SPREAD OF ESCHERICHIA COLI O157:H7 DURING FLUME WASHING AND DRYING OF FRESH-CUT ROMAINE LETTUCE  By  Siyi Wang !!!!!!!!!!!!A THESIS    Submitted to Michigan State University in partial fulfillment of the requirements for the degree of  Food Science Ð Master of Science  2016        ABSTRACT  SPREAD OF ESCHERICHIA COLI O157:H7 DURING FLUME WASHING AND DRYING OF FRESH-CUT ROMAINE LETTUCE  By  Siyi Wang  The microbiological safety of leafy greens remains a concern as evidenced from recent outbreaks. This study assessed the spread of E. coli O157:H7 during washing and drying of fresh-cut romaine lettuce. Radicchio was spot-inoculated at 10-1, 101 and 103 CFU/leaf and mixed with uninoculated romaine lettuce to obtain 5 kg batches with inoculated vs uninoculated ratios of 0.5:100, 1:100, 5:100 and 10:100. After 90 s of sanitizer-free flume washing followed by shaker table and centrifugal dryer, the radicchio was removed and the lettuce was divided into 225 g samples to test presence/absence of E. coli O157:H7 using a GeneQuence assay. Based on triplicate trials, lower inoculation levels led to decreased E. coli O157:H7 transfer to romaine lettuce (P < 0.05). All lettuce samples yielded E. coli O157:H7 when radicchio was inoculated at 103 CFU/leaf. At 101 CFU/leaf, the percentage of positive samples decreased from 96.8% to 93.7%, 81.0% and 63.5% while at 10-1 CFU/leaf, 22.2%, 6.3%, 4.8% and 6.3% were positive at 10:100, 5:100, 1:100 and 0.5:100 ratios. Within each inoculation level, there were no significant differences (P>0.05) among four product ratios. These findings are critical to predict the extent of cross-contamination under realistic conditions and will provide important data for improving exposure assessment in risk assessments for leafy greens.   iii                    To my mom Yan Cheng, my dad Xiaodong Wang                       iv ACKNOWLEDGEMENTS  Here I would like to say thank you to my major advisor, Dr. Elliot Ryser, who brought me into the food safety field. He offered me the opportunity to volunteer in the lab as an undergraduate student assistant, which opened the door to my commitment to scientific study and learning. It is my great pleasure to learn with and work for him. Without his support and guidelines, I would not have achieved all these accomplishments by myself. I also would like to thank my committee members Dr. Cornelius Barry and Dr. John Linz for their advice and support keeping me on the right track for my research.  I want to thank Haley Smolinski and Lin Ren for all the assistance and being great lab partners and friends. Without their help, it would have taken much longer to finish the project. As well, I would like to especially thank Hamoud Alnughaymishi, Ryann Gustafson and all my lab mates for their help, sharing and friendship. Last, but not least, I would like to thank my parents for their love.                                                      Siyi Wang          v TABLE OF CONTENTS  LIST OF TABLES ...................................................................................................................... vii   LIST OF FIGURES ................................................................................................................... viii   KEY TO SYMBOLS AND ABBREVIATIONS ........................................................................ x   INTRODUCTION ........................................................................................................................ 1   CHAPTER 1: Review of Pertinent Literature .............................................................................. 4   1.1  Fresh-cut production and lettuce consumption É.ÉÉÉÉÉÉÉÉÉÉ....ÉÉÉÉ 5 1.2  Outbreaks and safety concerns associated with the fresh-cut industryÉÉ.ÉÉÉ..É.. 8 1.3  Escherichia coli O157:H7ÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...É... 11 1.4  Sources of contamination during pre-harvest and post-harvest handling of lettuce ÉÉ12 1.4.1 Pre-harvest contaminationÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ....ÉÉ13 1.4.2 Post-harvest contaminationÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉ.É.. 14 1.5  Post-harvest processing and sources of contamination ÉÉÉÉ..ÉÉÉÉÉÉ..ÉÉ16  1.5.1 ShreddingÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..É.ÉÉ...17 1.5.2 ConveyingÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...ÉÉÉ..18 1.5.3 Flume WashingÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..É.ÉÉ. 18 1.5.4 Dewatering and DryingÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..É.ÉÉ. 20   1.6  Previous microbial studies on fresh produce with spot inoculationÉÉÉÉÉÉÉ.. 20 1.7  Previous cross-contamination studies on pathogen transfer and redistribution during processing ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉ.. 22 1.8  Risk Analysis & Assessment ÉÉ..ÉÉÉÉÉÉ..ÉÉÉÉÉÉ.ÉÉÉÉÉ.É.... 23 1.9  FSMA and the influences on food safetyÉÉÉÉ..ÉÉÉÉ..ÉÉÉÉÉÉÉÉÉ 24 1.10 Overall goalsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉ..É.. 25  CHAPTER 2: Spread of Escherichia coli O157:H7 during Flume Washing and Drying of Fresh!Cut Romaine Lettuce ......................................................................................................... 26   2.1  OBJECTIVE.................................................................................................................... 27    2.2  MATERIALS AND METHODS .................................................................................... 28  2.2.1 Overall experimental design........................................................................................ 28  2.2.2 Produce..................................................................................................................... 29  2.2.3 Bacterial strains used................................................................................................... 29  2.2.4 Inoculation of radicchio............................................................................................. 30    vi 2.2.5 Processing line .......................................................................................................... 31  2.2.6 Romaine lettuce processing and sample collection..................................................... 32  2.2.7 Chemical sanitizersÉÉÉÉÉÉ.............................................................................. 35  2.2.8 Microbiological analysis............................................................................................. 35 2.2.9 GeneQuence Assay..................................................................................................... 36 2.2.10 Statistical analysisÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...... 38 2.3  RESULTS.............................................................................................................. 39 2.3.1 Romaine lettuceÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉ.ÉÉ....É.ÉÉ 39 2.3.2 RadicchioÉÉÉÉÉÉÉÉÉ........ÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉ.ÉÉ... 42 2.3.3 WaterÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉ.ÉÉ.. 45 2.3.4 Sanitizer trialsÉÉÉÉÉÉÉÉ..ÉÉ...ÉÉÉÉÉÉÉÉ.ÉÉÉ....ÉÉÉ. 45 2.4  DISCUSSION................................................................................................................. 47  CHAPTER 3: Conclusions and Future Recommendations......................................................... 53  APPENDIX................................................................................................................................... 57   BIBLIOGRAPHY....................................................................................................................... 67              vii LIST OF TABLES  Table 1.1: Fresh produce including leafy green-associated E. coli O157:H7 outbreaks since 2006 based on Foodborne Outbreak Online Database..ÉÉÉÉ...ÉÉÉÉÉÉÉÉÉÉÉ..É.. 9  Table 2.1: Numbers and percent of positive samples among ~20 total samples with different inoculation levels and product ratios ÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉ.ÉÉ...É 41  Table 2.2: E. coli O157:H7 populations on radicchio before washing and E. coli O157:H7 log reductions on radicchio after processingÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉ 44  Table 2.3: Comparison between sanitizer-free trials and 60 ppm sanitizer trials at the same inoculation level (103 CFU/leaf) and inoculated:uninoculated ratio (10:100) ÉÉ.É...ÉÉÉ. 46  Table AI.1: Percent of samples detected as positive for E. coli O157:H7 due to the E. coli O157:H7 transfer to 225 g samples of romaine lettuce after 90 sec of flume washing in sanitizer-free waterÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 59  Table AI.2: E. coli O157:H7 populations on radicchio leaves before processing (log CFU/g) ..... 60  Table AI.3: E. coli O157:H7 population on radicchio leaves after processing (log CFU/g)É. 61  Table AI.4: Table AI.4: Percent of E. coli O157:H7 shed from radicchio leaves after processing ÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ62   Table AI.5: E. coli O157:H7 populations in wash water sample taken at the end of processing (log CFU/100 ml) ÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 63  Table AI.6: E. coli O157:H7 populations in water sample for each centrifugal drying batch taken (log CFU/100 ml) ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.É.64  Table AI.7: Summary of the results for experiments using 60 ppm sanitizer ÉÉÉÉÉ...É.. 65     viii LIST OF FIGURES  Figure 1.1: Percentage of Vegetable and Legume Availability in 2013 ÉÉÉÉÉÉ...É.ÉÉ 7  Figure 1.2: Lettuce U.S. imports ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..É 7  Figure 2.1: Lettuce shreddingÉÉÉÉÉÉÉ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..É 33  Figure 2.2: Flume washing and dewateringÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉ..É 33  Figure 2.3: CentrifugationÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.É..É 34  Figure 2.4: Sample collectionÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.É..É.. 34  Figure 2.5: The Stat Fax 4200 microplate reader and Stat Fax 2600 semi-automatic microplate washer ÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 37  Figure 2.6: GeneQuence kitÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 37  Figure 2.7: Percent of samples detected as positive for E. coli O157:H7 due to the E. coli O157:H7 transfer to 225 g samples of romaine lettuce after 90 sec of flume washing in sanitizer-free water based on different inoculation levels. Mean values with different letters are significantly different (P0.05) within the same product ratio (10:100, 5:100, 1:100, 0.5:100)ÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉ..É.ÉÉÉ..É.É 40   Figure 2.8: Percent of samples detected as positive for E. coli O157:H7 due to the E. coli O157:H7 transfer to 225g samples of romaine lettuce after 90 sec of flume washing in sanitizer-free water based on different product inoculated:uninoculated ratios. Mean values with same letter are not significantly different (P > 0.05) within the same inoculation level ..É 41  Figure 2.9: E. coli O157:H7 population on radicchio leaves before and after processing based on different inoculated:uninoculated product ratios. The log reductions after washing are not significantly different within the same inoculation level (P > 0.05) ÉÉÉÉÉÉÉÉ 43       ix Figure 2.10: E. coli O157:H7 population on radicchio leaves before and after processing based on different inoculation level. At 10-1 CFU/leaf, E. coli populations are below limit of detection (1 CFU/25g) so percent loss is not applicable at this inoculation level. The log reductions after washing are not significantly different within the same produce ratio (P > 0.05) É. 43  Figure AI.1: Growth curves in GeneQuence enrichment during 24 h incubation with different product:enrichment ratio (1:2 vs 1:9)ÉÉÉÉÉ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 66                                   x KEY TO SYMBOLS AND ABBREVIATIONS  ANOVA Analysis of Variance AgMRC Agriculture Marketing Resource Center CDC Centers for Disease Control and Prevention CFU Colony forming unit(s) CFSAN Center for Food Safety and Applied Nutrition LGMA California Leafy Green Products Handler Marketing Agreement  cm centimeter(s) CSPI Center for Science in the Public Interest ERS Economic Research Service EHEC Enterohemorrhagic Escherichia coli FDA Food and Drug Administration g gram(s) FSMA Food Safety Modernization Act GAPs Good Agricultural Practices GFP-labeled Green Fluorescent Protein-labeled GMPs Good Manufacturing Practices HUS Hemolytic uremic syndrome kg kilometer(s) L liter(s) LOD limit of detection ml milliliter(s)   xi PBS Phosphate Buffered Saline ppm Parts per million QPRAM Quantitative Predictive Risk Assessment Model RTE Ready-to-eat RH Relative Humidity s second(s) SD Standard Deviation ELISA Sandwich Enzyme-linked Immunosorbent Assay SSOPs Sanitation Standard Operation Procedures USDA United States Department of Agriculture TSA-YE Trypticase Soy Agar with 0.6% Yeast Extract TSB-YE Trypticase Soy Broth with 0.6% Yeast Extract US United States of America µl microliter(s)   !1 INTRODUCTION                 !2 Nowadays, fresh produce consumption in the United States has increased significantly. As an important source of vitamins, nutrients and fiber, the high demand for fresh produce can be explained by the increased consumer interest in developing healthy eating habits. Due to advances in preservation technology and benefits of global trade, many varieties of fresh produce are available all year long (Olaimat and Holley, 2012). Edible coatings and films with added antimicrobial compounds are also being used more frequently in the food industry to preserve fresh fruits and vegetables and increase shelf life (Silvia et al., 2011). Even though consumers benefit from the rapid growth of fresh produce consumption by having a healthier lifestyle, at the same time, no one can ignore the fact that there are more foodborne illness outbreaks associated with fresh produce consumption, especially leafy greens (Warriner et al., 2009).  In recent years, the contamination of leafy greens associated with E. coli O157: H7 has drawn significant attention from government and industry, as well as researchers (Danyluk and Schaffner, 2011). Leafy greens consumed raw, such as lettuce, are most likely to be connected with diseases resulting from E. coli O157:H7 contamination (Franz et al., 2009). According to the Centers for Disease Control and Prevention (CDC), multiple outbreaks of E. coli O157:H7 infections have been linked to leafy greens, which causes a huge economic loss and human illness. For example, one multistate outbreak of E. coli O157:H7 infection associated with ready-to-eat salads resulted in a total of 33 infections including 13 hospitalizations in four states, with 7 hospitalizations and 0 death (CDC, 2016). To prevent these foodborne disease outbreaks, it is necessary to understand the mechanism of cross-contamination in the food chain.  !3 Cross-contamination may occur at any point from farm to fork. The sources of contamination include contaminated irrigation water and soil, as well as improper human handling (Beuchat, 2002). When one leaf of lettuce is contaminated with E. coli O157:H7 in the field, this pathogen can transfer to other leaves, the wash water or equipment during commercial processing in the plant, leading to large quantities of cross-contaminated product.  As food supply chains are getting more complex and global, the Food and Drug Administration (FDA) uses risk analysis to minimize the risk of contamination and illness (FDA, 2016). To ensure food safety, risk assessments are being used to better understand the interactions between hazards, foods and human hosts. Besides pre-harvest cross-contamination, it is also important to understand how contamination can be spread in the processing pathway. Since the extent to which cross-contamination occurs is not well characterized, additional research to quantify pathogen redistribution during post-harvest processing is needed in order to fill the current data gaps.  Consequently, the objectives of this study were to: 1. Quantify the spread of Escherichia coli O157:H7 during pilot-scale sanitizer-free washing and drying of fresh-cut romaine lettuce. 2. Assess the spread of Escherichia coli O157:H7 during processing with chemical sanitizers.    !4 CHAPTER 1:   Review of Pertinent Literature                         !5 1.1 Fresh-cut production and lettuce consumption In recent years, consumption of fresh produce has increased significantly due to healthy eating trends. As a very important portion of the Americans diet, fresh produce provides a high level of vitamins and minerals that contribute to human health. Consequently, the demand for fresh-cut leafy green vegetables such as lettuce has continued to expand because more consumers want to achieve healthy diets and convenience at the same time (Weng et al., 2016). According to the United States Department of Agriculture (USDA) (2016), fresh lettuce, including head, romaine and leaf lettuce, was one of the top three vegetables consumed in the US. Based on the Percentage of Vegetable and Legume Availability Chart in 2013 (Figure 1.1), the consumption was 25.5 pounds per person in 2013. With $12 billion in annual sales in the past few years in the US, the fresh-cut sector of the produce industry has become the fastest growing segment (FDA, 2008).  In general, there are two types of lettuce: head lettuce and leaf lettuce, which includes romaine, butter-head and leaf types. Even though lettuce is produced in all 50 states, the top two states, California and Arizona, accounted for 98 percent of the leaf lettuce in 2013 nationwide. Other than local production, US imports of lettuce have increased in recent years according to the Vegetables and Pulses Data in Figure 1.2 (ERS, 2016). As fresh products, almost all head lettuce is field-packed for bulk sale or transported to salad processing facilities (Agriculture Marketing Resource Center (AgMRC), 2016). To be minimally processed, lettuce must be washed, chopped into small pieces, mixed with other leafy greens, sorted into bags and sold as  !6 salad products in the market. Compared to raw vegetables, minimally processed produce is more perishable with a relatively short shelf life (Siroli et al., 2015).             !7  Figure 1.1: Percentage of Vegetable and Legume Availability in 2013 (USDA, 2013) Figure 1.2: Lettuce U.S. imports (ERS, 2016)    !8 1.2 Outbreaks and safety concerns associated with the fresh-cut industry Normally, since there is no inactivation step before consuming fresh-cut products such as ready-to-eat (RTE) salads, this type of product is likely to become a potential source of human pathogens (Castro-Ibanez et al., 2015). As the market continues to grow, various challenges have emerged along the production chain from the raw material to its consumption to ensure food quality and safety. Due to changes in food production and consumption patterns, produce-associated outbreaks have been reported more frequently. Because the fresh-cut industry moved from small local farms to large-scaled central processing facilities, the negative impact on the industry can be broader (Buchholz et al., 2014).  From 1996 to 2006, there were 72 foodborne illness outbreaks associated with the consumption of fresh-cut produce. It is also noted that lettuce and other leafy greens have been implicated in multiple foodborne disease outbreaks especially associated with E. coli O157:H7 (Jensen et al., 2014). The outbreaks of fresh produce, including leafy greens associated with E. coli O157:H7 since 2006, are summarized in Table 1.1 (CDC, 2016).  In 2015, there was a multistate outbreak of listeriosis associated with packaged salads produced in an Ohio processing plant, which caused 19 hospitalizations and one  death (CDC, 2016). According to the Center for Science in the Public Interest (CSPI), the number of outbreaks linked to fresh produce consumption, especially lettuce, has increased since 2000 (CSPI, 2009). Those outbreaks not only affect public health, but also lead to economic losses for the society at the same time.   !9 Table 1.1: Fresh produce including leafy green-associated E. coli O157:H7 outbreaks since 2006 based on Foodborne Outbreak Online Database (CDC, 2016). Year Location Illnesses reported Produce 2006 Multistate 238(3 deaths) Spinach 2006 Multistate 77 Lettuce 2006 Multistate 80 Lettuce 2007 AL 26 Salads 2008 WA 10 Spinach 2008 Multistate 13 Spinach 2009 Multistate 16 Lettuce 2009 Multistate 22 Lettuce 2010 Multistate  31 Romaine Lettuce 2011 Multistate  26 Lettuce 2011 Multistate  60 Romaine Lettuce 2012 CA  12 Lettuce 2012 Multistate  58 (3 developed HUS) Romaine Lettuce  !10 Table 1.1 (contÕd) 2012 Multistate  33 Packaged leafy greens 2012 Multistate  16 Lettuce 2013 Multistate  33 Salads 2016 Multistate  9 Alfalfa Sprouts  Even though eating vegetables raw helps maintain the nutrients in fresh produce, this practice may also can be risky due to the presence of pathogens. Vegetables can become contaminated during cultivation, harvest or postharvest with foodborne pathogens. Two major pathogens associated with vegetables are Salmonella and Shiga toxin-producing Escherichia coli, which can be introduced through soil, irrigation water, insects and human handling (Delbeke et al., 2014). With improper handling or storage conditions, such contamination can lead to serious consequences.  