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Recovery Study of Salmonella spp. off the Surfaces of Tomatoes and Packing Line Materials


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A RECOVERY STUDY OF Salmonella SPP. FROM THE SURFACES OF TOMATOES AND PACKING LINE MATERIALS By RAINA LENEVE ALLEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Raina Leneve Allen

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To my parents, for without your love and ne ver-ending support none of this would have been possible. Your guidance and belief in me allowed me to get this far. Dad, I thank you for always wanting something better for your children. You have sacrificed to no end for me. Mom, your faith has always made you strong in my eyes. You were always here to lean on and so many things make you great.

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ACKNOWLEDGMENTS I would like to give special thanks to my committee chair, Dr. Keith R. Schneider, for the opportunities he has given me and the guidance he has offered me throughout the past two years. I admire all he does and I am thankful to have obtained my masters degree under his direction. I would also like to thank the members of my graduate committee, Dr. Douglas Archer and Dr. Steve Sargent, for their help and assistance with this project. I thank my family. Their love and support have inspired me to succeed. I thank my brother, for always having an encouraging word for my ear. I want to thank my colleagues Ben Warren and Tom Ballesteros. I am blessed to have earned this degree with such wonderful friends. I also want to thank my friends, Kim Saranko, Michelle Burtch, Christen McGinnis, the Tuccis, Meg Mizell and Liz Benz. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTERS 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................5 Foodborne Illnesses Associated with Fresh Produce....................................................7 Salmonella Species.....................................................................................................10 Salmonellosis Outbreaks Involving Fresh Tomatoes.................................................12 Tomatoes and Salmonellae.........................................................................................13 Tomato Industry..........................................................................................................16 Postharvest Handling of Tomatoes.............................................................................17 Extrinsic Factors Influencing Microbial Viability......................................................21 Attachment of Microorganisms to Various Surfaces..................................................24 Microbiological Recovery Methods Involving Fresh Produce...................................26 3 MATERIALS AND METHODS...............................................................................29 Selection of Temperature and Relative Humidity Combinations...............................29 Acquisition and Maintenance of Salmonella Cultures...............................................30 Growth Levels of Salmonella Serovars after a 20-Hour Incubation..........................31 Preparation of Inoculum.............................................................................................32 Inoculation Procedures...............................................................................................33 Inoculation of Tomatoes......................................................................................33 Inoculation of Packing Line Materials................................................................34 Salmonella Recovery off Tomato Surfaces and Packing Line Surfaces....................35 Statistical Analysis......................................................................................................36 4 RESULTS...................................................................................................................38 Growth Levels of Salmonella Serovars after a 20-Hour Incubation..........................38 v

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Recovery of Salmonella off Tomato Surfaces............................................................39 Tomatoes Subjected to Spring Parameters..........................................................40 Tomatoes Subjected to Fall/Winter Parameters..................................................41 Tomatoes Subjected to Ripening Room Parameters...........................................41 Comparison of Tomato Recovery Studies...........................................................42 Recovery of Salmonella off Packing Line Surfaces...................................................42 Stainless Steel Surfaces Subjected to Spring Parameters....................................42 Stainless Steel Surfaces Subjected to Fall/Winter Parameters............................43 Comparison of Stainless Steel Recovery Studies................................................44 PVC Surfaces Subjected to Spring Parameters...................................................44 PVC Surfaces Subjected to Fall/Winter Parameters...........................................44 Comparison of PVC Recovery Studies...............................................................45 Sponge Rollers Subjected to Spring Parameters.................................................46 Sponge Rollers Subjected to Fall/Winter Parameters.........................................46 Comparison of Sponge Roller Recovery Studies................................................47 Conveyor Belt Surfaces Subjected to Spring Parameters....................................48 Conveyor Belt Surfaces Subjected Fall/Winter Parameters................................48 Comparison of Conveyor Belt Recovery Studies................................................48 Unfinished Oak Surfaces Subjected Spring Parameters......................................49 Unfinished Oak Surfaces Subjected to Fall/Winter Parameters..........................50 Comparison of Unfinished Oak Recovery Studies..............................................51 5 DISCUSSION.............................................................................................................52 Recovery of Salmonella off Tomato Surfaces............................................................54 Recovery of Salmonella off Packinghouse Surfaces..................................................57 6 CONCLUSION...........................................................................................................66 LIST OF REFERENCES...................................................................................................68 BIOGRAPHICAL SKETCH.............................................................................................76 vi

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LIST OF TABLES Table page Table 3-1. Temperature and relative humidity combinations selected to simulate a ripening room environment (20C/90%RH) and a fall/winter (20C/60%RH) and spring (30C/80%RH) tomato production conditions..............................................30 Table 3-2. Salmonella enteritidis serovars obtained from Dr. Linda J. Harris at the University of California, Davis: wild types* and rifampicin-resistant serovars listed with source...............................................................................................................31 Table 3-3. Surface area dimensions of each type of packing line material that was inoculated with a five serovar rifampicin-resistant Salmonella cocktail.................34 vii

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LIST OF FIGURES Figure page Figure 4-1. Average log 10 counts of five Salmonella serovars (rif+) after a 20-hour incubation.................................................................................................................39 Figure 4-2. Salmonella recovery (log 10 CFU/ml) from tomato surfaces in ripening room parameters (20C/90%RH) and spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days......................................................................40 Figure 4-3. Salmonella recovery (log 10 CFU/ml) from stainless steel surfaces in spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days...................43 Figure 4-4. Salmonella recovery (log 10 CFU/ml) from PVC surfaces in spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days...................45 Figure 4-5. Salmonella recovery (log 10 CFU/ml) from sponge roller surfaces in spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days...................47 Figure 4-6. Salmonella recovery (log 10 CFU/ml) from conveyor belt surfaces in spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days...................49 Figure 4-7. Salmonella recovery (log 10 CFU/ml) from unfinished oak surfaces in spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days...................50 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science A RECOVERY STUDY OF Salmonella SPP. FROM THE SURFACES OF TOMATOES AND PACKING LINE MATERIALS By Raina Leneve Allen December 2003 Chair: Keith R. Schneider Major Department: Food Science and Human Nutrition Salmonellosis is a common gastrointestinal foodborne illness that is caused by the bacterium Salmonella. Every year, approximately 40,000 culture confirmed cases of salmonellosis are reported in the United States. Multi-state salmonellosis outbreaks have occurred due to the consumption of contaminated raw tomatoes. This study was designed to evaluate the recovery of Salmonella spp. from tomato, stainless steel, polyvinyl chloride (PVC), sponge roller, conveyor belt and unfinished oak surfaces. Fruit and material surfaces were maintained at specific temperatures and relative humidity (RH), 30C/80%RH, 20C/60%RH and 20C/90%RH for 28 days. Different temperature and relative humidity combinations had a significant effect on the survival of Salmonella on tomato and packing line surfaces. An ambient temperature (20C) combined with 90%RH or 60%RH seemed to better facilitate the survival of Salmonella as compared to an elevated temperature (30C) combined with 80%RH. ix

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Log 10 values of recovered Salmonella from tomato surfaces decreased over time in all three simulated environments. Tomatoes stored at 20C/60%RH and 20C/90%RH had an approximate 4.0 log 10 CFU/ml reduction of Salmonella over 28 days. A lower amount of Salmonella was recovered from tomatoes stored at 30C/80%RH over 28 days (1.0 log 10 CFU/ml by Day 14). Salmonella was recovered from stainless steel and PVC surfaces stored at 20C/60%RH for all sampling intervals. Salmonella was only recovered from stainless steel and PVC surfaces on Days 0, 1, 3 and 7 while contained at 30C/80%RH. No Salmonella was recovered from conveyor belt surfaces stored at 30C/80%RH after Day 3 or from sponge roller surfaces stored at 30C/80%RH after Day 0. Salmonella was recovered from conveyor belt surfaces stored at 20C/60%RH until Day 14 (0.60 log 10 CFU/ml). No Salmonella was recovered from sponge roller surfaces held at 20C/60%RH after Day 3. Recovery of Salmonella from unfinished oak surfaces was variable. Salmonella was recovered from oak surfaces held under 20C/60%RH at approximately 2.0 log 10 CFU/ml on Day 28. Salmonella recovery fluctuated over 28 days for oak surfaces stored at 30C/80%RH. On Days 3 and 14 there were increases in Salmonella recovery (approximately 3.0 log 10 CFU/ml and 1.0 log 10 CFU/ml, respectively). On Days 7, 11, 21 and 28, no Salmonella was recovered from oak surfaces. It is suspected that oak pieces harbored and protected Salmonella in its matrix. Results show the importance of a regular sanitation program for surfaces, since Salmonella could survive for weeks on tomato and packing line surfaces in an accommodating environment, thus increasing the risk of foodborne illness in fresh-market tomatoes. x

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CHAPTER 1 INTRODUCTION During the past two decades, an increase in consumption of fresh produce has occurred in the United States (Tauxe et al. 1997). Greater distribution distances for fresh produce from new geographic sources have allowed a variety of fresh produce to be readily available to consumers year round. Increased availability of fresh produce accompanied with increased demand of fresh produce has resulted in an elevation of produce-associated foodborne illness outbreaks in the U.S. (Tauxe et al. 1997). The Centers for Disease Control and Prevention (CDC) report that the number of produce-associated outbreaks has doubled between the periods of 1973 to 1987, and 1988 to 1991, and that the number of cases associated with these outbreaks has more than doubled (Tauxe et al. 1997). In January of 1997, President Clinton announced a Food Safety Initiative in response to a report he received from the U.S. Department of Health and Human (DHHS) Services, the U.S. Department of Agriculture (USDA) and the U.S. Environmental Protection Agency (EPA). This report announced domestic produce as an area of concern for food safety in the U.S. (Rajkowski and Baldwin 2003). Later that year, a plan entitled Produce & Imported Foods Safety Initiative was announced in hopes to provide further assurance for higher health and safety standards for fruits and vegetables consumed by the American public (FDA 1999). In 1999, a survey was conducted by the Food and Drug Administration (FDA) concerning imported produce, and 40 out of 1000 samples (4%) tested positive for bacterial pathogens, of which 35 of 1

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2 these samples (80%) tested positive for Salmonella contamination and 9 (20%) with Shigella (CFSAN-FDA 2001). Fresh fruits and vegetables were traditionally considered safe to eat raw, straight from the field, but now pathogenic microorganisms may contaminate fresh commodities. Fruits and vegetables can become contaminated with pathogenic microorganisms by the way of many mechanisms. Contamination can occur in fields or orchards, through contaminated irrigation water, harvesting, postharvest handling, processing, distribution and preparation in food service or home settings (Beuchat 1995). All varieties of produce have the potential to harbor pathogenic microorganisms. If contaminated commodities enter a packinghouse facility, cross-contamination of processing equipment and other produce is likely to occur (Brackett 1999). The survival or growth of pathogens found on or in raw produce are affected by environmental surroundings as well as pathogens metabolic capabilities. These metabolic capabilities are greatly influenced by intrinsic and extrinsic ecological factors naturally present in the produce or imposed during production, processing, distribution and preparation at the site of consumption (Beuchat et al. 2001). Two very important environmental characteristics that can greatly affect fresh commodities are temperature and relative humidity. The impact of these two extrinsic factors will affect endogenous microflora and pathogen populations that may be present on fresh commodities (Brackett 1987). Bean sprouts, watermelon, cantaloupe, honeydew, green grapes and tomatoes are fresh commodities that have been associated with foodborne salmonellosis (Tauxe et al. 1997). Salmonellosis is a common gastrointestinal foodborne illness that is caused by the

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3 bacterium called Salmonella. The role of Salmonella in foodborne disease was first documented in the late 1800s, whereas the human clinical disease, typhoid fever, dates back to the beginning of that century (Cox 2000). Worldwide, Salmonella is the second most causative agent of foodborne illness (Cox 2000). Every year, approximately 40,000 cases of salmonellosis are reported in the United States (CDC 2001). Foodborne outbreaks of Salmonella spp. are most commonly linked to animal derived foods; however plant derived foods have also served as sources of illness (Cox 2000; Nguyen-The and Carlin 1994; Tauxe et al. 1997; Brackett 1999). Recent surveys of fresh produce have identified several Salmonella serotypes as the causative agents in human foodborne illness (CFSAN-FDA 2001). Large outbreaks of salmonellosis have been caused by consumption of contaminated raw tomatoes. Three multi-state outbreaks of foodborne illness were caused by the consumption of raw tomatoes contaminated with Salmonella Javiana in 1992, Salmonella Montevideo in 1993 and Salmonella Baildon in 1999 (CFSAN-FDA 2001). These outbreaks were all traced to Salmonella-contaminated packinghouse facilities where the tomatoes were minimally processed. In June of 2002, Salmonella Javiana was the cause of an outbreak at the 2002 United States Transplant Games in Orlando, Florida. The origin of the outbreak was identified as raw, diced tomatoes (CDC 2002). This recovery study evaluates the survival and recovery of Salmonella spp. from the surfaces of tomatoes and typical tomato packing line materials. Materials that were evaluated included stainless steel, conveyor belt, polyvinyl chloride (PVC), sponge rollers and unfinished oak wood. Fruit and material surfaces were inoculated with a

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4 known amount of a rifampicin resistant five-serovar Salmonella cocktail. Salmonella recovery off the various surfaces was assessed by a vigorous rub-shake recovery method. Inoculated fruit and material surfaces were subjected to specific temperature and relative humidity combinations for 28 days. The temperature and relative humidity combinations were selected to imitate Florida fall/winter and spring tomato production season conditions and ripening room parameters for mature green tomatoes during ethylene treatment and storage.

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CHAPTER 2 LITERATURE REVIEW The United States Centers for Disease Control and Prevention (CDC) claims that more than 200 diseases are known to be transmitted through food consumption (Bryan 1982). It is estimated that foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations and 5,000 deaths annually in the United States (Mead et al. 1999). Tauxe et al. (1997) report that due to a shift in diet toward greater consumption of fresh fruits and vegetables and farther distribution distances from new geographic sources, there are more reported illnesses involving fresh produce. The United States Food and Drug Administration (FDA) has conducted surveys on both imported and domestic produce, and a report on domestic products revealed a 1.6% contamination rate on sampled produce (Rajowski and Baldwin 2003). In the United States from 1988 to 1992, 64 outbreaks of foodborne diseases were attributed to the consumption of fresh fruits and vegetables; nine deaths resulted from the outbreaks (Bean et al. 1997). Worldwide, many pathogens have been identified as the causative agents of foodborne disease associated with the consumption of contaminated produce. Non-typhoidal Salmonella spp., Shigella spp., Listeria monocytogenes, Yersinia spp., Aeromonas spp., Campylobacter spp., Staphylococcus aureus and Escherichia coli O157:H7 are all bacterial pathogens that have caused foodborne infections (Nguyen-The and Carlin 1994; Beuchat 1995). The presence of pathogens on fresh produce alone can cause an outbreak; replication of pathogens on or in fresh produce does not have to occur. However, extensive research has documented that human pathogens are capable of 5

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6 replication on many types of undamaged or specifically wounded produce (Beuchat 1995). The FDA states that the survival of pathogens on fresh fruits and vegetables at low infective doses can initiate foodborne disease in the elderly, children and immunocompromised, but for healthy individuals a higher infective dose would be necessary (FSIS 2001). Temperature and relative humidity are extrinsic factors that influence the persistence and survival capacity of microorganisms on the surfaces of fruits and vegetables. Storage of healthy fruits and vegetables kept at optimum temperature, relative humidity, and atmospheric gas composition will yield maximum sensory and preservation attributes. However, optimum storage settings do not always result in minimizing the growth of microorganisms found on the produce (Beuchat 1992). Studies have shown that a variety of lettuce types, leafy greens and fruit can support postharvest multiplication of pathogenic bacteria under conditions of permissive temperature and relative humidity increasing the risk of foodborne illness (Abdul-Raouf 1993). Many types of vegetables and low-acid fruits are capable of supporting rapid multiplication of pathogens at temperatures ranging between 15 to 25C (Suslow 2002). Microbial quality of fresh produce is a large safety issue in the produce processing industry. A blanching or thermal kill step cannot be applied to fresh-market produce to eliminate bacteria (Hurst and Schuler 1992). Fresh produce facilities rely heavily on proper temperature control and good plant and employee sanitation to uphold quality and safety. Fresh fruits and vegetables are very nutritious and overall are categorized as safe foods (Harris et al. 2002). There is potential for fresh produce to become a risk in the food chain if postharvest techniques are abused. Produce quality can be judged from

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7 aesthetic factors (color, texture, aroma), but presence of foodborne pathogens are not so simple to detect. Preventing contamination of fresh-market produce from pathogens is crucial in assuring wholesome foods for safe human consumption (Harris et al. 2002). Fresh-market produce can be sold as whole entities or produce can be prepared and processed to a greater extent. Fresh-cut products have grown rapidly during the past decade (Cantwell and Suslow 2002). These fruit and vegetable products are prepared and handled to maintain freshness while offering convenience to consumers. Preparation of fresh-cut produce involves cleaning, washing, trimming, coring, slicing, shredding and other similar steps. These steps increase perishability of the produce items. Examples of fresh-cut produce are mixed salads, broccoli florets, diced onions and sliced and diced tomatoes. Fresh-cut produce items usually only have a shelf-life of 10-14 days. Higher respiration rates indicate a very active metabolism and a faster deterioration rate (Cantwell and Suslow 2002). Foodborne Illnesses Associated with Fresh Produce World-wide, the per capita consumption of fresh and lightly processed fruits and vegetables has increased over the last decade. With an increase in consumption of fresh produce, a heightened amount of human foodborne disease outbreaks involving fresh produce have resulted (Beuchat 1995). There are many pathogenic microorganisms that have been associated with foodborne disease resulting from contaminated produce. Pathogens of great concern are Salmonella spp., Shigella spp., E. coli O157:H7 and L. monocytogenes. Poultry, eggs and dairy products are most commonly associated with salmonellosis outbreaks. In recent years, Salmonella has been linked to many produce-associated outbreaks. Raw bean sprouts were the causative agents in salmonellosis outbreaks that

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8 occurred in the United Kingdom and Sweden in the late 1980s (Beuchat 1995). Salmonella Saintpaul was identified as the epidemic serovar in many cases of foodborne infection. Melons contaminated with Salmonella have also been causative agents of foodborne disease. As early as 1955, S. Miami and S. Bareilly were linked to the consumption of fresh-cut watermelon (Gayler et al. 1955). S. Javiana and S. Oranienburg were identified to have been the cause of salmonellosis outbreaks associated with the consumption of watermelon (CDC 1979; Blostein 1991). Studies have demonstrated that Salmonella (a five-serovar cocktail of S. Anatum, S. Chester, S. Havana, S. Poona and S. Seftenberg) can grow on rind-free pieces of watermelon, cantaloupe and honeydew (Golden et al. 1993). Over a 24-hour period, Salmonella populations exhibited multiple log-unit increases on melon varieties maintained at 23C. Tomatoes have also been documented as vehicles of foodborne disease. The consumption of raw tomatoes contaminated with Salmonella led to two separate multi-state outbreaks in 1992 and 1993 (Hedburg et al. 1999). S. Javiana implicated the outbreak in 1992 and S. Montevideo implicated the outbreak in 1993. All four species of the genus Shigella are pathogenic to humans. Shigella spp. has been responsible for many outbreaks involving contaminated raw vegetables. Lettuce and leafy greens have been documented vehicles of Shigella-contaminated produce. S. sonnei was responsible for an outbreak involving contaminated lettuce in Texas and shredded lettuce was responsible for another outbreak involving 347 cases of S. sonnei gastroenteritis (Davis et al. 1988). Two U.S. midwestern outbreaks of S. flexneri infections where linked to green onions that were harvested from a single farm in Mexico (Cook et al. 1995). Melons and tropical fruits have also been reported to harbor Shigella.

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9 Escatrin et al. (1989) reports that Shigella spp. can grow and survive on the surfaces of fresh-cut pieces of watermelon, papaya and jicama in slightly acidic condition (less than pH 6.0). E. coli O157:H7 is considered an emerging foodborne pathogen (Beuchat 1995). Cattle are primary reservoirs of this pathogen and an extensive amount of foodborne disease outbreaks have been linked with undercooked beef and dairy products. Fresh produce can also harbor this microorganism. E. coli O157:H7 has repeatedly been connected with unpasteurized apple cider, salad bars and melons. It has been documented that E. coli O157:H7 rapidly multiplies in watermelon and cantaloupe cubes at 8C (Del Rosario and Beuchat 1995). In 1994, culture confirmed E. coli O157:H7 infections were traced back to raw broccoli served on a salad bar. It was concluded that the broccoli was cross-contaminated with raw ground beef during the preparation of the vegetable (Beuchat 1995). L. monocytogenes can grow on fresh produce stored at refrigeration temperatures (4C). Controlled atmosphere storage does not seem to affect or influence the growth of the microorganism (Beuchat 1995). L. monocytogenes is prevalent on plant vegetation (Beuchat et al. 1990). In 1981, a large listeriosis outbreak was attributed to the consumption of contaminated coleslaw. The outbreak was traced back to a cabbage farmer who used a combination of composted and fresh sheep manure to fertilize cabbage fields (Schlech et al. 1983). L. monocytogenes has been detected in bean sprouts, leafy vegetables and cut cucumbers (Arumugaswamy et al. 1994). It has also been reported that this pathogen can survive on the surface of tomatoes held at 21C (Beuchat and Brackett 1991).

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10 The epidemiology of foodborne diseases is constantly changing. Reoccurrence of well-recognized pathogens are observed in outbreaks and newly recognized foodborne pathogens also emerge. Fresh produce has been extensively documented as potential vehicles for foodborne disease. Worldwide, fresh fruits and vegetables are an essential part of diets and minimizing the occurrence of foodborne disease associated with contaminated produce is essential. Salmonella Species Documentation of the human clinical disease caused by Salmonella, typhoid fever, dates back to the early 1800s (Cox 2000). Historically, Salmonella has been documented as causing foodborne disease since the late 1800s. The bacteria were discovered by an American veterinary pathologist, Dr. Daniel E. Salmon, who isolated the microorganism from hog cholera infected swine. In the 1900s, the genus Salmonella was created in Dr. Salmons honor after similar organisms were isolated from outbreaks of foodborne disease (Cox 2000). The bacterium is widely associated with food animals and their production environment. The genus Salmonella exists within the family of Enterobacteriaceae. According to the Encyclopedia of Food Microbiology (Cox 2000), the genus consists of one species; Salmonella enterica. Salmonella are Gram-negative facultative, oxidase-negative, catalase-positive, anaerobic rod-shaped bacilli (Bergeys Manual of Determinative Bacteriology 1994). Most strains are motile and ferment glucose. Biochemical tests can further characterize the genus into specific serogroups and serovars. These tests characterize two antigens, the O or somatic antigen, and the H antigen or flagellin antigen. The O antigen designates differences in epitopes of lipopolysaccharide (LPS), which is the major component of the outer membrane of Gram-negative bacteria. The H

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11 antigen differentiates strains into serovars that are based on the variation in flagellins or subunit proteins in the flagella (Bergeys Manual of Determinative Bacteriology 1994). Salmonellosis is the illness that Salmonellae induce in humans, usually by ingestion the bacteria through contaminated food products. According to the Centers for Disease Control and Prevention (CDC), salmonellosis has been a reportable disease in the United States since 1943. Physicians must report cases of infection to local health departments that report to state health departments that ultimately report annual totals to the CDC (Tauxe et al. 1997). Salmonellosis is one of the most frequently reported causes of foodborne gastroenteritis and is estimated to cause 1.4 million cases each year in the United States, of which 40,000 cases are culture confirmed (CDC 2000). The CDC has estimated that 95% of Salmonella infections originate from foodborne sources (Frenzen et al. 1999). Two serotypes of Salmonella cause over half of the reported salmonellosis cases: Salmonella Enteritidis and Salmonella Typhimurium (CDC 2000). Infection is initiated by the ingestion of a dose of Salmonella effective enough to surpass primary host defenses. The bacteria proceed to colonize the gastrointestinal tract. Infectious doses are determined by physiological characteristics of the ingested strain and the physiological state of the host. Typical infectious doses usually range between 10 6 -10 8 CFU and epidemiological evidence has demonstrated that an infectious dose may be as little as a few (10) cells (Cox 2000). A range of environmental conditions affect the survival, growth or death of Salmonella. The optimum growth temperature for this microorganism is 37C, but it has been observed to grow between 2-54C (Cox 2000). Generally, Salmonellae are heat labile, but exposure of Salmonella to adverse conditions generally increases the resistance

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12 of the microorganism to heat. Optimum pH levels range from 6.5-7.5 and as temperature increases the sensitivity to low pH increases. Salmonella can grow at water activity (a w ) values between 0.999 and 0.945 in laboratory media and at a low a w value of 0.93 in foods (Cox 2000). Growth of the microorganism has not been documented at a w lower than 0.93, but survival time of the microorganism has been noted to increase as a w decreases (Cox 2000). In low-moisture foods, survival of Salmonella can be measured in months. Salmonellosis Outbreaks Involving Fresh Tomatoes Tomatoes have been the sources of several foodborne illness outbreaks. The microorganism responsible for these outbreaks is usually identified as Salmonella. A large multi-state outbreak occurred in 1990 that resulted in 176 cases of S. Javiana infections. A restaurant and child care center reported illnesses associated with consuming raw tomatoes. The outbreak was traced to a repacking facility. The tomatoes were distributed to various restaurants and grocery stores. No other potential sources were associated with this outbreak (Hedburg et al. 1999). Another large outbreak occurred in 1993. There were 100 reported cases of foodborne illness that resulted from this multi-state outbreak. The causative agent of the outbreak was S. Montevideo. The outbreak was traced back to the same repacking facility from which the 1990 outbreak was traced. Mature-green tomatoes were picked by hand, and transported in field bins which contained approximately 1,500 pounds of tomatoes each. The lots of tomatoes were dumped into a common water bath (dumptank) where they were contamination most likely occurred (Hedburg et al. 1999). In 1999, another multi-state outbreak of salmonellosis was attributed to the consumption of raw tomatoes. The origin of contamination was traced to two tomato

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13 grower/packer cooperatives. The lots of tomatoes were handpicked and transported to the facility in covered plastic bins (Cummings et al. 2001). Most recently in June of 2002, in Orlando, Florida there were two reported cases of S. Javiana infections. The illnesses were contracted from contaminated pre-packaged diced Roma tomatoes. Efforts are underway to identify the routes of contamination (CDC 2002). Tomatoes and Salmonellae The increased frequency of salmonellosis outbreaks involving fresh tomatoes has prompted researchers to investigate Salmonella in and on tomatoes. Many researchers and food scientists have conducted experiments focusing on recovery of the pathogen from tomato surfaces and tomato matrices, survival of the pathogen on and in tomatoes, and the effectiveness of sanitizers on the pathogen. In a study conducted by Guo et al. (2001), the survival of salmonellae brushed onto tomato plants was investigated. Flowers and stems on tomato plants were inoculated with a five serovar Salmonella cocktail before and after fruit set. Twenty-one to 49 days elapsed between the date of inoculation and sampling. Forty-three sound, red, ripe tomatoes were harvested from inoculated plants and plants that were not inoculated. All plants were evaluated for the presence of Salmonella. Plants that were not inoculated produced tomatoes that were not contaminated with Salmonella. However, 11 of 30 tomatoes (37%) harvested from inoculated plants were positive for Salmonella (confirmed by polymerase chain reaction (PCR) assay). Stem-inoculated plants were positive for Salmonella before and after flower set at 43% and 40%, respectively. Twenty-five percent of Salmonella-positive tomatoes were harvested from plants that were inoculated on the flower. The surface of the tomatoes and the stem scars tissues of

