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Fate of Escherichia coli O157

Permanent Link: http://ufdc.ufl.edu/UFE0025049/00001

Material Information

Title: Fate of Escherichia coli O157 H7 and Salmonella spp. on Fresh and Frozen Cut Mangoes, Papayas and Pineapples
Physical Description: 1 online resource (133 p.)
Language: english
Creator: Strawn, Laura
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: coli, escherichia, mango, o157, papaya, pineapple, salmonella
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Escherichia coli O157:H7 and Salmonella infection has been associated with consumption of a number of fruits and vegetables. Although the fate of E. coli O157:H7 and Salmonella on many of these products is well studied, little is known about their behavior on cut mango, papaya and pineapple. Mangoes, papayas and pineapples have all been associated with outbreaks of salmonellosis. Three documented Salmonella outbreaks in the U.S. have been associated with consumption of raw mangoes, occurring in 1998, 1999 and 2001 from Salmonella Oranienburg, Salmonella Newport and Salmonella Saintpaul, respectively. A large outbreak in 1996 of Salmonella Weltevreden occurred in Jurong, Singapore. Salmonella Weltevreden was isolated from papaya and pineapple obtained at a local fruit stall, implicating these fruit as a possible source for the outbreak. Most recently in 2006, an outbreak of Salmonella Litchfield occurred in Australia due to the consumption of fresh cut papaya. The objective of this study was to evaluate the fate of E. coli O157:H7 and Salmonella on fresh (23degreeC, 12degreeC and 4degreeC) and frozen (-20degreeC) cut mangoes, papayas and pineapples. Cut mangoes, papayas and pineapples were spot inoculated with either a four strain or five strain cocktail of E. coli O157:H7 or Salmonella, respectively. Inoculated samples were air dried, placed in containers and stored at 23 plus or minus 2, 12 plus or minus 2, 4 plus or minus 2 and -20 plus or minus 2degreeC to simulate various potential storage conditions. Temperature and relative humidity was monitored and recorded by sensors in each container. Samples were enumerated following stomaching on selective and nonselective media at days 0, 1, 3, 5 and 7 (23plus or minus2degreeC); 0, 1, 3, 5, 7, 10, 14, 21 and 28 (12plus or minus2 and 4plus or minus2degreeC); and 0, 7, 14, 21, 28, 60, 90, 120, 150 and 180 (-20plus or minus2degreeC). Population levels in log CFU/g were calculated. Fruit had visually spoiled by day 3, 5 or 10 at 23plus or minus2, 12plus or minus2degreeC or 4plus or minus2degreeC, respectively. Spoilage was not observed in frozen cut samples. E. coli O157:H7 and Salmonella have the potential to grow on temperature abused fresh cut mangoes and papayas held at 23degreeC. At 12degreeC, Salmonella populations can grow on cut mangoes and papayas; however, E. coli O157:H7 populations remained stable. E. coli O157:H7 and Salmonella can also survive for extended periods of time on refrigerated mangoes and papayas. E. coli O157:H7 and Salmonella, inoculated onto pineapple, did not grow but survived at population levels able to cause foodborne illness up to the shelf life of each storage temperature. E. coli O157:H7 and Salmonella can survive on frozen cut mangoes, papayas and pineapples for up to 180 days. This work indicates that fresh and frozen cut mangoes, papayas and pineapples have the potential to be vectors for E. coli O157:H7 and Salmonella transmission and preventive procedures should be in effect during production and postharvest processing.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Laura Strawn.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Danyluk, Michelle D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025049:00001

Permanent Link: http://ufdc.ufl.edu/UFE0025049/00001

Material Information

Title: Fate of Escherichia coli O157 H7 and Salmonella spp. on Fresh and Frozen Cut Mangoes, Papayas and Pineapples
Physical Description: 1 online resource (133 p.)
Language: english
Creator: Strawn, Laura
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: coli, escherichia, mango, o157, papaya, pineapple, salmonella
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Escherichia coli O157:H7 and Salmonella infection has been associated with consumption of a number of fruits and vegetables. Although the fate of E. coli O157:H7 and Salmonella on many of these products is well studied, little is known about their behavior on cut mango, papaya and pineapple. Mangoes, papayas and pineapples have all been associated with outbreaks of salmonellosis. Three documented Salmonella outbreaks in the U.S. have been associated with consumption of raw mangoes, occurring in 1998, 1999 and 2001 from Salmonella Oranienburg, Salmonella Newport and Salmonella Saintpaul, respectively. A large outbreak in 1996 of Salmonella Weltevreden occurred in Jurong, Singapore. Salmonella Weltevreden was isolated from papaya and pineapple obtained at a local fruit stall, implicating these fruit as a possible source for the outbreak. Most recently in 2006, an outbreak of Salmonella Litchfield occurred in Australia due to the consumption of fresh cut papaya. The objective of this study was to evaluate the fate of E. coli O157:H7 and Salmonella on fresh (23degreeC, 12degreeC and 4degreeC) and frozen (-20degreeC) cut mangoes, papayas and pineapples. Cut mangoes, papayas and pineapples were spot inoculated with either a four strain or five strain cocktail of E. coli O157:H7 or Salmonella, respectively. Inoculated samples were air dried, placed in containers and stored at 23 plus or minus 2, 12 plus or minus 2, 4 plus or minus 2 and -20 plus or minus 2degreeC to simulate various potential storage conditions. Temperature and relative humidity was monitored and recorded by sensors in each container. Samples were enumerated following stomaching on selective and nonselective media at days 0, 1, 3, 5 and 7 (23plus or minus2degreeC); 0, 1, 3, 5, 7, 10, 14, 21 and 28 (12plus or minus2 and 4plus or minus2degreeC); and 0, 7, 14, 21, 28, 60, 90, 120, 150 and 180 (-20plus or minus2degreeC). Population levels in log CFU/g were calculated. Fruit had visually spoiled by day 3, 5 or 10 at 23plus or minus2, 12plus or minus2degreeC or 4plus or minus2degreeC, respectively. Spoilage was not observed in frozen cut samples. E. coli O157:H7 and Salmonella have the potential to grow on temperature abused fresh cut mangoes and papayas held at 23degreeC. At 12degreeC, Salmonella populations can grow on cut mangoes and papayas; however, E. coli O157:H7 populations remained stable. E. coli O157:H7 and Salmonella can also survive for extended periods of time on refrigerated mangoes and papayas. E. coli O157:H7 and Salmonella, inoculated onto pineapple, did not grow but survived at population levels able to cause foodborne illness up to the shelf life of each storage temperature. E. coli O157:H7 and Salmonella can survive on frozen cut mangoes, papayas and pineapples for up to 180 days. This work indicates that fresh and frozen cut mangoes, papayas and pineapples have the potential to be vectors for E. coli O157:H7 and Salmonella transmission and preventive procedures should be in effect during production and postharvest processing.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Laura Strawn.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Danyluk, Michelle D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025049:00001


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FATE OF ESCHERICHIA COLI O157:H7 AND SALMONELLA SPP. ON FRESH AND FROZEN CUT MANGOES, PAPAYAS AND PINEAPPLES By LAURA KATHRYN STRAWN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009 1

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2009 Laura Kathryn Strawn 2

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To MDD. You are more than just a mentor and ro le model to me, but also a friend. This thesis and my time here at Florida is a direct result of your influence. You have provided me with the skill set to be successful in my Ph D and the life lessons to be a better person. For the past five years you have been my anchor. No matter what went down you never walked away from supporting and guiding me past it. You have always pushed me to challenge myself and remember who I am. My apprenticeship with you comes to an end with my departure to Cornell, but I will never forget everything you have taugh t me. How could I ever forget my Gretzky of Food Micro 3

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ACKNOWLEDGMENTS I would like to thank Dr. Linda J. Harris for the opportunity to work as an undergraduate in her food microbiology laboratory at UC Davis. Without that opportunity I might never have realized what my true passions were or met Dr. Michelle D. Danyluk. Dr. Danyluk completely changed my professional life. She taught me every basic microbiology skill I now take for granted and always gave me th e confidence to succeed. The two years I have spent under her direct supervision completing my masters degree were incredible I can only hope one day to be the kind of researcher, mentor and human being she is. I would also like to thank my supervisory committee members: Dr. Rene GoodrichSchneider and Dr. Steve Sargent. Thank you both for your time, support and input, which was essential to the success of my project. E ach of you provided a unique twist; Dr. GoodrichSchneider for pushing me to explore the processi ng and production side of my project and Dr. Sargent for inspiring me to think outside of be ing just another microbiologist. Dr. Sargent the postharvest field trip was one of the most beneficial academic experiences I have ever had: thank you. I am grateful to Dr. Keith Schneider and Dr Ed Exteberria who always had an open door for me. Dr. Schneider the opportunities you provided me with were so generous and will always be extremely appreciated. Additionally, thank y ou to Dr. Jesse Gregory for teaching me how to really think and not just answer test questions. Overall, I am th ankful to everyone listed above. You have all made an impact on how I see the sc ience field and allowed me to grow into the researcher I am today. I am blessed to have the family and frie nds I do. I would like to thank everyone for dealing with costly cell phone bill s. I would like to especially th ank my parents and brothers for always grounding me and reminding me that there wa s a life outside of lab work. Their constant 4

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emotional support cannot be expressed in words. A ll of my friends from my various journeys in life: Kathryn Sullivan, Karina Dyk, Cindy Wallace, Kevin Furmanek, Becky Wong, Lyle Farrell, Jenna Marina, Jennette Villeda, Leann Manley, Marianne Fatica, Rachel McEgan, Huy Huynh and Jeremy Chenu. The encouragement I received from all of you, allowed me to complete this, even when I wanted to give up. My entire project would not have been feas ible without the assist ance of the Citrus Research and Education Center, CREC, and tech nical support of Loretta M. Friedrich and Gwen Lundy. I am thankful to all of the CREC staff fo r help with posters and being so friendly. All the CREC graduate students as well, this place wouldnt have been as fun without you all. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................9LIST OF FIGURES .......................................................................................................................11ABSTRACT ...................................................................................................................... .............12 CHAPTER 1 INTRODUCTION ................................................................................................................ ..142 LITERATURE REVIEW .......................................................................................................18Tropical Fruit Overview .........................................................................................................18Tropical Fruit Heal th Benefits ................................................................................................19Foodborne Illness ....................................................................................................................20Major Pathogens of Concern ..................................................................................................21Bacterial Pathogens ................................................................................................................21Campylobacter ................................................................................................................21Pathogenic E. coli ............................................................................................................21Listeria monocytogenes ...................................................................................................22Salmonella s pp. ................................................................................................................22Shigella spp. ....................................................................................................................24Sporeformers .................................................................................................................. .24Staphylococcus ................................................................................................................25Vibrio ...............................................................................................................................25Viral Pathogens .......................................................................................................................26Hepatitis A .......................................................................................................................26Norovirus .........................................................................................................................26Parasitic Pathogens .................................................................................................................27Characteristics of Selected Foodborne Pathogens ..................................................................27E. coli O157:H7 ...............................................................................................................27Salmonella .......................................................................................................................28Sources of Contamination ...................................................................................................... .30Production Environment ..................................................................................................30Postharvest Handling .......................................................................................................31Human Hygiene ...............................................................................................................31Current Foodborne Pathogen Research on Tropical Fruits ....................................................32Aai .......................................................................................................................... ........32Acerola ............................................................................................................................32Avocado ...........................................................................................................................33Banana ........................................................................................................................ .....35Caj .......................................................................................................................... ........37 6

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Coconut ....................................................................................................................... .....37Guava ...............................................................................................................................39Kiwifruit ..................................................................................................................... .....39Mamey ......................................................................................................................... ....40Mango ......................................................................................................................... .....40Papaya ........................................................................................................................ ......41Passion Fruit ....................................................................................................................43Pineapple ..................................................................................................................... ....45Pitanga .............................................................................................................................46Pathogen Prevention ...............................................................................................................47Regulatory Programs .......................................................................................................47Handling Parameters .......................................................................................................48Alternative Technologies .................................................................................................49Research Objectives ........................................................................................................... .....503 MATERIALS AND METHODS ...........................................................................................53Preliminary Tests ............................................................................................................. .......53Tropical Fruit ..........................................................................................................................54Bacterial Strains and Culture Conditions ...............................................................................54Inoculum Preparation ..............................................................................................................55Acid Adaption of Salmonella spp. Strains ..............................................................................56Inoculum Concentrations ....................................................................................................... .56Preparation of Tropical Fruit ................................................................................................. .57Tropical Fruit Inoculation .......................................................................................................57Storage Conditions ..................................................................................................................57Spoilage ..................................................................................................................................58Enumeration of Pathogens ...................................................................................................... 58Enrichment .................................................................................................................... ..........59E. coli O157:H7 ...............................................................................................................59Salmonella .......................................................................................................................60Statistics .................................................................................................................... ..............604 RESULTS ..................................................................................................................... ..........64Preliminary Results ........................................................................................................... ......64Storage Temperature a nd Relative Humidity .........................................................................64Background Microflora ..........................................................................................................64Pathogen Enumeration .......................................................................................................... ..65Inoculum Concentration .........................................................................................................65Fate of E. coli O157:H7 on Cut Mangoes ..............................................................................66Fate of E. coli O157:H7 on Cut Papayas ................................................................................67Fate of E. coli O157:H7 on Cut Pineapples ............................................................................68Fate of Salmonella on Cut Mangoes .......................................................................................69Fate of Salmonella on Cut Papayas ........................................................................................72Fate of Salmonella on Cut Pineapples ....................................................................................73Fate of Acid Adapted Salmonella on Cut Pineapples .............................................................75 7

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5 DISCUSSION .................................................................................................................. .....1076 FUTURE WORK ................................................................................................................. .119LIST OF REFERENCES .............................................................................................................122BIOGRAPHICAL SKETCH .......................................................................................................133 8

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LIST OF TABLES Table page 2-1 Selected Fruits Common ly Accepted as Tropical ..............................................................512-2 Outbreaks of foodborne illness associated with the consumption of tropical fruit ............523-1 E. coli O157:H7 Strains .....................................................................................................623-2 Salmonella Serovars...........................................................................................................634-1 Background Microflora enumerated on PCA following incubation at 23 2C ..............784-2 Background Microflora enumerated on PCA following incubation at 12 2C ..............794-3 Background Microflora enumerated on PCA following incubation at 4 2C ................804-4 Background Microflora enumerated on PCA following incubation at -20 2C .............814-5 E. coli O157:H7 on Fresh Cut Mangoes enumerated on TSANP and SMACNP following incubation at 23 2C .......................................................................................824-6 E. coli O157:H7 on Fresh Cut Mangoes enumerated on TSANP and SMACNP following incubation at 12 2C .......................................................................................834-7 E. coli O157:H7 on Fresh Cut Mangoes enumerated on TSANP and SMACNP following incubation at 4 2C .........................................................................................844-8 E. coli O157:H7 on Frozen Cut Mangoes enumerated on TSANP and SMACNP following incubation at -20 2C .....................................................................................854-9 E. coli O157:H7 on Fresh Cut Papayas enumerated on TSANP and SMACNP following incubation at 23 2C .......................................................................................864-10 E. coli O157:H7 on Fresh Cut Papayas enumerated on TSANP and SMACNP following incubation at 12 2C .......................................................................................874-11 E. coli O157:H7 on Fresh Cut Papayas enumerated on TSANP and SMACNP following incubation at 4 2C .........................................................................................884-12 E. coli O157:H7 on Frozen Cut Papaya enumerated on TSANP and SMACNP following incubation at -20 2C .....................................................................................894-13 E. coli O157:H7 on Fresh Cut Pineapples enumerated on TSANP and SMACNP following incubation at 23 2C .......................................................................................904-14 E. coli O157:H7 on Fresh Cut Pineapples enumerated on TSANP and SMACNP following incubation at 12 2C .......................................................................................91 9

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4-15 E. coli O157:H7 on Fresh Cut Pineapples enumerated on TSANP and SMACNP following incubation at 4 2C .........................................................................................924-16 E. coli O157:H7 on Frozen Cut Pineapples enumerated on TSANP and SMACNP following incubation at -20 2C .....................................................................................934-17 Salmonella spp. on Fresh Cut Mangoes enum erated on TSANP and BSANP following incubation at 23 2C .......................................................................................944-18 Salmonella spp. on Fresh Cut Mangoes enum erated on TSANP and BSANP following incubation at 12 2C .......................................................................................954-19 Salmonella spp. on Fresh Cut Mangoes enum erated on TSANP and BSANP following incubation at 4 2C .........................................................................................964-20 Salmonella spp. on Fresh Cut Mangoes enum erated on TSANP and BSANP following incubation at -20 2C .....................................................................................974-21 Salmonella spp. on Fresh Cut Papayas e numerated on TSANP and BSANP following incubation at 23 2C .......................................................................................984-22 Salmonella spp. on Fresh Cut Papayas e numerated on TSANP and BSANP following incubation at 12 2C .......................................................................................994-23 Salmonella spp. on Fresh Cut Papayas e numerated on TSANP and BSANP following incubation at 4 2C .......................................................................................1004-24 Salmonella spp. on Fresh Cut Papayas e numerated on TSANP and BSANP following incubation at -20 2C ...................................................................................1014-25 Salmonella spp. on Fresh Cut Pineapples enumerated on TSANP and BSANP following incubation at 23 2C .....................................................................................1024-26 Salmonella spp. on Fresh Cut Pineapples enumerated on TSANP and BSANP following incubation at 12 2C .....................................................................................1034-27 Salmonella spp. on Fresh Cut Pineapples enumerated on TSANP and BSANP following incubation at 4 2C .......................................................................................1044-28 Salmonella spp. on Fresh Cut Pineapples enumerated on TSANP and BSANP following incubation at -20 2C ...................................................................................1054-29 Acid Adapted Salmonella spp. on Fresh Cut Pineapples enumerated on TSANP and BSANP following incubation at 23 2C .......................................................................106 10

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LIST OF FIGURES Figure page 4-1 Average surface and center temperatures dur ing cooling of cut mango flesh stored at 4 2C over 180 min (n = 9). ............................................................................................764-2 Average surface and center temperatures duri ng cooling of cut papaya flesh stored at 4 2C over 180 min (n = 9). ............................................................................................77 11

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Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science FATE OF ESCHERICHIA COLI O157:H7 AND SALMONELLA SPP. ON FRESH AND FROZEN CUT MANGOES, PAPAYAS AND PINEAPPLES By Laura Kathryn Strawn August 2009 Chair: Michelle D. Danyluk Major: Food Science and Human Nutrition Escherichia coli O157:H7 and Salmonella infection has been associated with consumption of a number of fruits and vegetables. Although the fate of E. coli O157:H7 and Salmonella on many of these products is well studied, little is known about their behavior on cut mango, papaya and pineapple. Mangoes, papayas and pineapples have a ll been associated with outbreaks of salmonellosis. Three documented Salmonella outbreaks in the U.S. have been associated with consumption of raw mangoes, occurring in 1998, 1999 and 2001 from Salmonella Oranienburg, Salmonella Newport and Salmonella Saintpaul, respectively. A large outbreak in 1996 of Salmonella Weltevreden occurred in Jurong, Singapore. Salmonella Weltevreden was isolated from papaya and pineapple obtained at a local fr uit stall, implicating these fruit as a possible source for the outbreak. Most recently in 2006, an outbreak of Salmonella Litchfield occurred in Australia due to the consumption of fresh cut papa ya. The objective of this study was to evaluate the fate of E. coli O157:H7 and Salmonella on fresh (23C, 12C and 4C) and frozen (-20C) cut mangoes, papayas and pineapples. Cut ma ngoes, papayas and pineapples were spot inoculated with either a four st rain or five strain cocktail of E. coli O157:H7 or Salmonella respectively. Inoculated samples were air drie d, placed in containers a nd stored at 23 2, 12 2, 4 2 and -20 2C to simulate various poten tial storage conditions. Temperature and relative 12

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13 humidity was monitored and recorded by sensors in each container. Samples were enumerated following stomaching on selective and nonselective media at days 0, 1, 3, 5 and 7 (23C); 0, 1, 3, 5, 7, 10, 14, 21 and 28 (12 and 4C); and 0, 7, 14, 21, 28, 60, 90, 120, 150 and 180 (20C). Population levels in log CFU/g were cal culated. Fruit had visually spoiled by day 3, 5 or 10 at 23, 12C or 4C, respectively. Spoilage was not observed in frozen cut samples. E. coli O157:H7 and Salmonella have the potential to grow on temperature abused fresh cut mangoes and papayas held at 23C. At 12C, Salmonella populations can grow on cut mangoes and papayas; however, E. coli O157:H7 populations remained stable. E. coli O157:H7 and Salmonella can also survive for extende d periods of time on refrigerated mangoes and papayas. E. coli O157:H7 and Salmonella, inoculated onto pineapple, di d not grow but survived at population levels able to cause foodborne illness up to the shelf life of each storage temperature. E. coli O157:H7 and Salmonella can survive on frozen cut mangoes, papayas and pineapples for up to 180 days. This work indicates that fresh and frozen cut mangoes, papayas and pineapples have the potential to be vectors for E. coli O157:H7 and Salmonella transmission and preventive procedures should be in effect during production and postharvest processing.

