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1 SALMONELLA TRANSFER POTENTIAL DURING HAND HARVESTING OF TOMATOES UNDER LABORATORY CONDITIONS By PARDEEPINDER KAUR BRAR A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE RE QUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011
2 2011 Pardeepinder Kaur Brar
3 I would like to dedicate this work to my family and friends for being the source of inspiration and pride for me.
4 ACKNOWLEDGMENTS For their support and guidance through my graduate studies, I wish to express sincere appreciation to my graduate committee: Dr. Michelle Danyluk Dr. Keith Schneider, and Dr. Max Teplitski Especially, I would like to thank Dr. Michelle Danyluk for accepting me as graduate research assistant, encouraging and believing in me, and providing a great environment during my education in Food Safety I thank all who helped me with my research, including Rachel McEgan, Lorrie Freidrich Gwen Lundy, Joshua Vandamm, an d Luis Martinez Thanks a lot for helping me in learning various laboratory skills and giving me tons of moral support and appreciation time to time. I really appreciate all your time and support. This research would not have possible without the help of C itrus Research and Education Center CREC. I want to thanks the entire CREC group for their open doors and assistance. Finally, I would like to extend my special appreciation to my parents, my fiance Prabhjot, my uncle Mr. Amarjit Singh Dhaliwal, and my great friends Maninder and Sushila for their love and support.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 2 LITERATURE REVIEW ................................ ................................ .......................... 19 Fresh Produce ................................ ................................ ................................ ........ 19 Consumption of Fresh Produce ................................ ................................ ........ 19 Fresh Produce Related Outbreaks ................................ ................................ ... 20 Pathogens Associated with Raw Produce Outbreaks ................................ ...... 22 Sources of Raw Produce Contamination ................................ .......................... 24 Pre harvest ................................ ................................ ................................ 25 Harvesting ................................ ................................ ................................ .. 26 Post h arvest ................................ ................................ ............................... 26 Possible Reasons behind Increase in Raw Produce Outbreaks ....................... 27 Cost Associated with Foodborne Outbreaks ................................ .................... 28 Salmonella ................................ ................................ ................................ .............. 29 Salmonella Nomenclature ................................ ................................ ................ 30 Growth and Survival Characteristics ................................ ................................ 32 Diseases Caused by Salmonella ................................ ................................ ...... 33 Typhoid Salmonella ................................ ................................ ................... 33 Non ty phoid Salmonella ................................ ................................ ............. 34 Salmonella in Non host Environment ................................ ............................... 35 Salmonella and Tomatoes ................................ ................................ ................ 37 Salmonella on surface of tomato ................................ ................................ 38 Salmonella in tomatoes ................................ ................................ .............. 40 Salmonella Disinfection on Produce ................................ ................................ 42 Bacterial Transfer ................................ ................................ ............................. 46 Tomato Production ................................ ................................ ................................ 48 Tomato Growing in Florida ................................ ................................ ............... 50 Tomato Harvesting in Florida ................................ ................................ ........... 52 Tomato Good Agricultural Practices and Best Management Practices ............ 54 Exemption from T GAPs and T BMPs Requirements ................................ ...... 58 Gloves ................................ ................................ ................................ ..................... 58 Focus of Research ................................ ................................ ................................ .. 61
6 3 MATERIAL AND METHODS ................................ ................................ .................. 63 Preliminary Experiments ................................ ................................ ......................... 63 Carrier Medium ................................ ................................ ................................ 63 Inoculum Drying Times ................................ ................................ ..................... 63 Culture Media ................................ ................................ ................................ ... 64 Dirty Reusable Gloves ................................ ................................ ...................... 64 Tomatoes ................................ ................................ ................................ ................ 65 Gloves ................................ ................................ ................................ ..................... 65 Salmonella Strains ................................ ................................ ................................ .. 65 Cocktail Preparation ................................ ................................ ............................... 66 Inoculum Procedures ................................ ................................ .............................. 66 Transfer Scenarios ................................ ................................ ................................ 67 Clean Reusable or Single use or Dirty Reusable Gloves to Tomatoes ....... 67 Tomatoes to Clean Reusable or Single use Gloves ................................ ...... 67 Clean Reusable or Single use or Dirty Reusable to Many Tomatoes .......... 67 Enumeration of Pathogen ................................ ................................ ....................... 67 Enrichment ................................ ................................ ................................ .............. 68 Transfer Coefficients ................................ ................................ ............................... 69 Statistics ................................ ................................ ................................ ................. 69 4 RESULTS ................................ ................................ ................................ ............... 70 Preliminary Experiments ................................ ................................ ......................... 70 Carrier Media ................................ ................................ ................................ .... 70 Dry ing Time ................................ ................................ ................................ ...... 70 Culture Media ................................ ................................ ................................ ... 71 Dirty Glove Protocol ................................ ................................ ......................... 71 Salmonella Transfer from Clean Reusable Gloves to Tomatoes ............................ 72 Salmonella Transfer from Single use Gloves to Tomatoes ................................ ..... 72 Salmonella Transfer from Dirty Gloves to Tomatoes ................................ .............. 73 Salmonella Transfer from Tomatoes to Clean Reusable Gloves ............................ 74 Salmonella Transfer from Tomato to Single use Gloves ................................ ........ 74 Salmonella Transfer from Clean Reusable Gloves to Twenty five Tomatoes ......... 75 Salmonella Transfer from Singl e use Gloves to Twenty five Tomatoes ................. 76 Salmonella Transfer from Dirty Reusable Gloves to Ten Tomatoes ....................... 78 5 DISCUSSION ................................ ................................ ................................ ....... 126 Single Touch ................................ ................................ ................................ ......... 127 Subsequent Touches ................................ ................................ ............................ 132 6 FUTURE WORK ................................ ................................ ................................ ... 135 LIST OF REFERENCES ................................ ................................ ............................. 138 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 154
7 LIST OF TABLES Table page 4 1 Salmonella transfer from inoculated single use glove to tomato using different carrier mediums n = 3 12. ................................ ................................ .. 79 4 2 Salmonella transfer from inoculate d single use glove to tomatoes at different drying times using 0.1% peptone as a carrier media n=6 12. .......................... 80 4 3 Salmonella transfer from inoculated single use glove to tomatoes using TSAR, BS AR and XLDR media with water as a carrier medium n=3. .............. 81 4 4 Salmonella transfer from inoculated dirty gloves to tomatoes at 0 h, 1 h and 24 h of inoculum drying, following a 5 s touch n=3 ................................ .......... 82 4 5 Salmonella transfer from inoculated tomatoes to dirty gloves at 0 h, 1h and 24 h of inoculum drying, following a 5 s touch n=3. ................................ .......... 83 4 6 Salmonella transfer from inoculated dirty gloves to tomatoes at 0 h and 1 h inoculum drying, following 5 s touch n=3. ................................ ......................... 84 4 7 Salmonella transfer from inoculated dirty glove s to tomatoes at 0 h and 1 h inoculum drying, following 5 s touch n=3. ................................ ......................... 85 4 8 Salmonella transfer from inoculated clean reusable gloves to tomatoes after 0 h, 1 h and 24 h of inoculum drying following a 5 s touch n=9 18. ................. 86 4 9 Salmonella transfer from inoculated single use gloves to tomatoes after 0 h, 1 h and 24 h of inoculum drying, following a 5 s touch n=9 18. ........................ 87 4 10 Salmonella transfer from inoculated dirty reusable gloves to tomatoes after 0 h and 1 h of inoculum drying, following a 5 s touch n=9. ................................ .. 88 4 11 Salmonella transfer from inoculated tomatoes to clean reusable gloves after 0 h, 1 h and 24 h of inoculum drying, following a 5 s touch n=9 18. ................. 89 4 12 Salmonella transfer fr om inoculated tomatoes to single use gloves after 0 h, 1 h and 24 h of inoculum drying, following a 5 s touch n=9 18. ........................ 90 4 13 Salmonella transfer from inoculated clean reusable glove to twen ty five tomatoes touched subsequently with wet inoculum n=9. ................................ 91 4 14 Salmonella transfer from inoculated clean reusable glove to twenty five tomatoes touched subsequently with an hour dried inoculums n=9. ................ 92 4 15 Salmonella transfer from inoculated single use glove to twenty five tomatoes touched subsequently with wet inoculums n=9. ................................ ............... 93
8 4 16 Salmonella transfer from inoculated single use glove to twenty five tomatoes touched subsequently with an hour dry inoculums n=9. ................................ ... 94 4 17 Salmonella transfer fro m inoculated dirty reusable glove to ten tomatoes touched subsequently with wet inoculum n=9. ................................ ................. 95 4 18 Salmonella transfer from inoculated dirty reusable gloves to ten tomatoes touched subseq uently with an hour dried inoculum n=9. ................................ .. 96
9 LIST OF FIGURES Figure page 4 1 Comparison of different carrier mediums for transfer from inoculated glo ve single use to tomatoes with it after 0 h black bar and 1 h light grey bar drying time n=3 12. ................................ ................................ .......................... 97 4 2 Comparison of transfer after different drying times of inocula using 0.1% pepton e water as carrier medium by performing enumerations on TSAR black bar and BSAR light grey bar media n=6 12. ................................ ...... 98 4 3 Comparison of different culture media; TSAR black bar, BSAR light g rey bar and XLDR dark grey bar for recovery of Salmonella from tomatoes. Water was used as carrier medium for inocula n=3. ................................ ........ 99 4 4 Population of Salmonella inoculated onto clean reusable g love black color bar and transferred to a tomato light grey color bar following a 5 s touch n=9 18.. ................................ ................................ ................................ .......... 100 4 5 Distribution of Salmonella transfer coefficients TCs from reusable gloves t o tomatoes with 0 h dried inoculum using TSAR media n=9. ............................ 101 4 6 Distribution of Salmonella transfer coefficients TCs from reusable gloves to tomatoes with 0 h dried inoculum using BSAR me dia n=9. ............................ 102 4 7 Distribution of Salmonella transfer coefficients TCs from reusable gloves to tomatoes with 1 h dried inoculum using TSAR media n=9. ............................ 103 4 8 Distribution of Salmonella transfer coefficients TCs from reusable gloves to tomatoes with 1 h dried inoculum using BSAR media n=9. ............................ 104 4 9 Population of Salmonella inoculated onto clean single use glove black color bar and transferred to a tomato light grey color bar following a 5 s touch n=9 18.. ................................ ................................ ................................ .......... 105 4 10 Distribution of Salmonella tra nsfer coefficients TCs from single use gloves to tomatoes with 0 h dried inoculum using TSAR media n=9. ........................ 106 4 11 Distribution of Salmonella transfer coefficients TCs from single use glov es to tomatoes with 0 h dried inoculum using BSAR media n=9. ........................ 107 4 12 Distribution of Salmonella transfer coefficients TCs from single use gloves to tomatoes with 1 h dried inoculum using TSAR media n=9. ........................ 108 4 13 Distribution of Salmonella transfer coefficients TCs from single use gloves to tomatoes with 1 h dried inoculum using BSAR media n=9. ........................ 109
10 4 14 Population of Salmonella inoculated onto dirty gloves black color bar and transferred to tomatoes light grey color bar following a 5 s touch n=9. The solid line on the graph is the limit of detection 1.3 log CFU/glove or tomato. 110 4 15 Distribution of Salmonella transfer coefficients TCs from dirty reusbale gloves to tomatoes with 0 h dried inoculum using TSAR media n=9. ............. 111 4 16 Distribution of Salmonella transfer coefficients TCs from dirty reusbale gloves to tomatoes with 0 h dried inoculum using BSAR media n=9. ............. 112 4 17 Population of Salmonella inoculated onto tomatoes light grey color bar and transferred to a clean reusable glove black color bar following a 5 s touch n=9 18.. ................................ ................................ ................................ .......... 113 4 18 Distribution of Salmonella transfer coefficients TCs from tomatoes to reusbale gloves with 0 h dried inoculum using TSAR media n=9. .................. 114 4 19 Distribution of Salmonella transfer coefficie nts TCs from tomatoes to reusbale gloves with 0 h dried inoculum using BSAR media n=9. ................. 115 4 20 Distribution of Salmonella transfer coefficients TCs from tomatoes to reusbale gloves with 1 h dried inoculum using TSAR media n=9. .................. 116 4 21 Distribution of Salmonella transfer coefficients TCs from tomatoes to reusbale gloves with 1 h dried inoculum using BSAR media n=9. ................. 117 4 22 Population of Salmonella inoculated onto tomatoes light grey color bar and transferred to a clean single use glove black color bar following a 5 s touch n=9 18.. ................................ ................................ ................................ .......... 118 4 23 Distribution of Salmonella transfer coefficients TCs from tomatoes to single use gloves with 0 h dried inoculum using TSAR media n=9. .......................... 119 4 24 Distribution of Salmonella transfer coefficients TCs from tomatoes to single use gloves with 0 h dried inoculum using BSAR media n=9. ......................... 120 4 25 Distribution of Salmonella transfer coefficients TCs from tomatoes to single use gloves with 1 h dried inoculum using TSAR media n=9. .......................... 121 4 26 Distribution of Salmonella transfer coefficients TCs from tomatoes to single u se gloves with 1 h dried inoculum using BSAR media n=9. ......................... 122 4 2 7 Population of Salmonella inoculated onto clean reusable gloves first bars and transferred to subsequently touched tomatoes remaining bars after 0 h black color bars and 1 h light grey color bars of inoculums drying n=9.. ... 123
11 4 28 Population of Salmonella inoculated onto clean single use gloves first bars and transferred to subsequently touched tomatoes remaining bars after 0 h black color bars and 1 h light grey color bars of inoculums drying n=9. .... 124 4 29 Population of Salmonella ino culated onto dirty reusable gloves first bars and transferred to subsequently touched tomatoes remaining bars after 0 h black color bars and 1 h light grey color bars of inoculums drying n=9. .... 125
12 Abstract of Thesi s Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science S ALMONELLA T RANSFER D URING H AND H ARVESTING O F TOMATOES U NDER L ABORATORY C ONDITIONS By Pardeepi nder Kaur Brar August 2011 Chair: Michelle Danyluk Major: Food Science and Human Nutrition Tomatoes have associated with many multistate outbreaks of Salmonella and h arvesting is suspected as a potential source of contamination. W orkers prefer to wear g loves during tomato harvesting and may reuse them multiple times E xperiments were performed using mature green ro und tomatoes with two types of gloves reusable an d single use and two hygienic conditions of reusable glove clean and dirty. Transfer sce narios used during experiments were glove clean and dirty to tomato, tomato to glove clean only and glove clean and dirty to many tomatoes. Dirty gloves reusable only were prepared by rubbing with a fresh tomato leaf f o r 20 s. Uninoculated surface was touched with inoculated surface 6 log CFU/surface for 5 s following 0 h, 1 h and 24 h drying Tomatoes, 25 for clean and 10 for dirty gloves, were also touched subsequently with gloves after 0 h and 1 h drying time. All the clean glove samples were p laced in sterile sampling bags with 20 ml of Butterfield s Phosphate Buffer; 0.1% Tween 20 was added to t he dirty reusable glove samples Following stomaching gloves or rubbing tomatoes, samples were enumerated on non selective and selective agar havin g r ifampicin. Enrichments were performed when samples fell below
13 the limit of detection N o signi ficant differences in TCs were obtained between clean reusable and single use as well as clean a nd dirty reusable gloves at three drying times 0 h, 1 h and 24 h on touching single tomato with an inocul ated glove surface. Similarly, n o significant differences were observed upon touching gloves with inoculated tomatoes at 0 h and 1 h drying time Drying the inoc ulum on tomato surface for 24 h results in signifi cantly more number of Salmonella positive reusable glove samples. Differences between 0 h and 1 h TC s were significant only when tomatoes were touched with inoculated gloves. When 25 tomatoes were touched with clean reu sable and single use gloves, TC s redu ced significantly from third tomato a nd seventh tomato, respectively, while in case of dirty reusable gloves, no significant reductions were observed for all the 10 tomatoes This study provides valuable insight into Salmonella transfer between gloves and mature, green tomatoes
14 CHAPTER 1 INTRODUCTION Consumption of fresh produce has increased in comparison to previous years USDA 2008a, b. Health consciousness is one of the main driving forces behind this trend National Cancer Institute, 1991. Availabi lity of produce has also increased due to better transportation, storage facilities and import from other countries. Ahvenainen, 1999. Along with increasing consumption, increase in produce linked o utbreaks has also been reported Mead et al., 1999; Scal lan et al., 2011 Produce is available in many forms, examples include raw produce, minimally processed produce, or fresh cut produce, which is harvested, packed and shipped to the stores without undergoing any kind of pathogen reduction step. M inimally p rocessed produce and fresh cut produce also known as r eady to use produce, i s a growing segment in grocery stores and restaurants Tauxe et al., 1997. Fresh fruits and vegetables are minimally processed and typically consumed raw. Once contaminated, re moval of pathogens fr om fresh produce is a difficult task, attention at all points from farm to fork is critical to prevent contamination of fresh produce. Risk of contamination is linked to fresh produce while still on plants, in fields or orchards, or du ring harvesting, transport, processing, distribution, marketing, at retai l, or in home while preparing. A variety of fresh fruits and vegetables ha ve been involved in foodborne outbreaks. Performing trace back investigations is also difficult in produce ou tbreaks due to short shelf life. Prevention of contamination is believed to be the key for controlling produce linked outbreaks. Guide to minimize microbial food safety hazards for fresh fruits and vegetables is a guidi ng document issued by Food and D rug Administration FDA which covers the general safety features that can be adopted by
15 industry to reduce the microbial risk. Despite the availability of guiding documents, foodborne outbreaks related to fresh produce still occur, infecting thousands and c ausing deaths. Non typhoidal Salmonella NTS is the leading cause of food related hospital ization and deaths in the U.S. ; occurring due to foodborne infections Scallan et al., 2011. Approximately 11% 1.03 million of the foodborne illnesses, 35% 19,58 6 of hospitalizations and 2 8% 378 of deaths in the U.S. Scallan et al., 2011. Salmonella has been linked to many produce items including tomatoes. Outbreaks have found to be associated with cut tomatoes as well as intact tomatoes. In 1990, 176 illness es caused by Salmonella Javiana were reported in four states. These illnesses were linked to raw tomatoes CDC, 1991; Hedberg et al., 1999. In 1993, 100 reported cases of salmonellosis caused by sv. Montevideo were part of a thr ee state outbreak that was linked to raw tomatoes Hedberg et al., 1999. In January 1999, Salmonella Baildon was recovered from 86 infected persons in eight states. Many restaurants across several states were involved, suggesting the tomatoes were likely contaminated in the beginni ng, at the farm or during packing, and before distribution Cummings et al., 2001. In July 2002, an outbreak of salmonellosis caused by Salmonella Javiana occurred associated with attendance at the 2002 U.S. Transplant Games held in Orlando, Florida durin g late June of that year. The outbreak investigation ultimately identified 141 ill persons. The epidemiological investigation impl icated fresh, pre package diced Roma tomatoes as the probable vehicle for the outbreak CDC, 2002. During August and Septembe r 2002, a n outbreak of salmonellosis cause by sv. Newport affecte d the East Coast. Approximately 404 confirmed cases were identified in
16 over 22 states. Epidemiological analysis indicated that tomatoes were the most likely vehicle, and were traced back to t he toma to packing facility in the mid a tlantic region. Inspections of that packing facility revealed numerous violations of the Good Agricultural Practices GAPs and Good Manufacturing Practices GMPs published by the FDA Greene et al., 2008. In early July 2004, an outbreak of salmonellosis associated with Roma tomatoes, o ccurred in many states of U.S. and Canada. Five separate serotypes of Salmonella were eventually associated with the outbreak and those were Salmonella Javiana, Salmonella Typhimurium, Salmonella Anatum, Salmonella Thompson, and Salmonella Mu e nche n CDC, 2005. In 2005 2006 four large multistate outbreaks associated with Salmonella and tomatoes occurred and were linked to cut tomatoes served in restaurants. Investigations of this outbre ak confirmed that tomatoes involved were supplied either whole or pre cut from tomato fields in Florida, Ohio and Virginia CDC, 2007. In 2008, an outbreak of salmonellosis caused by sv. Sanitpaul infected numerous people. Investigators suspected raw toma toes as a source of outbreak in the beginning and warn the consumers against raw tomatoes, which was later found to b e associated with jalapeo and S errano pepper s This cost Florida tomato industry c.a. $100 million CDC, 2008. All the above outbreaks sh owed that Salmonella Tomato outbreaks have infected many people all over the country in past two decades. Most of the tomatoes involved in the outbreaks discussed above came from either Florida or Virginia. Florida is the main pr oducer of tomatoes in the U .S. due to its warm climate VanSickle and Hodges, 2008. The occurren ce of many multistate outbreaks led the Florida tomato industry, in cooperation with the Florida Department of Agriculture and Consumer Services FDACS to develop and enforc e Tomato Goo d
17 Agr icultural Practices T GAPs Tomato Best Management Practices T BMPs for the safe production, harvesting, processing and distribution of tomatoes. DACS, 2007 These practices are mandatory in Florida for growers, packers, repackers and other agenci es involved in tomatoes production. Salmonella contamination of tomatoes and other fresh produce has been suspected to occur through contact with contaminated irrigation water, manure, animal intrusion in field, worker s hands, contaminated utensils, cloth es and others Beuchat, 1996; Sargent et al., 1989; Bloomfield and Scott, 1997; Scott and Bloomfield, 1993 All these sources provide a potential route for Salmonella contamination during tomato production. Salmonella has shown survival on the surface of tomatoes and inside tomatoes, provided adequate time and appropriate conditions exist Shi et al., 2007; Wei et al., 1995, Asplund and Nurmi, 1991. Salmonella is believed to enter the tomato plant through flowers, cracks and crevices on the surface of pla nt and tomatoes Guo et al., 2001. Harvesting is one of the different potential sources of tomatoes contamination. Mechanical harvesting is not common in Florida due to high cost and less expertise, thus most tomatoes are harvested manually Zahara and Jo hnson, 1981 As per T GAPs, workers are required either to wear gloves during harvesting or wash their hands frequently to prevent potential contamination or spread of contamination. Tomato fields are sprayed with different pesticides during growing phase. To reduce the exposure of skin to various pesticides present on the tomato surface and to protect cuts on hands while harvesting, most of the workers prefer to wear gloves. As the harvesting progress, workers can use the same gloves for whole day and wash them with water
18 and sanitizer at the end of the day. The same gloves are then used on the next day of harvesting. This research focuses on the risk of Salmonella cross contamination between gloves and mature green, round tomatoes during hand harvesting. C lean and dirty reusable gloves were selected to perform experiments. Experiments were also performed with clean single use gloves used in tomato packinghouses to sort and pack tomatoes in cartons, after washing with sanitizer. Comparison between two differ ent types of gloves reusable and single use and two different hygienic conditions of reusable gloves clean and dirty will help in quantifying the risk of Salmonella cross contamination during harvesting and post harvesting of mature green, round tomato es.
