Fate of Escherichia Coli O157

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Fate of Escherichia Coli O157 h7, Listeria Monocytogenes, and Salmonella Spp. on Fresh-Cut Celery
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1 online resource (108 p.)
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english
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Vandamm,Joshua P
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Degree:
Master's ( M.S.)
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University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Danyluk, Michelle D.
Committee Members:
Goodrich, Renee M
Sargent, Steven A

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Subjects / Keywords:
bag -- behavior -- celery -- coli -- container -- cut -- escherichia -- food -- fresh -- listeria -- microbiology -- monocytogenes -- salmonella -- surface -- temperature
Food Science and Human Nutrition -- Dissertations, Academic -- UF
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Food Science and Human Nutrition thesis, M.S.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Electronic Thesis or Dissertation

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Abstract:
Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella infections have been associated with the consumption of numerous fresh produce items. In 2010, an outbreak of listeriosis was traced back to fresh-cut celery, with contamination believed to have occurred during processing. While no E. coli O157:H7 or salmonellosis infections have been associated with celery, both pathogens have been isolated from celery sold at market. Little is known about the fate of these pathogens on celery, or the effect of consumer handling practices on the potential risk. The objective of this study was to determine the fate of E. coli O157:H7, L. monocytogenes, and Salmonella on fresh-cut celery at refrigeration (4 degrees C), abuse (12 degrees C), and ambient temperatures (22 degrees C) under different storage conditions. Fresh-cut celery samples were spot inoculated at 3 log CFU/g with a cocktail of one of the pathogens onto either cut or uncut surfaces and held at 4 ? 2 degrees C, 12 ? 2 degrees C, or 22 ? 2 degrees C, and kept in either sealed bags or closed containers. Samples were enumerated following stomaching on selective and nonselective media after storage for 0, 1, 3, 5 and 7 days (when held at 4 ? 2 degrees C and 12 ? 2 degrees C), and after 0, 0.33, 0.71, 1, and 2 days (when held at 22 ? 2 degrees C). At 4 degrees C, each of the three pathogens populations declined by ca. 0.5-1.0 log CFU/g over 7 days. At 12 degrees C, E. coli O157:H7 and Salmonella populations did not change, while L. monocytogenes populations increased over 7 days by ca. 0.5 log CFU/g. At 22 degrees C, E. coli O157:H7 populations increased by ca. 1 log CFU/g, Salmonella populations increased by ca. 1.5 to 2.0 log CFU/g, and L. monocytogenes by ca. 0.3 log CFU/g, with the majority of growth in all three pathogens occurring after the first 0.71 days. Significant differences were observed between samples inoculated on cut versus uncut surfaces, but not between container types. This work indicates that E. coli O157:H7, L. monocytogenes and Salmonella spp. can grow on improperly stored fresh-cut celery, and demonstrates the importance of cooling and maintaining proper refrigeration during the distribution and handling of fresh-cut celery.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Joshua P Vandamm.
Thesis:
Thesis (M.S.)--University of Florida, 2011.
Local:
Adviser: Danyluk, Michelle D.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-02-29

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1 FATE OF ESCHER I CHIA COLI O157:H7, LISTERIA MONOCYTOGENES AND SALMONELLA SPP. ON FRESH CUT CELERY By JOSHUA PETER VANDAMM A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 Joshua Peter Vandamm

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3 To my family for being the source of inspiration and pride for me, and for supporting me throughout my academic life. And to my grandfather, Dr. Michael J. Begab, whose example demonstrated just how rich and fulfilling a career in science could be.

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4 ACKNOWLEDGMENTS I would like to express my sincere thanks to my instructor, advisor, and mentor Dr. Michelle D. Danyluk. I have learned a tremendous amount from Dr. Danyluk and her incredible research team. Dr. Danyluk is an excellent mentor and continues to inspire an d amaze me with her insights, depth of knowledge, and professional productivity. I have matured greatly as a scientist under her tutelage, and owe her a debt of gratitude for taking me on as a student. I thank my supervisory committee members: Dr. Ren e M. Goodrich Schneider and Dr. Steve Sargent. Thank you both for lending your time, expertise, and insights, which led me to explore my research in different ways and were essential to the success of this project. Thank you to Dr. Goodrich for teaching me so much about food s afety and helping to cultivate my interests and career aspirations. I am grateful to Dr. Keith Schneider for giving me a place to work in his lab during my time in Gainesville. Dr. Schneider has been an inspirational example for the positive academic, social, and economic impact that extension work can bring. His insights i nto the realities faced by the food i ndustry have been enormously helpful. Additionally, I would like to give special thanks to Dr. Jesse Gregory for his in struction in food chemistry. I learned more from Dr. Gregory during a single course than I previously thought possible. My research would not have been possible without the help and resources of the Citrus Research and Education Center (CREC). I woul d like to give special thanks to everyone in my lab for their instruction, technical support, and assistance: Lorrie Freidrich, Pardeepinder Brar, Rachel McEgan, Gwen Lundy, and Luis Martinez. They are an amazing group of people and it has been a pleasure working with them I have learned a great deal of laboratory skills them and they all have my sincere gratitude.

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5 TABLE OF CONTENTS ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 7 ABSTRACT ................................ ................................ ................................ ................................ ..... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 11 2 LITERATURE REVIEW ................................ ................................ ................................ ....... 15 Celery ................................ ................................ ................................ ................................ ..... 15 Background ................................ ................................ ................................ ..................... 15 Nutritional Profile ................................ ................................ ................................ ........... 15 Production ................................ ................................ ................................ ....................... 16 Harvest and Processing ................................ ................................ ................................ ... 18 Marketing of Celery in the United States ................................ ................................ ....... 19 Economic Impact ................................ ................................ ................................ ............ 20 Price of a Recall to Industry ................................ ................................ ........................... 21 Biological Hazards Associated with Fresh Produce ................................ ....................... 22 Sources of Contamination on Fresh Produce ................................ ................................ 22 High Risk Consumer Handling Behaviors Associated with Fresh Produce .................. 23 In Home Hazard Intervention Strategies ................................ ................................ ........ 25 Escherichia coli ................................ ................................ ................................ ..................... 26 Biology of Escherichia coli ................................ ................................ ............................ 26 Nomenclature and Classification of Pathogenic Escherichia coli ................................ 28 Clinical Features of EHECs ................................ ................................ ............................ 31 Virulence Factors and Pathogenesis of EHECs ................................ .............................. 33 Epidemiology of Escherichia coli O157:H7; Focus on Fresh Produce ......................... 34 Ecological Dissemination and Spread of EHECs ................................ ........................... 35 Listeria monocytogenes ................................ ................................ ................................ ......... 36 Biology of Listeria monocytogenes ................................ ................................ ................ 36 Phylogenics and Subtyping of Listeria monocytogenes ................................ ................. 39 Clinical Features of Listeriosis ................................ ................................ ....................... 40 Pathogenesis of Listeria monocytogenes ................................ ................................ ........ 42 Epidemiology of Listeriosis ................................ ................................ ........................... 43 Listeriosis Outbreaks Associated with Fresh Produce ................................ ................... 44 Reservoirs of Listeria monocytogenes ................................ ................................ ............ 45 Human Carriage of Listeria monocytogenes ................................ ................................ .. 46 Listeria monocytogenes in Food ................................ ................................ ..................... 47 Listeria monocytogenes in Celery and Other Fresh Produce ................................ ......... 49 Listeria monocytogenes, Industrial Control Points ................................ ........................ 50 Food Regulations for Listeria monocytogenes ................................ ............................... 51 Salmonella ................................ ................................ ................................ ............................. 52

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6 Biology of Salmonella ................................ ................................ ................................ .... 52 Salmon ella Pathogenesis ................................ ................................ ................................ 54 Genetic Determinants of Virulence in Salmonella Spp. ................................ ................. 56 Etiology and Clinical Features of Human Salmonellosis: Nontyphi Serotypes ............. 57 Epidemiology of Salmonellosis Associated with Produce ................................ ............. 59 3 MATERIALS AND METHODS ................................ ................................ ........................... 61 Strain Information ................................ ................................ ................................ .................. 61 Preliminary Experiments ................................ ................................ ................................ ....... 62 Celery ................................ ................................ ................................ ................................ ..... 62 Antibiotic and Growth Medium Preparation ................................ ................................ ......... 63 Inoculum Preparation ................................ ................................ ................................ ............. 63 Inoculation and Storage ................................ ................................ ................................ ......... 64 Enumeration ................................ ................................ ................................ ........................... 64 Statistics ................................ ................................ ................................ ................................ 65 4 RESULTS ................................ ................................ ................................ ............................... 67 Background Microflora ................................ ................................ ................................ ......... 67 Escher ichia coli O157:H7 ................................ ................................ ................................ ..... 67 Listeria monocytogenes ................................ ................................ ................................ ......... 68 Salmonella ................................ ................................ ................................ ............................. 69 5 DISCUSSION ................................ ................................ ................................ ......................... 76 6 CONCLUSIONS AN D FUTURE WORK ................................ ................................ ............. 83 LIST OF REFERENCES ................................ ................................ ................................ ............... 86 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 108

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7 LIST OF TABLES Table Page 3 1 Escherichia coli O157:H7 serotypes used, confirmed original source of isolate, original isolate designation, and laboratory identification code. ................................ ....... 66 3 2 Listeria monocytogenes strain used, confirmed original source of isolate, original isolate designation, and laboratory identification code. ................................ ..................... 66 3 3 Salmonella serovar used, confirmed original source of isolate, original isolate designation, and laboratory identification code. ................................ ................................ 66 4 1 Recovery of Escherichia coli O157:H7 from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 4 2C for up to 7 days. ................................ ................................ ................................ ....... 71 4 2 Recovery of Escherichia coli O157:H7 from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 12 2C for up to 7 days ................................ ................................ ................................ ........ 71 4 3 Recovery of Escherichi a coli O157:H7 from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 22 2C for up to 2 days. ................................ ................................ ................................ ....... 72 4 4 Recovery of Listeria monocytogenes from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 4 2C for up to 7 days ................................ ................................ ................................ ............ 72 4 5 Recovery of Listeria monocytogenes from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and store d in either a bag or container held at 12 2C for up to 7 days. ................................ ................................ ................................ ........... 73 4 6 Recovery of Listeria monocytogenes from celery inoculate d at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 22 2C for up to 2 days. ................................ ................................ ................................ ........... 73 4 7 Recovery of Salmonella from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 4 2C for up to 7 days ................................ ................................ ................................ ................................ .... 74 4 8 Recovery of Salmonella from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 12 2C for up to 7 days. ................................ ................................ ................................ ................................ 74

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8 4 9 Recovery of Salmonella from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 22 2C for up to 2 days. ................................ ................................ ................................ ................................ 75

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9 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Masters of Science FATE OF ESCHER I CHIA COLI O157:H7, LISTERIA MONOCYTOGENES AND SALMONELLA SPP. ON FRESH CUT CELERY By Joshua Peter Vandamm August 2011 Chair: Michelle D. Danyluk Major: Food Science and Human Nutrition Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella infection s have been associated with the consump tion of numerous fresh produce items. In 2010, an outbreak of listeriosis was traced back to fresh cut celery, with contamination believed to have occurred d uring processing. While no E. coli O157:H7 or salmonellosis infections have been associated with celery, both pathogens have been isolated from celery sold at market. Little is known about the fate of these pathogens on celery, or the effect of consumer handling practices on the potential risk The objective of this study was to determine the fate o f E. coli O157:H7, L. monocytogenes and Salmonella on fresh cut celery at refrigeration (4C), abuse (12C), and ambient temperatures (22C) under different storage conditions. Fresh cut celery samples were spot inoculated at 3 log CFU/g with a cocktail of one of the pathogens onto either cut or uncut surfaces and held at 4 2C, 12 2C, or 22 2C, and kept in either sealed bags or closed containers. Samples were enumerated following stomaching on selective and nonselective media after storage for 0 1, 3, 5 and 7 days (when held at 4 2C and 12 2C), and after 0, 0.33, 0.71, 1, and 2 days (when held at 22 2C). At 4C, each of the three pathogens populations declined by ca 0.5 1.0 log CFU/g over 7 days At 12C, E. coli O157:H7 and Salmonella populations did not change, while L. monocytogenes populations increase d over 7 days by ca. 0.5

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10 log CFU/g. At 22C E. coli O157:H7 populations increased by ca. 1 log CFU/g, Salmonella populations increased by ca. 1.5 to 2.0 log CFU/g, and L. m onocytogenes by ca. 0.3 log CFU/g, with the majority of growth in all three pathogens occurring after the first 0.71 days. Significant differences were observed between samples inoculated on cut versus uncut surfaces, but not between container types. Thi s work indicates that E. coli O157:H7, L. monocytogenes and Salmonella spp. can grow on improperly stored fresh cut celery, and demonstrates the importance of cooling and maintaining proper refrigeration during the distribution and handling of fresh cut ce lery.

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11 CHAPTER 1 INTRODUCTION Americans have substantially increased their consumption of fresh produce. The U.S. Department of Agriculture (USDA) (2008) reported that between 1970 and 2008, the U.S. per capita consumption of fresh vegetables increased f rom 49 to 82 kg (approximately 67%). Data from the Produc e Marketing Association (PMA) (2007) indicates retail sales of fresh produce increased from 32.5 to $56.3 billion between 1998 and 2006. In 2006, U.S. domestic celery sales grossed $920 million, 1. 6% of the gross profit from produce sales (PMA, 2007) The U.S. is a net exporter of celery, shipping more the 2.5 million 100 lb. crates overseas in 2009 ( Huntrods, 2010; Lucier and Jerardo, 2005) The U.S. also imports celery, especially during the winter months. In 2009, ca. 21,204 MT of fresh celery was imported from producers in Mexico (Huntrods, 2010) In 1997, the 12 existing celery farms in Florida produced 4,114 acres of celery, the second largest crop in the U.S., after California (USDA, 1999) In 1992, the last year in which data was coll ected, Florida produced 315.4 million lbs of celery, with an average yield of 41,500 lbs. per acre. The total value of the 1992 harvest was $39.1 million (USDA, 1999) Although not a major plate vegetable, versatility in both fresh and cooked forms, utility as a food ingredient and nutritional properties have made celery a staple in the produce a isle. During 2009, U.S. per capita consumption of fresh celery was 6.1 lbs. (USDA, 2011 ; Huntrods, 2010) The introduction of prepackaged fresh cut products, including celery, over the past two decades has likely helped increase consumption of celery (Lucier and Jerardo, 2005) Celery is comm only consumed in two manners, either as an ingredient or as raw snack. Nearly the entire Florida crop of celery is destined for fresh market as full stalks, celery hearts, or fresh cut

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12 (USDA, 1999) The value added segment of fresh cut celery sticks is gaining in popularity and there is a growing market for new washed and ready to eat products (Freedonia Group, 2010) Fruits and vegetables have gained notoriety in recent years as a vehicle of human disease (D'Aoust and Maurer, 2007) The U.S. Centers for Disease Control (CDC) estimates that in the 1990s, at least 12% of foodborne d isease outbreaks were associated with the consumption of fresh produce (FDA, 2004) The raw nature of fresh cut produce items contributes to its potential risk. Although the incidence of foodborne illness associated with the fresh produce is low in comparison to the total volume consumed per capita in the U.S., recent increases in consumption have coincided with an increase in reported outbreaks associated with their consumption (CAST, 2009) Because most vegetable crops are cultivated in an open, natural environment, they are vulnerable to contamination. Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. are among the top 6 disease causing foodborne pathogens (CDC, 2011) and are found throughout the natural environment (Austin, 1991) Contamination can occur at any point in the supply chain from production through processing and distrib ution to preparation in the home or food service kitchen. Major processing cross contamination points include cutting, trimming, washing, and cooling of produce (Doyle and Erickson, 2008) In the consumer kitchen, specific sources of produce contamination are often more difficult to trace, but are primarily believed to be the result of surface transfer from contact with contaminated meat or from improperly sanitized cutting utensils and cleaning s urfaces, or contaminated wash water (Chai et al., 2008; Chen et al., 2001; De Wit et al., 1979; Doyle and Erickson, 2008; Kusumaningrum et al., 2003) It is important to note that consumer refrigerators differ in the ir ability to maintain a constant and uniform temperature. In a recent study of 200 home refrigerators, average temperatures fluctuated

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13 between the top shelf (1.9C), bottom shelf (3.3C), and door (5.2C) (Godwin, 2007) Tempe ratures also rose above the danger zone (4C) for over 2 h per day in the top shelf (in 33%), middle shelf (in 45%), and door (in 80% of refrigerators). Between 1973 and 1997, 190 produce associated outbreaks involving 16,058 illnesses and 8 deaths were reported to the U.S. CDC (Sivapalasingam et al., 2004) Celery has been associated with three outbreaks of foodborne disease. The first was an outbreak of Typhoid fever in 1899, at a Massachusetts asylum which afflicted 40 inmates over two weeks (Morse, 1899) In 1991, a point source outbreak of acute gastroenteritis at the U.S. Air Force Academy resulting from the consumption of chicken salad afflicted approximately 1,4 40 cadets, 105 of which required intravenous rehydration. The outbreak was originally attributed to Salmonella however norovirus was eventually identified as the cause, due to the exposure of the celery component to non potable water (Warner et al., 1991) Most recently in October, 2010, chopped celery was the confirmed cause of a listeriosis outbreak in Texas that sickened 6 to 10 people, 4 to 5 of whom died as a result ( FDA, 2 010 ) To our knowledge, no studies have explored the fate of E. coli O157:H7 on celery. No E. coli O157:H7 outbreaks have been associated with fresh cut celery; however, it has been isolated from 23 of 89 (27%) celery samples sold in Mexican markets (Zepeda Lopez et al., 1995) Listeria monocytogenes has been isolated from celery in several market surveys, and grows on asparagus, broccoli, and cauliflower stored at 4 C (Besser et al., 1999) and on lettuce at 5C (Beuchat and Brackett, 1990; Steinbruegge et al., 1988) In one survey on the microbial safety of fresh cut RTE celery and other vegetables, L. monocytogenes was detected on 8 of 120 (6.7%) fresh cut celery samples stored at 10C, but n ot (0 of 175 samples) on produce stored at

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14 4C (Odumeru et al., 1997) A Chilean survey detected L. monocytogenes on fresh cut celery in supermarket prepared salads in 2 of 13 (15.4%) samples taken (Cordano and Jacquet, 2009) Several studies have detected Salmonella on celery, although its fate has not been de termined. Garcia Villanova et al. (1987) found 2 of 26 (8%) celery samples obtained at Spanish markets positive for Salmonella species. Viswanathan and Kaur (2001) sampled 120 fruits and vegetables procured at markets in Spain and found that out of eight celery samples, five (63%) were positive for Salmonella Typhi Recently, in Mexico City, Quiroz Santiago et al. (2009) confirmed 3 of 100 (3%) celery samples taken from a central supply station to be positive for Salmonella species. Currently, very little is known about the behavior of E. coli O157:H7, L. monocytogenes or Salmonella on fresh cut celery, or the impact of consumer handling practices on the potential risk. Our objective was to determine the fate of these pathogens on fresh cut celery under different storage conditions. We hypothesize that: (i) E. coli O157:H7 and Salmonella populations on fresh cut celery will decrease at refrigeration temperatures and increase with increasing temperature throughout the range encountered in typical produce transportation and storage environments, (ii) L. monocytogenes will grow at typical storage temperatures, and grow optimally on temperature abused fresh cut celery, (iii) all three pathogen grow th rates will be higher on cut than on uncut celery surface in oculation sites, and (iv) the type of storage container (bag or container) will affect the behavior of all three pathogens.

