BACILLALES INFLUENCE QUALITY AND SAFETY OF DAIRY PRODUCTS A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Sarah Marie Beno December 2017 © 2017 Sarah Marie Beno BACILLALES INFLUENCE QUALITY AND SAFETY OF DAIRY PRODUCTS Sarah Marie Beno, Ph. D. Cornell University 2017 Bacillales, an order of Gram-positive bacteria, are commonly isolated from dairy foods and at various points along the dairy value chain. Three families of Bacillales are analyzed in this work: (i) Listeriaceae (represented by Listeria monocytogenes), (ii) Paenibacillaceae (represented by Paenibacillus), and (iii) Bacillaceae (represented by the Bacillus cereus group). These families impact both food safety and food quality. Most Listeriaceae are non- pathogenic, but L. monocytogenes has one of the highest mortality rates of foodborne pathogens. Listeria spp. are often reported in food processing environments. Here, 4,430 environmental samples were collected from 9 small cheese-processing facilities and tested for Listeria and L. monocytogenes. Prevalence varied by processing facility, but across all facilities, 6.03 and 1.35% of samples were positive for L. monocytogenes and other Listeria spp., respectively. Each of these families contains strains capable of growth at refrigeration temperatures. To more broadly understand milk spoilage bacteria, genetic analyses were performed on 28 Paenibacillus and 23 B. cereus group isolates. While no specific genes were significantly associated with cold-growing Paenibacillus, the growth variation and vast genetic data introduced in this study provide a strong foundation for the development of detection strategies. Some species within the B. cereus group have previously been shown to grow at refrigeration temperatures, but the genetic analyses provided here will be of importance for the development of screening tools and to more successfully assess spoilage risk. Overall, the work presented here covers groups of bacteria that are common challenges for the dairy industry and provides large data sets to encourage additional research surrounding these topics. BIOGRAPHICAL SKETCH Sarah Marie Beno was born on January 17, 1991 in Marietta, Georgia as the oldest of three children to Paul and Kathleen Beno. Sarah attended Meredith College from 2009-2013 and graduated from the Honors Program with a B.S. in Biological Sciences and a B.A. in Chemistry. At Meredith College, Sarah participated in undergraduate research, where she studied the African legume, Tylosema esculentum. Throughout her coursework, she gained an interest in microbiology and decided to pursue food microbiology in graduate school. Sarah began a Ph.D. program in Food Science, Microbiology, and International Development following graduation. Her committee, Drs. Martin Wiedmann, Randy Worobo, and Ralph Christy, provided many opportunities to connect with industry members and to apply this knowledge internationally. Sarah spent three months working with the International Potato Center (Centro Internacional de la Papa; CIP) in Nairobi, Kenya to develop a food safety training program and laboratory that can be used in junction with the sweetpotato value chain. Other international experiences relevant to her doctoral work include a two-week trip looking at value addition in food processing throughout India, and a trip to Uppsala, Sweden to participate in the Global Challenges University Alliance summer program focused on food quality, food safety, and food security. Sarah will next begin an IRACDA-MERIT fellowship program at the University of Alabama-Birmingham where she will train in both biomedical research and teaching, in preparation for a career in academia. Outside of academia, Sarah enjoys horseback riding, volunteering at the local SPCA (from where she adopted her fluffy companion, Lister), and experiencing new places. iii To my family and friends for all of their love and support throughout this journey. iv ACKNOWLEDGMENTS I would like to thank Martin Wiedmann, my committee chair, advisor, and the principal investigator of the research presented here. Martin is challenging, yet generous, and I have benefitted extraordinarily from the lessons, support, and network that Martin shares. I also thank Kathryn Boor for always providing advice and encouragement. Kathryn is a true role model- hard working, successful, and kind. I would also like to thank my committee members, Randy Worobo and Ralph Christy, for their time, support, and useful connections (especially for my international trips). I owe many teaching and presenting opportunities, trips, and Taco Tuesdays to the Cornell Dairy Extension Team. The Dairy Extension Team has been instrumental in gaining teaching and extension opportunities throughout graduate school. I would especially like to thank Rob for helping me get started with the artisan cheese project, Kim for traveling with me frequently and teaching me about the world of auditing, and Louise for always seeking to improve our program. You have all been wonderful friends to me. Furthermore, the FSL/MQIP