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14 the tomatoes harbored higher percentages of the pathogen compared to the pulp of the tomatoes. PCR fingerprinting patterns revealed that S. Montevideo was the most persistent and dominant serotype detected on positive tomatoes. The serovar was isolated 49 days after inoculation of the tomato plants. In a study by Luasik et al. (2001), the elution, detection and quantification of seeded viruses and bacteria (Salmonella Montevideo) were investigated from the surfaces of strawberries and tomatoes. Mature, red Roma tomatoes were inoculated with Salmonella Montevideo on artificial surface scars, stem and blossom scars, and intact tomato surfaces. Results indicated a higher recovery of the pathogen from the stem, surface and blossom scars than pathogen recovery from smooth intact surfaces of tomatoes. It was also observed that when the tomatoes were immersed in Salmonella Montevideo-contaminated water, more attachment of the pathogen occurred in the stem scar area, followed by the blossom scar area, surface scars, and the intact tomato surface. It was hypothesized that the surface area and hydrophilicity of the rough areas evaluated (surface, stem and blossom scars) may affect microbial attachment. Tomatoes do not have lenticels, or pores, on their surface like many fruits, thus restricting gas exchange between the internal tissues of the fruit and the atmosphere. Lenticels also allow the infiltration of liquids. Pores do exist in the corky tissue of the stem scar area of tomatoes. Bacteria are more likely to attach and infiltrate into the interior of this rough portion of the fruit than the smooth epidermal surface. In a study conducted by Zhuang et al. (1995), survival patterns of S. Montevideo on and in raw tomatoes were evaluated as affected by temperature and chlorine treatment. Mature green tomatoes were dip inoculated with S. Montevideo and inoculated tomatoes

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15 were stored up to 18 days at different temperatures in combination with 45-60% relative humidity (RH). The stem scar tissues and core tissues of the tomatoes were analyzed for Salmonella populations and dipped in various chlorine concentrations. The survival and growth pattern of S. Montevideo was also examined in chopped, ripe tomatoes stored at various temperatures. Results of this study suggested that the persistence and viability of S. Montevideo on the surfaces and cores of tomatoes stored at 10C parallel the potential for Salmonella survival on and in tomato fruits during transport and storage. The populations of Salmonella inoculated on the surfaces of tomatoes held at 10C did not significantly change over the 18-day period. S. Montevideo was also observed to grow well in chopped ripe tomatoes stored at 20 or 30C. Chlorine concentration studies revealed that S. Montevideo was not totally eliminated from tomatoes when subjected to a disinfection treatment at 320ppm. This study clearly indicates that Salmonella serotypes contaminating fresh tomatoes pose a risk for potential foodborne salmonellosis outbreaks. A study conducted by Guo et al. (2002) demonstrated that water and soil serve as reservoirs of Salmonella that can potentially contaminate mature green tomatoes. Salmonella was observed to survive at high numbers in moist soil for at least 45 days. It was also observed that cells of Salmonella were able to infiltrate fruits via stem scars and enter the tomato pulp upon contact with moist, contaminated soils. Survival patterns of Salmonella on tomato surfaces were also investigated. Spot-inoculated tomatoes evaluated over a 14-day storage period (20C) showed a decrease in Salmonella populations over time. Populations decreased by approximately 4 logs over the entire storage period. Results obtained from this study differ from survival patterns of

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16 Salmonella reported by Zhuang et al. (1995). Zhuang et al. (1995) reported an increased amount of Salmonella on whole, intact tomatoes over time. Differences could be attributed to the different inoculation procedures used. A dip inoculation, as used by Zhuang et al. (1995), could result in cells becoming lodged in tissue areas that could enhance the survival and growth of cells during a prolonged storage period. Tomato Industry Two tomato industries exist in the United States. The fresh-market and processing tomato industries are separate markets and each possesses distinguishing characteristics. Tomato varieties are specifically bred to meet requirements of either the fresh or processing markets. All fresh-market tomatoes are picked by hand whereas, tomatoes bound for processing can be mechanically harvested (ERS 2000). Fresh-market tomatoes are widely produced and sold on the open market with higher and more variable prices than processing tomatoes (ERS 2000). According to the Economic Research Service (ERS), California and Florida comprise two-thirds of the acres used to grow fresh tomatoes in the United States. In the United States, this industry estimates that fresh-market tomato retail value exceeds $4 billion (ERS 2000). Florida leads the domestic market in the production of fresh-market tomatoes. Florida produced 42% of the fresh-market tomatoes in the United States during 1997-1999 and brought in $5.4 million of the states total farm value of vegetables (ERS 2000). Floridas tomato season extends from October to June. Most tomato production occurs during the months of April to May and again from November to January. Fresh-market tomatoes are available year-round in the United Stated because of imports and Floridas winter crops. Imported commodities are usually shipped to

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17 markets in the western states and Floridas winter crops are shipped to the eastern half of the nation (ERS 2000). The ERS (2000) reported that Americans consumed 4.8 billion pounds, or 17.8 pounds per person, of fresh-market tomatoes in 1999. Tomatoes rank third in consumer preference vegetables at the retail level and are only surpassed by potatoes and lettuce (Florida Tomato Committee 2002). Consumption of fresh-market tomatoes in the United States has most likely increased due to the increasing popularity of salads, salad bars and sandwiches dressed with tomatoes (Lucier et al. 2000). Tomatoes are very nutritious fruits and contain approximately half of the recommended daily allowance of vitamin C and 20% of the recommended daily allowance of vitamin A. Tomatoes also contain the compound lycopene which has been shown to reduce prostate cancer in men who consume at least 10 servings of tomatoes or tomato-based foods per week (Florida Tomato Committee 2002). Postharvest Handling of Tomatoes Tomatoes bound for the fresh-market are harvested by hand at a mature-green stage. Internally, a mature-green tomato will have a jellylike matrix in all locules, but maturity is difficult to determine from external examination. At a mature stage, tomato seeds will be sufficiently developed when a knife slices the fruit and the seeds are not penetrated by the cut (UF/IFAS 1998). When tomatoes are harvested, pickers place the fruits into plastic buckets or wooden field bins that usually hold up to 40-50 pounds of tomatoes. The buckets are carried to field trucks and emptied into pallet bins or gondolas. Next, tomatoes are transported to the packinghouse and dumped into a chlorinated dump tank. Dump tanks contain heated, chlorinated water to wash the fruits. Wash water should be maintained at a pH of 7 (neutral) and contain a recommended level

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18 of chlorine range of 100 to 150 parts per million (ppm) of chlorine (Sargent et al. 2001). Water temperature of dump tanks should be elevated 10 degrees above the pulp temperature of the tomato. Bartz and Showalter (1981) demonstrated that warm tomatoes (26C to 40C) immersed in cold water (approximately 18 degrees colder than incoming fruit) for 10 minutes or longer infiltrated water and any bacteria present in the water. Infiltration through the stem scar is associated with a negative temperature differential between the water and the tomato therefore; warm water is used in dump tanks to reduce the extent of infiltration of water into the tomato. Failure to maintain adequate chlorine levels in dump tanks can lead to increased microbial populations. It has been reported that Enterobacteriaceae populations increased on tomatoes washed in water containing 114 ppm chlorine and populations decreased once tomatoes were subjected to water containing 226 ppm (Beuchat 1992). Tomatoes exit the dump tank and travel over a series of perforated conveyor belts. Conveyor belts play an integral part in the functions of a packinghouse. Belts are fabricated from rubber compounds and they transport tomatoes at several points during handling. Conveyor belts are used to pre-size, cull, sort and size tomato fruits. Undersized tomatoes will fall through holes in the belts and travel to a cull chute. Sponge rollers also serve a very important role in tomato packinghouse operations. Tomato fruits are susceptible to injury and bruising. Sponge rollers buff and cushion the fruits as they proceed along the processing lines. Tomatoes will also contact sponge rollers after washing and absorb water off the fruit surface; as a result the sponges are constantly moist. Many Florida tomatoes are waxed with a food grade wax that increases

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19 the shine of the tomato and reduces water loss during marketing. Contamination of tomatoes has been known to occur during waxing procedures (Beuchat 1992). Sorting and grading of tomatoes is a laborious process. Color sorting occurs first, which separates tomatoes possessing any red color from fruits that are completely green in color. The fruits are then separated into grades that meet specific requirements for the U.S. No. 1, U.S. Combination, U.S. No. 2 or U.S. No. 3 of the U.S. Standards for Grades of Fresh Tomatoes (Florida Tomato Committee 2002). Following sorting and grading, tomatoes are mechanically sized by passing over continuous conveyor belts containing increasingly larger round holes that sort tomatoes by maximum allowable diameter for each designated size. In February of 1998, the Florida Tomato Committee (2002) ordered the following sizing classifications: 6x7 (formerly medium), 6x6 (formerly large) and 5x6 (formerly extra large). The sizing dimensions (diameter of fruit is measured in inches) are categorized by a minimum and maximum range for each size class. Graded and sized tomatoes are transported via conveyor belts to automatic fillers where the fruits are jumble-packed into corrugated fiberboard containers to a designated weight (UF/IFAS 1998). Boxes of tomatoes are then palletized and moved by units. Wooden pallets are used to transport unitized loads of tomato boxes and are used in many packinghouse facilities. Pallets are usually constructed from unfinished oak wood. Usually, mature-green tomatoes are immediately subjected to a ripening treatment. Ethylene is a natural ripening hormone that is released in ripening rooms. Ripening rooms are capable of holding many pallets of tomatoes at one time, and are maintained at very specific parameters. Precise optimum conditions of a typical ripening room are kept at 20C, 85-95%RH with a concentration of up to 150 ppm ethylene (UF/IFAS 1998).

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20 Tomatoes are susceptible to extensive water loss through the stem scar so a high relative humidity is necessary (UF/IFAS 1998). Constant air exchange is provided in ripening rooms to supply tomatoes with a continuous ripening-effective blend of ethylene and air to avoid the accumulation of carbon dioxide. Mature-green tomatoes are usually subjected to ethylene for 3 days. Once tomatoes are at a minimal color stage of breaker, the first sign of external yellow or pink color at the blossom end of the fruit, ethylene will not further accelerate the ripening process since the fruits are producing their own ethylene (Cantwell and Kasmire 2002). A constant supply of air also prevents carbon dioxide buildup when tomatoes respire. Carbon dioxide inhibits the ripening process and is an unwanted byproduct (Reid 2002). Ripe tomatoes are susceptible to chilling injury at temperatures below 10C (Cantwell and Kasmire 2002). However, ripening tomatoes develop chilling injury below 13C (Maul et al. 2000). Low temperatures inhibit development of full color and flavor in green mature fruit and the fruits are more susceptible to Alternaria decay (UF/IFAS 1998). Tomatoes are tropical commodities and must be maintained at warm temperatures. If tomatoes are held above 30C (85-86F) the fruits will develop more orange pigments than the desirable red pigments (UF/IFAS 1998). In 1994 and 1995, Rushing et al. (1996) tested tomatoes bound for the fresh-market for the presence of Salmonella spp. and verified that a proposed Hazard Analysis Critical Control Point (HACCP) program was effective in controlling the risk of contamination in the packinghouse. This study revealed that contamination seemed more likely to occur at the packinghouse where minimally processed fruits were dumped into a water bath, transported across conveyor belts and hand sorted prior to being packed into cartons.

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21 Packinghouse operations are designed to preserve and package fresh produce in a timely manner. Packinghouse facilities are currently included under the Good Agricultural Practices (GAP) guidelines and are exempt from Good Manufacturing Practices (GMP) regulations. GAP guidelines are generic and do not contain specific testing and monitoring guidelines (CFSAN-FDA 2001). The potential risk of contamination can be controlled by employee training and traceback plans. The Guide To Minimize Microbial Food Safety Hazards For Fresh Fruits and Vegetables (FDA 1998) has become a valuable tool for focusing on crucial areas of presumptive risk potential for fresh produce handling (CFSAN-FDA 2001). Extrinsic Factors Influencing Microbial Viability Growth and survival of microorganisms on fresh produce are influenced by the characteristics of the surrounding environment (Tauxe et al. 1997). Foodborne diseases that occur from contaminated produce often involve fruits and vegetables that have been subjected to nonthermal, minimal processing prior to time/temperature combinations permitting pathogens to survive and grow (Tauxe et al. 1997). The exteriors of produce act as physical barriers to protect from internalization of bacteria present on a commoditys surface. Temperature and relative humidity are two environmental factors that can affect microbial populations on produce. For minimally processed fruits and vegetables, two factors should be considered when evaluating the effect of temperature and growth rates of bacteria. First, storage temperature determines respiration rates of a commodity and the behavior of microorganisms may be influenced by changes in the gaseous atmosphere. Secondly, temperature can also influence the rate of senescence of a commodity therefore modifying the environment for microorganisms (Nguyen-The and Carlin 1994).

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22 Improper refrigeration during storage and preparation and poor product quality can enhance the survival of pathogens. Growth of pathogens on fresh, minimally processed fruits and vegetables has been reported in many studies. It was observed by Maxcy (1978), that E. coli, S. Typhimurium and Staphyloccocus aureus grew on shredded lettuce at room temperature (22-24C). Yu et al. (2001) reported the growth of E. coli O157:H7 on both externally and internally inoculated strawberries. A study by Zhuang et al. (1995) revealed that S. Montevideo populations significantly increased on tomato tissues after storage at 20 and 30C. Refrigeration temperatures limit the growth of most foodborne pathogens, but some pathogenic microorganisms will survive at lower temperatures. S. Typhimurium declined rapidly in apple juices stored at 4C, but managed to survive in the juices for a significant amount of time (Goverd et al. 1979). In the study by Yu et al. (2001), it was observed that E. coli O157:H7 populations were also recovered from externally and internally inoculated strawberries. However, there was a significant reduction in the populations recovered from the outside of the strawberry fruits than from the inside of the fruits at 5C. Another key environmental factor in determining the survival of bacteria is relative humidity. Traditionally, human pathogens are considered poor survivors in the natural plant surface environment (Suslow 2002). Beattie and Lindow (1994) state that the death of cells subjected to low relative humidity conditions is rather fast and viability of cells can decrease very close to first-order kinetics. In a study conducted by Guo et al. (2001), tomatoes inoculated with Salmonella were stored at 20C for one day with at a relative humidity of 70%. It was reported that a reduction of approximately one log 10 CFU per tomato occurred and the population slowly decreased by an additional 3 logs

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23 between days 1 and 14. In the same study, tomatoes were stored in contact with moist Salmonella inoculated soil for 14 days. An increase of approximately 2.5 log 10 CFU per tomato occurred during the first 4 days of storage. Similar counts remained constant for days 4 though 10 and the incidence of decay on tomatoes stored 10 days or more could not be analyzed for populations of Salmonella. There have been studies focusing on the incidence of Salmonella associated with bacterial soft rots and/or physical injury. Bacterial soft rot is the leading cause of postharvest losses of potatoes, tomatoes along with other types of fresh produce. Bacterial soft rot caused by group of plant pathogens which are harmless to humans, that includes Erwinia carotovora (subspecies carotovora and atroseptica), pectolytic Pseudomonas fluorescens and Pseudomonas viridiflava (Lund 1983). Infected tissues are broken down resulting in the softening and liquefaction of the internal fruit tissues and spreads bacteria over other commodities and food-handling equipment (Wei et al. 1995). E. carotovora is the most common of the soft rotting bacterial complex and is a member of Enterobacteriaceae of which Salmonella is also a member (Wells and Butterfield 1997). A study by Wells and Butterfield (1997) involving over 500 samples of healthy and soft rotted commodities collected from retail markets showed the incidence of suspected Salmonella was twice that on soft rotted samples than of healthy samples. Another study conducted by Wells and Butterfield (1999) showed that unlike bacterial soft rotted commodities, fungal rotted commodities (Alternaria tenuis, Bortrytis cinerea, Geotrichum candidum or Rhizopus stolonifer) showed no greater risk of elevated Salmonella populations.

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24 Attachment of Microorganisms to Various Surfaces Bacteria can be introduced to fresh commodities in the field through irrigation water, sewage, or contaminated soil and introduced into packinghouse environments. Attachment of bacteria to food processing surfaces is possible and can easily lead to product contamination (Zottola 1994). Some typical food contact surfaces found in processing facilities include stainless steel, rubber (conveyor belts), wood and plastic. Microorganisms on contaminated produce can easily attach to a variety of these surfaces in short contact times. It was observed by Mafu et al. (1990) that L. monocytogenes attached to stainless steel, glass, polypropylene and rubber surfaces after a brief contact time. Contact times ranged from 20 minutes to 1 hour. Attachment of the pathogen was reported at both 20C and 4C for all surfaces. Sanitizers were applied to each of the surfaces after attachment of L. monocytogenes and it was observed that porous surfaces (rubber surfaces in this study) seemed to protect the bacteria whereas sanitizers were more effective on nonporous surfaces. Wood is another porous material that is used in the form of field bins, pallets or containers to hold fresh produce. A study conducted by Boucher et al. (1998) observed the enhanced survival of Campylobacter jejuni cells when incubated at 30C in nutrient broth. The physical structure of wooden cubes acted as a protective environment for the bacteria. Plastic cubes were evaluated in the same manner as the wooden cubes, but enhanced survival of Campylobacter jejuni cells was not observed. Microorganisms can irreversibly attach themselves to surfaces and form biofilms. Biofilm-associated cells produce an extracellular polymeric substance (EPS) and have a defined architecture. Microbial biofilms have been known form on food processing surfaces. Pathogenic microorganisms such as Campylobacter, Salmonella and E. coli

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25 have been known to form strong biofilms on various surfaces (Somers et al. 1994). A study conducted by Joseph et al. (2001) was also in agreement reporting that Salmonella strains will form biofilms on plastic, stainless steel and cement. Biofilms are much more resistant to sanitizers as compared to planktonic cells and serve as a source of contamination for foods (Somers et al. 1994). Hood and Zottola (1997) inoculated stainless steel surfaces with S. Typhimurium, L. monocytogenes, and E. coli O157:H7 and reported that all pathogens adhered to the surface when grown in media, but adherence levels often did not increase after 1 hour. In a study conducted by Ronner and Wong (1993), it was found that the behaviors of biofilm cells were greatly influenced by surface type. Buna-n rubber (nitrile rubber), a gasket material commonly used in the food industry, had a bacteriostatic effect on S. Typhimurium and L. monocytogenes. The bacteriostatic effect of the rubber was most pronounced under lower nutrient conditions. S. Typhimurium was less affected by the bacteriostatic component than L. monocytogenes. It is easier to prevent the formation of biofilms and microbial contamination than to eliminate a biofilm from a surface after establishment. Attachment of bacteria to food processing equipment and contact surfaces can easily lead to contamination of product. Sanitation procedures and environmental awareness in food processing facilities can reduce the incidence of foodborne illness. Equipment parts and food contact surfaces such as stainless steel, PVC, conveyor belts, sponge rollers and wood surfaces are widely used in tomato processing facilities. Limited studies on these types of surfaces have been reported. Microorganisms attached to surfaces are a hazardous source of potential contamination for any material coming in

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26 contact with the surfaces. Factors such as temperature, relative humidity, nutrient level of the growth medium, type of attachment surface and species or strain of bacteria can influence the amount of adherence to surfaces. Microbiological Recovery Methods Involving Fresh Produce The increase in foodborne illnesses associated with fresh produce in the past decade has resulted in the increase of testing commodities for the presence and enumeration of pathogens (Burnett and Beuchat 2001). Conventional methods of detection, enumeration, identification and characterization of microorganisms are described in such reference books as Compendium of Methods for the Microbiological Examination of Foods (CMMEF), FDA Bacteriological Analytical Manual (BAM), Official Methods of Analysis of the AOAC, and Standard Methods for the Examination of Dairy Products (Fung 2001). Methods for analyzing foods of animal origin and thermally processed food of plant origin for both spoilage and pathogenic microorganisms have been clearly defined in such reference manuals. Methods for selecting and preparing samples of raw fruits and vegetables for analysis of microorganisms are less defined (Burnett and Beuchat 2001). Procedures for preparing and isolating Salmonella for 18 food groups are outlined in the FDA BAM (Andrews et al. 1998). Currently, a specific protocol for preparing samples of raw produce is not defined. Microbiologists and food scientists are currently utilizing a wide variety of procedures to prepare whole and fresh-cut fruit and vegetables to enumerate pathogens. Sample weight/diluent volume ratios, diluent composition, type of processing and time used to process samples all greatly vary. Some types of processing include blending, stomaching, homogenizing, macerating, rubbing and shaking (Burnett and Beuchat 2001).

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27 It is essential for standard methods to be defined in order to accurately determine the presence and populations of pathogenic microorganisms on fresh fruits and vegetables. Development and validation of standard methods can be applied to determine survival and growth characteristics in challenge studies and the efficacy of antimicrobial treatments in eliminating pathogens on fresh produce (Beuchat et al. 2001). One single protocol would be ideal, but is not feasible for all produce types. An optimum protocol for produce depends upon the site of retrieval of pathogens. Analysis from a surface, tissue or both will vary in methodology, but a basic analytical method for each procedure would form standard guidelines to optimize the recovery of pathogens (Beuchat et al. 2001). An acceptable method for evaluating whole fruits and vegetables is a process in which the whole intact produce is vigorously hand massaged or hand rubbed for a period of time which can ranges from 40 seconds to two minutes (Beuchat et al. 2001; Burnett and Beuchat 2001; Harris et al. 2001; Zhuang et al. 1995). Inoculation procedures for fruits and vegetables usually occur by either spot inoculation and dipping or spraying. The major problem with inoculation via dipping or spraying is that the number of cells applied to the produce is unknown. Spot inoculation allows a known volume of inoculum and a known cell density that is applied to the produce. Spot inoculation is superior to dip or spray inoculation and this type of inoculation imitates contamination of the produce from a source such as contact with soil, workers hands, or equipment surfaces (Beuchat et al. 2001). In studies determining the efficiency for retrieval of cells, the applied inoculum should be dried at a standard temperature and relative humidity for a specific amount of time before recovery of cells or treatment is administered (Beuchat et al. 2001).

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28 Due to the increased frequency of documented outbreaks of foodborne disease attributed to fresh produce, many researchers are focused upon pathogenic microorganisms on raw fruits and vegetables. Standard methods that accurately determine the presence and numbers for a wide variety of pathogenic microorganisms associated with fresh produce are needed so studies conducted on this subject can be compared without controversy. The following objectives were explored in this recovery study. Establish growth characteristics for five rifampicin-resistant Salmonella serovars. Recover inoculated Salmonella from surfaces of tomatoes and packinghouse materials. Determine if a specific temperature and relative humidity combination affect the survival of Salmonella spp. on the surfaces of tomatoes and packing line materials.

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CHAPTER 3 MATERIALS AND METHODS Three separate temperature and relative humidity environments were simulated using an environmental humidity chamber. Tomato surfaces and packing line material surfaces were inoculated with a Salmonella cocktail comprised of five rifampicin-resistant serovars. Salmonella-inoculated fruit and material surfaces were subjected to specific environmental conditions inside the chamber for 28 days. Simulated environments mimicked standard tomato ripening room parameters and Florida fall/winter and spring tomato production seasons. Recovery of Salmonella from tomato surfaces and packing line material surfaces for each simulated environment was monitored on Days 0, 1, 3, 7, 11, 14, 21 and 28. Tomato ripening room parameters were simulated to evaluate only Salmonella-inoculated tomato fruits for 28 days. Both Salmonella-inoculated tomatoes and Salmonella-inoculated packing line materials were evaluated in environments paralleling typical Florida fall/winter and spring tomato production environments. Selection of Temperature and Relative Humidity Combinations The selected temperature and relative humidity settings for Florida fall/winter and spring tomato production seasons were based upon weather archives obtained from the Florida Automated Weather Network (FAWN) (University of Florida Institute of Food and Agricultural Sciences 2003) (Table 3-1). The average documented temperature and relative humidity were accumulated for the 2001 and 2002 fall/winter and spring tomato 29

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30 production seasons in Quincy, FL. The chosen parameters for each production season were used to simulate an open-air packinghouse environment. Table 3-1. Temperature and relative humidity combinations selected to simulate a ripening room environment (20C/90%RH) and a fall/winter (20C/60%RH) and spring (30C/80%RH) tomato production conditions. Simulated Environment Temperature (C) Relative Humidity (%) Standard tomato ripening room 90 20 Florida spring tomato production season 80 30 Florida fall/winter tomato production season 60 20 Acquisition and Maintenance of Salmonella Cultures Salmonella serovars were obtained through Dr. Linda J. Harris at the University of California, Davis, Department of Food Science and Technology. The five Salmonella enteritidis serovars used in this study were Agona, Gaminara, Michigan, Montevideo, and Poona (Table 3-2). The serovars obtained were adapted to the antibiotic rifampicin at the University of California, Davis. The serovars were adapted to rifampicin (rif+) by methods described in a study conducted by Lindeman and Suslow (1987). The five Salmonella serovars (rif+) were transferred to PROTECT Bacterial Preservers (Scientific Device Laboratories, Des Plaines, IL) upon arrival to the laboratory (summer of 2002) and stored at -70C. Rifampin is synonymous with rifampicin. This antibiotic inhibits protein synthesis of mammalian cells and it is freely soluble in methanol (Merck Index 2001). A 10,000 ppm (1%) stock solution of rifampicin was utilized throughout this study. The stock solution was prepared by dissolving 0.1 g of rifampin (Fisher #BP267925, Fisher Scientific, Pittsburg, PA) dissolved in 10 ml of high performance liquid chromatography

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31 (HPLC) grade methanol (Fisher, Fair Lawn, NJ). The stock solution was filter sterilized. Rifampicin is light-sensitive therefore, the stock solution was protected from light and was stored at room temperature. The media used to recover Salmonella off inoculated surfaces, Tryptic Soy Agar (TSA) (Difco, Sparks, MD), was supplemented with 80g/ml rifampin (rif+) antibiotic. The antibiotic-resistant serovars allowed differentiation from natural micoflora or non-rifampicin resistant bacteria that may have been present on the matrices evaluated; enabling the sole isolation of Salmonella serovars (rif+) (Beuchat et al. 2001; Lukasik et al. 2001). Table 3-2. Salmonella enteritidis serovars obtained from Dr. Linda J. Harris at the University of California, Davis: wild types* and rifampicin-resistant serovars listed with source. Serovar Designation Serovar Name Origin LJH517* LJH618 Agona Alfalfa sprouts LJH518* LJH616 Gaminara Orange juice LJH521* LJH615 Michigan Cantaloupe LJH519* LJH614 Montevideo Human isolate from tomato outbreak LJH630* LJH631 Poona Human isolate from tomato outbreak Growth Levels of Salmonella Serovars after a 20-Hour Incubation Growth studies were conducted to determine the rate of growth for each of the five serovars after a 20-hour incubation period. Growth rates were determined so the Salmonella cocktail would consist of equivalent quantities (CFU/ml) of each serovar, as one or more serovars would not dominate the inoculum suspension. The five Salmonella serovars (rif+) were revived off PROTECT Bacterial Preservers by aseptically transferring one bacterial preserver into 10 ml of Tryptic Soy Broth (TSB) (Difco, Sparks, MD) supplemented with 80 l of rifampin. The cultures were then incubated in a

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32 shaking incubator (Queue Systems, Asheville, NC) at 30 rotations per minute at 37C for 24 hours. The cultures were successively transferred for three days in TSB (rif+) to obtain uniform cell type (Beuchat et al. 2001). Each of the five serovars were transferred into 10 ml of fresh TSB (rif+) and incubated at 37C for 20 hours. Following the incubation period, three replicates of each serovar was serially (1:10) diluted in 9 ml tubes of sterile Phosphate Buffered Saline (PBS) (ICN Biomedicals Inc., Aurora, OH). Appropriate dilutions were plated out by pour plate technique using TSA (rif+). Plates were statically incubated at 37C for 48 hours. Colony forming units (CFU) were counted and recorded. Serovars were taken off PROTECT Bacterial Preservers at the beginning of each 28-day experiment. Two 20-hour growth studies were conducted to ensure growth rates for all five serovars were successively similar upon revival off bacterial preservers. Preparation of Inoculum Three days prior to each experiment, the five Salmonella serovars were revived from bacterial preservers. Overnight transfers were performed using 10 ml tubes of TSB (rif+) each day. On the day of the experiment, an 18-hour culture of each serovar was harvested via centrifugation (2,000 x g, 15 minutes at 22C). Cells were washed twice with PBS. Equivalent aliquots of the five serovars at approximately 1.0 x 10 8 CFU/ml were combined as a Salmonella cocktail. The cocktail was maintained at room temperature for one hour. If the time between preparation of the inoculum and inoculation of the surfaces exceeded one hour the inoculum was stored at 4C until the surfaces could be inoculated that day. The inoculum was serially diluted using 9 ml tubes of PBS to confirm cell concentration. The dilutions were plated in triplicate via pour plate technique using TSA (rif+).