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15 CHAPTER 2 LITERATURE REVIEW Celery Background First cultivated about 3,000 years ago, celery ( Apium graveolens ) is a member of the parsley family native to brackish marshlands of southern Europe and North Africa that border the Mediterranean Sea (Ozores Hampton et al., 2011) Celery is a cold season, biennial crop grown as an annual (Huntrods, 2010) A popular herb and vegetable in North America and Europe, celery varieties with fleshy stems are most popular, being served raw or diced and cooked as an important ingredient in ma n y soups, sauces, stews, and gumbos. Modern cult i vars are larger, more succulent, and less stringy than their ancestral wild varieties (Lucier and Jerardo, 2005) A stalk of celery (or head) is a modified leaf bundle, consist ing of several in dividual fleshy stems (or petioles ) Celery hearts are ma de by trimming off the outer petioles of a stalk, and are therefore composed of only the inner and most tender petioles (Lucier and Jerardo, 2005) A lthough not a major plate vegetable, versatility in both fresh and cooked forms and nutritional properties of celery have made it a staple in the produce isle (Lucier and Jerardo, 2005) Long an important ingredient in vegetable platters, the introduction of prepackaged fresh cut products over the past two decades has likely helped expand the reach of celery into the diet of consumers (Lucier and Jerardo, 2005) The leaves and roots are also used as flavoring herbs, most often in European cuisine. Celery seed and salt (ground celery seeds mixed with salt) are also important flavoring herb s used worldwide (Huntrods, 20 10) Nutritional P rofile Originally believed to be a sedative and cultured for its purported medicinal properties, use of celery as a food item was not recorded until 1623, in France (Huntrods, 2010) A low

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16 calorie, high fiber food contain ing anti inflammatory polysaccharide compounds diet s high in celery have been shown to improve serum lipid profiles (Ovodova et al., 2009; Tourkostani et al., 2009) The major bioactive antioxidants and polyphenolic flavonoid (typically glycosylated flavonoid malonates) constituents of celery include caffeic acid, p courmaric acid, and ferulic acid, and apigenin, luteolin, chrysoeriol, and kaempferol, respectively (Lin et al., 2007; Yao et al., 2010) Celery is particularly high in luteolin, which in animals models and human cell cult ure studies inhibit ed angiogenesis, induce d cancer cell apoptosis, and prevent ed carcinogenesis, as well as hyper sensitize tumor cells to cytotoxic chemotherapeutic agents (Lopez Lazaro, 2009) Luteolin can inhibit microglial, hippocampal production of IL 6, and thus may mitigiate neuro inflammation and improve working memory, particularly in the elderly (Jang et al., 2010; Jang et al., 2008) Celery also contains 10, 4, 15, and 2% of the recommended daily allowance (RDA) of vitamin A, calcium, vitamin C, and iron, respectively and contains only 15 calories (mostly from sugars and polyols) per 110 g serving (FDA, 2009) The major sugar and polyol constituents of celery stalks include sucrose, glucose, fructose, and mannitol, the latter representing 33.5 to 39.3% of the total bioavailable carbohydrates (Ruperez and Toledano, 2003) Celery oil from ground seeds or roots is currently marketed as a dietary supplement, having demonstrated anti rheumatic properties and hypolipidemi c promotion of healthy blood pressure (Cheng et al., 2010; Huntrods, 2010; Lin et al., 2011) Production In the United States, the majority of celery is grown in Florida, California, and Michigan (Lucier and Je rardo, 2005) Over the last two decades, the industry has become increasingly concentrated in California, which, in 2005, accounted for 81% of the national celery acreage; up from 63% in 1992 (Lucier and Jerardo, 2005) The most common variety of celery in the United

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17 States is the Pascal type (Huntrods, 2010) In Florida, common agricultural varieties include Floribelle M9, June Belle, and Florida 683 (Ozores Hampton et al., 2011) Soil preparation is more i mportant in celery than in most other crops (USDA, 1999) Celery in Florida is grown on heavily fertilized, muck soils. Fields are plowed, disked, and leveled, then alternately flooded and dried during the summer to manage nematodes, insects, and other parasites (USDA, 1999) After flooding, the plowing and leveling process is rep eated, followed by a sub soil mole draining operation to aid in sub surface irrigation prior to planting (Bertelson et al., 1994) Cutting transplant ation is the primary method of planting, since it is difficult to obtain uniform stands from direct seeding ( Masabni and Lillard 2011 ) Seedlings are spaced in double rows about 6 12 inches apart in row, with 18 40 inches between rows on 40 inch raised beds (Masabni and Lillard 2011 ; Ozores Hampton et al., 2011) Celery is grown from seed on mineral soils enriched with phosphorous oxide ( P 2 O 5 ) micronutrients, and 20 to 25% nitrogen, and pot assium oxide ( K 2 O ) before transplanting to histosol soil (Ozores Hampton et al., 2011) The seeding mineral soil is side dressed in two applications, one 2 to 3 weeks after planting, and the second 6 to 8 weeks after planting. The histosol soils are b roadcasted with a variety P 2 O 5 and micronutrient fertilizers before planting. Cultivation time from transplanting to maturity is 75 to 90 days (Ozores Hampton et al., 2011) The average population of celery crop per acre is 58,080. Several applications of potassium and nitrogen are made throughout the growth season (October to April) (Ozores Hampton et al., 2011) A variety of herbicides, insecticides, and antibiotics are heavily rel ied upon by celery producers to help ensure a productive harvest (Ozores Hampton et al., 2011) Celery is more difficult to grow than most fruits and vegetables, requiring a longer growth season, large quantities of water on par with carrot crops, an d cooler temperatures

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18 (optimal 15.6 21.1C) (Bertelson et al., 1994; Huntrods, 2010; Masabni and Lillard 2011 ; USDA, 1999) One hundred percent of the celery acreage in Florida is irrigated B ecause of its shallow root system, celery is very sensitive t o changes in water level (Huntrods, 2010) If the specific microcl imate requirement s are not met, mature celery stalks can become dry and stringy (Huntrods, 2010) At the higher temperature s celery crop s becomes more susceptible to disease and insect damage, as well internal, physiological problems (USDA, 1999) In juvenile crops, if ambient temperature drop below 10C fo r between 10 and 14 days, premature bolting can occur, leaving the stalk with no commercial value (USDA, 1999) Harvest and P rocessing Fresh celery must be harvested within a few days after reaching marketable size to avoid quality deteriorati on (Huntrods, 2010) Growers stagger planting to develop uniform lo ts that reach marketable size weekly (Huntrods, 2010) Harvested c elery plants must be carefully handled and never stacked more than four stalks high with in storage or shipping crates (Huntrods, 2010) Most Florida celery is harvested by hand, where labors trim, size, and wash in 1 ppm free Chlorine ( Personal com munication May 2011) and pack the crop on site (USDA, 1999) Often, stalks are packed upright in crates and require constant storage and shipping tempe rature and humidity; ca. 32 34F and 85 95% RH (Huntrods, 2010; Masabni and Lillard 2011 ; Suslow and Cantwell, 2009) Cartons or crates of celery weigh 50 to 60 lbs (Masabni and Lillard 2011 ) Harvested celery is brought to a hydrocooling facility and loaded onto refrigerated trucks for transport, usually to market. About 8% of all U.S. celery is harvested for processing. Only celery destined for processing can be mechanically harvested (Lucier and Glaser, 2005) T he market is dominated by a handful of large producers, which have matured an infrastructure that possesses the

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19 processing and handling capabilities necessary to quickly deliver this relatively perishable crop to market (Huntrods, 2010) Celery that that will be cut or diced may be machine harvested, then taken to a pac kinghouse, hydrocooled, and stored at 34 38F for not more than 48 h ( Personal communication May 2011) Cutting of celery stick s is often done by water jet, and dicing by machine using metal blades. Peracetic acid (PAA) may be used as a sanitizing agent in processing lines in either a flume or drenching processing. Processed celery is then packed into small (ca. 1.6 oz) bags or large (ca. 1,200 lb) bins for industrial use ( Personal communication May 2011) Within 24 h after processing, it is loaded onto temperature controlled trucks for transport (Bertelson et al., 1994; Bewick, 1994) The entre process may take up to 3 days before export from the packinghouse Marketing of C elery in the United States While most U.S. celery is sold in the fresh market, a portion is also used in prepared foods including soups, juices and premade entrees (Lu cier and Jerardo, 2005) Established in 1957, there are three grades of celery based on uniformity, size, and defects (USDA, 1959) From highest quality to least these grades are; U.S. Extra, U.S. #1, and U.S. #2 (Suslow and Cantwell, 2009; USDA, 1959) (Suslow and Cantwell, 2009) Fresh and fresh cut celery is primarily sold to retailer wholesalers, terminal market brokers, wholesale handlers and the military (Bertelson et al., 1994; Bewick, 1994) The majority of fresh celery sold is eventually consumed at home (76% of all market sales), while restaurant consumption account for about 14% of total per capita consumption (Lucier and Jerardo, 2005) Distributers have established only a small niche in the expanding fast food

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20 market, which represents only 4% of celery consumption in the U.S. (Lucier and Jerardo, 2005) Consumers eat >90% of processed celery products at home. In 1997, in Florida, the 12 existing farms produced 4,114 acres of celery, the second largest crop in the U.S., after California (USDA, 1999) Nearly the entire Florida crop of celery is destined for fresh market sale as full stalks, celery hearts, or fre sh cut (USDA, 1999) The total cultivation cost including labor, machinery, nutrients, insecticides, herbicides, and fungicides of celery between 1993 and 1994 was $4,580 per acre, with pest management representing 42 % of the cost (US DA, 1999) In 1992, the last year in which data was collected, Florida produced 315.4 million lbs. of celery, with an average yield of 41,500 lbs. per acre. The total value of the 1992 Florida harvest was $39.1 million (USDA, 1999) The demographic profile of celery consumptions are skewed towards older population s, with U.S. citizens under 20 accounting for 30% of the total population, but only consuming 17% of the fresh celery sold at market (Lucier and Jerardo, 2005) Economic I mpact A steady, ample supply from a relatively efficient industry has kept U.S. market prices low and limited the reach of foreign competitors into the American and Global markets (Lucier and Jerardo, 2005) In 2006, within the U.S., celery sales alone grossed $920 million, 1.6% of the gross profit from produce sales (PMA, 2007) Also according to the PMA (2007), about 50% of all North American households purchased celery 3 times or more per year in 2005. During 2009, U.S. consumers used a per capita average of 6.1 lbs. of fresh celery (USDA, 2011 ; Huntrods, 2010) This per capita consumption has held relatively unif orm over the past 90 years, ranging from ca. 6.1 to 8.2 lbs/year, which peaked during the 1950s ( USDA, 2000 ) Between 2003 and 2004, the average gross annual value of a celery farm in the U.S. was ca. $261 million (Lucier and Jerardo, 2005)

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21 The United States is a net exporter of celery, having shipped more the 2.5 million cwt ( 100 lb. crates) overseas in 2009 (Brown et al., 20 02 ; Huntrods, 2010; Lucier and Jerardo, 2005) Between 2002 and 2004, exported fresh celery totaled $48 million, while imported celery was valued at only $9 million (Lucier and Jerardo, 2005) Also during this time, 13% of the U.S. celery supply was exported annually. Canada is the largest single importer, having received 80% of total exports between 2002 and 2004, and a bout 93,970 metric tons (MT) in 2009 (Huntrods, 2010) Despite be ing a major producer and exporter, the U.S. also imports celery. In 2009, ca. 21,204 MT of fresh celery was imported from producers in Mexico, a practice which increases during the winter months (Huntrods, 2010) Unlike many storable commodities, fresh market celery exhibits only a weak season al price pattern, reflecting the relatively consist season to season marketing (Lucier and Jerardo, 2005) Celery prices are known to follow a pronounced 3 year cycle, which may reflect recurring weather patterns (Lucier and Jerardo, 2005) Price of a Recall to I ndustry Over the last decade, the number or at least the public pr ofile of food recalls has increased dramatically, with Salmonella, L. monocytogenes, and pathogenic E. coli having been associated with some of the largest scale recalls. Given the large number of variables involved, it is difficult to estimate the costs of these events to industry and shareholders with any accuracy, but catastrophic and devastating may in some cases be considered fair de scriptors. In one example, the well publicized baby spinach recalled in 2006 attributed to E. coli O157:H7 cost Dole $350 million including lawsuits and refunds, not accounting for lost customers and business relationships ( NRI 2010) As a result of the Salmonella related peanut recall of 2009, expert testimony from industrial insiders and government official s estimated that

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22 the confirmed $1 billion loss in production and sales of peanuts could be just the beginning, as consumer demand for any product containing peanuts subsequently plummeted. Among the multitude of costs incurred by food companies in the event of a recall include removal and disposal of both stored and distributed products, loss of trust and quite possibly business with l arge buyers, loss of consumer faith for the foreseeable future, often hefty lawsuits from those consumers struck ill from contaminated products, government mandated closure of processing plants, and possibly government imposed penalties if it is found that codes have been violated (Associated Press, 2009) Biological Hazards Associated w ith Fresh P roduce Fruits and vegetables have gained notoriety in recent years as a vehicle of human disease (D'Aoust and Maurer, 2007) The fact that fresh cut produce items are often consumed raw contributes to the potential risk. Although the incidence of foodborne illness associated with fresh produce is low in comparison to the total volume consumed per capita in the United States, recent increases in consumption have coincided with an increase in reported outbreaks associated with their consumption (CAST, 2009) This increase in incidence is believed to have developed partially as a result of inc reases in global export of fresh produce from countries with tropical and subtropical climate s (D'Aoust and Maurer, 2007) Production conditions in these countries do not always meet minimum standards and can facilitate contamination (D'Aoust and Maurer, 2007) Because fresh produce is often consumed raw, foodborne disease p revention efforts must focus on good sanitary practices throughout the supply chain. Sources of Contamination o n Fresh P roduce Escherichia coli O157:H7, L. monocytogenes, and Salmonella are found throughout the natural environment, and contamination can occur at any point in the supply chain from production through processing and distribution to preparation in the home or food service

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23 kitchen. Because most produce is cultivated in an op en, natural environment, it is highly vulnerable to contamination. At the field production level, Good Agricultural Practices (GAPs) have been implemented by many producers to minimize the risk of microbial cross contamination from various environmental sources/vectors including farm laborers, soil, fertilizers, spray and irrigation water, wild and domestic animals, and insects. Contamination may also arise during cultivation by infection through an open portal such as damage d plant tissues (e.g. herbivo re damage) or scar tissue, internalization during embryogenesis, or from natural uptake of pathogens through the root system (Guo et al., 2001) tho ugh this likely is not a common occurrence. During further processing, several modes of produce cross contamination have been identified. Major processing cross contamination points include cutting, trimming, washi ng, and cooling of produce (Doyle and Erickson, 2008) In a consumer kitche n, specific sources of produce contamination are often more difficult to trace, but are primarily believed to be the result of surface transfer from contact with conta minated meat, from improperly sanitized cutting and cleaning surfaces, or contaminated wa sh water (Chai et al., 2008; Chen et al., 2001; De Wit et al., 1979; Doyle and Erickson, 2008; Kusumaningrum et al., 2003) High Risk Consumer Handling B ehaviors As sociated w ith Fresh P roduce Fresh cut produce may bec ome contaminated in the kitchen through cross contamination during final preparation or serving of the product. Due to the notorious difficulty in identifying a causal relationship between outbreaks and risky behaviors, the degree of risk associated with consumer behavior is an open question. For example, it has been shown that temperature abuse can increase the growth rate of pathogens in many fresh produce items (e.g. iceberg lettuce ) (FDA, 2008; Koseki and Isobe, 2005) but it is not certain whether this behavior in the home has

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24 contributed to illnesses associated with produce. Other risky consumer behaviors believed to contribute to produce related outbreaks that have occurred in t he home kitchen include inadvertent contact with raw meat (e.g. melon) (Harris et al. 2003) a nd from the contaminated hands of food handler s (Ayiek et al., 2004; De Roever, 1998; Iversen et al., 1987; Lues and Van Tonder, 2007; Shojaei et al., 2006) Reports also sug gest that consumers often keep fresh cut produce in the refrigerator well after the expiration date, and may regularly consume spoiled items ( CDHS 2007) It should also be noted that spreading of bacterial contaminants from outer organic surfaces (e.g. melon rind) can be facil itated by slicing (Chai et al., 2008) although this phenomenon has less bearing on consumer practices for already cut produce. Much of the available data used to quantify consumer behavior in the home kitchen is obtained through national mail surveys. One such survey of 2,000 Americans that focused on consumer handling of fresh produce (but not necessarily fresh cut) indicated that 6% of those surveyed seldom or never wash fresh produce before consumption (Li Cohen and Bruhn, 2002) The same survey also showed that almost half of respond ents d id not consistently wash their hands before handling fresh produce. Further, 5% of respondents indicated that they did not use wash water, and 24% only wash produce with water after contact with meat products. Li Cohen and Bruhn (2007) conducted focus groups to observe first hand consumer practices related to fresh produce (not specifically fresh cut). The results demonstrated a variety of frequent and potentially hazardous handling behaviors. Those who washed lettuce with water most o ften either rinsed leaves under running water or submerged them in a filled sink or washing basin Most used plain water, although some added salt, vinegar or bleach. After washing, leaves were either shaken and directly served or left to drain in a cola nder, in the dish rack, or on paper towels or cloth. Few used a salad spinner, and some placed leaves in a

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25 pillowcase and spun dry in a laundry dryer. Certain items including cut carrots and cut melons were not typically washed. Bruhn and Li Cohen (2007) also indicated that consumer refrigerator cleaning frequency ranged from once per week to once per year, and was dependent on whether the inside appeared dirty or stained. Common methods of cleanin g the refrigerator included dish detergent or soap, bleach, baking soda, antibacterial soap, and cleaning solution. It is important to note that consumer refrigerators differ in their ability to maintain a constant and uniform temperature. In a recent s tudy of 200 home refrigerators, average temperatures fluctuated between the top shelf (1.9C), bottom shelf (3.3C), and door (5.2C) (Godwin, 2007) Temperatures also rose above the danger zone (4C) for over 2 h per day in the top shelf (in 33%), middle shelf (in 45%), and door (in 80% of refrigerators). In Home Hazard Intervention S trategies Although washing of fresh cut produce is one of the most common hazard mitigation strategies, there is limited data available to compar e the relative effectiveness of many common washing methods employed in the home. Data published by Parnell and Harris (2003) on the efficacy of consumer washing methods of apples has been used to provide standard consumer guidance for washing fresh produce that involves rinsing and rubbing where possible under running water and drying with a clean towel. Parnell et al. (2005) studied the effects of washing cantaloupe and honeydew melons, which also provided critical information on the efficacy of washing smooth and netted rind melons. The latter study also demonstrated a potential for the transfer of pathogens from one site to another on the rind during washing with a volume of untreated (unchlorinated) water.