19 CHAPTER 2 LITERATURE REVIEW Fresh P roduce Consumption of Fresh P roduce Demand for fresh fruits and vegetables has incre ased significantly in the U.S. in past decade. Between 1970 and 2008, U.S. per capita consumption of fresh vegetables increased app roximately 67%, from 49 to 82 kg 107.9 to 180.5 lb per year USDA 2008a and from 1976 to 2007, U.S. per capita consumption of fresh fruit increased approximately 19%, from 38 .2 to 45.5 kg 84.2 to 100.2 lb per year USDA 2008b. Per capita expenditure on fresh produce is expected to increase more than per capita expenditure on any ot her foods by 2020 Clemens, 2004. The driving force behind the increase in consumption of fresh produce is health. The National Cancer Institute s NCI and Produce for Bet ter Health Foundation PBHF five a day program encourages Americans to consume at least five servings of fruits and vegetables per day National Cancer Institute, 1991. This program increase d the consumption of produce by Americans from 23% to 26% from 1991 to 1998. To strengthen the program, NCI formed a partnership with U. S. Department of Agriculture USDA in April 2002 National Cancer Institute, 1991. This partnership resulted in increased consumer awareness about the benefits of fresh produce, p romotion of healthier lifestyles by public health officials, and the presence of new produce items not previously available; all hav ing an impact in changing the diet of the American people Berger et al., 2010; Beuchat, 1996. Consumption of fruits and v egetables is encouraged in many parts of the world due to the high fiber, vitamins, minerals and low fat content found in these items which is believed to help in preventing cancer and
20 cardiovascular diseases Ness and Powles, 1997. New techniques to impr ove shelf life of fresh produce as better storage facilities, improved marketing facilities and innovative packaging facilities have made availability of fresh produce feasible throughout the year Ahvenainen, 1999. To meet the growing dem and of fresh pro duce, the U.S. has signed trade agreements with other countries to import fresh produce Clemens, 2004. As a result, between the period of 1980 to 2001, import of fresh fruits and vegetables increased by 155 and 265 percent, respectively Clemens, 2004. In 2001, U S fresh fruit imports accounted for 38.9%, up from 24.2% in 1980 and fresh vegetables accounted for 11.6%, up from 5.5% in 1980 Clemens, 2004. In 2002, about 27% 859, 502 metric tons of tomatoes consumed in U.S were imported from two count ries, Mexico and Canada. Mexico 84.2% i.e ., 723, 425 metric tons was the larges t supplier of tomatoes to U.S. in 2002 11.7% i.e ., 100, 499 metric tons Clemens, 2004. As is true with tomatoes, the import of other fruits and vegetables has also increas ed since 1980. Produce from different countries of the world Ecuador, Guatemala, Mexico, Canada, Chile, Costa Rica and others are commonly available in U.S Clemens, 2004. Fresh Produce Related O utbreaks A foodborne outbreak is defined as an incident in which two or more persons experience a similar illness after ingesting a common food, which epidemiological analysis implicates as the source of the illness FDA, 2006. Historically, f resh fruits and vegetables were never believed to support growth of bacteri a; currently fresh produce are associated with a number of foodborne outbreaks annually Scallan et al., 2011; N ACMC F ood 1999; De Roever, 1998; Francis et al., 1999; Nguyen the, 1994; Tauxe, 1997. The reason for an increased number of produce li nked foodborne illness
21 outbreaks occurring in summer months is not yet clear, but it may be attributed to the fact that more and more fresh fruits and vegetables are consumed in hot weather and the presence of high temperature conditions in summer also fav ors the growth of pathogens Hedberg et al., 1994 From 1987 to 1992 a constant number of produce related outbreaks was discovered annually, but between 1993 and 1997, a five fold increase in the number of produce related outb reaks was observed Bean et al., 1997 Etiological agents could not be determined from more than 50% of the outbreaks during these periods and the identified etiological agents outbreaks were predominantly due to bacterial pathogens, primarily Salmonella Bean et al., 1997 During the period of 1995 and 1998, nine outbreaks occurre d from the consumption of fresh vegetable sprouts. Salmonella and E scherichia coli O157:H7 were the causative agents behind these foodborne illnesses NACMCF, 1999. T he Center for Science in Public Interest CSPI, performed a comprehensive survey of all the outbreaks with identified food vehicle in U.S. and found produce accounts for 13% of outbreaks and 21% of illnesses during the period of 1990 to 2005 CSPI, 2005. C SPI I n ter n et s database of 5,000 foodborne illness outbreaks, indicates that between 1 990 and 2003, 554 outbreaks with nearly 28,315 cases have been linked to produce and produce dishes. Among these, vegetables were linked to 205 outbreaks with 10,358 cases, while fruits were identified as the vehicle in 93 outbreaks with 7,799 cases. Of th e 93 fruit associated outbreaks, 15 were linked to berries and 25 were linked to melons. Produce dishes were implicated in 256 out breaks involving 10,158 cases.
22 According to the estimate of Scallan et al. 2011 contaminated food including fresh produce i s responsible for 9.4 million illnesses, 55,961 hospitalization, 1,351 deaths annually in the U.S The y further estimate that produce related outbreaks have decreased by 20% during the past decade Scallan et al., 2011. The various reasons for observing d ecreased produce linked outbreaks may b e diffe rent analysis method by Scallan et al. in comparison with the previous Mead et al. 1999 study, a smaller sample size used for study 1/5 th of previous study, a smaller proportion of Norovirus outbreaks estim ated as foodborne and of cour se the safer food supply. The current estimate of foodborne illnesses was done by considering 31 known agents of foodborne illnesses Scallan et al., 2011. The data were collected from the period of 2000 2008 and estimates wer e based on U.S population in the year of 2006 Scallan et al., 2011. Pathogens Associated with Raw Produce O utbreaks Produce associated outbreaks can be caused by bacteria, viruses and parasites. From 1973 1987, around 90% of the foodborne illnesses from all sources, including produce were reported to be associated with bacterial pa thogens Bean et al., 1990. Similarly in the next decade 1990 2000, bacterial pathogens causes maximum foodborne illnesses associated with all the sources Mead et al., 199 9. On the contrary, the recent analysis done by Scallan et al. 2011 have estimated that Norovirus 58% is the etiological agent behind most of the foodborne illnesses in the U.S. T he current estimate of number of foodborne illnesses caused by Norovirus is lower than the previous estimate 40% to 26% as less proportion of norovirus outbreak s are estimated to be foodborne under current estimate Scallan et al., 2011. Specifically emphasizing all produce linked outbreaks, as per the investigations done b y CSPI, from 1990 to 2005, Norovirus was the top cause of outbreaks 40%,
23 followed by Salmonella 18%, Escherichia coli 8% and Clostridium 6%. The main hazards associated with fruits were Norovirus 39%, Salmonella 28%, and Cyclospora 8%. Among vegetable outbreaks, the major pathogens were Norovirus 26%, Salmonella 21%, and C lostridium 12%. The major pathogens in produce dish outbreaks were Norovirus 51%, Salmonella 13%, E. coli 6% and Shigella 6%. According to the above data, Norov irus and Salmonella were more frequently associated with produce linked outbreaks than other pathogens during 1990 2005. Specific produce items reported to be involved with salmonellosis outbreak are alfalfa sprouts Proctor et al., 2001; CDC, 2009, jalap eno pepper Klontz et al., 2010, tomatoes Cumming et al., 2001, bean sprouts O Mahony et al., 1990, cantaloupes Mohle Boetani et al., 1999, and waterm elons Gayler et al., 1955 The risk of foodborne pathogen transmission from fresh produce is asso ciated with imported as well as domestic produce. In March 1999, FDA analyzed 1000 imported and domestic fresh produce commodities Broccoli, cantaloupe, celery, cilantro, culantro, loose leaf lettuce, parsley, green onion, strawberries and tomatoes for S almonella E. coli O157:H7 and some for Shigella spp. Among all the imported samples, 44/1003 was found positive for either Salmonella 35/44 or E. coli O157:H7 9/44 while 11/1028 domestic samples were found positive for either Shigella spp. 5/11 or S almonella 6/11 FDA, 2001; FDA 2003 Many bacterial pathogens can be isolated from raw produce Beuchat, 1996. Clostridium botulinum Bacillus cereus and Listeria monocytogenes are the bacteria which are normally present in soil whereas Salmonella, Shi gella E. coli and Camp ylobacter are believed to originate from enteric environment and contaminate raw produce through feces, sewage water, or untreated
24 irrigation water contact. Enteric pathogens primarily Salmonella and E. coli O157:H7 are among the g reatest concern as they may grow in or on fresh fruits and vegetables under favorable conditions. Growth and survival of pathogens on fresh produce depends upon factors like the type of organism, type of produce, pH, moisture, oxygen, acid, and various oth er intrinsic and extrinsic factors. Foodborne illness occurs when produce is contaminated with pathogen and sufficient number of pathogen cells remain ing viable until consumption of produce Harris et al., 2003. The minimum number of cells required to cau se illness in humans depends upon the health, susceptibility and age of individual amongst other factors Hence the term infective dose is very relative and rather dose response model s should be used to predict the probability of risk. A d ose response m odel for Salmonella was prepared by FAO/WHO in 2002, and describes the relationship between n umber of bacteria or dose ingested and lik elihood of infection. The elderly children, the women who are pregnant and immunocompromised individuals are more prone to infections. Some pathogens like Salmonella multiply inside the human body and then cause infection ; t his is called a food i nfection Harris et al., 2003. To prevent outbreaks associated with fresh produce there is need to do research with various po tential pathogens, sources and other variables influencing contami nation of fresh produce. Many pathogenic microbes have been isolated from variety of fresh fruits and vegetables at variable frequency. Prevention of contamination is the likely key required to co ntrol produce linked outbreaks. Sources of Raw Produce C ontamination Produce comes from different sources and countries and is often eaten without further processing. It is quite susceptible to contamination and must be handled
25 carefully during produ ction, harvesting, packaging, transportation, storage and cons umption. In most of the produce associated outbreaks, the exact source of contamination is not identified and different pre and post harvest sources are suspected to be the most likely causes of contamination. Pre harvest Among the pre harvest sources, soil feces, irrigation water, green or inadequately composted manure, air, wild and domestic animals are considered as likely sources of contamination Beuchat, 1996; Doyle and Erickson, 2008. Th e land or soil where the crop is grown can harbor pathogens from previous crop and can potentially transmit these pathogens to the next crop. The literature also suggests that moist soil can harbor Salmonella for 45 days Guo et al., 2002. Animal or human feces containing pathogens can likely contaminate crop fields through contact with irrigation water and manure applied in fields Cox et al., 2005; Holley et al., 2006 ; Marina and Odumeru, 2004 Properly treated or composted manure from animals is applie d as fertilizers to improve and maintain productive soils and helps in stimulating plant health. On the contrary, inadequately decomposed manure applied to soil can possibly contaminate the crops grown in the same soil Solomon et al., 2002 Salmonella in oculated into hog manure treated soil at 5 log cfu/g survived up to 180 days This observation supports the hypothesis that human pathogens can persist in animal manure Holley et al., 2006. Irrigation water is another potential route through which microb ial contaminants can come in contact with fruits and vegetables. Applying pesticides with contaminated water can highly likely contaminate plants to which it is applied. Contamination of plants through airborne transmission is not well studied However, in livestock sheds, transfer of pathogens through air has been documented which supports the concept of plant
26 contamination through airborne transmission Dowd et al., 2004. Along with airborne transmission, transmission of pathogens through animals, birds, insects contacting the outside grown produce are also po ssible sources Beuchat, 1996. Harvesting Human contacts Montville et al., 2001, gloves Jimenez et al., 2007, shirts, handling containers Sargent et al., 1989, clothes Bloomfield and Scott, 1 997; Scott and Bloomfield, 1993 used in harvesting are the various potential sources of contamination during harvesting. Infected or ill workers can transfer pathogens directly to produce by touching Todd et al., 2009 Unclean hands or harvesting tools used by healthy workers can also contaminate produce. Gloves, which are used as barrier for preventing contamination of food by handlers, can become permeable to bacteria through their subsequent use and can spread the contamination Montville et al., 2001 According to a study, gloved hands transfer Salmonella to and from fresh green bell peppers due to smooth surface of both Jimenez et al., 2007. A worker s shirt that touches produce during or immediately after harvest can be a potential source of cros s contamination. A dditionally dirty bins and buckets used to hold fresh produce like tomatoes can be additional potential sources of cross contamination during har vesting Sargent et al., 1989. Post harvest Depending upon the commodity, produce is either packed in the field and sent to destination market or it is placed in bins and sent to a packing facility. Employees, equipment cold storage units, packaging material, transport container, wash/rinse water used for washing or cross contamination from oth er food items stored in same storage unit are the various sources that can potentially contaminate produce after harvesting
27 Beuchat, 1997. Although, gloves, hair nets are worn by employees working in packing houses, ill workers, improper boot cleaning st ation, inadequate bathroom facilities are sources that can potentially contaminate the produce in a packing house. Food contact surfaces such as conveyor belts and dump tanks c an contaminate produce if not sanitized properly. Improper storage conditions i mproper temperature and moisture used to hold or transport fresh produce can be considered a point where pathogens may grow on product USDA, 1997. In the food preparation environment, cross contamination of salads with Campylobacter and Salmonella from chicken carcass via. kitchen surfaces are documented Kusumaningrum et al., 2004 The use of same utensils for fresh produce and meat products highly increases the chances of cross con tamination Chen et al., 2001. Possible Reasons behind Increase in Raw Produce O utbreaks Madden 1992 categorizes the fresh produce in the list of potentially hazardous food which includes food products that contain the nutrients necessary to support rapid and progressive growth of infectious or toxigenic microorganisms Despite the numerous prevention efforts by industry foodborne outbreaks associated with raw produce remain persistent problem in the U.S. Increased importation of fresh produce items, increased consumption of fresh produce, globalization of food supply, c hange in agronomic practices, change in eating habits, increase in susceptibility of individuals, and improved surveillance are all believed to contribute to fresh produce being linked foodborne illness outbreaks Guo et al., 2000; Nyachuba, 2010; Beuchat, 2002. I n 200 2, consumers spent ca. 46.1% of their food income outside home due to busy schedules Marriot t and Gravani, 2006. Nearly 70% of illnesses associated with tomatoes were found to be linked with restaurants where tomatoes were used primarily
28 in salads Klein et al., 2009. Increase in fresh produce consumption is observed in all the seasons, which led to an increase in importation of products from different parts of world where sometimes the safety standards are below U.S. safety standards. An i ncrease in outbreaks may also be linked to various unhygienic practices followed from field through consumption Beuchat, 1996; Beuchat and Ryu, 1997; D Aoust, 1994 The identification process or traceback, for the source and pathogen responsible for pro duc e related outbreak is slow and can lead to an increased number infect ed person due to continued consumption of the contaminated crop The foremost reason for the delay in i dentifying and controlling produce related outbreaks is the complex distribution system of produce. The complicated distribution system and the perishable nature of fresh produce, made the traceability of outbreak very difficult Harris et al., 2003. By the time the outbreak is confirmed, the produce associated with the outbreak is ra rely available to verify pathogen presence. In addition, produce is often served al ong with other ingredients that make the search of finding exact source even more difficult Tauxe et al., 1997. On an average, it generally takes several weeks to identify the source of outbreak can lead to an increased number of persons getting ill Harris et al., 2003. Cost Associated with Foodborne O utbreaks Foodborne outbreaks do not merely affect the people who suffer from infection but also cause huge loss to the res ponsible food company in terms of money and reputation. The costs involved in foodborne diseases are the medical treatment costs hospital services, physician services and drugs, business losses, loss of productivity, loss of quality of life death, pain, suffering and functional disability and costs to others in society e.g. costs to insurance companies that pay medical expenses Dalton et al.,
29 1996; Scharff, 2010. The recent estimate of economic cost of foodborne illness in the U.S. is quite compreh ensive and included all the sources of illnesses. Approximately, $152 billion is estimated to be the cost associated with foodborne illnesses, with an average cost per case being $1,850 Scharff, 2010. Data from Foodborne Disease Outbreak Surveillance s ys tem estimated that produce is responsible for $39 billion of health related costs in the U.S. every year. One illness case from contaminated produce costs on an average of $1,960, which is more than cost associated with one illness case from any other sour ce $1,850. The cost of one foodborne illness case involving non typhoidal Salmonella is $9,146, while the overall burden of Salmonella outbreaks in U.S. in one year is estimated at $14 billion Scharrf, 2010. The ten states having highest cost per case are Hawaii, Florida, Connecticut, Pennsylvania, South Carolina, the District of Columbia, Mississippi, New York, Massachusetts, and New Jersey Scharff, 2010. This cost estimate illustrates the magnitude of problem and burden of foodborne illnesses on the U.S. and the need for more efforts and research in the field of food safety. Salmonella Salmonella belongs to family Enterobacteriaceae and was named after Daniel E. Salmon, an American bacteriologist who isolated strain enterica or choleraesuis from int estine of pigs suffering from a disease with symptoms similar to those of human cholera in 1885 Bell and Kyriadides, 2000 Salmonella is a gram negative, rod shaped 0.7 1.5 2.0 5.0 m, non spore forming, oxidase negative and catalase positive bacteri a that can grow under both aerobic and anaerobic conditions Maurer and D Aoust, 2007. Most of the strains are motile in nature. Salmonellae reside in the intestinal tract of warm and cold blooded animals This enteric bacterium catabolizes D
30 glucose and other carbohydrates and produces acid and gas Maurer and D Aoust, 2007. Salmonella N omenclature Salmonella species have been known for more than 100 years and are prevalent worldwide. Nomenclature which is widely used these days is based on biochemical t esting and recognizes only two species of Salmonella i.e Salmonella enteric a and Salmonella bongori, with Salmonella enterica having six subspecies, Salmonella enterica subsp. enterica I Salmonella enterica subsp. salmonae II, Salmonella enterica subs p. arizonae IIIa, Salmonella enterica subsp. diarizonae IIIb, Salmonella enterica subsp houtenae IV and Salmonella enterica subsp. indica VI. Subspecies of Salmonella enterica are designated by Roman numerals as specified. Further sub classificati on into serotypes serovars is done at state public health laboratories D Aoust, 2007; Tindall, 2005. The serovars of Salmonella were initially named on the basis of their specific host e.g ., Typhimurium causes mouse typhoid fever. After recognizing tha t host specificity did not exist for many serovars, new strains were named based on the location from where they were isolated. To overcome the problem of long names, serovars are abbreviated however this nomenclature is also no longer used. Kauffmann Whi te scheme, also known as antigenic formula for Salmonella serovars, was the first attempt to systematically classify Salmonella into serovars or serotypes. Salmonella strains are classified into serovars or serotypes based on antigens they possess. As of 2002, more than 2,541 serovars of Salmonella ha ve been differentiated as per this scheme and the number is increasing every year D Aoust, 1989. Different Salmonella antigens include: O antigen determined on the basis of