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CHAPTER 1 INTRODUCTION The current food climate drives health concer ns in all ages of the population. Some of the fastest growing food compan ies are ones that focus on hea lthy alternatives. In 2005-2006, Jamba Juice, a fruit smoothie restaurant, was lis ted as one of the fastest growing restaurant chains with a 25% growth per centage (Sloan, 2007). Jamba Juice is just one of the many companies cashing in on the emerging market place of tropical fruits. The average American consumes 13 pounds more fresh fruit annually than he or she did twenty years ago (Huang and Huang, 2007). There are over 3,000 species of tropical fruits with approximately 140 million tons produced each y ear worldwide (Faylon et al., 2006). This emerging tropical fruit market in the United States can be contri buted to an increase in global imports, immigrant population and consumer familia rity. The U.S. climate, excluding Hawaii and parts of Florida, does not support the growth of most tropical fruit crops. Thus, the U.S. tropical fruit market is estimated to be appr oximately 80% imports (Yusuf and Salau, 2007). Developing countries report an estimated 98% of tropical fruit production (Yusuf and Salau, 2007). The United States has recently increased the number of import permits to these countries, in order to supply the U.S. ma rket with tropical fruits (Huang and Huang, 2007). The U.S. population includes many people who are from regi ons like South Ameri ca, Asia and India where mango consumption is common and extensive. Mangoes are known as the king of fruits in India and papayas are highly consumed in a va riety of ways in Brazil (Tharanathan et al., 2006; Penteado and Leitao, 2004). In the past, immigr ants to the U.S. had to rely on specialty stores or markets to find tropi cal fruits; however, with increas ing immigrant populations to the U.S. and the melding of cultures tropical fru it is now commonly found in grocery stores all across the country. Consumer perception of tropical fruits has ch anged as well. Tropical fruits 14

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used to be exotic and unfamiliar, but now are widely identified and consumed by all ages. Consumers can find a wide variety of tropical fruits in grocery store produce sections and on restaurant menus. Tropical fruits are a fixture in the lives of mo st Americans in food items such as fruit smoothies, juice blends, fruit salads and the growing sector of fresh cut produce. Each year there are approximately 76 million cases of foodborne disease, 325,000 hospitalizations, and 5,000 deaths in the U.S. (Mead et al., 1999). Produce was listed as the top vector of foodborne outbreaks from 1990 to 2005 (CSPI, 2006). Pathogens associated with produce outbreaks are Norovirus (33%), Salmonella (23%), Cyclospora (11%), Shigella (8%), Escherichia coli, E. coli (6%), Clostridium (5%), Hepatitis (5%), Campylobacter (2%), Staphylococcus (1%), Bacillus (1%), and others (5%) (CSPI 2006). Pathogens such as Vibrio and Listeria are included in the category of others. The U.S. Food and Drug Administration, FDA, conducted an imported produce survey in 1999 and found 40 of 1000 samples that tested positive for bacterial pathogens. In particul ar, 35 of the 40 samples tested positive for Salmonella (FDA, 2001a). Over the last twenty years an increase in produce related outbreaks has occurred. Factors contributi ng to this rise in foodborne dis ease in produce could be due to increased consumption, availability, health department awareness, government outbreak surveillance systems, distributi on and distance traveled. Produce outbreaks have been associated with a large variety of fruits and vegetables and in various forms, such as lettuce, tomato, cantaloupe, watermelon, sprouts (alfalfa), apple ju ice, orange juice, frozen strawberries, mango, papaya, pineapple and many other vegetables and fruits (Ackers et al., 1998; Hedberg et al., 1999; Ries et al., 1990; Blostei n, 1993; Mohle-Boetani et al., 2001; Cody et al., 1999; Cook et al., 1998; Hutin et al., 1999; Beatty et al., 2004; Gibbs et al ., 2009; and Ooi, 1997). 15

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Produce is often consumed raw or uncooked, increasing the risk of foodborne disease unless food safety is maintained throughout the fa rm to fork process. The possibility for pathogen contamination exists during the in itial farming, handling, processing, packaging, distribution, consumer preparati on and storage. Food safety re gulating bodies within the FDA and United States Department of Agriculture, USDA, have im plemented several preventive strategies to combat foodborne disease and future outbreaks. So me of these preventive programs include Good Agriculture Practices, GAPs, G ood Manufacturing Practices, GMPs, Standard Operating Procedures, SOPs, and Hazard Analys is Critical Control Points, HACCP. All are designed to limit the contamination of pathoge ns onto the raw produce commodities. Strict record keeping and monitoring allows for programs to be inspected and regulating bodies and companies to make corrective actions if required. Preliminary research is crucial to the development to these preventive programs. Understanding the ecology of pathogens on produce can help regulatory bodies, companies and consumers quantify risks associated with particular commodities, as well as understand how to handle the commodities in the safest manner to limit foodborne disease. There is limited research on pathogens and their behavior on tropical fruits. The objective of this thesis is to increase data avai lable in this currently lim ited research niche. This study evaluates the growth and survival of E. coli O157:H7 and Salmonella on fresh and frozen cut tropical fruits at various temperatures. The tropical fruits selected were mangoes, papayas, and pineapples due to their global popularity and consumer demand. Outbreaks have been noted in each fruit. The majority of bacterial foodborne outbreaks are caused by Salmonella Shigella or E. coli species. Shigella contamination, most co mmonly associated with infected food handlers, was excluded from this study. In the last 20 years an increase in outbreaks of foodborne illness associated with E. coli O157:H7 and Salmonella from fresh fruits 16

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17 and juices has occurred (Brandl, 2006). Cut mangoes, papayas and pineapples were spot inoculated with either a five st rain or four strain cocktail of E. coli O157:H7 or Salmonella respectively. Inoculated samples were air drie d, placed in containers a nd stored at 23 2, 12 2, 4 2 and -20 2C to simulate various cond itions from farm to fork. Temperature and relative humidity was monitored and recorded by sensors in each container. Samples were enumerated following stomaching on selective a nd nonselective media at days 0, 1, 3, 5 and 7 (23C); 0, 1, 3, 5, 7, 10, 14, 21 and 28 (12 and 4C); and 0, 7, 14, 21, 28, 60, 90, 120, 150 and 180 (-20C). Population levels in log CFU per g of fruit flesh were calculated. This thesis research will hopefully yield useful information on the behavior of potential pathogens on tropical fruit in order to aid in future food safety and sanitation programs.

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CHAPTER 2 LITERATURE REVIEW There are many definitions for tropical fruits. The universal consensus is that a tropical fruit originates from a warm climate called the tr opics. The tropics are generally considered to be approximately 25 north (Tropic of Cancer) and 25 south (Tropic of Capricorn) of the equator. This tropic region is frequently void of classical seas ons with temperatures remaining relatively constant throughout the ye ar. This environment is ideal for tropical fruit since another definition classifies tropical fruit as intolerant to frost. A sele cted list of tropical fruits is provided in table 2-1. Tropical Fruit Overview Amongst commonly accepted tropical fruits, avocado, banana, mango, papaya and pineapple have the highest worldwide production (Faylon et al., 2006). The United States is amongst the largest importer of tropical fruit commodities. In 2004, the U.S. accounted for approximately 25% of imported bananas, 35% of imported mangoes and 50% of imported papayas worldwide (Faylon et al., 2006). The majori ty of the tropical fruits come into the U.S. following production in other regions of the world where no uniform standards exist for production and post-harvest practices, thus the safe ty of these products is a potential concern. Worldwide tropical fruit consumption per capita has increased 1/3 over th e last twenty years (FAO, 2004). This increase in fresh fruit cons umption is due primarily to the growing popularity of tropical fruit. Tropical fruit imports into the U.S. have doubled over the past 20 years from 7% to 15% (Huang and Huang, 2007). Forecasts predict tropical fruit trade and world production to expand over the next ten years (Yusuf and Salau, 2007). Mango and papaya consumption in the U.S. is specifically increasing due to the expanding immigrant popul ation and a push by commodity organizations 18

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such as the National Mango Board. The Na tional Mango Board, founded in May 2007, has a variety of mango recipes and preparation (cutting and slicing) techniques for consumers to experiment with. Mango is predicted to have the greatest impor t growth demand; by 2010 mango importation into the U.S. is estimated to grow an additional 7% (FAO, 2003). Papaya is predicted to have a 5.6% increase in total export volume worldwide according to the Food Agriculture Organization, FAO, by 2014 (Faylon et al., 2006). The pr ice of tropical fruit in the U.S. has also decreased due to the vast dive rsity of global tropical fruit production, making tropical fruit more available to all types of markets and consumers, throughout the year (FAO, 2003). Outside the U.S., it a familiar practice to pur chase fresh-cut fruit from street vendors. These fresh cut fruit providers do not adhere to the same permit regulations as U.S. food vendors. Normally, street vendors or markets cut fresh fruits in th e morning and sell to consumers throughout the day. Many times these fres h cut fruits are temper ature abused for the entire day from cutting to consumption. Common practice is to squirt lemon juice on the fresh cut fruit, such as papaya in order to preven t pathogen growth (Escartin et al., 1989). The outbreak surveillance systems and pathogen detecti on capabilities in many of these countries do not exist or are so poor that many tropical fruit outbreaks or illnesses could be severely underreported. Tropical Fruit Health Benefits Nutritional and health benef it claims related to consumption are hypothesized to have contributed to the rise in popular ity of tropical fruits to the American consumer. The health benefits of fruit consumption are well documen ted (Bazzano et al., 2002). Recent studies are continuing to add evidence related to how beneficial to consumers health eating fr uits, including tropical fruits, can be. Diets high in fruits can reduce risk of obesit y, stroke, cardiovascular 19

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disease, diabetes and cancers (Cooke et al., 2003) The Dietar y Guidelines for Americans published by the USDA recommends five servings of fruit and vegetable daily and in 2005 listed fruit consumption as one of its food groups to encourage (USDA, 2005). Tropical fruit can be sources of vitamin C, carotenoids and provi tamin A (Knee, 2002). Cooked green banana consumption by children (6-24 months) over five days has been found to significantly reduce colonic inflammation and severity of Shigellosis (Rabbani et al., 2003). Recently guava was found to contain high contents of dietary fiber an d antioxidants (Jimenez-Escrig et al., 2001), and high antioxidant activity (Huang et al., 2004). Consumption of fruits with these characteristics are believed to decrease the risk associated with diseases such as cardiovascular and cancers (Jimenez-Escrig et al., 2001). Subjects who ate diets of kiwifruit had significantly higher stool production along with bulkier and softer stools pr omoting healthier digestion and bowel function (Rush et al., 2002). Kiwifruit might prove to have a market in digestive health as they are agreeable to most of the populati on. Acerola has also been found to be an extremely good source of carotenoids and is being market ed in fruit juice drink blends similar to tomatoes in V8. In the last year many emerging tropic al fruits, like acerola are be coming common staples in healthy juice blends. Foodborne Illness Food safety is an important aspect of government regulation, company policy and consumer health. Each year there are approximately 76 million cases of foodborne disease, 325,000 hospitalizations and 5,000 deaths in the United States (Mead et al., 1999). Most cases of foodborne illnesses are neve r diagnosed so this number is speculated to be grossly underreported. Foodborne infections in healthy adults are typically se lf-limiting without the need for hospitalization. Death, when reporte d, usually occurs in the young, elderly, and immune comprised (Mead et al., 1999). 20

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Major Pathogens of Concern Bacterial, viral and parasitic pathogens have all been associated with fruit consumption. Most fruit outbreaks (54%) are never associated with a particular pat hogen due to the difficult nature of traceback and underreporti ng (Sivapalasingam et al., 2004). Salmonella hepatitis A and Cyclospora are the most common bacterial, vira l and parasitic agents respectively (Sivapalasingam et al., 2004). It is estimated even fewer than 10 organisms contaminating a serving of fruit would be sufficient to cause illness. Once cut, fruits typically contain ample fermentable sugars and a high water activity su pplying the necessary conditions for microbial growth and survival on. Research demonstrates replication can occur rapidly on cut, damaged or wounded fruit (Beuchat, 1996). Outbreaks have o ccurred due to the consumption of various tropical fruits as li sted in Table 2-2. Bacterial Pathogens Campylobacter Campylobacter has the highest number of estimated cases out of all the bacterial foodborne diseases with approximately 2,500,000 (Mead et al., 1999). The instance of hospitalization or death in Campylobacter infections in the U.S. is rare, unless the infected person is high risk, such as the elderly or immune compromised. Campylobacter infections are commonly associated with poultry; however, the risk between cross cont amination of infected poultry and fruit in delicatesse ns can occur (Beuchat, 1996). Two documented outbreaks of Campylobacter jejuni have been associated with tropical fruit; pineapple and guacamole (avocado) occurred in 1999 and 2002, respectiv ely in the U.S. (CDC, 1998 and CDC, 2002). Pathogenic E. coli The FDA has recognized E. coli O157:H7 as an emerging fo odborne pathogen since the 1980s. It is estimated each year that 75,000 cases occur (Mead et al., 1999). The symptoms are 21

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very similar to Salmonellosis; however, diarrhea is bloody, and long term sequella can include hemolytic uremic syndrome (HUS) and neurologi cal disorders. During the past ten years, E. coli O157:H7 has been linked to consumption of ra w produce including leafy greens, apple cider, alfalfa sprouts and cantaloupe (Sivapalasingam et al., 2004). An outbreak of non-O157:H7 E. coli (O11:H43) was documented in the U.S. due to pineapple consumption by Sivapalasingam et al. (2004) in 1994. There have been no other documented E. coli outbreaks associated with tropical fruits. Listeria monocytogenes There are an estimated 2,500 cases each year of Listeria monocytogenes, with the highest hospitalization rate (0.92 2) of the foodborne pathogens discussed here (Mead et al., 1999). L. monocytogenes is of huge concern to pregnant women as it may lead to stillbirths or spontaneous abortions. L. monocytogenes is commonly found on plants and both the plant production and processing environment (Beuchat et al., 1990). These factors make it a likely contaminant of raw tropical fruit commodities due to its hi gh environmental prevalence. Additionally, L. monocytogenes is known to grow on fresh produce at refr igeration temperatures (Beuchat, 1996). There have been no documented cases of L. monocytogenes outbreaks on tropical fruit commodities; however, with the growing sector of fresh cut produce at refrigerated storage, preventive food safety programs should incorporate L. monocytogenes as a pathogen of risk. Salmonella s pp. Salmonella is estimated to cause approximately 1,500,000 cases each year (Mead et al., 1999). Typical salmonellosis symptoms include non-bloody diarrhea and severe cramping. Most people will recover completely without hospitalization by rest and keeping hydrated. Multiple serovars of Salmonella can cause foodborne illness from consumption of fresh produce (Beuchat, 1996). Salmonella outbreaks have been associat ed with the following tropical 22

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products: raw mangoes, frozen mamey, papaya, pin eapple and various types of coconut (Table 22). Four documented Salmonella outbreaks have been associated with mangoes, occurring in 1998, 1999, 2001 and 2003 from Salmonella Oranienburg, Newport, Saintpaul and Saintpaul, respectively (PHAC, 1998; Sivaapalas ingam et al., 2003; Beatty et al., 2004; CDC, 2003). In all cases it was determined that imported raw mango es were the source of the outbreak. In 1998-99 an outbreak of typhoid fever occurred in South Florida resulting in at least 16 illnesses. Salmonella Typhi was isolated from 15 patients and th e source of the outbreak was traced back to a frozen fruit smoothie containing mamey. The frozen mamey fruit was reported to have been imported from Guatemala. This was the firs t typhoid fever outbreak in the U.S. due to commercially imported food (Katz et al ., 2002). In 1996, a large outbreak of Salmonella Weltevreden occurred in Jurong, Singapore and involved at least 116 shipyard workers. Papaya and pineapple were both implicat ed as possible sources of the Salmonella outbreak. Fruit samples (honeydew, papaya, pineapple and watermel on) taken from a local fruit stall were found to be positive for Salmonella Weltevreden (Ooi, 1997). It has been long known that raw, unprocessed coconut supports the growth of Salmonella (Schaffner et al., 1967). Kovacs (1959) first isolated Salmonella from dried coconut in 1959. Salmonella outbreaks have occurred in many forms of coconut, such as deshelled coc onut and dried coconut. An outbreak of typhoid fever and salmonellosis was traced back to dried coconut in South Wales and spanned several months (Wilson and Mackenzie, 1955). In 1960-1961 a Salmonella outbreak was traced directly back to coconut in Liverpool, E ngland (Semple, 1962). An outbreak of 167 cases of paratyphoid fever A occurred in Singapore and was linked to c onsumption of deshelled coconuts (Teoh et al., 1997). 23