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26 Escherichia coli Biology of Escherichia coli Escherichia coli was first discovered in 1885 by the German pediatrician and bacteriologist, T heodor Escherich (Feng et al., 2002) All E. coli can be classified into four groups based on ecological features and virulence potential: nonpathogenic mutualists; commensal opportunists (pathogenic un der the right conditions); parasitic pathogens; and occasional symbionts ( i.e. not common to gut microflora) (Gritsenko and Bukharin, 2000) Mutualistic E. coli can benefit their hosts by producing vitamin K 2 (Bentley and Meganathan, 1982) and by preventing the establ ishment of ingested pathogenic bacteria through spatial resource competition (Reid et al., 2001) Escherichia coli is one of the most well studied sequenced (Blattner et al., 1997) As normal microbiota, E. coli is the primary facultative anae robe in the human gut, assisting in the breakdown of polysaccharides (Salyers, 1985) Escherichia coli has been shown water, or from individuals handling the child. Once in th e colon, it adheres to the mucosa. Interestingly, enhanced food digestibility may not be the only mutualistic benefit from E. coli and other normal intestinal micrbiota to humans, and the hypothesis of a coevolutionary human adaptive immune system is well reasoned (Lee and Mazmanian, 2010) It is only when these commensal organisms acquire virulence genes through horizontal transfer that they become parasitic. In fac t, Mutaflor, or E. coli Nissle 1917, is a probiotic proven in numerous clinically trials to be effective in alleviating symptoms of various irritable bowel diseases (IBDs) including irritable bowel syndrome, ulcerative colitis (UC), chronic constipation, C pouchitis, in addition to its utility as a bacterial prophylactic and immunological enhancer in

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27 newborns (Hancock et al., 2010; Konturek et al., 2008; Konturek et al., 2009; Kruis et al., 1995; Midtvedt, 2009) Escherichia coli are gram negative, rod shaped, non sporulating, facultative anaerobes. The se bacteria can thrive on a variety of substrates, and uses mixed acid fermentation under anaerobic conditions, p roducing lactate, succinate, ethanol, acetate, and carbon dioxide (Meng et al., 2007) During cellular respiration, E. coli uses several respiratory pathways and a variety of redox pairs, including the oxidation of pyruvic acid, for mic acid, amino acids and hydrogen, with reducible substrates including oxygen, nitrate, dimethyl sulfoxide and trimethylamine N oxide (Ingledew and Poole, 1984) Some strains of E. coli possess peritrichous flagella, while others are immobile (Darnton et al., 2007) A mesophile, E. coli optimal growth temperature is 35 37C, although some strains have been shown to survive temperatures up to 49C (Brock et al., 1994) Despite their mesophilic nature, E. coli are not always confined to the intestine, and can survive for months outside the body in many open environments (van Elsas et al., 2011) making them somewhat useful indicator organisms f or fecal contamination (Duris et al., 2006; Feng et al., 2002) The minimum pH for growth of E. coli O157:H7 is 4.0 to 4.5 (Meng et al., 2007) although it can survive much lower pH. Studies have shown that hot acetic, citric, and lactic acid sprayed at concentrations up to 1.5% onto meat inoculated with E. coli were not sufficient to appreciably effect population size (Brackett et al., 1994) When inoculated at high concentrations, E. coli survives fermentation, drying, and storage of sausage at pH 4.5 for up to 2 months at 4C (Glass et al., 1992) in mayonnaise at pH 3.6 to 3.9 for up to 7 weeks at 5C and for 3 weeks at 20C (Zhao and Doyle, 1994) and in apple cider at pH 3.6 to 4 for up to 31 or 3 days at 8 or 25C, respectively (Zhao et al., 1993)

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28 Enteric pathogens such as virulent E. coli must pass through the gastric acid pH barrier with pH level as low as 1.5 to 2.5 to cause gastrointestinal disease. Specifically, E. coli O157:H7 has evolved several amino acid dependent systems that control the production of the decarboxylase isozymes GadA, GadB, and AdiA; pyridoxal phosphate containing enzymes that replace the alpha carboxyl groups of their amino acid substrates with H + recruited from the cytoplasm (Castanie Cornet et al., 1999; De Biase et al., 1999; Hersh et al., 1996; Smith et al., 1992) Acid resistance also provides cross protection to E. coli O157:H7, increasing tolerance to heat, radiation, and antimicrobials (Meng et al., 2007) Biochemically, MacConkey and Eosin Methylene Blue (EMB) agar are selective for E. coli On MacConkey agar, deep red col onies are produced through the fermentation of lactose, which causes a drop in pH, leading to a darkening of the medium, and a pink halo of bile salts is confirmative for E. coli On Levine EMB agar, E. coli produces colonies with a characteristic greenis h black metallic sheen. E. coli is also lysine positive, and will grow on TSI slants with an A/AG+H 2 S profile. Additionally, IMViC is {+ + }, as E. coli is indole positive (red ring), methyl red positive (bright red), Voges Proskauer negative, and ci trate negative. Nomenclature and C lassification of P athogenic Escherichia coli Escherichia coli is a gamma proteobacteria of the Enterobacteriaceae family. Although not necessarily based on evolutionary relatedness, the serotype subdivision system used i s based on three major surface antigens, including the O antigen (part of the lipopolysaccharide layer), the H antigen (a flagellin), and the K antigen (capsular) (Orskov et al., 1977) As of November 2005, 173 O antigens, 56 H antigens, and 103 K antigens have been identifie d ( Stenut z et al., 2006 ) Established nomenclature dictates that it is necessary only to determine the O and H antigens to serotype strains of diarrheagenic E. coli with the O antigen identifying serogroup and the H antigen identifying serotype (Meng et al., 2007) Although certain serogroups often fall

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29 into one category of diarrheagenic E. coli some such as O55, O111, O126, and O128 appear in more than one category (Meng et al., 2007) Further, diarrheagenic E. coli isolates are categorized into six pathotypes based on virulence factors, pathogenic biomechanics, associated clinical syndromes, as well as O:H serotype s. These pathotypes include enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), diffuse adhering E. coli (DAEC), enteroaggregative E. coli (EAEC), and enterohemorrhagic E. coli (EHEC; Shiga toxin producing E. co li STEC). Of these groups, the pathogens belonging to STEC cause the most frequent and severe illness (Meng et al., 2007) The first E. coli pathotype to be described, EPEC is featured by severe diarrhea in infants, especially in developing countries (Meng et al., 2007) The major serogroups associated with this pathotype include O55, O86, O111ab, O119, O125ac, O126, O127, O128ab, and O142. EPEC induce attaching and effacing (A/E) lesions, and can invade epithelial cells (Nataro and Kaper, 1998) ETEC are also a major cause of infantile diarrhea in developing countries, and are the (Meng et al., 2007) E scherichia coli of this pathotype colonize the proximal small intestine by fimbrial colonization factors (CFA) and produce both heat stable and heat labile enterotoxins. Serogroups members include O6, O8, O15, O20, O25, O27, O63, O78, O85, O115, O128ac, O148, O159, and O167 (Nataro and Kaper, 1998) EIEC is featured by nonbloody diarrhea and dysentery similar to that caused by Shigella species. With infection principally focused in the colon, EIEC invade and prolife rate in epithelial cells, destroying host cells in the process. Serogroup members include O28ac, O29,

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30 O112, O124, O126, O136, O143, O144, O152, O162, and O167, with O124 being most common (Nataro and Kaper, 1998) The epidemiological profile of DAEC is age related, manifesting in young children under 5 years old, and over 1 year old, with relativ e risk increases with age. Features are typically mild and include diarrhea with or without fecal leukocytes. Pathogenically DAEC strains attach to host cells via a diffuse adherent pattern invitro. They do not invade epithelial cells, synthesize heat stable or heat labile enterotoxins, or Shiga toxin, and do not possess EPEC adherence factors. Serogroup members include O1, O2, 021, and O75 (Meng et al., 2007; Nataro and Kaper, 1998) EAEC manifests in infants and children, and is characterized by an aggressive and biomechanically unique pathogenic pattern of adherence. In vitro, EAEC adhere on the surface of HEp 2 cells in a stack ed brick appearance (Meng et al., 2007) Serotype members include O3, O15, 044, 077, O86, O92, O111, and O127 ( Meng et al., 2007; Nataro and Kaper, 1998) STEC were first recognized as foodborne pathogens in 1982, when E. coli O157:H7 was the determined cause of two outbreaks of hemorrhagic colitis, a clinical feature unique to this pathotype. Escherichia coli O157:H7 has since been associated with many foodborne outbreaks and is the predominate STEC associated disease (Meng et al., 2007) Other serogroup members include O26, O45, O103, O111, O121, O145 and the sorbitol fermenting O157:N M serotype. The pathogenic profile of STEC includes the production of Shiga toxins (Stx), or Shiga like verotoxins. VT is so named due to its cytotoxicity to African green monkey kidney (Vero) cells, and Shiga toxins (Stx) due to their similarity with th e Shiga toxin produced by Shigella dysenteriae type 1 (Obrig, 1997) Stx and VT are structurally and functionally identical (Hoffmann et al., 2010; Takao et al., 1988)

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31 To test for shiga toxin production, E. coli isolates can be exposed to mammalian tissue culture (Paton and Paton, 1998) Other methods for the detection of E. coli O157:H7 include real time PCR, ELISA tests, colony immunoblots with verotoxin antibodies, direct fluorescence microscopy enzyme bioassay, and immunomagnetic capture techniques (De Boer and Heuvelink, 2000; Fedio et al., 2007; Lauer et al., 2009 ; Sharma et al., 2008; Song and Kwon, 2009) Escherichia coli O157:H7 strains are distinguished by an inability to grow at 44.5C, ferment sorbitol within 24 h, or produce Beta glucuronidase through hydrolysis of 4 methlyumbelliferyl D glucuronide (MUG), and the possession of the locus of enterocyte effacement (LEE) pathogenicity island, as well as a 60 MDa plasmid (Meng et al., 2007) Evidence suggests that only 20% of the genome is common to all E. coli strains (Lukjancenko et al., 2010) Different strains of E. coli are often host specific, which can make the original source of fecal contamination from environmental samples traceable. For example, strain typing E. coli sampled from a water source may allow researcher s to make assumptions about whether the original source of contamination wa s human, another mammal, or bird species (Feng et al., 2002) Clinical F eatures of EHECs Although the precise infectious dose of E. coli O157:H7 is unknown it has been estimated at < 100 cells, and possibly as little as 10 cells in high risk populations (Meng et al., 2007) Food poisoning from E. coli O157:H7 is usually caused by eating unwashed vegetables or undercooked meat, although there are many other pote ntial sources (Rangel et al., 2005) Ingestion of the bacteri a is typically followed by a 3 to 4 day incubation period, but can range from 2 to 12 days, during which time colonization in the large intestine occurs (Meng et al., 2007) Illness typically onsets with nonbloody diarrhea and severe abdominal cramping for 1 to

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32 2 days, progressing in the 2 nd or 3 rd day to bloody diarrhea that last from 4 to 10 days (Besser et al., 1999; Tarr et al., 2005) Symptoms usually resolve after 1 week. While most often mild, the severity of illness caused by pathogenic E. coli varies considerably. Virulent strains of E. coli typically cause gastroenteritis and urinary tract infection, which are unpleasant for adults, but can often lead to serious cond itions such as hemolytic uremic syndrome (HUS) and neonatal meningitis in children, particularly those of the developing world (Nataro and Kaper, 1998) Epidemiological studies suggest that the highest age specific incidence of E. coli O157:H7 infection occurs in children between 2 and 10 years old (Meng et al., 2007) T he more virulent strains such as E. coli O157:H7, O121 and O104:H21 produce potentially lethal toxins and can cause serious illness or death in the elderly, very young, and immunocompromised populations (Nataro and Kaper, 1998) About 6% of all E. coli O157:H7 cases progress to HUS (most frequent in children), 75% of which require blood transfusi ons, and 50% requiring dialysis (Karmali et al., 2004; Meng et al., 2007) HUS is the primary cause of renal failure in children. Peritonitis, which can be fatal wit hout intervention, can manifest when virulent E. coli penetrate the intestinal epithelium through a pre existing gap in the barrier such as an ulcer, ruptured appendix, or surgery and enter the abdominal cavity. The precise role of Stxs in colonic diseases, HUS, and neurological disorders has not been fully elucidated, although the presence of Stxs in neurons has been verified by immunoelectron microscopy (Meng et al., 2007) In HUS, it ha s been found that endothelial dysfunction is the triggering event, and that bloodborne Stxs invade the renal microvasculature causing thrombogenesis (Zoja et al., 2010) Histopathic examinations have also revealed structural alterations in the glomeruli of HUS patients, which suggests that subepithelial cell can also serve as target sites of opportunity for STEC attack (Meng et al., 2007)

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33 Escher ichia coli are extremely sensitive to some antibiotics including streptomycin and gentamicin. However, caution is always warranted since E. coli is known to quickly acquire multidrug resistance (Amaya et al., 2011; Aslani et al., 2011; Cuevas et al., 2011) The American Gastroenterological Association Foundation (AGAF) recommends that all clinical stool specimens be routinely tested for E. coli O157:H7 (Tarr, 1995) Virulence Factors and P athogenesis of EHECs EHEC have evolved the abi lity to cause disease by adhering to host intestinal epithelium cell membranes with A/E lesions and then producing and secreting one or more Stxs. All A/E lesion associated proteins known which include a type III secretion system (TTSS), an outer membrane adhesin (intimin), and translocating intimin receptors (Tir) and other effector proteins are encoded on the LEE pathogenicity island (Meng et al., 2007) s of approximately 20 different genes, and is used by STEC to secrete virulence factors from the bacteri um into a host cell (Garmendia et al., 2005) Following effector translocation, the TTSS complex is disengaged to allow closer bacterial attachment through intimin Tir interaction s (Garmendia et al., 2005) Transmembrane intimin receptor (Tir) forms an extracellular loop that interacts with intimin in a reticular array between host and bacteri um STEC serotypes contain a unique 60 MDa plasmid (pO157) that is thought to play a role in pathogenesis (Meng et al., 2007) The pO157 is composed of putative virulence genes with 7 insertion sequences located directly upstream These genes including those encoding for the adhesin ToxB, EHEC hemolysin, the serine protease EspP, a catalase peroxidase encoded by katP (role in defense from oxidative attacks), and the metalloprotease StcE (Meng et al., 2007) STEC can express both the heat stable Stx2 and the heat labile Stx1 enterotoxins (Rasooly and Do, 2010) Stx holotoxins are structurally and functionally similar to cholera

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34 toxin, with the B subunits assisting in host intestinal epithelium cell adhesion and entry while the A subunit is cleaved by trypsin into an enzymatic A1 fragment, which is released into the cytosol causing cell death and preventing host epithelial cell wa ter uptake, leading to diarrhea (Tauschek et al., 2002) Rather than transfer via the TTSS involved in A/E lesion formation, Stxs are translocated via a Type II secretion pathway (Tauschek et al., 2002) B subunit receptor binding induces membr ane invagination and the holotoxin is endocytosed and endosomally transferred via the trans Golgi network and endoplasmic reticulum (Sandvig et al., 2010; Utskarpen et al., 2010) It is during this trafficking that the A subunit is released to the cytosol. Once within the host cell cytosol, the A subunit acts toxicokinetically by inhibiting protein synthesis at translation (Sandvig, 2001) The cytosolic A subunit undergoes partial proteolysis by host cell proteases and is split into a 27 kDa active N glycosidase (A1) bridged to the remaining 4 kDa fragment (A2) in a disulfide bond. The A 1 target site of action is the 28S rRNA of the 60S ribosomal subunit. A1 cleaves the N glycoside bond in one adenosine position, thereby inhibiting elongation factor dependent binding to ribosomes by aminoacyl bound tRNA (Sandvig and VanDeurs, 1996) Epidemiology of Escherichia coli O157:H7; Focus on Fresh P roduce In most non O157 STEC illness outbreaks, the modes of transmission are unknown, and only a few have been positively traced back to a food and/or water source (Brooks et a l., 2005; Johnson et al., 1996) In contrast, E. coli O157:H7 has many times been identified as the cause of disease in outbreaks of severe illness worldwide. In 1996 in Japan, more than 11,000 cases of E. coli O157:H7 illness were reported (Meng et al., 2007) In the United States, between 1982 and 2002, 350 outbreaks and 8,598 cases of E. coli O157:H7 were documented (Rangel e t al., 2005) Of these outbreak related cases, 61% were foodborne. Also of these, 1,493 (17.4%)