31 lipolysaccharide, present on th e outer membrane of bacteria, H antigen, determined on basis of peritrichous flagella, Vi antigen, also known as capsule antigen occurs only in serovars Typhi, Paratyphi C and Dublin Maurer and D Aoust, 2007. Serotype is mainly based on the immunoreactiv ity of two surface structures, O antigen and H antigen. O antigen is a carbohydrate also called a polysaccharide. It is a polymer of O subunits ; each O subunit is typically composed of four to six sugars depending on the O antigen. Different sugars and d ifferent linkages between sugars produce the different antigen and are designated by mainly numbers or letters. H antigen is the filamentous portion of the bacterial flagella. H antigen is made up of protein subunits called flagellin. The ends of flagell in are conserved and give the filament its characteristic structure. H antigens are typically designated by lower case letter and numbered z s. Each serovar possess a unique combination of antigens, known as antigenic formula. Apart from serological and bi ochemical classification, phage typing is also gaining importance. The serovars are further differentiated based on their cell sensitivity to lytic activity of selected bacteriophages into phage type PT or definitive type DT Maurer and D Aoust, 2007; Bell and Kyriakides, 2000. Most nearly 1,500 of the Salmonella strains associated with foodborne illness are present under Salmonella enterica subsp. enterica, Maurer and D Aoust, 2007; Brenner et al., 2000 only 20 serovars are present under Salmonell a bongori. Some serovars of Salmonella are ubiquitous while some are rare Maurer and D Aoust, 2007 For example, S almonella Enteritidis and S almonella Typhimurium are the most common serovars associated with human illness while Salmonella Mjordan is rar ely reported Maurer and D Aoust, 2007; D Aoust, 1989 Some Salmonella serovars cause diseases
32 in one host only, for example, S almonella Gallinarum is prevalent in poultry, S almonella Cholerasuis in swine and S almonella Typhi and S almonella Paratyphi A ar e common among humans Bell and Kyriadides, 2000 The serovars which are restricted to one host only, are known as host restricted, e.g S almonella Gallinarum and S almonella Typhi; serovars that are common in one host but has potential to cause illness i n others also, are called host adapted, e.g. S almonella Dublin. Those serotypes who have broad range of hosts are called unrestricted, e.g. S almonella Typhimurium and S almonella Enteritidis Uzzau et al. 2000. Growth and Survival C haracteristics Salmonella can ad apt to wide range of growth conditions. It can grow at low temperature conditions of 2C and elevated temperature of 54C, although most of the serotypes are unable to grow below 7.0C Baker et al., 1986; Droffner and Yamamoto, 1992; Bell and Kyriadides, 2000 The growth is slow below 10C and optimum around 35 37C. Another study showed that viable cells of Salmonella species can be isolated from milk at 60 67.5C D Aoust, 1994 Salmonella has shown potential to tolerate high temperature conditions in low water activity or high fat content food materials Bell and Kyriakides, 2000. The range of pH at which Salmonella can grow is also very wide i.e ., 3.8 Asplund and Nurmi, 1991 to 9.5 Holley and Proulx, 1986, however, very few serotypes grow below p H 4.5. The optimum growth of Salmonella is observed in the pH range of 6.5 to 7.5. The water activity required for the growth of Salmonella ranges from 0.94 to 0.99 or greater. Some salmonellae are found to tolerate dry environments e.g. S. Agona D Aoust 1989. The addition of 3 4% sodium chloride generally inhibits the growth of Salmonella although salt tolerance is found to increase with increase in temperature Maurer and D Aoust, 2007. All the above facts show that Salmonella has
33 potential to grow under poor climatic conditions contribute to it as an important foodborne pathogen. Salmonella also exhibit ability to survive in frozen or low water activity products for months and sometime years, although growth is rarely observed under such conditions but viable cells can be obtained Bell and Kyriakides, 2000. Research has shown the persistence of Salmonella in dry environments and outbreak with chocolates is one of many examples Werber et al., 2004. Diseases C aused by Salmonella Salmonella can caus e two main illnesses in humans, one being typhoid or enteric fever, occurred due to ingestion of Salmonella in bloodstream and is caused by Salmonella Typhi or Salmonella Paratyphi. The o ther is gastroenteritis or non bloody diarrheal disease, occurred due to foodborne infection. It has reported in many Salmonella linked multistate outbreaks. Salmonella mainly causes self limiting illnesses but can also cause chronic diseases like reactive arthritis, osteomyelitis, enteric fever, cardiac inflammation or ne ural disorders D Aoust, 1994 Typhoid Salmonella Typhoid fever occurs due to the ingestion of food or water contaminated with S Typhi and is mainly prevalent in developing countries. Approximately, 400 typhoidal cases occur in the U.S. and nearly 70% o f these cases are associated with international travel to developing countries of Asia, Africa or Latin America Schneider and Goodrich, 2008. Typhoidal fever is typically associated with humans. Humans nearly 4% can be unapparent chronic carriers and c an shed this bacterium in their stool. The only source of typhoid fever is through fecal oral route Schneider and Goodrich, 2008. S. Typhi has potential to overcome the defense mechanism of human intestine. Upon ingestion of contaminated food or water, S almonella starts multiplying inside the body with the
34 p hagocytes Immune system cells responsible for killing bacteria, spread into the bloodstream and cause destruction of internal organs of the body as liver, spleen, bone marrow, intestines and the mese nteric lymph nodes which can potentially lead to death in humans. Symptoms of typhoid fever include fever, abdominal cramps in the first week of infection and watery diarrhea, abdominal pain, headache, nausea, loss of appetite, rose spots across abdomen, c onstipation in the second week. The use of antibiotics can gradually resolve the symptoms in subsequent weeks but infection can also turn into meningitis, osteomyelitis or other serious problems D Aoust, 1994. Incubation period for typhoid fever ranges f rom 8 to 28 days and symptoms generally persists for 4 to 8 weeks approximately in untreated individuals D Aoust, 1994; Schneider and Goodrich, 2008 Non t yphoid Salmonella Salmonellosis is a major cause of bacterial enteric illness in both humans and an imals. It is quite prevalent in many countries Notermans et al., 1992; Sewell et al., 2001; Todd et al., 2009; Werber et al., 2005. Non typhoid Salmonella NTS refers to any serotype under Salmonella, except Typhi and Paratyphi, is responsible for salmo nellosis. Although NTS infections mainly cause mild to moderate self limiting gastroenteritis, serious cases can result in death Most 90% of the illnesses, hospitalizations an d deaths in the U.S. are estimated to be caused by seven major pathogens Salm onella Norovirus, Campylobacter Toxoplasma, E. coli O157, Listeria, Clostridium perfringens Among above seven, around 11% 1.03 million of foodborne illnesses, 35% 19,586 of hospitalizations and 28% 378 of deaths in the U.S. were caused by non typ hoidal Salmonella Scallan et al., 2011. Hence, NTS is the leading cause of estimated hospitalization and deaths in U.S. as per the recent report on
35 foodborne illnes s es Scallan et al., 2011. NTS pass to humans through contaminated food and water contact just as S. Typhi does Salmonella is often believed to spread through the fecal oral route Maurer and D Aoust, 2007 It can cause serious infection among children and persons greater than 65 years of age. Individuals recovering from Salmonella have pot ential to shed the ba cteria up to three months. Non t yphoid Salmonella has been linked to animal contact, animal based foods eggs, beef especially undercooked meat products, dairy products, fresh produce and others. Many Salmonella serovars are found to be associated with produce related outbreaks Beuchat, 1996. The incubation period for salmonellosis ranges from 6 72 hours usually 12 36 hours, however, incubation periods longer than three days have been documented Rafatellu, 2008. The duration of d isease is approximately less than 10 days un like typhoid fever. Salmonellosis is localized infection colonizing intestine and the mesenteric lymph nodes as NTS rarely overcome the intestinal defense Rafatellu, 2008. Once NTS overcomes the intestinal defe nse, it can cause systemic and deadly infection. It leads to chronic clinical condition similar to typhoidal infection after migrating from intestine to other organs of the body D Aoust, 2004. Salmonella in N on host E nvironment Salmonella resides mainly in the gastrointestinal tracts of animal hosts including animals, birds, reptiles and humans for long period of time in their life Maurer and D Aoust, 2007 ; Sanyal et al., 1997 Animal hosts are considered as the primary habitat in the life of this bacte rium which provides all the required conditions sugars and free amino acids for the survival and growth of Salmonella. A fter being shed by its host species Salmonella comes into environment and can pass on to different things. The wide host range of bac terium aids its persistence in the environment. Salmonella has
36 shown ability to survive on fresh produce Beuchat, 1996, in water Boring et al., 1971; Cherry et al., 1997, soil, manure Natvig et al., 2002 and dry food products Bell and Kyriakides, 20 02. Studies have demonstrated the persistence of Salmonella in poultry, pig and cow farms. It has been collected from cow herd, ground meat of cow, cattle feed, raw materials, waste slurry from infected pigs and from various other sites Baloda et al., 20 01; Hurd et al., 2002; Millemann et al., 2000. The rate of survival in aquatic conditions and saline conditions is quite high Salmonella Chao et al., 1987. Salmonella has been found in bathrooms, toilets and have infected people indicating the potential of transmission and survival in air Barker, 2000. In addition, pigs have also shown symptoms of salmonellosis after inhaling Salmonella Fedorka Cray, 1995. Salmonella has also been shown to adhere to mineral particles Stenstorm et al., 1989. During its life cycle, s almonellae actively move between its host species and non host environment Winfield and Groisman, 2003. Salmonella can survive in non host environment for long period of time Winfield and Groisman, 2003. Salmonella experiences harsh ph ysiochemical conditions on plant surfaces Temperature, moisture, osmotic conditions fluctuate very frequently in short period of time, unlike its host environment. Lack of nutrients availability and exposure to ultraviolet radiations makes its survival ev en more difficult Hirano et al., 2000; Lindow et al., 2003. A study conducted to compare the population of human enteric pathogens with the population of leaf associated bacteria, shows that under wet and warm conditions, human enteric pathogens can grow at higher rate than leaf associated pathogens Brandl, 2002; O Brien et al., 1989. Apart from constantly fluctuating conditions experienced on plants, carbon sources found on plants help in the survival of
37 enteric pathogens. Different fruits and vegetabl es provide unique ecological atmosphere due to differences in surface morphology, internal tissue composition, metabolic activities of leaves, stem, flower roots, and tubers allowing wide variety of microbes to survive and grow on different produce Beuc hat, 2002. Intrinsic and extrinsic ecological factors naturally present in produce or imposed during different operation, greatly affect the potential of pathogens to stay and manifest their capabilities Beuchat, 2002. The knowledge and understanding of microbial ecology on fresh produce is very important for the development of strategies to prevent pathogen contamination of produce. All the above factors show the ubiquitous nature of Salmonella and its ability to survive or p ersist in non host environme nt. Salmonella and Tomatoes In the list of top ten riskiest produce items in the U.S. tomatoes stand at eighth place Klein et al., 2009. Tomatoes have been implicated in 14 multistate foodborne outbreaks of Salmonella recently from the period of 1996 to 2008 CDC, 2007; CDC, 2009; Cummings et al., 2001; Greene et al., 2008; Hedberg et al., 1999; Tauxe, 1997. According to USDA ARS nearly 1 840 cases of Salmonella have been associated with tomatoes consumption during the period of 1998 to 2006 and approx imately, 1 503 cases of salmonellosis were associated with both tomatoes and peppers consumption in 2008 Hedberg et al., 1999 Most of the outbreaks linked to tomatoes have been traced to the product originating from Virgi nia or Florida, according to FDA Tomato Safety Initiative, 2007. Salmonella strains involved in these outbreaks include Salmonella Baildon, Salmonella Thompson, Salmonella Montevideo, Salmonella Anatum, Salmonella Braenderup, Salmonella Javiana, Salmonella Newport, Salmonella Typhimurium and Salmonella Saintpaul CDC, 2008a. The trace back investigations
38 could not find the exact source of contamination and pre harvest contamination is thought to be a probable source for these outbreaks. Various suspected pre harvest sources of Salmonella contamination in tomatoes are irrigation water, soil, manure, insects, flowers and human sources Beuchat, 1996 Contamination during harvesting is also suspected as a potential source of outbreaks. F ield packed tomatoes have higher chances of contaminat ion due to direct human contact Sargent et al., 1989. The behavior of Salmonella on the surface of tomato, inside the tomato and decontamination with different sanitizers has been described in numerous studies. Salmonella on surface of t omato The behavio r of Salmonella is different on the surface of tomato and inside tomato. Outside tomato, it has to tolerate the har sh environmental conditions that do not support the growth of Salmonella in most of the times. Previous research Salmonella survival and grow th on tomato surface under suitable temperature, relative humidity, atmospheric gas composition, and other required conditions has been docu mented Maintaining high humidity and temperature during tomato storage has shown increase in growth of Salmonella on the surface of tomatoes Iturriaga et al., 2003 The persistence and growth of Salmonella on intact tomato es depends upon serovar Shi et al., 2007, inoculation dose Wei et al., 1995, inoculation site Das et al., 2006, temperature Zhuang et al., 1995, medium used to deliver bac terial cells Wei et al., 1995 and method and drying time of inoculation Lang et al., 2004. Some serovars persist longer than others Shi et al., 2007. Serovars like Hadar, Montevideo or Newport showed more persistence than Enteritidis, Typhimurium and Dublin on the surface of ripened or red tomatoes while in case of immature or green tomatoes, all the above mentioned serovars showed same persistence. Some of above serovars are
39 from tomato associated outbreaks and other s are from animal or clinical isolates Shi et al., 2007. According to one study, on the surface of mature, green tomatoes, Salmonella Montevideo showed significant growth within 7 days and 1 day at 25C and 30C storage temperature respectively, while a t 10C, it persisted on tomato surface for 18 days without any significant reduction in population Zhuang, 1995. Salmonella was found to survive on t omato skin for approximately 20 hours in distilled water Wei et al., 1995. Surface inoculation of Salmo nella on intact tomatoes through spot inoculation method was r eported as the best method when comparing dip, spot and spray inoculation. Same researchers has reported better Salmonella recovery after 1 h drying of inoculated tomatoes as compared to 24 h dr yin g indicating that method of inoculation and drying time does affect the recovery of Salmonella from tomato surface Lang et al., 2004. Inoculation site also effect the survival and growth of Salmonella Salmonella inoculated at stem scars and growth cr acks showed better survival than on tomato skin Wei et al., 1995. Stem scars are considered the location on the tomato plant where the most Salmonella infiltration can occur Das et al., 2006. Along with, intact tomatoes, Salmonella serovars showed pers istence on dry tomato leaflets and tomato stem, as well Guo et al., 2001; Rathinasabapathi, 2004. Salmonella Montevideo showed no significant reduction in population from 6.6 log CFU per leaflet on providing hydroponic nutrient and 100% relative humidity Although, approximately, 3 .0 to 3.5 log reduction in population was observed, when leaflets were dried after 48 h incubation at 60% relative humidity Rathinasabapathi, 2004. Studies related to sources of intact tomato contamination with Salmonella reve al that the transfer and growth of Salmonella on the tomato surface is possible through contact
40 with contaminated moist soil Guo et al., 2002. Controlled atmosphere packaging and modified atmosphere packaging have proved quite effective in reducing Salmo nella population from the surface of tomatoes Das et al., 2006. Salmonella in to matoes The exterior of produce is thought to be a physical barrier, preventing bacteria from penetrating to the inside of the product Tauxe et al., 1997. The outer surface of plant has a protective layer made up of cu tin and wax called cuticle that is first defense of microbial pathogens Beldin et al., 1998. The presence of outer layers including, the skin, rind, or peel on the fruit makes the permeability of pathogens dif ficult Tauxe et al., 1997. Once inside the produce, pathogens are exposed to nutrient rich environment in opposite to the surface of produce. The growth of Salmonella has been reported in many produce items like in papaya, mangoes, watermelons, pineapple s Penteado and Leitao, 2004; Strawn and Danyluk, 2010. Salmonella internalization and colonization of tomato fruit is possible through its contact with tomato stem, flowers, and cuts on skin surface Guo et al., 2001. A study conducted to identify the i rrigation water and seed stock as the possible sources of internal contamination of tomato fruit have demonstrated that contaminat ed irrigation water 7 l og CFU/ml of Salmonella Montevideo and seed stock seeds soaked in 8 l og CFU/ml of Salmonella Montevi deo for 24 h were not able to contaminate tomato fruit Miles et al., 2009. Similar results with contaminated irrigation water 5 Log CFU/ml were obtained by Jablasone et al. 2004 Conversely hydroponically grown tomatoes have shown Salmonella presen ce inside tomato after sucking up Salmonella contaminated water Guo et al., 2002 and international outbreak of Salmonella with alfalfa sprouts was reported to be caused by
41 seed contamination Mahon et al., 1997. This shows that more research and work is required to find out the ways and the sources of contamination of tomato fruit. Once Salmonella internalized tomato plant, its elimination is very difficult except cooking or any other similar kill step. Fully ripe tomatoes have low pH 3.9 4.4 which is un supportive for the growth of Salmonella Beuchat, 2002. When inside the tomato, Salmonella has to combat with its low pH for its sustenance. The potential of this bacterium to survive as well as grow in tomatoes under optimum temperature and moisture cond itions has been documented several times and was firstly r eported by Asplund and Nurmi 1991 Salmonella enterica serovars Typhimurium, Infantis, Baildon ha ve been show n to grow in tomatoes Asplund and Nurmi, 1991; Zhuamg et al., 1995; Weissinger et al., 2000. When the surface of produce is cut or bruised, fluids containing nutrients or antimicrobials are excreted, this can enhance or retard the growth of pathogens Beuchat, 2002. In case of cut or diced tomatoes, the temperature plays an important role in the growth of bacteria e.g. Salmonella Montevideo population in tomatoes at 5C, does not change for 9 days but increased significantly at 20C or 30C in 22 hours Zhuang et al., 1995. Similar l y, Salmonella Baildon showed growth on diced tomatoes at temperature conditions of 21C or 30C, and died when stored at 4C Weissinger et al., 2000. Growth of Salmonella in tomatoes was found to be enhanced by the presence of proteolytic mo ld that increases the pH of tomatoes and gives better environment for bacteria to proliferate Wade et al., 2003; Wells et al., 1999. Once inside the tomatoes, Salmonella was found to resist sanitizer treatments Burnett and Beuchat, 2007.