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33 Inoculation Procedures Inoculation of Tomatoes Domestic-market mature green tomatoes (Florida 47) were supplied by DiMare (Tampa, Inc., Tampa, FL) for all experimental studies. Tomato samples were extracted from the processing lines prior to the waxing process. Size classification of the tomatoes, according to the Florida Tomato Committee, was 6x7 (formerly medium) (Florida Tomato Committee 2002). For fruit inoculation, tomatoes were aseptically placed onto sterile fiberglass trays with the stem scars facing down. Ten 10 l drops of inoculum suspension, for a total of 100 l of inoculum suspension per whole tomato, were placed around the blossom scar area using a Repeater Plus pipette (Eppendorf AG, Germany). The inoculum suspension was not placed directly onto the blossom scar. Immediately after inoculation, the tomatoes were placed under a hood (LABCONCO Corporation, Kansas City, MO) at room temperature (approximately 22C) and the inoculated surfaces were allowed to completely dry for a maximum of 2 hours. Dried samples were placed in a Caron 6030 (Caron, Marietta, OH) environmental humidity chamber. The Caron humidity chamber was equipped with a Caron CRS 101 (Caron, Marietta, OH) water supply system to deliver distilled water to the humidify the chamber. A Whatlow Series 96 temperature and relative humidity controller (Whatlow, Winona, MN) installed in the environmental chamber continuously monitored, displayed and controlled the temperature and relative humidity output inside the chamber. Periodically, a calibrated humidity meter (Control Company, Friendswood, TX) was placed inside the chamber to verify the relative humidity reading on the output panel. A magnetic thermometer (Fisherbrand by ERTCO, West Paterson, NJ) was placed on the inside wall of the chamber to verify the temperature output on the panel.

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34 Inoculation of Packing Line Materials Recovery studies included the following packinghouse materials: stainless steel (type 304, no.4 finish), conveyor belt, polyvinyl chloride (PVC) rollers, sponge rollers, and wood (unfinished oak). The packinghouse materials were obtained from Tri-Pak Machinery, Inc. (Harlingen, TX). Tri-Pak Machinery, Inc. is a Texas-based retailer and manufacturer of materials and equipment used in tomato packinghouses. The unfinished oak pieces were supplied by Lowes Home Improvement Warehouse (Gainesville, FL). The materials were chosen based upon contact surfaces that fresh-market tomatoes encounter from harvest (into wooden field bins) to various other surfaces encountered by tomatoes on a typical packing line. Stainless steel surfaces and conveyor belt surfaces were cut into coupons by Tri-Pak Machinery, Inc. (Table 3-3). Polyvinyl chloride (PVC) cylindrical rollers and sponge rollers were received as whole entities from the manufacturer (Table 3-3). The PVC cylinders were cut into equivalent pieces by the Mechanical Engineering Department at the University of Florida (Table 3-3). The sponge rollers were cut into equivalent sections by laboratory personnel (Table 3-2). The wood pieces were cut into cubes of equivalent dimensions by Lowes Home Improvement Warehouse (Table 3-3). Table 3-3. Surface area dimensions of each type of packing line material that was inoculated with a five serovar rifampicin-resistant Salmonella cocktail. Packing Line Material Dimensions of each Inoculated Surface Stainless Steel 2.5cm x 2.5cm Conveyor Belt 2.5cm x 2.5cm PVC 2.5cm x 2.5cm Wood 2.5cm x 2.5cm Sponge roller 2.5cm x 2.5cm

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35 The pre-cut stainless steel coupons were immersed in methanol (Fisher, Fair Lawn, NJ) overnight to remove any oil residue. The next day, stainless steel coupons were thoroughly rinsed with deionized water (University of Florida). All packinghouse material pieces were autoclaved for 20 minutes at 121C, 15 psi (Consolidated Stills and Sterilizers, Boston, MA) to achieve sterility. Autoclaved material pieces were aseptically placed onto sterile fiberglass trays. Sponge roller pieces were dampened with sterile deionized water prior to inoculation due to the wet nature of sponge rollers found along the processing lines in tomato packinghouses. Each type of material was marked with a single dot made by a Sharpie Permanent Marker (Sanford, Bellwood, IL) on the area where a tomato would most likely be encountered. All materials were inoculated with ten 10-l spots of inoculum suspension near the marked area of each piece. The inoculated materials were placed under a hood until completely dry. The trays containing the dried inoculated materials were placed in the Caron 6030 environmental chamber. Salmonella Recovery off Tomato Surfaces and Packing Line Surfaces Tomatoes and packinghouse materials were extracted at pre-determined time intervals from the environmental chamber and recovery studies were performed. Each recovery study involved sampling at 0, 1, 3, 7, 11, 14, 21 and 28 days for each surface. Each sampling period consisted of three single-fruit or single-packinghouse material replicates. On Day 0, the samples were aseptically removed from the fiberglass tray prior to being placed into the environmental chamber and placed into sterile Stomacher (Fisherbrand, Fair Lawn, NJ)) bags containing 100 ml of sterile PBS. For all other sampling days (1 through 28), the tomatoes or packinghouse material pieces were aseptically removed from the environmental chamber and individually placed into sterile

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36 Stomacher bags containing 100 ml of sterile PBS on the appropriate days. Tomato samples were constantly rubbed and shaken for one minute (Burnett and Beuchat 2001; Harris et al. 2001; Zhuang et al. 1995). Rubbing action was concentrated around the inoculated blossom scar area of the tomatoes to loosen any reversibly attached bacteria. The packinghouse materials were also rubbed and vigorously shaken for one minute in 100 ml of PBS. Vigorous rubbing was specifically applied to the dotted area on each piece of material. The PBS diluent was squeezed in and out of the sponge as well as rubbed and shaken to try and recover any Salmonella that may have migrated into the sponge matrix. The sample diluent from each Stomacher bag was then serially (1:10) diluted using sterile PBS dilution tubes. The serial dilutions were then pour plated using TSA (rif+). A negative control for the TSA (rif+) was poured to make certain the media was not contaminated. The plates were statically incubated at 37C for 48 hours. Tomato fruits and pieces of each of the materials that were not inoculated with the Salmonella cocktail were sampled for control purposes. The control samples were rubbed and shaken for one minute in 100 ml of PBS and serially (1:10) diluted as previously described for the inoculated samples. The serial dilutions were pour plated using TSA (rif+) and statically incubated at 37C for 48 hours. Statistical Analysis Results from the 20-hour growth studies were evaluated using a Students t test with an level of 0.05. All results from recovery studies were averaged counts (CFU/ml) of recovered Salmonella. Statistical analyses were performed using the Statistical Analysis System (SAS; SAS Institute, Cary, NC). The GLM procedure in SAS was used to analyze changes of bacterial populations between replications in each experiment. Multiple comparisons were performed using the Least Squares Mean adjusted by the

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37 Bonferroni method for the tomato and material data. Results that yielded P values of < 0.05 were considered significant in this recovery study.

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CHAPTER 4 RESULTS Recovery studies were designed to assess the recovery of Salmonella from the surfaces of tomato fruits and packing line materials. Inoculated fruit and material surfaces were subjected to three separate temperature/relative humidity environments for 28 days. The simulated environments were traditional tomato ripening room parameters, Florida fall/winter tomato production parameters and Florida spring tomato production parameters. Salmonella-inoculated fruit and material samples were periodically extracted from the simulated environments and evaluated for the survival of Salmonella. The recovery of Salmonella off fruit and material surfaces were assessed to determine if a specific temperature and relative humidity combination would affect Salmonellae survival over a prolonged period of time. Each type of surface was sampled in triplicate for all observation intervals; Day 0, 1, 3, 7, 11, 14, 21 and 28. The recovered Salmonella from each replicate was averaged and data was compiled into graphs. Graphs depict the relationship between log 10 CFU/ml Salmonella survivors and time (days) for all surfaces in each simulated environment. Growth Levels of Salmonella Serovars after a 20-Hour Incubation Two 20-hour growth studies were conducted for each of the five rifampicin-resistant serovars. No significant differences in growth rates were found to exist for any of the five serovars growth rates observed between the two studies (P <0.05). Results from these preliminary studies ensured that serovar growth rates were equivalent to one another and a consistent inoculum could be created (Figure 4-1). Prior to each 38

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39 experiment, the cell concentration of each Salmonella cocktail was estimated. This was accomplished by pour plating appropriate dilutions for each cocktail in triplicate using TSA (rif+). No significant differences (P <0.05) were found to exist between any inocula suspensions used for any recovery studies (data not shown). 012345678910S. AgonaS. GaminaraS. MichiganS. MontevideoS. PoonaAverage Log10 CFU/m l Study 1 Study 2 Figure 4-1. Average log 10 counts of five Salmonella serovars (rif+) after a 20-hour incubation. Recovery of Salmonella off Tomato Surfaces Mature green tomatoes (Florida 47) were inoculated with a five serovar Salmonella cocktail and stored separately for 28 days in all simulated environments. It should be noted that the simulated ripening room parameters did not include the addition of ethylene. Commercially, ethylene is typically applied during the ripening process of mature green tomatoes. Tomatoes not inoculated with the Salmonella cocktail were sampled at the beginning of each experiment to ensure the rifampicin-supplemented TSA eliminated all background microflora present on the fruits. All controls were found to be negative.

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40 Tomatoes Subjected to Spring Parameters Tomatoes subjected to spring production parameters, 30C and 80%RH, showed an overall decrease in log 10 values of Salmonella for Day 0 to Day 21, but a slight increase in Salmonella recovery was observed between Day 21 and Day 28 (Figure 4-2). The inoculum applied to tomato surfaces was estimated at 8.26 log 10 CFU/ml. On Day 0, 5.08 log 10 CFU/ml of the applied inoculum was recovered from tomato surfaces. At Day 21, no Salmonella was recovered from tomato surfaces. The greatest average reduction of recovered Salmonella was observed between Day 3 and Day 7 at 2.55 log 10 CFU/ml. Unexpectedly, a 1.17 log 10 CFU/ml increase was then observed on Day 28. This was unexpected because no Salmonella was recovered on Day 21. This increase was found to be significant (P <0.05). As the experiment progressed and tomatoes ripened, fruits held in this regime (30C/80%RH) appeared more orange in color than the tomatoes held at a lower temperature (20C). 0.001.002.003.004.005.006.000481216202428DaysLog10 CFU/ml Survivor s 80RH/30C 90RH/20C 60RH/20C Figure 4-2. Salmonella recovery (log 10 CFU/ml) from tomato surfaces in ripening room parameters (20C/90%RH) and spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days.

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41 Tomatoes Subjected to Fall/Winter Parameters Tomatoes subjected to Florida fall/winter tomato season parameters, 20C and 60%RH, also showed an overall log 10 reduction of Salmonella on the surfaces of tomatoes over 28 days (Figure 4-2). The inoculum applied to tomato surfaces was estimated at 8.59 log 10 CFU/ml. On Day 0, 4.01 log 10 CFU/ml of Salmonella cocktail was recovered. A 1.00 log 10 CFU/ml reduction was observed between Day 0 and Day 1, but a 0.66 log 10 CFU/ml increase of recovered Salmonella was observed from Day 1 to Day 3. This slight increase was found to be insignificant (P <0.05). Again, a significant decrease in log 10 CFU/ml was observed from Day 3 to Day 11 at 2.31 log 10 CFU/ml. From Day 11 to Day 14, another insignificant increase of Salmonella was observed at 0.44 log 10 CFU/ml. For the remainder of the 28-day period (Day 14 to Day 28) a slight reduction in CFU/ml recovery was observed. This minimal reduction in Salmonella over these days was not significant (P <0.05). Tomatoes Subjected to Ripening Room Parameters Tomatoes subjected to ripening room parameters, 20C and 90%RH, exhibited an overall reduction of Salmonella on the surfaces of tomatoes for a 28-day period (Figure 4-2). The inoculum suspension applied to the fruits was estimated at an average value of 8.15 log 10 CFU/ml. After the applied inoculum was allowed to completely dry, three tomatoes were sampled for Day 0. An average value of 4.64 log 10 CFU/ml of recovered Salmonella was observed on Day 0. The average log 10 value for Day 1 exhibited a slight increase of 0.7 log 10 CFU/ml in recovered Salmonella from Day 0. This increase was found to be insignificant (P <0.05). From Day 1 to Day 28, the average recovered Salmonella off tomato fruits exhibited a significant decrease in value over time. On Day 28, 1.42 log 10 CFU/ml of Salmonella was recovered from tomato surfaces.

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42 Comparison of Tomato Recovery Studies Recovery observations showed that Salmonella was recovered on final sampling interval (Day 28) in all three simulated environments. The levels of recovered Salmonella at the end of the three experiments were not significantly different from one another (P <0.05). The largest log 10 value reduction of Salmonella was observed for tomatoes held at 30C and 80%RH for 28 days. Tomatoes that were held at 20C and 60%RH had variable recovery that exhibited two separate increases in log 10 values for between sampling periods of Day 1 and 3, and Day 11 and 14. The increases were found to be insignificant; nonetheless it was unexpected that a slightly greater amount of Salmonella was recovered on Day 14 than Day 11. Tomatoes held at 20C and 90%RH exhibited a very linear pattern of reduction for log 10 values between Day 1 and Day 28 (R 2 = 0.9965). Salmonella was able to survive in all simulated environments, but survival patterns were very different. Day 21 in spring parameters (30C/80%RH) was the only sampling interval for any environment where no Salmonella was recovered. Recovery of Salmonella off Packing Line Surfaces Fresh-market tomato packinghouses are typically open-air facilities. The environments simulated for all materials paralleled spring and fall/winter parameters for tomato production seasons in Florida. Each type of material was subjected to both simulated environments for 28 days. Stainless Steel Surfaces Subjected to Spring Parameters Stainless steel surfaces held at 30C and 80%RH showed a total log 10 reduction at Day 11 (Figure 4-3). The inoculum applied to stainless steel surfaces was estimated at 8.01 log 10 CFU/ml. A value of 4.39 log 10 CFU/ml of Salmonella was recovered on Day 0. No significant log 10 reduction was observed between Day 0 and Day 1. A significant log

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43 reduction of 4.34 log 10 CFU/ml was observed from Day 1 to Day 11. On Day 11, no Salmonella was recovered. The reduction in Salmonella followed a linear pattern (R 2 = 0.9875). On Days 11, 14, 21 and 28 no Salmonella was recovered. Day 7 was the last sampling interval where Salmonella was recovered (1.29 log 10 CFU/ml) from the surfaces of stainless steel. 0.001.002.003.004.005.000481216202428DaysLog10 CFU/ml Survivor s 80RH/30C 60RH/20C Figure 4-3. Salmonella recovery (log 10 CFU/ml) from stainless steel surfaces in spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days. Stainless Steel Surfaces Subjected to Fall/Winter Parameters Stainless steel surfaces held at 20C and 60%RH did not exhibit a total log 10 value reduction of Salmonella at the conclusion of the sampling period (Figure 4-3). For the entire 28-day period, an overall log value reduction of 3.67 log 10 CFU/ml was observed. The inoculum applied to stainless steel surfaces was estimated at 8.59 log 10 CFU/ml. A value of 4.41 log 10 CFU/ml of Salmonella was recovered on Day 0. From Day 0 to Day 11, a 2.96 log 10 CFU/ml reduction of Salmonella was observed. On Day 14, the amount of recovered Salmonella was similar to the log 10 value recovered on Day 11. A 0.46 log 10

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44 CFU/ml reduction was observed between Day 14 and Day 28. This reduction was not significant (P <0.05). On Day 28, 0.74 log 10 CFU/ml of Salmonella was recovered. Comparison of Stainless Steel Recovery Studies The average log 10 values of Salmonella recovered on Day 0 for each experiment were not significantly different from one another (P <0.05). In the simulated spring environment, it was observed that Salmonella did not survive past Day 11 on stainless steel surfaces. For fall/winter environments, it was observed that Salmonella survived on stainless steel surfaces for the entire 28-day period. Recovered Salmonella survival off the stainless steel was significantly higher at 20C and 60%RH than recovered Salmonella at 30C and 80%RH (P <0.05). PVC Surfaces Subjected to Spring Parameters The inoculum applied to PVC surfaces was estimated at 8.01 log 10 CFU/ml. A value of 5.13 log 10 CFU/ml of Salmonella was recovered on Day 0. PVC surfaces subjected to spring production parameters showed a total log 10 reduction (5.13 log 10 CFU/ml) by Day 11 (Figure 4-4). A linear pattern of total Salmonella reduction was observed from Day 0 to Day 11 (R 2 = 0.9636). The last detection of Salmonella on PVC surfaces occurred on Day 7 with an average log 10 value of 1.00 log 10 CFU/ml. PVC Surfaces Subjected to Fall/Winter Parameters Salmonella was recovered from the surfaces of PVC surfaces for every sampling interval over a 28-day period (Figure 4-4). The inoculum applied to stainless steel surfaces was estimated at 8.59 log 10 CFU/ml. A value of 5.14 log 10 CFU/ml of Salmonella was recovered on Day 0. A significant decrease in Salmonella reduction observed over the 28-day period occurred from Day 0 to Day 1 with an average log 10 reduction of 1.19 log 10 CFU/ml. Overall, there was an average 4.57 log 10 CFU/ml

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45 reduction observed from Day 1 to Day 28. On Day 28, an average of 0.573 log10 CFU/ml of Salmonella was recovered from the surfaces of PVC. 0.001.002.003.004.005.006.000481216202428DaysLog10 CFU/ml Survivor s 80RH/30C 60RH/20C Figure 4-4. Salmonella recovery (log 10 CFU/ml) from PVC surfaces in spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days. Comparison of PVC Recovery Studies The average log 10 values recovered on Day 0 for each experiment were not significantly different from one another (P <0.05). The most significant decrease in Salmonella recovery in fall/winter parameters was observed in Days 0 through Day 11 with a 3.50 log 10 CFU/ml reduction. For the final three sampling periods (Day 14, 21 and 28), Salmonella only exhibited a 1.07 log 10 CFU/ml reduction. Salmonella was recovered off PVC surfaces held in spring parameters for the first four sampling intervals (Day 0-Day 7). Salmonella was recovered for a longer period of days from PVC surfaces at a lower temperature/relative humidity combination than at a temperature/higher relative humidity combination. Salmonella was not recovered from PVC surfaces held in spring parameters after Day 7. However, Salmonella was recovered off PVC surfaces held in fall/winter parameters for the entire 28-day period. The survival of Salmonella on PVC

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46 surfaces held in 20C and 60%RH was significantly higher than Salmonella on PVC surfaces at 30C and 80%RH (P <0.05). Sponge Rollers Subjected to Spring Parameters The inoculum applied to sponge roller surfaces was estimated at 8.01 log 10 CFU/ml. A value of 4.97 log 10 CFU/ml of Salmonella was recovered on Day 0 (Figure 4-5). Sponge rollers held at spring parameters exhibited a complete log 10 value reduction of 4.97 log 10 CFU/ml by Day 1. Salmonella was only recovered on Day 0. Salmonella was not able to be recovered from sponge rollers once they had entered the simulated environment at 30C and 80%RH. Sponge Rollers Subjected to Fall/Winter Parameters The inoculum applied to sponge roller surfaces was estimated at 8.59 log 10 CFU/ml. A value of 4.06 log 10 CFU/ml of Salmonella was recovered on Day 0. Sponge rollers held at fall/winter parameters exhibited a complete log 10 value reduction of 4.06 log 10 CFU/ml by Day 7 (Figure 4-5). Salmonella was only recovered on Days 0, 1 and 3. On Day 3, the average log 10 value of Salmonella recovered was 0.30 log 10 CFU/ml. A very linear and significant log 10 value reduction of Salmonella was observed between Day 0 and Day 3 (R 2 = 0.9999).

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47 0.001.002.003.004.005.006.000481216202428DaysLog10 CFU/ml Survivo r 80RH/30C 60RH/20C Figure 4-5. Salmonella recovery (log 10 CFU/ml) from sponge roller surfaces in spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days. Comparison of Sponge Roller Recovery Studies The log 10 values recovered on Day 0 for each experiment were not significantly different from one another (P <0.05). Significant reduction of Salmonella was observed from Day 1 to Day 3 off sponge rollers held in fall/winter parameters. Significant reduction of Salmonella was observed from Day 0 to Day 1 from sponge rollers held in spring parameters. Salmonella was recovered in one more sampling interval (Day 3) in the simulated fall/winter parameters than in spring parameters. Salmonella was recovered only at Day 0 from sponge rollers held in spring parameters. No Salmonella was recovered from any of the two environments at Days 7, 11, 14, 21 and 28. On Day 3, Salmonella recovery from surfaces of sponge rollers held at 20C and 60%RH was significantly higher than the Salmonella recovery on sponge rollers held at 30C and 80%RH (P <0.05 ). Sponge rollers were dampened with sterile, distilled water when inoculated. Sponge surfaces did not remain moist over the 28 day sampling intervals.

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48 Conveyor Belt Surfaces Subjected to Spring Parameters Salmonella was only recovered off conveyor belt surfaces held at spring parameters (30C/80%RH)on Days 0, 1 and 3. The inoculum applied to conveyor belt surfaces was estimated at 8.01 log 10 CFU/ml. A value of 4.10 log 10 CFU/ml of Salmonella was recovered on Day 0 (Figure 4-6). A linear and significant reduction in the recovery of Salmonella was observed between Day 0 and Day 3 (R 2 = 0.9803). Salmonella was last recovered on Day 1 at 2.26 log 10 CFU/ml from conveyor belt surfaces. Conveyor Belt Surfaces Subjected Fall/Winter Parameters The inoculum applied to conveyor belt surfaces was estimated at 8.59 log 10 CFU/ml. A value of 4.25 log 10 CFU/ml of Salmonella was recovered on Day 0. Conveyor belt surfaces stored at 60%RH and 20C showed a log 10 value reduction of 1.4 log 10 CFU/ml between Day 0 and Day 1 (Figure 4-6). Between Day 1 and Day 21, a 2.85 log 10 CFU/ml reduction was observed. Salmonella was last recovered from conveyor belt surfaces on Day 14 at an average log 10 value of 0.60 log 10 CFU/ml. Comparison of Conveyor Belt Recovery Studies The average log 10 values recovered on Day 0 for each experiment were not significantly different from one another (P <0.05). Salmonella was recovered for a longer period of days from conveyor belt surfaces in 20C and 60%RH than at 30C and 80%RH. Salmonella was only recovered for Day 0 and Day 1 in the simulated spring environment, whereas Salmonella recovery was observed until Day 14 in the simulated fall/winter environment. Salmonella recovery from conveyor belt surfaces in 20C and 60%RH was significantly higher than Salmonella recovery from conveyor belt surfaces in 30C and 80%RH (P <0.05).

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49 0.001.002.003.004.005.00048121620242DaysLog10 CFU/ml Survivor 8 s 80RH/30C 60RH/20C Figure 4-6. Salmonella recovery (log 10 CFU/ml) from conveyor belt surfaces in spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days. Unfinished Oak Surfaces Subjected Spring Parameters The inoculum applied to unfinished oak surfaces was estimated at 8.01 log 10 CFU/ml. A value of 4.73 log 10 CFU/ml of Salmonella was recovered on Day 0. Unfinished oak surfaces held in spring parameters exhibited a total log 10 value reduction by Day 21 of the 28-day sampling period (Figure 4-7). A significant decrease of 2.62 log 10 CFU/ml was observed from Day 0 to Day 1. From Day 1 to Day 3, a 0.89 log 10 CFU/ml increase was observed. This increase was found to be insignificant (P <0.05). On Day 3, no Salmonella was recovered and this trend continued until Day 14. On Day 14, Salmonella was recovered from oak surfaces at 1.00 log 10 CFU/ml. Salmonella was not recovered from unfinished oak surfaces for the final two sampling periods, Day 21 and Day 28.

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50 0.001.002.003.004.005.006.000481216202428DaysLog10 CFU/ml Survivor s 80RH/30C 60RH/20C Figure 4-7. Salmonella recovery (log 10 CFU/ml) from unfinished oak surfaces in spring (30C/80%RH) and fall/winter (20C/60%RH) regimes over 28 days. Unfinished Oak Surfaces Subjected to Fall/Winter Parameters The inoculum applied to unfinished oak surfaces was estimated at 8.59 log 10 CFU/ml. A value of 3.22 log 10 CFU/ml of Salmonella was recovered on Day 0. Salmonella was recovered off the surfaces of unfinished oak at every sampling interval (Figure 4-7). An initial 0.12 log 10 CFU/ml reduction of recovered Salmonella was observed from Day 0 to Day 1. From Day 1 to Day 3, a 0.33 log 10 CFU/ml increase in recovered Salmonella was observed. This slight increase was determined to be insignificant (P <0.05). A 1.65 log 10 CFU/ml reduction was observed from Day 3 to Day 11. Unexpectedly, Salmonella was recovered at a log 10 value on Day 14 at 0.18 log 10 CFU/ml. The decrease log 10 values observed from Day 14 to Day 21 was not significant (P <0.05). A 0.20 log 10 CFU/ml reduction of Salmonella was observed during the final two sampling periods.

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51 Comparison of Unfinished Oak Recovery Studies The log 10 values recovered on Day 0 for each experiment were not significantly different from one another (P <0.05). Viable Salmonella recovered from unfinished oak surfaces was recovered in greater amounts and for a longer period of days in spring parameters than at fall/winter parameters. Survival of Salmonella on oak surfaces stored at fall/winter parameters was significantly higher (P <0.05) than the survival of Salmonella on oak surfaces held in spring parameters. Salmonella recovery off of unfinished oak surfaces was variable for both simulated environments.

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CHAPTER 5 DISCUSSION Consumption of fresh fruits and vegetables has significantly increased over the past ten years. The industry is constantly challenged with the concern of microbial food safety hazards. Many steps are taken to harvest, process and distribute fresh produce and with each step the opportunity for potential pathogenic contamination increases. Environmental factors such as temperature and relative humidity have a large impact on the quality of fruits and vegetables along with the survival capacity of present pathogens. Effective intervention strategies have been implemented in packinghouses, such as chlorinated dump tanks, but these strategies cannot totally eliminate all microbiological dangers associated with the consumption of raw produce. It is also necessary that packinghouse equipment receive regular cleaning and disinfecting. In recent years, multiple foodborne illnesses associated with consumption of Salmonella-contaminated tomatoes have been traced to packinghouse facilities. In this study, a five serovar rifampicin-resistant Salmonella cocktail was administered to tomato and packing line surfaces. The various surfaces were subjected to different temperature and relative humidity combinations that simulated conditions encountered during tomato growing, packing and ripening. Recovery of Salmonella from the surfaces was performed by placing the surfaces into 100 ml of PBS and applying a rub-shake method as previously described. It has been recommended that a minimum of five strains at approximately equal populations be selected for the inoculum (CFSAN-FDA 2001). The five Salmonella 52

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53 enterica serovars selected for this study were S. Agona, S. Gaminara, S. Michigan, S. Montevideo and S. Poona. These serovars were obtained from Dr. Linda J. Harris, University of California Davis, and were marked with 80g/ml rifampicin. Rifampicin was selected because it is a stable marker and is particularly effective for isolating pathogens from inoculated fruits that have significant natural background microflora and adhering soil. S. Agona, Gaminara and Michigan serovars were isolated from fresh produce or produce products (orange juice). S. Montevideo and Poona serovars were human isolates linked to fresh produce outbreaks. Growth characteristics for all five serovars were evaluated by conducting 20-hour growth studies. Two studies were conducted on each of the five serovars. The population of each serovar at the end of a 20-hour incubation period was found to be insignificantly different (Students t test, = 0.05) from one another (Figure 4-1). S. Poona was observed to have the highest population at the end the 20-hour incubation period (37 o C), but there was less than a 0.5 log 10 CFU/ml difference between S. Poonas population and the serovar with the lowest growth level. This was the case in both growth studies. It was determined that all five serovars achieved counts of at least 1.0 x 10 8 CFU/ml after 20 hours of incubation. It was then concluded that acceptable inocula could be prepared from the five rifampicin-resistant Salmonella serovars. Prior to each recovery study, appropriate serial dilutions of the inoculum were pour-plated to determine the viable population of Salmonella. These counts for each prepared inoculum for all experiments conducted also showed little variation between one another. All inocula were determined to contain viable Salmonellae populations at 1.08 x 10 8 CFU/ml.