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35 resulted in hospitalization. The total number of outbreaks associated with E. coli increased dramatically from 1982, peaking during 2000. A variety of fo ods have been implicated as the vehicle of transmission in E. coli O157:H7 cases including ground meat, roast beef, cooked meats, jerky, salami, raw milk, pasteurized milk, yogurt, cheese, ice cream bars, lettuce, unpasteurized apple cider or juice, cantal oupe, potatoes, radish sprouts, alfalfa sprouts, lettuce, spinach, fruit salad, vegetable salad, and cake (Meng et al., 2007; Patel et al., 2010) The first outbreak of E. coli O157:H7 infection related to fresh produce was reported in 1991 (Rangel et al., 2005) From 1982 to 2002, in cases where a source was determined, 21% of reported cases were produce associated. Annual produce associated outbreak size averaged 20, with a range from 2 to 736 incidences per outbreak (Rangel et al., 2005) In 1996 in Japan, the cause of multiple outbreaks of E. coli O157:H7 infection involving 11,826 cases was traced back to white radish sprouts (Me ng et al., 2007) Of all E. coli O157:H7 outbreaks reported on in the 1982 to 2002 study, 29% occurred in communities, 28% in restaurants, and 16% in schools (Rangel et al., 2005) According to both passive and active (i.e. Foodnet) investigations, outbreaks of E. coli O157:H7 are seasonal, peaking during the warmest months (Rangel et al., 2005) This trend may be the result of an increased survival and prevalence of the pathogen due to the increased ambient temperature, the (Meng et al., 2007) Ecolog ical Dissemination and S pread of EHECs Pathogenic E. coli are carried in the gastrointestinal tracts of many mammals and other thermoregulators including humans, cattle and other livestock, domestic pets, rodents, birds, and other wildlife, and can be dis tributed to soil and water through fecal shedding and the agricultural application of manure (Beutin et al., 1993; Galiero et al., 2005; Meng et al., 2007;

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36 Nielsen et al., 2004) Although the prevalence of STEC is generally highest in sheep (Kudva et al., 1996) in the human food chain, cattle are believed to be the primary source and ground beef is impl icated in the majority of outbreaks (Meng et al., 2007) In 2002, a USDA national study found that 38.5% of dairy farms had at least 1 cow positive for fecal E. coli O157:H7, and that overall, 4.3% of individual cows were shedders of E. coli O157:H7 (Meng et al., 2007) Because E. coli O157:H7 is primar il y a human pathogen, animals that carr iers are not typically ill. However, evidence exists that E. coli O157:H7 can cause diarrhea and A/E lesions in neonatal calves (Naylor et a l., 2003) Studies have shown that STEC are present in up to 60% of bovine herds, but in most cases, prevalence rate s range from 10 to 25% (Meng et al., 2007) In human patients, fecal shedding of E. coli O157:H7 usually lasts no more than 21 days following the onset of symptoms (Meng et al., 2007) The fecal carriage and dissemination of E. coli O157:H7 directly (from person to person), or indirectly (from person to food to person), has been repeatedly implicated in outbreak scenarios (Meng et al., 2007) Contributing to the significance of person to person transfer of E. coli O157:H7 is its extraordinarily low infectious dose. It should be noted that asymptomatic long term car riage of pathogenic E. coli has not been observed (Meng et al., 2007) Interestingly, studies of dairy farm families have reveal elevated antibody titers against surface antigens of E. coli O157:H7, although there was no evidence o f fecal contamination (Meng et al., 2007) Listeria monocytogenes Biology of Listeria monocytogenes Listeria monocytogenes was discovered in 1926 by both E.G.D. Murray and James Pirie independently, and named after Lord Joseph Lis ter (Rocourt, 1996) Between 1930 and 1950, only a few cases of listeriosis were reported. However, in the past 30 years following a 1981 outbreak in Nova Scotia that was traced back to contaminated coleslaw (Schlech et al., 1983)

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37 lis teriosis has emerged as a foodborne pathogen of major public health concern. The 1981 event was the first definitively documented case of foodborne transmission of listeriosis, confirmed through the use of a case control study in combination with strain t yping (Schlech et al., 1983) In the time since the Nova Scotia outbreak, there have been hundreds of cases of listeriosis reported annually (Rocourt and Jacquet, 1992) Listeria monocytogenes is a gram positive, non spore forming rod shaped, facultative anaerobe with motility via peritrichous flagella at <30C. However, the bacteria do not (Grundling et al., 2004) When viewed under a light microscope, L. monocytogenes exhibits a characteristic tumbling motility (Farber and Losos, 1988) An intracellular pathogen, L. monocytogenes moves within eukaryotic cells at body Several Lis teria spp., including L. monocytogenes are catalase positive and oxidase negative. The further biochemical identification of L. monocytogenes is limited, but includes hemolysis via the CAMP test to differentiate from non pathogenic Listeria species, and ac id production from D xylose (Swaminathan et al., 2007) To a greater extent than most other foodborne pathogens, L. monocytogenes is widely distributed in the natural environment, resistant to low pH and hi gh NaCl environments, is microaerobic, and psychrotrophic. Listeria monocytogenes can grow in moderate salt environments (6.5% sodium chloride), can grow in the presence of up to 12% NaCl, and will survive for long periods under higher salinity (Swaminathan et al., 2007) Listeria monocytogenes is particularly problematic due to its extreme environmental durability, survival, and ubiquity, and its ability to grow at low temperatures (Swaminathan et al., 2007) With a growth temperature range of 2 to 45C, the average generation times of 39 different L.

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38 monocytogenes strains were 43, 6.6, and 1.1 h, at 4, 10, and 37C, respectively (Barbosa et al., 1994) Respective lag times were 151, 48, and 7.3 h. Listeria monocytogenes does not survive over 50C, and is preserved or moderately inactivated at temperatures below 0C (Swaminathan et al., 2007) In one study, the ltrB gene associated with low temperature growth was found to be unique to serotype 4b strains (Zheng and Kathariou, 1997) The pH range for the growth of L. monocytogenes is a function of temperature and acid type (Swaminathan et al., 2007) Although previously thought to have a pH growth rang e between 5.6 and 9.6, on media, L. monocytogenes will grow at values as low as pH 4.4. Below pH 4.3, Listeria spp. can survive, but not multiply. When exposed to 0.1% acetic, citric, and lactic acids in TSB, growth is inhibited, and inhibition is invers ely proportional to temperature (Swaminathan et al., 2007) The type of acid used influences the fate of L. monocytogenes with lactic and citric acids being less bacteriocidal than acetic acid and equival ent pH values (Swaminathan et al., 2007) Generally, the minimum water activity (a w ) for growth of most strains of L. monocytogenes is 0.93, with an optimum a w >0.97. However, some strains with gro w at a w values as low as 0.9, and most strains will survive at a w 0.83 (Shahamat et al., 1980 ) Of importance to food manufacturers who use low a w to preserve their products, the thermal kill temperature of L. monocytogenes has an inverse relationship with a w (Sumner et al., 1991) The genome of virulent L. monocytogenes strains encodes a large number of surface, secretory, transport system, and re gulatory proteins. This suggests a high degree of adaptability, and is in keeping with the wide variety of environmental survivability of L. monocytogenes (Glaser et al., 2001) The vast array of proteins at the disposal of L. monocytogenes also appears

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39 to b e a critical feature in the evolution of virulence and unique infectious process (Doumith et al., 2004) Phylogenics and S ubtyping of Listeria monocytogenes The genus Listeria includes six species; L. monocytogenes L. ivanovii L. innocua L. welshimeri L. seeligeri and L. grayi (Swaminathan et al., 2007) While both L. ivanovii and L. monocytogen es are pathogenic to some animals, L. monocytogenes is the only Listeriae species considered pathogenic to humans. While there are 13 serotypes of L. monocytogenes that are all pathogenic, more than 90% of human cases (in which type has been identified) b elong to serotype 1/2a, 1/2b, or 4b (Salova et al., 2005; Seeliger, 1986) Since 2005, multiplex PCR based methods have been established for identifying serotype between different L. monocytogenes isolates (Doumith et al., 2005) In recent years, several molecular typing methods have been successfully used to identify specific strains within a given L. monocytogenes serotype. These molecular tools include multilocus enzyme electrophoresis, ribotyping, DNA microrestriction, DNA macrorestriction, and random amplification of polymorphic DNA (Graves et al. 1999) In the United States, PulseNet, a division of the U.S. Center for Disease Control and Preventio n (CDC) is a network of laboratories that routinely uses highly standardized molecular methods to subtype foodborne pathogens in order to rapidly detect disease clusters that may have a common source. Since 1999, PulseNet has used pulsed field gel electro phoresis (PFGE) to subtype L. monocytogenes and their exact protocol has been available since 2006 (Graves and Swaminathan, 2006) Worldwide, all of the major foodborne outbreaks of listeriosis that occurred during 1980s, and 33 to 50% of sporadic cases, were caused by serotype 4b. Intriguingly, also during this period, most isolates recovered from food were strains of 1/2a and 1/2b serotypes (Swaminathan et al., 2007) This observation suggests th at serotype 4b may be the most virulent

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40 of the three. DNA from several strains of serotype 4b associated with different outbreaks have demonstrated a host mediated resistance to Sau3AI and other restriction enzymes, some of which are likely associated wit h virulence factor genes (Zheng and Kathariou, 1997) and a presumed restriction modification system (85M, 85R, and 85S) specific to these strains has been identified (Zhang et al., 2003) Wiedmann et al. ( 1997) defined three distinct lineages for L. monocytogenes with different pathogenic potential. Lineage I isolates included serotypes 1/2b and 4b, and were l ater confirmed as the most predominate human pathogen of the three, through meta analysis of studies examining clinical isolates of L. monocytogenes also from the Wiedmann laboratory (Gaillard et al., 1991; Gray et al., 2004; Jeffers et al., 2001; Norton et al., 2001; Swaminathan et al., 2007) Lineage II isolates were of serotype 1/2a and 1/2c, and lineage III contained only animal isolates. Clinical F eatures of L isteriosis Although the number of annual cases is fairly low, listeriosis is a major public health concern due to the severity of the disease, which can cause meningitis, septicemia, and fetal abortion, resulting in a high fatality rate (20 30% of cases). These and other severe disease manifestations are often preceded by influenza like symptoms including pers istent fever, and gastrointestinal disturbances such as nausea, vomiting, and diarrhea (Gray, 1962 ) The onset time from early symptoms to more serious listeric pathology ranges from a few days up to 5 weeks (Swaminathan et al., 2007) Listeriosis mainly presents in individuals pre conditioned with impaired T cell mediated immunity (Swam inathan et al., 2007) The most severely immunocompromised patients include those with predisposing statuses such as malignancy, organ post transplant, immunosuppressive therapy, human immunodeficiency virus (HIV), and the more advanced elderly. Liste riosis is 300 times more likely to occur in AIDS patients than in

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41 the general population (Schuchat et al., 1991) High risk populations also include pregnant women, neonates, immunocomprom ised adults, and the elderly. The infectious dose and course of disease caused by L. monocytogenes is a function of host immunological status, in combination with microbial virulence factors (Swaminat han et al., 2007) The dose necessary for gastrointestinal listeriosis to afflict low risk populations is believed to very high. In five outbreak investigations involving the gastrointestinal listeriosis (non bacteremic) infection of low risk persons, ingested food contamination levels ranged from 1.9 X 10 5 to 1.6 X 10 9 CFU (Swaminathan et al., 2007) Incubation time for the enteric form of listeriosis is short, typically between 18 and 27 h (Swaminathan et al., 2007) Whether the L. monocytogenes strains that cause gastrointestinal listeriosis in low risk populations possess additional virulence factors similar to common enteric pathogens is not known. Animal exposure studies suggest that severity of disease features is inversely proportional to dosage level (Farber et al., 1991) However, animal models do not aid in the accurate determination of the min imum infective dose in humans. Based on observational data obtained during outbreak and sporadic case investigations, it appears that the infective dose for listeriosis in at risk populations (Swaminathan et al., 2007) In nonpregnant, immunocompromised adult, li steriosis primarily presents with septicemia, meningitis, and meningeocephalitis. Less common manifestations include endocarditis (in patients with preexisting cardiac lesions), and other types of focal infections including endoophthalmitis, septic arthrit is, osteomyelitis, pleural infection, and peritonitis (Sl utsker and Schuchat, 1999) Mortality in this disease population is between 20 and 30% (Swaminathan et al., 2007)

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42 In pregnant women infected with Listeria although disease features are often mild and mainly febrile in nature, L. monocytogenes is teratogenic and readily crosses the materno fetal (i.e. placental) barrier, resulting in feto maternal infection. Most cases of fetal infection result in spontaneous abortion or stillbirth. Survivin g neonates of feto maternal listeriosis less than 7 days old present with pneumonia and sepsis, while those over 7 days old manifest meningitis and sepsis (Slutsker and Schuchat, 1999) Compelling evidence from two investigated outbreaks support the idea that gastrointestinal listeriosis can also occur in ind ividuals having no predisposing condition, with febrile gastroenteritis being the most consistent symptoms (Aureli et al., 200 0; Dalton et al., 1997; Slutsker and Schuchat, 1999) The outbreak investigated by Aureil et al. (2000) exclusively involved adolescent school children. Pathogenesis of Listeria monocytogenes The unique pathogenesis of L. monocytogenes is such that it is able to cross t hree host barriers; the intestinal barrier, the blood brain barrier (BBB), and the materno fetal (placental) barrier. Listeria monocytogenes is an intracellular, neurotropic pathogen that can cause a variety peripheral local infections, including infectio ns of CNS (Drevets et al., 2008; Vazquez Boland et al., 2001) There are many gaps in our understanding of the precise mechanisms of L. monocytogenes pathogene sis (Swaminathan et al., 2007) but much of what is known comes from cell kinetic and animal model studies. Internalin A (InlA) and InIB are the two main L. monocytogenes invasion proteins, with t he transmembrane protein E cadherin being the host cell receptor site for InlA (Cossart and Toledo Arana, 2008) InlB interacts with several host cell receptors including gCiqR/p33, Met/HGFR (hepatocyte growth factor receptor), and GAGs (glycosaminoglycans). gC1qR/p32 is a peripheral membrane bound protein known to interact with many viral proteins (Cossart and

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43 Toledo Arana, 2008) Met, a tyrosine kinase, is the receptor for HGF (hepatocyte growth factor), and likely the most important receptor for InlB (Cossart and Toledo Arana, 2008) Both InlA and InlB functionally mimic the host signaling apparatus to exploit receptor mediated endocytosis and gain cell entry. Once within the host cell, L. monocytogenes neutralizes host cell vacuolar hydrogen peroxide through the production of superoxide dismutase. Vacuole escape may be through the co opting of phagosomal machinery (Archambaud e t al., 2006) After penetrating the gastrointestinal epithelium, L. monocytogenes forces its path from cell to adjoining cell within the host using an actin based propulsion system (Ramaswamy et al., 2007) Once monocytes, macrophages, or polymorphic leukocytes are invaded, the host becomes septicemic. Because L. monocytogenes is able to penetrate m onocytes, it can be transported across the blood brain barrier (BBB) (Drevets et al., 2008; Ramaswamy et al., 2007) Specifically in mice, L. monocytogenes cr osses the BBB through transport within the Ly 6C high monocyte subpopulation (Drevets et al., 2008) Listeriosis is not an exclusively foodborne disease. Although uncommon, nosocomial outbreaks of neonatal cross infection, both through equipment cross contamination and by cutaneous route infection associated with the use of contaminated m ineral oil, have been documented (Pejaver et al., 1993; Schuchat et al ., 1991) Focal cutaneous infections by L. monocytogenes have also been observed in persons whom occupationally handle live stock, such as farmers and veterinary workers (Swaminathan et al., 2007) Epidemiology of L isteriosis Pregnant women and prenatal infants are probably the most at risk subpopulations for listeriosis (Jackson et al., 2010) The recent emergence of Listeria monocytogenes as a major pathogen of public health concern is likely due in part to the convergence of many medical,

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44 industrial, and social factors th at have resulted in larger high risk populations (Swaminathan et al., 2007) Medical factors include increased life expectancy from medical and public health improvements, the AIDS epidemic, and in creased use of immunosuppressive therapies. Industrial and social factors include the centralization and consolidation in food production and processing, the global expansion of food distribution, an increase in the use of refrigeration, changes in handli ng practices, an expanding market for frozen convenience foods (or RTE; read to eat) and fresh produce (Swaminathan et al., 2007) Improved diagnostic methods and active public health surveillance m ay also contribute to the increase in reported incidences of listeriosis (Swaminathan et al., 2007) Since the first confirmed foodborne outbreak of listeriosis in 1981, investigation of over 30 outbreaks have repeatedly concluded that consumption of contaminated food is the primary mode of listeriosis transmission (Swaminathan et al., 2007) While outbreaks get the most publicity, many cases of h uman listeriosis are sporadic, although some cases thought to be sporadic may share an unrecognized common source (Swaminathan et al., 2007) Adding to the difficulty inherent to outbreak investiga tion s non enteric listeriosis can manifest after a very long incubation time (up to 5 weeks), which can make obtaining accurate food histories especially difficult (Swaminathan et al., 2007) This practical difficulty, along with the inability to examine incriminated foodstuffs after such an extended period, often makes L. monocytogenes traceback an impossible task. For this reason, although the majority of cases of listeriosis are thought to be f oodborne, it is often impossible to positively identify a source (Swaminathan et al., 2007) Listeriosis Outbreaks Associated with Fresh P roduce In 1979 an outbreak of L. monocytogenes associated with raw produce specifically raw celery and lettuce served as garish with three foods was suspected, although the food source

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45 was not positively confirmed (Ho e t al., 1986) In the 1981 Nova Scotia outbreak which afflicted 34 pregnant women and 7 nonpregnant adults coleslaw was the suspected vehicle of transmission. The epidemic strain was later isolated from an unopened package of this locally prepared prod uct, made with cabbage that had been fertilized with the manure of diseased sheep being the likely source (Schlech et al., 1983) In the above case, harvested cabbage had been stored in an unheated shed over winter and spring; a possible environmental advantage for the survival of this psychrophile. In 2010, an outbreak of l isteriosis occurred in Texas involving 10 cases and 5 de aths over an 8 month period. Sangar Fresh Cut Produce Company was ordered by the Texas Department of State Health Services (DSHS) to recall all products shipped since January 2010, after their investigation discovered chopped celery positive for L. monocy togenes Genetic subtyping of isolates from the implicated celery linked it to 6 of the 10 cases of listeriosis. At the Sangar processing plant, inspectors cited several sanitation problems including condensation above food products, soil on a preparatio n table, and hand washing lapses ( FDA 2010) Reservoirs of Listeria monocytogenes Listeria monocytogenes has been isolated from fecal matter of healthy birds, deer, ruminates, and many other animals (Swaminathan et al., 2007; Weis and Seeliger, 1975) L isteria monocytogenes has been found in grasses and silage associated with transmission of animal l isteriosis as well as on various other naturally decaying vegetation (Husu et al., 1990; Weis and Seeliger, 1975 ; Welshimer, 1968) In the Netherlands, Listeria spp. have been found in samples from canal surface water, in lakes, in polder ditch water, and in California sewage and freshwater bay tributaries (Colburn et al., 1990; Di jkstra, 1982; Geuenich et al., 1985) In 2010, a survey conducted at Finger Lakes National Forest in New York confirmed the presence of four Listeriae isolates with phylogenetic relatedness close to, but clearly distinct from L.