42 Salmonella Disinfection on P roduce O nce the produce is infected with Salmonella it becomes very difficult or almost impossible to eliminate contamination until cooked or any such heat treatment is given. Implementation of decontamination steps is imperative to reduce the risk from little to great extent. Fresh produce does not undergo p athogen reduction steps unless they are irradiated Saroj et al., 2006. The use of hot water to remove pathogenic microbes from the surface of whole or cut fresh produce has been practiced by food industry but the adverse effects on color, texture and fla vor limit the use of this treatment. The efficacy of disinfectants depends upon the nature of fruits and vegetables treated, pH and temperature of solution, bacteria targeted, concentration and time of exposure of disinfectant Parish et al., 2003. Many d isinfectants with varied success have been investigated on to matoes. Irradiation is an effective tool in controlling pathogens on the surface of raw fruits and vegetables Saroj et al., 2006. Pulse UV light has been tried and tested on raspberries and str awberries with positive outcome against enteric bacteria, E. coli O 157:H7 and Salmonella with no adverse effect on fruits Bialka and Demirci, 2008. Although many researchers have emphasized the use of irradiation for decontaminating raw produce, yet the reluctance of consumers and high cost required for irradiations are the factors affecting its popularity in food industry. Chlorine remains a convenient and inexpensive sanitizer, and is quite commonly used in food environments. It is available in three f orms sodium hypochlorite, calcium hypochlorite and chlorine gas. The recommendation for chlorine is 100 to 150 ppm free chlorine at pH 6.5 7.5 Ritenour et al., 2002; Par ish et al., 2003. At pH 7, 80% of chlorine is in hypochlorous form the active form t hat actually kills bacteria Ritenour et al., 2002. The pH of fresh Florida waters is 8 and is decreased by adding citric acid
43 mainly before adding chlorine to it Ritenour et al., 2002. Depending upon concentration of hypochlorous acid exposure time required to obtain reduction in microbial population varies typically increasing the concentration, decreases the exposure time required and vice versa. Keeping the concentrations of hypochlorous acid high is considered effective in controlling cross con tamination even when exposure time is less than 1 min Ritenour et al., 2002; Felkey, 2006. According to Wei et al. 1995 Salmonella inoculated at stem scars, unbroken surface and wounds was not disinfected with the application of 100 ppm of chlorine fo r 2 min. Zhuang et al. 1995, however, reported the effectiveness of chlorine treatment in reducing bacterial population and concluded that dipping mature green tomatoes in 60 to 110 ppm chlorine solution for 2 min can significantly reduce the Salmonella population from the surface and the core tissue. Although, increasing the concentration of chlorine did not result in complete inactivation Similarly, trisodium phosphate was found to be very effective in controlling Salmonella on the surface and core tis sues of mature, green tomatoes according to a study by Zhuang and Beuchat 1996 Significant reductions were found from the surface a nd core tissues of tomatoes on dipped in 1% and 4 15% solution of trisodium phosphate respectively. Complete elimination of Salmonella from surface was obtained by dipping tomatoes in 15% solution Zhuang and Beuchat, 1996. Chlorine was found effective in reducing Salmonella population on seeds as well. Alfalfa sprout seeds inoculated with Salmonella Stanley showed reductio n in population at chlorine concentrations of 100 and 290 ppm in 10 min. More reduction was observed in case of higher chlorine concentration. However, further increase in concentration did not led to any further reduction in population of Salmonella Jaqu ette et al., 1996. In
44 another study, alfalfa sprouts were inoculated with a five strain mixture of Salmonella and dipped in chlorine solution of concentration 200, 500 or 2,000 ppm. Population of Salmonella was reduced to undetectable levels after treatme nt with 2,000 ppm chlorine. Reduction in population was similar with the use of 200 and 500 ppm of chlorine or 2% and 5% of hydrogen peroxide, respectively when dipped for 2 min Beuchat, 1997. The major challenge in chlorine use is to maintain its concen tration in a free form. Presence of high organic matter in wash water and inaccurate pH can decrease the effectiveness of chlorine and cause increase in microbial population Ritenour et al., 2002. A study was conducted by Senter at al. 1985 to determine the microbiological changes in fresh market tomatoes during packing operations. They observed higher population of Salmonella on tomatoes washed in 114 ppm chlorine solution as compared to the controls indicating that degradation of chlorine can lead to i ncrease in bacterial population. However, increase in concentration of chlorine to 226 ppm did decre ase the enterobacteriacae count An issue raised by Garg et al. 1990 is the difference in effectiveness of chlorine rinses in laboratory and industrial en vironment which can also be attributed to the fact of inadequacy of chlorine in industrial environment at certain moments. Above all, the formation of chlorinated organic compounds such as trihalomethanes from chlorine have raised safety concerns Parish e t al., 2003. Due to all of the se issues with the use of chlorine, alternate treatments have been studied to eliminate microbial population with different rate of success on fresh produce. Antimicrobial activity was found to increase with the addition of b romine into solution containing chlorine Shere et al., 1962. Use of ozone in the elimination of
45 Salmonella from fruit, egg shell and berry surfaces is also an effective treatment Rodriguez et al., 2004. Another finding by Perry et al. 2008 emphasized the sequential use of heat and ozone as they proved to be more effective than either of them alone Ozone is also found to be effective for the treatment of fruit juices and water Patil et al., 2010; Restaino et al., 1995. Mattson et al. 2010 studied t he efficacy of four plant derived antimicrobials namely, carvacrol, trans cinnamaldehyde, eugenol and resorcylic acid in tomato wash solutions against Sa lmonella spp Although, sensory or quality analys e s were not performed during this study, reduction s in Salmonella populations were observed with the use of plant derived antimicrobials Mattson et al., 2010 After harvesting tomatoes are dumped in water to remove the field heat. Tomatoes have potential to uptake Salmonella cells and water from dump tank Bartz and Showalter, 1981. Infiltration is observed to be more from stem end scar. It occurs only when water pressure overcomes the internal gas pressure Zhuang et al., 1995. To avoid infiltration of Salmonella cells by tomatoes, the temperature of du mp tank is recommended to be 10F greater than the fruit pulp temperature. Zhuang et al. observed significantly high uptake by core tissue of tomato at 25C on dipping in 10C as compared to 25 or 37C Zhuang et al., 1995. As the temperature difference between harvested tomatoes and water is responsible for this water movement, maintaining water temperature higher than fruit pulp temperature helps in preventing this problem. Salmonella can escape disinfectants by various ways. Oliver et al. 2005, dete rmined chlorination can induce viable but not culturable state VBNC in bacterial
46 cells which can pose serious threat to wastewater decontamination methods used. Both uncombined free and combined chlorine were tested on E. coli and S. Typhimuri um and VB NC cells were obtained Oliver et al., 2005. In addition to the potential for VNBC cells, microbes can also dwell in cracks, crevices, pockets, inaccessible to chlorine and add to lack of chlorine s effectiveness. Another important issue is ingestion of b acterial cells by protozoa. Salmonella resistance to free chlorine is increased after getting ingested by protozoa. T. pyriformis provided 50 fold more resistance to many bacterial pathogens upon ingestion; bacterial pathogens can survive in chlorinated wa ter if they are inside protozoa King et al., 1988. Infiltration of bacterial pathogens into raw produce also provides shield to bacterial pathogens and they can potentially escape disinfection Ritenour et al., 2002. Bacterial T ransfer Most of the bacte rial transfer studies performed to date have been conducted in processing and handling environments considering different food contact surfaces as a potential source to transfer pathogens to and from food. Microbial transfer to any surface depends upon man y factors like bacteria involved Mackintosh et al., 1984, type of surfaces Chen et al., 2001, moisture level Gill et al., 2002 and inoculation dose involved Montville et al., 2003. All the mentioned factors affect the cross contamination rates betw een surfaces. A study conducted by Lin et al. 1997 demonstrated a strong relationship between inoculation dose of bacteria used and their transfer to knife used to cut tomatoes. Salmonella Rifampicin resistant inoculated with high dose onto the stem sc ar end was able to contaminate the center and the bottom of the tomato, while low inoculum dose was unable to contaminate the center of tomato. The knife used to cut the tomatoes was found to be contaminated as well and had
47 potential to transfer this conta mination to subsequent tomatoes cut with the knife Lin et al., 1997. Anoth er study conducted by Fravalo et al. 2009 regarding Campylobacter transfer from chicken thighs to cutting board demonstrates that the transfer of Campylobacter from naturally con taminated chicken thighs to cutting boards is inversely related to the initial l oad Transfer rates for Camp y lobacter to cutting boards were determined by dividing the ratio of bacterial cells present on the blade to the bacterial cells natur ally present o n the skinned poultry Fravalo et al., 2009 Escherichia coli transfer to iceberg lettuce through blade portion of field coring device Tao r mina et al ., 2009 and Listeria monocytogenes transfer to salami through slicer A a rnisalo et al., 2007 ; Sheen, 200 8 also demonstrated bacterial transfer potential between food and food contact surfaces. The study on transfer of Listeria to salmon during slicing was conducted by A a rnisalo et al. 2007 and after observing all the possible patterns of surface transfer, empirical equations were developed which depends on inoculation dose and slicing number Sheen 2008 developed a model to predict the transfer of Listeria monocytogenges to sal ami during slicing with a blade. Bacterial transfer studies related t o gloves have also been documented Montville et al. 2001 used a model for obtaining bacterial transfer rates between chicken an d bare hands, chicken and gloved hands, bare hands to lettuce and hands to lettuce with gloves on The transfer rate observed from chic ken to bare hands was in between 0.61 to 10.43% and chicken to gloved hands was from 0.0001 to 0.24% The transfer rate from gloved hands to lettuce ranged from 0.0003 to 0.0545%. This research also illustrated a point that bacteria can transfer from food to gloved hands and then again to
48 food from those gloved hands, which is in agreement with other finding s that gloves are permeable to bacteria and ha ve potential to transfer pathogens Tomato Production Tomatoes Solanum lycopersicum belong to the family Solanaceae and origin ate from the west coast of South America in the areas of Peru and Ecuador Olson, 2009. Botanically, tomato is a fruit because it is a mature ovary, but has always considered as vegetable because of its use in meals or as salad inste ad of desert. In 1893, U. S. Supreme Court declared it a vegetable. Tomatoes are mainly water, in 100 g of tomato fruit, 93.76 g is water. Tomatoes are rich in magnesium, potassium and phosphorous. They are consider ed as good source of lycopene, v it amin C, v itamin A and folate UDSA, 2004. One medium sized tomato provides 40% of the RDA of vitamin C ascorbic acid, 20% of the RDA of vitamin A, significant amounts of potassium, dietary fiber, calcium, plus some other vitamins in lesser amount with as few a s 35 calories Sargent, 1989. Hence, tomatoes are considered as highly nutritious vegetable. The U.S. is one of the top leading tomato producing countries. The prod uction of tomatoes in the U. S. increased by 25% from 1991 to 2007 VanSickle and Hodges, 2008. Approximately 4.1 billion lb of fresh tomatoes were produced in the U.S. for domestic supply in 2007 Vansickle and Hodges, 2008 Along with increase in production, the consumption of tomatoes has also increased in the U.S. According to USDA Econom ic Research Services, the consumption of fresh tomatoes increased by 40% in the period of 1997 1999 over the 1977 1978 period Lucier et al., 2000. Per capita consumption of raw and processed tomatoes estimated for the decade of 1990 2000 in U.S. is 16.7 lb and 75.2 lb respectively P rocessed tomato products, including
49 sauces, catsup, pastes, and salsa, account for 81% of the total tomato consumption Lucier et al., 2000. Increase in domestic consumption has increased importation of fresh produce, which accounts for 56% of total domestic consumption. Importation of tomatoes is mainly from Mexico and Canada, with Mexico being the main supplier after petitioning U.S International Trade C ommission in 1996 Vansickle, 2007. Switch in demand was observed in 2005, when the demand of greenhouse tomatoes increased among consumers. Green house tomatoes have become more popular than field grown tomatoes and make up the majority of imported tomatoes. The southeastern areas of U.S. Alabama, Florida, Georgia, North Carolina, South Carolina, Tennessee, and Virginia and California are the main tomato producing areas. The southeastern areas produces more than half 57% of the tomatoes of the Unites States which is around 2.11 million lb as per 2006 data, whereas Cali for nia produces 1.23 million lb which is 33% of total tomato production in the U.S. VanSickle, 2008. Th ese data showed that around 90% of the tomato production in the U.S. is concentrated in above mentioned regions, while the rest of the nation produces less than 10% of total tomatoes of U.S The seasons for tomatoes production in southeast U.S. and California are fall, winter, spring and spring, and summer and fall, respectively VanSickle, 2008. Florida is the largest producer of fresh market tomatoes and produced a pproximately 1.45 billion lb of fresh tomatoes in 2007 34% of U.S. total production Vansickle and Hodges, 2008 Among all the vegetable crops grown in Florida, t omatoes are the third most important crop after potato and lettuce, accountin g for more than 22% of total cash receipt FASD, 2008 Round tomatoes are more popular in
50 Florida but along with round tomatoes, R oma tomatoes, cherry tomatoes and grape tomatoes are also grown on significant acreage in Florida Roka, 2010; Van Sickle, 20 08. Florida e xports around 1.1 billion lb of tomatoes of fresh produce to the Canada and other countries. In late 1980s and early 1990s, land under tomato cultivation was more than 50 ,000 ac that decreased to in between 43 to 45,000 ac until after 200 4 05 and further to 31,500 ac in 2007 08 FTC, 1992; FTC, 2008. Florida tomato industry contributes $997 million annually to the state economy including both direct tomato sale $464 million and indirect and induced effect from these sales FAS D, 2008. It c reates 8,231 direct and indirect jobs and around $299 million goes to tomato labor income in Florida VanSickle, 2008. The main tomato growing areas in Florida are: Miami Dade county, Southwest Florida, Palm Beach Fort Pierce regions, Tampa bay area and F lorid a Panhandle area west of Tallaha ssee Sargent, 1989. The average yield in Florida is about 1 400 25 lb carton per ac Olson, 2009. Tom ato G rowing in Florida In Florida, tomato plants are first planted in greenhouses and about five weeks later seedl ings ar e transplanted to fields. One pound of seeds produces ca 140,000 tomato plants. The planting period for Florida tomatoes specifically is July Aug15 and Feb Apr15 in North Florida, Aug Sept and Jan Feb in central Florida and Aug Feb in South Florida Oslon, 2009. Dista nce between rows and plants kept in Florida for t omato production is 48 72 in and 12 31 in respectively Optimum plan t to plant distance is 27 in decreasing the distance can overcrowd the plants Olson, 2009. Most of the production is through indete rminate varieties and 7 to 8 f t stakes are used for the tall plants. Though staking increases the production costs, it improves the yield and overall quality of fruit as well. Selection of proper variety is very important to obtain good
51 yi eld. The required characteristics of good cultivar are: it should be high yielding and disease resistant. Along with this, horticultural quality of fruit like shape, size, color, smoothness, resistance to defects, adaptability and market acceptance which i ncludes ripening ability, firmness and flavor of tomato fruit are also considered Olson, 2009. Different cultural practices like bed preparation, fumigation, nutrient management, pruning, staking are required for better yield Sargent, 1989. Tomatoes ar e harvested around 100 to 120 days after the seeds are planted. The six different maturity and ripening stages as per U.S. standards for mature tomatoes: green, breaker, turning, pink, l ight red and red. Tomatoes are considered green when the tomato surfac e is green. The shade of green can vary from light to dark. Green tomatoes are not very sweet and require ripening to develop the red color and sweetness. Being a climacteric fruit, similar to cantaloupe, avocado, mango, and papaya; tomatoes can ripen afte r harvesting T aking advantage of this property of tomatoes, harvested mature green tomato can be kept in specialized storage room with the supply of natural hormone ethylene that aids in ripening of tomatoes Sargent, 1989. If harvested at proper mature green stage and given proper treatment, high quality tomatoes are developed. Mature green, round tomato is the main variety of tomato harvested in Florida, representing 90% of total tomato production. In 2002 03, 73% of Florida field tomato sales by weigh t were mature green tomatoes, down from 86 percent in 1997. The decrease of market share of mature, green tomatoes is due to availability of Canadian and European tomato products VanSickle, 2008. The second ripening stage is called breaker stage where 1 0% or less of tomato surface show definite break of color from green to tannish yellow, pink or red. Third stage is called
52 Turning stage, when greater than 10% but less than 30% of tomato surface show Tannish yellow, pink or red color. Fourth stage is pink when greater than 30% but less than 60% of tomato surface show pink or red color. Fifth stage is light red where more than 60% and less than 90% of tomato surface show red color. The final stage is red when more than 90% of surface show s a red color Sarg ent, 1989. Tomatoes harvested at red stage are very vulnerable to bruises and damages and have a shorter shelf life H arvesting of tomatoes at a stage from breaker to red is called as vine ripened tomatoes Sargent, 1989. Tomato Harvesting in Florida Fre sh tomatoes are handpicked in Florida and transferred to buckets and then to field crates. Various parameters to check the ripening of fruit are: position on the plant, size, shape, surface appearance, and presence of brown corky tissue on the stem scar. A part from these external parameters, checking internal condition of a few tomatoes is another parameter. A few tomatoes should be picked from field prior to harvesting and sliced to check its internal condition Sargent, 1989. Harvesting starts at late mo rning to protect crop from disease spread. Depending upon the market conditions, length of harvesting day can be more or less Roka, 2010. Harvesting is done by crew s with one crew having 6 categories of workers: pickers, checkers, bucket handlers, dumpe rs, row boss, and tractor drivers Zahara and Johnson, 1981. Pickers pick the right fruit and put them in buckets. After filling two buckets, tomatoes are shifted to gondolas, handlers take buckets and dump them and return the empty buckets to pickers Za hara and Johnson, 1981. Gondolas or bins should be placed close to workers to avoid the walking distance and to reduce chan ces of injury. Gondolas should not be overfilled as that can impart bruises or other defects in tomatoes. Bins, gondolas, buckets or other