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54 Recovery of Salmonella off Tomato Surfaces Tomato surfaces were subject to three simulated environments: ripening room parameters (20 o C/90% RH), fall/winter tomato production season parameters (20 o C/60% RH) and spring tomato production season parameters (30 o C/80%RH). Surface recovery was assessed by applying a rub-shake method, as previously described. Tomatoes have a fairly firm surface that can withstand moderate rubbing and agitation. This rub-shake method of recovery was chosen because presently, it seems to be the most effective protocol for removing microorganisms from the surfaces of whole fruits and vegetables like tomatoes (CFSAN-FDA 2001). Whole, unblemished tomatoes were specifically chosen for inoculation studies. It has been researched that microbial cells that contact the surface of produce and interact with organic acids or other antimicrobials that are naturally found in plant tissue fluid or ruptured cells as a result of mold or insect invasion, cellular death may occur (Sofos et al. 1998). The rub-shake method is a simple surface wash that recovers surface bacteria without rupturing any plant cells that might interact with the inoculated pathogen. Spot inoculation was utilized because it enables the measurement of a known number of cells adhering to the produce. Dip or spray inoculation procedures do not allow the measurement of a known amount of inoculum. Results from this study were in agreement with Guo et al. (2002) in that Salmonella populations decreased over time on tomato surfaces in all simulated environments. The lowest quantities of Salmonella were recovered from tomatoes held in the spring season parameters (30C/80%RH). Viable Salmonella populations seemed to die-off or enter a nonculturable state by Day 21 of the recovery study. On Day 28, an unexpected increase in log 10 value was observed. Salmonella was recovered from two of the three tomato replicates sampled on Day 28, but log 10 values were significantly higher

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55 than Day 21 where no recovery of Salmonella was observed. Between Day 7 and Day 11, a log 10 value decrease of 2.55 log 10 CFU/ml was observed. This was the largest log 10 value decrease of Salmonella seen between any two sampling periods for all simulated tomato environments. Tomatoes held in ripening room (20C/90%RH) and fall/winter production parameters (20C/60%RH) seemed to exhibit similar patterns of Salmonella recovery. The highest log 10 values of Salmonella populations were recovered from tomato surfaces held in ripening room parameters. Slightly more Salmonella was recovered on Day 1 than on Day 0. This increase was insignificant, but a possible explanation for this phenomenon could be that some cells of Salmonella did not survive the drying process while others could have survived, but were shocked and could not be recovered by conventional culture methods (CFSAN-FDA 2001). For Days 1 through Day 28, a linear reduction of Salmonella was observed with the largest decrease in recovered log 10 values seen between Day 21 and Day 28 at 1.0 log 10 CFU/ml. Tomato surfaces held in fall/winter parameters showed the most Salmonella reduction between Day 0 and Day 1 when compared to the other environments. On Day 3, an increase in Salmonella recovery was observed. The same phenomenon for injured cells could have occurred as previously mentioned. The lower amount of moisture (60%RH) in the environment could explain the delayed recovery of injured cells on Day 3 instead of Day 1 as seen in ripening room parameters (90%RH). The availability of more moisture could have possibly allowed Salmonella to recover at a faster rate. An approximate 2.0 log 10 CFU/ml reduction in recovered Salmonella was observed from Day

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56 3 to Day 11 and again, on Day 14 a slight increase in recovered Salmonella was observed. Very similar levels of Salmonella were recovered from tomato surfaces for the last three sampling intervals. All three simulated environments exhibited Salmonella recovery on Day 28 at very equivalent log 10 values (approximately 1.5 log 10 CFU/ml). Salmonella survival patterns were different for every simulated environment, but the final sampling interval yielded similar log 10 values of recovered Salmonella. Overall, Salmonella was recovered more in an environment where the temperature was maintained at 20C and the relative humidity was at a high level. It has been documented by many researchers that bacterial populations have a greater chance of survival and growth in the presence of free moisture on leaves, from precipitation, dew or irrigation. Essentially, a higher level of humidity enhances the survival of bacterial cells (Beattie and Lindow 1999). Salmonella was still observed to survive very well at 20 o C in conjunction with a slightly lower relative humidity. Salmonella seemed less likely to survive in an elevated temperature (30C). Salmonella was consistently recovered from tomato surfaces at a greater log 10 value than any of the packinghouse surfaces while utilizing the rub-shake method of recovery. Tomatoes are organic surfaces that respire and participate in gas exchange with the surrounding atmosphere. The tomatoes may have experienced different rates of respiration at 20 o C than at 30 o C and this could have had some effect on the Salmonella. Viable Salmonella could have aggregated in the stem scar of the fruit or have become irreversibly attached to the fruits surface and was not recovered. Over time, Salmonella

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57 could have entered a nonculturable state do to nutrient depletion, injury or environmental stress. After harvest, pathogens seem to survive but not proliferate on the outer surface of tomato fruits, especially in a high humidity and an ambient temperature (20 o C). Pathogen levels were observed to decline on the outer surface of tomatoes over time and the rate of reduction seemed to be strongly related to temperature. Growth on intact surfaces was not observed. Foodborne pathogens do not produce the necessary enzymes to destroy the protective outer barriers on most produce, thus restricting the availability of nutrients. Salmonella was recovered from tomato surfaces in all three simulated environments and this indicates that the pathogen can survive on tomato surfaces for a significant amount of time and should be a concern in the fresh produce industry. If contaminated fruits enter a packinghouse facility it is very probable that cross-contamination is likely upon the contact of processing equipment. Recovery of Salmonella off Packinghouse Surfaces Five types of packing line materials were inoculated with a five serovar rifampicin-resistant Salmonella cocktail and subjected to fall/winter and spring tomato production season parameters. Typically, Florida packinghouse facilities are open-sided, shed-like buildings that shelter the minimal processing of fresh produce harvested in near by fields. The five materials that were evaluated in recovery studies were stainless steel (type 304, no. 4 finish), polyvinyl chloride (PVC), sponge rollers, conveyor belts and unfinished oak surfaces. Stainless steel is commonly found in most food processing facilities. It is a smooth, easily sanitized surface that is widely recognized as an excellent material for the food industry (Midelet and Carpentier 2002). Dump tanks, processing lanes and much

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58 equipment in tomato packinghouses are made of stainless steel. Overall, Salmonella was recovered at greater log 10 values and over a longer period of days off stainless steel surfaces held in fall/winter parameters than stainless steel surfaces held in spring parameters. Salmonella was only recovered from stainless steel surfaces held at 80%RH and 30C for the first four sampling intervals (Days 0, 1, 3 and 7). Salmonella was last recovered at a log 10 value of 1.29 log 10 CFU/ml on Day 7. On Days 11, 14, 21 and 28 no Salmonella was recovered. Significantly more Salmonella was recovered from stainless steel surfaces held at 20C and 60%RH when compared to surfaces held at 30C and 80%RH. The most reduction of Salmonella for both environments was observed from Day 0 to Day 11 off surfaces held in spring parameters. After Day 11 for surfaces held in spring parameters, the recovered populations of Salmonella did not significantly decrease. On Day 28, Salmonella was recovered at 0.74 log 10 CFU/ml from surfaces held in spring parameters. Polyvinyl chloride (PVC) is another commonly used material in the food industry. It is a polymer that is typically used to cover roller bars that move tomato fruits along processing lines. PVC rollers allow tomatoes to be smoothly transported so extensive bruising and injury is minimal. Recovery patterns of Salmonella from PVC surfaces for both simulated environments were very similar to recovery patterns from stainless steel surfaces. Salmonella was only recovered on Days 0, 1, 3 and 7 from PVC surfaces held in spring parameters. The most significant amount of Salmonella reduction (2.0 log 10 CFU/ml) observed in spring parameters was seen between Days 3 and 7. Similar to recovery patterns from stainless steel surfaces, more log 10 values of Salmonella were recovered from PVC surfaces at 60%RH and 20 o C than from surfaces held at 30C and

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59 80%RH. Salmonella was recovered for every sampling period from PVC surfaces held in fall/winter parameters. It was very clear that viable Salmonella cells were able to survive on PVC surfaces for a prolonged period of days at 60%RH and 20 o C. Stainless steel and PVC are both hydrophobic surfaces (Midelet and Carpentier 2002). Stainless steel is a nonporous surface but is often marked by grooves and crevices. This was clearly seen in scanning electron photomicrographs taken of stainless steel type 304, no. 4 finish (Mafu et al. 1990). Stainless steel is also very resistant to wear. PVC is a dense polymer with smooth surface, but contains microscopic holes and crevices. PVC is also resistant to wear, but is more likely to bend or accumulate cracks or holes than stainless steel surfaces. Of all the surfaces tested in this recovery study, stainless steel and PVC were the least porous materials. The most amounts of Salmonella were recovered from these two surfaces when compared to the other surfaces that were tested. It is most likely that very few salmonellae infiltrated into the matrix of these two surface types. Hydrophobic qualities accompanied with the dense nature of the materials most likely prevented bacteria from migrating very far from the point of inoculation. It was evident that more Salmonella was recovered off both of these surface types at 20 o C than at 30C. Salmonella was not recovered from surfaces held at 30 o C for a prolonged period of time. Viable cells of Salmonella were not recovered from either surface past Day 7 at 30 o C and 80%RH. It is possible that Salmonella, at 30C, had entered a nonculturable state due to environmental stresses. Biofilm formation is yet another possibility, although this is not likely because no planktonic cells were recovered from either surface during the final four sampling intervals. It has been documented that bacteria can readily and irreversibly attach to many surface types upon very short contact

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60 times, even within one minute (Mafu et al. 1990). Surface types that have been exposed to bacteria over short contact times and have been observed to form biofilms are glass, rubber, stainless steel, and many types of plastics (Ronner and Wong 1993; Mafu et al. 1990). Conveyor belts play an integral part in the functions of a packinghouse. Belts are rubber compounds (composition not disclosed by manufacturer) that transport tomatoes to all areas in the facility. Conveyor belts are used to pre-size, cull, sort and size tomato fruits. Rubber surfaces are smooth to the touch, but scanning electron pictographs show that particles, crevices and holes appear on the surface (Mafu et al. 1990). Salmonella was recovered from conveyor belt surfaces held at 30C and 80%RH on Days 0 and 1. No Salmonella was recovered for any other sampling intervals for spring parameters. However, Salmonella was recovered at a significantly higher amount from conveyor belt surfaces held at 20C and 60%RH. Recovery was observed on Days 0 through 14. A sharp reduction of Salmonella was seen between Day 0 and Day 1. Between Days 1 and 11, an approximate 2.0 log 10 CFU/ml reduction of Salmonella was observed. The reported log 10 values of recovered Salmonella on Days 11 and 14 were very similar, almost no reduction was seen. For the final two sampling intervals, no Salmonella was recovered from conveyor belt surfaces held at 20C and 60%RH. The patterns of recovered Salmonella from conveyor belt surfaces differed from those of stainless steel and PVC because no Salmonella was recovered from conveyor surfaces held at 20C and 60%RH after Day 14. Salmonella was recovered from stainless steel and PVC surfaces until Day 28 for surfaces held at 20C and 60%RH. It was still evident that Salmonella survived for a longer period of days on conveyor

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61 surfaces held at 20C than surfaces held at 30C. It is possible that the composition of the conveyor belts had a slight bacteriostatic affect on Salmonella. The pathogen was recovered at very low amounts on Days 11 and 14 and no survival was observed to occur at Days 21 and 28. An extreme reduction was also recorded between Day 0 and Day 1. It has been documented that some types of rubber surfaces have a strong bacteristatic effect on pathogens. Buna-n rubber (nitrile rubber) is a gasket material typically used in food processing environments. It has been documented that material has a slight bacteriostatic effect on Salmonella Typhimurium and a strong bacteristatic effect on Listeria monocytogenes under low nutrient conditions. It also inhibited the growth of several other pathogens to varying degrees (Ronner and Wong 1993). Sponge rollers also serve a very important role in tomato packinghouse operations. Tomato fruits are susceptible to injury and bruising. Sponge rollers buff and cushion the fruits as they proceed along the processing lines. Sponge rollers absorb dumptank water off the fruit surface and the sponges are constantly moist. Thus, sponge roller samples were dampened with sterile, distilled water prior to inoculation. The surface and matrix of the rollers were extremely porous and small holes were clearly visible. Sponge roller surfaces were hydrophilic and absorbed the inocula whereas previous surfaces were hydrophobic. Very little Salmonella was recovered from sponge rollers held in fall/winter or spring parameters. Approximately 5.0 log 10 CFU/ml of Salmonella was recovered from sponge rollers held at 30C and 80%RH on Day 0. Day 0 was the only sampling interval in which Salmonella was recovered for spring parameters. Fall/winter parameters allowed Salmonella to be recovered from sponge rollers for a longer period of days than

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62 spring parameters. Recovery was observed on Days 0, 1 and 3 for rollers held in fall/winter parameters. Day 1 was the only sampling interval that was found to be significantly different between the two simulated environments. On Day 1, a significantly higher log 10 value of Salmonella was recovered from sponge rollers held at 20C and 60%RH. On Day 3, a very low log 10 value of Salmonella was recovered from surfaces held in fall/winter parameters and the difference was determined to be insignificant when compared to the recovery on Day 3 for sponges in spring parameters. Overall, the two recovery patterns for the two simulated environments were very similar. The composition of the sponge rollers (not disclosed by the manufacturer) seemed to have a strong bacteriostatic effect on Salmonella. Even with the extreme reduction of Salmonella seen in both environments, the pathogen was recovered for a longer time period at 20C and 60%RH. Wooden pallets are used to transport unitized loads of tomato boxes and are used in many packinghouse facilities. Wooden field bins are sometimes used to collect harvested tomatoes, although plastic field bins are more common. Pallets are usually constructed from unfinished oak wood. Wood surfaces are very rough and porous. The oak surfaces were hydrophilic and inocula quickly soaked into the surfaces. Salmonella recovery was extremely variable off unfinished oak surfaces. Overall, recovery of the pathogen did follow the pattern of recovery from the other surfaces. It was evident that Salmonella was recovered more from wooden surfaces held at 20C and 60%RH. Surfaces held in fall/winter parameters seemed to facilitate the survival of Salmonella over the entire 28 day period. An approximate 1.0 log 10 CFU/ml reduction of Salmonella was observed over the entire experiment. This amount of Salmonella

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63 reduction was much lower than any reduction observed for other surface types. Recovery values did fluctuate between sampling intervals in both environments. On two separate sampling intervals, an increase in Salmonella recovery was observed rather than an anticipated decrease. For the final sampling interval (Day 28) in fall/winter parameters, Salmonella recovered from oak surfaces was significantly higher than the recovered Salmonella in spring parameters. The most variable recovery pattern observed throughout the entire recovery study was unfinished oak surfaces held at 30C and 80%RH. From Day 0 to Day 1 a large reduction in Salmonella was observed. On Day 3, Salmonella recovery increased by 1.0 log 10 CFU/ml. For the next two sampling periods, Days 7 and 11, no Salmonella was recovered. For all other materials, recovery patterns followed a trend. When no Salmonella had been recovered during one sampling interval, no other sampling intervals yielded the recovery of Salmonella. Unfinished oak surfaces did not follow this trend. On Day 14, Salmonella was recovered from wood surfaces at 1.0 log 10 CFU/ml. On Days 21 and 28, the pathogen was not recovered. Unfinished oak surfaces are known to be very coarse and irregular. It is suspected that the inocula seeped into the matrix of the wood samples. When the samples were rubbed and shaken for recovery purposes, it was evident that the recovery of Salmonella was extremely variable. Salmonella was most likely harbored by the matrix of the wood. Once the pathogen had migrated into the wood matrix recovery methods utilized were not able to extract the pathogen very easily. These results were in agreement with Boucher et al. (1998). Campylobacter jejuni was observed to exhibit enhanced survival on cubes of wood when compared to survival on cubes of plastic. Bacteria were observed to be sealed inside the porous membrane of the wood cubes. The physical structure was

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64 necessary for the protection of Campylobacter jejuni and soluble free-radical scavengers from the wood were not responsible for the observed protection. Deeply scored plastic cubes did not offer enhanced survival in aerated broths. Scanning electron microscopy was utilized to determine the size of the openings within the wood in relation to the bacterial cells. Holes and crevices in the wood were noted to be larger than the bacterial cells allowing the cells to enter the wood matrix. It was established that the physical structure of the wood, rather than its chemistry was responsible for the woods protective effect. It is postulated that the unfinished oak surfaces behaved similarly to the wood cubes examined in the previously described experiment. It is very likely that viable Salmonella was harbored inside the oak pieces and were not recovered. Proliferation of Salmonella was not observed on any surface type. As previously stated, growth of pathogens on intact surfaces of fruit is not common because foodborne pathogens do not produce the enzymes necessary to breakdown the outer barriers that protect the produce (CFSAN-FDA 2001). The availability of nutrients and moisture is therefore limited. However, after harvest, pathogens are able to survive on the outer surfaces of fresh fruits and vegetables, especially if the humidity is high. This indicates that temperature was an important variable for the survival of Salmonella on the various surfaces evaluated in this study. High relative humidity was present in ripening room parameters and spring parameters, but it was seen that Salmonella was recovered in greater quantities at a lower temperature (20C). It should be noted that bacterial soft rot microorganisms commonly infect tomatoes and the incidence of Salmonella increases in infected fruits (this was not a factor in this study).

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65 The two temperatures selected for this recovery study were 20C and 30C. The ambient temperature (20C) seemed to allow Salmonella to survive for a longer period of days. The warmer temperature (30C) seemed to inhibit the ability of Salmonella to survive as well on various surfaces. Salmonella most likely exhausted all resources very quickly at 30C. The microorganism grows very well at 37C, but nutrient depletion encountered by Salmonella over the 28 days most likely inhibited survival over time. This trend was observed for every recovery study performed. Lower temperatures seem to facilitate the survival of Salmonella rather than higher temperatures. It has also been reported that certain strains of salmonellae can survive for longer periods of time under refrigeration temperatures than at room temperature (Parish 1997; Zhao et al. 1993). A further study might explore the recovery of Salmonella off various surfaces under refrigeration temperatures (4C) to see if survival of the pathogen is enhanced. Recovery of Salmonella at low levels is still an important concern. Lower levels of the pathogen were still recovered as time increased (up to 28 days for tomatoes). The infectious dose of salmonellae ranges from 10 to 100,000 cells (CFSAN-FDA 2001). This indicates that even low levels of Salmonella in favorable conditions can facilitate a foodborne disease outbreak. This is a chief concern for the fresh produce industry due to the fact that edible horticultural crops are consumed without a treatment to help eliminate any pathogenic microorganisms that may be present.

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CHAPTER 6 CONCLUSION All objectives of this study were accomplished. Growth rates of five rifampicin resistant Salmonella serovars were established and it was determined that an appropriate cocktail could be made. Salmonella was successfully recovered from tomatoes and all material surfaces. It was observed that the pathogen survived longer on all surface types in the simulated fall/winter regime (20C/60%). Salmonella has the capability to survive over a prolonged period of time in certain temperature and relative humidity combinations on tomato fruits and various equipment surfaces. Results showed that Salmonella populations on tomato surfaces held at 20C were observed to decline over time (approximately a 4.0 log 10 CFU/ml reduction over 28 days). Of all simulated environments, spring tomato production parameters (30C/80%RH) yielded the lowest recovery of Salmonella over 28 days. Stainless steel and PVC surfaces had similar recovery patterns of Salmonella at 20C and 60%RH over 28 days. No Salmonella was recovered after Day 7 at 30C and 80%RH off these surfaces. No Salmonella was recovered from conveyor belt and sponge roller surfaces after 21 and 7 days, respectively. Salmonella recovery off wood surfaces exhibited the most variability. Wood surfaces maintained at 60%RH and 20C exhibited the most Salmonella recovery of any surface type at the end of 28 days. An ambient temperature (20C) combined with a higher (90%RH) and moderate (60%RH) relative humidity seemed to facilitate Salmonella survival better than an elevated temperature (30C) 66

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67 combined with a high (80%RH) relative humidity. Temperature seemed to be an important factor affecting the survival of Salmonella on various surface types. Surface types of the materials also seemed to affect Salmonella recovery and survival over time. More Salmonella was recovered from the smooth and nonporous surfaces like stainless steel and PVC. Rough and porous surfaces, like the wood surfaces, seemed to harbor Salmonella in this matrix better than smoother surfaces. Sponge rolls and conveyor belt surfaces also showed a possible bacteriostatic effect on Salmonella over time. Sponge rollers did not allow Salmonella survival for longer than Day 1 in the spring regime and Salmonella was not recovered from the rollers after Day 3 in the fall/winter regime. Salmonella was not recovered after Day1 in the spring regime from conveyor belt surfaces. Salmonella did survive on conveyor surfaces until Day 14 in the fall/winter regime, but it was recovered at very low levels.

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71 U.S. Food and Drug Administration [FDA]. April 1999. The food safety initiative. Center for Food and Applied Nutrition. http://www.fda.gov. Accessed 2003 September. Food Safety and Inspection Service [FSIS]. 2001. Salmonella questions and answers. http://www.fsis.usda.gov. Accessed 2003 May. Florida Tomato Committee. 2002. Florida tomato facts and sizing. http://www.floridatomatoes.org/facts.html Accessed 2003 May. Frenzen, P., T. Riggs, J. Buzby, T. Breuer, T. Roberts, D. Voetsch, S. Reddy, and the FoodNet Working Group. 1999. Salmonella cost estimate update using FoodNet data. Food Review. 22(2):10-15. Fung, D.Y.C. 2001. Rapid methods of microbiological analysis: Update. Pp.63-74. In: Wilson, C.L. and S. Droby. (Ed.), Microbial Food Contamination. CRC Press. Gayler, G.E., R.A. MacCready, J.P. Reardon and B.F. McKernan. 1955. An outbreak of salmonellosis traced to watermelon. Public Health Rep. 70:311-313. Golden, D.A., E.J. Rhodehamel and D.A. Kautter. 1993. Growth of Salmonella spp. in cantaloupe, watermelon and honeydew melons. J. Food Prot. 56:194-196. Goverd, K.A., F.W. Beech, R.P. Hobbs and R. Shannon. 1979. The occurrence and survival of coliforms and salmonellas in apple juice and cider. J. Appl. Bacteriol. 46:521. Guo.X., J. Chen, R.E. Brackett and L.R. Beuchat. 2001. Survival of samonellae on and in tomato plants from the time of inoculation at flowering and early stages of fruit development through fruit ripening. Appl. Environ. Microbiol. 67(10):4760-4764. Guo, X., J. Chen, R.E. Brackett and L.R. Beuchat. 2002. Survival of Salmonella on tomatoes stored at high relative humidity, in soil and on tomatoes in contact with the soil. J Food Prot. 65(2):274-279. Harris, L.J., D. Zagory and J.R. Gorny. 2002. Safety Factors. Pp.301-313. In: Adel A. Kader (Ed.) 3 rd ed., Postharvest Technology of Horticultural Crops. University of California, Agriculture and Natural Resources, Publication 3311. Hedburg, C.W., F.J. Angulo, K.E. White, C.W. Langkop, W.L. Schell, M.G. Stobierski, A. Schutat, J.M. Besser, S. Dietrich, L. Helsel, P.M. Griffin, J.W. McFarland, M.T. Osterholm and the Investigation Team. 1999. Outbreaks of salmonellosis associated with eating uncooked tomatoes: Implications for public health. Epidemiol. Infect. 122:135-393.

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73 Midelet, G. and B. Carpetier. 2002. Transfer of microorganisms, including Listeria monocytogenes from various materials to beef. App. Environ. Microbiol. 68(8):4015-4024. Nguyen-The, C. and F. Carlin. 1994. The microbiology of minimally processed fresh fruits and vegetables. Crit. Rev. Food Sci. Human Nutr. 34(4):371-401. Parish, M.E. 1997. Public health and nonpasteurized fruit juices. Crit. Rev. Microbiol. 23(2):109-19. Rajkowski, K.T and E.A. Baldwin. 2003. Concerns with minimal processing in apple, citrus and vegetable products. Pp.35-52. In: John Novak, Gerald Sapers, Vijay Juneja (Ed.), Microbial Safety of Minimally Processed Foods, CRC Press. Reid, M.S. 2001. Ethylene in postharvest technology. Pp.149-162. In: Adel A. Kader (Ed.), Postharst Technology of Horticultural Crops 3 rd ed., University of California, Agriculture and Natural Resources, Publication 3311. Ronner, A.B. and A.C.L. Wong. 1993. Biofilm development and sanitizer inactivation of Listeria monocyogenes and Salmonella typhimurium on stainless steel and Buna-n rubber. J. Food Prot. 56(9):750-758. Rushing, J.W., F.J. Angulo and L.R. Beuchat. 1996. Implementation of a HACCP program in a commercial fresh-market tomato packinghouse: a model for the industry. Dairy Food Environ. Sanit. 16(9):549-553. Sargent, S.A., M.A. Ritenour and J.K. Brecht. 2001. Handling, cooling and sanitation techniques for maintaining postharvest quality. EDIS (Extension Digital Information Source) publication, University of Florida, Cooperative Extension Service. http://edis.ifas.edu/CV115 Accessed 2003 November. Schlech, W. F., P.M. Lavigne, R.A. Bostolussi, A.C. Allen, E.V. Haldane, A.J. Wort, A.W. Hightower, S.E. Johnson, S.H. King, E.S. Nicholls and C.V. Broome. 1983. Epidemic listeriosis-evidence for transmission by food. N. Engl. J. Med. 308:203-206. Sofos, J. N., L. R. Beuchat, P. M. Davidson and E.A. Johnson. 1998. Naturally occurring antimicrobials in food. Council for Agric. Sci. Technol. Task Force. Report 132. Pp.103. Somers, E.B., S.L. Schoeni and A.C.L. Wong. 1994. Effect of trisodium phosphate on biofilm and planktonic cells of Campylobacter jejuni, E. coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium. Int. J. Food Microbiol. 22:269-276.