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46 monocytogenes in the soil, as well as in stagnate, and flowing waters (Graves et al., 2010) Listeria spp. have also been found on alfalfa and other crops grown in soil enriched with sewage sludge (Alghazali and Alazawi, 1990) In one study involving radishes growing in soil inoculated with L. monocytogenes 50% of recovered samples were confirmed positive after 3 months (Vanrenterghem et al., 1991) The apparent abundance of L. monocytogenes in soil is like due to contamination through detritus and fecal matter, encouraged by the cool, moist soil ecosystem with nutrients being supplied from the decaying organic materials (Fenlon, 1999) Human C arriage of Listeri a monocytogenes Although listeriosis does not generally afflict healthy individuals, the healthy human population are possible asymptomatic carriers of L. monocytogenes and may aid in its environmental distribution through fecal shedding (Swaminathan et al., 2007) Listeria monocytogenes has been isolated from up to 6% of fecal samples from health individuals (Slutsker and Schuchat, 1999) and 21.6% of symptomatic patients (Jensen, 1993) Fecal specimens from patients with listeriosis were much higher than asymptomatic carriers, with 21% carrying 4 CFU/g fecal matter. Supporting the notion that L. monocytogenes is subject to gastrointestinal carriage and spread between people, one study involving six sporadic cases found that 21% of symptomatic patients and 18% of asymptomatic household contacts defecate the same serotype and multilocus enzyme type of L. monocytogenes as the patient (Schuchat et al., 1993) However, it should be noted that in some of these cases, an undetermined shared food vector between symptomatic and asympt omatic contacts may have been responsible for the intestinal carriage of both. Also of note, the prevalence of L. monocytogenes in human stool is low, and the duration of fecal shedding is short, which argues against the appropriateness of routine screeni ng of workers whom handle high risk foods as a prevention tool for the fecal

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47 spread of th is pathogen, and possible transmission to food, or to at risk worker contacts (Grif et al., 2003; Sauders et al., 2005) Listeria monocytogenes in F ood Considering the high degree of environmental adaptability and ubiquity of L. monocytogenes there are many potential routes of transmi ssion between ecosystems; animal fecal transport and water carriage being particularly suspect (Swaminathan et al., 2007) By whatever means distributed in nature, the transmission of L. monocytogenes to f ood processing plants and into food products can be attributed to worker contamination, transport equipment, from animal feces or hides, from raw plant or animal tissue, and in produce from contaminated food crops themselves, pre harvest (Swaminathan et al., 2007) Since the growth of L. monocytogenes is favored by the presence of water (though requirements are small) and nutrients, its detection in moist areas of food processing plants such as water drains, residues, and in processing equipment, is little surprise (Cox et al., 1989) The bacteria can strongly attach to many kind of surfaces including stainless steel, glass, and rubber, and is kno wn to form single as well as multi species biofilms on food contact surfaces at 10 and 21C (Jeong and Frank, 1994a; Jeong and Frank, 1994b) making it particularly difficult to eliminate from processing facilities (Gravani, 1999) Contaminated contact surfaces and dicing machinery can repeatedly contaminate RTE foods (Lunden et al., 2002) Listeriae have a lso been observed to remain on human hands after washing, and can be aerosolized (Swaminathan et al., 2007) The presence of L. monocytogenes inside food processing plants is so common that they co uld be considered a reservoir, as contaminated effluents from these plants can noticeably increase environmental spread of this species (Swaminathan et al., 2007) Epidemiological investigations o f outbreaks, along with active surveillance of sporadic cases have demonstrated that some ready to eat (RTE) foods are high risk vehicles for the

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48 transmission of listeriosis within susceptible populations (Swa minathan et al., 2007) These foods are typically preserved th rough refrigeration and supply L. monocytogenes with an appropriate environment for growth during manufacture, transportation, and storage. Such foods include fresh cut produce, soft unferm ented cheeses, minimally reheated frankfurters, certain delicatessen meats, foods mixed with mayonnaise, and some seafoods (Farber, 2000; Swaminathan et al., 2007) Multiple risk assessments for L. monocytogenes in RTE foods have been performed ( Crepet et. al., 2007 ; Erickson, 2010; Farber, 2000; FDA, 2004; Harris et al 2003 ; McLauchlin et al., 2004 ; WHO, 2004 ) Canadian regulatory policy mandates inspection and compliance action on RTE foods that are known to support the growth of L. monocytogenes with the highest priority for high risk foods that have a shelf life greater than 10 days (Farber, 2000) In 2003, the FDA in collaborat ion with the USDA Food Safety and Inspection Service (FSIS) and the CDC, published a table documenting the results of a risk assessment for RTE foods, with each food type ranked in order of risk. According to the table, delicatessen meats were the greates t risk of all foods tested, and was predicted to be the vector for over 1,598 cases of listeriosis, annually (Swaminathan et al., 2007) Also according to this study, fruits and vegetables were con sidered low risk vectors for the transmission of human listeriosis, with a median of less than one case per year. However, it should be noted that this does not account for the further handling, processing, packaging, and shipping that is involved with th e commercial distribution of fresh cut vegetables. To model this risk assessment, five factors were determined to affect consumer exposure to L. monocytogenes during food consumption; 1) the amount and frequency of consumption of each food thought to pose a potential risk, 2) the frequency and level of the bacteria in each type of RTE food, 3) the growth potential of L. monocytogenes in a food during refrigeration, 4)

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49 storage temperature, and 5) duration of storage prior to consumption (Swaminathan et al., 2007) By changing the parameters of the risk assessment model, the researchers were able to estimate the impact of control strategies. For example, according to the model, the number of estimate d cases of listeriosis could be reduced by 69% if all home refrigerators consistently operated at or below 7.2C. It was also estimated that the median number of case of listeriosis among the elderly could be reduced by 13.6% if the maximum storage time o f deli meat was reduced from 28 to 14 days (Swaminathan et al., 2007) Listeria monocytogenes in Celery and Other Fresh P roduce Once contaminated, fresh vegetables carrying L. monocytogenes are ofte n eaten without cooking, irradiation, or chemical treatment, making listeriosis a serious problem, particularly with regard to high risk populations. The relatively small number of outbreaks for which L. monocytogenes is responsible is likely due to the l ow number of high risk persons. Produce, and especially fresh cut RTE produce items have been increasingly linked to foodborne outbreaks (Sivapalasingam et a l., 2004) Crop contamination occurs both pre and post harvest, with cross contamination documented to occur through both water and harvest equipment, though the most frequent contamination v ector is the incoming product (Erickson, 2010) Pathogen survival during storage is dependent o n storage conditions and product type. Chemical intervention can effectively reduce the potential for cross contamination from harvest and processing equipment or through water, but its ability of remove p athogens from the product is quite limited (Erick son, 2010) In 2007, a Bayesian approach meta analysis of 165 studies designed to estimate the prevalence and concentration of L. monocytogenes on fresh vegetables (including celery) contaminated with more than 1, 2, and 3 log/CFU were observed in 1. 44, 0.63, and 0.17% of all samples respectively (Crepet et al., 2007) In North America, Europe, and Asia, L.

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50 monocytogenes has been isolated from many vegetables, including celery, bean sprouts cabbage, cucumbers, leafy greens, potatoes, prepackaged salads, radishes, and tomatoes (Audurier et al., 1980; Crepet et al., 2007) In many cases of foodborne human listeriosis, food contamination does not occur on the production and processing end of the supply chain, but thr ough persistent L. monocytogenes strains in retail environments (Vazquez Boland et al., 2001) With the myriad of pos sible cross contamination points, it is often impossible to ascertain the precise contamination point in any given case or outbreak. In one survey on the microbial safety of fresh cut RTE celery and other vegetables, L. monocytogenes was detected on 8 of 120 (6.7%) fresh cut celery samples stored at 10C, with 0 of 175 samples positive at 4C (Odumeru et al., 1997) One Chilea n survey detected L. monocytogenes on fresh cut celery in supermarket prepared salads in 2 of 13 (15.4%) samples taken (Cordano and Jacquet, 2009) Listeria monocytogenes Industrial Control P oints Much discussion of the problem of L. monocytogenes in various food pr ocessing environments, as well as possible control methods were provided by Gravani (Gravani, 1999) In 2002, rese archers proposed a six step Listeria control program for processing plants (Tompkin, 2002) The steps included, 1) pr evention of establishment of Listeria spp. i n niches and other sites where the bacteria may take hold, 2) implementation of sampling programs, 3) rapid and effective response to positive sampling, 4) follow up verification, 5) short term assessment of the most recent samplings to facilitate early detection and determine trends or growth patterns, and 6) long term assessment to identify scattered contamination events and measure progress. The environmental sampling design and response to positive findings d etermines the effectiveness of the Listeria control program in food processing facilities (Tompkin, 2002)

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51 Modern methods of food preservation including the use of chemical preservatives, vacuum packaging, and modified atmosphere packaging (MAP) may not be sufficient hurdles to the foodborne transmission of listeriosis (Swaminathan et al., 2007) Bacteria derived antibiotic agents such as pediocins, bacteriocins, and lactic acid can inhibit or destroy L. monocytogenes cells to varying degrees. However, caution is warranted in the use of bacteriocins as anti listerial agents since it is known that L. monocytogenes can become antibiotic resistant to bavaricin A and nisin (Davies and Adams, 1994; Davies et al., 1996; Larsen and Norrung, 1993) Certain essential oils such as carvacrol, thymol, eugenol, perillaldehyde, cinnamaldehyde, and cinnamic acid have demonstrated anti listerial activity by partitioning lipid membranes and thereby increasing cell permeability and osmolytic poten tial (Burt, 2004) L. monocytogenes on fresh produce (Prakash et al., 2000; Swaminathan et al., 2007) As well, heat stress can reduce the virulence of L. monocytogenes (Swaminathan et al., 2007) Food R egulations for Listeria monocytogenes Due to its frequent occurrence of in foods and processing environments, and the impracticality of maintaining zero tolerance, regulatory agencies in some cou ntries including Canada and France have legislated policies which direct inspection and compliance to set tolerance levels for the L. monocytogenes in high risk foods (Swaminathan et al., 2007) In the U.S.), despite their acknowledgement of the widespread distribution of L. monocytogenes and difficulty in the production of RTE foods free from contaminati on (Shank et al., 1996) This L. monocytogenes would require more data than is currently available on the number of Listeriae which do not cause human disease (Shank et al., 1996)

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52 Salmonella Biology of Salmonella Salmonella is widely distributed throughout nature and has been isolated from soil, water, foods, and animal gastrointestinal tracts ( Anderson and Ziprin, 2001; Nisbet and Ziprin, 2001 ) Coupled with this widespread distribution, intensive husbandry practices used in the meat, poultry, fish, and shellfish industries along with the recycling of offals and inedible raw materials into animal feed has favored the persistence of Salmonella in the global food chain (D'Aoust, 1994; Doyle, 1989) In 1885, Salmon and Smith were the first t o isolate and identify Salmonella (S almonella enterica serovar Choleraesuis) from swine suffering from hog cholera (Le Minor, 1981) In the early 1900s, serological advances led to methods for detection of the somatic (O) and flagellar (H) antigens of Salmonella through exploitation of antigen ant ibody specificity (Le Minor, 1981) Subsequently in 1941, White and Kauffma nn devised an antigenic scheme for Salmonella classification the Kauffmann White scheme now consisting of more than 2,500 serovars (Popoff et al., 2004) Salmonellae grow optimally at 37C, but have a growth range of 2 54C (D'Aoust et al., 1975) They are gram negative, rod shaped facultative anaerobes of the family Enterobacteriaceae. Salmonellae are usually mobile via peritrichous flagella, with serovar Pullorum and Gallinarum the afl agullate exceptions, along with various mutant strains (D'Aoust and Maurer, 2007) Salmonellae are chemoorganotrophs with both respiratory and fermentative metaboli c pathways. Often identified through presumptive biochemistry, salmonellae produce acid and gas byproducts from carbohydrate catabolism on Triple Sugar Iron growth media. Generally, during Salmonella metabolism of D Glucose, thiosulfate is utilized as a terminal electron acceptor, being reduced to hydrogen sulfide gas (D'Aoust and Maurer, 2007) H 2 S can

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53 also react with ferrous sulfate to form ferrous sulfide, a bl ack precipitate. Additionally, most salmonellae enzymatically decarboxylate lysine to cadaversine in a lysine iron media, are oxidase negative, catalase positive, can metabolize citrate as a sole carbon source, and do not hydrolyze urea (D'Aoust and Maurer, 2007) Although the above describes a template for biochemical identification, Salmonella is far from a metabolically homogeneous genus, and most serovars canno t be specifically distinguished through biochemical testing. Distorting the diagnostic landscape is the increasing occurrence of biotypes that can hydrolyze urea, produce indole from tryptophan, are not inhibited by KCN (potassium cyanide) and do not dec arboxylate lysine ( D'Aoust and Maur er, 2007) Because Salmonella metabolism of lactose and sucrose is plasmid mediated and therefore subject to genetic transfer and exchange, genetically atypical serovars and strains may escape detection by disaccharide nutrient source plating (Doyle, 1989) For this reason, bismuth sulfide agar is the preferred medi a, which utilizes secondary metabolic production of H 2 S gas as an indicator (Doy le, 1989) However, when serovar specific identification is needed, targeted biomolecular analysis aimed at identifying stable genetic loci or protein products may be required. Salmonellae demonstrate high inter and intraspecific variability and are a daptable to extreme environments. Although considered mesophiles, some strains can grow at elevated (Droffner and Yamamoto, 1991) while others exhibit psychrotrophic properties in their ability to grow on foods at refrigeration temperature (2 to 4C) (D'Aoust et al., 1975) which raises concerns regarding the efficacy of cold induced bacteriostasis. The adaptability of salmonellae has also been demonstrated in their ability to grow at pH values ranging from 4.5 to 9.5, with an optimum growth pH of 6.5 to 7.5 ( D'Aoust and Maurer 2007) In addition, the ecological adaptability inherent to a facultatively anaerobic nature contributes to

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54 salmonellae adaptability. Salmonella has been shown to grow under modified atmospheric conditions containing low levels CO 2 (20 to 50% [vol/vol]), on raw bee f and cooked crabs meat (Bergis et al., 1994; Ingham et al., 1990) Salmonella Pathogenesis To cause infection, Salmonella must survive and surpass an array of non specific host defense hurtles including lactoperoxidase in saliva, gastric acidity, mucoid sec retions from intestinal goblet cells (which containing IgA antibodies), intestinal peristalsis, sloughing off of luminal epithelial cells, and various nonspecific phagocytes, coupled with adaptive immunity associated with T and B lymphocytes (particularly immunologically innate complement system of pathogen inactivation (D'Aoust and Maurer, 2007) Invading Salmonella must also outcompete the normal intestinal microflora for resources and the space necessary for enterocyte penetration. Diarrhea is a physiological response to Salmonella infection and is the result of enterocolitis coinciding with increased goblet cell mucus s ecretion, and extensive leukocyte translocation into the infected tissues, and release of leukocytic prostaglandins triggering mucosal inflammation as well as activating epithelial cell adenyl cyclase, and signaling increased fluid secretion into the intes tinal lumen (Guttman and Finlay, 2008; Kaufmann et al., 2001; Po lotsky et. al., 1994) Failure of host defense systems can result in septicemia and degenerate int spondylitis (D'Aoust and Maurer, 2007) Upon contact with luminal epithiu m surface, Salmonella are induced to synthesize type 1 fimbriae, and transient, external, membrane bound proteinaceous invasion appendages (Collazo et al., 1995; Ginocchio et al., 1994) Once attached, signal transduction leads to the host cell