53 equipment used during harvesting should be sanitized quickly to avoid cross contamination. Highly trained workers are required to do harvesting at proper stage and cause minimum damage to tomatoes. Immature, dead, decayed, defective tomatoes should be left in the field only Roka, 2010; Sargent 1989. After picking tomatoes are divided on the basis of size and color. Mechanical harvesting started in 1977, but is not popular among harvester s in Florida due to the high cost involved, more incidences o f injury of tomatoes and dirt problems on the conveyor belt Zahara and Johnson, 1981. Tomato fru it has very thin epidermis that increases its chances of getting bruised, wounded or punctured Bartz and Showlater, 1981. After harvesting, tomatoes are bro ught to packing house and dumped into chlorinated dump tanks. Adequacy of chlorine in dump tank is important to prevent cross contamination. Bruised or punctur e d tomatoes can suck up water from dump tank if the temperature of dump tank water is lower than t he temperature of pulp of tomatoes. M aintaining a dump tank s water temperature 10C higher than the tomato pulp temperature will help to control this problem Sargent, 1989. The rate dumping of tomatoes is also important as it determines the effective re moval of all the dead, bruised or unwanted toma toes from the lot Three different classifications are used for Florida tomatoes 67, 66 and 56 and larger. The minimum and maximum sizes under these classifications are 2 9/32 in and 2 19/32 in 2 17/32 in and 2 29/32 in 2 25/32 in and none, respectively FTC, 2004. After sorting green tomatoes are packed in fiberboard carton single or double layer with net weight of 2 5 lb All the cartons are staked on standard pallets, with 80 25 lb carton o n each pa llet Sargent, 1989. Pallets are then shifted to ripening room s
54 with controlled temperature 68 to 72F and relative humidity 85 to 95 % conditions that facilitate ripening. Proper temperature maintenance is very important for good color development. Hi gher temperature inhibits the red color and low temperature slows down the process. Lower temperature s can also results in chilling injury Sargent, 1989. Ethylene, a natural plant hormone that helps in ripening is used at the concentration of 150 ppm. Pr oper air circulation is must in ripening room to avoid accumulation of carbon dioxide. Accumulation of more than 1% carbon dioxide can adversely affect the ripen ing process. Around 24 72 h of ethylene exposure is required by green tomatoes to develop the a ppropriate color. Immature fruit sometimes do not develop the desired red color even after 5 days of exposure Sargent, 1989. The recommended storage temperature of tomatoes is 55F; storing tomatoes below this temperature can result in poor quality Sarg ent, 1989. Tomato Good Agricultural Practices and Best Management Practices T omatoes related outbreaks in the U.S. raised a question on the safety procedures adopted by tomato growers, packers and re packers. Contamination of tomatoes is believed to occur at every step of production. Therefore, in order to minimize the likelihood of contamination of fresh market tomatoes by human pathogens, several preventive steps are required to be taken. Keeping this in mind, T GAPs and T BMPs were developed to help the Florida tomato industry in producing safe tomato crop. T GAPs and T BMPs are considered mandatory by the State of Florida and are still voluntary in California. Florida is the first state in U.S. to implement frequent mandatory government inspection and a udit of tomato handling, production and packing to verify adherence to T GAP and T BMP practices. T GAPs and T BMPs are mandatory standards imposed
55 by the state of Florida used to ensure the safety of fresh tomatoes produced, packed, repacked, distributed and sold in Florida or from Florida DACS, 2007. The main purposes of these practices are enhancing safety of tomatoes by safely doing different operations and educating and training the workers at all levels. T GAPs and T BMPs are enforced by the co oper ative effort between Florida Department of Agricultural and Consumer Services FDACS and Florida Tomato industry and supported by scientific research DACS, 2007. Good agricultural practices course s sanitation of dump tanks and packing lines workshop, w orkshop on in plant sanitation, worker hygiene and field and plant sanitation are the major courses recommended for growers, packers, re packers and workers under T GAPs and T BMPs. These practices became effective from July 1, 2008 DACS, 2007 T GAPs ar e specifically used for field and green house production of tomatoes. T GAPs require safe distance of fields from any animal operation or animal farms where tomatoes are grown Keeping a safe distance from animal farms reduce the risk of runoff and possib le chance s of contamination. Domestic animals are required to be excluded from the tomato field during growing and harvesting as they can shed pathogens in fields A ny kind of residual material that can harbor insects or pest s should be excluded as well. F ields or green houses where tomatoes are grown are monitored on a regular basis and record s are kept. Along with the safe field conditions, all the inputs that are given to field to grow tomatoes are also regulated under GAPs. Irrigation water used for cro ps is required to be non contaminated and in case it is contaminated then proper treatments should be done before application to fields. These steps will eliminate any chance of contamination of fiel d through irrigation water that is highly
56 suspe cted sourc e of tomatoes contamination Water us ed for pesticides spray is required to be potable water having appropriate microbial standards. All the tests are recorded and given to inspectors on request. GAPs have considered worker s hygiene as a crucial part for the safety of crop s Worker s hygiene and training is a big part of these practices. Workers suffering from illness es are not allowed to do activities involving food contact and proper monitoring and documentation of worker s hygiene, training and educatio n is also required. Pesticides and fertilizers are used according to their instruction s and requirements only. Manu re used in field should be properly composted as inefficiently composted manure can harbor pathogens and contaminate the crop. Record of date of compositing, methods used for compositing and date of application of manure to field are kept for future uses under T GAPs DACS, 2007 Harvesting of tomatoes is an operation where workers c o me in to direct contact with the tomato fruit. Presence of pa thogens on raw produce during the time of harvest can directly cause human infection Beuchat, 2008. A long with the worker hygiene, all the equipment they use is also required to be free of pathogens. Harvesting crew s are required to sanitize the harvesti ng containers at least weekly or more frequently, if necessary and the use of final pack containers in the field is prohibited. The food contact surface any equipment, container that touches the produce is cleaned and sanitized routinely with permitted s anitizers. Good sanitation not only prevents infection of crops, but also reduces decay during shipping and storage. Keeping records of good sanitation practices is also crucial to show adherence to a food safety plan and to help identify potential problem areas. Good recordkeeping helps in trace back investigations and quick actions, if necessary. During harvesting, harvesting crew s are required to
57 remove any kind of dirt, debris associated with the tomatoes and dead, decayed or damaged fruit are left in f ield s only DACS, 2007 Tomato Best Management Practices T BMPs are used for packing house operations and post harvest handling of tomatoes. Packing house s and processing facilities are required to be constructed according to the requirements. The only d ifference between packing and processing facility is packing facility does not require closed structures as with processing facilities. The use of potable water meeting all the microbial standards with daily change and cleaning of dump tank is recommended under these practices. The temperature of dump tank is required to be maintained 10F higher than temperature of fruit pulp to minimize the water and microorganisms intrusions into the fruit. Diligently removal of all the dead, damaged and decayed tomatoes is mandatory. T he disinfectants should be used at proper concentration and for proper time to achieve maximum possible decontamination in processing facilities. Timely monitoring of disinfectants is required to ensure that the adequacy of disinfectant is maintained. Ensure proper hand washing and toilet facility fo r workers that are required to be cleaned and sanitized regularly. T BMPs require proper storage area for tomatoes with written sanitation procedure for coolers and storage space. Animal and pest exclusion from the area where tomatoes are handled and packed is also required. Proper pest control programs are required to be documented. Only permitted chemi cals and pesticides are to be used inside the facility. Record keeping of all the products pack ed, shipped, handled, store d transported, standard operating procedures, sanitation standard operating procedures, sanitization monitoring records, sanitation monitoring records is also required. Packing containers are required to have the
58 address of the packer or grower and tomatoes are required to have positive lot identification number. Packing of fruit in unsanitized or the incoming containers and mixing of produce from different producers is prohibited in repacking facilit ies The cleanliness of trans porting vehicles needs to be ensured before loading and requires transporters to have positive lot identification required for trace back. T BMPs require trace back provision from all who handle tomatoes at certain point during the whole process DACS, 200 7. Ex emption from T GAPs and T BMPs R equirements An i ndividual grower producing tomatoes no more than two twenty five pound boxes for one customer or selling these boxes to local farmers market are not required to follow T GAPs and T BMPs by law. Along wi th this, charitable contributions are also exempted from these practices DACS, 2007. Gloves Hands are a potential intermediate source for spreading the pathogens to food items. A survey of 81 foodborne illnesses linked to food workers conducted by Guzewi ch and Ross 1999 illustrates that 89% of the outbreaks are due to transmission of pathogens through hands. Another survey by Bean et al. in 1997 found around 36% of all the foodborne disease that occurred during between 1988 1992 2,423 outbreaks were d ue to p oor personal hygiene of workers. Workers can transmit pathogens to food from a contaminated surface, from another food, or from hands contaminated with organisms from their gastrointestinal tract. Workers who no longer show symptoms of salmonellosis can continue shedding Salmonella in their stools for five weeks as asymptomatic carrier, and may constitute significant risk. The resident microflora that is almost always present on the skin is not of major concern in term of human infections
59 however transient pathogenic microorganisms that temporarily stay on the hands or the skin are of greater concern Paulson, 1996. Pathogens believed to transmit through workers are E. coli Hepatitis A virus, Salmonella spp., Shigella spp., and Clostridium perfri ngens Restaino and Wind, 1990; Synder, 1997. Salmonella present on surface even in very low number 120 organisms/cm 2 has potential to transfer to finger tips of workers and further to food touched with contaminated fingers Scott and Bloomfield, 1990; P ether and Glibert, 1971. After contamination from worker hands, multipli cation of pathogens can occur under favorable conditions of temperature or mois ture To prevent the cross contamination gloves are recommended by researchers to act as a barrier to b are hand contact with food. Research on gloves is mainly conduc ted in healthcare settings that are not similar to the food service or food production and processing In food establishments, gloves are used for the purpose of preventing pathogens from trans ferring to food via hand s contact from workers Paulson, 1996. Gloved hands are considered more protective than bare hands in transferring bacteria as per the various studies conducted. A stud y conducted by Montville at al. 2001 emphasized the importan ce of gloves in reducing microbial transfer from bare hands to chicken and from chicken to bare hands. During harvest, g loves also help in protecting harvesting crew from coming in contact with the different chemicals sprayed on tomato crop. Many pesticide s, insecticides and fungicides are sprayed to tomato crop during production period and most workers wish to limit their exposure to these chemicals. G loves are recommended for use during picking or harvesting of tomatoes. Although gloves are found to be mo re protective as compared to bare hands they cannot be considered completely safe. Transfer of pathogens from food to gloves
60 hands has also been documented Mont ville et al., 2001. This shows that gloves are permeable and bacteria ha ve the potential to p ass t hrough them. In addition, gloves promote a false sense of security among workers. Contaminated gloves transfer pathogens to different patients in hospital settings Ehrenkranz, 1992. Glove use can also promote poor hand washing and more accumulation of pathogens on hands. Washing the hands prior to and after glov e use is highly recommended. W earing gloves without hand washing may lead to the contaminat ion of both the inner and the outer part of glove. Avoiding washing hands and not wearing gloves can provide good moist and warm environment for the transient pathogens present on the surface of hands and can lead to their growth Larson et al., 1989. Loose fitting and very tight gloves enhances the chances of growth and more transfer of microorganisms Larson et al., 1989 Different types of gloves are used in food establishments including disposable and reusable gloves. Disposable gloves are typically very thin 4 8 mils thick. They provide poor chemical resistance but better touch sensitivity. They s hould never be reused and changed frequently. They are not very protective against hazardous chemicals and bio hazardous material. Disposable gloves can tear very easily. Reusable gloves, on the other hand are typically 18 28 mils thick. They are much bett er than disposable gloves against strong chemicals and hazard s Reusable gloves have a long cuff made of same kind of material which can provide extra protection. Reusable gloves should be washed and sanitized properly before reused. Reusable rubber gloves are mainly used for cleaning purposes Imperial College London 2005. Latex gloves use in medical facilities has higher chances of retaining the microorganism because of its three dimensional lattice structure responsible for elasticity also. Washing wit h
61 several different antimicrobials has little effect in the removal of bacteria from latex surface. This provides very useful information that microorganisms can adhere to the latex gloves and cannot be easily remove d from them Doebbeling et al., 1992. T omatoes have been linked to several outbreaks in the recent years. T ransfer of human pathogens following human contact infected employees during harvesting has been documented Todd et al., 2009. S etting up and maintaining an appropriate sanitation prog ram throughout handling is important. Unde r GAPs g love use Disposable or Reusable or bare hands with frequent hand washing is recommended dur ing tomato harvesting. When single use or disposable gloves are used the washing and sanitizing prior to the us e of glove s is suggested FDA, 1998. In Florida tomato packing house, following T BMPs disposable gloves are mainly preferred and changed very frequently for example after touch ing h air, mouth or any other surface except tomato. Disposable gloves are no t reused in toma to packing houses in Florida. Reusable gloves are required to be made of easily cleaned or sanitized material as pre FDA guidelines and are required to be cleaned and sanitized when required. Reusable gloves are required to be kept at safe a nd sanitized place when not used by employees. Reusable gloves are used in Florida during harvesting of tomatoes and reused next day after sanitizing FDA, 1998. T GAP s do not have any recommendatio n to change the gloves during harvesting, in turn, permit ting workers to keep using gloves until torn Focus of Research The level of Salmonella transfer from harvesting surfaces to or from tomatoes is a n understudied topic needing more attention and research. In this research we have tried to quantify the risk associated with gloves, which is considered as the potential routes of cross contamination during tomato harvest. The two types of gloves were
62 tested single use latex gloves and reusable rubber gloves. Reusable gloves were also made dirty to evaluate if the risk associated with glove use changes over the course of the day as gloves become soiled. The outcomes obtained from this work are helpful in providing science based recommendations to tomato growers and packers regarding their gloves use policies
63 C HAP TER 3 MATERIAL AND METHODS Preliminary Experiments Preliminary experiments were performed to gain insight into experimental procedures an d to further define variables. Preliminary e xperiments were performed to determine the carrier medium required for p reparing Salmonella cockta il, the drying time for inoculum on the surface the culture media for recovering Salmonella cells and to make gloves dirty similar to real tomato harvesting situation. Carrier M edium The following carrier medium s were evaluated: water, 0.1% Peptone water Difco, Becton Dickinson, MD, Tryptic Soy Broth TSB Difco, Becton Dickinson, Sparks, MD, 5% Horse serum HS and Butterfield s phosphate buffer BPB Hardy Diagnostics, CA. The carrier mediums were used to prepare Salmonell a cocktail see below and experiments were performed by inoculating single use gloves and touching with mature green, round tomatoes, imme diately and after 1 h inoculum drying Each group consisted of three samples, and only one replication was performed for all the carrier mediums n=3 except for 0.1% peptone water, which was re plicated for four times n=12. Inoculum Drying T imes To evaluate the inoculum drying time on surface of gloves different drying times were selected including immediate sampling, 30 min, 1 h, 1 h 30 min, 2 h and 3 h. Experiments were performed by inoculating single use glove s see below and touching with tomatoes. Peptone water 0.1% was used as a carrier medium for the trial. Each
6 4 replication consisted of three samples and two replications n=6 were performed at ea ch drying time except 1 h, for which four replications were performed n=12. Culture M edia Tri a l experiments were also performed to determine the suitable culture media for the experiments. Tryptic soy agar TSA ; Dif co, Becton Dickinson, Sparks, MD ; non selective, Bismuth sulfite agar BSA ; Difco, Becton Dickinson, Sparks, MD ; selective and Xylose lysine deoxycholate agar XLD ; Difco, Becton Dickinson, Sparks, MD ; selective supplemented with rifampicin 100 g/ml; TSAR, BSAR, XLDR Fisher Bioreagents, Fair Lawn, NJ were used to enumerate bacterial population duri ng trial. The main comparison was being made between the two selective media BSAR and XLDR. Single use gloves, mature green, round tomatoes and water as a carrier medium we re used to obtain the data. O ne replication with t hree samples was performed for all the culture media n=3. The inoculum was dried in bio safety cabinet for 0 min, 15 min, 30 min, 45 min and 60 min The purpose was to evaluate the tw o selective media types BSAR and TSAR under the combination of carrier medium, and inoculum drying time to evaluate where maximum Salmonella transfer and subsequent enumeration occur. Dirty Reusable G loves To soil reusable gloves similar to real tomato harvesting field, different quantities of soil 0.5 g, 0.3 g, 0.1 g, different volumes of tomato internal tissue 1/4 TSP, 1/8 TSP, a drop and a tomato leaf were rubbed on glove piece for 5 s to 30 s with 5 s interval. Based on appearance, 0.1 g of soil, one drop of tomato internal tissue and a tomato leaf were selected to rub on glove piece for 20 s. Experiments were also performed with differently inoculated dirty glove pieces. One set was immediately
65 inoculated after making dirty, while other set was d ried for half an hour and then inoculated. The purpose was to observe, if any, differences in Salmonella transfer from differently dried and inoculated gloves to tomatoes. Tomatoes Round, mature green tomatoes were purchased from local sources and stored a t 40F before use. After washing with cold water, tomatoes were stored overnight at room temperature prior to inoculation. A circle of 2 to 3 cm diameter was drawn on the surface of toma toes on the day of inoculation. Gloves To perform experiments with glo ves, clean gloves Reusable or Single use were purchased from local sources and were cut into 5 cm 5 cm square size pieces with sterile scissors. For experiments with dirty reusable gloves, clean reusable gloves were cut into 5 cm 5 cm and rubbed with a tomato leaf for 20 s. Salmonella S trains Five rifampicin resistant Salmonella strains used in experiments were: Salmonella Michigan MDD 251 ; Cantaloupe, Salmonella Montevideo MDD 236 ; Almond survey Danyluk et al. 2007, Salmonella Newport MDD 314 ; Tomato outbreak Greene et al. 2008 Salmonella Poona MDD 237 ; Cantaloupe outbreak CDC, 1991, and Salmonella Saintpaul MDD 295 ; Orange juice. A stock solution of rifampicin was prepared by adding 1 g of rifampicin in 20 ml of methanol. The solution w as then filter sterilized Nalgene 0.20 m pore size, Rochester, NY, wrapped in foil and stored in refrigerator at 42C until used. In 1 L media, 2 ml of stock solution of rifampicin was added to obtain the final concentration.