According to the FDA, processing whole fresh produce into fresh-cut products increases the risk of bacterial growth and contamination. This is because the natural exterior barrier of the produce is broken during processing. When produce is chopped or shredded, the release of plant cellular fluids provides nutrients for bacterial growth. In addition, the cross-contamination that occurs during fresh-cut processing may put large volumes of product at risk (FDA, 2008).    !11 1.3 Escherichia coli O157:H7  Escherichia coli is a Gram-negative, rod-shaped, facultative anaerobic bacterium. While most E. coli strains colonize the GI tract of humans and animals harmlessly, there are some pathogenic strains which acquire virulence factors through plasmids, transposons, bacteriophages, and/or pathogenicity islands (Lim et al., 2010). As a subgroup of E. coli, enterohemorrhagic E. coli (EHEC) may cause bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) (Goswami et al., 2015).  Among EHEC strains, E. coli O157:H7 is a leading cause of foodborne and waterborne disease outbreaks in the US with other outbreaks associated with E. coli O157:H7 having been reported worldwide including Australia, Canada, Japan and various countries in Europe and southern Africa (Huang et al., 2014). As one of the major virulence factors involved in E. coli O157:H7 pathogenesis, the Shiga toxins (Stx) are classified as either Stx1 or Stx 2 based on their immunoreactivity (Yin et al., 2015). Other than E. coli O157:H7, many other serotypes of STEC cause disease including E. coli O145. Limited public health surveillance data exists for these other serotypes with many such infections going unreported or undiagnosed (CDC, 2015).  E. coli O157:H7 was first recognized in 1982 as a human pathogen associated with outbreaks of bloody diarrhea in Oregon and Michigan (Lim et al., 2010). The first outbreak occurred in Oregon with 26 cases of which 19 were hospitalized. After three months, the second outbreak was reported in Michigan with 21 cases and 14 hospitalizations (Institute of Food Technologists, 1997). In 2006, a multistate outbreak associated with E. coli O157:H7 linked to  !12 spinach consumption was identified, causing over 200 cases and three deaths (Gelting et al., 2011).  Based on information published by the FDA, the infective dose is still unknown but may be similar to that of Shigella spp. with as few as 10 organisms inducing illness (FDA, 2014). Human illness may be observed even after ingestion of 1 CFU and disease in humans may develop without prior multiplication in food (Boqvist et al., 2015). Symptoms observed from most identified cases include severe diarrhea and abdominal cramps. Even though the target populations include people of all ages, young children and the elderly seem to be most susceptible to developing more serious symptoms. Particularly for children under five years old, the infection may cause a complication called hemolytic uremic syndrome, which is a serious disease in which red blood cells are destroyed and the kidneys fail due to platelet aggregation in renal arteries and glomerular capillaries (NY State Department of Health, 2006). 1.4 Sources of contamination during pre-harvest and post-harvest handling of lettuce Even though spoilage bacteria, yeasts and molds predominate on fresh produce such as leafy greens, fresh produce can still be contaminated with human pathogens, parasites and viruses from the stages of growing, harvesting, postharvest handling, processing and distribution (Gil et al., 2015; Olaimat and Holley, 2012). As we know, there are various sources of contamination that occur at pre-harvest or post-harvest steps before and after transferring to processing facilities.   !13 1.4.1 Pre-harvest contamination From 1996 to 2008, almost half of the fresh produce outbreaks were traced to leafy greens containing E. coli O157:H7 with implications of pre-harvest contamination (DÕLima and Suslow, 2009). At the pre-harvest stage of lettuce production, sources of contamination may include soil, feces, irrigation water, insecticides, dust, insects, inadequately composted manure and wild or domestic animals (Beuchat, 2002). As well, improper human handling such as using unclean tools in the field can also introduce contamination. For instance, when lettuce is cut and cored by workers in the field, blades of field coring devices may contact contaminated soil and transfer pathogens to lettuce tissues, which causes cross-contamination. According to a simulated field coring study, when using the same contaminated field coring device to consecutively cut the stems of the lettuce heads, E. coli O157:H7 can be transferred from one head to the next (Taormina et al., 2008). Soil can be considered as a natural environment for human pathogens especially when animal wastes are added for fertilizers. Based on multiple reports, E. coli O157:H7 is able to survive in soil for 7 to 25 weeks, depending on the soil type and conditions, which makes cross-contamination more likely to occur (Lang and Smith, 2007; Olaimat and Holley, 2012). Microbial safety of lettuce can be affected by the quality of irrigation water and type of irrigation system (Olaimat and Holley, 2012). As reported by Solomon et al. (2002), spray irrigation represents a higher level of risk for lettuce plants to be contaminated compared to surface irrigation. As a result, when pathogens transfer from soil or water, surface contamination  !14 of the edible lettuce leaves occurs, which increases the chance of foodborne disease (Delaquis et al., 2007).  In addition, airborne insect pests such as fruit flies that harbor E. coli on their bodies or guts can contaminate the surface of lettuce and serve as primary vectors of human pathogens. Insects may also affect the behavior of pathogens and damage the lettuce surface indirectly through their feeding activities (Erickson et al., 2010).  The survivability of pathogens depends on environmental factors and weather conditions including predation, competition, water stress, temperature, UV radiation, pH, inorganic ammonia and organic nutrients (Rogers and Haines, 2005). In order to minimize contamination of fresh produce such as lettuce by foodborne pathogens, both contamination routes and environmental factors should be considered when analyzing causes of cross-contaminations (FDA, 2006; Park et al., 2014). 1.4.2 Post-harvest contamination After harvesting, lettuce is typically hand-cut, occasionally field cored, packed in the field and placed in large bulk bins for transport to the precooling or processing facilities (Delaquis et al., 2007). Any improper handling after harvest may lead to safety concerns. From previous studies, leafy greens such as lettuce can be contaminated during storage and transport (Gil et al., 2015). Consequently, lettuce should be stored in adequate facilities and transported in vehicles to minimize damage and access by pests (CAC/RCP 53, 2003). Any lettuce showing signs of decay  !15 should be removed and discarded before transport and storage to minimize the risk of getting human pathogens associated with decay or damage (Brandl, 2008; FDA, 2006). During transport and storage, the preferred method used for precooling lettuce is vacuum cooling, considering its high surface-to-volume ratio. In vacuum cooling, produce is placed inside a vacuum chamber after which a vacuum is used to evaporate the water from the produce surface, lowering the temperature of the product (Ezeike and Hung, 2009). Optimum storage of lettuce is as close to 0 o C as possible with 98 to 100% relative humidity (RH), but the freezing injury may occur when stored at  -0.2 oC (Saltveit, 2016; UC Davis, 2013).  After transport from the field to the facility, lettuce should be stored as soon as possible. In general, if lettuce is stored at 5 o C or below, it will help prevent the growth of E. coli O157:H7. However, temperature abuse that occurs during storage and transportation may lead to problems (Khalil and Frank, 2010).  Several studies have focused on the effect of temperature on the survival of E. coli O157: H7. Li et al. (2001) found a decline in E. coli O157:H7 populations on shredded iceberg lettuce at 5oC, and an increase in growth during storage at 15oC of 2.0 and 3.0 log CFU/g after 7 and 14 days, respectively. In another study conducted by Francis and OÕBrien (2001), the researchers reported that there was no change in growth of E. coli O157:H7 populations in cut iceberg lettuce when stored at 4 and 5oC, while an increase of 1.0 log CFU/g was recorded at 8 and 10oC after 12 days and 120 h, respectively. Hence, it is necessary to track temperature changes in order to minimize microbial growth during periods of temperature abuse. Additionally, good  !16 hygiene, cleaning and sanitation practices during postharvest storage are necessary to ensure food safety (FDA, 2008).  1.5 Post-harvest processing and sources of contamination  A significant amount of lettuce is processed into ready-to-eat, fresh-cut salads to fulfill consumersÕ healthy eating desires. However, contamination of a small batch of lettuce can spread to large quantities of product during further processing, and may lead to foodborne disease outbreaks that affect the health of consumers.  According to a recent outbreak investigation, the equipment used for cutting and shredding leafy greens in a processing plant was identified as one source of contamination (Stafford, 2002). Based on previous research conducted in a pilot plant-scale facility, one contaminated batch of leafy greens can easily contaminate subsequent uncontaminated batches if no effective antimicrobial interventions are applied (Buchholz et al., 2012). To prevent and minimize the chance of contamination, fresh produce processors should follow the FDA Food Safety Modernization Act (FSMA) Produce Safety rules, which provide standards for the growing, harvesting, packing, and holding of produce for human consumption (FDA, 2016), as well as the food safety guidelines for leafy greens as outlined in the California Leafy Green Products Handler Marketing Agreement (LGMA). In addition, sanitation programs such as Good Manufacturing Practices (GMPs), Good Agricultural Practices (GAPs), and Sanitation Standard Operation Procedures (SSOPs) should also be followed (FDA, 2006). Moreover, it is important  !17 to understand how pathogens transfer during processing so preventive actions can be applied at each step to minimize contamination.  