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Shigella s pp. All Shigella species are pathogenic to humans (Beuchat, 1996). Salmonella and E. coli which can have animal or environmental hosts, Shigella only has human hosts. Shigellosis can occur from consumption of contaminated raw products like salads, following contamination from direct human contamination (Beuchat, 1996). Unlike Shigella contamination on foods can be prevented by proper human sanitation, such as campaigns for employee hand washing. The CDC estimates almost 500,000 cases each year are due to Shigella infections (Mead et al., 1999). In 1991, a shigellosis outbreak associated with a coconut milk dessert occurred at a school in Suan Phung, Thailand. Approximately 200 patients became ill over a 2-month period with Shigella dysenteriae type 1 (Hoge et al., 1995). Furthermore, two Shigella outbreaks have occurred in guacamole (avocados) in 1998 and 2002 (CDC, 1998 and CDC, 2002). Both outbreaks were contributed to improper employee sanitation. Sporeformers Major spore forming foo dborne pathogens include Bacillus cereus, Clostridium botulinum and Clostridium perfringens. No documented outbreaks have been associated with these organisms; however, as many tropical fruits are packaged in controlled or modified atmosphere packaging they may become a future concern. The control of oxygen can create a partially or fully anaerobic environment that c ould possibly enhance the growth and survival of both sporeforming bacteria (Beuchat, 1996). Bacillus cereus causes approximately 30,000 illnesses each year in the U.S. (Mead et al., 1999). Clostridium species cause an approximate 280,000 illnesses each year in the U.S., with the majority 90% being cases of C. perfringens (Mead et al., 1999). 24

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Staphylococcus Staphylococcus is a common bacterial inhabitant of the human skin, and only certain species (S. aureus) are hazardous foodborne pathogens. S. aureus is a foodborne intoxication meaning it produces a toxin on the food. The cons umer then consumes the toxin by eating the food to get ill. E. coli Salmonella and Shigella are common foodborne infections. Foodborne infections cause a person to become ill by cons uming the food, which contains the bacterial pathogen. The bacterial pathogen once inside the body then colonizes the intestinal tract to cause illness. Each year there are approximately 185,000 cases of Staphylococcus foodborne poisoning and only two deaths in the U.S. (Mead et al., 19 99). The number of cases is estimated to be underreported by 38 times due to th e short illness duration and its flu like symptoms (Mead et al., 1999). Three outbreaks from tropical fruit consumption associated with S. aureus are linked to banana products. In 2002, 2003 and 2004 S. aureus outbreaks were documented in plantains, banana pudding and banana pudding respectively (C SPI, 2006; CDC, 2003; CDC, 2004). Vibrio Each year there are approximately 8,000 Vibrio infections and 60 deaths in the U.S. (Mead et al., 1999). Vibrio is a waterborne pathogen that is commonly associated with seafood products like oysters. Pathogens in the Vibrio species include V. cholerae V. parahaemolyticus and V. vulnificus. Vibrio infections are very serious and us ually require hospitalization due to the risk of septicaemia (Mead et al., 1999). Specifically V. cholera has an estimated 54 cases each year in the U.S. (Mead et al ., 1999). In Maryland, an outbreak of V. cholera was traced back to imported frozen coconut milk (Taylo r et al. 1993). This outbreak highlights the importance of securing safe tropical fruit products from the global marketplace. 25

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Viral Pathogens Hepatitis A Hepatitis A has been linked to several outbreak s in frozen fruits, su ch as raspberries and strawberries (Beuchat, 1996). Tropical fruits are commonly found frozen in smoothie bars and for home smoothie use. Hepatitis A has a significantly higher frequency of hospitalizations and case fatalities than Norovirus. Each year there are appr oximately 83,000 cases reported, 11,000 hospitalizations and 83 deaths (M ead et al., 1999). The symptoms of Hepatitis A are nausea, vomiting, diarrhea, fever and possibly jaundice (Mead et al., 1999). There is only one documented Hepatitis A outbreak related to trop ical fruit consumption in 2000 due to guacamole (avocado) (CDC, 2000). Norovirus Norovirus is the most common foodborne di sease in the U.S. with approximately 23,000,000 reported cases, 50,000 hospitalizations and 300 deaths each year (Mead et al., 1999). The symptoms include nausea, diarrhea and abdomi nal pain or cramps (Mead et al., 1999). It has been associated with outbreaks in avo cados, bananas and pineapples. One norovirus outbreak has occurred due to consumption of raw avocados and three to guacamole in 2001, 2005, 2005 and 2006 respectively (CSPI, 2001; CD C, 2005; CDC, 2005; CDC, 2006). A norovirus outbreak in banana pudding occurred in 2002 and another in an unspecified banana product in 2005 (CDC, 2002; CDC, 2005). Four norovirus outbreaks have been documented in unspecified pineapple products in 1999, 2001, 2002 and 2003, as well as one norovirus outbreak related to the consumption of fresh cut pi neapple in 2001 (CDC, 1999; CDC, 2001; CDC, 2002; CDC, 2003; CDC, 2001). 26

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Parasitic Pathogens Cyclospora has caused the majority of parasite outbreaks associated from produce between 1973 and 1997 (Sivapalasingam et al., 2004) Each year it is estimated that 16,000 cases of Cyclospora are reported in the U.S. (Mead et al., 1999). Giardia lambia accounts for the most reported parasitic illnesses with approximately 2,000,000 each year, while Toxoplasma gondii accounts for the most parasitic deaths eac h year with approximately 750 (Mead et al., 1999). The importance of water quality is extremel y crucial to preventing parasite outbreaks in produce. Industry should have proper sanitation plans in orde r to ensure their production and processing lines have water that is contamina tion free. There have been no tropical fruit outbreaks related to paras itic pathogens, but the potential remains. Characteristics of Selected Foodborne Pathogens E. coli O157:H7 E. coli is a common component of human and animal digestive tract microbial flora (Meng et al., 2007). For th e healthy human and animal, E. coli is rarely harmful as it has a symbiotic relationship with the diges tive tract microbial flora. Some E. coli strains have over time acquired virulence characteristic s. These virulence factors cause E. coli to be pathogenic to humans and animals and may cause diarrhea, urinary tract infections, sepsis, meningitis and others (Meng et al., 2007). One of the fr equently discussed virulent strains of E. coli is O157:H7. In 1982, E. coli O157:H7 was first identified as a foodborne pathogen, in an outbreak traced back to Oregon from eating unde rcooked hamburgers (Meng et al., 2007). E. coli isolates are categorized with three antige ns: the somatic (O), flagella (H) and capsule (K) antigens. When identifying different isolates of E. coli the only antigens needed are the O and H (Meng et al., 2007). The O antigen identifies the serogroup of a strain, while the H antigen identifies its serotype. E. coli O157:H7 is also within the family Enterobactiaceae and is noted for its ability 27

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to produce the Shiga toxin and acid resistance (M eng et al., 2007). There are two major groups of Shiga toxins: Stx1 and Stx2. Shiga toxins work by inhibiting protein s ynthesis in the host and can induce colonic disease, HUS and neur ological diseases (Meng et al., 2007). E. coli O157:H7 must be able to withstand the acidic gastric environment of the digestive tract (Meng et al., 2007). A study by Lin et al. (1996) found that three mechanisms exist for E. coli O157:H7 acid residence: acid induced oxida tive, an acid induced arginine -dependent, and a glutamatedependent system. The minimum pH for growth of E. coli O157:H7 is approximately 4.0-4.5 (Meng et al., 2007). Th e highest incidence of E. coli O157:H7 outbreaks are in the warmest months (Meng et al., 2007). A study by Ra ngel et al. (2005) found that 89% of the 350 outbreaks in the United States occurred May to N ovember. This is of note, as most domestic tropical fruit, in particular mango, are harves ted during the summer months (May-September). Papayas, which are harvested year around, have tw ice as much production in the summer months (May-September). One of the propos ed reasons for the increase in E. coli O157:H7 outbreaks during this time period is inappropriate temper ature control in the shipping and packing processes. Salmonella Salmonella was documented as a food borne disease in the 1800s (Cox, 2000). The genus Salmonella was not officially named until 1900 after a number of outbreaks from infected animals were isolated (Cox, 2000). Salmonella is within the Enterobactiaceae family. This family consists of gram negative, rod shap ed and facultative anaer obes approximately 1.5-2.0 m in size (Cox, 2000). The optimum growth of Salmonella bacteria are body temperature (37C). They are noted by their ability to utilize both respiration and fermentative pathways and produce gas and acid. It is generally recognized th at there is one main species in the genus: Salmonella enterica (Cox, 2000). Salmonella species are classified by serology and there are over 2,541 28

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known serovars (Popoff et al., 2004). The identification of Salmonella is accomplished by the tenuous procedure of serological confirmation, which involves th e agglutination of bacterial surface antigens with salmonella-specific antib odies (Maurer and DAoust, 2007). This procedure utilizes two main antigens: somatic (O) lipopolysaccharides (L PS) and flagellin (H) antigens, as well as capsular (K) antigens (Maurer and DAoust, 2007). The somatic (O) lipopolysaccharides (LPS) antigens ar e heat stable and part of the outer surface of gram negative bacteria, which can be classified as major or minor antigens. The flagellin (H) antigens are heatlabile and determine serovar type by variation in the flagella. Salmonella flagellar can be monophasic, diphasic or a lternate between monophasic and dipha sic (two sets of antigens). Capsular (K) antigens are limited to the Vi antigen found in Salmonella serovars Typhi, Paratyphi C and Dublin (Maurer and DAoust, 2007). Salmonella is most commonly found in animal and environmental reservoirs. It is extremely resilient and has been found to adapt to severe enviro nmental or processing conditions (Maurer and DAoust, 2007). Some exam ples of the severe conditions that Salmonella retains the ability to grow are at temperatures as lo w at 2C and as high as 54C (Baker et al., 1986; Kim et al., 1989), low pH foods (pH 3.99 and pH 4.05) (Asplund and Nurmi, 1991; Chung and Goepfert, 1970) and water activities as low as 0.93 (Troller, 1986). A study by Airoldi and Zottola (1988) determined that the preconditioning of Salmonella cells to low temperatures can increase the growth and survival of those cells in refrigerated foods It has also been determined that that preconditioning of Salmonella cells to low pH environmen ts can increase the growth and survival of those cells in acidic foods (Huhtanen, 1975). The growth of Salmonella in acidic environments can increase with increasing temp erature (DAoust, 1989; Fluit and Schmitz, 1999; and Thomas et al., 1992). Bearson et al. ( 1998) concluded that ther e is an interrelated 29

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relationship with acid tolerance and virulence in Salmonella Salmonella is a particularly important food safety risk due to its persistent survival. Salmonella can survive on foods for extended periods of time in anywhere from fr eezing to ambient conditions (DAoust, 1989). Sources of Contamination Consumers of tropical fruits have many choices as tropical fruits can be sold whole, dried, fresh cut and frozen cut. Most consumers will consume tropical fruits raw or without a processing kill step. Since tropical fruits are consumed raw proper postharvest processing, handling and storage are essential to providing the safest product. The three poten tial sources of pathogen contamination in tropical fruit are the production environment, postharvest handling and human hygiene. Production Environment Marth (1969) concluded that original contamination on co conuts wasnt due to human carriers or polluted waters, but fr om contact with soils containing Salmonella Salmonella is known to survive and be found in the environment, in such places like soil, water reservoirs and feces (Beuchat, 1996; Guo et al., 2002). Currently, the U.S. requires all impor ted mangoes to undergo a postharvest disinfestations procedure involving heat treatment to remove the potential for introduction of the tephritid fruit fly (Code of Federal Regulations, 2001). This heat treatment is often followed by rapid cooling of the mangoes in water. Penteado et al. (2004) determined that Salmonella can be internalized into intact mango flesh during th ese types of postharvest heat treatments. Additionally, Bordini et al. (2007) reported a similar conclusion that Salmonella can indeed internalize, survive and grow in the interior flesh of mangoes. Ov erall, tropical fruits have the potential to become vehicles of foodborne disease and the ecology of major pathogens of concern should be investigated. 30

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Postharvest Handling Processing preparation in the packinghouse is crucial to removing contaminants picked up from the environment at the farm level. I ndustry and consumers alike employ a washing step to aid in this removal of contaminants, which co uld be potential pathogens. The washing step removes soil and microorganisms if done properly; it has been shown in potable water to achieve an approximate 1 log reduction (Beuchat, 1996) Produce at an indus try level is commonly washed in water containing 50-200 mg L-1 of chlorine (Parish et al., 2001). In the fresh cut fruit industry after the washi ng step the fruits are usually cut. It is imperative to cut fruits in a pathogen free environment because cutting fruits increases the potential to transfer pathogens from the outer surface of the fruit to the nutrient abundant flesh. The nutrient abundant flesh of tropical fruit has been shown by researchers to provide an ample growing substrate for pathogen proliferati on. A study by Penteado and Leitao (2004) found Salmonella to replicate in 4 hours on fresh cut pa payas held at ambient temperature. Additionally, another study provi ded similar findings in that Shigella and Salmonella were both found to replicate in 3 hours on fresh cut papaya s held at room temperature (Escartin et al., 1989). Human Hygiene Human hygiene can attack the safety of fruits at many different stages from farm to fork. Many pathogens are passed directly fr om human to human contact, such as Shigella Workers must have proper instruction and facilities to wash their hands, use the restroom and consume meals. It is important to note that to eliminate these types of pathogens human hygiene is a necessity. Many companies also utilize the outsid e sources of University Extension to provide lectures and talks discussing the impo rtance of proper human hygiene. 31

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Current Foodborne Pathogen Research on Tropical Fruits Aai Aai is one of the newest tropical fruits to be featured in the U.S. marketplace, in such forms as health drinks or weight loss smoothies. It is native to Centra l and South Am erica with its largest production in Brazil (Per eira et al., 2008). Numerous outbreaks in Brazil have been associated with aai juice consumption since 2005 due to wild triatomines, alternatively known as the kissing bugs (Pereira et al., 2008). Tr iatomines are common carriers of the protozoan Typanosoma cruzi which in humans causes Chagas diseas e. Chagas disease has two phases, the first phase can be asymptoma tic or include symptoms of fe ver, edema, hepatosplenomegaly and or meningoencephalitis, while the second phase can produce comprised heart or digestive function (Pereira et al., 2008). There are two proposed pathways for triatomines contamination of aai juice. The two pathways are triatomine s may be grinded with the aai berries into the juice due to their attraction to the light used on the juic ing machine or from poor sanitation during transport or fruit processing (Pereira et al., 2008). Aai ju ice has been labeled a high risk food for Chagas disease; however, with proper sanitation and safety protocols in place the number of outbreaks may be reduced. Acerola Considered an emerging tropical fruit, acerola is native to the Caribbean islands, Central America and the Amazonian region (A ssis et al., 2008). Acerola is a small cherry like fruit with a pH of 3.24. It contains hi gh levels of vitamin C and studies have found the human body to absorb acerola produced vitamin C be tter than synthetic asco rbic acid (Assis et al., 2008). In the U.S. the market for acerola is mostly in th e pharmaceutical industry; however, because of its vitamin C content more products could be seen with acerola in the future. 32

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As an emerging tropical fruit, few studi es have been done on the foodborne disease potential of acerola. E. coli O157:H7 can survive in acerola pulp for periods of 7-11 days depending on the strain in refrigeration temperatur es (Marques et al., 2001). This survival is important to note as acerola is imported into the U.S. under refrigerated conditions to slow respiration and metabolism, and main tain the quality of the fruit. Avocado Mexico is the worlds biggest producer and e xporter of avocado (FAO, 2003). The total world production of avocados was approximate ly 3.5 million tons in 2004 (FAO, 2003). The U.S. is the leading importer of avocados acc ounting for 30% of the to tal import share (FAO, 2003). Avocados imported to the U.S. are mainly used in the production of guacamole, which is a mixture of diced cilantro, tomato, chili, lime ju ice, and salt (Iturriaga et al., 2002). Avocados are susceptible to bacterial contamination from other fresh commodities that have been associated with foodborne outbreaks ( E. coli O157:H7 outbreak in cilantro; Zepeda-Lopez et al., 1995 and Salmonella Javiana outbreak in tomatoes; Hedburg et al., 1999) their high water activity (0.98) and pH (6.5). A survey of guacamole from street vendors and restaurants from Mexico found positive samples of S. aureus E. coli Salmonella and L. monocytogenes. The incidence of positive samples was higher in street vendor guacamole samples than restaurants. S. aureus was found in guacamole samples 4.3% and 10.3%, E. coli in 54.3% and 69%, Salmonella in 0% and 3.4%, and L. monocytogenes in 15.2% and 17.2% for rest aurant and street vendor locations respectively (Arvizu-Medr ano et al., 2001). This survey indicates that avocado can be an acceptable substrate for pathogens. Further work should be conducted to prevent foodborne illness from avocadoes (subsequently outbreaks associated with guacamole). Various food pathogens have been found to grow in avocado pulp and juice. Salmonella E. coli O157:H7, S. aureus, and L. monocytogenes all exhibit growth in avocado pulp or juice 33

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over a range of temperature conditions. Arvizu-Medrano et al. (2001) found that Salmonella E. coli O157:H7, and S. aureus all displayed growth in avocado pulp when inoculated at low (1.5 log CFU/ml) or high (3.5 log CFU/ml) levels in am bient storage conditions w ithin the first 24 h. Population increases were independent of initial inoculum levels, except for S. aureus A 6 and 5 log CFU/ml population increase was observed for Salmonella and E. coli O157:H7 respectively within the first 24 h in avocado pulp stored at 2025C. Population increases for S. aureus were dependent on initial inoculum concen tration. Avocado pulp inoculated at 3.5 log CFU/ml observed a S. aureus population increase of 2 log CFU/ml over the 24 h storage period, while pulp inoculated at 1.5 log CFU/ml obser ved a population increase of 4 log CFU/ml. While, S. aureus appears to be less of a concern in avo cado pulp it should be mentioned that one of the major sources of S. aureus contamination is by an infect ed food handler, so it should not be ignored as a potential risk of foodborne illness (ArvizuMedrano et al. 2001). A study by Iturriago et al. (2002) found L. monocytogenes to grow on avocado pulp stored at ambient temperatures (22C) as well. Avocado pulp sa mples were inoculated at approximately 2 log CFU/g and held for either 2 days. L. monocytogenes increased 4 log CFU/g within the first 24 h and a total of 7 log CFU/g in the 48 h held at 22C. The lag time for L. monocytogenes on avocado pulp was found to only be 3 h in am bient temperature conditions, illustrating the importance of temperature control. E. coli O157:H7 and S. aureus in avocado pulp held at refr igeration temperatures (4C) for up to 2 weeks exhibited no growth, but surviv al for the duration of the experiment (ArvizuMedrano et al. 2001). Alternatively, Salmonella and L. monocytogenes populations on avocado pulp held at refrigeration temperat ures (4C) did exhibit growth. Salmonella populations had a slight increase before maintaining a steady population count for the 2-week storage period 34