66 Cocktail P reparation Sal monella cultures, stored at 80C in cryogenic vials were obtained from Danyluk laboratory culture collection prior to each experiment, thawed and then were streaked onto Tryptic Soy Agar plates supplemented with rifampicin 100 g/ml; TSAR followed by ov ernight incubation 24 2 h at 372C. One isolated colony was transferred to 10 ml of tryptic soy broth supplemented with rifampicin 100 g/ml; TSBR, and incubated for 24 h at 372C. Following this incubation, a 10 l loopful was transferred into 10 ml of fresh TSBR and incubated for 24 h at 372C. Cultures were collected by centrifugation Allegra X 12, Beckman Coulter, Fullerton, CA at 3 000 rpm for 10 min. The cells were washed two times by pouring the supernatant and suspending in 10 ml of BPB. Washed cells were then suspended in BPB with half the initial volume 5 ml. The resultant cultures had ca. 10 9 CFU/ml bacterial population Serial dilutions were performed twice in 9 ml of 0.1% peptone to achieve concentration of ca. 10 7 CFU/ml. Equal vol ume of each Salmonella strain was collected in to a cocktail and vortexed. The Salmonella cocktail was then suspended in 1 ml size micropipettes and microcentrifuged Eppendorf, Minispin Hauppauge, NY at 13,400 rpm for 10 min. The supernatant was dumped o ff from all micropipettes and 1 ml of 0.1 % peptone water carrier medium was added to each. The cocktail was vortexed and stored on ice for up to 1 h, prior to inoculating gloves or tomatoes. Inoculum P rocedures Tomatoes or glove pieces were inoculated i n bio safety cabinet with the Salmonella cocktail. A 100 l cocktail of bacterial cells were distributed in 6 to 8 drops on the glove pieces or circled area of tomatoes Iturriaga et al., 2006. Inoculum was dried for 0 h, 1 h or 24 h prior to transfers
67 T ransfer S cenarios Cle an Reusable or Single use or Dirty Reusable Gloves to T omatoes The uninoculated tomatoes were touched with the inoculated glo ve surfaces, for less than 5 s and placed into a sterile sampling bag 17.78 cm 30.48 cm Fisher brand, Pittsburg, PA. Samples were taken at three different drying times: 0 h wet inoculum, 1 h and 24 h along with one control sample. Each replication consisted of three inoculated gloves samples for each drying time and three samples for control. Three add itional glove samples were placed in each group and sampled with similar protocol as tomatoes, to determine the population of Salmonella on inoculated surface. A total of six replication s were performed n=18. Tomatoes to Clean Reusable or Single use G l oves Inoculated tomatoes were touched with g love pieces, for less than 5 s and placed in sterile sampling bags in similar way as explained in the above transfer scenario. Sampling method, sampling time and number of replications were also similar to the gl oves to tomato scenario described above Cle an Reusable or Single use or Dirty Reusable to Many T omatoes Inoculated glov es were touched with 25 sequential tomatoes clean gloves and 10 sequential tomatoes dirty gloves, one after the other and placed in sampling bags. Samples were taken immediately after inoculation 0 h a nd after 1 h drying of inoculum. Experiments were replicated three times with three samples at one time point n=9. Enumeration of P athogen Twenty milliliters of BPB was added to t he sampling bags containing the originally inoculated surface and the transfer surface with clean gloves were used. For all work involving dirty gloves, 20 ml BPB with 0.1% Tween 20 was added to the sampling bags.
68 Tween 20 was added to help in removal of cells from soiled surfaces Raiden et al., 2003. Sampling bags with tomato samples subjected to a gent l e rub shake rub for 1 min and glove samples were stomached Smasher, AES Lab, Cranbury, NJ for 1 min prior to enumeration. Serial dilutions were prepar ed using 0.1% peptone water 9ml and liquid in bags was surface plated 0.25 ml in quadruplicate and 0.1 ml in duplicate onto both TSAR and BSAR media to increase the limit of detection The colonies present after incubation at 372C for 242 h TSAR o r 482 h BSAR incubations were counted. Enrichment When counts fell below the limit of detection 1.3 log CFU/surface, enrichment for Salmonella was conducted by the US Food and Drug Administration Bacteriological Analytical Manual FDA BAM protocol fo r produce FDA, 2007. Briefly f or Salmonella 20 ml of double strength lactose broth Difco, Becton Dickinson was added to the sterile sampling bags and incubated at 37 2C for 24 h. One hundred microliters and 1 ml of mixture was then transferred to 9.9 ml tubes of Rappaport Vassiliadis R10 RV; Difco, Becton Dickinson and tetrathionate TT; Difco, Becton Dickinson broths, respectively. Test tubes were incubated for 48 h at 42 2C for RV and 24 h at 37 2C for TT. A 10 l loopful was streaked onto BSA, XLD, and Hektoen Enteric agar HE; Difco, Becton Dickinson, and incubated at 37 2C for 24 h. Salmonella positive colonies are black with metallic sheen on BSA, red with black centers on XLD, and blue green with or without a black center on H E. Positive colonies were selected and transferred 10 l needle to triple sugar iron agar TSI; Difco, Becton Dickinson slants and lysine iron agar LIA; Difco, Becton Dickinson. Slants were incubated at 37 2C
69 for 24 h. A confirmed Salmonella enri chment results in TSI slants have a pink top and black bottom with gas formation and LIA tubes are black or no color change. Transfer Coefficients Transfer coefficients are calculated by the following equation: TC = P Where P t = Salmonella pop ulation on touched surface which can be tomato or glove CFU/surface, P i = Salmonella population on inoculated surface CFU/surface. Statistics All the results obtained were appropriately averaged to get the final counts. Transfer coefficients obtained w ere analyzed using Statistical analysis system SAS 9.2; SAS institute inc., Cary, NC, USA for analysis of variance ANNOVA and significance was determined by least square significant test at p<0.05.
70 CHAPTER 4 RESULTS Preliminary Experiments Carrier M ed ia The population of Salmonella obtained immediately after inoculat ion and after 1 h of inoculum drying with 0.1% peptone water as carrier medium were 5.1 0.2 log CFU/tomato and 5.0 0. 0 log CFU/ tomato, which were higher than 5% Horse Serum 3.00.6 log C FU/ tomato and 2.50.0 log CFU/ tomato and TSB 4.90.2 log CFU/tomato and 4.80.0 log CFU/tomato at both drying times Table 4 1; Figure 4 1. The population of Salmonella obtained from 0.1% peptone water was similar to water 5.1 0.2 log CFU/tomato when inoculum was wet, however, after drying the inoculum for 1 h, Salmonella population obtained from water 4.9 0.1 log CFU/ tomato was lower than the population obtained from 0.1% peptone water Table 4 1; Figure 4 1 Comparing BPB 5.00.1 log CFU/tomato and 5.00.1 log CFU/tomato and 0.1% peptone water demonstrated that population of Salmonella obtained from wet inoculum was lower in case of BPB. Hence, 0.1 % peptone water was selected as carrier medium for further experiments. Drying T ime Transfer of Sa lmonella to tomatoes was not observed after 1 h of inoculum drying on gloves as it dried out with the exception of one experiment with 5% Horse Serum, where it transferred up to 2 h Table 4 2; Figure 4 2. Salmonella population obtained after 15 min of inoculum drying 5.20.2 log CFU/tomato, 30 min 5.10.2 log CFU/tomato and 45 min 5.00.2 log CFU/tomato was almost equal to the population obtained from wet inoculum 5.10.2 log CFU/tomato Table 4 2; Figure 4 2. However
71 drying the inoculum for 1 h showed lesser Salmonella recovery 4.80.2 log CFU/tomato. Thus, future experiments included immediate sampling upon inoculation and 1 h of inoculum drying. Culture M edia On comparing results obtained from TSAR, BSAR and XLDR Table 4 3; Figure 4 3. X LDR media showed almost 1 log lower Salmonella recovery from wet inoculum 4.60.3 log CFU/tomato as well as 1 h dried inoculum 4.50.3 log CFU/tomato as compared to TSAR 5.10.2 log CFU/tomato and 5.10.1 log CFU/tomato and BSAR 4.90.5 log CFU/toma to and 4.90.4 log CFU/tomato. TSAR and BSAR were selected as culture media for the research. Dirty Glove P rotocol There were no significant differences in the recovery of Salmonella log CFU/tomato from tomatoes touched with four different dirty gloves D1, D2, D3 and D4 Table 4 4 or from two different dirty gloves D1 and D2 touched with inoculated tomatoes p "e0.05 Table 4 5 For further experiments gloves were made dirty by rubbing a tomato leaf with it for 20 s. After one hour of inoculum drying, the population of Salmonella recovered from tomatoes touched with inoculated dirty gloves was below the detection limit in all the four cases D1, D2, D3 and D4. However, the population of Salmonella obtained from two dirty gloves Log CFU/glove touched with inoculated tomatoes did not differ significantly between wet and dry inoculum conditions p "e0.05. Experiments were also performed with two differently inoculated dirty gloves. One set of dirty gloves were made dirty by rubbing a tomato leaf for 20 s and then immediately inoculated Table 4 6, while others were made dirty in simila r pattern and dried at room temperature for half an hour before inoculating Table 4 7. No significant
72 differences were observed in recovery of Salmonella from tomatoes touched with differently inoculated dirty gloves p "e0.05. All the further experiments were performed without drying t he dirty gloves for 30 min Salmonella Transfer from Clean Reusable Gloves to T omatoes When the inoculum was wet, Salmonella population obtained from inoculated clean reusable gloves was 5.9 0.1 log CFU/glove, while 5.20.1 log CFU/tomato was obtained from tomatoes touched with gloves Table 4 8; Figure 4 4. Transfer coefficients obtained from wet inoculum were 0.250.1 i.e ., 25%. On drying the inoculum for 1 h, ca 5.40.2 log CFU/glove and 5.10.1 log CFU/tomato were obta ined from inoculated clean reusable gloves and tomatoes, respectively. Transfer coefficients obtained after 1 h of inoculum drying were 0.480.5 i.e ., 48%. TCs obtained after 1 h were significantly different than TCs obtain ed from wet inoculum p "d0.05. Distribution of TCs can be seen in tables x y, demonstrating a near normal distribution for results received. Statistical analysis performed for TCs obtained from two different media TSAR and BSAR at different inoculum drying times, showed no significant differences p "e0.05. Hence, all the rest of analysis was performed using TSAR values only. Drying the inoculum for 24 h, recovered <2.20.7 log CFU/glove and <1.40.2 log CFU/tomato of Salmonella population from inoculated clean reusable gloves and tomat oes, respectively. Among all samples, one inoculated glove sample and six tomato samples were below the detection limit after 24 h of inoculum drying. Salmonella Transfer from Single use Gloves to T omatoes Salmonella population transferred from wet 0 h i noculated single use gloves and tomatoes was 6.00.1 log CFU/glove and 5.40.3 log CFU/tomato and the transfer coefficients were 0.320.1 i.e ., 32% ; Table 4 9 ; Figure 4 27 After drying the
73 inoculum in biosafety cabinet for 1 h, 5.90.4 log CFU/glove a nd 5.40.1 log CFU/tomato of Salmonella population was obtained from inoculated single use gloves and tomatoes, respectively. Transfer coefficients obtained after 1 h inoculum drying were 0.290.2 i.e. 29% and were significantly different than TCs obtai ned from we t inoculum p "d0.05. After drying the inoculum on glove surface for 24 h, 2.50.5 log CFU/glove of Salmonella population was obtained from single use gloves, while all the tomato samples touched with inoculated gloves were below the detection limit upon en umeration Table 4 9; Figure 4 27 There were no significant differences between TCs obtained from tomatoes touched with clean reusable and single use gloves at different drying times 0 h and 1 h p "e0. 05 Table 4 8; F igure 4 4; Table 4 9; Figure 4 27 Drying the inoculum on glove surface for 24 h, showed similar results, n o significant differences in number of Salmonella positive tomato samples were obtained between clean reusable and single use gloves after 24 h of inoculum drying p "e 0.05 However, T Cs obtained from 1 h dried inoculum were significantly higher than TCs obtained from wet inoculum p "d0.05, which declined after 24 h drying, when statistical analysis was performed with both the gloves together. Salmonella Transfer from Dirty Gloves to T o matoes When the inoculum was wet 0 h drying, Salmonella population obtained from inoculated dirty gloves and tomatoes were 5.50.2 log CFU/glove and 5.00.2 log CFU/tomato, respectively Table 4 10 ; Figure 4 14 TCs obtained from wet inoculum were 0.41 0.3 i.e ., 41 % Table 4 10 Drying the inoculum on dirty glove surface for 1 h reduced the recovery of Salmonella from inoculated gloves as well as from tomatoes touched with dirty gloves. Population obtained from dirty gloves after 1 h inoculum
74 drying w as 3.40.2 log CFU/glove, while for tomato samples, enrichments were performed and five tomato samples touched with inoculated gloves came out positive Table 4 10; Figure 4 14 There were no significant differences in TCs calculated for dirty and clean r eusable gloves, when the inoculum was wet p "e0.05 Table 4 8; Fi gure 4 4; Table 4 10; Figure 4 14 Drying the inoculum on clean and dirty glove surface for 1 h gave similar results. N o significant differences in number of Salmonella positive tomato samples were obtained between inoculated clean a nd dirty reusable gloves p "e0.05. Salmonella Transfer from Tomatoes to Clean Reusable G loves Salmonella population obtained from inoculated tomatoes and transferred to clean reusable gloves on immediate 0 h sampling was 6.00.1 log CFU/tomato and 5.40. 3 log CFU/glove, respectively Table 4 11; Figure 4 1 7 TCs obtained from wet inoculum were 0.180.0 i.e ., 18% Table 4 11 After drying the inoculum on tomato surface for 1 h, 5.60.4 log CFU/tomato and 5.40.1 log CFU/glove of Salmonella was obtained from tomatoes and clean reusable gloves. TCs after 1 h of inoculum drying were 0.380.2 38% and was not significantly different from TCs from wet inoculum p "e0.05 Table 4 11. After drying the inoculum on tomato surface for 24 h, 4.10.2 log CFU/tomato of Salmonella was obtained from inoculated tomatoes while <2.80.1 log CFU/glove was found to transfer to glove surface Table 4 11 ; Figure 4 1 7 Two glove samples out of total 9 samples were below the limit of detection upon enumeration after 24 h of drying Table 4 11. Salmonella Transfer from T omato t o Single use G loves Salmonella population obtained from inoculated tomatoes at 0 h was 5.70.4 log CFU/tomato and that recovered from gloves was 5.40.2 log CFU/ glove Table 4 12 ;
75 Figure 4 22 TCs obtained from wet inoculum were 0.370.2 i.e ., 37 % Ta ble 4 12. After drying the inoculum for 1 h, Salmonella recovered from inoculated tomato and gloves were 5.40.4 log CFU/tomato and 5.20.5 log CFU/glove, respectively Table 4 12; Figure 4 22 TCs obtained after 1 h drying 0.390.2 were not significan tly diffe rent from those obtained from wet inoculum p "e0.05. When the inoculum was dried for 24 h on tomato surface, 3.40.6 log CFU/tomato of Salmonella population was obtained from inoculated tomato surface, while all the glove samples were below the limit of de tection upon enumeration Table 4 12; Figure 4 22 There were no significant differences in TCs obtained from clean reusable and single use gloves touched with inoculated tomatoes at different drying times 0 h and 1 h p "e0.05 Table 4 11; Figure 4 1 7; Ta ble 4 12; Figure 4 22 However, after drying the inoculum on tomato surface for 24 h, significant differences in number of Salmonella positives were observed between clean reusable and single use glove samples, with clean reusable gloves showing more po sit ives p "d0.05. Drying the inoculum on tomato surface for 1 h did not significantly affect the Salmonella transfer from inoculated tomatoes to clean gloves p "e0.05. Salmonella Transfer from Clean Reusable Gloves to Twenty five T omatoes Transfer coefficient s obtained from first tomato touched with inoculated clean reusable gloves wet inoculum were 0.610.6 and decreased continuously up to ninth tomato 0.010. 0 Table 4 13 ; Figure 4 27 The remaining tomatoes touched with inoculated clean reusable gloves w et inoculum yield <0.01 transfer From tomato 10 to tomato 15, six or seven samples out of nine total samples were positive upon enrichment for Salmonella Table 4 13 ; Figure 4 27 From the 16 th to 23 rd tomato, at least two and at most five samples were p ositive upon enrichment Table 4 13 ; Figure
76 4 27 and for last two tomatoes 24 th and 25 th touched with gloves, none of the sample came out positive. Transfer of Salmonella was observed up to 23 tomatoes touched subsequently with inoculated clean reusable gloves wet inoculum. Significant differences in TCs were observed between first tomato touched with gloves and third tomato up to ninth tomato, when the inoculum was wet p "d0.05. On drying the inoculum for 1 h, TCs obtained from first tomato were 0.861.7 and decreased for subsequently touched tomatoes Table 4 14 ; Figure 4 27 TCs obtained from the fifth tomato touched with one hour dried inoculum were 0.12 0.3 Table 4 14 ; Figure 4 27 From tomatoes six to 16 tomato, at least three and at most seven samples out of nine were positive upon enrichment Table 4 14 ; Figure 4 27 Tomatoes 17 to 19 showed two positive samples Table 4 14 ; Figure 4 27 The remaining tomatoes touched with inoculated clean reusable gloves tomatoes 20 25, only the only positive enrichment was one of nine samples from the 22 nd tomato Table 4 14 ; Figure 4 27 There were no significant differences between TCs obtained from tomatoes touched with wet 0 h and dry 1 h Salmonella inoculum, comparing TCs obtained from first five tomatoes only p "e0.05 Table 4 13; Figure 4 27 ; Table 4 14 Salmonella Transfer from Single use Gloves to Twenty five T omatoes When the inoculum wa s wet on single use gloves, TCs obtained from first to tenth tomato, decreased from 0 .330.4 to 0.010.0 Table 4 15 ; Figure 4 28 From the 11 th to 15 th tomato, at least five and at most eight samples were positive upon enrichment. From the 16 th to 23 rd t omato, at least two and at most four samples were positive for Salmonella The last two tomatoes touched with inoculated single use gloves were positive with one out of nine samples Table 4 15; Figure 4 28 Significant differences
77 in TCs were obtained be tween first tomato and seventh tomato up to ninth tomato touched with inoculated gloves p "d0.05. When the inoculum was dried for 1 h, TCs obtained from first tomato were 0.490.3 and decreased to 0.010.0 up to 8 th tomato Table 4 16 ; Figure 4 28 For th e ninth tomato, eight out of nine an d for tenth tomato, six out of nine samples, were positive for Salmonella upon enrichment. For all the rest of the tomatoes 11 to 25 at least one sample and at most four samples were positive upon Salmonella enrichme nt Table 4 16 ; Figure 4 28 There were no significant differences in TCs obtained from wet and 1 h dried inoculum, comparing TCs obtained from five tomatoes only p "e0.05 Table 4 15; Figure 4 28; Table 4 16 Since no significant differences were obtained between wet and dry inocula, comparisons between clean reusable and single use gloves were made using wet inoculum TCs only. St atistical analysis showed that TCs obtained from first two tomatoes T1 and T2 touched with clean inoculated reusable gloves were significantly higher than all the tomatoes touched with inoculated single use gloves, expect first tomato Table 4 13 ; Fig ure 4 27; Table 4 15; Figure 4 28 This implies that Salmonella transfer from clean reusable gloves to first two tomatoes was higher, which after that was similar as single use gloves. However, from single use gloves, statistically insignificant TCs were obt ained up to seventh tomato. Thus, single use gloves transfer less Salmonella ca. 33% to more number of tomatoes touched, while clean reusable gloves transferred more Salmonella 61% to fewer tomatoes. Salmonella positive tomato samples obtained after en richment of the 10 th to 25 th tomato from both clean reusable and single use gloves were not significantly different from each other p "e0.05.