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74 Suslow, T.V. 2002. Production practices affecting the potential for persistent contamination of plants by microbial foodborne pathogens. Pp. 241-256. In: Lindow, S.E., E.I. Hecht-Poinar, and V.J. Elliot (Ed.), Phylloshpere Microbiology. American Phytopathological Society Press, Minnesota. Tauxe, R.V., H. Kruse, C. Hedburg, M. Potter, J., Madden and K. Wachsmuth. 1997. Microbial hazards and emerging issues associated with produce, a preliminary report to the National Advisory Committee on microbiologic criteria for foods. J. Food Prot. 60(11):1400-1408. University of Florida/ Institute of Florida Agricultural Sciences [UF/IFAS]. Sargent, Steve. 1998. Handling Florida Vegetables-Tomato. Florida Cooperative Extension Service, EDIS publication: SS-VEC-928. http://edis.ifas.ufl.edu Accessed 2003 May. United States Department of Agriculture/ Natural Agricultual Statistics Service [USDA/NASS]. 2001. Fruit and vegetable agricultural practices-1999, June. U.S. Department of Agriculture/National Agricultural Statistics Service, Washington, D.C. http://www.usda.gov/nass/pubs/rpts106.htm Accessed 2003 May. University of Florida Institute of Food and Agricultural Sciences. 2003. http://fawn.ifas.ufl.edu Accessed 2003 May. Wei, C. I., T. S. Huang, J.M. Kim, W.F. Lin, M.L. Tamplin and J.A. Bartz. 1995. Growth and survival of Salmonella Montevideo on tomatoes and disinfection with chlorinated water. J. Food Prot. 58:829-836. Wells, J.M. and J.E. Butterfield. 1997. Salmonella contamination associated with bacterial soft rot of fresh fruits and vegetables in the marketplace. Plant Dis. 81:867-872. Wells, J.M. and J.E. Butterfield. 1999. Incidence of Salmonella on fresh fruits and vegetables affected by fungal rots or physical injury. Plant Dis. 83:722-726. Yu, K., M.C. Newman, D.D. Archbold and T.R. Hamilton-Kemp. 2001. Survival of Escherichia coli O157:H7 on strawberry fruit and reduction of the pathogen population by chemical agents. J. Food Prot. 64(9):1334-40. Zhao T., M.P. Doyle and R.E. Besser. 1993. Fate of enterohemorrhagic Escherichia coli O157:H7 in apple cider with and without preservatives. Appl. Environ. Microbiol. 59(8):2526-30. Zhuang, R.Y., L.R. Beuchat and F.J. Angulo. 1995. Fate of Salmonella montevideo on and in raw tomatoes as affected by temperature and treatment with chlorine. Appl. Environ. Microbiol. 61:2127-2131.

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75 Zottola, E.A. 1994. Microbial attachment and biofilm formation: A new problem for the food industry. Food Tech. 48:107-114.

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BIOGRAPHICAL SKETCH Raina Leneve Allen was born in Tampa, FL, on July 28, 1979. In 2001, she received her Bachelor of Science from the University of Florida in food science and human nutrition. Upon graduation, she was accepted into the University of Floridas food science masters program. In this program, her specialization focused on food microbiology with a special interest in microbial safety of fresh-market produce. Upon receiving her masters degree, Raina plans to pursue a career in the food industry and continue working in the area of food safety. 76


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Title: Recovery Study of Salmonella spp. off the Surfaces of Tomatoes and Packing Line Materials
Physical Description: Mixed Material
Copyright Date: 2008

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A RECOVERY STUDY OF Salmonella SPP. FROM THE SURFACES OF
TOMATOES AND PACKING LINE MATERIALS















By

RAINA LENEVE ALLEN


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


2003

































Copyright 2003

by

Raina Leneve Allen




























To my parents, for without your love and never-ending support none of this would have
been possible. Your guidance and belief in me allowed me to get this far. Dad, I thank
you for always wanting something better for your children. You have sacrificed to no
end for me. Mom, your faith has always made you strong in my eyes. You were always
here to lean on and so many things make you great.















ACKNOWLEDGMENTS

I would like to give special thanks to my committee chair, Dr. Keith R. Schneider,

for the opportunities he has given me and the guidance he has offered me throughout the

past two years. I admire all he does and I am thankful to have obtained my master's

degree under his direction. I would also like to thank the members of my graduate

committee, Dr. Douglas Archer and Dr. Steve Sargent, for their help and assistance with

this project.

I thank my family. Their love and support have inspired me to succeed. I thank

my brother, for always having an encouraging word for my ear.

I want to thank my colleagues Ben Warren and Tom Ballesteros. I am blessed to

have earned this degree with such wonderful friends. I also want to thank my friends,

Kim Saranko, Michelle Burtch, Christen McGinnis, the Tucci's, Meg Mizell and Liz

Benz.
















TABLE OF CONTENTS
page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .............................................. .. ............... ........... .. vii

LIST OF FIGURES ............................. ... .. ........ ... ........ ... ............. .. viii

ABSTRACT .............. .................. .......... .............. ix

CHAPTERS

1 INTRODUCTION ............... ................. ........... ................. ... .... 1

2 LITER A TU R E REV IEW ............................................................. ....................... 5

Foodborne Illnesses Associated with Fresh Produce............................ .....................7
Salm onella Species .... .......... .... .......... ..................... ........ ............... 10
Salmonellosis Outbreaks Involving Fresh Tomatoes ...........................................12
T om atoes and Salm onellae .................................................................... ............... 13
T om ato Industry .................................................. .....................16
Postharvest H handling of Tom atoes.................................... ............................ ....... 17
Extrinsic Factors Influencing Microbial Viability ..................................................21
Attachment of Microorganisms to Various Surfaces...............................................24
Microbiological Recovery Methods Involving Fresh Produce................................26

3 M ATERIALS AND M ETHODS ........................................ ......................... 29

Selection of Temperature and Relative Humidity Combinations..................................29
Acquisition and Maintenance of Salmonella Cultures .............................................30
Growth Levels of Salmonella Serovars after a 20-Hour Incubation ..........................31
P reparation of Inoculum .................................................................................... ........ 32
Inoculation P procedures .............................. ........................ .. ...... .... ............ 33
Inoculation of Tom atoes............................................................... ............... 33
Inoculation of Packing Line M materials .................................. ...... ................34
Salmonella Recovery off Tomato Surfaces and Packing Line Surfaces ..................35
Statistical A nalysis................................................... ...... 36

4 R E S U L T S ............................................................................. 3 8

Growth Levels of Salmonella Serovars after a 20-Hour Incubation ..........................38









Recovery of Salmonella off Tomato Surfaces............ .................................39
Tomatoes Subjected to Spring Parameters ...... .... ............ ....................... 40
Tomatoes Subjected to Fall/Winter Parameters ................. ..................41
Tomatoes Subjected to Ripening Room Parameters ................ ............ .....41
Comparison of Tomato Recovery Studies................................ ............... 42
Recovery of Salmonella off Packing Line Surfaces................... .............. 42
Stainless Steel Surfaces Subjected to Spring Parameters................ ........ 42
Stainless Steel Surfaces Subjected to Fall/Winter Parameters..........................43
Comparison of Stainless Steel Recovery Studies..................... ..... ..........44
PVC Surfaces Subjected to Spring Parameters ................................................44
PVC Surfaces Subjected to Fall/Winter Parameters .......................................44
Comparison of PVC Recovery Studies .................................... ............... 45
Sponge Rollers Subjected to Spring Parameters ............................................46
Sponge Rollers Subjected to Fall/Winter Parameters .................................. 46
Comparison of Sponge Roller Recovery Studies ............................................. 47
Conveyor Belt Surfaces Subjected to Spring Parameters ..............................48
Conveyor Belt Surfaces Subjected Fall/Winter Parameters ............................48
Comparison of Conveyor Belt Recovery Studies.....................................48
Unfinished Oak Surfaces Subjected Spring Parameters............... ......... 49
Unfinished Oak Surfaces Subjected to Fall/Winter Parameters........................50
Comparison of Unfinished Oak Recovery Studies.................. ................51

5 D ISC U SSIO N ............................................................................. ................. 52

Recovery of Salmonella off Tomato Surfaces.............. ............ ...............54
Recovery of Salmonella off Packinghouse Surfaces................... .............. 57

6 C O N C L U SIO N ......... ......................................................................... ........ .. ..... .. 66

LIST OF REFEREN CES .................................................................... ............... 68

BIO GRAPH ICAL SK ETCH .................................................. ............................... 76















LIST OF TABLES


Table p

Table 3-1. Temperature and relative humidity combinations selected to simulate a
ripening room environment (20C/90%RH) and a fall/winter (20C/60%RH) and
spring (30C/80%RH) tomato production conditions. ...........................................30

Table 3-2. Salmonella enteritidis serovars obtained from Dr. Linda J. Harris at the
University of California, Davis: wild types* and rifampicin-resistant serovars listed
w ith source. .......................................... ............................ .. 31

Table 3-3. Surface area dimensions of each type of packing line material that was
inoculated with a five serovar rifampicin-resistant Salmonella cocktail. ...............34















LIST OF FIGURES


Figure page

Figure 4-1. Average logo counts of five Salmonella serovars (rif+) after a 20-hour
incubation ............................................................................39

Figure 4-2. Salmonella recovery logoo CFU/ml) from tomato surfaces in ripening room
parameters (200C/90%RH) and spring (300C/80%RH) and fall/winter
(200C/60% RH) regimes over 28 days ........................................... ............... 40

Figure 4-3. Salmonella recovery logoo CFU/ml) from stainless steel surfaces in spring
(300C/80%RH) and fall/winter (200C/60%RH) regimes over 28 days. .................43

Figure 4-4. Salmonella recovery logoo CFU/ml) from PVC surfaces in spring
(300C/80%RH) and fall/winter (200C/60%RH) regimes over 28 days. .................45

Figure 4-5. Salmonella recovery logoo CFU/ml) from sponge roller surfaces in spring
(300C/80%RH) and fall/winter (200C/60%RH) regimes over 28 days. .................47

Figure 4-6. Salmonella recovery logoo CFU/ml) from conveyor belt surfaces in spring
(300C/80%RH) and fall/winter (200C/60%RH) regimes over 28 days. .................49

Figure 4-7. Salmonella recovery logoo CFU/ml) from unfinished oak surfaces in spring
(300C/80%RH) and fall/winter (200C/60%RH) regimes over 28 days. .................50















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

A RECOVERY STUDY OF Salmonella SPP. FROM THE SURFACES OF
TOMATOES AND PACKING LINE MATERIALS

By

Raina Leneve Allen

December 2003

Chair: Keith R. Schneider
Major Department: Food Science and Human Nutrition

Salmonellosis is a common gastrointestinal foodborne illness that is caused by the

bacterium Salmonella. Every year, approximately 40,000 culture confirmed cases of

salmonellosis are reported in the United States. Multi-state salmonellosis outbreaks have

occurred due to the consumption of contaminated raw tomatoes.

This study was designed to evaluate the recovery of Salmonella spp. from tomato,

stainless steel, polyvinyl chloride (PVC), sponge roller, conveyor belt and unfinished oak

surfaces. Fruit and material surfaces were maintained at specific temperatures and

relative humidity (RH), 300C/80%RH, 200C/60%RH and 200C/90%RH for 28 days.

Different temperature and relative humidity combinations had a significant effect

on the survival of Salmonella on tomato and packing line surfaces. An ambient

temperature (20C) combined with 90%RH or 60%RH seemed to better facilitate the

survival of Salmonella as compared to an elevated temperature (30C) combined with

80%RH.









Logio values of recovered Salmonella from tomato surfaces decreased over time in

all three simulated environments. Tomatoes stored at 200C/60%RH and 200C/90%RH

had an approximate 4.0 logo CFU/ml reduction of Salmonella over 28 days. A lower

amount of Salmonella was recovered from tomatoes stored at 300C/80%RH over 28 days

(1.0 logo CFU/ml by Day 14).

Salmonella was recovered from stainless steel and PVC surfaces stored at

200C/60%RH for all sampling intervals. Salmonella was only recovered from stainless

steel and PVC surfaces on Days 0, 1, 3 and 7 while contained at 300C/80%RH. No

Salmonella was recovered from conveyor belt surfaces stored at 300C/80%RH after Day

3 or from sponge roller surfaces stored at 300C/80%RH after Day 0. Salmonella was

recovered from conveyor belt surfaces stored at 200C/60%RH until Day 14 (0.60 logo

CFU/ml). No Salmonella was recovered from sponge roller surfaces held at

200C/60%RH after Day 3. Recovery of Salmonella from unfinished oak surfaces was

variable. Salmonella was recovered from oak surfaces held under 200C/60%RH at

approximately 2.0 logo CFU/ml on Day 28. Salmonella recovery fluctuated over 28

days for oak surfaces stored at 300C/80%RH. On Days 3 and 14 there were increases in

Salmonella recovery (approximately 3.0 logo CFU/ml and 1.0 logo CFU/ml,

respectively). On Days 7, 11, 21 and 28, no Salmonella was recovered from oak

surfaces. It is suspected that oak pieces harbored and protected Salmonella in its matrix.

Results show the importance of a regular sanitation program for surfaces, since

Salmonella could survive for weeks on tomato and packing line surfaces in an

accommodating environment, thus increasing the risk of foodborne illness in fresh-

market tomatoes.














CHAPTER 1
INTRODUCTION

During the past two decades, an increase in consumption of fresh produce has

occurred in the United States (Tauxe et al. 1997). Greater distribution distances for fresh

produce from new geographic sources have allowed a variety of fresh produce to be

readily available to consumers year round. Increased availability of fresh produce

accompanied with increased demand of fresh produce has resulted in an elevation of

produce-associated foodborne illness outbreaks in the U.S. (Tauxe et al. 1997). The

Centers for Disease Control and Prevention (CDC) report that the number of produce-

associated outbreaks has doubled between the periods of 1973 to 1987, and 1988 to 1991,

and that the number of cases associated with these outbreaks has more than doubled

(Tauxe et al. 1997). In January of 1997, President Clinton announced a Food Safety

Initiative in response to a report he received from the U.S. Department of Health and

Human (DHHS) Services, the U.S. Department of Agriculture (USDA) and the U.S.

Environmental Protection Agency (EPA). This report announced domestic produce as an

area of concern for food safety in the U.S. (Rajkowski and Baldwin 2003). Later that

year, a plan entitled Produce & Imported Foods Safety Initiative was announced in hopes

to provide further assurance for higher health and safety standards for fruits and

vegetables consumed by the American public (FDA 1999). In 1999, a survey was

conducted by the Food and Drug Administration (FDA) concerning imported produce,

and 40 out of 1000 samples (4%) tested positive for bacterial pathogens, of which 35 of









these samples (80%) tested positive for Salmonella contamination and 9 (20%) with

.l/ngelu (CFSAN-FDA 2001).

Fresh fruits and vegetables were traditionally considered safe to eat raw, straight

from the field, but now pathogenic microorganisms may contaminate fresh commodities.

Fruits and vegetables can become contaminated with pathogenic microorganisms by the

way of many mechanisms. Contamination can occur in fields or orchards, through

contaminated irrigation water, harvesting, postharvest handling, processing, distribution

and preparation in food service or home settings (Beuchat 1995). All varieties of produce

have the potential to harbor pathogenic microorganisms. If contaminated commodities

enter a packinghouse facility, cross-contamination of processing equipment and other

produce is likely to occur (Brackett 1999).

The survival or growth of pathogens found on or in raw produce are affected by

environmental surroundings as well as pathogens' metabolic capabilities. These

metabolic capabilities are greatly influenced by intrinsic and extrinsic ecological factors

naturally present in the produce or imposed during production, processing, distribution

and preparation at the site of consumption (Beuchat et al. 2001). Two very important

environmental characteristics that can greatly affect fresh commodities are temperature

and relative humidity. The impact of these two extrinsic factors will affect endogenous

microflora and pathogen populations that may be present on fresh commodities (Brackett

1987).

Bean sprouts, watermelon, cantaloupe, honeydew, green grapes and tomatoes are

fresh commodities that have been associated with foodborne salmonellosis (Tauxe et al.

1997). Salmonellosis is a common gastrointestinal foodborne illness that is caused by the









bacterium called Salmonella. The role of Salmonella in foodborne disease was first

documented in the late 1800's, whereas the human clinical disease, typhoid fever, dates

back to the beginning of that century (Cox 2000). Worldwide, Salmonella is the second

most causative agent of foodborne illness (Cox 2000). Every year, approximately 40,000

cases of salmonellosis are reported in the United States (CDC 2001). Foodborne

outbreaks of Salmonella spp. are most commonly linked to animal derived foods;

however plant derived foods have also served as sources of illness (Cox 2000; Nguyen-

The and Carlin 1994; Tauxe et al. 1997; Brackett 1999). Recent surveys of fresh produce

have identified several Salmonella serotypes as the causative agents in human foodborne

illness (CF SAN-FDA 2001).

Large outbreaks of salmonellosis have been caused by consumption of

contaminated raw tomatoes. Three multi-state outbreaks of foodborne illness were

caused by the consumption of raw tomatoes contaminated with Salmonella Javiana in

1992, Salmonella Montevideo in 1993 and Salmonella Baildon in 1999 (CFSAN-FDA

2001). These outbreaks were all traced to Salmonella-contaminated packinghouse

facilities where the tomatoes were minimally processed. In June of 2002, Salmonella

Javiana was the cause of an outbreak at the 2002 United States Transplant Games in

Orlando, Florida. The origin of the outbreak was identified as raw, diced tomatoes (CDC

2002).

This recovery study evaluates the survival and recovery of Salmonella spp. from

the surfaces of tomatoes and typical tomato packing line materials. Materials that were

evaluated included stainless steel, conveyor belt, polyvinyl chloride (PVC), sponge

rollers and unfinished oak wood. Fruit and material surfaces were inoculated with a






4


known amount of a rifampicin resistant five-serovar Salmonella cocktail. Salmonella

recovery off the various surfaces was assessed by a vigorous rub-shake recovery method.

Inoculated fruit and material surfaces were subjected to specific temperature and relative

humidity combinations for 28 days. The temperature and relative humidity combinations

were selected to imitate Florida fall/winter and spring tomato production season

conditions and ripening room parameters for mature green tomatoes during ethylene

treatment and storage.














CHAPTER 2
LITERATURE REVIEW

The United States Centers for Disease Control and Prevention (CDC) claims that

more than 200 diseases are known to be transmitted through food consumption (Bryan

1982). It is estimated that foodborne diseases cause approximately 76 million illnesses,

325,000 hospitalizations and 5,000 deaths annually in the United States (Mead et al.

1999). Tauxe et al. (1997) report that due to a shift in diet toward greater consumption of

fresh fruits and vegetables and farther distribution distances from new geographic

sources, there are more reported illnesses involving fresh produce. The United States

Food and Drug Administration (FDA) has conducted surveys on both imported and

domestic produce, and a report on domestic products revealed a 1.6% contamination rate

on sampled produce (Rajowski and Baldwin 2003). In the United States from 1988 to

1992, 64 outbreaks of foodborne diseases were attributed to the consumption of fresh

fruits and vegetables; nine deaths resulted from the outbreaks (Bean et al. 1997).

Worldwide, many pathogens have been identified as the causative agents of

foodborne disease associated with the consumption of contaminated produce. Non-

typhoidal Salmonella spp., ./ngel//l spp., Listeria monocytogenes, Yersinia spp.,

Aeromonas spp., Campylobacter spp., Staphylococcus aureus and Escherichia coli

0157:H7 are all bacterial pathogens that have caused foodborne infections (Nguyen-The

and Carlin 1994; Beuchat 1995). The presence of pathogens on fresh produce alone can

cause an outbreak; replication of pathogens on or in fresh produce does not have to occur.

However, extensive research has documented that human pathogens are capable of









replication on many types of undamaged or specifically wounded produce (Beuchat

1995). The FDA states that the survival of pathogens on fresh fruits and vegetables at

low infective doses can initiate foodborne disease in the elderly, children and

immunocompromised, but for healthy individuals a higher infective dose would be

necessary (FSIS 2001).

Temperature and relative humidity are extrinsic factors that influence the

persistence and survival capacity of microorganisms on the surfaces of fruits and

vegetables. Storage of healthy fruits and vegetables kept at optimum temperature,

relative humidity, and atmospheric gas composition will yield maximum sensory and

preservation attributes. However, optimum storage settings do not always result in

minimizing the growth of microorganisms found on the produce (Beuchat 1992). Studies

have shown that a variety of lettuce types, leafy greens and fruit can support postharvest

multiplication of pathogenic bacteria under conditions of permissive temperature and

relative humidity increasing the risk of foodborne illness (Abdul-Raouf 1993). Many

types of vegetables and low-acid fruits are capable of supporting rapid multiplication of

pathogens at temperatures ranging between 15 to 250C (Suslow 2002).

Microbial quality of fresh produce is a large safety issue in the produce processing

industry. A blanching or thermal kill step cannot be applied to fresh-market produce to

eliminate bacteria (Hurst and Schuler 1992). Fresh produce facilities rely heavily on

proper temperature control and good plant and employee sanitation to uphold quality and

safety. Fresh fruits and vegetables are very nutritious and overall are categorized as safe

foods (Harris et al. 2002). There is potential for fresh produce to become a risk in the

food chain if postharvest techniques are abused. Produce quality can be judged from









aesthetic factors (color, texture, aroma), but presence of foodborne pathogens are not so

simple to detect. Preventing contamination of fresh-market produce from pathogens is

crucial in assuring wholesome foods for safe human consumption (Harris et al. 2002).

Fresh-market produce can be sold as whole entities or produce can be prepared and

processed to a greater extent. Fresh-cut products have grown rapidly during the past

decade (Cantwell and Suslow 2002). These fruit and vegetable products are prepared and

handled to maintain freshness while offering convenience to consumers. Preparation of

fresh-cut produce involves cleaning, washing, trimming, coring, slicing, shredding and

other similar steps. These steps increase perishability of the produce items. Examples of

fresh-cut produce are mixed salads, broccoli florets, diced onions and sliced and diced

tomatoes. Fresh-cut produce items usually only have a shelf-life of 10-14 days. Higher

respiration rates indicate a very active metabolism and a faster deterioration rate

(Cantwell and Suslow 2002).

Foodborne Illnesses Associated with Fresh Produce

World-wide, the per capital consumption of fresh and lightly processed fruits and

vegetables has increased over the last decade. With an increase in consumption of fresh

produce, a heightened amount of human foodborne disease outbreaks involving fresh

produce have resulted (Beuchat 1995). There are many pathogenic microorganisms that

have been associated with foodborne disease resulting from contaminated produce.

Pathogens of great concern are Salmonella spp., .\/ngel// spp., E. coli 0157:H7 and L.

monocytogenes.

Poultry, eggs and dairy products are most commonly associated with salmonellosis

outbreaks. In recent years, Salmonella has been linked to many produce-associated

outbreaks. Raw bean sprouts were the causative agents in salmonellosis outbreaks that









occurred in the United Kingdom and Sweden in the late 1980's (Beuchat 1995).

Salmonella Saintpaul was identified as the epidemic serovar in many cases of foodborne

infection. Melons contaminated with Salmonella have also been causative agents of

foodborne disease. As early as 1955, S. Miami and S. Bareilly were linked to the

consumption of fresh-cut watermelon (Gayler et al. 1955). S. Javiana and S. Oranienburg

were identified to have been the cause of salmonellosis outbreaks associated with the

consumption of watermelon (CDC 1979; Blostein 1991). Studies have demonstrated that

Salmonella (a five-serovar cocktail ofS. Anatum, S. Chester, S. Havana, S. Poona and S.

Seftenberg) can grow on rind-free pieces of watermelon, cantaloupe and honeydew

(Golden et al. 1993). Over a 24-hour period, Salmonella populations exhibited multiple

log-unit increases on melon varieties maintained at 230C. Tomatoes have also been

documented as vehicles of foodborne disease. The consumption of raw tomatoes

contaminated with Salmonella led to two separate multi-state outbreaks in 1992 and 1993

(Hedburg et al. 1999). S. Javiana implicated the outbreak in 1992 and S. Montevideo

implicated the outbreak in 1993.

All four species of the genus .\/nlgl//t are pathogenic to humans. \/ngel//t spp. has

been responsible for many outbreaks involving contaminated raw vegetables. Lettuce

and leafy greens have been documented vehicles of .\/nge//ll-contaminated produce. S.

sonnei was responsible for an outbreak involving contaminated lettuce in Texas and

shredded lettuce was responsible for another outbreak involving 347 cases of S. sonnei

gastroenteritis (Davis et al. 1988). Two U.S. midwestern outbreaks of S. flexneri

infections where linked to green onions that were harvested from a single farm in Mexico

(Cook et al. 1995). Melons and tropical fruits have also been reported to harbor ./Nhge/\l









Escatrin et al. (1989) reports that .\/ngell/ spp. can grow and survive on the surfaces of

fresh-cut pieces of watermelon, papaya and jicama in slightly acidic condition (less than

pH 6.0).

E. coli 0157:H7 is considered an emerging foodborne pathogen (Beuchat 1995).

Cattle are primary reservoirs of this pathogen and an extensive amount of foodborne

disease outbreaks have been linked with undercooked beef and dairy products. Fresh

produce can also harbor this microorganism. E. coli 0157:H7 has repeatedly been

connected with unpasteurized apple cider, salad bars and melons. It has been

documented that E. coli 0157:H7 rapidly multiplies in watermelon and cantaloupe cubes

at 80C (Del Rosario and Beuchat 1995). In 1994, culture confirmed E. coli 0157:H7

infections were traced back to raw broccoli served on a salad bar. It was concluded that

the broccoli was cross-contaminated with raw ground beef during the preparation of the

vegetable (Beuchat 1995).

L. monocytogenes can grow on fresh produce stored at refrigeration temperatures

(4C). Controlled atmosphere storage does not seem to affect or influence the growth of

the microorganism (Beuchat 1995). L. monocytogenes is prevalent on plant vegetation

(Beuchat et al. 1990). In 1981, a large listeriosis outbreak was attributed to the

consumption of contaminated coleslaw. The outbreak was traced back to a cabbage

farmer who used a combination of composted and fresh sheep manure to fertilize cabbage

fields (Schlech et al. 1983). L. monocytogenes has been detected in bean sprouts, leafy

vegetables and cut cucumbers (Arumugaswamy et al. 1994). It has also been reported

that this pathogen can survive on the surface of tomatoes held at 21C (Beuchat and

Brackett 1991).









The epidemiology of foodborne diseases is constantly changing. Reoccurrence of

well-recognized pathogens are observed in outbreaks and newly recognized foodborne

pathogens also emerge. Fresh produce has been extensively documented as potential

vehicles for foodborne disease. Worldwide, fresh fruits and vegetables are an essential

part of diets and minimizing the occurrence of foodborne disease associated with

contaminated produce is essential.

Salmonella Species

Documentation of the human clinical disease caused by Salmonella, typhoid

fever, dates back to the early 1800's (Cox 2000). Historically, Salmonella has been

documented as causing foodborne disease since the late 1800's. The bacteria were

discovered by an American veterinary pathologist, Dr. Daniel E. Salmon, who isolated

the microorganism from hog cholera infected swine. In the 1900's, the genus Salmonella

was created in Dr. Salmon's honor after similar organisms were isolated from outbreaks

of foodborne disease (Cox 2000). The bacterium is widely associated with food animals

and their production environment.

The genus Salmonella exists within the family of Enterobacteriaceae. According to

the Encyclopedia of Food Microbiology (Cox 2000), the genus consists of one species;

Salmonella enterica. Salmonella are Gram-negative facultative, oxidase-negative,

catalase-positive, anaerobic rod-shaped bacilli (Bergey's Manual of Determinative

Bacteriology 1994). Most strains are motile and ferment glucose. Biochemical tests can

further characterize the genus into specific serogroups and serovars. These tests

characterize two antigens, the O or somatic antigen, and the H antigen or flagellin

antigen. The O antigen designates differences in epitopes of lipopolysaccharide (LPS),

which is the major component of the outer membrane of Gram-negative bacteria. The H









antigen differentiates strains into serovars that are based on the variation in flagellins or

subunit proteins in the flagella (Bergey's Manual of Determinative Bacteriology 1994).

Salmonellosis is the illness that Salmonellae induce in humans, usually by

ingestion the bacteria through contaminated food products. According to the Centers for

Disease Control and Prevention (CDC), salmonellosis has been a reportable disease in the

United States since 1943. Physicians must report cases of infection to local health

departments that report to state health departments that ultimately report annual totals to

the CDC (Tauxe et al. 1997). Salmonellosis is one of the most frequently reported causes

of foodborne gastroenteritis and is estimated to cause 1.4 million cases each year in the

United States, of which 40,000 cases are culture confirmed (CDC 2000). The CDC has

estimated that 95% of Salmonella infections originate from foodborne sources (Frenzen

et al. 1999). Two serotypes of Salmonella cause over half of the reported salmonellosis

cases: Salmonella Enteritidis and Salmonella Typhimurium (CDC 2000). Infection is

initiated by the ingestion of a dose of Salmonella effective enough to surpass primary

host defenses. The bacteria proceed to colonize the gastrointestinal tract. Infectious

doses are determined by physiological characteristics of the ingested strain and the

physiological state of the host. Typical infectious doses usually range between 106-

108CFU and epidemiological evidence has demonstrated that an infectious dose may be

as little as a few (10) cells (Cox 2000).