After transport to processing facilities, the production of fresh-cut lettuce involves shredding, flume washing in water with sanitizers, shaker table dewatering, centrifugal drying to remove remaining water and packing into small bags (Buchholz et al., 2014). The processing equipment used can vary based on the size and product type of the facility, but for the most part it includes a shredder, conveyer, flume tank, shaker table and centrifugal dryer where cross-contamination may occur from any piece of equipment. 1.5.1 Shredding During the shredding step, lettuce is mechanically shredded using high-speed machines (Barry-Ryan and OÕBeirne, 1998). While shredding reduces the size of the product, the damage during cutting irrevocably alters biochemical characteristics of tissues and provides opportunities for microbial invasion (Delaquis et al., 2007). A study conducted by Khalil and Frank (2009) showed that the populations of E. coli O157:H7 on shredded lettuce pieces were significantly larger than the populations on intact leaves.  Researchers in Japan found that fresh-cut vegetable samples from examined factories were heavily contaminated with E. coli O157:H7 from equipment surfaces used for trimming and slicing (Kaneko et al., 1999). Buchholz et al. (2012) assessed the transfer of E. coli O157:H7 from equipment surfaces to fresh-cut produce. During pilot plant- scale processing, E. coli O157:H7 populations decreased 0 to 3.6 log CFU/100 cm2 on the equipment surface after  !18 processing with the greatest losses seen for the shredder. These findings suggest a high likelihood of cross-contamination between shredders and produce. 1.5.2 Conveying Conveyors can be used at multiple points in the processing line to transport produce between different steps. After shredding, the shredded lettuce is step-conveyed and transported to the flume washer. There are various types of materials such as polypropylene and acetal that can be used for conveyor belts (Buchholz et al., 2010). While the surfaces of these conveyor belts tend to be smooth to maximize cleaning and sanitizing efficiency, there are still chances for bacteria to settle and proliferate.   In a study conducted by Mafu et al. (1990) crevices and holes were observed on the surfaces of conveyor belts made of polypropylene, rubber and stainless steel, using scanning electron microscopy. Allen et al. (2005) showed that the Salmonella can persist up to 28 days on stainless steel, polyvinylchloride, and wooden surfaces during fall and winter (20 o C/60% RH) with undetectable levels on sponge rollers and conveyor belts after 7 and 21 days, respectively. Considering the long period of pathogen survival, proper sanitation programs should be followed closely to prevent contamination.  1.5.3 Flume Washing In general, fresh-cut lettuce is washed in cold water with sanitizers. This important step in processing removes dirt and exudates, improves product appearance, and reduces the microbial load, but can transfer pathogens to the water (Haute et al., 2015; Munther et al., 2015). Proper  !19 decontamination procedures such as washing with sanitizers are critical to protect the safety of fresh-cut produce (Qadri et al., 2015). Among numerous types of sanitizers, chlorine is most widely used because of its low cost and ease of use. The antimicrobial activity of chlorine depends on the amount of free chlorine in the solution, the pH, the temperature, and the amount of organic matter (Suslow, 2001). Even though widely used, chlorine has been criticized for lacking efficacy against pathogens in certain conditions. Efficacy is limited in the presence of high organic loads, increased temperature, or light exposure (Gonzalez et al., 2004). For example, the chlorine compounds can be consumed with organic and inorganic constituents rapidly so that the efficacy of chlorine-based sanitizers is reduced (Zhou, 2013). Since more foodborne outbreaks have been associated with fresh-cut produce processing in recent years, many studies have focused on comparing and improving the antimicrobial activity of sanitizers. Lopez-Galvez et al. (2009) conducted an experiment to compare the efficacy of chlorine, Tsunami, Citrox and Purac against non-pathogenic E. coli on fresh-cut lettuce and the processing water used. The results showed that Citrox (5000 mg/l) and Purac (20000 mg/l) at the recommended doses did not prevent E. coli transfer between contaminated and non-contaminated produce while chlorine (40 mg/l) and Tsunami (500 mg/l) prevented cross-contamination during processing.  Even though washing helps reduce the microbial load, the water used for washing can serve as a vehicle for cross-contamination. Pathogens can penetrate into crevices, cut surfaces or  !20 intercellular spaces of lettuce, which become barriers for disinfection strategies (Buchholz et al., 2012). 1.5.4 Dewatering and Drying Following the washing step, fresh-cut lettuce is dewatered on a shaker table and dried using a centrifugal dryer to remove remaining water. This is an essential processing step because excess moisture may increase microbial growth (Cantwell and Suslow, 2004). In addition, this step must be performed with care to avoid tissue damage, product moisture content decrease and cell leakage that may lead to microbial growth on the produce (Artes and Allende, 2005).  For different types of products, key parameters of the dewatering system such as time and speed of centrifugation must be adjusted properly. Especially for leafy greens, damage of tissues may occur due to high speed of centrifugation and cause products to leak fluids which reduces the quality (Artes and Allende, 2005; UC Davis, 2016). If the products are too delicate, then a forced air method should be applied (UC Davis, 2016). 1.6 Previous microbial studies on fresh produce with spot inoculation Based on many previous studies focused on microbial contamination of fresh produce, there are three common inoculation methods including spot inoculation, dip inoculation and spray inoculation. In spot inoculation, drops of a bacterial suspension are placed directly onto the produce surface using a micropipette (Koseki et al., 2003).  Compared to the other inoculation methods, spot inoculation allows researchers to know the number of cells placed on the surface regardless of the weight or size of the product being tested  !21 (Mukhopsdhyay et al., 2013). In dip inoculation, controlling the total volume of inoculum adhering to the produce surface remains difficult, which means the number of cells applied on the surface is unknown. In addition, spray inoculation also has issues regarding the entire amount of the spray contacting the surface, especially for smaller items (Lang et al., 2003).  In one study, Lang et al. (2004) investigated how the survival and recovery of E. coli O157:H7 was affected by different methods of inoculation for lettuce and parsley. Compared to dip and spray inoculation, more consistent initial populations of pathogens were obtained after spot inoculating leafy greens, which allowed for more accurate measurement of microbial reductions.  Another study that evaluated the efficacy of sanitizers concluded that the effectiveness of sanitizers against E. coli O157:H7 was affected by the inoculation method. It is possible that E. coli cells adhered less tenaciously to the surface with spot inoculation so treatments were more effective compared to spray or dip inoculation (Singh et al., 2002). Similarly, the experiment conducted on the evaluation of sanitizer efficacy of cantaloupes also showed that bacteria on spot-inoculated cantaloupes were more vulnerable due to the looser attachment to surfaces, which indicated that the bacteria can be detached more easily by washing treatments with sanitizers (Annous et al., 2005). Considering the advantages of spot inoculation discussed above and to better mimic field contamination of lettuce, spot inoculation was applied in this study.    !22 1.7 Previous cross-contamination studies on pathogen transfer and redistribution during processing There are numerous studies indicating that cross-contamination can occur during fresh produce processing (Buchholz et al., 2012; Buchholz et al., 2014; Davidson et al., 2013; Wang and Ryser, 2013). Pathogens introduced by contaminated produce at earlier stages are likely to transfer and redistribute to pieces of equipment and the wash water upon further processing of, uncontaminated produce. It is important to track down how pathogens transfer during processing and better understand the extent of cross-contamination so that preventative actions can be developed and implemented.  A quantitative study conducted by Holvoet et al. (2014) observed the cross-contamination processes and the transfer of E. coli from water to lettuce and vice versa without using sanitizers. This work showed the likelihood of cross-contamination at the flume washing step. Based on multiple experiments conducted in our laboratory, the cross-contamination pathway for leafy greens was determined through pilot plant trials. In one study by Buchholz et al. (2014), 45 kg of uninoculated lettuce was processed, followed by 9.1 kg of radicchio contaminated by dip inoculation with E. coli O157:H7 and finally by 907 kg of uninoculated iceberg lettuce, which was then collected into 40 bags (~22.7 kg per bag). Based on the results of direct plating with or without membrane filtration, E. coli was detected in lettuce, radicchio, and water samples and also found on equipment surface. Those findings demonstrate the potential for contaminated lettuce to spread pathogens to larger batches during processing.  !23 In other work quantifying the transfer of E. coli O157:H7 to equipment during lettuce processing, 22.7 kg of iceberg lettuce was inoculated with different levels of E. coli O157:H7 and processed. The report showed that the produce contact surfaces of the shredder, conveyor, flume tank, shaker table and centrifuge were all contaminated to various degrees after processing (Buchholz et al., 2012). 1.8 Risk Analysis & Assessment According to the FDA, food safety decisions must be made based on available scientific data and systematic analysis to prevent contamination and illness (FDA, 2016). As food supplies become more global and complex, it is better to test large quantities of food to make sure there are no pathogens present. However, such large-scale testing is impossible in practice. Therefore, conducting quantitative microbial risk assessments using models and tools becomes a solution to enhance food safety (Rijgersberg et al., 2010).  