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(Arvizu-Medrano et al. 2001). L. monocytogenes on avocado pulp exhibited steady growth over the 2-week storage period reaching 4, 6 and 8 log CFU/g at 3, 7 and 14 days respectively (Iturriago et al., 2002). Refrigerat ion is commonly used in the food industry to delay or inhibit growth of spoilage and pathogenic organisms. However, refrigeration should not be considered an effective tool against th e growth and survival of L. monocytogenes in avocado pulp. L. monocytogenes in avocado pulp held at freezing temp eratures was recovered over a 60-week storage period at consistent levels of 3.5 log CFU/g (Iturriago et al 2002). E. coli O157:H7 and Salmonella can grow in avocado juice at both ambient and refrigeration temperatures. Avocado juice was filtered, steamed and inoculated with E. coli O157:H7 populations of 3.5 log CFU/ml. E. coli O157:H7 populations stored at ambient temperatures observed growth increases of 3, 2, and 1 log CFU/ml at days 1, 2, and 3 respectively. After day 3, E. coli O157:H7 populations reached a maximum of 9.5 log CFU/ml and exhibited a decline for the rest of the e xperiment duration (Muta ku et al., 2005). Avocado juice steamed and inoculated with either 3.3 or 2.8 log CFU/ml E. coli or Salmonella respectively, reached a maximum level of ca 7.5 log CFU/ml within the first 16 h at 37C. Both then proceeded to decline over the 2-d storage period (Yigeremu et al., 2001). In avocado juice held at 4C, Salmonella populations increased ca. 1.0 log CF U/ml in the first 24 h before declining, while E. coli populations had a longer lag time before increasing approximately 1.01.5 log CFU/ml on day 2 (Yigeremu et al., 2001). Mutaku et al. (2005) found E. coli O157:H7 populations stored at refriger ation temperatures displayed a steady growth increase of approximately 0.8 log CFU/ml each day over a 5-d time span (Mutaku et al. 2005). Banana Banana is the most recognized tropical fruit. Over the last te n years world banana exports have expanded by 28% (FAO, 2009). Projections for 2010 have the U.S. going entirely 35

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towards relying on imported bananas (FAO, 2009). Ecuador is the leading exporter in bananas followed by Costa Rica and Colombia. This gradual shift may be due to lower prices of bananas from outside countries, as well as the U.S. being below the level required to feed the consumer demand. Bananas are high in dietary fiber, potas sium, and digestible ca rbohydrates that make bananas a healthy part of the human diet (Rabani et al., 2003). Little research exists on cut bananas (pH ca. 5.5) and pat hogen proliferation as it is not a common means of banana consumption. Further re search evaluating transfer of pathogens on the banana peel to the internal fr uit flesh is warranted. Behrsi ng et al. (2003) inoculated high concentrations (ca. 105-106 CFU/ml) of L. innocua, Salmonella and E. coli on to the inedible skin (peel) of banana and observed its behavior over 13 days at 18C. No growth, only survival was observed by all three pathogens on the banana peel in this study. Banana puree and yoghurt can support the surviv al of pathogens for extended periods of storage at refrigeration and freezing temperatures. E. coli O157:H7, L. monocytogenes and Salmonella when inoculated in banana puree held at freezing temperatures (-23C) exhibit survival for up to 12 weeks (Oyarzabal et al., 20 03). Samples of banana puree were inoculated with a 5-strain cocktail of each bacterium at an initial inoculum concentration of 4 log CFU/g. Pathogen recovery took place every 6 and 24 h, once a week for 4 weeks, and biweekly for the rest of the storage period Within the first 6 h, L. monocytogenes had dropped below the limit of detection, but remained positive upon enrichment for the 12-week storage period. E. coli O157:H7 and Salmonella survived better than L. monocytogenes in frozen banana puree with population counts stabilizing after the first week at 3 and 2.5 l og CFU/g, respectively, for the 12week storage period. A lower in itial inoculum concentration did not affect survival of E. coli O157:H7 in frozen banana puree (Oyarzabal et al., 2003). A study by Sabreen and Korashy 36

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(2001) found L. monocytogenes to survive in banana yoghurt when held at refrigeration (5C). Banana yoghurt samples were inoculated with L. monocytogenes populations of ca. 7.5 log CFU/ml and declined over the 7-d storage peri od to ca. 3.5 log CFU/ml. Bananas alone do not have a low pH; however, have considerably lower pH values due to being in yogurt. Over the 7 day storage period the pH of th e banana yoghurt inoculated with L. monocytogenes decreased from 4.79 to 3.98 giving a possible reason for L. monocytogenes declining (Sabreen and Korashy, 2001). Caj Caj (sometimes spelled kaj) is a minor tr opical fruit grown prim arily in Brazil (Souza, 1998). The fruit resembles a mango, but much smaller. Caj has an attractive appearance, strong nutritional profile, sweet smelling odor an d tasty composition. Caj is high in betacryptoxanthin, known for its positi ve effects on retinal function in the body (Rodriquez-Amaya et al., 2006). In South American countries the fruit can be found in markets as fresh cut, pulp, juice, and even ice cream (Souza, 1998). Pathogen growth and survival on caja is relatively unknown due to its small market and previous theories about the ab ility of foodborne pathogens to survive at extremely low pH values. The pH of caj is ca. 2.65. Marques et al. (2001) found that bo th acid adapted and non acid adapted E. coli O157:H7 strains survived for up to 11 da ys in refrigerated caj pulp. Some strain to strain variability was observed, stra ins from calfs renal swab and a human isolate associated with an apple cider outbreak surviv ed for 11 days where a strain from hamburger survived only 4 days before reaching unde tectable levels (Mar ques et al. 2001). Coconut Coconut is one of the most useful tree fruits in the world, with almo st every part of the coconut utilized. World production of coconut has increased steadily over the past fifty years, 37

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accelerating in the last 20 years, with Indonesia as the leading producer (FAO, 2002). The white meat of the coconut (pH ca. 6) can be eaten raw or shredded and dried to be used as an ingredient in a wide variety of foods from cakes to bevera ges. Shelled fresh coconuts can be consumed for up to a month if refrigerated. A survey looking at fresh coconut slices being sold by street vendors and markets in India found 58% of coconut slices to be positive for enterotoxigenic S. aureus and 15% of coconut slices to contain Shigella species dysenteraie (type 1 and 5) and flexneri (type 2a) (Ghosh et al., 2007). The survey comprised of 150 coconut slices collected from 75 vendors (50 fixed stalls and 25 mobile st alls) around New Delhi and Patiala City, India. The researchers observed vendors who sold coconut slices to have unclean contact surfaces and a lack of potable water. De sserts containing fresh coconut have a significantly higher contamination rate of S. aureus than desserts without fresh co conut (Suklampoo et al., 2003). One hundred twenty samples of Thai desserts were collected from department stores and fresh markets in Bangkok, Thailand and split into two groups : half with fresh coconut and half without fresh coconut. Overall, 74.2% of desse rts collected were contaminated with S. aureus S. aureus contamination was 83.3% in desserts with fres h coconut, compared to only 65% in desserts without fresh coconut (Suklampoo et al., 2003). The higher incidence of S. aureus contamination in desserts with fresh coconut im plies that fresh coconut might be a possible vehicle for foodborne disease. Since coconut is co mmonly used as an ingredient the behavior of pathogens is rarely studie d alone as a substrate. L. monocytogenes is able to grow in fr esh-cut coconut held at va rious temperatures (2, 4, 8 and 12C), environments (air or modified atmosphere, 65% N2, 30% CO2, and 5% O2), and inoculum sizes (low inoculum, 2 log CFU/g; high inoculum, 5.7 log CFU/g) (Sinigaglia et al., 2006). Coconuts were purchased from local grocery st ores in Italy, deshelled and cut into slices. 38

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Coconut slices kept in ai r with high inoculums of L. monocytogenes populations exhibited growth within 48 regardless of st orage temperature. Similar results were seen for modified air packaging; however, storage at 2 C delayed growth for 5 days. Co conut slices kept in air with low inoculums of L. monocytogenes grew within 24 h at 8 and 12C and following 5 days at 2 and 4C. Modified air packaging led to growth of L. monocytogenes at all storage temperatures within 48 h. These results corre spond to other studies (Farber et al., 1998) demonstrating that modified atmosphere packaging does not control the growth of L. monocytogenes. Guava Guavas are commonly produced in South and Ce ntral America, Mexico and India. In the U.S. they are normally processed into juices jams and purees. However, around the world, guavas are consumed fresh and are eaten like an apple; including the ri nd, flesh and seeds all consumed. The rind of a guava alone has more v itamin C than an entire orange (Jimenez-Escrig et al., 2001). Various organisms in the Bacillus genus have been isolated from guava, most notably B. cereus. S. aureus was also isolated in the same study (Valentin-Ramos, 1959). E. coli O157:H7, when inoculated at low inoculum levels of 2 log CFU/ml survived in guava pulp (pH 2.37) for up to 13 days when stored at either 6 or -10C (Leite et al., 2002). However, little research has been investigated, including pathogen survival of the rinds of guava. Kiwifruit The top producers of kiwifruit are Italy, Ne w Zealand and Chile (FAO, 2005). The U.S. imports the majority of kiwifruit from these th ree producers. The fruits are extremely high in vitamin C (comparable to an orange) and dietary fi ber. The pH of kiwifruits is approximately 3.6. 39

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Little research exists on the food safety concerns in ki wifruit. Roller and Seedhar (2002) determined the application of carvacrol or cinna mic acid (1 mM) to kiwifruit can delay spoilage for up to 5 days when kept at chill temper atures (4 and 8C) without adverse sensory characteristics, such as browning. Both carvacr ol and cinnamic acid are generally regarded as safe (GRAS). Carvacrol is a main component of the essential oils thyme and oregano and cinnamic acid occurs in cinnamon (Roller and Se edhar, 2002). While, no studies have been done how these compounds affect pathogens or pathogen survival on kiwifruit, the general microflora of kiwifruit is reduced using either of these compounds. Mamey Mamey is a popular tropical fr uit in Honduras, Guatemala and Cuba (Katz et al., 2002). It is typically consumed raw or as the main ingr edient in milkshakes, smoothies and ice cream. The texture of mamey is similar to that of an avocado, but with a sweet flavor. No research is currently available on the behavior of foodborne pathogens on the surface or flesh of mamey. A Salmonella typhi outbreak due to the consumption of frozen mamey occurred in the U.S. in 1999. Other tropical fruit with similar pH to th at of mamey (pH ca. 5.7) have allowed pathogen growth. Mango Mango has recently become a familiar commodity in the market place. The leading exporters in the world of mangoes are Mexico (41%), the Philippine s (7.8%) and Pakistan (7.6%) (FAO, 2001). The production of mangoes has increased tremendous ly, with the price dropping. Mango juice is an extremely popular dr ink in Taiwan, Japan and Southeast Asia (Hsin-Yi and Chou, 2001). The U.S. obtains most of its mangoes from Mexico and South America. 40

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Work has been shown that Salmonella can internalize into intact mango flesh during these types of postharve st heat treatments and Salmonella outbreaks are hypothesized to be due to Salmonella internalization (Penteado et al., 2004). Little research exists on the growth and survival of Salmonella or other pathogens in mangoes. E. coli O157:H7 can survive in mango juice and pulp. E. coli O157:H7, when inoculated at inoculum levels of 2 log CFU/ml, can survive in mango pulp for up to 13 d when stored at either 6 or -10C (Le ite et al., 2002). Two strains of acid adapted and non acid adapted E. coli O157:H7 were found to survive in mango juice held at ambient (25C) and refrigeration (7C) temperatures for 6 and 8 days, respectively (Hsin-Yi and Chou, 2001). The survival of E. coli O157:H7 was better in refrigerated storage with population counts remaining ca. 4.2 log CFU/ ml for both acid adapted and non acid adapted strains after 8 d. Regardless of strain or acid adaptation, E. coli O157:H7 population counts quickly declined at ambient temp erature after 3 days until the end of the 6-d storage period. Though, acid adapted E. coli O157:H7 did give consistently highe r counts at each sample period compared to non acid adapted E. coli O157:H7 at ambient temperatures the same general trend was observed over storage (Hsin-Yi and Chou, 2001). Papaya Papaya is widely consumed in Mexico and South America. There has been a three-fold increase in papaya production in the world since 1965 with Brazil the leading exporter (FOA, 2001). World consumption of papaya has also stead ily increased over the last thirty years (FOA, 2001). Papaya has a higher pH (ca. 5.7) than most tropical fruits and allows for abundant pathogen growth in ambient temper ature conditions (ca. 23C). A survey of fresh cut papayas sold by street vendors in Calcutta, India found high aerobic plate c ounts, positive coliform detection and the pres ence of enteric pathogens (Mukhopadhyay et al., 2002). Out of the 30 500g samples of papaya taken over a 3-month period (Apr-June), 48% tested positive for E. coli, 41

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17% for coagulase-positive S. aureus 3% for Salmonella and 3% for V. cholerae Higher total aerobic plate counts indicated th e likely contamination with enteric pathogens (Mukhopadhyay et al., 2002). Salmonella and Shigella have been found to grow on fresh cut papaya, while Campylobacter jejuni has shown survival in fresh cut papaya. Salmonella typhi was inoculated onto fresh cut papaya cubes with and without lem on at room temperature (25C) and held for 6 h. At the end of the 6 h, populations increase d 1.4 and 0.8 log CFU/cube on papaya without lemon and with lemon, respectiv ely. Various serogroups of Shigella were found to grow within 6 h of being inoculated onto fresh cut cube s of papaya at room temperature (25C). S. sonnei S. flexneri, and S. dysenteriae populations increased approxima tely 2.2, 2.0, and 1.6 log CFU/cube respectively. A subsequent study was done on S. sonnei inoculated into suspensions of papaya and water held at 22C for 24 h. S. sonnei populations increased ca. 4 log CFU/ml over the 24 hr in all papaya suspensions (Escartin et al., 1989). C. jejuni has also been shown to survive on fresh cut papaya for up to 6 hours (Castillo and Escartin, 1994). Fresh papaya were aseptically cut, half treated with 0.5 ml lemon juice, inoculated with ca. 3 log CFU/cube of C. jejuni and held at ambient storage conditions (25-29C). Lemon juice was added to lower the pH of the fresh-cut papaya. Over the 6-h storage period no growth was observed in fresh cut papaya with or without the addition of lemon; however, a decrease was seen following lemon juice addition. The pH of papaya with lemon added was ca. 3.0, which has been reported by Doyle and Roman (1981) to be lethal to C. jejuni C. jejuni did not grow on papaya with or without lemon juice, but did survive within acceptable population ra nges to cause foodborne illness (Castillo and Escartin, 1994). 42

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E. coli O157:H7 and Salmonella were also observed to grow in papaya ju ice and pulp when held at ambient and refrigeration temperatures. A study by Muta ku et al., (2005) found that filtered and steamed pa paya juice inoculated at E. coli O157:H7 populations 3.5 log CFU/ml held at ambient temperatures (20-25C) exhi bited large growth incr eases during the 5 day storage period. On day 3, E. coli O157:H7 populations reached a maximum of 8.5 log CFU/ml before gradually decreasing. E. coli O157:H7 in papaya juice held at refrigeration temperatures (4C) grew for the entire 5-d stor age period. On day 3, refrigerated E. coli O157:H7 populations had increased 2 log CFU/ml and remained appr oximately 5.5 log CFU/ml throughout the storage period (Mutaku et al., 2005). Similar results are seen for papaya juice inoculated with E. coli O157:H7, where populations had reached a maximum of 9 log CFU/ml after 16 h at 37C and remained steady over the 2 day storage. E. coli O157:H7 populations at refrigeration increased ca. 1 log CFU/ml in the first 24 h before declin ing slowly (Yigeremu et al., 2001). Penteado et al. (2004) found Salmonella Enteritidis to grow on papaya pulp held at 10, 20 and 30C. Salmonella Enteritidis from an initial inoculum of 2 log CFU/g increased 1.8, 6 and 7 log CFU/g, for 7, 2 and 1 days at 10C, 20C and 30C, respectively (Penteado et al., 2004). Salmonella inoculated in papaya juice (ca. 3.7 log CFU/ml) e xhibited large growth increases when stored at 37C and mild growth at 4C over a 2 day stor age period (Yigeremu et al., 2001). Within the first 16 h at 37C, Salmonella populations had reached 9 log CFU/ml and remained as such throughout storage. Papaya juice held at 4C, observed a Salmonella population increase of 1 log CFU/ml in 24 h before slowly declining (Yigerem u et al., 2001). Passion Fruit Passion fruit is extremely popular in Brazil, requiring im portation from other South American countries (FAO, 2001). In Hawaii pa ssion fruit juice is generally referred and marketed to consumers as passion fruit nectar. In a survey of microflora in frozen (-20C) 43

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passion fruit nectar it was found that Bacillus and Achromobacter were the predominant bacteria recovered; however no pathogenic bacteria to humans were ever recovered. Bacillus could be recovered for storage periods of up to 540 days (Aea and Bushnell, 1962a). Aea and Bushnell (1962b) reported that Salmonella enterica can survive for up to 90 days in passion fruit nectar when held in frozen storage (-20C), and passion fruit nectar at ambient temperatures (25C) had a lethal effect on E. coli Salmonella and Shigella when inoculated at concentrations of 4-5 log CFU/ml. This study shows the ability of frozen storage to aid in pot ential pathogen long term survival (Aea and Bushnell, 1962b). Fruit pulps are commonly frozen in Brazil to extend the shelf life. Consumers then add water and refrigerate in order to consume fruit juice at affordab le prices. A study by Marques et al. (2001) evaluated the survival of acid adapted and non-acid adapted E. coli O157:H7 strains inoculated at 6 log CFU/ml in refrigerated (4C) passion fruit pulp over a 30 day storage period. The same general survival patt ern exists between acid adapted and non-acid adapted cells. E. coli O157:H7 populations steadily decreased over storage on the refrigerated passion fruit pulp until counts were undetectable between days 12-21 days. Passion fruit is also commonly cut in half and the fruit flesh eaten out of the rind like eating out of a bowl. Behrsing et al. (2003) inoc ulated the rind of passion fruit with ca. 5-6 log CFU/ml with L. innocua, Salmonella and E. coli and observed the behavior at 10C over a 6 d period. At the end of the 6 d storage only L. innocua survived by direct plating; S. Salford and E. coli were recovered by enrichment. Possible explanat ions for the lack of bacterial growth were the smooth nature and waxed coating of the passion fruit skin. Fruits that have uneven surfaces have shown better pathogen survival. This allo ws for the hypothesis that uneven surfaces allow for better pathogen protection or atta chment (Behrsing et al., 2000). 44