A range of environmental conditions affect the survival, growth or death of

Salmonella. The optimum growth temperature for this microorganism is 37C, but it has

been observed to grow between 2-540C (Cox 2000). Generally, Salmonellae are heat

labile, but exposure of Salmonella to adverse conditions generally increases the resistance









of the microorganism to heat. Optimum pH levels range from 6.5-7.5 and as temperature

increases the sensitivity to low pH increases. Salmonella can grow at water activity (aw)

values between 0.999 and 0.945 in laboratory media and at a low aw value of 0.93 in

foods (Cox 2000). Growth of the microorganism has not been documented at aw lower

than 0.93, but survival time of the microorganism has been noted to increase as aw

decreases (Cox 2000). In low-moisture foods, survival of Salmonella can be measured in

months.

Salmonellosis Outbreaks Involving Fresh Tomatoes

Tomatoes have been the sources of several foodborne illness outbreaks. The

microorganism responsible for these outbreaks is usually identified as Salmonella. A

large multi-state outbreak occurred in 1990 that resulted in 176 cases of S. Javiana

infections. A restaurant and child care center reported illnesses associated with

consuming raw tomatoes. The outbreak was traced to a repacking facility. The tomatoes

were distributed to various restaurants and grocery stores. No other potential sources

were associated with this outbreak (Hedburg et al. 1999).

Another large outbreak occurred in 1993. There were 100 reported cases of

foodborne illness that resulted from this multi-state outbreak. The causative agent of the

outbreak was S. Montevideo. The outbreak was traced back to the same repacking

facility from which the 1990 outbreak was traced. Mature-green tomatoes were picked

by hand, and transported in field bins which contained approximately 1,500 pounds of

tomatoes each. The lots of tomatoes were dumped into a common water bath

("dumptank") where they were contamination most likely occurred (Hedburg et al. 1999).

In 1999, another multi-state outbreak of salmonellosis was attributed to the

consumption of raw tomatoes. The origin of contamination was traced to two tomato









grower/packer cooperatives. The lots of tomatoes were handpicked and transported to

the facility in covered plastic bins (Cummings et al. 2001).

Most recently in June of 2002, in Orlando, Florida there were two reported cases of

S. Javiana infections. The illnesses were contracted from contaminated pre-packaged

diced Roma tomatoes. Efforts are underway to identify the routes of contamination

(CDC 2002).

Tomatoes and Salmonellae

The increased frequency of salmonellosis outbreaks involving fresh tomatoes has

prompted researchers to investigate Salmonella in and on tomatoes. Many researchers

and food scientists have conducted experiments focusing on recovery of the pathogen

from tomato surfaces and tomato matrices, survival of the pathogen on and in tomatoes,

and the effectiveness of sanitizers on the pathogen.

In a study conducted by Guo et al. (2001), the survival of salmonellae brushed onto

tomato plants was investigated. Flowers and stems on tomato plants were inoculated

with a five serovar Salmonella cocktail before and after fruit set. Twenty-one to 49 days

elapsed between the date of inoculation and sampling. Forty-three sound, red, ripe

tomatoes were harvested from inoculated plants and plants that were not inoculated. All

plants were evaluated for the presence of Salmonella. Plants that were not inoculated

produced tomatoes that were not contaminated with Salmonella. However, 11 of 30

tomatoes (37%) harvested from inoculated plants were positive for Salmonella

(confirmed by polymerase chain reaction (PCR) assay). Stem-inoculated plants were

positive for Salmonella before and after flower set at 43% and 40%, respectively.

Twenty-five percent of Salmonella-positive tomatoes were harvested from plants that

were inoculated on the flower. The surface of the tomatoes and the stem scars tissues of









the tomatoes harbored higher percentages of the pathogen compared to the pulp of the

tomatoes. PCR fingerprinting patterns revealed that S. Montevideo was the most

persistent and dominant serotype detected on positive tomatoes. The serovar was isolated

49 days after inoculation of the tomato plants.

In a study by Luasik et al. (2001), the elution, detection and quantification of

seeded viruses and bacteria (Salmonella Montevideo) were investigated from the surfaces

of strawberries and tomatoes. Mature, red Roma tomatoes were inoculated with

Salmonella Montevideo on artificial surface scars, stem and blossom scars, and intact

tomato surfaces. Results indicated a higher recovery of the pathogen from the stem,

surface and blossom scars than pathogen recovery from smooth intact surfaces of

tomatoes. It was also observed that when the tomatoes were immersed in Salmonella

Montevideo-contaminated water, more attachment of the pathogen occurred in the stem

scar area, followed by the blossom scar area, surface scars, and the intact tomato surface.

It was hypothesized that the surface area and hydrophilicity of the rough areas evaluated

(surface, stem and blossom scars) may affect microbial attachment. Tomatoes do not

have lenticels, or pores, on their surface like many fruits, thus restricting gas exchange

between the internal tissues of the fruit and the atmosphere. Lenticels also allow the

infiltration of liquids. Pores do exist in the corky tissue of the stem scar area of tomatoes.

Bacteria are more likely to attach and infiltrate into the interior of this rough portion of

the fruit than the smooth epidermal surface.

In a study conducted by Zhuang et al. (1995), survival patterns of S. Montevideo on

and in raw tomatoes were evaluated as affected by temperature and chlorine treatment.

Mature green tomatoes were dip inoculated with S. Montevideo and inoculated tomatoes









were stored up to 18 days at different temperatures in combination with 45-60% relative

humidity (RH). The stem scar tissues and core tissues of the tomatoes were analyzed for

Salmonella populations and dipped in various chlorine concentrations. The survival and

growth pattern of S. Montevideo was also examined in chopped, ripe tomatoes stored at

various temperatures. Results of this study suggested that the persistence and viability of

S. Montevideo on the surfaces and cores of tomatoes stored at 100C parallel the potential

for Salmonella survival on and in tomato fruits during transport and storage. The

populations of Salmonella inoculated on the surfaces of tomatoes held at 100C did not

significantly change over the 18-day period. S. Montevideo was also observed to grow

well in chopped ripe tomatoes stored at 20 or 300C. Chlorine concentration studies

revealed that S. Montevideo was not totally eliminated from tomatoes when subjected to

a disinfection treatment at 320ppm. This study clearly indicates that Salmonella

serotypes contaminating fresh tomatoes pose a risk for potential foodborne salmonellosis

outbreaks.

A study conducted by Guo et al. (2002) demonstrated that water and soil serve as

reservoirs of Salmonella that can potentially contaminate mature green tomatoes.

Salmonella was observed to survive at high numbers in moist soil for at least 45 days. It

was also observed that cells of Salmonella were able to infiltrate fruits via stem scars and

enter the tomato pulp upon contact with moist, contaminated soils. Survival patterns of

Salmonella on tomato surfaces were also investigated. Spot-inoculated tomatoes

evaluated over a 14-day storage period (200C) showed a decrease in Salmonella

populations over time. Populations decreased by approximately 4 logs over the entire

storage period. Results obtained from this study differ from survival patterns of









Salmonella reported by Zhuang et al. (1995). Zhuang et al. (1995) reported an increased

amount of Salmonella on whole, intact tomatoes over time. Differences could be

attributed to the different inoculation procedures used. A dip inoculation, as used by

Zhuang et al. (1995), could result in cells becoming lodged in tissue areas that could

enhance the survival and growth of cells during a prolonged storage period.

Tomato Industry

Two tomato industries exist in the United States. The fresh-market and

processing tomato industries are separate markets and each possesses distinguishing

characteristics. Tomato varieties are specifically bred to meet requirements of either the

fresh or processing markets. All fresh-market tomatoes are picked by hand whereas,

tomatoes bound for processing can be mechanically harvested (ERS 2000). Fresh-market

tomatoes are widely produced and sold on the open market with higher and more variable

prices than processing tomatoes (ERS 2000).

According to the Economic Research Service (ERS), California and Florida

comprise two-thirds of the acres used to grow fresh tomatoes in the United States. In the

United States, this industry estimates that fresh-market tomato retail value exceeds $4

billion (ERS 2000). Florida leads the domestic market in the production of fresh-market

tomatoes. Florida produced 42% of the fresh-market tomatoes in the United States

during 1997-1999 and brought in $5.4 million of the state's total farm value of vegetables

(ERS 2000). Florida's tomato season extends from October to June. Most tomato

production occurs during the months of April to May and again from November to

January. Fresh-market tomatoes are available year-round in the United Stated because of

imports and Florida's winter crops. Imported commodities are usually shipped to









markets in the western states and Florida's winter crops are shipped to the eastern half of

the nation (ERS 2000).

The ERS (2000) reported that Americans consumed 4.8 billion pounds, or 17.8

pounds per person, of fresh-market tomatoes in 1999. Tomatoes rank third in consumer

preference vegetables at the retail level and are only surpassed by potatoes and lettuce

(Florida Tomato Committee 2002). Consumption of fresh-market tomatoes in the United

States has most likely increased due to the increasing popularity of salads, salad bars and

sandwiches dressed with tomatoes (Lucier et al. 2000). Tomatoes are very nutritious

fruits and contain approximately half of the recommended daily allowance of vitamin C

and 20% of the recommended daily allowance of vitamin A. Tomatoes also contain the

compound lycopene which has been shown to reduce prostate cancer in men who

consume at least 10 servings of tomatoes or tomato-based foods per week (Florida

Tomato Committee 2002).

Postharvest Handling of Tomatoes

Tomatoes bound for the fresh-market are harvested by hand at a mature-green

stage. Internally, a mature-green tomato will have a jellylike matrix in all locules, but

maturity is difficult to determine from external examination. At a mature stage, tomato

seeds will be sufficiently developed when a knife slices the fruit and the seeds are not

penetrated by the cut (UF/IFAS 1998). When tomatoes are harvested, pickers place the

fruits into plastic buckets or wooden field bins that usually hold up to 40-50 pounds of

tomatoes. The buckets are carried to field trucks and emptied into pallet bins or

gondolas. Next, tomatoes are transported to the packinghouse and dumped into a

chlorinated dump tank. Dump tanks contain heated, chlorinated water to wash the fruits.

Wash water should be maintained at a pH of 7 (neutral) and contain a recommended level









of chlorine range of 100 to 150 parts per million (ppm) of chlorine (Sargent et al. 2001).

Water temperature of dump tanks should be elevated 10 degrees above the pulp

temperature of the tomato. Bartz and Showalter (1981) demonstrated that warm tomatoes

(260C to 400C) immersed in cold water (approximately 18 degrees colder than incoming

fruit) for 10 minutes or longer infiltrated water and any bacteria present in the water.

Infiltration through the stem scar is associated with a negative temperature differential

between the water and the tomato therefore; warm water is used in dump tanks to reduce

the extent of infiltration of water into the tomato. Failure to maintain adequate chlorine

levels in dump tanks can lead to increased microbial populations. It has been reported

that Enterobacteriaceae populations increased on tomatoes washed in water containing

114 ppm chlorine and populations decreased once tomatoes were subjected to water

containing 226 ppm (Beuchat 1992).

Tomatoes exit the dump tank and travel over a series of perforated conveyor belts.

Conveyor belts play an integral part in the functions of a packinghouse. Belts are

fabricated from rubber compounds and they transport tomatoes at several points during

handling. Conveyor belts are used to pre-size, cull, sort and size tomato fruits.

Undersized tomatoes will fall through holes in the belts and travel to a cull chute.

Sponge rollers also serve a very important role in tomato packinghouse operations.

Tomato fruits are susceptible to injury and bruising. Sponge rollers buff and cushion the

fruits as they proceed along the processing lines. Tomatoes will also contact sponge

rollers after washing and absorb water off the fruit surface; as a result the sponges are

constantly moist. Many Florida tomatoes are waxed with a food grade wax that increases









the shine of the tomato and reduces water loss during marketing. Contamination of

tomatoes has been known to occur during waxing procedures (Beuchat 1992).

Sorting and grading of tomatoes is a laborious process. Color sorting occurs first,

which separates tomatoes possessing any red color from fruits that are completely green

in color. The fruits are then separated into grades that meet specific requirements for the

U.S. No. 1, U.S. Combination, U.S. No. 2 or U.S. No. 3 of the U.S. Standards for Grades

of Fresh Tomatoes (Florida Tomato Committee 2002). Following sorting and grading,

tomatoes are mechanically sized by passing over continuous conveyor belts containing

increasingly larger round holes that sort tomatoes by maximum allowable diameter for

each designated size. In February of 1998, the Florida Tomato Committee (2002)

ordered the following sizing classifications: 6x7 (formerly medium), 6x6 (formerly large)

and 5x6 (formerly extra large). The sizing dimensions (diameter of fruit is measured in

inches) are categorized by a minimum and maximum range for each size class.

Graded and sized tomatoes are transported via conveyor belts to automatic fillers

where the fruits are jumble-packed into corrugated fiberboard containers to a designated

weight (UF/IFAS 1998). Boxes of tomatoes are then palletized and moved by units.

Wooden pallets are used to transport unitized loads of tomato boxes and are used in many

packinghouse facilities. Pallets are usually constructed from unfinished oak wood.

Usually, mature-green tomatoes are immediately subjected to a ripening treatment.

Ethylene is a natural ripening hormone that is released in ripening rooms. Ripening

rooms are capable of holding many pallets of tomatoes at one time, and are maintained at

very specific parameters. Precise optimum conditions of a typical ripening room are kept

at 200C, 85-95%RH with a concentration of up to 150 ppm ethylene (UF/IFAS 1998).









Tomatoes are susceptible to extensive water loss through the stem scar so a high relative

humidity is necessary (UF/IFAS 1998). Constant air exchange is provided in ripening

rooms to supply tomatoes with a continuous ripening-effective blend of ethylene and air

to avoid the accumulation of carbon dioxide. Mature-green tomatoes are usually

subjected to ethylene for 3 days. Once tomatoes are at a minimal color stage of

"breaker", the first sign of external yellow or pink color at the blossom end of the fruit,

ethylene will not further accelerate the ripening process since the fruits are producing

their own ethylene (Cantwell and Kasmire 2002). A constant supply of air also prevents

carbon dioxide buildup when tomatoes respire. Carbon dioxide inhibits the ripening

process and is an unwanted byproduct (Reid 2002).

Ripe tomatoes are susceptible to chilling injury at temperatures below 10C

(Cantwell and Kasmire 2002). However, ripening tomatoes develop chilling injury

below 130C (Maul et al. 2000). Low temperatures inhibit development of full color and

flavor in green mature fruit and the fruits are more susceptible to Alternaria decay

(UF/IFAS 1998). Tomatoes are tropical commodities and must be maintained at warm

temperatures. If tomatoes are held above 30C (85-86F) the fruits will develop more

orange pigments than the desirable red pigments (UF/IFAS 1998).

In 1994 and 1995, Rushing et al. (1996) tested tomatoes bound for the fresh-market

for the presence of Salmonella spp. and verified that a proposed Hazard Analysis Critical

Control Point (HACCP) program was effective in controlling the risk of contamination in

the packinghouse. This study revealed that contamination seemed more likely to occur at

the packinghouse where minimally processed fruits were dumped into a water bath,

transported across conveyor belts and hand sorted prior to being packed into cartons.









Packinghouse operations are designed to preserve and package fresh produce in a

timely manner. Packinghouse facilities are currently included under the Good

Agricultural Practices (GAP) guidelines and are exempt from Good Manufacturing

Practices (GMP) regulations. GAP guidelines are generic and do not contain specific

testing and monitoring guidelines (CFSAN-FDA 2001). The potential risk of

contamination can be controlled by employee training and traceback plans. The Guide

To Minimize Microbial Food Safety Hazards For Fresh Fruits and Vegetables (FDA

1998) has become a valuable tool for focusing on crucial areas of presumptive risk

potential for fresh produce handling (CFSAN-FDA 2001).

Extrinsic Factors Influencing Microbial Viability

Growth and survival of microorganisms on fresh produce are influenced by the

characteristics of the surrounding environment (Tauxe et al. 1997). Foodborne diseases

that occur from contaminated produce often involve fruits and vegetables that have been

subjected to nonthermal, minimal processing prior to time/temperature combinations

permitting pathogens to survive and grow (Tauxe et al. 1997). The exteriors of produce

act as physical barriers to protect from internalization of bacteria present on a

commodity's surface. Temperature and relative humidity are two environmental factors

that can affect microbial populations on produce.

For minimally processed fruits and vegetables, two factors should be considered

when evaluating the effect of temperature and growth rates of bacteria. First, storage

temperature determines respiration rates of a commodity and the behavior of

microorganisms may be influenced by changes in the gaseous atmosphere. Secondly,

temperature can also influence the rate of senescence of a commodity therefore

modifying the environment for microorganisms (Nguyen-The and Carlin 1994).









Improper refrigeration during storage and preparation and poor product quality can

enhance the survival of pathogens. Growth of pathogens on fresh, minimally processed

fruits and vegetables has been reported in many studies. It was observed by Maxcy

(1978), that E. coli, S. Typhimurium and Staphyloccocus aureus grew on shredded lettuce

at room temperature (22-240C). Yu et al. (2001) reported the growth ofE. coli 0157:H7

on both externally and internally inoculated strawberries. A study by Zhuang et al.

(1995) revealed that S. Montevideo populations significantly increased on tomato tissues

after storage at 20 and 300C. Refrigeration temperatures limit the growth of most

foodborne pathogens, but some pathogenic microorganisms will survive at lower

temperatures. S. Typhimurium declined rapidly in apple juices stored at 40C, but

managed to survive in the juices for a significant amount of time (Goverd et al. 1979). In

the study by Yu et al. (2001), it was observed that E. coli 0157:H7 populations were also

recovered from externally and internally inoculated strawberries. However, there was a

significant reduction in the populations recovered from the outside of the strawberry

fruits than from the inside of the fruits at 5C.

Another key environmental factor in determining the survival of bacteria is

relative humidity. Traditionally, human pathogens are considered poor survivors in the

natural plant surface environment (Suslow 2002). Beattie and Lindow (1994) state that

the death of cells subjected to low relative humidity conditions is rather fast and viability

of cells can decrease very close to first-order kinetics. In a study conducted by Guo et al.

(2001), tomatoes inoculated with Salmonella were stored at 200C for one day with at a

relative humidity of 70%. It was reported that a reduction of approximately one logo

CFU per tomato occurred and the population slowly decreased by an additional 3 logs









between days 1 and 14. In the same study, tomatoes were stored in contact with moist

Salmonella inoculated soil for 14 days. An increase of approximately 2.5 logo CFU per

tomato occurred during the first 4 days of storage. Similar counts remained constant for

days 4 though 10 and the incidence of decay on tomatoes stored 10 days or more could

not be analyzed for populations of Salmonella.

There have been studies focusing on the incidence of Salmonella associated with

bacterial soft rots and/or physical injury. Bacterial soft rot is the leading cause of

postharvest losses of potatoes, tomatoes along with other types of fresh produce.

Bacterial soft rot caused by group of plant pathogens which are harmless to humans, that

includes Erwinia carotovora (subspecies carotovora and atroseptica), pectolytic

Pseudomonasfluorescens and Pseudomonas viridiflava (Lund 1983). Infected tissues are

broken down resulting in the softening and liquefaction of the internal fruit tissues and

spreads bacteria over other commodities and food-handling equipment (Wei et al. 1995).

E. carotovora is the most common of the soft rotting bacterial complex and is a member

of Enterobacteriaceae of which Salmonella is also a member (Wells and Butterfield

1997). A study by Wells and Butterfield (1997) involving over 500 samples of healthy

and soft rotted commodities collected from retail markets showed the incidence of

suspected Salmonella was twice that on soft rotted samples than of healthy samples.

Another study conducted by Wells and Butterfield (1999) showed that unlike bacterial

soft rotted commodities, fungal rotted commodities (Alternaria tenuis, Bortrytis cinerea,

Geotrichum candidum or Rhizopus stolonifer) showed no greater risk of elevated

Salmonella populations.









Attachment of Microorganisms to Various Surfaces

Bacteria can be introduced to fresh commodities in the field through irrigation

water, sewage, or contaminated soil and introduced into packinghouse environments.

Attachment of bacteria to food processing surfaces is possible and can easily lead to

product contamination (Zottola 1994).

Some typical food contact surfaces found in processing facilities include stainless

steel, rubber (conveyor belts), wood and plastic. Microorganisms on contaminated

produce can easily attach to a variety of these surfaces in short contact times. It was

observed by Mafu et al. (1990) that L. monocytogenes attached to stainless steel, glass,

polypropylene and rubber surfaces after a brief contact time. Contact times ranged from

20 minutes to 1 hour. Attachment of the pathogen was reported at both 200C and 40C for

all surfaces. Sanitizers were applied to each of the surfaces after attachment of L.

monocytogenes and it was observed that porous surfaces (rubber surfaces in this study)

seemed to protect the bacteria whereas sanitizers were more effective on nonporous

surfaces. Wood is another porous material that is used in the form of field bins, pallets or

containers to hold fresh produce. A study conducted by Boucher et al. (1998) observed

the enhanced survival of Campylobacterjejuni cells when incubated at 300C in nutrient

broth. The physical structure of wooden cubes acted as a protective environment for the

bacteria. Plastic cubes were evaluated in the same manner as the wooden cubes, but

enhanced survival of Campylobacterjejuni cells was not observed.

Microorganisms can irreversibly attach themselves to surfaces and form biofilms.

Biofilm-associated cells produce an extracellular polymeric substance (EPS) and have a

defined architecture. Microbial biofilms have been known form on food processing

surfaces. Pathogenic microorganisms such as Campylobacter, Salmonella and E. coli









have been known to form strong biofilms on various surfaces (Somers et al. 1994). A

study conducted by Joseph et al. (2001) was also in agreement reporting that Salmonella

strains will form biofilms on plastic, stainless steel and cement. Biofilms are much more

resistant to sanitizers as compared to planktonic cells and serve as a source of

contamination for foods (Somers et al. 1994). Hood and Zottola (1997) inoculated

stainless steel surfaces with S. Typhimurium, L. monocytogenes, and E. coli 0157:H7

and reported that all pathogens adhered to the surface when grown in media, but

adherence levels often did not increase after 1 hour. In a study conducted by Ronner and

Wong (1993), it was found that the behaviors of biofilm cells were greatly influenced by

surface type. Buna-n rubber nitrilee rubber), a gasket material commonly used in the

food industry, had a bacteriostatic effect on S. Typhimurium and L. monocytogenes. The

bacteriostatic effect of the rubber was most pronounced under lower nutrient conditions.

S. Typhimurium was less affected by the bacteriostatic component than L.

monocytogenes.

It is easier to prevent the formation of biofilms and microbial contamination than to

eliminate a biofilm from a surface after establishment. Attachment of bacteria to food

processing equipment and contact surfaces can easily lead to contamination of product.

Sanitation procedures and environmental awareness in food processing facilities can

reduce the incidence of foodborne illness.

Equipment parts and food contact surfaces such as stainless steel, PVC, conveyor

belts, sponge rollers and wood surfaces are widely used in tomato processing facilities.

Limited studies on these types of surfaces have been reported. Microorganisms attached

to surfaces are a hazardous source of potential contamination for any material coming in









contact with the surfaces. Factors such as temperature, relative humidity, nutrient level

of the growth medium, type of attachment surface and species or strain of bacteria can

influence the amount of adherence to surfaces.

Microbiological Recovery Methods Involving Fresh Produce

The increase in foodborne illnesses associated with fresh produce in the past

decade has resulted in the increase of testing commodities for the presence and

enumeration of pathogens (Burnett and Beuchat 2001). Conventional methods of

detection, enumeration, identification and characterization of microorganisms are

described in such reference books as Compendium of Methods for the Microbiological

Examination ofFoods (CMMEF), FDA Bacteriological Analytical Manual (BAM),

Official Methods of Analysis of the AOAC, and Standard Methods for the Examination of

Dairy Products (Fung 2001). Methods for analyzing foods of animal origin and

thermally processed food of plant origin for both spoilage and pathogenic

microorganisms have been clearly defined in such reference manuals. Methods for

selecting and preparing samples of raw fruits and vegetables for analysis of

microorganisms are less defined (Burnett and Beuchat 2001). Procedures for preparing

and isolating Salmonella for 18 food groups are outlined in the FDA BAM (Andrews et

al. 1998). Currently, a specific protocol for preparing samples of raw produce is not

defined. Microbiologists and food scientists are currently utilizing a wide variety of

procedures to prepare whole and fresh-cut fruit and vegetables to enumerate pathogens.

Sample weight/diluent volume ratios, diluent composition, type of processing and time

used to process samples all greatly vary. Some types of processing include blending,

stomaching, homogenizing, macerating, rubbing and shaking (Burnett and Beuchat

2001).









It is essential for standard methods to be defined in order to accurately determine

the presence and populations of pathogenic microorganisms on fresh fruits and

vegetables. Development and validation of standard methods can be applied to determine

survival and growth characteristics in challenge studies and the efficacy of antimicrobial

treatments in eliminating pathogens on fresh produce (Beuchat et al. 2001). One single

protocol would be ideal, but is not feasible for all produce types. An optimum protocol

for produce depends upon the site of retrieval of pathogens. Analysis from a surface,

tissue or both will vary in methodology, but a basic analytical method for each procedure

would form standard guidelines to optimize the recovery of pathogens (Beuchat et al.

2001). An acceptable method for evaluating whole fruits and vegetables is a process in

which the whole intact produce is vigorously hand massaged or hand rubbed for a period

of time which can ranges from 40 seconds to two minutes (Beuchat et al. 2001; Burnett

and Beuchat 2001; Harris et al. 2001; Zhuang et al. 1995).

Inoculation procedures for fruits and vegetables usually occur by either spot

inoculation and dipping or spraying. The major problem with inoculation via dipping or

spraying is that the number of cells applied to the produce is unknown. Spot inoculation

allows a known volume of inoculum and a known cell density that is applied to the

produce. Spot inoculation is superior to dip or spray inoculation and this type of

inoculation imitates contamination of the produce from a source such as contact with soil,

workers' hands, or equipment surfaces (Beuchat et al. 2001). In studies determining the

efficiency for retrieval of cells, the applied inoculum should be dried at a standard

temperature and relative humidity for a specific amount of time before recovery of cells

or treatment is administered (Beuchat et al. 2001).









Due to the increased frequency of documented outbreaks of foodborne disease

attributed to fresh produce, many researchers are focused upon pathogenic

microorganisms on raw fruits and vegetables. Standard methods that accurately

determine the presence and numbers for a wide variety of pathogenic microorganisms

associated with fresh produce are needed so studies conducted on this subject can be

compared without controversy.

The following objectives were explored in this recovery study.

Establish growth characteristics for five rifampicin-resistant Salmonella serovars.

Recover inoculated Salmonella from surfaces of tomatoes and packinghouse
materials.

Determine if a specific temperature and relative humidity combination affect the
survival of Salmonella spp. on the surfaces of tomatoes and packing line materials.














CHAPTER 3
MATERIALS AND METHODS

Three separate temperature and relative humidity environments were simulated

using an environmental humidity chamber. Tomato surfaces and packing line material

surfaces were inoculated with a Salmonella cocktail comprised of five rifampicin-

resistant serovars. Salmonella-inoculated fruit and material surfaces were subjected to

specific environmental conditions inside the chamber for 28 days. Simulated

environments mimicked standard tomato ripening room parameters and Florida

fall/winter and spring tomato production seasons. Recovery of Salmonella from tomato

surfaces and packing line material surfaces for each simulated environment was

monitored on Days 0, 1, 3, 7, 11, 14, 21 and 28. Tomato ripening room parameters were

simulated to evaluate only Salmonella-inoculated tomato fruits for 28 days. Both

Salmonella-inoculated tomatoes and Salmonella-inoculated packing line materials were

evaluated in environments paralleling typical Florida fall/winter and spring tomato

production environments.

