotypes that are regularly associated with listeriosis foodborne illness outbreaks, serotypes 1/2a and 4b. AMR profiling, though, indicated that all recovered Listeria spp. strains were sensitive to antibiotics commonly used in the treatment of foodborne listeriosis. Collectively, our data suggest that there is an increased risk of environmental contamination for PHP facilities that function as packinghouses and handle multiple types of raw produce, though additional studies including diverse PNW PHF facilities are needed to support this hypothesis. Antibiotic resistance in L. monocytogenes food chain isolates should be continuously monitored, including further genomic characterization of isolates to better understand overall strain relatedness, pathogenicity and AMR potential. ©Copyright by John Jorgensen September 13, 2019 All Rights Reserved Prevalence and Characterization of Listeria spp. Recovered from Pacific Northwest Produce Handling and Processing Facilities by John Jorgensen A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented September 13, 2019 Commencement June 2020 Master of Science thesis of John Jorgensen presented on September 13, 2019 APPROVED: _________________________________________________________________________________________________________ Co-Major Professor, representing Food Science and Technology _________________________________________________________________________________________________________ Co-Major Professor, representing Food Science and Technology _________________________________________________________________________________________________________ Head of the Department of Food Science and Technology _________________________________________________________________________________________________________ Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. _________________________________________________________________________________________________________ John Jorgensen, Author ACKNOWLEDGEMENTS I am very grateful for the opportunity to be have been a graduate student and research assistant at Oregon Student University. I can’t thank the University and FST Department enough for the opportunity to further my education, as well as the Oregon Department of Agriculture and United States Department of Agriculture for funding my research, covering my tuition and living stipend. To my advisors, Dr. Joy Waite-Cusic and Dr. Jovana Kovacevic, thank you so much for the expertise, mentorship and guidance that you provided me with over the last two years. I appreciate everything and cannot thank you enough. You have helped me grow as a student of food safety and microbiology and I hope to mimic the professionalism each of you presented yourself with each day. Thank you to my lab mate Cara Boucher for all you help and keeping me motivated through your consistent hard work. To my former lab mates, peers and interns; Danton Batty, Alex Emch, Tony Paris, Quintin Ferris, Sebastian Ramirez, Jj Stull and Clara Szalay, thank you so much for your help and support. I am so appreciative of my parents, Jim and Michel Ruud, for their support through my academic career. I would not be here today without everything they have done for me throughout my entire life, including their extended support throughout these last two years. I also thank God for giving me strength through all those long nights and early mornings. Finally, and most importantly, thank you to my wife Kelsey Swieca. You have been there for me every moment and were always willing to drop everything to help me with my research and thesis. I could not have done this without you and love you dearly. TABLE OF CONTENTS Page Chapter 1: Literature Review ........................................................................................................ 1 1.1 Summary of situation ................................................................................................................ 1 1.2 Foodborne illness linked to produce ................................................................................. 3 1.3 Listeria spp. and listeriosis ..................................................................................................... 4 1.4 Characterization of Listeria spp. .......................................................................................... 5 1.4.1 Serotyping and lineages ............................................................................................. 6 1.4.2 Molecular characterization techniques for Listeria spp. .............................. 8 1.4.3 Antimicrobial resistance ............................................................................................ 9 1.5 Food safety outbreaks and recalls .................................................................................... 10 1.5.1 Overview ........................................................................................................................ 10 1.5.2 Listeriosis outbreaks and Listeria spp. recalls in produce ....................... 11 1.6 Listeria spp. prevalence in produce associated environments ............................ 18 1.7 Transient and persistent strains ....................................................................................... 19 1.8 Environmental monitoring: guidance and control of Listeria spp. .................... 20 1.9 Summary of research approach .......................................................................................