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Pineapple The largest producer of pineapple is Braz il followed by Thailand, the Philippines and Costa Rica (FAO, 2001). Nearly all of the U. S. pineapple consumption is from imported pineapples. Pineapples are consumed in many diff erent ways, such as fresh cut, juice, or as a food ingredient. Pineapples have a low pH (ca. 3.8) and very active proteases, like bromelain (Mynott et al., 1999). Pineapple is not a good substrate for pathogenic microorganism growth. Fresh cut pineapples inoculat ed with initial Salmonella Enteritidis and E. coli O157:H7 population concentrations of 4.5 log CFU/g we re incapable of growth when stored at 4C, 10C, and 20C for the 2 day storage period (Nazuka et al., 2004). Populations of Salmonella and E. coli O157:H7 were found to be relatively stable, with no significant decrease observed. The limitation of this study is the short length of time it observes. It is well known that fresh cut pineapples stored at 4C and 10C have shelf lives of longer than 2 days. Mutaku et al. (2005) determined that E. coli O157:H7 does not grow in pineapple juice when held at ambient (20-25C) or refrigeration (4) temperatures for up to three days. While small reductions of approximately 0.1-0.5 log CFU/ml were observed between initial and final counts, there were no significant differences determined over the three-day storage period (Mutaku et al., 2005). In previous studies, E. coli O157:H7 has been shown to exhibit enhanced survival at lower temperatures in low pH foods (Mil ler and Kasper, 1994; Marques et al., 2001); however, this was not the case in pineapple juice. Yigeremu et al. (2001) also found that pineapple juice is a poor substrate for E. coli stored at 37C and 4C. Pineapple juice inoculated with E. coli was below the limit of detection within the first 16 h for 37C, as well as not detectable for 4C after 16 h (Yigeremu et al., 2001). 45

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Yigeremu et al. (2001) also found pineappl e juice to be a poor growth medium for Salmonella stored at 4C over 40 h. Pineapple juice was inoculated with 2.3 log CFU/ml Salmonella populations, which became undetectable after 24 h. Salmonella populations in pineapple juice stored at 37C increased ca. 2 log CFU/ml within the first 24 h (Yigeremu et al., 2001). Various pathogenic microorganisms can exhib it long term survival in frozen (-23C) pineapple juice concentrate (Oyarz abal et al., 2003). Pineapple juice concentrate was inoculated with 5 strain cocktails of acid adapted E. coli O157:H7, L. monocytogenes and Salmonella at initial populations of 4 log CFU/g and subse quently stored at -23C for 12 weeks. L. monocytogenes survived better than E. coli O157:H7 and Salmonella ; maintaining populations of ca. 3.3 log CFU/g. Both E. coli O157:H7 and Salmonella populations exhibited sharp decreases within the first 6 h. However, while Salmonella populations continued to slightly decrease until stabilizing at ca.1.5 log CFU/g for th e duration of storage, where as E. coli O157:H7 populations oscillated around 2.5 log CFU/g. When inoculated at a lower initial c oncentration (1.95 log CFU/g), E. coli O157:H7 populations showed survival in frozen pineapple juice concentrate for the 4 week long storage period (Oyarzabal et al., 2003). Leite et al. (2002) found similar results for E. coli O157:H7 in frozen pineapple pulp. When inoculated at low inoculum levels of 2 log CFU/ml, E. coli O157:H7 can survive for up to 13 days when stored at either 6 or -10C (Leite et al., 2002). Pitanga Pitanga is a relatively unknown fr uit to most U.S. consumers. It is high in vitamin A and has a low pH (ca. 2.8). It is commonly cons umed in Brazil and thought to have therapeutic properties ( Oliveira et al., 2005). 46

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Acid-adapted E. coli O157:H7 strains exhibited surviv al in refrigerated pitanga pulp surviving for 11 days each compared to non-acid adapted strains that survived between 4-7 days (Marques et al., 2001). Pathogen Prevention Regulatory Programs Pathogens can grow and survive on tropical fru it in a wide range of temperatures for time frames long enough to cause illness. Tropical fruit are an extensive and diverse group and cannot be categorized or regulated under the same umbrella. The potential for major outbreaks exists and preventive strategies are needed to en sure the safety of thes e products. Preventive strategies should be implemented and well doc umented from farm to fork, including good agricultural practices (GAPs) during harvest; good manufacturing practi ces (GMPs), standard operating procedures (SOPs) and hazard analysis critical control points (HACCP) in postharvest handling or processing facilities. GAPs aim to prevent contamination in the field. In the case of tropical fruit farms various GAPs might focus on irrigation waters and harvesting protocols. A farm will have to meet certain irrigation water standards or mandate workers not to pick up fruit off the dirt. GMPs are in place so contamination does not come from the packinghouse or processing facility. They primarily focus on worker hygiene, equipment and overall facility and ground maintenance. For instance, GMPs will set protocols for scheduled cleaning and sanitizing of food contact surfaces and equipments, as well as waste removal from the plant. HACCP programs are tailored to an individual plant to id entity risks and eliminate there occurrence. For example in juice processing if pasteurization stan dards are not met then ju ice is rerouted back into the pasteurization unit again. 47

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Most of the tropical fruits consumed in the U.S. are imported; global food safety standards are essential. These standards could for example ensure all the various mango farms comply with safe farming practices. There is currently no global food safety organization or standards; however, there are global GAPs. Glob alGAP is an international body that has tried to set voluntary standards for the safety of agriculture practices around the world. Handling Parameters Consumers can prevent foodborne illness by following simple guidelines. Whole tropical fruit should be stored at room te mperature to prevent chilling inju ry (above 12C). Whole fruits should be cut using the following steps: fruit should be hand washed in potable water first and cut on a clean surface. Cross contamination can oc cur from using cutting boards that were used for raw chicken or meat products. Washing fru it can reduce bacterial pathogens on the surface of the fruit by 10-100 cells (Beuchat, 1996). Fresh cut tropical fruits should always be kept at refrigeration temperatures (2-5C). Pathogens ar e less likely to grow if temperatures are not abused, while storage at warmer temperatures results in prolific pathogen growth (Penteado and Leitao, 2004). The shelf life on fresh cut fruit is approximately 3 days Frozen cut tropical fruits should be kept at constant frozen temperatures ( -18C). These temperatures greatly reduce generation for pathogens; however, do not result in significant pathogen reduction (Penteado and Leitao, 2004). If temperature abuse occurs frozen cut fruit should be th rown out as pathogens can grow if thawing occurs. Consumers can follow the below guidelines to minimize risks. Whole fruit should be washing thoroughly before cutting or eating. Tropical fruit that has visible bruising or soft spots should not be purchased. When cooking at home, consumers should be careful not to cross contaminate surfaces or utensils with raw poultry or meat products that could come in contact with tropical fruit. Tropical fruit should be stor ed at cool temperatures instead of being left out 48

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on the counter so pathogen growth does not occur. Frozen tropical fruit should be monitored since thawing can result in pathogen growth. Alternative Technologies Due to consumer concern and overall food safe ty of fruit and vegetable juices, the U.S. Food and Drug Administration (FDA) mandated a la w in 2001 to which processors are required to achieve a 5-log CFU/ml reduction of the most resistant pathogen to th at specific commodity (FDA, 2001b). Conventional fruit smoothie proce ssing is a thermal heat pasteurization of 72C for 15 s that results on average of a 6.3 log CF U/ml pathogen reduction (W alkling-Ribeiro et al., 2008). Pulsed electric field (PEF ) is a non-thermal processing tec hnique that has been used on a wide variety of liquid foods. PEF disrupts the cell membrane of bacteria by electroporation causing reversible or irreversib le damage based on a number of process variables (WalklingRibeiro et al., 2008). PEF can be used alone or in combination with other processing techniques (combination processing). Combination processing has become extremely popular over the past decade because of its ability to hit a broader spectrum than using single technologies used to target one specific objective. Using a fruit sm oothie made of pineapples, bananas, apples, oranges and coconut milk it was observed that a mild heat treatment (moderate preheating to 55C over a period of 15 s followed with cooling) and PEF (34 kV/cm at 650 kJ/L) was just as effective in reducing E. coli K12 as the conventional fruit sm oothie pasteurization technique. The combination of mild heat and PEF resu lted in a ca. 7 log CFU/ml reduction in E. coli K12 populations. The results of this experiment by Walkling-Ribeiro et al. (2008) prove PEF with combination processing can be used on fruit smooth ies in order to meet the FDAs 5 log CFU/ml reduction final rule. Another alternative to th ermal processing is high hydrostatic pressure, HHP. High hydrostatic pressure (100-1000 MPa) allows for the preservation of f ood without altering the 49

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quality. Buzrul et al. (2008) determined that kiwifruit and pi neapple juice inoculated with E. coli and L. innocua could obtain the FDA 5 log CFU/ml reduction by HHP (350 MPa) at all storage temperatures, (4, 20 and 37C) except 4C for pin eapple juice. It has been observed by Miller and Kaspar (1994) that low pH (pineappl e pH 3.7) enhances the survival of E. coli. Research Objectives Overall, little research exists on the behavior of pathogens on tropical fruit. This thesis aims to fill in part of this research void. The major thesis objective is to examine the fate of E. coli O157:H7 and Salmonella on fresh and frozen cut mangoes, papayas and pineapples. Major outbreaks have been associated with each of these fruits, and they all rank in the top five tropical fruits imported annually. My hypothesis is that E. coli O157:H7 and Salmonella on cut mangoes, papayas and pineapples will grow at 23C and 12C and survive at 4C and 20C. The results of this thesis will begin to address the current lack of research on tropical fruits and formulate new directions for future tropical fruit studies. 50

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Table 2-1. Selected Fruits Co mmonly Accepted as Tropical Common N ame Scientific Name Botanical Type p H.2 Origin (s) Aai E uterpe oleracea Tree; Palm 3.0 Central and South America Acerola Malpighia punicifolia Tree 3.2 Caribbean and Central America Avocado P ersea americana Tree 6.2 Mexico Banana Musa acuminata Herb 5.5 Southeast Asia Caj A nacardium giganteum Tree 2.7 Amazon region and the Guianas Coconut Cocos nucifera Tree; Palm 6.0 Asia Guava P sidium guajava Tree 2.4 N orthern and Central America Mamey P outeria sapota Tree 5.7 Mexico and South America Mango Mangifera indica Tree 4.2 Southern Asia and Eastern India Papaya Carica papaya Herb 5.7 Mexico and Central America Passion fruit P assif l ora herbertiana Vine 2.9 N orthwest Australi a Pineapple A nanas comosus Bromeliad 3.6 Eastern South America Pitanga E ugenia uniflora Tree 2.8 Eastern South America 51

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Table 2-2. Outbreaks of foodborne illness associ ated with the consumption of tropical fruit Fruit Product Pathogen Y ear Location Reference(s) Avocado Guacamole Campylobacter jejuni 2002 United States CDC, 2002 Hepatitis A 2000 United States CDC, 2000 Norovirus 2005 United States CDC, 2005 2005 United States CDC, 2005 2006 United States CDC, 2006 Salmonella Typhimurium 2003 United States CDC, 2003 2005 United States CDC, 2005 Shigella boydii 1998 United States CDC, 1998 Shigella sonnei 2002 United States CDC, 2002 Whole Norovirus 2001 United States CSPI, 2006 Banana Pie Norovirus 2005 United States CDC, 2005 Plantain Staphylococcus aureus 2002 United States CSPI, 2006 Pudding a Norovirus 2002 United States CDC, 2002 Salmonella Heidelberg 2004 United States CDC, 2004 Staphylococcus aureus 2003 United States CDC, 2003 2004 United States CDC, 2004 Unspecified Norovirus 2005 United States CDC, 2005 Coconut Deshelled Salmonella Paratyphi A 1997 Singapore Teoh et al ., 1997 Desiccated Salmonella serovar Typhi, Salmonella Senftenburg, and possible others 1953 Australia Wilson and Mackenzie, 1955 Salmonella Paratyphi B 1960 England Anderson, 1960 Salmonella serovar Java PT Dundee 1999 United Kingdom Ward et al ., 1999 Milk Shigella 1991 Thailand Hoge et al. 1995 Vibrio cholerae 1991 United States Taylor et al ., 1993 Mamey Frozen Smoothie Salmonella Typhimurium 1998-99 United States Katz et al ., 2002 Mango Raw Salmonella Newport 1999 United States Sivaapalasingam et al. 2003 Salmonella Oranienburg 1998 United States PHAC, 1998 Salmonella Saintpaul 2001 United States Beatty et al ., 2004 Unspecified Salmonella Saintpaul 2003 United States CDC, 2003 Papaya Fresh-cut Salmonella Litchfield 2006 Australia Gibbs et al., 2009 Salmonella Weltevreden 1996 Singapore Ooi, 1997 Pineapple Fresh-cut Norovirus 2001 United States CDC, 2001 Salmonella Weltevreden 1996 Singapore Ooi, 1997 Unspecified Campylobacter jejuni 1998 United States CDC, 1998 Escherichia coli O11:H43 1994 United States Sivapalasingam et al. 2003 Norovirus 2001 United States CDC, 2001 2002 United States CDC, 2002 2003 United States CDC, 2003 Norwalk virus 1999 United States CDC, 1999 a The Source of the Salmonella was never identified other than banana pudding. 52

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CHAPTER 3 MATERIALS AND METHODS Preliminary Tests Studies were conducted on tropical fruit to determine cooling temperature profiles using probes located on the surface and in the center of cut fruit pieces. Two tropical fruits were used: mango and papaya. The whole mangoes and papaya s were obtained from a local grocery store immediately before testing. Mangoes and papayas were rinsed with potable water, peeled and cut into 2.5 by 2.5 cm cubes in an aseptic environmen t with a sterile peeler and knife. Cut cubes were placed in sterile weigh boa ts and left out uncovered at ambient room temperature (23 2C) for 2 h. These conditions were selected to simulate inoculation conditions in the actual tropical fruit experiments. After 2 h time at ambi ent temperature, fruit cubes were separated into two groups for probe application: surface and cen ter. The surface group was outfitted with temperature probes (Squirrel data logger mode l 1206, Grant, Hillsborough, NJ) just under the flesh of the tropical fruit cube. The center gr oup was outfitted with temperature probes in the approximate center of the fruit flesh (0.5 cm from either top or bottom). Fruit cubes were then placed in stomacher bags held at 4 2C (Whirl -pak; Nasco, Modesto, CA). Temperature of the fruit flesh was recorded every 5 min for 180 min (3 h). Samples were then placed in a low temperature incubator set to 4 2C. Each group consisted of three samples and three replications were preformed (n = 9). Followi ng completion, cooling curves for each group were plotted. A student t-test was performed to determine if sign ificant differences, existed between surface or center probes. Small scale trial experiments were completed before any replications began each of the tropical fruit. The small scale experiments serv ed to help decide inoculum levels and to minimize excess plating during recovery. These experiments, lasting only 7 days, followed the 53

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same procedure as the statically significant replications for each mango, papaya and pineapple discussed below. The results were not analyze d, due to low sample numbers and were intended only as a guideline for future work. Trials were run for E. coli O157:H7 and Salmonella at 23 2C, 12 2C and 4 2C for mangoes and pineapples. No test runs for papaya, as adequate literature on other ente ric pathogens (i.e. Shigella ) behavior in papaya mediums exists to anticipate growth and survival patterns. Tropical Fruit The tropical fruits used in this study were ripe mangoes, papayas and pineapples. Tommy Atkins mangoes were obtained from a producer in Canal Point, Florida (Erikson Farms). Red Lady papayas were obtained fr om a wholesaler in Tampa, Florida (Baird Produce). The country of origin for the papa yas that Baird Produce received was Ecuador. Del Monte Gold pineapples were obtained from a processor in Plant City, Florida (Del Monte). The country of origin for the pineapples that Del Monte received was Costa Rica. Bacterial Strains and Culture Conditions All bacterial strains were stored at -80C in glycerol bead stock cryogenic vials in the Danyluk laboratory culture co llection. Four strains of E. coli O157:H7 were used (Table 3-1): H1730 (isolate from lettuce outbreak; human fe ces), SEA-13B88 (isolate from apple cider outbreak; human feces), F4546 (isolate from sp rout outbreak; human feces), and 994 (isolate from fermented salami). Five strains of Salmonella were used (Table 3-2): Michigan (isolate from cantaloupe outbreak; human feces), Montev ideo (isolate from tomato outbreak; human feces), Munchen (isolate from orange juice outbreak; human feces), Newport (isolate from tomato outbreak; environmental) and Saintpaul (isolate from orange surface). All strains were adapted to grow in the presence of 50 g/ml na lidixic acid (NA; Sigma Aldrich, St. Louis, MO) to prevent background microflora through the use of a stepwise e xposure (Parnell et al., 2005). 54

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A 5,000 g/ml stock solution of nalidixic acid was made prior to each replication for the media. The stock solution was prepared by dissolving 0.5 g of nalidixic acid in 10 0 ml of deionized, DI, water. The solution was then filter sterilized (Nalgene (0.20 m pore size), Rochester, New York), dispensed into 50 ml sterile tubes (Falcon; Becton Dickinson, Cockeysville, MD) wrapped in foil and stored in the refrigerator at 4 2C, until use. When 1L media amounts were prepared 10 ml of nalidixic acid stock so lution was added to create the final concentration of 50 g/ml. Inoculum Preparation Prior to each experiment replication, the frozen culture was streaked onto tryptic soy agar (TSA; Difco, Becton Dickinson, Sparks, MD) with 50 g/ml of NA (TSAN) and incubated at 37C for 24 h. All strains of E. coli O157:H7 and Salmonella were transferred to tryptic soy broth (TSB; Difco, Becton Dickinson) with 50 g/ml of NA (TSBN) two tim es at 24-h intervals, incubated at 37C, prior to their use as inocula. Each strain was subjected to centrifugation at 3,000 x g for 10 min (Allegra X-12, Beckman Coulter, Fullerton, CA). The cells were washed two times by pouring the supernatant and suspendi ng the cell pellet in 10 ml of 0.1% peptone (Difco, Becton Dickinson). Washed cells were suspended in 0.1% peptone at half the original culture volume. Serial dilutions were car ried out in 0.1% peptone (9 ml) to make E. coli O157:H7 and Salmonella inocula concentrations. Each strain s serial dilution tubes were plated out onto TSAN to confirm cell concentration. Equal volumes (1 ml) of each E. coli O157:H7 or Salmonella strain were combined to make the inoculum concentrations: 108, 106 and 104 CFU/ml. Final inocula were stored on ice for up to 1 h, prior to inoculating cut mangoes, papayas and pineapples. 55

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Acid Adaption of Salmonella spp. Strains Frozen cultures of the above mentioned Salmonella strains were streaked onto TSAN and incubated at 37C for 24 h. All strains of Salmonella were transferred to TSBN with 1% glucose (Difco, Becton Dickinson) at 24-h intervals following incubation at 37C, prior to their use as inocula (Beuchat and Mann, 2008). The addition of glucose to the broth induces the Salmonella to produce acid creating an acidic en vironment, thus acid adapting the Salmonella Each strain was subjected to centrifugation at 3,000 x g for 10 min (Allegra X-12, Beckman Coulter). The cells were washed two times by pouring the supernatant and suspending the cell pellet in 10 ml of 0.1% peptone (Difco, Becton Di ckinson). Washed cells were suspended in 0.1% peptone at half the original culture volume. The pH was recorded using pH test strips (Fisher, Fair Lawn, NJ) to ensure proper acid adaptation. Serial diluti ons were carried out in 0.1% peptone (9 ml) to make Salmonella inocula concentrations. Each strains se rial dilution tubes we re plated out onto TSAN to confirm cell concentration. Equal volumes (1 ml) of each Salmonella strain were combined to make the inoculum concentrations: 108 and 106 CFU/ml. Final acid adapted inocula were stored on ice for up to 1 h, pr ior to inoculating cut pineapples. Inoculum Concentrations E. coli O157:H7 samples were inoculated at 3 and 5 log CFU/g (23 2C) and 5 log CFU/g (all other temperatures). Salmonella samples on mango and papaya were inoculated with a low (1 log CFU/g), medium (3 log CFU/g) a nd high (5 log CFU/g) initial inoculum for 23 2C, 12 2C and 4 2C samples to evaluate th e effects of various leve ls of contamination. Pineapple samples were inocul ated with only a 5 log CFU/g Salmonella inoculum, as were frozen cut samples for all fruits. 56