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55 pathogen pinocytation, followed by bacterial proliferation (Garcia Del Portillo and Finlay, 1994; Ginocchio et al., 1994; Jones et al., 1994) Confined to endocytotic vacuoles within the host cell, Salmonella replicate within hours following internalization (Garcia Del Portillo and Finlay, 1994) Infe cted vacuoles migrate from apical to basal pole, where Salmonella is exocytosed into the lamina propria mucosae (Isberg and Tran Van Nhieu 1994; Polotsky et al., 1994 ) Although the mechanism which allows Salmonella to migrate into deeper layers of tissue is poorly understood, it is likely that the proteolytic enzyme plasmin present on the bacterial cell surface fa cilitates further transcytosis (Sjobring et al., 1994) The putative and highly conserved Salmonella virulence factor that causes diarrhea is diarrheagenic enterotoxin. Release of the toxin into host epithelial cell cytoplasm p recipitates activation of membrane bound adenyl cyclase and increased cytoplasmic concentrations of cyclic AMP in host cells. A concurrent fluid efflux into the lumen results in increased concentration of Cl ions in the mucosa and decreased Na + absorption, increasing net fluid exsorption (D'Aoust, 1991) In addition to enterotoxin, Salmonella serovars generally manifest an ou ter membrane bound heat labile cytotoxin which functions pathogenically through the inhibition of host protein synthesis and lysis of host cells, promoting further salmonellae dissemination into host tissue (D'Aoust, 1991; Koo et al., 1984) Systemic migration exposes invading bacteria to phagocytes, leukocytes, and prevailing antibacterial conditions in the cytoplasm (D'Aoust, 1991) Host genotype, as much as the virulence of the invading salmonellae, determines whether a systemic infection will take hold and whether development of enteric fever occurs (Schwan et al., 2000; Skamene et al., 1998)

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56 Iron is an essential micronutrient found only in small concentrations within eukaryotic cells. Having inva ded a host, the virulence of Salmonella is proportional to its ability to outcompete host cells and scavenge intracellular iron through an extracellular membrane bound siderophoric receptor ( an enterochelin) (D'Aoust, 1991) Environmental factors such as low pO 2 and high osmolarity also enhance bacterial virulence by altering the superhelicity of chromosomes, which influences the level of transcription of invasion related genes such as invA (Falkow et al., 1992; Garcia Del Portillo and Finlay, 1994) It is interesting to note that these conditions are consistent with those found within mammalian intestinal epithelial cells (Falkow et al., 1992) Genetic Determinants of V irulence in Salmonella S pp. Comparative genomic studies (between E. coli and Salmonella spp.) have revealed that many Salmonella specific genes are clustered together in chromosomal loci known as pathogenicity islands. These islands gener ally map next to tRNA genes and are often bordered by phage gene remnants. Additionally, pathogenicity islands usually have a lower GC (guanine cytosine) content than is found in metabolic and housekeeping gene sequences, suggesting horizontal gene transf er was involved in the evolution of virulence in Salmonella spp. (Schmidt and Hensel, 2006) In Salmonella enterica, these genomic islands encode for virulence factors essential to pathogenesis, and are the defining characteristics of this genus and species (Ba umler et al., 1998) However, not all genes that enhance virulence are associated with pathogenicity islands. For example, genes of various fimbriae operons which are believed to be important contributors to Salmonella host cell colonization and exoge nous, gastrointestinal hydrogen sequestration and respiration which is thought to provide invading Salmonella with a competitive advantage are not localized within pathogenicity islands (Maier et al., 2004; van Der Velden et al., 1998) The Salmonella pathogenicity island SPI1 is highly conserved within

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57 the species and between serovars, making it a useful gene target for PCR based detection in foods (Chen et al ., 1997) There is a great deal of intraspecific, genetic variation in S. enterica with 2 to 8% of the Typhimurium LT2 gene complement (ca. 4,500 genes) absent in one or more serovars (Chan et al., 2003; Porwollik et al., 2004) Much of this variability is attributed to various virulence genes found mobile gene elements (i.e. phages, plasmids, and transposons) and pseudogenes (i.e. those derived from point mutation, insertion, or deletions that result in nonsense or frameshift mutations) and the distribution of these elements among serovars (Ahmer et al., 1999; Bacciu et al., 2004; Bruss ow et al., 2004; Edwards et al., 2002; Parkhill et al., 2001) Salmone lla spp. have evolved such that acquisition of mobile gene elements and pseudogenetic polymorphisms can have a profound impact on the behavior specific serovars in vivo (D'Aoust and Maurer, 2007) Epigenetically, subtle changes in expression specific to a serovar ( Monsieurs et al., 2005; Winfield and Groisman 2004) may have an even more profound impact on host adaptation than mobile element variation (Encheva et al., 2005; McClelland et al., 2004; Monsieurs et al., 2005; Porwollik et al., 2004; Porwollik et al., 2005; Winfield and Groisman, 2004) Subtle mutations in promoter and regulatory sequences may also explain differences in behavior that occur within bacterial populations (Koutsolioutsou et al., 2001; Robbe Saule et al., 2003) Etiology and Clinical Features of Human Salmonellosis: Nontyphi S erotypes Salmonellosis is the enteric or systemic disease caused by Salmonella Manifestation s of the disease can vary depending on serotype and host. Certain highly host adapted serotypes cause disease almost exclusively in their specific hosts, while others exhibit a broad range of host specificity ( Ziprin and Hume 2001) In humans, the most highly host adapted organism known to medicine is Salmonella Typhi, which causes typhoid fever ( Anderson and Ziprin 2001)

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58 Exclusive to humans, Salmonella Typhi is contracted from water or food contaminated by another human source ( Ziprin and Hume 2001 ; Ohashi, 1988) Salmonella Choleraesuis is a swine pathogen that also causes highly fatal systemic infections in humans (Ziprin, 1994) The high fatality rate of Salmonella Choleraesuis is due in part to the fact that the infection can cause mycotic aneurysms (infection and weakening of the arterial walls) in human hos ts. Fortunately the incidence of Salmonella Choleraesuis is low, which may be attributable to the fact that most people are aware of the importance of properly cooking pork in foodborne disease prevention. Salmonella Enteritidis and Salmonella Senftenbe rg are somewhat host adapted to chickens and turkeys, respectively (Ziprin, 1994) Both are capable of causing severe human illness and death. Salmonella Enteritidis is c onsidered to be one of the major problem organisms among human bacterial diseases ( Ziprin and Hume 2001) and of particular public health concern because chicken eggs are sometimes contaminated by this serovar, which can lead to infection if eggs are not properly cooked (Ziprin, 1994) Salmonella Dubl in is another host adapted serovar. Salmonella Dublin causes septicemia in cattle and can cause zoonotic infections in human beings through the consumption of unpasteurized milk. Except as noted above, most Salmonella serovars that infect humans are not host adapted, but are instead found in a wide range of animal products, fruits, vegetables, and processed foods ( Anderson 2001) Although these serovars tend to be less virulent, they are responsible for the majority of cases, primarily presenting as uncomplicated enterocolitis (Hunter, 1997; Underman, 1997; Ziprin, 1994) All human cases of nontyphoid salmonellosis share many common clinical features. Onset is rapid (but ranges from hours to days depending on dose), and symptoms typically

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59 include enterocolitis and diarrhea, although systemic infection s can occur (Hui, 2001) Death is possible, especially with more virulent strains, and is more likely to occur in high risk subpopulations (i.e. the very young, aged, and immunologically compromised). The etiological sources of non Typhi Salmonella spp. include food, water, animals and animal products and human feces (Hui, 2001) Although many cases of salmonellosis are self resolving, antibiotics such as choloramphenicol and ampicillin are the mainstay treatments in cases where medical intervention is necessary (Hui, 2001) Possibly because of global overuse of antibiotic therapy, the prevalence of multidrug resistant (MDR) strains is ever increasing (Hui, 2001) Epidemiology of S almonellosis Associated with P roduce Between 1990 and 2005, 18% of all cases of produce associated foodborne outbreaks were cau se by Salmonella (CSPI, 2009) In all cases from 1998 to 2002 where the causative agents were determined to be bacterial and were serologically identified, Salmonella enterica accounted for the greatest proportion (9%). In 2001, the CDC reported 46 confirmed outbreaks of Salmonella Enteritidis (Tucker, 2003) Those outbreaks resulted in 1,681 reported illnesses, 102 ho spitalizations, and no deaths. In 24 (52%) of the 2001 cases where phage typing was performed, type 8 accounted for 28%, despite being sourced to different geographic locations and food items. The dispersed character of these outbreaks from a common stra in suggests that contamination likely occurred at a critical point prior to distribution (i.e. during the production or processing stages). Of the 2001 outbreaks where a location of consumption was known, 61% occurred in commercial food establishments (e. g. restaurants, caterers, etc.), 2% in long term care facilities, 4% in correctional facilities, and 30% in the general community (i.e. private homes, day care facilities, community centers, and religious meeting places). From 1998 to 2002, 306 (31%) of o utbreaks of foodborne bacterial pathogens where etiology was sourced to a location of consumption occurred in the home (Lynch et al., 2006)

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60 Proportional to the total volume consumed, the amount of contaminated fresh produce is relatively small (CAST, 2009) However, th e total number of outbreaks involving fresh fruits and vegetables is significant and increasing. In the 1990s, at least 12% of foodborne outbreaks were associated with fresh produce (FDA, 2004) Between 1973 and 1997, 190 produce associated outbreaks involving 16,058 illnesses and 8 deaths were reported to the C DC (Sivapalasingam et al., 2004) The mean number of these outbreaks inc reased from 4.4 per year between 1973 and 1987, to 9.75 per year between 1987 and 1997 (Tauxe, 1997) As a percentage of all reported outbr eaks, those associated with produce increased from 0.7% in the 1970s to 6% in the 1990s (Sivapalasingam et al., 2004) Also during this time, the annual median number of reported incidences per outbreak related to fresh produce increased from 21 to 43. Continuing on this upward trend over the next 4 years from 1998 to 2002, the number of reported outbreaks increased to 279, with 10,533 illnesses and 7 deaths (Lynch et al., 2006) Therefore, despite significant effort and governmental resources dedicated to prevention (CAST, 2009) the frequency of produce related foodborne illness outbreak s has increased, and outbreaks tend to involve a larger numbers of people. According to De Roever (1998), these increases are due to, 1) centralization and amplification of production; 2) increased regional and global distribution; 3) increased consumptio n of fresh or minimally processed products; 4) increased popularity of salad bars and restaurant dinning; and 5) increased consumer preference for organically cultivated produce, which may increase risk of using improperly composted manure However, new technologies in biomol ecular traceback and serotyping are epidemiological confounding factors, contributing to the increase in documented outbreaks but not necessarily reflecting an actual increase in disease frequency.

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61 CHAPTER 3 MATERIALS AND METHOD S Strain I nformation Ant ibiotic resistant bacterial stock strains of E. coli O157:H7, L. monocytogenes and Salmonella from the Danyluk laboratory culture collection were used in all experiments. These cultures were stored at 80C in tryptic soy broth with 15% glycerol on 3 mm glass beads. A five strain cocktail of E. coli O157:H7, was used (Table 3 1). These strains included, H1730 (isolate from lettuce outbreak; human feces), F4546 (isolated from alfalfa sprout outbreak; human feces ) 223 (isolated from the Odwalla apple juice outbreak ) EC4042 (isolated from spinach outbreak), and F658 (isolated from cantaloupe outbreak). Four strains of L. monocytogenes were used (Table 3 2), including LCDC 81 861 (isolated f rom raw cabbage outbreak), SCOTT A (isolated from perinatally transmitted human milk outbreak), 101M (isolated from beef outbreak), and strain V7 (isolated from cow milk outbreak). Five serovars of Salmonella were used to create a cocktail (Table 3 3), whi ch included: Michigan (LJH 521, isolate from cantaloupe outbreak; human feces), Montevideo (G4639, isolate from tomato outbreak; human feces), Enteritidis phage type 30 (ATCC BAA 1045, isolated from raw almond outbreak; human feces), Agona (LJH 517, isolat ed from alfalfa sprout outbreak; human feces), and Gaminara (F2712 isolated from orange juice outbreak; human feces). All strains used were antibiotic resistant. E scherichia coli O157:H7 and Salmonella R if), while L. monocytogenes strains were

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62 exposure to increasing concentrations of antibiotic, for the purpose of inhibiting the growth of background microflora (Parnell et al., 2005) Preliminary E xperiments For each pathoge n, small, trial experiments were performed before full scale experiments in order to determine the necessary inoculum levels and to minimize excess plating during recovery. These experiments were performed at three temperatures (4, 12, and 22C) and follo wed the same procedure as the statistically significant replications discussed below. For purposes of increasing the level of detection, some trials varying the volume of recovery buffer were performed to determine the lowest volume necessary to achieve a dequate sample maceration. To determine whether atmospheric levels of O 2 and CO 2 within sealed bags and containers were altered by sample respiration, six uncontaminated celery samples (three in bags and 3 in containers) were held at 4, 12 or 22 C (N=18), with atmospheric prob readings recorded once every 24 h. Data were recorded in %O 2 and assumed %CO 2 accumulation was calculated as the difference from initial O 2 At 22 C data was collected over 3 days, and at 4 and 12 C data was collected ov er 7 days. Celery Prior to each experiments, bags of fresh to afternoon, within one day of store delivery and held a t 4 2C for up to 24 h. Immediately prior to inoculation, celery sticks were further cut to 10 1 g using a sterile, serrated knife and cutting board.

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63 Antibiotic and Growth M edium P reparation Stock solutions of NA were prepared by dissolving 0.5 g in 100 ml of deionized water, stored in the refrigerator (4 2C) until use. For L. monocytogenes experiments, 10 ml of stock NA was added to 1 L cooled (ca. 45C) m odified Oxford m edia (MOX), resulting in a final Stock solutions of R if were prepared by dissolving 1.0 g in 20 ml methanol, resulting in a sterilized as described ab ove, and stored in the refrigerator until use. For E. coli O157H7 and Salmonella experiments, 2 ml of stock Rif was added to 1 L E. coli O157:H7, the nonselective media used was tryptic s oy a gar (TSA), and sorbitol MacConkey agar (SMAC) the selective media. For Salmonella TSA was also the nonselective media used, and b ismuth s ulfite s gar (BSA) as the selective media. All growth media, were supplemented with one of the abo ve referenced antibiotics (TSAR, BSAR,SMACR, MOXN), with the exception of TSA destined for enumeration of background microflora. Inoculum P reparation Prior to each replication, frozen stock cultures of each strain were streaked onto TSAR or TSAN and incu bated at 35 2C for 24 h. A single colony from each strain was then transferred to 10 ml tryptic Rif (TSBN) two times at 24 h intervals, and incubated at 35 2C for 24 h, prior to their use as inocu la. Each culture was centrifuged at 3,000 g for 10 min. Cells were washed twice in 10 ml 0.1% peptone after pouring off the supernatant and resuspending. Washed cells were resuspended in half the original culture volume. Each strain was plated out on TSAR or TSAN to confirm cell

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64 concentration. All cocktail strains were then combined in equal volumes (2 ml of each strain ) to make final inoculum cocktail. The cocktail was then diluted in 0.1% peptone to arrive at a final inoculum concentration of ca. 1 0 7 CFU/ml. The cocktail was stored on ice for up to 1 h, prior to sample inoculation. Inoculation and S torage Celery was spot inoculated with 10 l (3 5 drops) of the cocktail on either the cut or the uncut surface, resulting in ca. 10 5 CFU/ 10 g sample prior to drying at ambient temperature for 1 h in a biosafety cabinet. Post drying concentrations were ca. 10 3 CFU/g. After drying, each sample was transferred to either a 16.5 x 8.25 cm polyethylene plastic press to seal snack bag (Great Value) or 4 (Gladware Side Dish) and stored at one of three temperatures: 4 2C, 12 2C, or 22 2C. Enumeration For samples held at 4 or 12 2C, populations were enumerated after 0, 1, 3, 5, and 7 days, and for samples held at 22 2C, populations were enumerated after 0, 0.33, 0.71, 1, and 2 days. At each sampling point, samples were transferred into 1.6 L whirl pak bags, combined with 40 ml 0.1% peptone solution, and placed in a stomacher at high speed fo r 1 minute. Serial dilutions where made in 0.1% peptone and surface plated (0.1 ml) in duplicate onto selective and nonselective media containing R if or NA. To increase the limit of detection to 0.6 CFU/ml, 1 ml of the lowest dilution was distributed ove r four plates (0.25 ml/plate) of nonselective and selective media. Control samples were plated onto nonselective and selective media supplemented with the antibiotic, as well as onto TSA to determine background microflora. C, nonselective for 24 hours and selective media for 48 hours. After incubation, colonies were counted by hand and population levels were calculated in log CFU/g celery.