Selection of Temperature and Relative Humidity Combinations

The selected temperature and relative humidity settings for Florida fall/winter and

spring tomato production seasons were based upon weather archives obtained from the

Florida Automated Weather Network (FAWN) (University of Florida Institute of Food

and Agricultural Sciences 2003) (Table 3-1). The average documented temperature and

relative humidity were accumulated for the 2001 and 2002 fall/winter and spring tomato









production seasons in Quincy, FL. The chosen parameters for each production season

were used to simulate an open-air packinghouse environment.

Table 3-1. Temperature and relative humidity combinations selected to simulate a
ripening room environment (20C/90%RH) and a fall/winter (20C/60%RH)
and spring (30C/80%RH) tomato production conditions.
Simulated Environment Temperature (oC) Relative Humidity (%)

Standard tomato ripening 90 20
room
Florida spring tomato 80 30
production season
Florida fall/winter tomato 6
60 20
production season


Acquisition and Maintenance of Salmonella Cultures

Salmonella serovars were obtained through Dr. Linda J. Harris at the University

of California, Davis, Department of Food Science and Technology. The five Salmonella

enteritidis serovars used in this study were Agona, Gaminara, Michigan, Montevideo,

and Poona (Table 3-2). The serovars obtained were adapted to the antibiotic rifampicin

at the University of California, Davis. The serovars were adapted to rifampicin (rif+) by

methods described in a study conducted by Lindeman and Suslow (1987). The five

Salmonella serovars (rif+) were transferred to PROTECT T Bacterial Preservers

(Scientific Device Laboratories, Des Plaines, IL) upon arrival to the laboratory (summer

of 2002) and stored at -700C.

Rifampin is synonymous with rifampicin. This antibiotic inhibits protein

synthesis of mammalian cells and it is freely soluble in methanol (Merck Index 2001). A

10,000 ppm (1%) stock solution of rifampicin was utilized throughout this study. The

stock solution was prepared by dissolving 0.1 g of rifampin (Fisher #BP267925, Fisher

Scientific, Pittsburg, PA) dissolved in 10 ml of high performance liquid chromatography









(HPLC) grade methanol (Fisher, Fair Lawn, NJ). The stock solution was filter sterilized.

Rifampicin is light-sensitive therefore, the stock solution was protected from light and

was stored at room temperature. The media used to recover Salmonella off inoculated

surfaces, Tryptic Soy Agar (TSA) (DifcoTM, Sparks, MD), was supplemented with

80[g/ml rifampin (rif+) antibiotic. The antibiotic-resistant serovars allowed

differentiation from natural micoflora or non-rifampicin resistant bacteria that may have

been present on the matrices evaluated; enabling the sole isolation of Salmonella serovars

(rif+) (Beuchat et al. 2001; Lukasik et al. 2001).

Table 3-2. Salmonella enteritidis serovars obtained from Dr. Linda J. Harris at the
University of California, Davis: wild types* and rifampicin-resistant serovars
listed with source.
Serovar Serovar Name Origin
Designation
LJH517* Agona Alfalfa sprouts
LJH618
LJH518* Gaminara Orange juice
LJH616
LJH521* Michigan Cantaloupe
LJH615
LJH519* Montevideo Human isolate from tomato
LJH614 outbreak
LJH630* Poona Human isolate from tomato
LJH631 outbreak
Growth Levels of Salmonella Serovars after a 20-Hour Incubation

Growth studies were conducted to determine the rate of growth for each of the

five serovars after a 20-hour incubation period. Growth rates were determined so the

Salmonella cocktail would consist of equivalent quantities (CFU/ml) of each serovar, as

one or more serovars would not dominate the inoculum suspension. The five Salmonella

serovars (rif+) were revived off PROTECT TM Bacterial Preservers by aseptically

transferring one bacterial preserver into 10 ml of Tryptic Soy Broth (TSB) (DifcoTM,

Sparks, MD) supplemented with 80 al of rifampin. The cultures were then incubated in a









shaking incubator (Queue Systems, Asheville, NC) at 30 rotations per minute at 370C for

24 hours. The cultures were successively transferred for three days in TSB (rif+) to

obtain uniform cell type (Beuchat et al. 2001). Each of the five serovars were transferred

into 10 ml of fresh TSB (rif+) and incubated at 370C for 20 hours. Following the

incubation period, three replicates of each serovar was serially (1:10) diluted in 9 ml

tubes of sterile Phosphate Buffered Saline (PBS) (ICN Biomedicals Inc., Aurora, OH).

Appropriate dilutions were plated out by pour plate technique using TSA (rif+). Plates

were statically incubated at 370C for 48 hours. Colony forming units (CFU) were

counted and recorded. Serovars were taken off PROTECTTM Bacterial Preservers at the

beginning of each 28-day experiment. Two 20-hour growth studies were conducted to

ensure growth rates for all five serovars were successively similar upon revival off

bacterial preservers.

Preparation of Inoculum

Three days prior to each experiment, the five Salmonella serovars were revived

from bacterial preservers. Overnight transfers were performed using 10 ml tubes of TSB

(rif+) each day. On the day of the experiment, an 18-hour culture of each serovar was

harvested via centrifugation (2,000 x g, 15 minutes at 220C). Cells were washed twice

with PBS. Equivalent aliquots of the five serovars at approximately 1.0 x 10 CFU/ml

were combined as a Salmonella cocktail. The cocktail was maintained at room

temperature for one hour. If the time between preparation of the inoculum and

inoculation of the surfaces exceeded one hour the inoculum was stored at 40C until the

surfaces could be inoculated that day. The inoculum was serially diluted using 9 ml tubes

of PBS to confirm cell concentration. The dilutions were plated in triplicate via pour

plate technique using TSA (rif+).









Inoculation Procedures


Inoculation of Tomatoes

Domestic-market mature green tomatoes (Florida 47) were supplied by DiMare

(Tampa, Inc., Tampa, FL) for all experimental studies. Tomato samples were extracted

from the processing lines prior to the waxing process. Size classification of the tomatoes,

according to the Florida Tomato Committee, was 6x7 (formerly medium) (Florida

Tomato Committee 2002). For fruit inoculation, tomatoes were aseptically placed onto

sterile fiberglass trays with the stem scars facing down. Ten 10 l1 drops of inoculum

suspension, for a total of 100 ul of inoculum suspension per whole tomato, were placed

around the blossom scar area using a Repeater Plus pipette (Eppendorf AG, Germany).

The inoculum suspension was not placed directly onto the blossom scar. Immediately

after inoculation, the tomatoes were placed under a hood (LABCONCO Corporation,

Kansas City, MO) at room temperature (approximately 220C) and the inoculated surfaces

were allowed to completely dry for a maximum of 2 hours. Dried samples were placed in

a Caron 6030 (Caron, Marietta, OH) environmental humidity chamber. The Caron

humidity chamber was equipped with a Caron CRS 101 (Caron, Marietta, OH) water

supply system to deliver distilled water to the humidify the chamber. A Whatlow Series

96 temperature and relative humidity controller (Whatlow, Winona, MN) installed in the

environmental chamber continuously monitored, displayed and controlled the

temperature and relative humidity output inside the chamber.

Periodically, a calibrated humidity meter (Control Company, Friendswood, TX)

was placed inside the chamber to verify the relative humidity reading on the output panel.

A magnetic thermometer (Fisherbrand by ERTCOTM, West Paterson, NJ) was placed on

the inside wall of the chamber to verify the temperature output on the panel.









Inoculation of Packing Line Materials

Recovery studies included the following packinghouse materials: stainless steel

(type 304, no.4 finish), conveyor belt, polyvinyl chloride (PVC) rollers, sponge rollers,

and wood (unfinished oak). The packinghouse materials were obtained from Tri-Pak

Machinery, Inc. (Harlingen, TX). Tri-Pak Machinery, Inc. is a Texas-based retailer and

manufacturer of materials and equipment used in tomato packinghouses. The unfinished

oak pieces were supplied by Lowe's Home Improvement Warehouse (Gainesville, FL).

The materials were chosen based upon contact surfaces that fresh-market

tomatoes encounter from harvest (into wooden field bins) to various other surfaces

encountered by tomatoes on a typical packing line. Stainless steel surfaces and conveyor

belt surfaces were cut into coupons by Tri-Pak Machinery, Inc. (Table 3-3). Polyvinyl

chloride (PVC) cylindrical rollers and sponge rollers were received as whole entities from

the manufacturer (Table 3-3). The PVC cylinders were cut into equivalent pieces by the

Mechanical Engineering Department at the University of Florida (Table 3-3). The

sponge rollers were cut into equivalent sections by laboratory personnel (Table 3-2). The

wood pieces were cut into cubes of equivalent dimensions by Lowe's Home

Improvement Warehouse (Table 3-3).

Table 3-3. Surface area dimensions of each type of packing line material that was
inoculated with a five serovar rifampicin-resistant Salmonella cocktail.
Packing Line Material Dimensions of each Inoculated
Surface
Stainless Steel 2.5cm x 2.5cm
Conveyor Belt 2.5cm x 2.5cm
PVC 2.5cm x 2.5cm
Wood 2.5cm x 2.5cm
Sponge roller 2.5cm x 2.5cm









The pre-cut stainless steel coupons were immersed in methanol (Fisher, Fair

Lawn, NJ) overnight to remove any oil residue. The next day, stainless steel coupons

were thoroughly rinsed with deionized water (University of Florida). All packinghouse

material pieces were autoclaved for 20 minutes at 1210C, 15 psi (Consolidated Stills and

Sterilizers, Boston, MA) to achieve sterility. Autoclaved material pieces were aseptically

placed onto sterile fiberglass trays. Sponge roller pieces were dampened with sterile

deionized water prior to inoculation due to the wet nature of sponge rollers found along

the processing lines in tomato packinghouses. Each type of material was marked with a

single dot made by a Sharpie Permanent Marker (Sanford, Bellwood, IL) on the area

where a tomato would most likely be encountered.

All materials were inoculated with ten 10-rl spots of inoculum suspension near

the marked area of each piece. The inoculated materials were placed under a hood until

completely dry. The trays containing the dried inoculated materials were placed in the

Caron 6030 environmental chamber.

Salmonella Recovery off Tomato Surfaces and Packing Line Surfaces

Tomatoes and packinghouse materials were extracted at pre-determined time

intervals from the environmental chamber and recovery studies were performed. Each

recovery study involved sampling at 0, 1, 3, 7, 11, 14, 21 and 28 days for each surface.

Each sampling period consisted of three single-fruit or single-packinghouse material

replicates. On Day 0, the samples were aseptically removed from the fiberglass tray prior

to being placed into the environmental chamber and placed into sterile StomacherT

(Fisherbrand, Fair Lawn, NJ)) bags containing 100 ml of sterile PBS. For all other

sampling days (1 through 28), the tomatoes or packinghouse material pieces were

aseptically removed from the environmental chamber and individually placed into sterile









StomacherTM bags containing 100 ml of sterile PBS on the appropriate days. Tomato

samples were constantly rubbed and shaken for one minute (Burnett and Beuchat 2001;

Harris et al. 2001; Zhuang et al. 1995). Rubbing action was concentrated around the

inoculated blossom scar area of the tomatoes to loosen any reversibly attached bacteria.

The packinghouse materials were also rubbed and vigorously shaken for one minute in

100 ml of PBS. Vigorous rubbing was specifically applied to the dotted area on each

piece of material. The PBS diluent was squeezed in and out of the sponge as well as

rubbed and shaken to try and recover any Salmonella that may have migrated into the

sponge matrix. The sample diluent from each StomacherTM bag was then serially (1:10)

diluted using sterile PBS dilution tubes. The serial dilutions were then pour plated using

TSA (rif+). A negative control for the TSA (rif+) was poured to make certain the media

was not contaminated. The plates were statically incubated at 370C for 48 hours.

Tomato fruits and pieces of each of the materials that were not inoculated with the

Salmonella cocktail were sampled for control purposes. The control samples were

rubbed and shaken for one minute in 100 ml of PBS and serially (1:10) diluted as

previously described for the inoculated samples. The serial dilutions were pour plated

using TSA (rif+) and statically incubated at 370C for 48 hours.

Statistical Analysis

Results from the 20-hour growth studies were evaluated using a Students t test

with an a level of 0.05. All results from recovery studies were averaged counts (CFU/ml)

of recovered Salmonella. Statistical analyses were performed using the Statistical

Analysis System (SAS; SAS Institute, Cary, NC). The GLM procedure in SAS was used

to analyze changes of bacterial populations between replications in each experiment.

Multiple comparisons were performed using the Least Squares Mean adjusted by the






37


Bonferroni method for the tomato and material data. Results that yielded P values of <

0.05 were considered significant in this recovery study.














CHAPTER 4
RESULTS

Recovery studies were designed to assess the recovery of Salmonella from the

surfaces of tomato fruits and packing line materials. Inoculated fruit and material

surfaces were subjected to three separate temperature/relative humidity environments for

28 days. The simulated environments were traditional tomato ripening room parameters,

Florida fall/winter tomato production parameters and Florida spring tomato production

parameters. Salmonella-inoculated fruit and material samples were periodically extracted

from the simulated environments and evaluated for the survival of Salmonella. The

recovery of Salmonella off fruit and material surfaces were assessed to determine if a

specific temperature and relative humidity combination would affect Salmonellae

survival over a prolonged period of time.

Each type of surface was sampled in triplicate for all observation intervals; Day 0,

1, 3, 7, 11, 14, 21 and 28. The recovered Salmonella from each replicate was averaged

and data was compiled into graphs. Graphs depict the relationship between logo CFU/ml

Salmonella survivors and time (days) for all surfaces in each simulated environment.

Growth Levels of Salmonella Serovars after a 20-Hour Incubation

Two 20-hour growth studies were conducted for each of the five rifampicin-

resistant serovars. No significant differences in growth rates were found to exist for any

of the five serovar's growth rates observed between the two studies (P <0.05). Results

from these preliminary studies ensured that serovar growth rates were equivalent to one

another and a consistent inoculum could be created (Figure 4-1). Prior to each









experiment, the cell concentration of each Salmonella cocktail was estimated. This was

accomplished by pour plating appropriate dilutions for each cocktail in triplicate using

TSA (rif+). No significant differences (P <0.05) were found to exist between any

inocula suspensions used for any recovery studies (data not shown).


10
9
8
7


24
3
2

0



S' Study 2

Figure 4-1. Average logo counts of five Salmonella serovars (rif+) after a 20-hour
incubation.

Recovery of Salmonella off Tomato Surfaces

Mature green tomatoes (Florida 47) were inoculated with a five serovar

Salmonella cocktail and stored separately for 28 days in all simulated environments. It

should be noted that the simulated ripening room parameters did not include the addition

of ethylene. Commercially, ethylene is typically applied during the ripening process of

mature green tomatoes. Tomatoes not inoculated with the Salmonella cocktail were

sampled at the beginning of each experiment to ensure the rifampicin-supplemented TSA

eliminated all background microflora present on the fruits. All controls were found to be

negative.









Tomatoes Subjected to Spring Parameters

Tomatoes subjected to spring production parameters, 300C and 80%RH, showed

an overall decrease in logo values of Salmonella for Day 0 to Day 21, but a slight

increase in Salmonella recovery was observed between Day 21 and Day 28 (Figure 4-2).

The inoculum applied to tomato surfaces was estimated at 8.26 logo CFU/ml. On Day 0,

5.08 logo CFU/ml of the applied inoculum was recovered from tomato surfaces. At Day

21, no Salmonella was recovered from tomato surfaces. The greatest average reduction

of recovered Salmonella was observed between Day 3 and Day 7 at 2.55 logo CFU/ml.

Unexpectedly, a 1.17 logo CFU/ml increase was then observed on Day 28. This was

unexpected because no Salmonella was recovered on Day 21. This increase was found to

be significant (P <0.05). As the experiment progressed and tomatoes ripened, fruits held

in this regime (300C/80%RH) appeared more orange in color than the tomatoes held at a

lower temperature (200C).


6.00
.-*-,,RH 2,,C
4 5.00 -

4.00 -- 1)--RH (--("

3.00

~ 2.00
1.00

0.00

0 4 8 12 Days 16 20 24 28

Figure 4-2. Salmonella recovery logoo CFU/ml) from tomato surfaces in ripening room
parameters (200C/90%RH) and spring (300C/80%RH) and fall/winter
(200C/60%RH) regimes over 28 days.









Tomatoes Subjected to Fall/Winter Parameters

Tomatoes subjected to Florida fall/winter tomato season parameters, 200C and

60%RH, also showed an overall logo reduction of Salmonella on the surfaces of

tomatoes over 28 days (Figure 4-2). The inoculum applied to tomato surfaces was

estimated at 8.59 logo CFU/ml. On Day 0, 4.01 logo CFU/ml of Salmonella cocktail

was recovered. A 1.00 logo CFU/ml reduction was observed between Day 0 and Day 1,

but a 0.66 logo CFU/ml increase of recovered Salmonella was observed from Day 1 to

Day 3. This slight increase was found to be insignificant (P <0.05). Again, a significant

decrease in logo CFU/ml was observed from Day 3 to Day 11 at 2.31 logo CFU/ml.

From Day 11 to Day 14, another insignificant increase of Salmonella was observed at

0.44 logo CFU/ml. For the remainder of the 28-day period (Day 14 to Day 28) a slight

reduction in CFU/ml recovery was observed. This minimal reduction in Salmonella over

these days was not significant (P <0.05).

Tomatoes Subjected to Ripening Room Parameters

Tomatoes subjected to ripening room parameters, 200C and 90%RH, exhibited an

overall reduction of Salmonella on the surfaces of tomatoes for a 28-day period (Figure

4-2). The inoculum suspension applied to the fruits was estimated at an average value of

8.15 logo CFU/ml. After the applied inoculum was allowed to completely dry, three

tomatoes were sampled for Day 0. An average value of 4.64 logo CFU/ml of recovered

Salmonella was observed on Day 0. The average logo value for Day 1 exhibited a slight

increase of 0.7 logo CFU/ml in recovered Salmonella from Day 0. This increase was

found to be insignificant (P <0.05). From Day 1 to Day 28, the average recovered

Salmonella off tomato fruits exhibited a significant decrease in value over time. On Day

28, 1.42 logo CFU/ml of Salmonella was recovered from tomato surfaces.









Comparison of Tomato Recovery Studies

Recovery observations showed that Salmonella was recovered on final sampling

interval (Day 28) in all three simulated environments. The levels of recovered

Salmonella at the end of the three experiments were not significantly different from one

another (P <0.05). The largest logo value reduction of Salmonella was observed for

tomatoes held at 300C and 80%RH for 28 days. Tomatoes that were held at 200C and

60%RH had variable recovery that exhibited two separate increases in logo values for

between sampling periods of Day 1 and 3, and Day 11 and 14. The increases were found

to be insignificant; nonetheless it was unexpected that a slightly greater amount of

Salmonella was recovered on Day 14 than Day 11. Tomatoes held at 200C and 90%RH

exhibited a very linear pattern of reduction for logo values between Day 1 and Day 28

(R2 = 0.9965). Salmonella was able to survive in all simulated environments, but

survival patterns were very different. Day 21 in spring parameters (300C/80%RH) was

the only sampling interval for any environment where no Salmonella was recovered.

Recovery of Salmonella off Packing Line Surfaces

Fresh-market tomato packinghouses are typically open-air facilities. The

environments simulated for all materials paralleled spring and fall/winter parameters for

tomato production seasons in Florida. Each type of material was subjected to both

simulated environments for 28 days.

Stainless Steel Surfaces Subjected to Spring Parameters

Stainless steel surfaces held at 300C and 80%RH showed a total logo reduction at

Day 11 (Figure 4-3). The inoculum applied to stainless steel surfaces was estimated at

8.01 logo CFU/ml. A value of 4.39 logo CFU/ml of Salmonella was recovered on Day 0.

No significant logo reduction was observed between Day 0 and Day 1. A significant log









reduction of 4.34 logo CFU/ml was observed from Day 1 to Day 11. On Day 11, no

Salmonella was recovered. The reduction in Salmonella followed a linear pattern (R2

0.9875). On Days 11, 14, 21 and 28 no Salmonella was recovered. Day 7 was the last

sampling interval where Salmonella was recovered (1.29 logo CFU/ml) from the surfaces

of stainless steel.


5.00

-*- n RH 3 C
E 4.00
.. -- --ORH 20C

2 3.00


2.00 -


3 1.00 -


0.00
0 4 8 12 Days 16 20 24 28
Days

Figure 4-3. Salmonella recovery logoo CFU/ml) from stainless steel surfaces in spring
(300C/80%RH) and fall/winter (200C/60%RH) regimes over 28 days.

Stainless Steel Surfaces Subjected to Fall/Winter Parameters

Stainless steel surfaces held at 200C and 60%RH did not exhibit a total logo value

reduction of Salmonella at the conclusion of the sampling period (Figure 4-3). For the

entire 28-day period, an overall log value reduction of 3.67 logo CFU/ml was observed.

The inoculum applied to stainless steel surfaces was estimated at 8.59 logo CFU/ml. A

value of 4.41 logo CFU/ml of Salmonella was recovered on Day 0. From Day 0 to Day

11, a 2.96 logo CFU/ml reduction of Salmonella was observed. On Day 14, the amount

of recovered Salmonella was similar to the logo value recovered on Day 11. A 0.46 logo









CFU/ml reduction was observed between Day 14 and Day 28. This reduction was not

significant (P <0.05). On Day 28, 0.74 logo CFU/ml of Salmonella was recovered.

Comparison of Stainless Steel Recovery Studies

The average logo values of Salmonella recovered on Day 0 for each experiment

were not significantly different from one another (P <0.05). In the simulated spring

environment, it was observed that Salmonella did not survive past Day 11 on stainless

steel surfaces. For fall/winter environments, it was observed that Salmonella survived on

stainless steel surfaces for the entire 28-day period. Recovered Salmonella survival off

the stainless steel was significantly higher at 200C and 60%RH than recovered

Salmonella at 300C and 80%RH (P <0.05).

PVC Surfaces Subjected to Spring Parameters

The inoculum applied to PVC surfaces was estimated at 8.01 logo CFU/ml. A

value of 5.13 logo CFU/ml of Salmonella was recovered on Day 0. PVC surfaces

subjected to spring production parameters showed a total logo reduction (5.13 logo

CFU/ml) by Day 11 (Figure 4-4). A linear pattern of total Salmonella reduction was

observed from Day 0 to Day 11 (R2 = 0.9636). The last detection of Salmonella on PVC

surfaces occurred on Day 7 with an average logo value of 1.00 logo CFU/ml.

PVC Surfaces Subjected to Fall/Winter Parameters

Salmonella was recovered from the surfaces of PVC surfaces for every sampling

interval over a 28-day period (Figure 4-4). The inoculum applied to stainless steel

surfaces was estimated at 8.59 logo CFU/ml. A value of 5.14 logo CFU/ml of

Salmonella was recovered on Day 0. A significant decrease in Salmonella reduction

observed over the 28-day period occurred from Day 0 to Day 1 with an average logo

reduction of 1.19 logo CFU/ml. Overall, there was an average 4.57 logo CFU/ml









reduction observed from Day 1 to Day 28. On Day 28, an average of 0.573 loglO

CFU/ml of Salmonella was recovered from the surfaces of PVC.


6.00
i 80RH/30C
5.00
5.00 60RH/20C
S4.00

3.00

S2.00

S1.00

0.00
0 4 8 12 16 20 24 28
Days

Figure 4-4. Salmonella recovery logoo CFU/ml) from PVC surfaces in spring
(300C/80%RH) and fall/winter (200C/60%RH) regimes over 28 days.

Comparison of PVC Recovery Studies

The average logo values recovered on Day 0 for each experiment were not

significantly different from one another (P <0.05). The most significant decrease in

Salmonella recovery in fall/winter parameters was observed in Days 0 through Day 11

with a 3.50 logo CFU/ml reduction. For the final three sampling periods (Day 14, 21 and

28), Salmonella only exhibited a 1.07 logo CFU/ml reduction. Salmonella was recovered

off PVC surfaces held in spring parameters for the first four sampling intervals (Day 0-

Day 7). Salmonella was recovered for a longer period of days from PVC surfaces at a

lower temperature/relative humidity combination than at a temperature/higher relative

humidity combination. Salmonella was not recovered from PVC surfaces held in spring

parameters after Day 7. However, Salmonella was recovered off PVC surfaces held in

fall/winter parameters for the entire 28-day period. The survival of Salmonella on PVC









surfaces held in 200C and 60%RH was significantly higher than Salmonella on PVC

surfaces at 300C and 80%RH (P <0.05).

Sponge Rollers Subjected to Spring Parameters

The inoculum applied to sponge roller surfaces was estimated at 8.01 logo

CFU/ml. A value of 4.97 logo CFU/ml of Salmonella was recovered on Day 0 (Figure 4-

5). Sponge rollers held at spring parameters exhibited a complete logo value reduction

of 4.97 loglo CFU/ml by Day 1. Salmonella was only recovered on Day 0. Salmonella

was not able to be recovered from sponge rollers once they had entered the simulated

environment at 300C and 80%RH.

Sponge Rollers Subjected to Fall/Winter Parameters

The inoculum applied to sponge roller surfaces was estimated at 8.59 logo

CFU/ml. A value of 4.06 logo CFU/ml of Salmonella was recovered on Day 0. Sponge

rollers held at fall/winter parameters exhibited a complete logo value reduction of 4.06

logo CFU/ml by Day 7 (Figure 4-5). Salmonella was only recovered on Days 0, 1 and 3.

On Day 3, the average logo value of Salmonella recovered was 0.30 logo CFU/ml. A

very linear and significant logo value reduction of Salmonella was observed between

Day 0 and Day 3 (R2 = 0.9999).










6.00
500 80RH/30C
8 5.00
-- 60RH/20C
5 4.00

^ 3.00

0 2.00

S1.00

0.00
0 4 8 12 16 20 24 28
Days

Figure 4-5. Salmonella recovery logoo CFU/ml) from sponge roller surfaces in spring
(300C/80%RH) and fall/winter (200C/60%RH) regimes over 28 days.

Comparison of Sponge Roller Recovery Studies

The logo values recovered on Day 0 for each experiment were not significantly

different from one another (P <0.05). Significant reduction of Salmonella was observed

from Day 1 to Day 3 off sponge rollers held in fall/winter parameters. Significant

reduction of Salmonella was observed from Day 0 to Day 1 from sponge rollers held in

spring parameters. Salmonella was recovered in one more sampling interval (Day 3) in

the simulated fall/winter parameters than in spring parameters. Salmonella was

recovered only at Day 0 from sponge rollers held in spring parameters. No Salmonella

was recovered from any of the two environments at Days 7, 11, 14, 21 and 28. On Day 3,

Salmonella recovery from surfaces of sponge rollers held at 200C and 60%RH was

significantly higher than the Salmonella recovery on sponge rollers held at 300C and

80%RH (P <0.05 ). Sponge rollers were dampened with sterile, distilled water when

inoculated. Sponge surfaces did not remain moist over the 28 day sampling intervals.









Conveyor Belt Surfaces Subjected to Spring Parameters

Salmonella was only recovered off conveyor belt surfaces held at spring

parameters (300C/80%RH)on Days 0, 1 and 3. The inoculum applied to conveyor belt

surfaces was estimated at 8.01 logo CFU/ml. A value of 4.10 logo CFU/ml of

Salmonella was recovered on Day 0 (Figure 4-6). A linear and significant reduction in

the recovery of Salmonella was observed between Day 0 and Day 3 (R2 = 0.9803).

Salmonella was last recovered on Day 1 at 2.26 logo CFU/ml from conveyor belt

surfaces.

Conveyor Belt Surfaces Subjected Fall/Winter Parameters

The inoculum applied to conveyor belt surfaces was estimated at 8.59 logo

CFU/ml. A value of 4.25 logo CFU/ml of Salmonella was recovered on Day 0.

Conveyor belt surfaces stored at 60%RH and 200C showed a logo value reduction of 1.4

logo CFU/ml between Day 0 and Day 1 (Figure 4-6). Between Day 1 and Day 21, a 2.85

logo CFU/ml reduction was observed. Salmonella was last recovered from conveyor belt

surfaces on Day 14 at an average logo value of 0.60 logo CFU/ml.