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Preparation of Tropical Fruit Raw tropical fruit rinds (mangoe s and papayas) were washed in sterile water to remove any residues prior to the peeli ng and cutting process. Mangoes and papayas were peeled and cut using a flame sterilized peeler and knife, respec tively on a sterilized cu tting board in a flume hood. Flesh was cut into slices (ca. 2.5 by 7.5 cm) and cubes (ca. 2.5 by 2.5 cm) for mangoes and papayas respectively. Pineapples were wash ed in 200 ppm chlorine and pre cut into cubes (ca. 2.5 by 2.5 cm) at the Del Monte processing faci lity (Plant City, FL). Mangoes, papayas and pineapples were weighed into 100 g samp les in preparation for inoculation. Tropical Fruit Inoculation Mangoes, papayas and pineapples sample s (100 g) were inoculated with 20 l of inoculum, distributed in 4-6 drops over the cut surface of the tropical fruits Tropical fruits were held in a biological hood for 20 min to allow the inoculum to dry. After dying tropical fruits were placed into sterile stomacher bags with in ternal filters (Whirl-pak ; Nasco, Modesto, CA). Each bag was folded over and placed in a la rge plastic container (Fashion Clears 28 qt, Rubbermaid, Fairlawn, OH) with a temperature and relative humidity sensor (Tale Temp 4, Sensitech, Beverly, MA). Stomacher bags were fo lded and not sealed to allow air movement. Air movement through bags was essential to prevent an anaerobic environment from occurring due to tropical fruit respiration. Lids were left off the containers, except for samples kept under freezing conditions (-20 2C). Cut inoculated tr opical fruit samples were then stored at 23 2C, 12 2C, 4 2C and -20 2C. Storage Conditions Storage conditions were 23 2C, 12 2C, 4 2C and -20 2C and selected based on the following criteria. Fresh cut tropical fruits before consumer consumption are commonly prepared at ambient temperatures (23 2C), th is also serves as a common abuse parameter, 57

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room temperature. Fresh cut tropical fruit sold in grocery stores are often kept in open case refrigerators, that maybe as warm as 12 2C. Fresh cut tropical fruits are processed, shipped and stored in facilities under refrigeration (4 2C) to maintain the cold chain. Frozen cut products are stored commercially at -20 2 C. Spoilage At every sample day, spoilage was visual ly observed and recorded. Spoilage was determined when the fresh cut tropical fruits would no longer be acceptable for consumption. Visual signs included mold or yeast growth, discoloration and or decay. Enumeration of Pathogens Fruit samples were enumerated on the following schedule: 23 2C, days 0, 1, 3, 5 and 7; 12 2 and 4 2C, 0, 1, 3, 5, 7, 10, 14, 21 and 28; and -20 2C, 0, 7, 14, 21, 28, 60, 90, 120, 150 and 180. Tropical fruit samples (100 g) were mixed with 100 ml Dey/Engley (DE; Fisher) buffer and placed in a stomacher (Bag Mixer, Interscience, Weymouth, MA) for 2 min in stomacher bags with internal filters. DE buffe r was used to negate any pH effects that the tropical fruit may have had on enumeration. Afte r stomaching, serial dilutions were made in 0.1% peptone solution and surface plated (0.1 ml ) in duplicate onto selective and nonselective media with 50 g/ml NA and 0.1% pyruvic acid (Fisher). Pyruvic acid was added to nonselective and selective media to aid in the recovery of inju red cells (Knudsen et al., 2001). TSA was used as the nonselective media for both E. coli O157:H7 and Salmonella enumerations. Sorbitol MacConkey agar (SMAC; Difco, Becton Dickinson) was used as the selective agar for E. coli O157:H7 enumeration and bismuth sulfite ag ar (BSA; Difco, Becton Dickinson) was used as the selective agar for Salmonella enumeration. To increase th e limit of detection, to 0.3 log CFU/g an additional 1 ml of the lowest dilution was plated onto four plates each (0.25 ml/plate) of nonselective and selective me dia. Control samples were plated onto nonselective and 58

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selective agars, as well as plate count agar (PCA; Difco, Becton Dickinson) to determine background microflora. Plates were incubated at 37C for 24 h (TSA, SMAC and PCA) or 48 h (BSA) incubations. After incubation colonies were counted by hand and E. coli O157:H7 or Salmonella population levels were expressed in log CFU/g of tropical fruit. Enrichment When counts fell below the limit of dete ction (0.3 log CFU/g), enrichment for E. coli O157:H7 and Salmonella was conducted by the proper U.S. Food and Drug Administration Bacteriological Analytical Ma nual, FDA BAM, protocol for produce (FDA, 2007). Samples (100 g fruit and 100 ml of DE buffer) were stom ached and in some samples plated onto TSAN, BSAN or SMACN before the enrichment proce dures began. The enrichment protocols for E. coli O157:H7 and Salmonella are discussed below. E. coli O157:H7 For E. coli O157:H7 enrichment, 100 ml of doubl e strength brain heart infusion broth (BHI; Difco, Becton Dickinson) was added to th e sample and incubation at 35 2C for 3 h. Samples then receive 200 ml of double strength tr yptone phosphate broth (TP; Difco, Becton Dickinson) and incubation at 44 2C for 20 h. Sa mples are then streaked (10 l loop, Fischer) onto SMAC (Difco, Becton Dickinson) and Eosi n methylene blue agar (EMB; Difco, Becton Dickinson) plates. Plates we re incubated at 37 2C for 24 h, and inspected for typical E. coli O157:H7 colonies (red on SMAC and dark cente red with metallic sheen on EMB). Positive colonies are transferred (10 l needle) to triple sugar iron (TSI ; Difco, Becton Dickinson) slants and incubated at 37 2C for 24 h. A confirmed E. coli O157:H7 enrichment results in TSI slants that have a yell ow top and bottom. 59

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Salmonella For Salmonella enrichment, 200 ml of double stre ngth lactose broth (Difco, Becton Dickinson) was added to the sample and incubation at 37 2C for 24 h. One hundred microliters and 1 ml of mixture was then transf erred to 10 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 then streaked onto BSA (Difco, Becton Dickinson), Xylose Lysine Deoxycholate agar (XLD; Difco, Becton Dickinson), 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 cente rs on XLD, and blue-green with or without a black center on HE. Positive colonies are selected and transferred (10 l needle) to TSI (Difco, Becton Dickinson) slants and lysine iron agar (LIA; Difco, Becton Dickinson). Tubes are incubated at 37 2C for 24 h. A confirmed Salmonella enrichment results in TSI slants that have a pink top and black bottom with gas format ion and LIA tubes are black or no color change. Statistics Results for the mango and papaya temperature profiles were analyzed using a students ttest with significance being determined by P 0.05. At least three replications of each experime nt were performed. Each replication was begun on a separate day. Each sample was analyzed in duplicate. All resu lts were average plate counts calculated in log CFU/g. Data were examined using statistical analysis software (Statistica; StatSoft, Tulsa, OK) An analysis of variance, ANOVA, was used to compare counts within each inoculum level to determine if significant differences were observed between the days for both nonselective and selective media. Comparisons were also made within each day for non selective and selective me dia to determine if a differen ce was observed between the two 60

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media. If no significant difference existed be tween nonselective and selective media on each day then nonselective data alone was analyzed further. For further comparison, a three way anova among mangoes, papayas and pinea pples was analyzed for each day. Differences between mean values were considered significant at P 0.05. 61

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Table 3-1. E. coli O157:H7 Strains Original Designation Lab Code Source H1730 MDD16 Lettuce outbreak/human feces SEA-13B88 MDD18 Apple cider outbreak/human feces F4546 MDD19 Sprout outbreak/human feces 994 MDD20 Fermented salami 62

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Table 3-2. Salmonella Serovars Serovars Lab Code Source Michigan MDD24 Cantaloupe outbreak/human feces Montevideo MDD22 Tomato outbreak/human feces Munchen MDD30 Orange juice outbreak/human feces Newport MDD314 Tomato outbreak/environment Saintpaul MDD226 Orange surface 63

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CHAPTER 4 RESULTS Preliminary Results Mangoes after 180 min at refrigeration storage conditions (4 2C) recorded an average temperature of 5.7 0.2C or 5.9 0.4C (Figure 4-1) for surface and center temperature probes, respectively. No significant difference was de termined between surfac e and center temperature probes on cut mangoes ( P 0.05). Papayas after 180 min at refrigeration storag e conditions (4 2C) recorded an average temperature of 6.7 1.1C or 7.4 0.7C (Figure 42) for surface and center temperature probes, respectively. No significant difference was de termined between surfac e and center temperature probes on cut papayas ( P 0.05). Storage Temperature and Relative Humidity After 7 days at 23 2C the average temperature was 23.5 0.6C and the relative humidity was 67.2 4.1%. After 28 days at 12 2 and 4 2C the average temperatures were 11.9 0.4 and 3.5 0.8C, and the relative humidities were 45 3.1% and 55 6.0%, respectively. After 180 days at -20 2C the average temperature was -20.5 1.2C and the relative humidity 71 5.9%. Background Microflora Each tropical fruit/pathogen/temperature cond ition included a control sample that was not inoculated with pathogens. The control samples were always prepared and sampled first to prevent any cross contamination. The control samples were plated on TSAN and either SMACN or BSAN; selective media for E. coli O157:H7 or Salmonella respectively. No growth was observed on any of the control plates, indi cating the NA supplemented media effectively 64

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eliminated the growth of backgr ound microflora, and that no cr oss contamination has occurred during recovery. To determine background microflora levels, control samples were also plated on PCA. Mangoes, papayas and pineapples all exhibited similar behavior for background microflora at each temperature. At 23 2C microflora continued to increase for the duration of the experiment (Table 4-1). At 12 2C microflora increased until day 5 and then remained steady at ca. 7.7 log CFU/g (Table 4-2). At 4 2C microflora counts rema ined relatively stable through the entire duration of th e experiment at ca. 5.5 log CFU/ g (Table 4-3). At -20 2C microflora decreased ca. 1 log CFU/g in the firs t week before slowly declining over the 180 day storage (Table 4-4). It was determined that visual spoilage occurred at day 3 for 23 2C, day 5 for 12 2C, day 10 for 4 2C and never for -20 2C for all tropical fruit samples (mango, papaya and pineapple). No significant differences were observed between mango, papaya and pineapple spoilage trends. Pathogen Enumeration The average log CFU/g values for nonsel ective (TSAN) and selective (SMACN for E. coli O157:H7 and BSAN for Salmonella ) media on each sampling day were not significantly different ( P 0.05), thus only TSAN results will be discussed. All tables display the data between log CFU/g pathogen ( E. coli O157 or Salmonella ) populations and time (days) for all tropical fruit/temperature conditions. Inoculum Concentration No significant differences in population concen trations were observed between any of the E. coli O157:H7 and Salmonella strains ( P 0.05), ensuring an equal conc entration of all strains in the final inoculum cocktail. 65

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Fate of E. coli O157:H7 on Cut Mangoes Cut mangoes held at 23 2C were inoculated with a 5 or 3 log CFU/g inoculum of E. coli O157:H7 (Table 4-5). E. coli O157:H7 populations inoculated at 5 log CFU/g increased significantly 1.5 log CFU/g within the first 24 h ( P 0.05). Population counts remained constant until day 3. At day 5 a 0.5 log CFU/g decrea se was observed, followed by another 0.4 log CFU/g decrease on day 7. However, these decrea ses from days 3 to 5 or 5 to 7 were not significantly different from each other ( P 0.05). E. coli O157:H7 populations inoculated at 3 log CFU/g followed a very similar trend to th e higher inoculum level. In this case, E. coli O157:H7 populations increased significantly 1.8 log CFU/ g within the first 24 h (P 0.05), then remained relatively unchanged until day 5 ( P 0.05). At day 7 a 0.4 log CFU/g decrease was observed; however, this decr ease was not significant ( P 0.05). Cut mangoes held at 12 2C were only inoc ulated with a 5 log CFU/g inoculum of E. coli O157:H7 (Table 4-6). E. coli O157:H7 populations inoculated at 5 log CFU/g remained relatively constant at ca. 4.5 log CFU/g for 10 days. There was no significant difference observed between each set of sample recovery from days 0 to 10 ( P 0.05). On day 14 a significant 1.3 log CFU/g reduction was observed ( P 0.05). A significant 1.2 log CFU/g decrease was also observed on day 21 (P 0.05). E. coli O157:H7 populations remained unchanged over the next week from days 21 to 28 ( P 0.05). Cut mangoes held at 4 2C were only inoc ulated with a 5 log CFU/g inoculum of E. coli O157:H7 (Table 4-7). E. coli O157:H7 populations inoculated at 5 log CFU/g remained relatively unchanged during the entire duration of the experi ment at ca. 4.0 log CFU/g ( P 0.05). Cut mangoes held at -20 2C were only inoc ulated with a 5 log CFU/g inoculum of E. coli O157:H7 (Table 4-8). E. coli O157:H7 populations inoculated at 5 log CFU/g significantly decreased 0.4 and 0.4 log CFU/g over the 7 and 14 days respectively (P 0.05). From day 14 to 66

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day 28, E. coli O157:H7 populations remained unchanged (P 0.05). A significant population decrease was observed on day 60 of 0.6 log CFU/g ( P 0.05). From day 90 to day 180, E. coli O157:H7 populations remained ca. 2.5 log CFU/g (P 0.05). Fate of E. coli O157:H7 on Cut Papayas Cut papayas held at 23 2C were inoculat ed with a 5 or 3 log CFU/g inoculum of E. coli O157:H7 (Table 4-9). When inoculated at 5 log CFU/g, E. coli O157:H7 populations increased significantly by 2 log CFU/g within the first 24 h (P 0.05). E. coli O157:H7 populations continued to in crease significantly by 0.6 log CFU/g on day 3 ( P 0.05). Populations remained relatively stable between days 3 and 5 ( P 0.05). At day 7 a sign ificant 0.5 log CFU/g decrease was observed ( P 0.05). E. coli O157:H7 populations inoc ulated at 3 log CFU/g increased significantly 3.4 log CFU/g within the first 24 h ( P 0.05). An additional significant increase of 1.1 log CFU/g was observed on day 3 ( P 0.05). Populations remained relatively unchanged the remainder of the experime nt duration at ca. 6.5 log CFU/g (P 0.05). Cut papayas held at 12 2C we re only inoculated with a 5 log CFU/g inoculum of E. coli O157:H7 (Table 4-10). E. coli O157:H7 populations inoculated at 5 log CFU/g significantly increased 1.1 log CFU/g within the first 24 h ( P 0.05). Another, significant E. coli O157:H7 population increase of 1.7 log CFU/g was observed on day 3 (P 0.05). Populations remained relatively constant up to da y 14 at ca. 6.5 log CFU/g ( P 0.05). Due to a pe rsonal complication samples were not recovered on day 21. On day 28, E. coli O157:H7 populations still remained very high at 4.3 log CFU/g; howev er, had significantly decreased from populations recovered on day 14 ( P 0.05). Cut papayas held at 4 2C we re only inoculated with a 5 log CFU/g inoculum of E. coli O157:H7 (Table 4-11). E. coli O157:H7 populations decreased 1.1 log CFU/g due to air dying. Previous reductions due to init ial drying were ca. 0.5 log CFU/g. E. coli O157:H7 populations 67

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remained ca. 3.7 CFU/g for the remainder of the experiment ( P 0.05). Again, due to a personal complication samples were not recovered on day 21; however, as populations remained consistent at day 14 and 28, it can be assumed the day 21 result would have been similar ( P 0.05). Cut papayas held at -20 2C were only i noculated with a 5 log CFU/g inoculum of E. coli O157:H7 (Table 4-12). E. coli O157:H7 populations signifi cantly decreased 1.1 log CFU/g within the first week ( P 0.05). From day 7 to day 180, E. coli O157:H7 populations remained unchanged at ca. 3.0 log CFU/g ( P 0.05). Fate of E. coli O157:H7 on Cut Pineapples Cut pineapples held at 23 2C were inocul ated with a 5 or 3 log CFU/g inoculum of E. coli O157:H7 (Table 4-13). Populations remained unchanged during the first 24 h, before they began to decline rapidly ( P 0.05). Significant population de creases of 0.8, 1.8 and 1.2 log CFU/g were observed on days 3, 5 and 7 ( P 0.05). A similar trend was observed for E. coli O157:H7 populations inoculated with a lower in oculum level. No significant difference was observed between days 0 and 1 for pineapple samples ( P 0.05). On day 3 a significant 1.4 log CFU/g decrease was observed (P 0.05). E. coli O157:H7 populations remained just above the limit of detection on day 5. There was no significant difference betw een population counts on days 3 and 5 ( P 0.05). Recovery on day 7 was c onducted using the U.S. FDA BAMs enrichment protocol for produce. All 6 samples on day 7 were found to be positive for enrichment indicating that while population counts were below the limit of detection (0.3 log CFU/g), E. coli O157:H7 was still present on the cut pineapples. Cut pineapples held at 12 2C were only inoculated with a 5 l og CFU/g inoculum of E. coli O157:H7 (Table 4-14). E. coli O157:H7 populations remained unchanged during the first 24 h ( P 0.05). Following day 1, E. coli O157:H7 populations began to decline over the next 14 68

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days. On day 3 a significant population decrease of 0.7 log CFU/g was observed ( P 0.05). No decrease was observed in populations between days 3 and 5 ( P 0.05). Significant E. coli O157:H7 population decreases of 0.7, 1.3 and 0.7 l og CFU/g were observed on days 7, 10 and 14 ( P 0.05). After day 14, the FDA BAMs enrichment protocol for produce was used. All 6 samples on day 21 were found to be positive for enrichment indicating that while population counts were below the limit of detection, E. coli O157:H7 was still presen t on the cut pineapples. On day 28, none of the 6 samples were found to be positive for enrichment indicating that most likely all E. coli O157:H7 populations were not detectable by enrichment. Cut pineapples held at 4 2C were only inoculated with a 5 log CFU/g inoculum of E. coli O157:H7 (Table 4-15). E. coli O157:H7 populations on cut pi neapples remained relatively unchanged during the first w eek at ca. 4.0 log CFU/g ( P 0.05). However, on day 10, a significant 1.2 log CFU/g decrease was observed ( P 0.05). Populations then stabilized until day 21 at ca. 2.5 log CFU/g ( P 0.05). On day 28, another significant E. coli O157:H7 population decrease of 0.8 log CFU/g was observed ( P 0.05). Cut pineapples held at -20 2C were only inoculated with a 5 l og CFU/g inoculum of E. coli O157:H7 (Table 4-16). E. coli O157:H7 populations decreas ed 0.5, 0.8 and 0.4 log CFU/g on days 7, 14 and 21 respectively ( P 0.05). From day 21 to day 180, E. coli O157:H7 populations remained unchange d at ca. 2.2 log CFU/g ( P 0.05). Fate of Salmonella on Cut Mangoes Cut mangoes held at 23 2C were inoculated with a 5, 3 or 1 log CFU/g inoculum of Salmonella (Table 4-17). Salmonella populations inoculated at 5 log CFU/g significantly increased 2 log CFU/g within the first 24 h ( P 0.05), and remained relativ ely constant at ca. 6.5 log CFU/g until day 3 before declining ( P 0.05). Significant Salmonella population decreases of 1.5 and 1.8 log CFU/g were observed on days 5 and 7 respectively ( P 0.05). When inoculated at 69