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65 Statistics Data were calculated in mean log CFU/g. Values were the average of duplic ate plate counts, and the average again of six replicates One experiment was performed at each of the three temperatures for each pathogen. adjusted ANOVAs using SAS 9.2 for Windows. For each patho gen, all experimental factors were compared simultaneously. Comparisons of mean log CFU/g were made between bagged and container stored samples, cut and uncut inoculation surfaces, media types, and between as well as within time intervals. Possible combi ned effects between storage container type and site of surface inoculation were also examined. Comparisons were also made between temperatures, but not between pathogens. Differences between mean values were considered significant at P

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66 Table 3 1 Escherichia coli O157:H7 serotypes used, confirmed original source of isolate, original isolate designation, and laboratory identification code Serotype Source Designation Lab code E. coli O157:H7 Odwalla outbreak 223 MDD 20 E. coli O157:H7 C linical cantaloupe F658 MDD 326 E. coli O157:H7 C linical lettuce H1730 MDD 18 E. coli O157:H7 C linical alfalfa sprout F4546 MDD 19 E. coli O157:H7 C linical spinach EC4042 MDD 321 T able 3 2 Listeria monocytogenes strain used, confirmed original source of isolate, original isolate designation, and laboratory identification code. Species Source Strain/Designation Lab code L. monocytogenes R aw cabbage LCDC 81 861 MDD 328 L. monocytogenes M ilk outbreak, human SCOTT A MDD 329 L. monocytogenes B eef outbreak 101M MDD 330 L. monocytogenes M ilk outbreak, bovine V7 MDD 331 Table 3 3. Salmonella serovar used, confirmed original source of isolate, original isolate designation, and laboratory identification code Serovar Source Designation Lab code Enteriti dis PT30 R aw almonds ATCC BAA 1045 MDD 2 Agona A lfalfa sprouts LJH 517 MDD 17 Gaminara O range juice F2712 MDD 21 Michigan C antaloupe outbreak LJH 521 MDD 24 Montevideo T omato outbreak G4639 MDD 22

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67 CHAPTER 4 RESULTS Background M icroflora Throughout all experiments, control samples were enumerated for background microflora on TSA Background micro flora ranged from ca. 6 to 10 log CFU/g of celery and were highly variable between sample s (data not shown). Escherichia coli O157:H7 Escherichia coli O157:H7 populations held at 4 2C decreased significantly over 7 days ( P 2. 0 log, with the greatest decrease observed in samples inoculated on uncut surfaces and stored in polyethylene containers (Table 4 1). No significant differ ences exist ed between populations on days 0 and 1 ( P population size decreased significantly at 4 2C under all conditions ( P mean population size from day 3 to 5 at 4 2C were insi gnificant ( P was observed in population decreases between days 5 and 7 ( P conditions, there was no significant difference observed between nonselective (TSAR) and selective (SMACR) media types ( P 0.05). Initial (0 d) mean population size was significantly different on cut versus uncut inoculation surfaces ( P celery stored in bags and containers on days 3, 5, and 7 at 4 2C, wi th bagged samples having larger mean population sizes than those in containers (all P At 12 2C, although under all conditions there was a significant increase from day 0 to day 1 ( P 3 or 5 ( P E. coli O157:H7 populations remained relatively stable over 7 days (Table 4 2). Initial (0 d) population

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68 size was smaller on uncut versus cut surfaces at 12 2C. No significant differences in mean population sizes were observed between bag s and containers (all P At 22 2C, mean E. coli O157:H7 populations did not change dramatical ly, although from day 0 to day 1 under all conditions there was a slight but significantly increase in samples stored in bags ( P 3). Between 0 and 0.33 days, overall population size did not change significantly ( P increase in population size ( P differences in population sizes (all P inoculated on cut surfaces had larger mean populations than those inoculated on uncut surfaces ( P ved between bags and containers at any sample point ( P Listeria monocytogenes At 4 2C over the course of the experiment, mean L. monocytogenes populations decreased significantly under all conditions ( P log CFU/g (Table 4 4) ; the only significant decrease between consecutive sample points was between days 0 and 1 ( P Relatively large standard deviations (SD) were seen for all the L. monocytogenes experiments. No significant differences were observed between containe r type or inoculation location ( P 0.05). Samples inoculated on cut surfaces and stored in bags always had larger mean populations than those inoculated on uncut surfaces and stored in containers ( P inoculated on uncut surfaces and stor ed in bag had larger mean populations than those inoculated on uncut surfaces and stored in container ( P The behavior of L. monocytogenes populations held at 12 2C was variable, with little significant change over 7 days (Table 4 5). The only significant changes in mean population size was a slight but significant increase between days 3 and 5 ( P

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69 condition other than samples inoculated on cut surfaces and stored in bags ( P samples showed signifi cantly larger mean populations than days 0, 1, or 3 ( P had significantly larger mean populations than days 0 or 1 ( P size was significantly smaller on uncut versus cut surfaces ( P nt differences were observed between inoculation sites ( P populations inoculated on cut surfaces were larger than those inoculated on uncut surfaces ( P 0.05). No significant differences were observed between b ags and containers at any sampling point ( P At 22 2C, a ca. 0.3 log CFU/g increase was observed in mean L. monocytogenes populations over 2 days, under all conditions (Table 4 6). This small increase was determined to be significant ( P =0.04), with mean populations on day 0 smaller than those on day 2 ( P Changes in population sizes were not linear. M ean population of samples inoculated on uncut surfaces decreased significantly ( P en increasing significantly to near initial size over 0.33 to 0.71 days ( P there was no significant change in mean population size (all P 0.71 and day 2, mean populations increased slightly, thoug h significantly ( P = 0.01). No significant differences in mean population size were observed between container type or inoculation sites at any sample point ( P Salmonella Cut celery held at 4 2C were inoculated with the Salmonella cocktail at ca. 3 log CFU/g. Populations decreased significantly under all conditions, by ca. 1 log CFU/g, at a rate of 0.1 0.2 log CFU/g/day ( P 7). There were no significant differences in survival between bags and containers ( P lations were significantly greater on cut verses uncut sample inoculation sites throughout the experiment ( P

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70 day 0 ( P was not significant ( P Salmonella populations remained relatively stable between 3.0 to 3.5 log CFU/g for 7 days at 12 2C, under all conditions (Table 4 8). Although there was a significant decrease between day 0 and day 7 ( P ive sample days were insignificant ( P most significant decrease was seen between days 1 and 5 in samples inoculated on uncut surfaces ( P significantly smaller on uncut versus cut surface s ( P between samples stored in bags and those in containers ( P selective media at any sampling point ( P combined effects were observed between exp erimental factors (all P All Salmonella populations increased significantly by ca. 1.5 2 log CFU/g over 2 d at 22 2C, under all conditions ( P 9). The greatest growth (2.1 log CFU/g over 2 days) was observed on the cut surface of samples stored in containers, which on day 7 were significantly greater than those stored in bags, regardless of inoculation site ( P all conditions, growth appears to manifest as a sinuous curve, with the majority occurring at earlier sa mpling points and beginning to taper off after 0.71 days. After 0.71 days, changes in population size were statistically insignificant ( P inoculation on day 0, population size was significantly greater on cut versus uncut surfaces ( P 0.05). With the exception of day 7, there were no significant differences between bags and containers ( P sampling point ( P

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71 Table 4 1. Recovery of Escherichia coli O157:H7 from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 4 2C for up to 7 days. Values are the mean a (log CFU/g) of duplicate samples (n=6) enumerated on TSAR and SMACR. Bag Container Cut Uncut Cut Uncut Days TSAR S MACR TSAR SMACR TSAR SMAR TSAR SMACR 0 4.50.1 A ,a 4.30.1 3.50.9 A,b 2.40.6 4.50.1 A 4.30.1 3.50.9 A,b 2.40.6 1 4.40.1 A 3.60.7 4.30.2 A 3.40.8 4.40.2 A 3.80.7 3.61.1 A 3.31.2 3 3.50.8 B ,1 2.80.8 3.41.3 B 2.21.3 3.20.8 B 2.40.5 1.91.4 B, 2 1.20.9 5 3.70.7 B, 3 2.60.2 2.61.4 B, 1.60.9 2.50.2 B,4 1.90.4 2.51.3 B 1.60.9 7 3.01.0 C ,5 2.20.6 2.51.1 B, 1.70.8 2.50.5 B,a, 6 1.80.6 1.00.5 C ,b 0.80.3 a Mean standard deviation of duplicate samples from each of six replications. Within columns, means with different capital letters ar e significantly different. Within rows, means with different lower case letters (cut vs uncut) or numbers (bag vs container) are significantly different ( P 0.05). Table 4 2 Recovery of Escherichia coli O157:H7 from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 12 2C for up to 7 days. Values are the mean a (log CFU/g) of duplicate samples (n=6) enumerated on TSAR and SMACR. Bag Container Cut Uncut Cut Uncut Days TSAR SMACR TSAR SMACR TSAR SMACR TSAR SMACR 0 4.70.1 A 4.60.1 4.20.7 A 3.30.9 4.70.1 A 4.60.1 4.20.7 A 3.30.9 1 5.10.1 B 5.10.1 4.90.2 B 4.90.3 5.10.1 B 5.00.1 4.60.2 A 4.50.2 3 4.90.6 B 4.80.5 4.30.4 A 4.20.4 4.50.8 A 4.40.7 4.20.5 A 4.30.3 5 4.60.6 A 4.30.7 4.20.6 A 4.10.6 4.20.4 A 4.40.3 4.50.2 A 4.40.3 7 4.70.6 A 4.50.5 4.30.6 A 4.30.6 4.40.3 A 4.20.2 3.90.6 A 4.00.2 a Mean standard deviation of duplicate samples from each of six replications. Within columns, means with different capital letters are significantly different. Within rows, means with different lower case letters (cut vs uncut) or numbers (bag vs container ) are significantly different ( P 0.05 ).

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72 Table 4 3. Recovery of Escherichia coli O157:H7 from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 22 2C for up to 2 days. Values are the mean a (log CFU/g) of duplicate samples (n=6) enumerated on TSAR and SMACR. Bag Container Cut Uncut Cut Uncut Days TSAR SMACR TSAR SMACR TSAR SMACR TSAR SMACR 0 4.60.1 A 4.60.1 4.40.1 A 3.50.8 4.60.1 A 4.60.1 4.40.1 A 3.50.8 0.33 4.70.5 A,a 4.80.6 4.10.4 A,b 4.00.4 4.90.3 A ,a 4.70.3 4.10.6 A ,b 4.00.6 0.71 5.00.2 B,c 4.80.2 4.50.5 A,d 4.40.5 5.10.4 A ,c 4.80.3 4.50.4 A ,d 4.40.4 1 5.30.5 B,e 5.10.4 4.80.2 B,f 4.60.1 5.20.4 A ,e 4.90.3 4.70.6 A ,f 4.30.6 2 5.00.3 B 4.80.3 4.80.3 B 4.50.6 4.90.4 A 4.70.4 4.70.5 A 4.40.5 a Mean standard deviation of duplicate samples from each of six replications. Within columns, means with different capital letters are significantly different. Within rows, means with different lower case letters (cut vs uncut) or numbers (bag vs container ) are significantly different ( P 0.05). Table 4 4. Recovery of Listeria monocytogenes from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 4 2C for up to 7 days. Values are t he mean a (log CFU/g) of duplicate samples (n=6) enumerated on MOXN. Bag Container Days Cut Uncut Cut Uncut 0 3.70.7 A 3.50.6 A 3.70.7 A 3.50.6 A 1 2.90.6 B 3.40.7 A 2.90.7 B 2.70.7 B 3 2.60.2 B,1 2.90.5 A 2.91.0 B 1.60.6 C,2 5 2.40.2 B 2.60.2 B 2.00.3 B 2.10.2 B 7 2.30.9 B, 3 2.40.2 B 2.20.4 B 1.70.2 C,4 a Mean standard deviation of duplicate samples from each of six replications. Within columns, means with different capital letters are significantly different. Within rows, means with different lower case letters (cut vs uncut) or numbers (bag vs container ) are significantly different ( P 0.05 ).

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73 Table 4 5. Recovery of Listeria monocytogenes from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 12 2C for up to 7 days. Values are the mean a (log CFU/g) of duplicate samples (n=6) enumerated on MOXN. Bag Contai ner Days Cut Uncut Cut Uncut 0 3.90.5 A,a 3.20.6 A,b 3.90.5 A ,a 3.20.6 A ,b 1 3.50.6 A 3.30.8 A 3.30.6 A 3.50.7 A 3 4.50.3 B,c 2.60.3 B,d 3.50.9 A 3.20.9 A 5 4.11.0 C 4.00.8 C 4.20.8 B 4.21.0 B 7 4.11.2 B ,C 4.00.9 C 4.51.1 B 4.20.7 B a Mean standard deviation of duplicate samples from each of six replications. Within columns, means with different capital letters are significantly different. Within rows, means with different lower case letters (cut vs uncut) or numbers (bag vs container ) are significantly different ( P 0.05). Table 4 6. Recovery of Listeria monocytogenes from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 22 2C for up to 2 days. Values are t he mean a (log CFU/g) of duplicate samples (n=6) enumerated on MOXN Bag Container Days Cut Uncut Cut Uncut 0 3.30.8 A 3.40.8 A 3.30.8 A 3.40.8 A 0.33 2.90.2 A 2.50.3 B 2.80.2 A 2.70.3 B .071 3.40.3 B 3.20.5 A 3.60.8 B 3.00.3 A 1 3.80.9 B 3.20.4 A 3.20.7 B 3.50.3 C 2 3.80.5 B 3.80.3 C 4.11.0 B 3.80.5 C a Mean standard deviation of duplicate samples from each of six replications. Within columns, means with different capital letters are significantly different. Within rows, means with different lower case letters (cut vs uncut) or numbers (bag vs container ) are significantly different ( P 0.05).

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74 Table 4 7. Recovery of Salmonella from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 4 2C for up to 7 days. Values are the mean a (log CFU/g) of duplicate samples (n=6) enumerated on TSAR and BSAR. Bag Container Cut Uncut Cut Uncut Days TSAR BSAR TSAR BSAR TSAR BSAR TSAR BSAR 0 3.50.1 A,a 3.70.2 3.30.2 A,b 2.80.4 3.50.1 A ,a 3.70.2 3.30.2 A ,b 2.80.4 1 3.50.2 A,c 3.40.1 2.70.1 B,d 2.50.3 3.50.3 A ,c 3.50.3 2.90.4 A ,d 2.70.4 3 3.00.2 B 3.00.3 2.70.5 B 2.40.5 3.00.5 A 3.40.4 2.40.5 A 2.40.4 5 3.20.2 B, e 3.00.1 2.50.7 B,f 2.30.7 2.90.6 A ,e 2.676 1.80.6 B ,f 1.70.5 7 2.50.3 C 2.50.2 2.00.5 B 1.90.6 2.80.4 B 2.80.5 2.10.6 B 2.00.6 a Mean standard deviation of duplicate samples from each of six replications. Within columns, means with different capital letters are significantly different. Within rows, means with different lower case letters (cut vs uncut) or numbers (bag vs container ) are significantly different ( P 0.05). Table 4 8. Recovery of Salmonella from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 12 2C for up to 7 days. Values are the mean a (log CFU/g) of duplicate samples (n=6) enumerated on TSAR and BSAR. Bag Container Cut Uncut Cut Uncut Days TSAR BSAR TSAR BSAR TSAR BSAR TSAR BSAR 0 3.50.1 A,a 3.60.2 3.00.3 A,b 3.00.3 3.50.1 A ,a 3.60.2 3.00.3 A,b 3.00.3 1 3.70.1 A,c 3.70.2 3.10.2 A,d 2.90.3 3.70.2 A,c 3.80.1 3.00.3 A,d 2.90.4 3 3.40.2 A 3.60.2 3.00.3 A 3.00.4 3.50.2 A 3.60.1 3.10.4 A 3.30.2 5 3.20.3 B 3.30.3 3.00.3 A 2.80.5 3.30.3 B 3.30.3 3.10.4 A 3.20.2 7 3.20.4 B 3.10.4 2.90.5 A 3.10.5 3.20.4 B 3.20.4 2.60.4 A 2.60.4 a Mean standard deviation of duplicate samples from each of six replications. Within columns, means with different capital letters are significantly different. Within rows, means with different lower case letters (cut vs uncut) or numbers (bag vs container) are significantly different ( P 0.05).

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75 Table 4 9. Recovery of Salmonella from celery inoculated at approximately 3 log CFU/g onto cut or uncut surface and stored in either a bag or container held at 22 2C fo r up to 2 days. Values are the mean a (log CFU/g) of duplicate samples (n=6) enumerated on TSAR and BSAR. Bag Container Cut Uncut Cut Uncut Days TSAR BSAR TSAR BSAR TSAR BSAR TSAR BSAR 0 3.60.1 A 3.80.1 3.40.2 A 3.40.1 3.60.1 A 3.80.1 3.40.2 A 3.40.1 0.33 4.80.1 B,a 4.60.4 3.40.3 A,b 3.50.3 4.80.2 B ,a 4.80.2 3.70.3 A ,b 3.70.8 0.71 5.20.5 C,c 5.30.4 4.60.3 B,d 4.60.5 5.40.2 C ,c 5.40.2 4.20.4 B ,d 4.50.0 1 5.10.5 C,e 5.00.4 4.50.0 B,f 4.50.0 5.40.3 C ,e 5.30.2 4.60.1 C ,f 4.60.1 2 5.20.4 C,g ,1 5.10.5 4.80.1 B,h 4.80.2 5.70.1 C, I, 2 5.70.1 4.90.1 D ,j 4.90.0 a Mean standard deviation of duplicate samples from each of six replications. Within columns, means with different capital letters are significantly different. Within rows, means with different lower case letters (cut vs uncut) or numbers (bag vs container ) are significantly different ( P 0.05 ).