group has been a pleasure to work with. I would like to thank Maureen Gunderson for always honoring media requests and keeping the lab going. Thank you all for your friendship and for always working together. This lab is truly something special. Go Wiedmann Booriors! Of course, I must thank my family and my friends for all of the support. Thank you to my parents for always encouraging me and helping me get here! To Conor, for enduring another four years of long distance and for always offering your support. To my favorite housemates- Rachel Evanowski, Mary Kate Koch, Sam Hempel, Courtney Smith, and Claire Burtch, for bringing me laughter every day. Thank you to my friends (both human and furry) at If Only Farm and the Tompkins County SPCA for the happiness and love. A special thanks to my favorite fluffy gray friends, Jiminy and Lister, for always making me smile. v TABLE OF CONTENTS Biographical Sketch (iii) Dedication (iv) Acknowledgements (v) List of Figures (vii) List of Tables (viii) CHAPTER 1: Introduction (1) CHAPTER 2: Development and Validation of Pathogen Environmental Monitoring Programs for Small Cheese Processing Facilities (4) CHAPTER 3: Characterization of Paenibacillus odorifer Reveals Psychrotolerance is not Associated with Phylogeny (44) CHAPTER 4: A Number of Genes are Associated with Psychrotolerant Bacillus cereus Group Isolates (92) CHAPTER 5: Conclusions (134) APPENDIX A: Supplemental Material from Chapter 2 (137) APPENDIX B: Supplemental Material from Chapter 3 (138) APPENDIX C: Supplemental Material from Chapter 4 (191) vi LIST OF FIGURES 2.1 XbaI PFGE patterns for selected Salmonella isolates obtained from samples associated with Facility C (18) 3.1 Cladogram for 58 Paenibacillus isolates, built based on percentage average nucleotide identity by BLAST (61) 3.2 Growth patterns of 25 P. odorifer and 3 closely related isolates, grouped by Quality Threshold Clustering (67) 3.3 Phylogenetic tree constructed from the core SNPs of 25 P. odorifer and 3 closely related isolates with Quality Threshold Clustering data (71) 4.1 Growth patterns of 23 B. cereus group isolates, grouped by Quality Threshold Clustering (109) 4.2 Quality Threshold Clustering results for 23 B. cereus group isolates shown by QTC cluster (111) 4.3 Phylogenetic tree constructed from the core SNPs of 23 B. cereus group isolates with Quality Threshold Clustering data (114) vii LIST OF TABLES 2.1 Summary of facilities sampled (9) 2.2 Occurrence of different sigB allelic types for Listeria spp. isolates from each processing facility (20) 2.3 Routine and validation prevalence of Listeria spp. by facility (25) 2.4 Prevalence of PFGE patterns in Listeria monocytogenes isolates (31) 3.1 Detailed information on the 58 Paenibacillus isolates and 3 Viridibacillus isolates characterized by whole genome sequencing (57) 3.2 Categorization of 25 P. odorifer and 3 closely related isolates based on growth patterns at 6C in Skim Milk Broth (SMB) over 21 days (63) 3.3 Regression output from quantification of growth at 6 and 10°C in BHI and SMB for 28 Paenibacillus isolates (69) 4.1 Twenty-three B. cereus group isolates representing 22 different rpoB ATs were selected for cold growth quantification over 21 days at 6°C in SMB and BHI broth (100) 4.2 Categorization of 23 B. cereus group isolates based on growth patterns at 6°C in BHI and SMB over 21 days (107) viii CHAPTER 1 INTRODUCTION Bacillales, an order of Gram-positive bacteria, is commonly associated with concerns for both food safety and food quality. These organisms include Listeria monocytogenes, the Bacillus cereus group, and Paenibacillus. Paenibacillus and Bacillus are the two most influential genera for Gram-positive dairy spoilage (Ivy et al., 2012) and L. monocytogenes is a well-characterized foodborne pathogen. These three genera are important challenges for the dairy industry. In 2012, Ivy et al. characterized 1,288 spore-formers from dairy-associated sources and found that 68.7% of the Paenibacillus isolates represented the species P. odorifer. While P. odorifer is often able to grow at refrigeration temperature and can cause off-flavors and curdling in fluid milk (De Jonghe et al., 2010; Ranieri et al., 2012), the B. cereus group interestingly has ties to both food safety and food quality; certain members of the B. cereus group are associated with spoilage. Some strains of the B. cereus group are able to grow at low temperatures (Lechner et al., 1998; Miller et al., 2016), just like Paenibacillus (Moreno Switt et al., 2014) and L. monocytogenes (Chan and Wiedmann, 2009), while others have toxins that have led to diarrhetic and emetic disease (Bottone, 2010). Illnesses caused by bacteria in the Bacillus cereus group are typically short-lived and do not result in serious long-term injury (Ehling-Schulz et al., 2004). On the other hand, L. monocytogenes has one of the highest mortality rates of all foodborne disease (some estimates as high as 24%), even though it is opportunistic and generally only affects a small population (Farber and Peterkin, 1991).