78 Salmonella Transfer from Dirty Reusable Gloves to Ten T omatoes From wet inoculum on dirty reusable gloves, TCs obtained decreased from 0.220.2 to <0.010.02 for first tomato up to ninth tomato Table 4 17 ; Figure 4 29 For the tenth tomato touc hed with inoculated wet dirty reusable gloves, 8 samples out of 9 were positive for Salmonella upon enrichment. There were no significant differences in TCs obtained from all the ten tomatoes touched subsequently with inoculated dirty reusable gloves wet i noculum p "e0 .05 Table 4 17; Figure 4 29 After drying the inoculum on g love surface for 1 h, at least two and at most five samples came out positive upon Salmonella enrichment for all the ten tomatoes touched subsequently Table 4 18; Figure 4 29 Dryi ng the inoculum on dirty reusable gloves for 1 h showed almost equal chances of Salmonella transfer to all the ten tomatoes touched subsequently. Significant differences were observed between TCs obtained from tomatoes contacted subsequently with inoculate d clean and dirty reusable gloves wet inoculum p "d0.05. Table 4 13 ; Figure 4 27; Table 4 18; Figure 4 29 Similarly to the case of single use gloves, the transfer coefficients obtained from first two tomatoes touched with clean reusable gloves were significantly different from the TCs obtained from all ten tomatoes touched with dirty reusable glove p "d0.05. When the inoculum was wet, clean reusable gloves transfer more Salmonella 0.610.6 to first two touched tomatoes, while the Salmonella transfer to next eight tomatoes touched with clean reusabl e gloves and all the ten tomatoes touched with dirty reusable gloves, is similar. Comparing clean and dirty reusable gloves after 1 h of inoculum drying showed significant differences in number of Salmonella positive tomato samples, with clean reusable glo ves showing more transfer p "d0.05.
79 Table 4 1: Salmonella transfer from inoculated single use glove to tomato using different carrier mediums n = 3 12. Salmonella population log CFU/tomato Carrier Media 0 min. wet inoculum 1 h dry inoculum Wate r 5.10.2 4.90.1 0.1% Peptone water 5.10.2 5.00.0 Butterfield s Phosphate Buffer 5.00.1 5.00.1 5% Horse Serum 3.00.6 2.50.0 Tryptic Soy Broth 4.90.2 4.80.0
80 Table 4 2: Salmonella transfer from inoculated single use glove to tomatoes at d ifferent drying times using 0.1% peptone as a carrier media n=6 12. Drying time min. TSAR BSAR 0 5.10.2 4.90.1 15 5.20.2 5.00.1 30 5.10.2 4.60.1 45 5.00.2 4.40.0 60 4.80.2 4.70.2 90 <2.0 <2.0 120 <2.0 <2.0 180 <2.0 < 2.0
81 Table 4 3: Salmonella transfer from inoculated single use glove to tomatoes using TSAR, BSAR and XLDR media with water as a carrier medium n=3. Drying time min. TSAR BSAR XLDR 0 5.10.2 4.90.5 4.60.3 15 4.70.0 4.80.0 4.50.0 30 4 .90.0 4.70.0 4.50.0 45 4.60.0 4.30.0 3.70.0 60 5.10.1 4.90.4 4.50.3
82 Table 4 4: Salmonella transfer from inoculated dirty gloves to tomatoes at 0 h, 1 h and 24 h of inoculum drying, following a 5 s touch n=3. 0 h 1 h 24 h TSAR BSAR TSAR BSAR TSAR BSAR Dirty1 a 5.20.1 5.20.2 <1.3 <1.3 <1.3 "d1.3 Dirty2 b 5.20.1 4.50.6 <1.3 <1.3 <1.3 <1.3 Dirty3 c 5.00.1 4.90.1 <1.3 <1.3 <1.3 <1.3 Dirty4 d 5.30.3 5.20.1 <1.3 <1.3 <1.3 <1.3 a 0.1g soil, a leaf and a drop of tomato g ut was rubbed on glove for 20 s b 0.1g soil, a leaf was rubbed on gl ove for 20 s c 0.1g soil, a leaf and a drop of tomato gut was rubbed on glove for 20 s and dried for half an hour d 0.1g soil, a leaf rubbed on gloves for 20 s and dried for half an hour
83 Table 4 5: Salmonella transfer from inoculated tomatoes to dirty gloves at 0 h, 1h and 24 h of inoculum drying, following a 5 s touch n=3. 0 h 1 h 24 h TSAR BSAR TSAR BSAR TSAR BSAR Dirty1 a 5.30.2 5.10.1 5.20.0 5.10.1 <1.9 <1.3 Dirty2 b 5.30.2 5.10.1 5.20.1 5.00.2 <2.3 <1.3 a 0.1g soil, a leaf and a drop of tomato g ut was rubbed on glove for 20 s b 0.1g soil, a le af was rubbed on glove for 20 s
84 Table 4 6: Salmonella transfer from inoculated dirty gloves to tomatoes at 0 h and 1 h in oculum drying, following 5 s touch n=3. Dirty glove a Tomato Drying time h TSAR BSAR TSAR BSAR 0 5.40.9 5.60.9 5.00.2 5.00.1 1 3.30.1 1.50.2 <1.3 <1.3 a gloves were made dirty by rubbing tomato leaf with it for 20 s and 0.1% tween 20 was added to buffer for better recovery
85 Table 4 7: Salmonella transfer from inoculated dirty gloves to tomatoes at 0 h and 1 h inoculum drying, following 5 s touch n=3. Dirty glove a Tomato Drying time h TSAR BSAR TSAR BSAR 0 5.90.1 5.70.3 5.00 .2 5.00.1 1 4.10.3 3.10.6 <1.3 <1.3 a gloves were made dirty by rubbing tomato leaf with it for 20 s and were dried for half an hour before inoculating, 0. 1% tween 20 was added to buffer
86 Table 4 8 : Salmonella transfer from inoculated clean reusable g loves to tomatoes after 0 h, 1 h and 24 h of inoculum drying, following a 5 s touch n=9 18. Clean reusable glove Tomato TC Time h TSAR BSAR TSAR BSAR Average Range 0 5.90.1 5.80.1 5.20.1 5.20.1 0.250.1 0.14 0.36 1 5.40.2 5.10.4 5.10 .1 4.90.2 0.480.5 0.22 0.86 24 <2.20.7 a <1.3 b <1.40.2 c <1.40.2 c 3/9 a n=9, one replication was below the limit of detection b n=9, all nine replications were below the limit of detection c n=9, six replications were below the limit of detection
87 Ta ble 4 9 : Salmonella transfer from inoculated single use gloves to tomatoes after 0 h, 1 h and 24 h of inoculum drying, following a 5 s to uch n=9 18. Single use glove Tomato TC Time h TSAR BSAR TSAR BSAR Average Range 0 6.00.1 5.80.1 5.40.3 5 .40.2 0.320.1 0.11 0.47 1 5.90.4 5.10.4 5.40.1 5.30.2 0.290.2 0.05 0.66 24 2.50.5 2.20.7 <1.3 a <1.3 b 0/9 a n=9, all the replications were below the limit of detection
88 Table 4 10 : Salmonella transfer from inoculated dirty reusable gloves t o tomatoes after 0 h and 1 h of inoculum dryin g, following a 5 s touch n=9. Dirty reusable glove Tomato TC Time h TSAR BSAR TSAR BSAR Average Range 0 5.50.2 5.10.2 5.00.2 5.00.2 0.410.3 0.14 0.86 1 3.40.2 2.10.5 "d1.3 a "d1.3 b 5/9 a fo ur replications were negative upon enrichment b all nine replications were below the limit of detection
89 Table 4 1 1 : Salmonella transfer from inoculated tomatoes to clean reusable gloves after 0 h, 1 h and 24 h of inoculum drying, following a 5 s touch n=9 18. Tomato Clean reusable glove TC Time h TSAR BSAR TSAR BSAR Average Range 0 6.00.1 6.00.2 5.40.3 5.40.2 0.180.0 0.12 0.21 1 5.60.4 5.60.4 5.40.1 5.30.2 0.380.2 0.11 0.76 24 4.10.2 3.80.3 <2.81.0 a <2.70.9 b 7/9 a n=9, two replications were below the limit of detection b n=9, one replication was below the limit of detection
90 Table 4 1 2: Salmonella transfer from inoculated tomatoes to single use gloves after 0 h, 1 h and 24 h of inoculum drying, following a 5 s touch n=9 18. Tomato Clean reusable glove TC Time h TSAR BSAR TSAR BSAR Average Range 0 5.70.4 5.70.4 5.40.2 5.20.5 0.370.2 0.22 0.87 1 5.40.4 5.30.5 5.20.5 5.10.5 0.390.2 0.11 0.62 24 3.40.6 3.10.7 <1.3 a <1.3 a 0/9 a n=9, all the replicati ons were below the detection limit
91 Table 4 1 3 : Salmonella transfer from inoculated clean reusable glove to twenty five tomatoes touched subsequently with wet inoculum n=9. Tomato TC Tomato no. TSAR BSAR Average Range Glove 5.70.4 5.90.1 T 1 5.30.2 5.20.4 0.610.6 0.11 1.8 T 2 4.61.2 4.41.3 0.460.6 0.00 1.8 T 3 4.31.2 4.11.4 0.270.3 0.00 5.1 T 4 4.01.2 3.81.5 0.260.6 0.00 1.8 T 5 3.51.2 3.21.3 0.060.1 0.00 0.34 T 6 3.11.4 3.11.4 0.030.1 0.00 0.06 T 7 3.11.1 3.01 .1 0.020.0 0.00 0.02 T 8 2.51.5 2.71.3 0.020.0 0.00 0.08 T 9 2.61.2 <2.91.1 c 0.010.0 0.00 0.08 T 10 <2.10.9 b 6/9 6/9 T 11 <1.81.1 a 7/9 7/9 T 12 <1.60.5 a 7/9 7/9 T 13 <1.90.9 a 7/9 7/9 T 14 <1.80.7 a 7/9 7/9 T 15 <2.10.8 a 7/9 7/9 T 16 5/9 5/9 5/9 T 17 4/9 4/9 4/9 T 18 3/9 3/9 3/9 T 19 5/9 5/9 5/9 T 20 4/9 4/9 4/9 T 21 2/9 2/9 2/9 T 22 2/9 2/9 2/9 T 23 2/9 2/9 2/9 T 24 0/9 0/9 0/9 T 25 0/9 0/9 0/9 a two replications were negative upon enrichment b three replications were negative upon enrichment c three replications were below the limit of detection
92 Table 4 14 : Salmonella transfer from inoculated clean reusable glove to twenty five tomatoes touched subsequently with an hour dried inoculums n=9. Tomato TC Tomato no. TSAR BSAR Average Range Glove 5.30.3 5.20.4 T 1 4.60.7 4.60.7 0.861.7 0.02 4.9 T 2 3.51.7 3.21.7 0.872.2 0.00 2.2 T 3 3.61.2 3.31.4 0.160.3 0.00 0.85 T 4 3.31.6 <3.51.4 c 0.350.7 0.00 1.9 T 5 2.81.5 <3.1 1.5 c 0.120.3 0.00 0.78 T 6 <2.31.6 a <2.51.3 c 7/9 T 7 <2.31.5 a 7/9 7/9 T 8 <2.11.3 b 6/9 6/9 T 9 4/9 4/9 4/9 T 10 5/9 5/9 5/9 T 11 4/9 4/9 4/9 T 12 4/9 4/9 4/9 T 13 4/9 4/9 4/9 T 14 3/9 3/9 3/9 T 15 4/9 4/9 4/9 T 16 3/9 3/9 3/9 T 17 2/9 2/9 2/9 T 18 2/9 2/9 2/9 T 19 2/9 2/9 2/9 T 20 0/9 0/9 0/9 T 21 0/9 0/9 0/9 T 22 1/9 1/9 1/9 T 23 0/9 0/9 0/9 T 24 0/9 0/9 0/9 T 25 0/9 0/9 0/9 a two replication were negative upon enrichment b three replicati ons were negative upon enrichment c two or three replications were below the limit of detection
93 Table 4 1 5 : Salmonella transfer from inoculated single use glove to twenty five tomatoes touched subsequently with wet inoculums n=9. Tomato TC Tomato n o. TSAR BSAR Average Range Glove 5.80.5 5.80.2 T 1 5.10.1 5.00.1 0.330.4 0.04 1.2 T 2 4.80.4 4.60.7 0.180.2 0.01 0.17 T 3 4.60.4 4.40.9 0.130.1 0.01 0.35 T 4 4.30.5 4.01.2 0.050.0 0.00 0.09 T 5 4.01.1 <4.20.6 d 0.050.1 0.00 0 .09 T 6 3.80.8 3.21.2 0.020.0 0.00 0.03 T 7 3.51.0 <3.41.0 d 0.010.0 0.00 0.02 T 8 3.01.4 <3.50.6 d 0.010.0 0.00 0.04 T 9 3.01.0 <2.90.9 d 0.010.0 0.00 0.03 T 10 2.51.0 <2.71.1 d 0.010.0 0.00 0.06 T 11 5/9 5/9 5/9 T 12 <1.40.8 a 8/ 9 8/9 T 13 <1.80.8 b 7/9 7/9 T 14 <1.70.8 b 7/9 7/9 T 15 <1.70.8 c 6/9 6/9 T 16 4/9 4/9 4/9 T 17 2/9 2/9 2/9 T 18 2/9 2/9 2/9 T 19 4/9 4/9 4/9 T 20 2/9 2/9 2/9 T 21 2/9 2/9 2/9 T 22 3/9 3/9 3/9 T 23 3/9 3/9 3/9 T 24 1/9 1/9 1/9 T 25 1/9 1/9 1/9 a one replication was negative upon enrichment b two replications were negative upon enrichment c three replications were negative upon enrichment d one or two replications were below the limit of detection
94 Table 4 16 : Salmonell a transfer from inoculated single use glove to twenty five tomatoes touched subsequently with an hour dry inoculums n=9. Tomato TC Tomato no. TSAR BSAR Average Range Glove 5.40.1 5.20.2 T 1 5.00.3 4.70.5 0.490.3 0.1 1.1 T 2 4.01.3 3.8 1.2 0.300.4 0.03 0.98 T 3 4.01.0 <4.10.7 a 0.260.5 0.00 1.5 T 4 3.51.3 3.21.4 0.370.6 0.00 1.4 T 5 2.91.0 <3.01.1 a 0.050.1 0.00 0.31 T 6 2.51.1 2.51.0 0.010.0 0.00 0.05 T 7 2.21.1 <1.90.7 b 0.010.0 0.00 0.01 T 8 2.21.1 <1.90.7 b 0.010.0 0.00 0.05 T 9 8/9 8/9 8/9 T 10 6/9 6/9 6/9 T 11 3/9 3/9 3/9 T 12 4/9 4/9 4/9 T 13 3/9 3/9 3/9 T 14 3/9 3/9 3/9 T 15 4/9 4/9 4/9 T 16 1/9 1/9 1/9 T 17 3/9 3/9 3/9 T 18 2/9 2/9 2/9 T 19 2/9 2/9 2/9 T 20 1/9 1/9 1/ 9 T 21 1/9 1/9 1/9 T 22 1/9 1/9 1/9 T 23 1/9 1/9 1/9 T 24 1/9 1/9 1/9 T 25 1/9 1/9 1/9 a one replication were below the limit of detection b three replications were below the limit of detection
95 Table 4 1 7 : Salmonella transfer from inoculate d dirty reusable glove to ten tomatoes touched subsequently with wet inoculum n=9. Tomato TC Tomato no. TSAR BSAR Average Range Glove 5.70.2 5.30.3 T 1 4.90.5 4.80.5 0.220.2 0.01 0.69 T 2 4.80.4 4.90.4 0.160.1 0.10 0.39 T 3 4.50.5 3.81.0 0.090.1 0.02 0.29 T 4 4.50.2 4.10.1 0.070.0 0.03 0.15 T 5 4.00.5 3.40.7 0.030.0 0.00 0.05 T 6 4.10.3 <3.30.9 b 0.030.0 0.00 0.06 T 7 3.41.1 <2.82.0 b 0.020.0 0.00 0.10 T 8 3.70.3 3.40.7 0.020.0 0.00 0.05 T 9 <3.10.9 a <2. 81.1 b <0.010.02 b 0.00 0.07 T 10 <2.71.1 a <2.41.0 b 8/9 a one replication was negative upon enrichment b one replication was below the limit of detection
96 Table 4 18 : Salmonella transfer from inoculated dirty reusable gloves to ten tomatoes touched subsequently with an hour dried inoculum n=9. Tomato TC Tomato no. TSAR BSAR Average Range Glove 4.30.3 <1.60.5 a T 1 4/9 4/9 4/9 T 2 3/9 3/9 3/9 T 3 3/9 3/9 3/9 T 4 2/9 2/9 2/9 T 5 3/9 3/9 3/9 T 6 3/9 3/9 3/9 T 7 5/9 5/9 5 /9 T 8 2/9 2/9 2/9 T 9 3/9 3/9 3/9 T 10 3/9 3/9 3/9 a four replications were below the limit of detection
97 Figure 4 1 : Comparison of different carrier mediums for transfer from inoculated glove s ingle use to tomatoes with it after 0 h black bar and 1 h light grey bar drying time n=3 12.
98 0 1 2 3 4 5 6 7 0 15 min 30 min 45 min 1 h log CFU/tomato Drying time min. Figure 4 2: Comparison of transfer after different drying times of inocula using 0.1% peptone water as carrier medium by performing enumerations on TSAR black bar and BSAR light grey bar media n=6 12.
99 0 1 2 3 4 5 6 7 0 15 30 45 60 Log CFU/tomato Drying time min. Figure 4 3: Comparison of different culture media; TSAR black bar, BSAR light grey bar and XLDR dark grey bar for recovery of Salmonella from tomatoes. Water was used as carrier medium for inocula n=3.
100 Figure 4 4 : Population of Salmonella inoculated onto clean reusable glove black color bar and transferred to a tomato light grey color bar following a 5 s touch n= 9 18. The solid line on the graph is the limit of detection 1.3 log CFU/glove or tomato.
101 Figure 4 5 Distribution of Salmonella transfer coefficients TCs from reusable gloves to tomatoes with 0 h dried inoculum using TSAR media n=9.
102 Figure 4 6 Distribution of Salmonella transfer coefficients TCs from reus able gloves to tomatoes with 0 h dried inoculum using BSAR media n=9.
103 Figure 4 7 Distribution of Salmonella transfer coefficients TCs from reusable gloves to tomatoes with 1 h dried inoculum using TSAR media n=9.
104 Figure 4 8 Distribution of Sa lmonella transfer coefficients TCs from reusable gloves to tomatoes with 1 h dried inoculum using BSAR media n=9.
105 Figure 4 9 : Population of Salmonella inoculated onto clean single use glove black color bar and transferred to a tomato light grey color bar following a 5 s touch n= 9 18. The solid line on the graph is the limit of detection 1.3 log CFU/glove or tomato.
106 Figure 4 10 Distribution of Salmonella transfer coefficients TCs from single use gloves to tomatoes with 0 h dried inoculu m using TSAR media n=9.
107 Figure 4 11 Distribution of Salmonella transfer coefficients TCs from single use gloves to tomatoes with 0 h dried inoculum using BSAR media n=9.
108 Figure 4 12 Distribution of Salmonella transfer coefficients TCs from single use gloves to tomatoes with 1 h dried inoculum using TSAR media n=9.
109 Figure 4 13 Distribution of Salmonella transfer coefficients TCs from single use gloves to tomatoes with 1 h dried inoculum using BSAR media n=9.
110 Fi gure 4 14 : Populati on of Salmonella inoculated onto dirty gloves black color bar and t ransferred to tomatoes light grey color bar following a 5 s touch n=9 The solid line on the graph is the limit of detection 1.3 log CFU/glove or tomato.
111 Figure 4 15 Distributio n of Salmonella transfer coefficients TCs from dirty reusbale gloves to tomatoes with 0 h dried inoculum using TSAR media n=9.
112 Figure 4 16 Distribution of Salmonella transfer coefficients TCs from dirty reusbale gloves to tomatoes with 0 h dried inoculum using BSAR media n=9.