One quantitative risk assessment published for Listeria monocytogenes in ready-to-eat food predicted the relative risk rankings among 23 food categories based on two public health metrics outputs (Chen et al., 2013). As a risk analysis tool developed by the FDA, quantitative predictive risk assessment models (QPRAM) are used to predict and characterize risks from fresh produce consumption, and to track each unit of produce providing a history of details (FDA, 2015). These risk analysis tools provide researchers and federal agencies with an important strategy in preventing future contamination events.  !24 The Codex Alimentarius points out that risk analysis is a framework used by food safety personnel to help with decision-making and to better understand the interactions between hazards, foods and human hosts (Demortain, 2012). Even though the FDA has developed a number of risk assessment tools such as the FDA-iRisk, due to the lack of available published data on cross-contamination studies of fresh-cut produce, large informational data gaps exist that limit the accuracy of the models being developed. Consequently, more data needs to be collected to improve the reliability of current risk assessments. 1.9 FSMA and the influences on food safety  In 2011, the FDA Food Safety Modernization Act (FSMA), a set of regulations which expands the scope of food safety laws, was signed into law by President Obama, aimed at better protecting public health by improving food safety systems in the US. With these regulations, the focus shifted from simply responding to preventing contamination events.  In 2016, the FDA made $19 million available to help states implement these food safety rules through activities such as educational programs, inspection and technical assistance (FDA, 2016; Pouliot, 2014). Specifically, FSMA encourages coordination and collaboration among federal, state and local agencies in the area of inspections and food safety (Tai, 2015).  One section of the final rule focuses on produce safety. Quoting from the FDA, Òthis rule establishes, for the first time, science-based minimum standards for the safe growing, harvesting, packing, and holding of fruits and vegetables grown for human consumptionÓ. In this case, the  !25 risk of contamination is more likely to be reduced for fresh produce production and processing if these standards are followed.  1.9 Overall goals The microbiological safety of fresh-cut leafy greens remains an ongoing concern as evidenced by scores of recalls and sporadic outbreaks. Consequently, this study was designed to assess the transfer and redistribution of realistically low levels of Escherichia coli O157:H7 during simulated commercial production of fresh-cut romaine lettuce.  Based on previous studies conducted in Dr. RyserÕs laboratory, a small portion of contaminated product may spread and contaminate a large batch during processing (Buchholz et al., 2014). Consequently, a better understanding of how cross-contamination occurs in the processing pathway and the extent to which the cross-contamination occurs is needed. These findings, which reflect real-world contamination levels, will be critical to improving current risk assessment tools under development.               !26 CHAPTER 2:   Spread of Escherichia coli O157:H7 during Flume Washing and Drying of Fresh Cut Romaine Lettuce                                !27 2.1 OBJECTIVE The objective of this cross-contamination study was to quantify Escherichia coli O157:H7 transfer and redistribution on fresh-cut romaine lettuce and wash water during post-harvest processing, both with and without sanitizers.                                 !28 2.2 MATERIALS AND METHODS 2.2.1 Overall experimental design. This study quantified the variability in redistribution of E. coli O157:H7 contamination after simulated commercial production of fresh-cut romaine lettuce. All transfer and redistribution data were obtained using Michigan State UniversityÕs pilot-scale production line for fresh-cut leafy greens. The results were based on three replicates giving a total 36 experiments.!Using inoculated radicchio as a colored surrogate for uninoculated romaine lettuce, radicchio leaves (~5 x 5 cm) were spot-inoculated one day ahead of processing and held overnight at 4 oC to achieve three inoculation levels (~10-1, 101 and 103 CFU/leaf). On the day of processing, the inoculated radicchio leaves were mixed with uninoculated romaine lettuce leaves (~5 x 5 cm) to obtain a 5 kg batch with inoculated:uninoculated product ratios of 0.5:100, 1:100, 5:100 and 10:100. After 90 seconds of flume washing in sanitizer-free water followed by shaker table dewatering and centrifugal drying, all radicchio leaves were removed from the lettuce before testing.  Each batch of romaine lettuce (5kg) was then divided into ~20 225-g samples which were analyzed for presence/absence of E. coli O157:H7 using the E. coli O157:H7 GeneQuence asssay (Neogen Corp, Lansing, MI). The percentage of E. coli O157:H7 lost from radicchio leaves during processing was determined by comparing E. coli O157: H7 populations on the radicchio samples before (control) and after washing using direct plating with or without membrane filtration, depending on the inoculation level. E. coli O157:H7 populations were also assessed in two 50 ml wash water samples; one was taken at the beginning as a control and  !29 another one was taken at the end of washing. One centrifugation water sample was also assessed for E. coli O157:H7 using membrane filtration. 2.2.2 Produce. Individually wrapped romaine lettuce (12 heads/case) and radicchio heads (9 heads/case) were obtained from a local wholesaler (Stan Setas Produce Co., Lansing, MI), stored in a 4o C walk-in cooler, and used within 3 days of delivery. On the day of use, the outer romaine lettuce leaves were discarded with the remaining leaves separated until the core was reached. For the processing experiments, one 5 kg batch of romaine lettuce was mechanically shredded using the Urschel shredder for each processing run, and then collected for flume washing. 2.2.3 Bacterial strains used. For safety purposes, all of this work was conducted using non-toxigenic, avirulent strains. Recent studies of bacterial attachment ability were conducted in our laboratory with a modification of the microtiter plate assay described by Jackson t al. (2002). Based on the results, these avirulent strains behaved similarly to a set of virulent strains linked to outbreaks involving these same commodities (Buchholz et al., 2012; Wang and Ryser, 2014). Four non-toxigenic, green fluorescent proteinÐlabeled (GFP-labeled), ampicillin-resistant strains of E. coli O157:H7 (ATCC 43888, CV2b7, 6980-2, and 6982-2) previously obtained from Dr. Michael Doyle (Center for Food Safety, University of Georgia, Griffin, GA) were used and prepared in trypticase soy broth (Difco, Becton Dickinson, Sparks, MD) containing 0.6% yeast extract (Difco, Becton Dickinson) and 100 ppm ampicillin (ampicillin sodium salt, Sigma  !30 Chemical Co., St. Louis, MO) (TSBYE-AMP) and 10% (v/v) glycerol (Sigma Chemical Co., St. Louis, MO) and stored at -80¡C until use.  Working cultures were prepared by streaking each stock culture on trypticase soy agar plates (Difco, Becton Dickinson) containing 0.6% yeast extract and 100 ppm ampicillin (TSAYE-Amp). After 18Ð24 h of incubation at 37¡C, a single colony was subjected to two successive transfers in 9 ml of TSBYE-Amp and incubated at 37o C for 24 h. After incubation, the cultures were combined in equal volumes to obtain a 4-strain cocktail containing ~109 CFU/ml, and then diluted in sterile phosphate buffer solution (PBS) to the levels needed for inoculation.  2.2.4 Inoculation of radicchio. In order to assess redistribution of the inoculum to previously uncontaminated product, the required quantities of radicchio (aseptically cut into pieces measuring ~ 5 x 5 cm with an average weight of 1 g per piece) were spot-inoculated with a total of 50 !l of the E. coli O157:H7 cocktail at multiple locations (normally 5-8 locations) on the leaf to obtain populations of 10-1, 101 or 103 CFU/leaf after overnight storage at 4oC. After 15 minutes of drying in a biosafety cabinet, these inoculated surrogate products were collected into sterile plastic boxes (PLA NatureWorks, NE) and stored overnight at 4oC to allow the inoculum to attach to the leaf surface to mimic pre-harvest contamination.  Just before processing, a 25 g radicchio sample was assessed to determine the starting inoculation level. Four different inoculated:uninoculated product ratios (0.5:100, 1:100, 5:100, 10:100) based on product weight were used to determine the differences in pathogen  !31 redistribution based on the amount of inoculated to uninoculated product processed for each experiment. . 2.2.5 Processing line. A pilot-scale processing line consisting of a lettuce shredder, step conveyer, flume tank, shaker table, and dewatering centrifuge was used for all experiments. The commercial lettuce shredder (model TRS 2500 Urschel TranSlicer, Valparaiso, IN) was operated at a feed belt/slicing wheel speed of 198 m/min and 905 RPM, respectively, to obtain a shred size of approximately 5 " 5 cm. A stainless steel non-refrigerated water recirculation tank (~1000 L capacity) was connected to a 3.6-m long stainless steel flume tank (Heinzen Manufacturing, Inc., Gilroy, CA) equipped with two overhead spray jets by a 4.1-m long, 10-cm diameter hard plastic discharge hose and a centrifugal pump (model XB754FHA, Sterling Electric, Inc., Irvine, CA) that circulated the water at ~15 L/sec. A custom-made stainless steel screen was installed at the end of the flume tank to retain product for 90 seconds of washing. The stainless steel shaker table for partial dewatering was operated by a 1 HP Baldor washdown duty motor (Baldor Electric Co., Ft. Smith, AR) at 1760 RPM. Water removed from the leafy greens during mechanical shaking passed through a fine mesh screen and was fed into the water holding tank by a water recirculation spout underneath the shaker table. A 22.7-kg capacity centrifugal Spin Dryer (model SD50-LT, Heinzen Manufacturing, Inc.) with three internally timed spin cycles totaling 60 s was used for centrifugal drying. The water used for this processing line was tap water at ~10oC.   !32 2.2.6 Romaine lettuce processing and sample collection. Uninoculated romaine lettuce leaves (5 kg) were hand-fed into the shredder (Figure 2.1) at a rate of approximately 0.5 kg/second, and mixed with the required amount of inoculated radicchio (0.5:100, 1:100, 5:100, 10:100 on a per weight basis). Thereafter, the leaves were manually dumped into the flume tank (Figure 2.2), washed in 890 L of recirculating sanitizer-free water (~10oC) for 90 s, released from the flume tank, partially dewatered on the shaker table, collected in a single centrifugation basket and then centrifugally dried (Figure 2.3).  