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3 and 1 log CFU/g Salmonella populations followed a very simila r trend to the higher inoculum level. When inoculated at 3 log CFU/g Salmonella populations significant ly increased 3.1 log CFU/g within the first 24 h (P 0.05), and remained relatively unchanged until day 3 at ca. 6.1 log CFU/g ( P 0.05). A significant decrease in Salmonella populations of 2.3 and 1.5 log CFU/g was observed on days 5 and 7 respectively ( P 0.05). Salmonella populations inoculated at 1 log CFU/g increased by 2.3 and 3 log CFU/ g on days 1 and 3 respectively ( P 0.05), before beginning to decline. Populati on decreases of 1.6 and 1 log CFU/g were observed on days 5 and 7 respectively. The decline between days 3 and 5 wa s significant ( P 0.05); however, the decline between days 5 and 7 was not significant ( P 0.05). Cut mangoes held at 12 2C were inoculated with a 5, 3 or 1 log CFU/g inoculum of Salmonella (Table 4-18). Salmonella populations inoculated at 5 log CFU/g significantly increased 1.4 log CFU/g within the first 24 h ( P 0.05), then remained constant until day 3 at ca. 5.8 log CFU/g, before beginning to decline ( P 0.05). A significant Salmonella population decrease of 1.2 log CFU/g was observed between day 3 and 5 ( P 0.05). Salmonella populations then remained ca. 4.2 log CFU/g until day 10 ( P 0.05). After day 10, an additional significant decline of 1.8 log CFU/g was observed (P 0.05). Between days 14 and 28 a slow decline continued with populations ranging from 1 to 0.6 log CFU/g (P 0.05). Large standard deviations toward the end of the experiment did not allow for any significant differences. Salmonella populations inoculated at 3 log CFU/g significantly increased by 0.5 and 3.1 log CFU/g on days 1 and 3 ( P 0.05), before beginning to declin e. A significant 1.9 log CFU/g population decrease on day 5 was observed ( P 0.05). No significant changes in Salmonella populations were determined between days 5 and 7 ( P 0.05). Salmonella populations inoculated at 1 log CFU/g remained relativel y unchanged within the first 24 h (P 0.05). Populations 70

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increased 0.6 log CFU/g on day 3 ( P 0.05), then remained rather unchanged for the remainder of the experiment at ca. 1.2 log CFU/g ( P 0.05). Samples were not enumerated after day 7 for the lower inoculum levels as it was beyond the anticipa ted shelf life of the fresh cut mangoes at this temperature. Cut mangoes held at 4 2C were inoculated with a 5, 3 or 1 log CFU/g inoculum of Salmonella (Table 4-19). Salmonella populations increased by 1 l og CFU/g within the first 24 h when inoculated with 5 log CFU/g ( P 0.05), then remained stable until day 3 at ca. 5 log CFU/g ( P 0.05). On day 5 a 0.8 log CFU/g si gnificant decrease was observed ( P 0.05), prior to stabilizing at ca. 3.9 log CFU/g fo r the next week (days 7 to 14; P 0.05). On day 21, an additional 0.6 log CFU/g significant decrease was observed ( P 0.05), prior to stabilizing at ca. 3.1 log CFU/g for the remainder of the experiment. When inoculated at 3 log CFU/g, Salmonella populations remained constant at ca. 2.8 log CFU/g for the first 24 hr ( P 0.05), prior to significant population decrea ses of 0.8 and 0.4 log CFU/g, respectively on days 3 and 5 ( P 0.05). Salmonella populations then stab ilized at ca. 1.7 log CFU/g from days 5 and 7 ( P 0.05). Salmonella populations inoculated at 1 log CFU/g remained unchanged following the initial air drying loss at 0.5 log CFU/ g for the week long experiment ( P 0.05). The experiment for Salmonella inoculated onto cut mangoes at 3 and 1 log CFU/g was not enumerated beyond day 7 as it was designed to observe general trends for Salmonella behavior at lower inoculum levels. Cut mangoes held at -20 2C were only inoc ulated with a 5 log CFU/g inoculum of Salmonella (Table 4-20). Populatio ns significantly decreased 1.3 and 0.7 log CFU/g on days 7 and 21 respectively (P 0.05), prior to stabi lizing on day 21 and remaining unchanged at ca 2.6 log CFU/g through day 180 ( P 0.05). 71

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Fate of Salmonella on Cut Papayas Cut papayas held at 23 2C were inoculated with a 5, 3 or 1 log CFU/g inoculum of Salmonella (Table 4-21). Salmonella populations inoculated at 5 log CFU/g significantly increased 2.3 log CFU/g within the first 24 h ( P 0.05), and remained stable at ca. 7.7 log CFU/g until day 5 before declining ( P 0.05). A significant population d ecrease of 1.1 log CFU/g was observed on day 7 ( P 0.05). Salmonella populations inoculated at 3 log CFU/g increased 3.6 and 1.2 log CFU/g on days 1 and 3 respectively ( P 0.05). Populations remained relatively unchanged until day 5 at ca. 7.3 log CFU/g (P 0.05). A significant decrease of 0.7 log CFU/g was observed on day 7 ( P 0.05). When inoculated at 1 log CFU/g, Salmonella populations increased 3.8 and 1.6 log CFU/g on days 1 and 3 respectively ( P 0.05). Populations remained relatively unchanged for th e duration of the experiment at ca. 6 log CFU/g (P 0.05). Cut papayas held at 12 2C were inoculated with a 5, 3 or 1 log CFU/g inoculum of Salmonella (Table 4-22). Salmonella populations inoculated at 5 log CFU/g increased 1.1, 1.8 and 0.7 log CFU/g on days 1, 3 and 5 respectively (P 0.05), then remained constant from days 5 to 7 at ca. 7.6 log CFU/g (P 0.05). After a week of growth, Salmonella populations on papayas then declined, by 0.6 and 0.8 log CFU/g on days 10 and 14, respectively ( P 0.05). Due to personal complications no sample recovery occurred on day 21. However, on day 28, Salmonella populations were 2.7 log CF U/g lower than on day 14. Salmonella populations inoculated at 3 log CFU/g si gnificantly increased 2.2, 0.8 and 1.6 log CFU/g on days 1, 3 and 5 ( P 0.05), then remained unchanged at ca. 7.2 log CFU/g from days 5 to 10 ( P 0.05). A significant 1.1 log CFU/g Salmonella population decrease on day 14 was observed ( P 0.05). On day 21 sample recovery was not enumerated due to a personal complication; however, on day 28, Salmonella populations were significantly lo wer ( 2.9 log CFU/g) on day 14. Salmonella populations inoculated at 1 l og CFU/g significantly increased 1.3, 2.6 and 1.5 log CFU/g on days 72

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1, 3 and 5 respectively ( P 0.05), but on day 7 had a significant decrease of 1.1 log CFU/g ( P 0.05). There was no significant difference between Salmonella populations on days 7 and 10 as populations remained stable at ca. 4.8 log CFU/g (P 0.05). After day 10 populations continued to decrease, by 1.2 and 1.1 l og CFU/g on days 14 and 28, respectively ( P 0.05). Due to personal complications no sample recovery occurred on day 21. Cut papayas held at 4 2C were inoculated with a 5, 3 or 1 log CFU/g inoculum of Salmonella (Table 4-23). Salmonella populations inoculated at 5 log CFU/g remained ca. 3.5 CFU/g during the first 2 weeks ( P 0.05). While, Salmonella populations were not significantly different between sampling days there was a genera l slow decline in overall log CFU/g counts. Samples were not recovered on day 21 due to a personal complication. On day 28 Salmonella populations were 3.1 log CFU/g. Salmonella populations inoculated at 3 log CFU/g remained relatively constant at ca. 2 l og CFU/g for the first 2 weeks ( P 0.05). Again, samples were not recovered on day 21. On day 28, Salmonella populations were 1.8 log CFU/g. Salmonella populations inoculated at 1 log CFU/g remained unchanged afte r the initial air dying loss at ca. 0.5 log CFU/g for the entire 28 day experiment ( P 0.05). Cut papayas held at -20 2C were only inoculated with a 5 log CFU/g inoculum of Salmonella (Table 4-24). Salmonella populations inoculated at 5 log CFU/g significantly decreased 1.1 and 0.6 log CFU/g at th e 7 and 21 day marks respectively ( P 0.05). From day 21 to day 180, Salmonella populations remained uncha nged at ca. 2.6 log CFU/g ( P 0.05). Fate of Salmonella on Cut Pineapples Cut pineapples held at 23 2C were inocul ated with a 5, 3 or 1 log CFU/g inoculum of Salmonella (Table 4-25). Salmonella populations at 5 log CFU/g remained unchanged during the first 24 hr at 4.1 log CFU/g ( P 0.05). After day 1, Salmonella populations began to decline. Significant population decreases of 1.5, 1.5 and 0.7 log CFU/g were observed on days 3, 5 and 7 73

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( P 0.05). A similar trend was observed for populations inoculated with lower inoculum levels. When inoculated at 3 log CFU/g, Salmonella remained 2.7 log CFU/g for the first 24 hr ( P 0.05). After day 1, populations began to decline on cut pineapple by 1.8 and 0.3 log CFU/g on days 5 and 7 respectively (P 0.05). Salmonella populations inoculated with 1 log CFU/g remained relatively unchanged within the first 24 h at 0.7 log CFU/g before falling below the limit of detection for the duration of the experiment ( P 0.05). All 6 samples were positive upon enrichment on both days 3 and 5 using the U.S. FDA BAMs enrichment protocol for produce. On day 7, none of the 6 samples were found to be positive for enrichment indicating that most likely all Salmonella populations were not det ectable by enrichment. Cut pineapples held at 12 2C were only inoculated with a 5 l og CFU/g inoculum of Salmonella (Table 4-26) and remained unchanged during the first 24 h ( P 0.05). After day 1, Salmonella populations began a steady decline, with decreases of 0.5, 1.2 and 1.3 log CFU/g on days 3, 5 and 7 ( P 0.05). All 6 samples were positive upon enrichment on both days 10 and 14 using the U.S. FDA BAMs enrich ment protocol for produce. On days 21 and 28, none of the 6 samples were found to be positive for enri chment indicating that most likely all Salmonella populations were not detect able by enrichment. Cut pineapples held at 4 2C were only inoculated with a 5 log CFU/g inoculum of Salmonella (Table 4-27), and remained relatively unchanged during the first 24 h (ca. 4.0 log CFU/g; P 0.05). However, over the next 10 days Salmonella populations slowly decreased, including a 0.5 log CFU/g decrease on day 3 ( P 0.05). Populations stabilized between days 3 and 5 at ca. 3 log CFU/g ( P 0.05). Salmonella population decreases of 0.6 and 1 log CFU/g were observed on days 7 and 10 ( P 0.05). Populations stabilized again between days 10 and 14 at ca. 1.3 log CFU/g ( P 0.05). All 6 samples were positive upon enrichment on day 21 using the 74

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U.S. FDA BAMs enrichment protocol for produ ce. On day 28, none of the 6 samples were found to be positive for enrichment indicating that most likely all Salmonella populations were not detectable by enrichment. Cut pineapples were only inoculated with a 5 log CFU/g inoculum of Salmonella (Table 4-28), and decreased 2 log CFU/ g within the first week ( P 0.05). From day 14 to day 180, Salmonella populations remained uncha nged at ca. 2.2 log CFU/g ( P 0.05). Fate of Acid Adapted Salmonella on Cut Pineapples Cut pineapples held at 23 2C were inoc ulated with a 5 or 3 log CFU/g inoculum of acid adapted Salmonella (Table 4-29). The average pH of acid adapted Salmonella serovars were 4.5, which is much lower than the normal Salmonella serovars grown in TSB (average pH 7; non acid adapted Salmonella serovars). Acid adapted Salmonella populations inoculated at 5 log CFU/g remained unchanged during the first 24 hr at ca. 3.9 log CFU/g ( P 0.05). After day 1, acid adapted Salmonella populations began to decline, with population decreases of 1.1, 1.2 and 0.7 log CFU/g were observed on days 3, 5 and 7 ( P 0.05). Acid adapted Salmonella populations inoculated with 3 l og CFU/g were unchanged within the first 24 h at ca. 2.7 log CFU/g ( P 0.05). After day 1, acid adapted Salmonella populations began to decrease, with decreases of 1.4 and 0.4 log CFU/g on days 3 and 5 respectively. All 6 samples were positive for enrichment on day 7 using the U.S. FDA BAMs enrichment protocol for produce. Overall, there were no significant differences between th e behavior of non acid adapted and acid adapted Salmonella on fresh cut pineapples for any of the 7 sample recovery days ( P 0.05). 75

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Figure 4-1. Average surface and center temperatures during cooling of cut mango flesh stored at 4 2C over 180 min (n = 9). Surface temperaure Center temperaure 76

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Figure 4-2. Average surface and center temperatures during cooling of cut papaya flesh stored at 4 2C over 180 min (n = 9). Surface temperaure Center temperaure 77

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Table 4-1. Background Microflo ra enumerated on PCA follo wing incubation at 23 2C Time (day) Mango Papaya Pineapple 0 a 5.3.3 4.0.3 3.9.2 1 7.0.5 6.7.4 5.1.4 3 b 7.7.4 8.6.2 6.9.3 5 8.0.5 8.9.3 7.6.2 7 7.8.5 9.1.7 8.0.5 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of six replications (n = 12) and followed by standard deviation. b Visual spoilage. 78

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Table 4-2. Background Microflo ra enumerated on PCA follo wing incubation at 12 2C Time (day) Mango Papaya Pineapple 0a 5.3.3 4.2.2 3.9.2 1 6.2.4 6.6.3 4.4.4 3 7.3.3 7.7.3 6.3.4 5 b 8.2.5 8.4.4 6.9.4 7 8.3.3 8.5.4 7.7.3 10 8.2.3 8.8.4 7.4.4 14 7.9.6 8.3.3 7.8.4 21 7.6.5 8.0.3 7.5.2 28 7.5.2 7.9.3 7.0.2 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of six replications (n = 12) and followed by standard deviation. b Visual spoilage. 79

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Table 4-3. Background Microflo ra enumerated on PCA follo wing incubation at 4 2C Time (day) Mango Papaya Pineapple 0a 5.3.4 4.1.2 3.8.2 1 5.4.3 4.4.4 3.7.3 3 5.4.2 4.5.3 4.2.2 5 5.8.4 4.7.3 4.8.3 7 5.8.3 5.2.4 5.5.3 10 b 5.6.2 5.4.3 6.2.5 14 5.2.2 5.8.4 6.4.4 21 5.0.3 5.6.5 5.8.3 28 4.9.4 5.4.3 5.5.3 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of six replications (n = 12) and followed by standard deviation. b Visual spoilage. 80

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Table 4-4. Background Microflo ra enumerated on PCA followi ng incubation at -20 2C Time (day) Mango Papaya Pineapple 0 a 5.4.3 4.1.3 3.9.2 7 4.9.3 3.6.3 3.4.5 14 4.6.4 3.8.3 3.8.4 21 4.6.6 4.2.4 4.4.3 28 4.1.5 4.2.3 4.0.4 60 4.3.5 4.1.3 4.0.4 90 3.8.3 3.9.4 4.1.5 120 3.5.5 4.0.5 4.2.3 150 3.0.5 3.7.4 3.8.4 180 2.8.3 4.0.4 4.5.6 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of six replications (n = 12) and followed by standard deviation. 81

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Table 4-5. E. coli O157:H7 on Fresh Cut Mangoes enumerated on TSANP and SMACNP following incubation at 23 2C ca. 5 log CFU/g ca. 3 log CFU/g Time (day) TSANP SMACNP TSANP SMACNP 0 a 4.5.2 4.5.2 2.9.1 2.8.1 1 6.0.4 5.9.3 4.7.3 4.2.3 3 5.7.5 5.5.3 4.5.3 4.3.2 5 5.2.4 5.1.3 4.4.5 4.1.9 7 4.8.5 4.8.4 4.0.1 4.0.2 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. 82

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Table 4-6. E. coli O157:H7 on Fresh Cut Mangoes enumerated on TSANP and SMACNP following incubation at 12 2C ca. 5 log CFU/g Time (day) TSANP SMACNP 0a 4.6.4 4.3.3 1 4.5.2 4.2.1 3 4.5.3 4.1.1 5 4.5.6 4.4.7 7 3.9.4 3.8.3 10 3.9.4 3.9.4 14 2.6.6 2.5.6 21 1.4.6 1.0.7 28 1.5.2 1.3.2 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. 83

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Table 4-7. E. coli O157:H7 on Fresh Cut Mangoes enumerated on TSANP and SMACNP following incubation at 4 2C ca. 5 log CFU/g Time (day) TSANP SMACNP 0a 4.5.4 4.4.3 1 4.5.3 4.3.1 3 4.3.2 4.3.2 5 4.2.1 4.1.1 7 4.2.1 4.1.2 10 4.1.2 4.0.1 14 3.9.1 3.9.4 21 3.8.3 3.8.5 28 3.6.4 3.5.5 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. 84

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Table 4-8. E. coli O157:H7 on Frozen Cut Mangoes enumerated on TSANP and SMACNP following incubation at -20 2C ca. 5 log CFU/g Time (day) TSANP SMACNP 0 a 4.5.4 4.2.1 7 4.1.1 3.9.2 14 3.7.2 3.5.3 21 3.1.7 3.0.7 28 3.7.3 3.2.2 60 3.1.3 2.9.1 90 2.7.4 2.5.2 120 2.3.7 2.2.5 150 2.1.5 2.0.3 180 2.2.4 2.0.4 a Values are expressed as log CFU per gram; values are the average of duplicate samples each of three replications (n = 6) and followed by standard deviation. 85

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Table 4-9. E. coli O157:H7 on Fresh Cut Papayas enumerated on TSANP and SMACNP following incubation at 23 2C ca. 5 log CFU/g ca. 3 log CFU/g Time (day) TSANP SMACNP TSANP SMACNP 0 a 4.6.3 4.5.2 2.6.1 2.5.2 1 6.6.2 6.5.2 6.0.4 5.8.3 3 7.2.3 7.0.4 7.1.3 7.0.4 5 6.9.4 6.9.2 6.7.4 6.5.3 7 6.4.2 6.2.2 6.3.3 6.1.2 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. 86