113 Figure 4 1 7 : Population of Salmonella inoculated onto tomatoes light grey color bar and transferred to a clean reusable glove black color bar following a 5 s touch n= 9 1 8. The solid line on the graph is the limit o f detection 1.3 log CFU/glove or tomato.
114 Figure 4 18. Distribution of Salmonella transfer coefficients TCs from tomatoes to reusbale gloves with 0 h dried inoculum using TSAR media n=9.
115 Figure 4 19 Distribution of Salmonella transfer coeffici ents TCs from tomatoes to reusbale gloves with 0 h dried inoculum using BSAR media n=9.
116 Figure 4 20 Distribution of Salmonella transfer coefficients TCs from tomatoes to reusbale gloves with 1 h dried inoculum using TSAR media n=9.
117 Figure 4 21 Distribution of Salmonella transfer coefficients TCs from tomatoes to reusbale gloves with 1 h dried inoculum using BSAR media n=9.
118 F igure 4 22 : Population of Salmonella inoculated onto tomatoes light grey color bar and transferred to a clean single use glove black color bar following a 5 s touch n= 9 18. The solid line on the graph is the limit of detection 1.3 log CFU/glove or tomato.
119 Figure 4 23 Distribution of Salmonella transfer coefficients TCs from tomatoes to single use glov es with 0 h dried inoculum using TSAR media n=9.
120 Figure 4 24 Distribution of Salmonella transfer coefficients TCs from tomatoes to single use gloves with 0 h dried inoculum using BSAR media n=9.
121 Figure 4 25 Distribution of Salmonella transfer coefficients TCs from tomatoes to single use gloves with 1 h dried inoculum using TSAR media n=9.
122 Figure 4 26 Distribution of Salmonella transfer coefficients TCs from tomatoes to single use gloves with 1 h dried inoculum using BSAR media n=9.
123 Figure 4 27 : Population of Salmonella inoculated onto clean reusable gloves first bars and transferred to subsequently touched tomatoes remaining bars after 0 h black color bars and 1 h light grey color bars of inoculums drying n=9. The soli d line on the graph is the limit of detection 1.3 log CFU/glove or tomato
124 Figure 4 28 : Population of Salmonella inoculated onto clean single use gloves first bars and transferred to subsequently touched tomatoes remaining bars after 0 h black c olor bars and 1 h light grey color bars of inoculums drying n=9. The solid line on the graph is the limit of detection 1.3 log CFU/glove or tomato.
125 Figure 4 29 : Population of Salmonella inoculated onto dirty reusable gloves first bars and trans ferred to subsequently touched tomatoes remaining bars after 0 h black color bars and 1 h light grey color bars of inoculums drying n=9. The solid line on the graph is the limit of detection 1.3 log CFU/glove or tomato
126 CHAPTER 5 DISCUSSION Tom ato farming is the third largest industry in Florida FASD, 2008. Florida tomato industry has suffered due to outbreaks of Salmonella illnesses associated with tomatoes. An o utbreak of salmonellosis from jalapeno/s errano pepper s which was first attributed to be lin ked to tomatoes, costs Florida tomato industry $100 million CDC, 2008. All these incidences have raised the need of more research and inputs towards tomato safety. This research evaluates risks associated with cross contamination of Salmonella between workers gloved hands and mature green, round tomatoes in simulated harvest and packinghouses upon single and sequential touches. No significant differences were seen between results on selective on non selective medias, and only results from non se lective media are discussed. Selective media promotes the growth of specific organism only and inhibit other organisms. For example, BSA, the sel ective media used in this study inhibits the growth of gram positive microorganisms. T he other, TSA is non sel ective media with casein and soybean meal the nutrient source. Addition of rifampicin as an antibiotic makes the rifampicin resistant Salmonella isolation, convenient. Rifampicin, an antibiotic, is used for experiments to eliminate the interference of any background flora, which cou ld possibly affect the results. Three different transfer scenarios were studied during experiments which were from i clean or dirty gloves to tomatoes ii tomato to clean gloves iii clean or dirty gloves to many tomatoes. Tw o different types of gloves reusable or single use, two di fferent hygienic conditions of gloves clean and dirty and three different drying time for inoculum 0 h, 1 h, 24 h were studied, except doing experiments with dirty single
127 use gloves and except drying inoculum for 24 h for gloves to many tomato transfer scenario. First transfer scenario simulates the transfer that could take place from gloves used by workers to the tomatoes touched with those gloved hands, and to see whether the transfer can tak e place from contaminated gloves of workers to the tomatoes. Second transfer scenario was selected to simulate transfer from contaminated tomatoes to worker s gloves. When the gloves are contaminated and used during harvesting, the number of tomatoes it ca n potentially contaminate was estimated through third transfer scenario. As single use gloves are changed ve ry frequently in tomato packing houses, no experiments were performed with dirty single use gloves. Single T ouch Transfer coefficients obtained on to uching green tomatoes with inoculated reusable gloves and single use gloves were 25% and 32%, and transfer coefficients from inoculated green tomatoes to reusab le and single use gloves were c a 18% and 3 7%, when the inoculum was wet. Similar results obtain ed from experiments performed from glove to tomato and tomato to glove with wet inoculum may be due to the smooth surface of gloves and tomatoes. Jimenez et al. 2007 also obtained high transference of Salmonella enterica serovar Typhimurium from inoculat ed bell pepper to gloved hands and from gloved hands to bell pepper as compared to bare hands, which are rough in comparison to gloved hands or bell pepper surface. Similar to our results, Jimenez et al. 2007 did not observe significant differences in tr ansfer from glove surface to bell pepper and bell pepper surface to gloves. Salmonella transfer from inoculated gloves to tomatoes after 1 h was significantly higher than wet inoculum p=0.04. The possible reason behind this can be the curved surface of t omato, which may have had varied contact with the 6 or 8 inoculum drops on
128 glove surface at one time, potentially leaving some of the inoculum untouched. All the visible drops on the surface of gloves were touched after 1 h inoculum drying, which might be the reason for no change in Salmonella population from un inoculated surface of tomatoes between 0 h and 1 h drying. However, drying the inoculum on flat glove surface did reduce the population of Salmonella due to drying or desiccation effect by less than 0.5 l og CFU/glove. Hence the numerator of transfer coefficients calculation equation was same for both cases but the denominator decreases, which led to h igher TCs after 1 h of drying. Salmonella transfer from inoculated tomatoes to gloves did not change significantly after 1 h drying which is in contrary with the previous study by Lang et al. 2004, who found significant decrease in population from spot inoculated tomatoes after drying in biosafety hood for 1 h. The difference between the two studies is the site of inoculation on tomato surface. Lang and colleagues inoculated Salmonella on tomatoes at blossom end, while in this study, inoculation was done at the equator region, which a very different surface from the blossom end of tomatoes. Previous rese arch by Rusin et al. 2002 has shown that Salmonella attachment to rough or porous surface is more than non porous or smooth surface, which affects its transfer as well. This concept can be used to understand the different transfer rates after 1 h drying between this study and study by Lang et al. 2004. Drying the inoculum on glove surface for 24 h, reduce the Salmonella population by ca. 3.7 log CFU/reusable gloves and ca. 4.5 log CFU/single use glove surface, likely due to desiccation stress. Enrichmen ts performed when counts fell bel ow the limit of detection gave three out of nine positive from re usable gloves, while none of the nine
129 samples were positives for single use gloves. Drying the inoculum on tomato surface reduced the population by 1.9 log CF U/ tomato for the experiment performed with reusable gloves and by 2.3 log CFU/tomato for the exp eriment with single use glove. Similar to this study, Lang et al. 2004 also observed reduction in Salmonella population by 2.2 l og CFU/tomato from spot inocu lated tomatoes after 24 h of drying. The enrichment results obtained from reusable and single use gloves touched with inoculated tomatoes were 7/9 and 0/9, respectively. Reusable gloves showed more positives in both cases; where it was inoculated and where it was touched with the inoculum. This may be due to the difference in material of reusable glove and single use gloves, affecting the rate of drying of inoculum or rate of transfer of Salmonella More reduction in Salmonella populations on the tomato sur face in experiments with single use glove than in the experiments with reusable glove may be the reason for more transfer from tomatoes to reusable glove. The different drying rates of inoculum on tomato surface for experiments with clean reusable and sing le use gloves may be due to the position under the biosafety cabinet or a variation in surface properties of two lots of tomatoes used during experiments. Biofilm formation is beyond the scope of this study; however the concept of biofilm can be used to pr ovide possible explanations for some of the results. Bacterial biofilms can be defined as an assemblage of microorganisms adherent to each other and/or to a surface and embedded in a matric of exopolymers Costerton et al., 1999. Bacterial adhesion to s urface depends on factors like surface composition, roughness of contact surface, charge and hydrophobicity of both the contact surface and the cells Costerton et al., 1999. Bacterial biofilms protect bacteria from harsh and hostile environments.
130 Ukuku a nd Sapers 2001 have observed the decreased efficacy of chlorine and hydrogen peroxide sanitizers, when Salmonella was allowed to stay on cantaloupe melon surface for 24 h. The reason they propose for the decreased efficacy is biofilm formation by Salmone lla on the fruit surface, whose strength increases with time during storage Ukuku and Fett, 2002. If this is the case, these findings can be used to explain the reason for less reduction during 24h of drying of Salmonella on a tomato surface as compared to glove surface. Since the produce surface is biotic in nature, possibly the attachment and biofilm formation of Salmonella on tomato surface is stronger than on glove surface. Stronger biofilm may have helped the bacterial cells survive desiccation on to mato surface. Glove surfaces are made up of rubber material and rubber material are hydrophobic in nature with contact angle of 108.21.0 Sinde and Carballo, 2000. High hydrophobicity of the glove surface may prevent attachment of Salmonella cells. Lack of attachment implies the presence of loose cells on the surface, which may die more easily than the adhered cells. Additionally, glove pieces used in experiments were flat, while the tomato surface was curved; the flat surface may have been more exposed the dry air and the drying effect of Salmonella on glove surface might be more pronounced. Reusable gloves were made dirty by using soil 0.5 g, 0.3 g, 0.1 g, internal tissue of tomato 1/8 TSP, TSP, 1 drop and one tomato leaf; rubbed on glove pieces f or 5 s to 30 s. Experiments were performed with different types of dirty gloves and no significant differences were observed. Hence, rubbing a tomato leaf on glove surface for 20 s was selected as the best representation of dirty gloves in tomato harvestin g field. In all the dirty gl oves experiments, 0.1% of Tween 20 was used as a surfactant.
131 Surfactant is a substance that helps in lowering the interfacial tension between solid and a liquid or between two liquids. The use of Tween 20 in experiments was expe cted to improve the recovery of Salmonella from inoculated dirty glove surfaces. Dirty glove results did not differ significantly from clean reusable glove results, when inoculum was wet. Under wet inoculum transfer scenario, dirty gloves were made dirty, inoculated and immediately samples. Lack of the time between inoculum and dirty glove surface might be the reason for similar results from clean and dirty reusable gloves. On drying the inoculum in biosafety cabinet for 1 h, clean reusable gloves generate more Salmonella positive tomato samples than dirty reusable gloves. The layer of tomato leaf extract on glove surface possibly restricts Salmonella and might not let it transfer to tomato upon touching. This behavior was observed after 1 h inoculum drying, but not during immediate sampling, where the contact time for Salmonella with leaf debris was less. More contact time may help in better attachment of bacterial cells to the rough surface as per the concept of biofilm formation. Since dirty gloves are sim ilar to rough surface while clean gloves are similar to smooth surface, 1 h drying might cause more attachment of Salmonella cells to dirty surface and prevent its transfer to tomatoes. Our results obtained after 1 h inoculum drying on dirty gloves are sim ilar with the results obtained by Flores et al. 2006. They demonstrated that TCs from cutting boards to beef and beef to cutting boards decreased with increased amounts of beef tissue present on high density polyethylene cutting boards surfaces. Transfe r of E. coli O157:H7 from contaminated board surfaces to beef tissues was studied under both wet and dry 5 min inoculum conditions. They concluded that the decrease in TCs is due to reduction of the number of cells left on the surface after each subseque nt contact.
132 The results obtained from touching single tomato with inoculated clean gloves after 0 h, 1 h drying time showed that Salmonella has potential to transfer from inoculated worker s gloves to tomatoes. After 24 h, transfer was observed from clean reusable gloves but not from single use gloves, which implies clean reusable gloves can transfer Salmonella that may not have been removed from the glove during the previous days cleaning and sanitizing. For dirty gloves, Salmonella transfer was similar to clean gloves, when inoculum was wet. As the time of contact between dirty gloves and inoculum increased to 1 h, we believe that Salmonella gets trapped in the layer of tomato leaf extract, thus decreasing its transfer. Salmonella also transferred from ino culated tomatoes to both types of clean gloves after 0 h and 1 h drying time. However, after 24 h, transfer occurred to clean reusable gloves only, while single use gloves did not report any transfer. Subsequent T ouches On touching tomatoes subsequently wi th inoculated clean reusable gloves, TCs decreased continuously until the tomatoes were enriched for Salmonella In case of clean reusable gloves, significant differences in TCs were observed between T1 and T3 to T9, while for single use gloves, significan t reductions were obtained between T1 and T7 to T9. Statistical analysis showed that TCs obtained from first two tomatoes T1 and T2 touched with clean inoculated reusable gloves were significantly higher than all the tomatoes touched with inoculated sing le use gloves, expect first tomato. This implies that Salmonella transfer from clean reusable gloves to first two tomatoes was higher, which was similar to transfer from single use gloves from third tomato. However, from single use gloves same TC s were obt ained up to 7 th tomato. Thus, single use gloves transfer less Salmonella to a higher number of tomatoes touched, while clean reusable
133 gloves transferred more Salmonella to a smaller number of tomatoes. In general, the results from clean gloves to many toma toes show that as more and more tomatoes are touched with inoculum, the Salmonella transfer decreases, which is in contrary with Fravalo et al. 2009 findings, who concluded that Salmonella transfer is inversely related to the initial load. For dirty reus able gloves, TCs did not change significantly for the ten tomatoes touched. Even after drying the inoculum for 1 h, almost same number of Salmonella positive samples was obtained from all the ten tomatoes touched subsequently with dirty reusable gloves, re plicated nine times. Similar to statistical analysis from single use gloves and clean reusable gloves, first two TCs obtained from clean reusable gloves differ significantly from all the TC s obtained from dirty reusable gloves. Two possible reasons behind less transfer from dirty reusable gloves can be either death of Salmonella cells or trapping of Salmonella cells in the leaf extract. Based on the transfer of Salmonella to the ten tomatoes touched with inoculated gloves at 0 h and 1 h, it is more likely t hat Salmonella is getting stuck in the layer and transferring to tomatoes at slower rate Worker s gloves are potential source of Salmonella transfer during tomato harvesting. As the day progr ess and gloves become dirtier transfer of Salmonella to tomatoe s takes place at lower rate, and the risk of using dirty gloves is no greater than using clean gloves. Our results have shown Salmonella transfer between reusable gloves and tomatoes even after it has dried on the surface for one day. Although the transfer was less than what was seen with wet or 1h dried inoculum, it cannot be
134 neglected and the use of adequate cleaning and sanitizing for washing of gloves at the end of the shift is an important step to eliminate the chances of cross contamination.
135 CHAPTER 6 FUTURE WORK The use of gloves is considered as a preventive step during tomato harvesting. The results of this research illustrate that gloves can be the potential source of cross contamination to tomatoes during harvesting and contaminated tomatoes can transfer Salmonella to gloves. As hypothized, dirty gloves did not transfer more Salmonella to tomatoes; rather the risk of Salmonella transfer was similar for clean and dirty gloves, under wet inoculum conditions. Results also showed that clean reusable g loves transfer more Salmonella to a fewer number of tomatoes touched, while single use gloves and dirty reusable gloves transfer less Salmonella to a greater number of tomatoes. The decision of which is risky transfer scenario among above two is a hard tas k. The results from this research left us with some unanswered questions, which need further research and inputs to ease the risk management solution. Determining the risk potential of different sources that may transfer Salmonella or other pathogenic orga nisms is an important concern for researchers. This research focused on the hand harvesting of matu re green, round tomatoes only. The surface of all fruits and vegetables are not identical, thus the wide application of the results obtained here to other pr oduce varieties without subsequent experiments to quantify the risk of glove use in bacterial transfer would be unwise. For example, the skin of Roma tomatoes is thinner as compared to round tomatoes; Salmonella transfer to Roma or grape tomatoes may be di fferent in comparison with round tomatoes. Similar transfer coefficient experiments should be performed to determine the risk of Salmonella transfer from and to other tomato varieties and other fruits and vegetables, mimicking glove and harvest conditions unique to each.
136 The gloved hands of workers are not the only potential point of cross contaminatio n during round tomato harvest. The study of different equipment used during harvesting should be another area of future study. Transfer from different equipme nt, including buckets, utensils, worker s shirts, that may contact tomatoes during harvest should be consider to understand the overall risk of Salmonella transfer during harvesting. These future studies would help strengthen any future risk assessment mod els that attempt farm to fork modeling including the risk of bacterial cross contamination during harvesting of different produce items. E quipment used during harvest, u tensils, gloves and other items can be cleaned and sanitized at the end of the day and reused again and again. The effectiveness different sanitizers used on these harvesting tools should be another area of research. The different sanitizers can be tested for different equipment, including, dirty gloves, buckets, etc. against various pathog en and a standard method for sanitizing harvesting equipment could be developed. Additionally, s ince the use of sanitizers may damage the equipment, the effect of sanitizer use on glove surface properties is an interesting topic for future study. The use o f sanitizers on same gloves again and again might have increasing their roughness, which in turn might enhance the bacterial attachment potential of glove surface. This research evaluated transfer of only Salmonella during tomato harvesting. No resear ch ha s been conducted to evaluate the transfer of other pathogenic microorganisms like E. coli O157:H7 or Listeria monocytogenes during harvesting of any produce items.
137 Fresh produce continues to emerge as an important field of research in Food Safety. Multista te outbreaks linked to fresh produce have raised the concern about their safe production, harvesting, handling, storage and distribution. No reduction steps are currently commonly used for fresh produce items and they are mainly consumed by people as raw. Continued research is required which can help us determine different sources of contamination, risk associated with those sources of contamination and the ways to reduce those risks effectively. This research focused on the harvesting of mature green, roun d tomatoes with gloved hands. Above mentioned future work will provide a broader view to look into different things, which will help in reducing risk associated with different fresh produce and help in providing a safe produce items to consumers.
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154 BIOGRAPHICAL SKETCH Pardeepinder Kaur Brar was born in the city of Punjab, India. Her father is working as revenue officer of Punjab state and mo ther is a s chool teacher She started her u ndergraduate degree program in a griculture at the Punjab Agricultural University Ludhiana, Punjab, India in 2004. Throughout her degree she was awarded with a University Merit Scholarship She r eceived an award in th e last year of her degree for highest Ov erall Credit Point Average in her major food science and t echnology. All through her college years, she participated in many campus activities and g ained a lot of honors in sports and folk dan ces In spring 2009, Pardeepinder started her Master of S cience in the food s cience program under the instruction of Dr. Michelle Danyluk at the University of Florida. She worked in a sho rt project in the beginning of master s degree to get familiarize wit h different skills Her short project focused on Efficacy of aqueous and alcohol based quaternary ammonium sanitizers for reducing Salmonella in dusts generated in almond hulling and shelling facilities She has participated in various depart mental activ ities at UF like College bowl and product development Pardeepinder is planning on continui ng her education in Food Safety through a PhD.