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76 CHAPTER 5 DISCUSSION The behavior of foodborne pathogens at refrigeration and ambient temperatures on a variety fresh and fresh cut produce is well established (Aruscavage et al., 2006; Heaton and Jones, 2008; Strawn et al., 2011) H owever little is known about the fate of human foodborne pathogens on celery. At 4 2C, populations of all three pathogens decreased by ca. 1 log CFU/g under all the conditions evaluated. This result was expected for E. coli O157:H7 and Salmonella. In a companion study of chopped romaine lettuce and shredded carrots (Zhao et al., 2010) populations of both E. coli O157:H7 and Salmonella behaved in a similar manner decreaseing by ca. 0.5 log CFU/g over 7 days when held at 4 C Listeria mono cytogenes was not evaluated by Zhao et al. (2010). Comparable behavior of Salmonella and E. coli O157:H7 has also been observed on minimally processed peaches, diced celery, leafy green s and herbs, zucchini squash, rutabaga, soybean sprouts, and alfalfa s prouts (Alegre et al., 2010; Prakash et al., 2010; Duffy et al., 2005; Weissinger et al., 2000; Hsu et al., 2006; Castro Rosa et al., 2010; E. coli O157:H7 and Salmonella populations declined slowly, but survived for 28 days (Strawn and Danyluk, 2010a; Strawn and Danyluk, 2010b) On mango held at 4C, Salmonella populations increased by ca. 1 log CFU/g after 1 day, but E. coli O157:H7 populations remained stable over 28 days (Strawn and Danyluk, 2010b) In contrast to the reduction we saw for L. monocytogenes on celery, L. innocula populations inoculated onto peaches held at 5C increased by ca. 0.5 log CFU/g over 6 days (Alegre et al., 2010). However, L. monocytogenes did not grow at 4C on rutabaga and shredded lettuce, and populations significantly decreased by 2 and 1.5 log CFU /g on dry coleslaw and

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77 soybean sprout, respectively On both shredded carrots and lettuce held at 4C, L. monocytogenes populations did not change (Kakiomenou et al., 1998 ) Survival of a pathogenic microorganism on produce is dictated by its metabolic capabilities, which can be greatly influenced by intrinsic ( e.g surface moisture) and extrinsic environmental factors ( e.g atmospheric changes) (Beuchat, 2002) Modified atmospheres develop within sealed storage devices containing fresh produce du e to plant cell respiration (Francis and O'Beirne, 2001) Atmospheric changes m ay affect the fate of some bacteria. Atmospheric conditions of up to 30% CO 2 do not inhibit the growth of E. coli O157:H7 on vegetables (Diaz and Hotchkiss, 1996; Francis and O'Beirne, 2001) but L. monocytogenes is known to be sensitive to certain environmental conditions (Belessi et al., 2011; Francis and O'Beirne, 2001) The concentrations of O 2 and CO 2 within packages can vary by food product rn e, 2001). In semipermeable (permeability to O 2 of 1200 ml/m 2 /day/atm and to CO 2 of 4000 ml/m 2 /day/atm) polypropylene packaging bags containing lettuce and ru tabaga, O 2 fell and CO 2 increased to achieve 6%/6% (O 2 /CO 2 ) and 4%/10%, respectively, over 7 days. With soybean sprouts and coleslaw mix, atmospheric changes were much greater, equilibrating at 0%/23% and 0%/25% (O 2 /CO 2 ), respectively, over 7 days. The r elatively modest atmospheric changes observed in packages containing lettuce and rutabaga were not found to affect the fate of L. monocytogenes However, growth of L. monocytogenes was suppressed by the atmospheric changes observed in packages containing soybean sprouts and coleslaw mix (Francis and O'Beirne, 2001) The inhibitory e ffect of high CO 2 concentrations on L. monocytogenes is related to decreased pH and interference with various metabolic machinery (Chen et al., 2003; Dixon and Kell, 1989; Hudson et al., 1994) However, we observed very l ittle passive

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78 accumulation of CO 2 at any temperature (data not shown), and do not believe that the respiration of fresh cut celery within sealed containers was sufficient to influenced pathogen behavior. The diversity and abundance of background microflora can also affects the growth of L. monocytogenes with lactic acid bacteria, enterobacteria, and pseudomonads being proven competitive inhibitors (Carlin et al., 199 5 ; Francis and O'Beirne, 1998a; Francis and O'Beirne, 1998b; Vescovo et al., 1996) The indigenous aerobic microflora of 13 different vegetables obtain from Spanish farms and markets has been evaluated and it was determined that celery ranked third highest in background cell concentration after beet leaves and artichoke, with average an background MPN of 2.2 x10 10 cells/100g celery (Garcia Villanova et al., 1987). S imilarly, we observed high populations (10 6 to 10 10 log CFU/g), and what visually appear ed to be diverse populations (noticeable differences in color and colony formation on TSA plates) of background microflora on control samples. This large background mi croflora inherent to celery may have contributed to the behavior of L. monocy togenes An atmosphere modified by sample respiration, large endogenous populations of microflora, and the relatively limited nutrient availability of celery (FDA, 2009) with likely differences in nutrient availability and water activity between cut and uncut surfaces ( Beuchat and Scouten, 2002; Sapers et al., 2006) are factors which may have contributed to the observed decrease in L. monocytogenes populations at 4C. Headspace atmosphere, sample pH, and background microflora have also been hypothesized as factors at tributing to the lack of L. monocytogenes growth on shredded carrots and lettuce (Kakiomenou et al., 1998) With a reported pH of 5.7 to 6.0 for fresh celery (FDA, 2007), w e do not believe that pH had an inhibitory role on pathogen behavior in this study. Less is known about the fate of foodborne pathogens on produce at abusive temperatures ( i.e above refrigeration but below normal room temperatures). For all three pathogens, we

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79 observed little to no change in population size at 12 2C, with L. monocyt ogenes having the greatest increase. At 12C, Salmonella populations inoculated at ca. 5 log CFU/g decreased on cut pineapple by ca. 2 to 3 log CFU/g over 7 days (Strawn and Danyluk, 2010a). Salmonella inoculated at ca. 3 log CFU/g increased by ca. 3.5 l og CFU/g on cut mango and papaya after 3 and 5 days, respectively (Strawn and Danyluk, 2010b) However, at 12C, E. coli O157:H7 pop ulations inoculated at 5 log CFU/g on cut pineapple decreased by ca. 4 log CFU/g over 14 days, but increased on papaya by ca. 3 log CF/g over 3 days, and did not change on mango over 10 days (Strawn and Danyluk 2010a; Strawn and Danyluk 2010b). Population s of L. innocua on fresh cut peaches stored at 10C increased by ca. 1.6 log CFU/g over 6 days (Alegre et al., 2010), while those of L. monocytogenes increased by ca. 1.5 log CFU/g on shredded lettuce and ca. 1 log CFU/g on rutabaga, did not change significa ntly on soybean sprouts, and decreased by 1.5 Escherichia coli O157:H7 populations had a similar behavior to L. monocytogenes on shredded lettuce, rutabaga, soybean sprouts, and packaged coleslaw over 5 days at 8 2001) On fresh cut celery at 22 2C, popu lations of all three pathogens increased. Escherichia coli O157:H7, Salmonella and L. monocytogenes populations increased by ca. 1.0, 0.5, and 2.0 log CFU/g, respectively, with the majority of growth occurring over the first 0.71 days in all cases. Salm onella and E. coli O157:H7 populations on both romaine lettuce and freshly shredded carrots held at 23C increased by 2.5 log CFU/g within 25 h of inoculation, similar to the results we see on celery (Zhao et al., 2010). Salmonella Montevideo populations inoculated onto raw tomatoes and stored at 30C increased from ca. 0.5 to 5 log CFU/g over 1 day before populations stabilized (Zhuang et al., 1995). In this study, similar to Z hao et al

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80 (2010) and Zhuang et al. (1995) maximum populations of Salmonella at 5 6 log CFU/g were observed, indicating a potential carrying capacity of Salmonella spp., as well as E. coli O157:H7 on some fresh produce items stored at or above ambient temp eratures. However, this pattern is certainly not a rule. Strawn and Danyluk (2010b) observed that Salmonella and populations on papaya inoculated at three different inoculum levels (1, 3, and 5 log CFU) held at 23C reached carrying capacity at ca. 7.2 l og CFU/g after 1 day. Under the same conditions, E. coli O157:H7 populations inoculated on papaya at two inoculum levels (3 and 5 CFU/g) reached a stable carrying capacity at 6.5 CFU/g after 3 days (Strawn and Danyluk, 2010b) On minimally processed peaches held at 25C, E. coli O157:H7 and Salmonella populations reached 8 log CFU/g within one day (Alegre et al., 2010a) H owever starting concentrations of 10 5 CFU/ml and storage in controlled atmosphere containers (semi oxygen permeable film) may have influenced th e results on peaches. The large numbers of competitive background microflora found on celery may also be a determining factor in the observed carryin g capacity for both E. coli O157:H7 and Salmonella spp. on celery. At 25C, Salmonella populations inoculated onto fresh Italian parsley increased from ca. 2.5 to 4.1 log CFU/g over 7 days (Duffy et al., 2005). These findings are similar those we observed on fresh cut celery. However, when held at 25C, Salmonella populations inoculated onto whole zucchini squash and zucchini slices decreased from ca. 7 to 1.5 log CFU/sample over 7 days, and increased from ca. 2.5 to 8 log CFU/sample over 2 days, respectiv ely (Castro Rosas et al., 2010). In the same study, a single strain of E. coli O157:H7 inoculated onto whole zucchini and zucchini slices decreased from ca. 7 to 5 log CFU/g, and increased from ca. 2.2 to 7 log CFU/sample, respectively, over 7 and 2 days, respectively (Castro Rosas et al., 2010). Compared with Salmonella spp. E. coli O157:H7 fared better on the skin of whole zucchini

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81 (Cas tro Rosas et al., 2010) On sliced, but not whole zucchini, E. coli O157:H7 and Salmonella behavior was similar to that observed on fresh cut celery. In our experiments, both E. coli O157:H7 and Salmonella populations were greater on cut versus uncut c elery surfaces. Microniches exist at epidermal cell junctions where cuticular waxes are less dense, water accumulates, and nutrients are more available than in other surface locations (Sapers et al., 2006) It has been hypothesized that microbial resource competition is particularly intense at these junctions (Castro Rosas et al., 2010) Salmonella populations inoculated onto alfalfa sprouts decrease over 1 day storage at 25 C by ca. 1.4 CFU/g (Gandhi et al., 2001). This observation is difficult to explain, but the researchers suggested that the waxy ou ter material of the sprout repelled the inoculum mixture, thereby repelling the bacteria. Another study on alfalfa sprouts found that at both 25 and 37C, pathogen population decreases were greater than that observed at 5C (Taormina and Beuchat, 1999) These observations are not consistent with those observed on fresh cut celery, and it is apparent that pathogen behavior is often unique to the produce item in question. Similar to the Castro Rosas et al. (2010) study comparing whole and sliced zucchini held at 25C, we frequently observed that samples inoculated with either E. coli O157:H7 or Salmonella populations on cut surfaces grew more than those inoculate d onto uncut surfaces. However, as a modified leaf bundle, celery petioles are not analogous to the epidermis of whole zucchini fruit, and some human pathogens may have the capability to invade through stomatal openings (Kroupitski et al., 2011). In an inoculation study of mechanically damaged lettuce, E. coli O157:H7 populations where shown to survive longer on damaged than on undamaged samples (Aruscavage et al., 2008) These findings are also consistent with our observations, since cut may be considered analogous to a damaged surface.

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82 Storage container type had little effect on pathogen fate. For celery inoculated with Salmonella held at 22 2C, L. monocytogenes held at 4 2C, and E. coli O157:H7 held at 4 2C, significant, combined effects were observed, where populations inoculated on cut celery surfaces and stored in b ags were larger than those inoculated on uncut surfaces and stored in containers. This suggests that storage type may have a small effect on pathogen behavior, and rigid containers may be preferable to bags in this respect.

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83 CHAPTER 6 CONCLUSIONS AND F UTURE WORK At 4C, populations of all three pathogens decreased by ca. 0.5 to 1.0 log CFU/g over 2 days, under all conditions Behavior at 4C was as expected for the mesophilic E. coli O157:H7 and Salmonella spp., but not for the psychrophilic L. monocyto genes The unexpected decline in populations of L. monocytogenes may have been due to a combination of environmental and ecological factors including passive MAP, interspecific competition with normal microflora, and low nutrient accessibility. At 12C, population sizes of E. coli O157:H7 and Salmonella spp. did not change over 7 days, under all conditions, while populations of L. monocytogenes increased by ca. 0.5 log CFU/g. Behavior at 12C was as expected, although more growth for L. monocytogenes po pulations was anticipated. At 22C, as expected, populations of E. coli O157:H7 increased by ca. 1 log CFU/g, L. monocytogenes increased by ca. 0.3 to 0.5 log CFU/g, and Salmonella increased by ca. 1.5 to 2 log CFU/g, with the majority of growth for all three pathogens occurring during the first 0.71 days. Significant differences in populations were frequently observed between inoculation location, with samples inoculated on cut su rfaces being larger than those inoculated on uncut surfaces. This result may have been due to differences in water activity and nutrient availability at the surface locations. Significant differences between container type did occur, but were infrequent. This result suggests only a minimal influence of the container types used on pathogen behavior. This work demonstrates that E. coli O157:H7, L. monocytogenes and Salmonella spp. can survive and grow on improperly stored fresh cut celery. We have also s hown that st rict maintenance of refrigeration temperatures can inhibit the growth of several pathogens on fresh

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84 cut celery. Therefore, we suggest that m aintaining the cold chain during transportation, distribution, storage, handling, in markets, and by co nsumers is essential to prevent disease proliferation. That pathogen populations did not fall below the limits of detection over 7 days, even at 4C, underscores the importance of implementation and strict adherence to preventative food safety programs at all stages of production and processing to mini mize the risk of contamination. Pathogen behavior has been shown to vary based on initial inoculum concentrations (Strawn and Danyluk 2010b). At 22C, pathogen populations recovered from tomato samples afte r 1 h of drying was significantly large than those obtained after 24 h drying time (Lang et al., 2004). That we observed some differences in the effect of drying time between samples inoculated on cut versus uncut surfaces is methodologically significant. During future experiments involving different locations of inoculation, we suggest varying drying times on the basis of site of inoculation to achieve a more precise initial inoculum load. Alternatively, different initial inoculum concentrations could b e applied to reach a uniform level of contamination between samples. Different strains of E. coli O157:H7, L. monocytogenes, Pseudomonas, and Salmonella attach to different regions of cut lettuce, suggesting different species specific (and possibly strain specific) attachment mechanisms (Tak euchi et al., 2000) This may also be true for celery, and future research on this topic is recommended to further develop our understanding of the behavior of various pathogens on celery. A greater understanding of the biomechanics of pathogen produce attachment and invasion could lead to more effective intervention and decontamination strategies, if these mechanisms can be chemically or biophysically disrupted (Teplitski et al., 2009)

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85 Spoilage and physiological changes in fresh cut celery occur over time (Robbs et al., 1996) The predominant spoilage bacteria was identified as Pseudomonas fluorescens and P. marginalis which caused water soaking, soft rot, and discoloration in celery tissues stored at 5 or 25C. Leuconostoc mesenteroides was also isolated and hypothesized to have been responsible for slime production (Robbs et al., 1996) Because these changes alter the microenvironment and likely affect water activity and nutrient availabi lity future studies on the effects of spoilage and natural microflora on pathogen behavior is suggested. Because of the growth potential of pathogens on fresh cut celery, further investigations into the factors influencing this behavior are warranted. Such studies could include an analysis of the abundance and behavior of natural microflora on fresh cut celery, as well as the respiration of cut celery in consumer storage containers and atmospheric conditions within the packaged product. Further examina tion of the effect of storage container type is also warranted, as the level of gas exchange may vary based on material permeability and seal integrity. Studies on the potential for cross contamination in the consumer kitchen as well as the efficacy of various prevention strategies (e.g. washing, chemical treatments, etc.) are suggested. While the fresh cut industry is generally known to practice strict food safety programs, once a consumer purchases a product, disease preven tion becomes a matter of proper consumer handling, and should be based on public education disseminated from an informed government.

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91 De Boer, E., Heuvelink, A. E., 2000. Methods for the detection and isolation of Shiga toxin producing Escherichia coli Soc. Appl. Microbiol. Symp. Ser. 29, 133 143 De Roever, C., 1998. Microbiologica l safety evaluations and recommendations on fresh produce. Food Control 9 321 347. De Wit, J.C., Broekhuizen, G., Kampelmacher, E. H., 1979. Cross contamination during the preparation of frozen chickens in the kitchen. J. Hyg Cambridge 83 27 32. Diaz, C., & Hotchkiss, J. H., 1996. Comparative growth of Escherichia coli O157:H7, spoilage organisms and shelf life of shredded iceberg lettuce stored under modified atmospheres. J. Sci. Food Agr. 70 433 438. Dijkstra, R. G., 1982. The occurrence of Listeria monocytogenes in surface water of canals and lakes, in ditches of one big polder, and in the effuents and canal s of a sewage treatement plant. Zbl. Bakt. Mik. Hyg. B 176 202 205. Dixon, N.M., Kell, D. B., 1989. The inhibition by CO2 of the grow th and metabolism of microorganisms. J. Appl. Bacteriol. 67 109 136. Doumith, M., Cazalet, C., Simoes, N., Frangeul, L., Jacquet, C., Kunst, F., Buchrieser, C., 2004. New aspects regarding evolution and virulence of Listeria monocytogenes revealed by com parative genomics and DNA arrays. Infect. Immun. 72 1072 1083. Doumith, M., Jacquet, C ., Gerner Smidt, P., Graves, L. M., Loncarevic, S., Mathisen, T., Martin, P., 2005. Multicenter validation of a multiplex PCR assay for differentiating the major Listeria monocytogenes Serovars 1/2a, 1/2b, 1/2c, and 4b: Toward an international standard. J. Food Protect. 68 2648 2650. Doyle, M. P. ,1989. Foodborne bacterial pathogens Marcel Dekker. New York, NY Doyle, M.P., Erickson, M. C., 2008. Summer meeting 20 07 the problems with fresh produce: an overview. J. Appl. Microbiol 105 317 330. Drevets, D.A., Dillon, M.J., Schawang, J.E., Leenen, P.J. M., 2008. Mechanisms of Ly 6C(high) Monocyte Recruitment to the Brain During Systemic Listeria monocytogenes Infe ction of Mice. Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother 48 60. Droffner, M.L., Yamamoto, N., 1991. Procedure for isolation of Escherichia, Salmonella, and Pseudomonas mutants capable of growth at the refractory temperature of 54 C. J. Microbiol. Meth. 14 201 206. Duffy E. A., Cisneros Zevallo s, L., Castillo, A., Pillai, S.D., Ricke, S.C., Acuff, G. R., 2005. Survival of Salmonella transformed to express green fluoresc ent protein on Italian parsley as affected by processing and storage. J. Food Protect 68, 687 695.

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108 BIOGRAPHICAL SKETCH Joshua Peter Vandamm was born in Silver Spring, Maryland to Michael and Deborah Van damm. Joshua attended Towson State University, where he graduated in 2008, with a B.S. degree in b iology with an emphasis in z oology. Joshua began his M.S. degree in 2009, at the University of Florida under the instruction of Dr. Michelle Danyluk. At th e University of Flor ida, Joshua studied food science, with an emphasis on food microbiology and s afety. Future plans include perusing a career in food safety and/or public health in industry or government.