Comparison of Conveyor Belt Recovery Studies

The average logo values recovered on Day 0 for each experiment were not

significantly different from one another (P <0.05). Salmonella was recovered for a

longer period of days from conveyor belt surfaces in 200C and 60%RH than at 300C and

80%RH. Salmonella was only recovered for Day 0 and Day 1 in the simulated spring

environment, whereas Salmonella recovery was observed until Day 14 in the simulated

fall/winter environment. Salmonella recovery from conveyor belt surfaces in 200C and

60%RH was significantly higher than Salmonella recovery from conveyor belt surfaces

in 300C and 80%RH (P <0.05).










5.00
S80RH/30C
8 4.00
.1 ^ 60RH/20C

S3.00


2.00


1.00


0.00 ..
0 4 8 12 Days 16 20 24 28

Figure 4-6. Salmonella recovery logoo CFU/ml) from conveyor belt surfaces in spring
(300C/80%RH) and fall/winter (200C/60%RH) regimes over 28 days.

Unfinished Oak Surfaces Subjected Spring Parameters

The inoculum applied to unfinished oak surfaces was estimated at 8.01 logo

CFU/ml. A value of 4.73 logo CFU/ml of Salmonella was recovered on Day 0.

Unfinished oak surfaces held in spring parameters exhibited a total logo value reduction

by Day 21 of the 28-day sampling period (Figure 4-7). A significant decrease of 2.62

logo CFU/ml was observed from Day 0 to Day 1. From Day 1 to Day 3, a 0.89 logo

CFU/ml increase was observed. This increase was found to be insignificant (P <0.05).

On Day 3, no Salmonella was recovered and this trend continued until Day 14. On Day

14, Salmonella was recovered from oak surfaces at 1.00 logo CFU/ml. Salmonella was

not recovered from unfinished oak surfaces for the final two sampling periods, Day 21

and Day 28.










6.00

5.00 4 80RH/30C
-. -[ 60RH/20C
3 4.00

3.00

2.00

1.00

0.00 .
0 4 8 12 16 20 24 28
Days

Figure 4-7. Salmonella recovery logoo CFU/ml) from unfinished oak surfaces in spring
(300C/80%RH) and fall/winter (200C/60%RH) regimes over 28 days.

Unfinished Oak Surfaces Subjected to Fall/Winter Parameters

The inoculum applied to unfinished oak surfaces was estimated at 8.59 logo

CFU/ml. A value of 3.22 logo CFU/ml of Salmonella was recovered on Day 0.

Salmonella was recovered off the surfaces of unfinished oak at every sampling interval

(Figure 4-7). An initial 0.12 logo CFU/ml reduction of recovered Salmonella was

observed from Day 0 to Day 1. From Day 1 to Day 3, a 0.33 logo CFU/ml increase in

recovered Salmonella was observed. This slight increase was determined to be

insignificant (P <0.05). A 1.65 logo CFU/ml reduction was observed from Day 3 to Day

11. Unexpectedly, Salmonella was recovered at a logo value on Day 14 at 0.18 logo

CFU/ml. The decrease logo values observed from Day 14 to Day 21 was not significant

(P <0.05). A 0.20 logo CFU/ml reduction of Salmonella was observed during the final

two sampling periods.






51


Comparison of Unfinished Oak Recovery Studies

The logo values recovered on Day 0 for each experiment were not significantly

different from one another (P <0.05). Viable Salmonella recovered from unfinished oak

surfaces was recovered in greater amounts and for a longer period of days in spring

parameters than at fall/winter parameters. Survival of Salmonella on oak surfaces stored

at fall/winter parameters was significantly higher (P <0.05) than the survival of

Salmonella on oak surfaces held in spring parameters. Salmonella recovery off of

unfinished oak surfaces was variable for both simulated environments.














CHAPTER 5
DISCUSSION

Consumption of fresh fruits and vegetables has significantly increased over the

past ten years. The industry is constantly challenged with the concern of microbial food

safety hazards. Many steps are taken to harvest, process and distribute fresh produce and

with each step the opportunity for potential pathogenic contamination increases.

Environmental factors such as temperature and relative humidity have a large impact on

the quality of fruits and vegetables along with the survival capacity of present pathogens.

Effective intervention strategies have been implemented in packinghouses, such as

chlorinated dump tanks, but these strategies cannot totally eliminate all microbiological

dangers associated with the consumption of raw produce. It is also necessary that

packinghouse equipment receive regular cleaning and disinfecting. In recent years,

multiple foodborne illnesses associated with consumption of Salmonella-contaminated

tomatoes have been traced to packinghouse facilities. In this study, a five serovar

rifampicin-resistant Salmonella cocktail was administered to tomato and packing line

surfaces. The various surfaces were subjected to different temperature and relative

humidity combinations that simulated conditions encountered during tomato growing,

packing and ripening. Recovery of Salmonella from the surfaces was performed by

placing the surfaces into 100 ml of PBS and applying a rub-shake method as previously

described.

It has been recommended that a minimum of five strains at approximately equal

populations be selected for the inoculum (CFSAN-FDA 2001). The five Salmonella









enterica serovars selected for this study were S. Agona, S. Gaminara, S. Michigan, S.

Montevideo and S. Poona. These serovars were obtained from Dr. Linda J. Harris,

University of California Davis, and were marked with 80[g/ml rifampicin. Rifampicin

was selected because it is a stable marker and is particularly effective for isolating

pathogens from inoculated fruits that have significant natural background microflora and

adhering soil. S. Agona, Gaminara and Michigan serovars were isolated from fresh

produce or produce products (orange juice). S. Montevideo and Poona serovars were

human isolates linked to fresh produce outbreaks.

Growth characteristics for all five serovars were evaluated by conducting 20-hour

growth studies. Two studies were conducted on each of the five serovars. The

population of each serovar at the end of a 20-hour incubation period was found to be

insignificantly different (Student's t test, a = 0.05) from one another (Figure 4-1). S.

Poona was observed to have the highest population at the end the 20-hour incubation

period (37C), but there was less than a 0.5 logo CFU/ml difference between S. Poona's

population and the serovar with the lowest growth level. This was the case in both

growth studies. It was determined that all five serovars achieved counts of at least 1.0 x

108 CFU/ml after 20 hours of incubation. It was then concluded that acceptable inocula

could be prepared from the five rifampicin-resistant Salmonella serovars. Prior to each

recovery study, appropriate serial dilutions of the inoculum were pour-plated to

determine the viable population of Salmonella. These counts for each prepared inoculum

for all experiments conducted also showed little variation between one another. All

inocula were determined to contain viable Salmonellae populations at 1.08 x 108 CFU/ml.









Recovery of Salmonella off Tomato Surfaces

Tomato surfaces were subject to three simulated environments: ripening room

parameters (20C/90% RH), fall/winter tomato production season parameters (20C/60%

RH) and spring tomato production season parameters (30C/80%RH). Surface recovery

was assessed by applying a rub-shake method, as previously described. Tomatoes have a

fairly firm surface that can withstand moderate rubbing and agitation. This rub-shake

method of recovery was chosen because presently, it seems to be the most effective

protocol for removing microorganisms from the surfaces of whole fruits and vegetables

like tomatoes (CFSAN-FDA 2001). Whole, unblemished tomatoes were specifically

chosen for inoculation studies. It has been researched that microbial cells that contact the

surface of produce and interact with organic acids or other antimicrobials that are

naturally found in plant tissue fluid or ruptured cells as a result of mold or insect

invasion, cellular death may occur (Sofos et al. 1998). The rub-shake method is a simple

surface wash that recovers surface bacteria without rupturing any plant cells that might

interact with the inoculated pathogen. Spot inoculation was utilized because it enables

the measurement of a known number of cells adhering to the produce. Dip or spray

inoculation procedures do not allow the measurement of a known amount of inoculum.

Results from this study were in agreement with Guo et al. (2002) in that

Salmonella populations decreased over time on tomato surfaces in all simulated

environments. The lowest quantities of Salmonella were recovered from tomatoes held

in the spring season parameters (300C/80%RH). Viable Salmonella populations seemed

to die-off or enter a nonculturable state by Day 21 of the recovery study. On Day 28, an

unexpected increase in logo value was observed. Salmonella was recovered from two of

the three tomato replicates sampled on Day 28, but logo values were significantly higher









than Day 21 where no recovery of Salmonella was observed. Between Day 7 and Day

11, a logo value decrease of 2.55 logo CFU/ml was observed. This was the largest logo

value decrease of Salmonella seen between any two sampling periods for all simulated

tomato environments.

Tomatoes held in ripening room (200C/90%RH) and fall/winter production

parameters (200C/60%RH) seemed to exhibit similar patterns of Salmonella recovery.

The highest logo values of Salmonella populations were recovered from tomato surfaces

held in ripening room parameters. Slightly more Salmonella was recovered on Day 1

than on Day 0. This increase was insignificant, but a possible explanation for this

phenomenon could be that some cells of Salmonella did not survive the drying process

while others could have survived, but were shocked and could not be recovered by

conventional culture methods (CFSAN-FDA 2001). For Days 1 through Day 28, a linear

reduction of Salmonella was observed with the largest decrease in recovered logo values

seen between Day 21 and Day 28 at 1.0 logo CFU/ml.

Tomato surfaces held in fall/winter parameters showed the most Salmonella

reduction between Day 0 and Day 1 when compared to the other environments. On Day

3, an increase in Salmonella recovery was observed. The same phenomenon for injured

cells could have occurred as previously mentioned. The lower amount of moisture

(60%RH) in the environment could explain the delayed recovery of injured cells on Day

3 instead of Day 1 as seen in ripening room parameters (90%RH). The availability of

more moisture could have possibly allowed Salmonella to recover at a faster rate. An

approximate 2.0 logo CFU/ml reduction in recovered Salmonella was observed from Day









3 to Day 11 and again, on Day 14 a slight increase in recovered Salmonella was

observed.

Very similar levels of Salmonella were recovered from tomato surfaces for the

last three sampling intervals. All three simulated environments exhibited Salmonella

recovery on Day 28 at very equivalent logo values (approximately 1.5 logo CFU/ml).

Salmonella survival patterns were different for every simulated environment, but the final

sampling interval yielded similar logo values of recovered Salmonella. Overall,

Salmonella was recovered more in an environment where the temperature was

maintained at 200C and the relative humidity was at a high level. It has been documented

by many researchers that bacterial populations have a greater chance of survival and

growth in the presence of free moisture on leaves, from precipitation, dew or irrigation.

Essentially, a higher level of humidity enhances the survival of bacterial cells (Beattie

and Lindow 1999). Salmonella was still observed to survive very well at 20C in

conjunction with a slightly lower relative humidity. Salmonella seemed less likely to

survive in an elevated temperature (300C).

Salmonella was consistently recovered from tomato surfaces at a greater logo

value than any of the packinghouse surfaces while utilizing the rub-shake method of

recovery. Tomatoes are organic surfaces that respire and participate in gas exchange with

the surrounding atmosphere. The tomatoes may have experienced different rates of

respiration at 20C than at 30C and this could have had some effect on the Salmonella.

Viable Salmonella could have aggregated in the stem scar of the fruit or have become

irreversibly attached to the fruit's surface and was not recovered. Over time, Salmonella









could have entered a nonculturable state do to nutrient depletion, injury or environmental

stress.

After harvest, pathogens seem to survive but not proliferate on the outer surface

of tomato fruits, especially in a high humidity and an ambient temperature (200C).

Pathogen levels were observed to decline on the outer surface of tomatoes over time and

the rate of reduction seemed to be strongly related to temperature. Growth on intact

surfaces was not observed. Foodborne pathogens do not produce the necessary enzymes

to destroy the protective outer barriers on most produce, thus restricting the availability of

nutrients. Salmonella was recovered from tomato surfaces in all three simulated

environments and this indicates that the pathogen can survive on tomato surfaces for a

significant amount of time and should be a concern in the fresh produce industry. If

contaminated fruits enter a packinghouse facility it is very probable that cross-

contamination is likely upon the contact of processing equipment.

Recovery of Salmonella off Packinghouse Surfaces

Five types of packing line materials were inoculated with a five serovar

rifampicin-resistant Salmonella cocktail and subjected to fall/winter and spring tomato

production season parameters. Typically, Florida packinghouse facilities are open-sided,

shed-like buildings that shelter the minimal processing of fresh produce harvested in near

by fields. The five materials that were evaluated in recovery studies were stainless steel

(type 304, no. 4 finish), polyvinyl chloride (PVC), sponge rollers, conveyor belts and

unfinished oak surfaces.

Stainless steel is commonly found in most food processing facilities. It is a

smooth, easily sanitized surface that is widely recognized as an excellent material for the

food industry (Midelet and Carpentier 2002). Dump tanks, processing lanes and much









equipment in tomato packinghouses are made of stainless steel. Overall, Salmonella was

recovered at greater logo values and over a longer period of days off stainless steel

surfaces held in fall/winter parameters than stainless steel surfaces held in spring

parameters. Salmonella was only recovered from stainless steel surfaces held at 80%RH

and 30C for the first four sampling intervals (Days 0, 1, 3 and 7). Salmonella was last

recovered at a logo value of 1.29 logo CFU/ml on Day 7. On Days 11, 14, 21 and 28 no

Salmonella was recovered. Significantly more Salmonella was recovered from stainless

steel surfaces held at 200C and 60%RH when compared to surfaces held at 300C and

80%RH. The most reduction of Salmonella for both environments was observed from

Day 0 to Day 11 off surfaces held in spring parameters. After Day 11 for surfaces held in

spring parameters, the recovered populations of Salmonella did not significantly

decrease. On Day 28, Salmonella was recovered at 0.74 logo CFU/ml from surfaces held

in spring parameters.

Polyvinyl chloride (PVC) is another commonly used material in the food industry.

It is a polymer that is typically used to cover roller bars that move tomato fruits along

processing lines. PVC rollers allow tomatoes to be smoothly transported so extensive

bruising and injury is minimal. Recovery patterns of Salmonella from PVC surfaces for

both simulated environments were very similar to recovery patterns from stainless steel

surfaces. Salmonella was only recovered on Days 0, 1, 3 and 7 from PVC surfaces held

in spring parameters. The most significant amount of Salmonella reduction (2.0 logo

CFU/ml) observed in spring parameters was seen between Days 3 and 7. Similar to

recovery patterns from stainless steel surfaces, more logo values of Salmonella were

recovered from PVC surfaces at 60%RH and 20C than from surfaces held at 300C and









80%RH. Salmonella was recovered for every sampling period from PVC surfaces held in

fall/winter parameters. It was very clear that viable Salmonella cells were able to survive

on PVC surfaces for a prolonged period of days at 60%RH and 20C.

Stainless steel and PVC are both hydrophobic surfaces (Midelet and Carpentier

2002). Stainless steel is a nonporous surface but is often marked by grooves and

crevices. This was clearly seen in scanning electron photomicrographs taken of stainless

steel type 304, no. 4 finish (Mafu et al. 1990). Stainless steel is also very resistant to

wear. PVC is a dense polymer with smooth surface, but contains microscopic holes and

crevices. PVC is also resistant to wear, but is more likely to bend or accumulate cracks

or holes than stainless steel surfaces. Of all the surfaces tested in this recovery study,

stainless steel and PVC were the least porous materials. The most amounts of Salmonella

were recovered from these two surfaces when compared to the other surfaces that were

tested. It is most likely that very few salmonellae infiltrated into the matrix of these two

surface types. Hydrophobic qualities accompanied with the dense nature of the materials

most likely prevented bacteria from migrating very far from the point of inoculation. It

was evident that more Salmonella was recovered off both of these surface types at 20C

than at 300C. Salmonella was not recovered from surfaces held at 300C for a prolonged

period of time. Viable cells of Salmonella were not recovered from either surface past

Day 7 at 30C and 80%RH. It is possible that Salmonella, at 300C, had entered a

nonculturable state due to environmental stresses. Biofilm formation is yet another

possibility, although this is not likely because no planktonic cells were recovered from

either surface during the final four sampling intervals. It has been documented that

bacteria can readily and irreversibly attach to many surface types upon very short contact









times, even within one minute (Mafu et al. 1990). Surface types that have been exposed

to bacteria over short contact times and have been observed to form biofilms are glass,

rubber, stainless steel, and many types of plastics (Ronner and Wong 1993; Mafu et al.

1990).

Conveyor belts play an integral part in the functions of a packinghouse. Belts are

rubber compounds (composition not disclosed by manufacturer) that transport tomatoes

to all areas in the facility. Conveyor belts are used to pre-size, cull, sort and size tomato

fruits. Rubber surfaces are smooth to the touch, but scanning electron pictographs show

that particles, crevices and holes appear on the surface (Mafu et al. 1990).

Salmonella was recovered from conveyor belt surfaces held at 300C and 80%RH

on Days 0 and 1. No Salmonella was recovered for any other sampling intervals for

spring parameters. However, Salmonella was recovered at a significantly higher amount

from conveyor belt surfaces held at 200C and 60%RH. Recovery was observed on Days

0 through 14. A sharp reduction of Salmonella was seen between Day 0 and Day 1.

Between Days 1 and 11, an approximate 2.0 logo CFU/ml reduction of Salmonella was

observed. The reported logo values of recovered Salmonella on Days 11 and 14 were

very similar, almost no reduction was seen. For the final two sampling intervals, no

Salmonella was recovered from conveyor belt surfaces held at 200C and 60%RH.

The patterns of recovered Salmonella from conveyor belt surfaces differed from

those of stainless steel and PVC because no Salmonella was recovered from conveyor

surfaces held at 200C and 60%RH after Day 14. Salmonella was recovered from

stainless steel and PVC surfaces until Day 28 for surfaces held at 200C and 60%RH. It

was still evident that Salmonella survived for a longer period of days on conveyor









surfaces held at 200C than surfaces held at 300C. It is possible that the composition of

the conveyor belts had a slight bacteriostatic affect on Salmonella. The pathogen was

recovered at very low amounts on Days 11 and 14 and no survival was observed to occur

at Days 21 and 28. An extreme reduction was also recorded between Day 0 and Day 1.

It has been documented that some types of rubber surfaces have a strong bacteristatic

effect on pathogens. Buna-n rubber nitrilee rubber) is a gasket material typically used in

food processing environments. It has been documented that material has a slight

bacteriostatic effect on Salmonella Typhimurium and a strong bacteristatic effect on

Listeria monocytogenes under low nutrient conditions. It also inhibited the growth of

several other pathogens to varying degrees (Ronner and Wong 1993).

Sponge rollers also serve a very important role in tomato packinghouse

operations. Tomato fruits are susceptible to injury and bruising. Sponge rollers buff and

cushion the fruits as they proceed along the processing lines. Sponge rollers absorb

dumptank water off the fruit surface and the sponges are constantly moist. Thus, sponge

roller samples were dampened with sterile, distilled water prior to inoculation. The

surface and matrix of the rollers were extremely porous and small holes were clearly

visible. Sponge roller surfaces were hydrophilic and absorbed the inocula whereas

previous surfaces were hydrophobic.

Very little Salmonella was recovered from sponge rollers held in fall/winter or

spring parameters. Approximately 5.0 logo CFU/ml of Salmonella was recovered from

sponge rollers held at 300C and 80%RH on Day 0. Day 0 was the only sampling interval

in which Salmonella was recovered for spring parameters. Fall/winter parameters

allowed Salmonella to be recovered from sponge rollers for a longer period of days than









spring parameters. Recovery was observed on Days 0, 1 and 3 for rollers held in

fall/winter parameters. Day 1 was the only sampling interval that was found to be

significantly different between the two simulated environments. On Day 1, a

significantly higher logo value of Salmonella was recovered from sponge rollers held at

200C and 60%RH. On Day 3, a very low logo value of Salmonella was recovered from

surfaces held in fall/winter parameters and the difference was determined to be

insignificant when compared to the recovery on Day 3 for sponges in spring parameters.

Overall, the two recovery patterns for the two simulated environments were very similar.

The composition of the sponge rollers (not disclosed by the manufacturer) seemed to

have a strong bacteriostatic effect on Salmonella. Even with the extreme reduction of

Salmonella seen in both environments, the pathogen was recovered for a longer time

period at 200C and 60%RH.

Wooden pallets are used to transport unitized loads of tomato boxes and are used

in many packinghouse facilities. Wooden field bins are sometimes used to collect

harvested tomatoes, although plastic field bins are more common. Pallets are usually

constructed from unfinished oak wood. Wood surfaces are very rough and porous. The

oak surfaces were hydrophilic and inocula quickly soaked into the surfaces.

Salmonella recovery was extremely variable off unfinished oak surfaces. Overall,

recovery of the pathogen did follow the pattern of recovery from the other surfaces. It

was evident that Salmonella was recovered more from wooden surfaces held at 200C and

60%RH. Surfaces held in fall/winter parameters seemed to facilitate the survival of

Salmonella over the entire 28 day period. An approximate 1.0 logo CFU/ml reduction of

Salmonella was observed over the entire experiment. This amount of Salmonella









reduction was much lower than any reduction observed for other surface types. Recovery

values did fluctuate between sampling intervals in both environments. On two separate

sampling intervals, an increase in Salmonella recovery was observed rather than an

anticipated decrease. For the final sampling interval (Day 28) in fall/winter parameters,

Salmonella recovered from oak surfaces was significantly higher than the recovered

Salmonella in spring parameters. The most variable recovery pattern observed

throughout the entire recovery study was unfinished oak surfaces held at 300C and

80%RH. From Day 0 to Day 1 a large reduction in Salmonella was observed. On Day 3,

Salmonella recovery increased by 1.0 logo CFU/ml. For the next two sampling periods,

Days 7 and 11, no Salmonella was recovered. For all other materials, recovery patterns

followed a trend. When no Salmonella had been recovered during one sampling interval,

no other sampling intervals yielded the recovery of Salmonella. Unfinished oak surfaces

did not follow this trend. On Day 14, Salmonella was recovered from wood surfaces at

1.0 logo CFU/ml. On Days 21 and 28, the pathogen was not recovered.

Unfinished oak surfaces are known to be very coarse and irregular. It is suspected

that the inocula seeped into the matrix of the wood samples. When the samples were

rubbed and shaken for recovery purposes, it was evident that the recovery of Salmonella

was extremely variable. Salmonella was most likely harbored by the matrix of the wood.

Once the pathogen had migrated into the wood matrix recovery methods utilized were not

able to extract the pathogen very easily. These results were in agreement with Boucher et

al. (1998). Campylobacterjejuni was observed to exhibit enhanced survival on cubes of

wood when compared to survival on cubes of plastic. Bacteria were observed to be

sealed inside the porous membrane of the wood cubes. The physical structure was









necessary for the protection of Campylobacterjejuni and soluble free-radical scavengers

from the wood were not responsible for the observed protection. Deeply scored plastic

cubes did not offer enhanced survival in aerated broths. Scanning electron microscopy

was utilized to determine the size of the openings within the wood in relation to the

bacterial cells. Holes and crevices in the wood were noted to be larger than the bacterial

cells allowing the cells to enter the wood matrix. It was established that the physical

structure of the wood, rather than its chemistry was responsible for the wood's protective

effect. It is postulated that the unfinished oak surfaces behaved similarly to the wood

cubes examined in the previously described experiment. It is very likely that viable

Salmonella was harbored inside the oak pieces and were not recovered.

Proliferation of Salmonella was not observed on any surface type. As previously

stated, growth of pathogens on intact surfaces of fruit is not common because foodborne

pathogens do not produce the enzymes necessary to breakdown the outer barriers that

protect the produce (CFSAN-FDA 2001). The availability of nutrients and moisture is

therefore limited. However, after harvest, pathogens are able to survive on the outer

surfaces of fresh fruits and vegetables, especially if the humidity is high. This indicates

that temperature was an important variable for the survival of Salmonella on the various

surfaces evaluated in this study. High relative humidity was present in ripening room

parameters and spring parameters, but it was seen that Salmonella was recovered in

greater quantities at a lower temperature (200C). It should be noted that bacterial soft rot

microorganisms commonly infect tomatoes and the incidence of Salmonella increases in

infected fruits (this was not a factor in this study).









The two temperatures selected for this recovery study were 200C and 300C. The

ambient temperature (200C) seemed to allow Salmonella to survive for a longer period of

days. The warmer temperature (300C) seemed to inhibit the ability of Salmonella to

survive as well on various surfaces. Salmonella most likely exhausted all resources very

quickly at 30C. The microorganism grows very well at 370C, but nutrient depletion

encountered by Salmonella over the 28 days most likely inhibited survival over time.

This trend was observed for every recovery study performed. Lower temperatures seem

to facilitate the survival of Salmonella rather than higher temperatures. It has also been

reported that certain strains of salmonellae can survive for longer periods of time under

refrigeration temperatures than at room temperature (Parish 1997; Zhao et al. 1993). A

further study might explore the recovery of Salmonella off various surfaces under

refrigeration temperatures (40C) to see if survival of the pathogen is enhanced.

Recovery of Salmonella at low levels is still an important concern. Lower levels

of the pathogen were still recovered as time increased (up to 28 days for tomatoes). The

infectious dose of salmonellae ranges from 10 to 100,000 cells (CFSAN-FDA 2001).

This indicates that even low levels of Salmonella in favorable conditions can facilitate a

foodborne disease outbreak. This is a chief concern for the fresh produce industry due to

the fact that edible horticultural crops are consumed without a treatment to help eliminate

any pathogenic microorganisms that may be present.














CHAPTER 6
CONCLUSION

All objectives of this study were accomplished. Growth rates of five rifampicin

resistant Salmonella serovars were established and it was determined that an appropriate

cocktail could be made. Salmonella was successfully recovered from tomatoes and all

material surfaces. It was observed that the pathogen survived longer on all surface types

in the simulated fall/winter regime (200C/60%).

Salmonella has the capability to survive over a prolonged period of time in certain

temperature and relative humidity combinations on tomato fruits and various equipment

surfaces. Results showed that Salmonella populations on tomato surfaces held at 200C

were observed to decline over time (approximately a 4.0 logo CFU/ml reduction over 28

days). Of all simulated environments, spring tomato production parameters

(300C/80%RH) yielded the lowest recovery of Salmonella over 28 days. Stainless steel

and PVC surfaces had similar recovery patterns of Salmonella at 200C and 60%RH over

28 days. No Salmonella was recovered after Day 7 at 300C and 80%RH off these

surfaces. No Salmonella was recovered from conveyor belt and sponge roller surfaces

after 21 and 7 days, respectively. Salmonella recovery off wood surfaces exhibited the

most variability. Wood surfaces maintained at 60%RH and 200C exhibited the most

Salmonella recovery of any surface type at the end of 28 days. An ambient temperature

(200C) combined with a higher (90%RH) and moderate (60%RH) relative humidity

seemed to facilitate Salmonella survival better than an elevated temperature (300C)









combined with a high (80%RH) relative humidity. Temperature seemed to be an

important factor affecting the survival of Salmonella on various surface types.

Surface types of the materials also seemed to affect Salmonella recovery and

survival over time. More Salmonella was recovered from the smooth and nonporous

surfaces like stainless steel and PVC. Rough and porous surfaces, like the wood surfaces,

seemed to harbor Salmonella in this matrix better than smoother surfaces. Sponge rolls

and conveyor belt surfaces also showed a possible bacteriostatic effect on Salmonella

over time. Sponge rollers did not allow Salmonella survival for longer than Day 1 in the

spring regime and Salmonella was not recovered from the rollers after Day 3 in the

fall/winter regime. Salmonella was not recovered after Day in the spring regime from

conveyor belt surfaces. Salmonella did survive on conveyor surfaces until Day 14 in the

fall/winter regime, but it was recovered at very low levels.
















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BIOGRAPHICAL SKETCH

Raina Leneve Allen was born in Tampa, FL, on July 28, 1979. In 2001, she

received her Bachelor of Science from the University of Florida in food science and

human nutrition. Upon graduation, she was accepted into the University of Florida's

food science master's program. In this program, her specialization focused on food

microbiology with a special interest in microbial safety of fresh-market produce.

Upon receiving her master's degree, Raina plans to pursue a career in the food

industry and continue working in the area of food safety.