After removing all the inoculated radicchio for separate testing, the entire 5 kg batch of romaine lettuce was subdivided into 225 g samples in separate Whirl-pak¨ bags for further analysis with the spent centrifugation water (Figure 2.4). A 25 g sample of inoculated radicchio was removed from the total removed and examined for numbers of E. coli O157:H7 after processing. One 50 ml wash water sample was collected from the water return spout above the recirculation tank before processing (control) and one 50 ml wash water sample was collected at the end of processing and tested for E. coli O157:H7. Finally, one 225g romaine lettuce sample per delivery was tested upon arrival using GeneQuence kit to ensure absence of E. coli O157:H7.      !33  Figure 2.1: Lettuce shredding    Figure 2.2: Flume washing and dewatering  !34  Figure 2.3: Centrifugation   Figure 2.4: Sample collection   !35 2.2.7 Chemical sanitizers. To mimic industrial practices more closely, romaine lettuce was also processed as above using flume water containing 60 ppm of available chlorine (XY-12, Ecolab, acidified to pH 6.50 with citric acid, Sigma-Aldrich, St. Louis, MO) (Davidson et al., 2013). However, considering that E. coli O157:H7 was expected to be non-detectable after processing, only the highest product inoculation level (103 CFU/leaf) and inoculated: uninoculated ratio (10:100) were assessed for romaine lettuce based on three replicates. 2.2.8 Microbiological analysis. All of the samples including 25 g of radicchio before processing (control), 25 g of radicchio after processing, three water samples and the ~20 225 g bags of romaine lettuce were qualitatively and quantitatively assessed for E. coli O157:H7 using direct plating and the GeneQuence assays (Neogen Corp., Lansing, MI). The 25 g radicchio samples were diluted in 50 ml sterile PBS and homogenized using a stomacher (Stomacher 400 Circulator, Seward, Worthington, UK) at 260 rpm for 2 min. Thereafter, 50 ml of the sample homogenate or 50 ml of wash and centrifugation water collected after processing were plated with or without prior membrane filtration on TSAYE-amp to quantify E. coli O157:H7 after 24 h of incubation at 37oC. The lettuce sample homogenate was enriched using the Neogen Reveal 20 h enrichment method (Neogen Corp., Lansing, MI) and examined for the presence/absence of E. coli O157:H7 using the GeneQuence assay according to the manufacturer. Due to the technical limitation in quantification of low levels of contamination, E. coli O157:H7 populations in each lettuce sample bag after processing were not assessed.  !36 2.2.9 GeneQuence Assay. The GeneQuence assay is a sandwich enzyme-linked immunosorbent assay (S-ELISA) in a microwell format, which can be used to screen various commodities for the presence of E. coli O157:H7 antigens (Neogen, 2016). The GeneQuence assay contains capture and detector DNA probes specific to the ribosomal RNA of E. coli O157:H7. A positive result occurs when both DNA probes bind to the target and the capture probe-coated well (Neogen, 2016). The instruments used for GeneQuence testing include a Stat Fax 4200 microplate reader and a Stat Fax 2600 automatic microplate washer provided by Neogen (Figure 2.5). The GeneQuence kits (Figure 2.6) were stored refrigerated in a 4o C walk-in cooler and equilibrated to room temperature before use (Neogen, 2016).              !37   Figure 2.5: The Stat Fax 4200 microplate reader and Stat Fax 2600 semi-automatic microplate washer  Figure 2.6: GeneQuence kit      !38 2.2.10 Statistical analysis. The quantitative results obtained from direct plating were converted to log CFU/g or log CFU/100 ml to calculate percent transfer based on the initial inoculum. In addition, the percentage of E. coli O157:H7 CFU lost during processing was determined from the inoculated product counts before and after processing. The qualitative results were analyzed by determining the percentage of bags positive for E. coli O157:H7 based on the total weight of product processed. Results based on triplicate experiments were averaged and subjected to an analysis of variance (ANOVA) using JMP 12.2 (SAS Institute Inc., Cary, NC). The Tukey- Kramer HSD test was used with P values of # 0.05 considered significantly different. Values of half the limit of detection were used when no E. coli O157:H7 were detected in the samples.                    !39 2.3 RESULTS 2.3.1 Romaine lettuce. After collecting and enriching samples overnight, GeneQuence testing was performed to determine the presence/absence of E. coli O157:H7 in the sample bags. Based on triplicate experiments, all lettuce samples yielded E. coli O157:H7 when radicchio was inoculated at 103 CFU/leaf. When radicchio was inoculated at 101 CFU/leaf, the percentage of positive samples decreased from 96.8% to 93.7%, 81.0% and 63.5% for inoculated: uninoculated ratios of 10:100, 5:100, 1:100 and 0.5:100, respectively. At 10-1 CFU/leaf, 22.2%, 6.3%, 4.8% and 6.3% of the samples were positive for the same inoculated:uninoculated ratios (Table 2.1). Based on triplicate experiments, when the inoculation level decreased from 103 CFU/leaf to 10-1 CFU/leaf, the populations of E. coli O157:H7 transferred from radicchio to romaine lettuce decreased as well (P < 0.05). Within each inoculation level, no significant differences (P > 0.05) were found among the four inoculated: uninoculated product ratios. The results are presented as graphs in Figures 2.7 and 2.8 below.                !40  Figure 2.7: Percent of samples detected as positive for E. coli O157:H7 due to the E. coli O157:H7 transfer to 225 g samples of romaine lettuce after 90 sec of flume washing in sanitizer-free water based on different inoculation levels. Mean values with different letters are significantly different (P 0.05) within the same product ratio (10:100, 5:100, 1:100, 0.5:100).  !41   Figure 2.8: Percent of samples detected as positive for E. coli O157:H7 due to the E. coli O157:H7 transfer to 225g samples of romaine lettuce after 90 sec of flume washing in sanitizer-free water based on different product inoculated:uninoculated ratios. Mean values with same letter are not significantly different (P > 0.05) within the same inoculation level.  Table 2.1: Numbers and percent of positive samples among ~20 total samples with different inoculation levels and product ratios. Inoculation level (CFU/leaf) Inoculated:uninoculated product ratio Positive samples/total samples Average % of positive samples ± SD    Rep 1     Rep 2  Rep 3  Mean (±SD) 103 10100 21/21 21/21 20/20 100.0%±0.0% 5100 21/21 20/20 21/21 100.0%±0.0% 1100 21/21 21/21 21/21 100.0%±0.0% 0.5100 21/21 21/21 20/20 100.0%±0.0% 101 10100 21/21 19/21 21/21 96.8%±10.0% 5100 17/21 21/21 20/20 93.7%±10.0% 1100 16/21 19/21 16/21 81.0%±10.0% 0.5100 18/21 6/21 16/21 63.5%±30.0% 10-1 10100 5/21 1/21 8/21 22.2%±20.0% 5100 2/21 2/21 0/21 6.3%±10.0% 1100 1/21 1/21 1/21 4.8%±0.0% 0.5100 4/21 0/20 0/21 6.3%±10.0% 0%10%20%30%40%50%60%70%80%90%100%10 310 110 -1Percentage of positive samplesInoculation level (CFU/leaf)10100510011000.5100""""####$$$$%&'%&%%&(% !42 2.3.2 Radicchio. When radicchio was inoculated at 103 CFU/leaf, the E. coli O157:H7 population decreased from 3.7 to 2.2, 3.9 to 2.3, 3.8 to 2.2, and 3.4 to 2.3 log CFU/g after processing for inoculated:uninoculated ratios of 10:100, 5:100, 1:100 and 0.5:100, respectively (Table 2.2). At 101 CFU/leaf, E. coli O157:H7 population decreased from 1.8 to 0.4, 1.9 to 0.1, 1.9 to 0, and 1.9 to 0.1 log CFU/g after processing (Figures 2.9 and 2.10). Since the E. coli O157:H7 populations in samples after processing were below limit of detection (1 CFU/25g) when radicchio was inoculated at 10-1 CFU/leaf, the percentage of CFUs lost could not be determined.                   !43  Figure 2.9: E. coli O157:H7 population on radicchio leaves before and after processing based on different inoculated:uninoculated product ratios. The log reductions after washing are not significantly different within the same inoculation level (P > 0.05).  Figure 2.10: E. coli O157:H7 population on radicchio leaves before and after processing based on different inoculation level. At 10-1 CFU/leaf, E. coli populations are below limit of detection (1 CFU/25g) so percent loss is not applicable at this inoculation level. The log reductions after washing are not significantly different within the same produce ratio (P > 0.05).  !44 Table 2.2: E. coli O157:H7 populations on radicchio before washing and E. coli O157:H7 log reductions on radicchio after processing.  Inoculation level (CFU/leaf) Inoculated: uninoculated ratio Radicchio before washing (log CFU/g) Radicchio after processing (log CFU/g) Reduction after processing (log CFU/g) 103 10100 3.7±0.0 2.2±0.2 1.5a 5100 3.9±0.1 2.3±0.4 1.6a 1100 3.8±0.2 2.2±0.2 1.6a 0.5100 3.4±0.3 2.3±0.1 1.1a 101 10100 1.8±0.4 0.4±0.1 1.4a 5100 1.9±0.1 0.1±0.5 1.8a 1100 1.9±0.1 0±0.3 1.9a 0.5100 1.9±0.1 0.1±0.1 1.8a 10-1 10100 -0.5±0.2 < 1 CFU/25g N/A 5100 -0.6±0.4 < 1 CFU/25g N/A 1100 -0.8±0.0 < 1 CFU/25g N/A 0.5100 -0.5±0.2 < 1 CFU/25g N/A                      !45 2.3.3 Water. Sanitizer-free wash water was used for this study. One 50 ml wash water sample was collected from the water return spout above the recirculation tank before and after processing and tested for E. coli O157:H7 along with one 50 ml water sample collected from the centrifugal dryer. E. coli O157:H7 was not detected in the water before processing (control), whereas populations varied from the limit of detection (0.3 log CFU/ 100 ml) to 1.2 log CFU/100 ml in wash water samples collected after processing. The lower inoculation level yielded more water samples with E. coli O157:H7 populations below the LOD. When the inoculation level was at 10-1 CFU/leaf, all the wash water samples were negative for E. coli O157:H7. Similarly, most of the centrifugation water samples were negative for E. coli O157:H7 with populations in the remaining centrifugation water samples ranging from the limit of detection (0.3 log CFU/ 100 ml) to 1.5 log CFU/100 ml. 2.3.4 Sanitizer trials. In order to mimic industrial practices more closely, romaine lettuce was also processed in wash water containing 60 ppm of available chlorine. However, only the highest inoculation level (103 CFU/leaf) and inoculated: uninoculated ratio (10:100) were investigated since E. coli O157:H7 was expected to be non-detectable under the other conditions.  Based on the three replicates, all lettuce samples were positive for E. coli O157:H7 (Table 2.3). The population of E. coli O157:H7 on radicchio decreased from 3.7 to 1.8 log CFU/g with 98.7% of CFUs loss after processing. In addition, all of the water samples were negative for E. coli O157:H7.    !46 Table 2.3: Comparison between sanitizer-free trials and 60 ppm sanitizer trials at the same inoculation level (103 CFU/leaf) and inoculated: uninoculated product ratio (10:100).   sanitizer-free  60 ppm sanitizer Inoculation level (CFU/leaf) 103 103 Inoculated: uninoculated ratio 10:100 10:100 positive samples among total 21 samples 21/21 21/21 % of positive samples/total samples 100% 100% log reduction on radicchio after processing 1.5±0.2 2.0±0.4 E. coli O157:H7 populations (CFU/100 ml) in wash water 29.3 0 E. coli O157:H7 populations (CFU/100 ml)  in centrifugation water 6.7 0                         !47 2.4 DISCUSSION This study investigated the probability and extent of E. coli O157:H7 redistribution between contaminated and uncontaminated products during pilot-plant scale processing. Even though non-pathogenic strains of E. coli O157:H7 were used, these same strains were previously shown to behave similarly in terms of attachment and growth to a set of produce-related outbreak (Buchholz, 2012).  Colored surrogates have been used in numerous studies to differentiate products after processing. For example, the red-leaf Lollo Rossa was used as a surrogate for green-leaf lettuce in a cross-contamination study because of the color contrast (Nou and Luo, 2010). Similarly, radicchio was used as a surrogate for romaine lettuce, which allowed physical separation between contaminated and uncontaminated products (Buchholz et al., 2014). In our study, radicchio, which was chosen due to its reddish purple leaves and similar surface structure to romaine lettuce, helped visually track the inoculated product through processing, and retrieve the the inoculated product from the romaine lettuce before analysis.  In this study, spot inoculation was used to mimic pre-harvest contamination from soil workersÕ hands or harvesting equipment (Xu et al., 2013). In another quantitative transfer study conducted in our laboratory, E. coli O157:H7 populations decreased 85.9% on dip-inoculated romaine after processing (Buchholz, 2012). Given the slightly greater E. coli reductions seen in our current study when radicchio was spot-inoculated, E. coli may have adhered less tenaciously to the surface since washing was more effective than with spray or dip inoculation (Singh et al.,  !48 2002). Similarly, E. coli O157:H7 populations were easier to decrease on spot-inoculated as compared to dip-inoculated green onions during washing (Xu et al., 2013). These observations can also be further explained by the increased infiltration of E. coli O157:H7 through cut or damaged leaves during dip inoculation (Koseki et al., 2003).  Sanitizer-free wash water was used to obtain quantitative transfer data since any sanitizer will inactivate the target organism, leading to falsely low transfer rates. Three inoculation levels (103,101 and 10-1 CFU/leaf) were used in this study to represent contamination scenarios with low inoculation levels. Based on previous field studies, either no or very low levels of contamination with pathogenic bacteria were found (Johnston, et al., 2005; Johnston, et al., 2006; Mukherjee et al., 2004). Johnston, et al. (2005) reported that the overall geometric mean E. coli counts were low for most fresh produce items including leafy greens (# 1.0 log CFU/g) and highest for cantaloupe (1.5 log CFU/g). Even though high levels of E. coli O157:H7 contamination due to Òsuper-sheddersÓ may occur in the field, it is more realistic to have low levels of contamination from various sources (Hoorfar, 2014; Munns et al., 2015). Moreover, several studies simulated cross-contamination of lettuce with E. coli O157:H7 to evaluate pathogen transfers strategies such as product-to-water, product-to-equipment transfer (Buchholz, 2012; Buchholz et al., 2014; Perez-Rodriguez, et al., 2011). However, this is the first study to assess pathogen transfer at realistic contamination and inoculated:uninoculated product ratios.   At the highest inoculation level of 103 CFU/leaf, all lettuce samples were positive regardless of the inoculated: uninoculated ratio. This high level of cross-contamination is the result of using  !49 of sanitizer-free water as well as direct surface contact between contaminated radicchio leaves and uncontaminated lettuce (FDA, 2008). When lower inoculation levels of 101 and 10-1 CFU/leaf were used, the percentage of positive bags decreased significantly (P < 0.05), showing that lower inoculation levels led to decreased E. coli O157:H7 transfer from radicchio to romaine lettuce during processing (P < 0.05). However, at 10 -1 CFU/leaf, a high product ratio did not guarantee more positive samples with a lower percentage of positive samples expected due to the relatively low inoculation level. Since some radicchio leaves will invariably degrade into small fragments during processing, the presence of any inoculated would give a high probability of detection in the GeneQuence assay. Four inoculated: uninoculated ratios were used to assess the impact of product volume on the extent of pathogen spread during washing. It is important to include product ratio as a variable in the DOE to evaluate how that will change cross-contamination based on the prevalence of contamination in the field. Based on previous field studies in the US and Canada, it indicates the prevalence of E. coli contamination is extremely low in the field. FDA reported (2003) 0 % of 1028 domestic samples of fresh produce were found to be contaminated with E. coli O157:H7, and several studies found no E. coli O157:H7 in fresh produce (Denis, et al., 2016; Johnston, et al., 2005; Mukherjee, et al., 2004). There were no significant differences between different ratios with same inoculation level (P > 0.05), it is possible to indicate the water spread may have a larger impact on the extent of pathogen transfer than the direct contact between radicchio and lettuce during washing, only if the direct contact at the step when mixed radicchio  !50 with lettuce after shredding can be ignored. In a previous study conducted using the same pilot-scale fresh-cut processing line, the potential for contaminated lettuce to contaminate previously uncontaminated lettuce during processing was investigated. A similar inoculated:uninoculated product ratio (approximately 1:100) was used but with a higher inoculation level of 106 CFU/ml compared to 103 CFU/m or less in our study. According to this previous study, most of samples (~38 bags) were positive for E. coli O157:H7 with an average of 1.3 log CFU/g, indicating large scale spread of the pathogen during processing when the inoculation level is high (Buchholz et al., 2015). In our study, lower inoculation levels were applied to assess cross-contamination in order to mimic more realistic conditions.  Water samples were taken at the end of processing and during the centrifugation step. E. coli O157:H7 was undetectable in the water after radicchio inoculated at 10-1 CFU/leaf was processed. However, since 890 L of water was used for washing, the target organism was likely still present, but at levels below the limit of detection. In contrast, at 103 CFU/leaf, E. coli O157:H7 was quantifiable in some of the water samples with more cells thus available to contaminate the lettuce during processing. For commercial processing of fresh-cut produce, sanitizers are commonly used to reduce microbial populations during washing. However, their efficacy in reducing populations on fresh-cut produce is normally limited to 1-3 logs (Haute et al., 2015; Sapers, 2001). In order to mimic industrial practices, the same experiment was conducted using wash water containing 60 ppm of available chlorine, but only at the highest inoculation level and ratio since E. coli  !51 O157:H7 was expected to be non-detectable for the other conditions. Based on triplicate experiments, lettuce samples were positive after processing in wash water containing a sanitizer, which indicated the limited efficacy of the sanitizer used for washing. Since recirculating wash water was used, the shredded lettuce increased the organic load of the water during processing. As learned from other studies, the efficacy of chlorine-based sanitizers is greatly reduced by the presence of high organic loads in the water (Allende. et al., 2008; Davidson et al., 2014; Shen. et al. 2012). Hence, commercial washing of fresh-cut produce in water containing a sanitizer does not ensure end-product safety.  According to previous studies and outbreak investigations, even though water can be used to wash produce to reduce potential contamination, wash water is also considered as a vehicle to spread pathogen contamination, as well as a direct source when the water does not meet quality standards (Holvoet. et al., 2014). Based on one study that evaluated the degree of microbial contamination during commercial processing in two Belgian fresh-cut produce processing facilities, the washing step was identified as a potential pathway to spread and introduce pathogens to the end product. Moreover, practices such as insufficient cleaning and use of high product/water ratios resulted in a rapid increase of E. coli indicating greater risk of cross-contamination, especially when sanitizer use is not allowed for fresh produce washing as in some European countries (Holvoet et al., 2012).  In summary, this study was conducted to better understand bacterial transfer and the different degrees of cross-contamination that can occur during washing and drying when realistic  !52 levels of E. coli O157:H7 and contaminated product are present. Being the first report focusing on fresh-cut processing with realistically low inoculation and inoculated:uninoculated product ratios, we have learned that when the inoculation is as low as 103 CFU/leaf, no matter what the product ratio is, the extent of cross-contamination can be high enough to contaminate large quantities of uncontaminated products. Even though the inoculation level is 10-1 CFU/leaf, there are still risks of cross-contamination during processing. We also found out that commercial washing of fresh-cut produce in water containing a sanitizer does not ensure end-product safety even when the inoculation level is relatively low. These findings provide more insights on E. coli O157:H7 transfer and redistribution during processing in realistic conditions. For commercial processing facilities, routine sampling of lettuce and wash water could be a way to minimize the risk of cross-contamination. Furthermore, these results will fill several critical data gaps in the current FDA risk assessment for fresh-cut leafy greens which is being designed to predict potential risks of fresh produce processing under different conditions.                !53 CHAPTER 3   Conclusions and Future Recommendations                                !54 Leafy greens have been implicated in multiple foodborne disease outbreaks and recalls involving E. coli O157:H7. Given this threat to public health, the FDA has shifted their strategy from responding to preventing potential contamination events, and has developed various risk assessment tools for better controlling and predicting food safety risks. Given these recent outbreaks, it is important to gain a clearer understanding of how cross-contamination occurs during commercial processing of leafy greens. This study aimed to fill an important risk assessment data gap identified by the FDA in regard to cross-contamination and redistribution of E. coli O157:H7 during pilot-scale processing of fresh-cut lettuce. One objective was to quantify E. coli O157:H7 transfer and redistribution during production of fresh-cut romaine lettuce using sanitizer-free wash water. Three inoculation level (103, 101, 10-1 CFU/leaf) and four inoculated: uninoculated product ratios were used to determine the extent of cross-contamination between contaminated and uncontaminated products. In this study, lower radicchio inoculation levels led to decreased E. coli O157:H7 transfer to romaine lettuce during processing. However, no significant differences were seen for the different product ratios at the same inoculation level.  In order to better simulate real world problems linked to E. coli O157:H7 contamination, relatively low E. coli O157:H7 inoculation and inoculated:uninoculated product ratios were used to simulate contamination levels in the field. When a small portion of produce is contaminated with low numbers of E. coli O157:H7 and processed with uncontaminated product, the percentage E. coli O157:H7-positive samples decreased after washing and drying, indicating the  !55 decreased food safety risk. Based on these results from sanitizer-free experiments, even though safety concerns still exist due to the presence of positive samples at the lowest inoculation level and with the lowest product ratio, the risk can be minimized using sanitizers during commercial processing. These findings are critical to predict the extent of cross-contamination under realistic conditions. The second objective was to assess E. coli O157:H7 transfer using a similar approach as above, but using wash water containing 60 ppm of free chlorine to mimic industrial practices more closely. Even when the sanitizer was used, all product samples were positive for E. coli O157:H7 after processing, indicating the limited ability of sanitizers to ensure the microbial safety of the finished product.  In the sanitizer experiments, all lettuce samples were positive for E. coli O157:H7 at 103 CFU/leaf and a product ratio of 10:100, which refutes our hypothesis that the level of E. coli O157:H7 population would be non-detectable. Therefore, conducting the same experiments at a lower inoculation level (101 CFU/leaf) with multiple product ratios may tell us under what conditions cross-contamination can be prevented during washing using a sanitizer. Other than that, future studies should focus on different sanitizers such as peroxyacetic acid-based sanitizers that can be applied during washing in order to test their efficacy to prevent cross-contamination during fresh produce processing, compared to chlorine-based sanitizer. Furthermore, since this study only focused on cross-contamination during washing and drying, other processing steps also need to be considered. Because shredding is an important processing step where  !56 cross-contamination may occur, assessing the extent of pathogen transfer and redistribution during shredding at a lower level of inoculation will provide realistic results to fulfill additional data needs for risk assessment.                              !57                  APPENDIX                    !58 Quantification and redistribution of Salmonella Typhimurium LT2 during simulated commercial production of fresh-cut baby spinach and cilantro By Haley S. Smolinski, Siyi Wang, and Elliot T. Ryser Abstract Recent outbreaks of illness traced to fresh-cut baby spinach and cilantro most likely resulted from low levels of bacterial pathogens spreading to subsequent product during processing. Consequently, this study aimed to assess the redistribution of Salmonella Typhimurium LT2 during simulated commercial production of fresh-cut baby spinach and cilantro. Four inoculated:uninoculated product weight ratios (0.5:100, 1:100, 5:100, and 10:100) and three different realistically low inoculation levels (10$, 10%, and 10-% CFU/g) were used with spot-inoculated red leaf lettuce serving as a colored surrogate for baby spinach and cilantro. 5 kg batches of product were flume-washed for 90 sec in a pilot scale leafy green processing line. After washing and removing the red leaf lettuce, all of the previously uninoculated product (~23 samples/225 g each) was assessed for presence/absence of Salmonella using the Salmonella GeneQuence Assay. Overall, 100% of the baby spinach samples with inoculated red leaf lettuce at 10$ CFU/g tested positive. When at 10% CFU/g, 100, 78, 61 and 22% of the samples yielded Salmonella at inoculated:uninoculated ratios of 10:100, 5:100, 1:100, and 0.5:100, respectively. At 10-% CFU/g, 8.0, 6.5, 4.5 and 0% of the samples yielded Salmonella with a similar trend seen for cilantro. Being the first study to assess the spread of expected levels of contamination on incoming leafy greens; these results should prove useful in refining current risk assessments.     !59 Table AI.1: Percent of samples detected as positive for E. coli O157:H7 due to the E. coli O157:H7 transfer to 225 g samples of romaine lettuce after 90 sec of flume washing in sanitizer-free water. Inoculation level(CFU/leaf) Inoculated:uninoculated product ratio positive samples / total samples  % of positive samples / total samples    Rep1  Rep2  Rep3 Mean SD 103 10:100 21/21 21/21 20/20 100.0% 0.0% 5:100 21/21 20/20 21/21 100.0% 0.0% 1:100 21/21 21/21 21/21 100.0% 0.0% 0.5:100 21/21 21/21 20/20 100.0% 0.0% 101 10:100 21/21 19/21 21/21 96.8% 10% 5:100 17/21 21/21 20/20 93.7% 10% 1:100 16/21 19/21 16/21 81.0% 10% 0.5:100 18/21 6/21 16/21 63.5% 30% 10-1 10:100 5/21 1/21 8/21 22.2% 20% 5:100 2/21 2/21 0/21 6.3% 10% 1:100 1/21 1/21 1/21 4.8% 0.0% 0.5:100 4/21 0/20 0/21 6.3% 10%                     !60 Table AI.2: E. coli O157:H7 populations on radicchio leaves before processing (log CFU/g) Inoculation level(CFU/leaf) Inoculated:uninoculated ratio E. coli O157:H7 populations on radicchio before processing-control (log CFU/g) Mean SD 103 10:100 3.7 3.7 3.6 3.7 0.0 5:100 3.8 4.0 3.9 3.9 0.1 1:100 3.9 4.1 3.6 3.8 0.2 0.5:100 3.4 3.7 3.1 3.4 0.3 101 10:100 2.0 1.3 2.0 1.8 0.4 5:100 2.0 1.8 2.1 1.9 0.1 1:100 1.8 1.8 1.9 1.9 0.1 0.5:100 2.0 2.0 1.8 1.9 0.1 10-1 10:100 -0.4 -0.4 -0.8 -0.5 0.2 5:100 -0.5 -0.4 -1.1 -0.6 0.4 1:100 -0.8 -0.8 -0.8 -0.8 0.0 0.5:100 -0.5 -0.6 -0.3 -0.5 0.2                        !61 Table AI.3: E. coli O157:H7 population on radicchio leaves after processing (log CFU/g).  Inoculation level ( CFU/leaf) Inoculated:uninoculated ratio E. coli O157:H7 populations on radicchio after processing (log CFU/g) Mean SD 103 10:100 2.1 2.5 2.1 2.2 0.2 5:100 2.4 1.8 2.6 2.3 0.4 1:100 2.4 2.1 2.1 2.2 0.2 0.5:100 2.2 2.3 2.3 2.3 0.1 101 10:100 0.3 0.5 0.4 0.4 0.1 5:100 -0.4 0.2 0.5 0.1 0.5 1:100 -0.4 0.3 0.0 0.0 0.3 0.5:100 0.1 0.1 0.0 0.1 0.1 10-1 10:100 N/A N/A N/A N/A N/A 5:100 N/A N/A N/A N/A N/A 1:100 N/A N/A N/A N/A N/A 0.5:100 N/A N/A N/A N/A N/A                       !62 Table AI.4: Percent of E. coli O157:H7 shed from radicchio leaves after processing.  Inoculation level (CFU/leaf) Inoculated:uninoculated ratio Percent of CFU loss on radicchio after processing (%) Mean SD 103 10:100 97.5% 94.3% 96.8% 96.2% 1.7% 5:100 96.6% 99.3% 94.7% 96.9% 2.3% 1:100 96.3% 98.9% 97.1% 97.4% 1.3% 0.5:100 94.2% 95.8% 98.2% 96.0% 2.0% 101 10:100 98.1% 86.8% 97.5% 94.1% 6.3% 5:100 99.6% 97.3% 97.2% 98.0% 1.3% 1:100 99.3% 97.2% 98.8% 98.4% 1.1% 0.5:100 98.7% 98.5% 98.3% 98.5% 0.2% 10-1 10:100 N/A N/A N/A N/A N/A 5:100 N/A N/A N/A N/A N/A 1:100 N/A N/A N/A N/A N/A 0.5:100 N/A N/A N/A N/A N/A N/A means not applicable for calculating percent of CFU loss on radicchio.                      !63 Table AI.5: E. coli O157:H7 populations in wash water sample taken at the end of processing (log CFU/100 ml). Inoculation level (CFU/leaf) Inoculated:uninoculated ratio E. coli O157:H7 populations from wash water at the end of  processing (log CFU/100ml) Mean SD 103 10:100 0* 1.9 0* 0.8 1.0 5:100 2.3 0.3 0* 0.9 1.2 1:100 1.8 1.7 0* 1.2 0.9 0.5:100 0* 1.1 0* 0.5 0.5 101 10:100 0* 0* 0.8 0.4 0.3 5:100 0* 0.3 0* 0.2 0.1 1:100 0* 0.3 0.6 0.4 0.2 0.5:100 0* 0* 0* 0.2 0.0 10-1 10:100 0* 0* 0* 0.2 0.0 5:100 0* 0* 0* 0.2 0.0 1:100 0* 0* 0* 0.2 0.0 0.5:100 0* 0* 0* 0.2 0.0  * Half of the LOD was used to calculate the mean log value for samples yielding no E. coli O157:H7 colonies by direct plating                    !64 Table AI.6: E. coli O157:H7 populations in water sample for each centrifugal drying batch taken (log CFU/100 ml). Inoculation level (CFU/leaf) Inoculated:uninoculated ratio E. coli O157:H7 populations  (log CFU/100ml) Mean SD 103 10:100 1.3 0* 0* 0.6 0.6 5:100 0* 2.0 2.3 1.5 1.1 1:100 1.1 1.2 0* 0.8 0.6 0.5:100 0* 0* 1.3 0.6 0.6 101 10:100 0* 0* 1.1 0.5 0.5 5:100 0* 0* 0* 0.2 0.0 1:100 0* 0* 0* 0.2 0.0 0.5:100 0* 0* 0* 0.2 0.0 10-1 10:100 0* 0* 0* 0.2 0.0 5:100 0* 0* 0* 0.2 0.0 1:100 1.1 0* 0* 0.5 0.5 0.5:100 0* 0* 0* 0.2 0.0  * Half of the LOD was used to calculate the mean log value for samples yielding no E. coli O157:H7 colonies by direct plating                    !65 Table AI.7: Summary of the results for experiments using 60 ppm sanitizer Sanitizer trials Three replicates Mean SD positive samples/total samples 21/21 21/21 21/21 100.0% 0.0  E. coli O157:H7 populations on radicchio before processing (log CFU/g) 3.5 3.8 3.8 3.7 0.2 E. coli O157:H7 populations (log CFU/g) on radicchio after processing  1.9 1.8 1.6 1.8 0.1 Percent of E. coli O157:H7 population lost during processing 97.7% 99.0% 99.3% 98.7% 0.9% E. coli O157:H7 populations (CFU/100 ml) in wash water 0.0 0.0 0.0 0.0 0.0 E. coli O157:H7 populations (CFU/100 ml)  in centrifugation water 0.0 0.0 0.0 0.0 0.0                            !66 Figure AI.1: Growth curves in GeneQuence enrichment during 24 h incubation with different product:enrichment ratio (1:2 vs 1:9)                         &)&*)&+)&,)&-)&%&)&%*)&&*+,-%&%**+./0123!4506!789:6;<=>?!43;./0123!#@/A?%*%B !67                    BIBLIOGRAPHY                     !68 BIBLIOGRAPHY  AgMRC, 2005. 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