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Table 4-10. E. coli O157:H7 on Fresh Cut Papayas enumerated on TSANP and SMACNP following incubation at 12 2C ca. 5 log CFU/g Time (day) TSANP SMACNP 0a 3.9.1 3.8.2 1 5.0.2 4.9.3 3 6.7.3 6.7.2 5 6.9.1 6.7.4 7 6.8.1 6.7.3 10 6.5.4 6.3.3 14 6.2.3 6.0.3 21 b 28 4.3.4 4.1.3 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. b Sample period was missed. 87

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Table 4-11. E. coli O157:H7 on Fresh Cut Papayas enumerated on TSANP and SMACNP following incubation at 4 2C ca. 5 log CFU/g Time (day) TSANP SMACNP 0a 3.9.1 3.8.2 1 4.0.2 4.1.1 3 4.0.2 3.9.3 5 3.8.4 3.7.3 7 3.8.3 3.6.4 10 3.6.3 3.5.2 14 3.2.5 3.0.4 21 b 28 3.5.3 4.3.2 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. b Sample period was missed. 88

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Table 4-12. E. coli O157:H7 on Frozen Cut Papaya enumerated on TSANP and SMACNP following incubation at -20 2C ca. 5 log CFU/g Time (day) TSANP SMACNP 0 a 4.8.3 4.6.2 7 3.7.2 3.5.3 14 3.5.2 3.3.4 21 3.4.4 3.3.2 28 3.4.4 3.3.3 60 3.4.3 3.2.3 90 3.1.2 3.0.2 120 3.0.3 2.8.3 150 2.7.4 2.6.2 180 2.6.2 2.4.2 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. 89

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Table 4-13. E. coli O157:H7 on Fresh Cut Pineapples enumerated on TSANP and SMACNP following incubation at 23 2C ca. 5 log CFU/g ca. 3 log CFU/g Time (day) TSANP SMACNP TSANP SMACNP 0 a 4.2.1 4.2.1 2.3.1 2.3.1 1 4.2.3 4.0.2 2.3.2 2.1.2 3 3.4.6 3.4.6 0.9.5 0.7.5 5 1.6.7 1.5.6 0.5.2 0.4.2 7 0.4.3 0.4.1 E+ b E+ a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. b Samples (6 of 6) positive upon enrichment. 90

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Table 4-14. E. coli O157:H7 on Fresh Cut Pineapples enumerated on TSANP and SMACNP following incubation at 12 2C ca. 5 log CFU/g Time (day) TSANP SMACNP 0a 4.2.1 4.2.1 1 4.5.4 4.0.2 3 3.8.4 3.7.4 5 3.5.3 3.4.3 7 2.8.4 2.9.2 10 1.5.7 1.3.6 14 0.8.2 0.6.3 21 E+ b E+ 28 E-c Ea Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. b Samples (6 of 6) positive upon enrichment. c Samples (0 of 6) negative upon enrichment. 91

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Table 4-15. E. coli O157:H7 on Fresh Cut Pineapples enumerated on TSANP and SMACNP following incubation at 4 2C ca. 5 log CFU/g Time (day) TSANP SMACNP 0a 4.2.1 4.2.1 1 4.1.2 4.0.2 3 4.2.2 4.0.2 5 4.2.2 4.1.2 7 4.0.1 4.0.1 10 2.8.8 2.6.7 14 2.6.3 2.5.6 21 2.0.6 1.9.4 28 1.2.5 1.0.4 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. 92

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Table 4-16. E. coli O157:H7 on Frozen Cut Pineapples enumerated on TSANP and SMACNP following incubation at -20 2C ca. 5 log CFU/g Time (day) TSANP SMACNP 0 a 4.2.1 4.2.1 7 3.7.3 3.6.2 14 2.9.4 2.8.3 21 2.5.1 2.4.2 28 2.4.3 2.3.2 60 2.3.3 2.2.3 90 2.3.3 2.1.2 120 2.1.4 2.1.2 150 2.0.3 1.9.4 180 1.9.3 1.7.4 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. 93

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Table 4-17. Salmonella spp. on Fresh Cut Mangoes enum erated on TSANP and BSANP following incubation at 23 2C ca. 5 log CFU/g ca. 3 log CFU/g ca. 1 log CFU/g Time (day) TSANP BSANP TSANP BSANP TSANP BSANP 0 a 4.7.3 4.5.2 2.9.1 2.7.1 0.6.2 0.5.2 1 6.7.3 6.5.2 6.0.3 5.9.3 2.9.1 2.9.1 3 6.3.2 6.3.3 6.2.3 5.9.2 5.9.4 5.8.4 5 4.8.4 4.5.2 3.9.5 2.5.9 4.3.8 4.2.8 7 3.0.5 3.0.4 2.4.1 2.2.2 3.6.5 3.2.3 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 18) and followed by standard deviation. 94

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Table 4-18. Salmonella spp. on Fresh Cut Mangoes enum erated on TSANP and BSANP following incubation at 12 2C ca. 5 log CFU/g ca. 3 log CFU/g ca. 1 log CFU/g Time (day) TSANP BSANP TSANP BSANP TSANP BSANP 0a 4.5.1 4.6.0 2.8.1 2.8.1 0.6.2 0.5.2 1 5.9.3 5.8.4 3.3.1 3.3.1 0.9.3 0.7.2 3 5.7.4 5.5.5 6.4.1 6.3.1 1.4.2 1.3.3 5 4.5.7 4.4.8 4.5.6 4.4.6 1.4.5 1.2.6 7 4.5.3 4.6.2 4.2.1 4.0.5 1.0.2 0.8.3 10 3.7.1 3.6.2 b 14 1.9.1 1.8.1 21 1.0.6 0.7.4 28 0.6.3 0.4.2 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. b Sample was not enumerated as it was beyond the anticipated shelf life of the product. 95

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Table 4-19. Salmonella spp. on Fresh Cut Mangoes enum erated on TSANP and BSANP following incubation at 4 2C ca. 5 log CFU/g ca. 3 log CFU/g ca. 1 log CFU/g Time (day) TSANP BSANP TSANP BSANP TSANP BSANP 0a 4.3.4 4.2.4 2.8.2 2.8.0 0.6.2 0.5.2 1 5.2.1 5.0.2 2.9.3 2.9.1 0.5.2 0.5.2 3 4.9.8 4.7.1 2.1.1 2.1.1 0.6.3 0.5.3 5 4.1.1 3.9.2 1.7.3 1.6.4 0.6.3 0.5.2 7 4.0.3 3.8.3 1.8.4 1.6.3 0.5.1 0.3.4 10 3.8.4 3.6.5 b 14 3.8.2 3.8.2 21 3.2.1 3.1.1 28 3.1.3 2.8.6 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. b Sample was not enumerated further as it wa s designed to observe th e general trends of Salmonella inoculated at lower inoculum levels. 96

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Table 4-20. Salmonella spp. on Fresh Cut Mangoes enum erated on TSANP and BSANP following incubation at -20 2C ca. 5 log CFU/g Time (day) TSANP BSANP 0 a 4.7.2 4.7.2 7 3.4.4 3.2.3 14 3.4.3 3.1.3 21 2.7.3 2.6.3 28 2.9.2 2.7.3 60 2.7.3 2.5.3 90 2.7.3 2.2.5 120 2.6.4 2.4.2 150 2.5.4 2.4.4 180 2.4.3 2.3.4 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. 97

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Table 4-21. Salmonella spp. on Fresh Cut Papayas e numerated on TSANP and BSANP following incubation at 23 2C ca. 5 log CFU/g ca. 3 log CFU/g ca. 1 log CFU/g Time (day) TSANP BSANP TSANP BSANP TSANP BSANP 0 a 4.7.2 4.6.1 2.6.2 2.7.2 1.1.1 0.9.2 1 7.0.1 6.9.3 6.2.3 6.1.2 4.9.1 4.6.1 3 7.7.8 7.5.5 7.4.5 7.2.4 6.5.5 6.5.3 5 7.8.3 7.6.2 7.3.3 7.2.4 6.2.2 6.1.4 7 6.7.2 6.7.3 6.6.2 6.5.3 5.8.5 5.7.2 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. 98

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Table 4-22. Salmonella spp. on Fresh Cut Papayas e numerated on TSANP and BSANP following incubation at 12 2C ca. 5 log CFU/g ca. 3 log CFU/g ca. 1 log CFU/g Time (day) TSANP BSANP TSANP BSANP TSANP BSANP 0a 4.1.1 4.0.2 2.4.1 2.2.2 0.6.2 0.4.1 1 5.2.1 5.2.2 4.6.3 4.5.4 1.9.1 1.8.3 3 7.0.2 6.9.4 5.4.3 5.2.3 4.5.5 4.3.2 5 7.7.5 7.5.3 7.0.2 6.9.3 6.0.2 5.9.5 7 7.6.4 7.4.3 7.4.4 7.4.6 4.9.1 4.7.4 10 7.0.3 6.8.4 7.3.4 7.0.3 4.7.5 4.6.2 14 6.2.3 6.0.2 6.2.2 6.0.4 3.5.0 3.3.5 21 b 28 3.5.5 3.4.5 3.3.2 3.3.4 2.4.1 2.2.2 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. b Sample period was missed. 99

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Table 4-23. Salmonella spp. on Fresh Cut Papayas e numerated on TSANP and BSANP following incubation at 4 2C ca. 5 log CFU/g ca. 3 log CFU/g ca. 1 log CFU/g Time (day) TSANP BSANP TSANP BSANP TSANP BSANP 0a 4.1.1 4.1.0 2.4.1 2.3.2 0.6.1 0.5.1 1 4.0.1 4.0.4 2.2.2 2.2.2 0.8.3 0.8.2 3 3.9.2 3.8.5 2.1.1 2.0.4 0.7.3 0.6.2 5 3.9.4 3.6.8 2.2.3 2.1.2 0.5.2 0.6.4 7 3.7.3 3.6.2 1.9.2 1.8.2 0.6.3 0.5.4 10 3.6.3 3.4.2 2.0.3 1.8.2 0.6.2 0.4.3 14 3.3.3 3.2.1 1.9.2 1.7.4 0.4.1 0.4.3 21 b 28 3.1.3 3.0.2 1.8.3 1.5.4 0.4.1 0.3.2 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. b Sample period was missed. 100

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Table 4-24. Salmonella spp. on Fresh Cut Papayas e numerated on TSANP and BSANP following incubation at -20 2C ca. 5 log CFU/g Time (day) TSANP BSANP 0 a 4.9.2 4.8.1 7 3.8.3 3.6.2 14 3.6.4 3.6.3 21 3.0.2 2.9.2 28 2.9.3 2.8.2 60 2.9.4 2.7.4 90 2.7.3 2.6.2 120 2.6.2 2.5.3 150 2.6.3 2.4.1 180 2.4.3 2.3.2 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. 101

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Table 4-25. Salmonella spp. on Fresh Cut Pineapples enumerated on TSANP and BSANP following incubation at 23 2C ca. 5 log CFU/g ca. 3 log CFU/g ca. 1 log CFU/g Time (day) TSANP BSANP TSANP BSANP TSANP BSANP 0 a 4.3.4 4.2.4 2.5.4 2.4.4 0.8.2 0.6.3 1 4.1.3 4.0.2 2.7.2 2.6.2 0.7.2 0.6.2 3 2.6.4 2.5.4 0.9.4 0.6.4 E+ b E+ 5 1.1.6 1.1.6 0.6.3 0.3.1 E+ E+ 7 0.4.5 0.4.2 E+ E+ E-c Ea Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 12) and followed by standard deviation. b Samples (6 of 6) positive upon enrichment. c Samples (0 of 6) negative upon enrichment. 102

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Table 4-26. Salmonella spp. on Fresh Cut Pineapples enumerated on TSANP and BSANP following incubation at 12 2C ca. 5 log CFU/g Time (day) TSANP BSANP 0a 4.3.4 4.2.4 1 4.0.2 3.9.2 3 3.5.5 3.4.4 5 2.3.3 2.3.3 7 1.0.3 0.9.4 10 E+ b E+ 14 E+ E+ 21 E-c E28 EEa Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. b Samples (6 of 6) positive upon enrichment. c Samples (0 of 6) negative upon enrichment. 103

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Table 4-27. Salmonella spp. on Fresh Cut Pineapples enumerated on TSANP and BSANP following incubation at 4 2C ca. 5 log CFU/g Time (day) TSANP BSANP 0a 4.3.4 4.2.4 1 3.8.4 3.8.5 3 3.2.5 3.2.5 5 2.8.5 2.7.4 7 2.2.1 2.2.1 10 1.2.3 1.1.3 14 1.3.3 1.2.3 21 E+ b E+ 28 E-c Ea Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. b Samples (6 of 6) positive upon enrichment. c Samples (0 of 6) negative upon enrichment. 104

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Table 4-28. Salmonella spp. on Fresh Cut Pineapples enumerated on TSANP and BSANP following incubation at -20 2C ca. 5 log CFU/g Time (day) TSANP BSANP 0 a 4.3.4 4.2.4 7 2.3.5 2.2.3 14 2.5.2 2.4.2 21 2.5.2 2.4.2 28 2.4.2 2.2.3 60 2.4.2 2.2.4 90 2.4.4 2.1.3 120 2.1.4 2.0.4 150 2.0.3 1.8.5 180 1.7.4 1.5.4 a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. 105

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106 Table 4-29. Acid Adapted Salmonella spp. on Fresh Cut Pineapples enumerated on TSANP and BSANP following incubation at 23 2C ca. 5 log CFU/g ca. 3 log CFU/g Time (day) TSANP BSANP TSANP BSANP 0 a 4.1.1 4.1.1 2.7.2 2.6.1 1 3.8.4 3.7.2 2.7.5 2.5.3 3 2.7.2 2.4.4 1.3.5 1.5.4 5 1.5.4 1.3.3 0.9.4 0.8.3 7 0.8.3 0.7.2 E+ E+ a Values are expressed as log CFU per gram; valu es are the average of duplicate samples from each of three replications (n = 6) and followed by standard deviation. b Samples (6 of 6) positive upon enrichment.

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CHAPTER 5 DISCUSSION Much is unknown about the potential for tropical fruits to be vehicles of foodborne disease. Diverse outbreaks have occurred due to tropical fruit consumption providing evidence that tropical fruit can serve as vectors for di fferent foodborne pathogens. Research on the behavior of pathogens in tropical fruits is limited. Growing tr ends in consumption of fresh fruits, especially tropical fruits, has created a need for mo re research in this area. Tropical fruits raise increased concerns as they are heavily imported from various developing countries. These concerns include a lack of one global food sa fety agency to regulate farming, harvesting, processing and shipping and vari ability seen in different re gions. Developing countries producing many tropical fruit have limited food safety agencies compared to the U.S. and unique challenges to overcome. Different routes of contamin ation of tropical fruit can be hypothesized, and data exist demonstrating Salmonella infiltration into mango flesh. No significant differences were observed between surface and center cooling of mangoes and papayas. This indicates that behavior of a surface spot inoculation on the flesh of the tropical fruit may be similar to what would be observed for internalized pathogens at 23C. Concerns that pathogens may begin to grow during the cutting and inocul ation at room temperature were found to not be of concern for this study. Following, 2 h at refrige ration temperature tropical fru it had reached 6C, within the range of 4 2C. Based trends obtained from the cooling curves and actual experimental time between fruit cutting, inoculation, and subsequent incubation, the worst case scenario is that tropical fruit were only exposed to ambient temp erature (23C) for a maximum 3 h. Escartin et al. (1989) found that the lag time for Salmonella growth on sliced papaya was approximately 3 h. No lag times for E. coli O157:H7 have been documented in tropical fruits. As 3 h represents 107

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the worst case scenario for which tropical fruits in this experiment were held above storage conditions, it is highly unlikely that this hold ti me affected the integrity of the experimental results. This is further supported by the obser vation that pathogen levels were within the expected range, assuming a 0.5 log CFU/g reduction due to the air dying of the inoculum, at time 0 (Knudsen et al., 2001). The visual spoilage timelines that were obs erved in mangoes, papayas and pineapples for each storage temperature was comparable to industr y spoilage expectations Spoilage in fresh cut fruits and vegetables can be caused by s poilage microorganisms like yeasts, molds and bacteria or by enzymatic reactions. Unlike pat hogenic organisms that make the food unsafe to consumer, microbial or enzymatic spoilage cause food quality to deteriorate, leading to the development of off colors, tastes, odors or textur es. At temperatures 23 2, 12 2 and 4 2C, spoilage visually determined on mangoes, papaya s and pineapples primarily due to discoloration and smell. At 3, 5 and 10 days, tropical fruit would begin to show br own or black spots or unpleasant odors for temperatures 23 2, 12 2 and 4 2C, respectively. Brown or black spots on tropical fruit could be possibly due to anthracnose. Anthracnose, usually caused by Colletotrichum gloeosporioides is one of the most common posth arvest spoilage diseases in mangoes and papayas (Pitt and Hocking, 1999). S poilage on mangoes, papayas and pineapples may also have been due to enzymatic ti ssue breakdown caused by polyphenol oxidase. Enzymatic reactions proceed faster at higher te mperatures and may be a contributing spoilage factor. Freezing (-20 2C) mangoe s, papayas and pineapples prevented visual spoilage for the duration of the experiment (180 days). Spoilage is prevented at freezing temperatures because microorganisms cannot grow, and enzymatic reac tions are significantly slowed. If freezing 108

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temperatures are not maintained, microbial a nd or enzymatic spoilage may occur on the cut mangoes, papayas and pineapples. Nalidixic acid was supplemented in all pathoge n enumeration media to inhibit the growth of background bacteria. It is an antibiotic th at is both effective against gram positive and negative bacteria. The addition of NA eliminated the enumeration of any background flora, which could potential comprise the results of this experiment. Only nonselective media (TSANP) data was disc ussed in the results section. Selective media was used to ensure results on nonselective media were representati ve of the specific pathogen being enumerated. Had there been a significant difference between the nonselective and selective media enumeration results then bot h media enumeration results would have been discussed. Significant differences between selective and non selective media may have alluded to several possibilities. These in clude cross contamination from inoculation, natural background microflora that is environmentally resistant to NA or other injured cells that are unable to grow on the selective media. Selective media is spec ifically tailored to promote specific organism growth, and may promote specific colony characteristics de velop to differentiate like pathogens. For example, SMAC is designed to identify E coli O157:H7 because it contains sorbitol as its fermentable sugar, which E. coli O157:H7 utilizes. This fermentation results in E. coli O157:H7 growth on SMAC as hot pink colonies. In contrast, Salmonella and other E. coli strains do not ferment sorbitol and grow into white colonies. Nonselective and selective media enumeration results were not significantly different in the results presen ted here, thus only nonselective (TSANP) results are discussed. Behavior of pathogens varied on cut mango, papaya and pineapple fruit surfaces. Mango, papaya and pineapple, while all classified as tropical fruit, provide unique intrinsic factors that 109

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affect microbial fate. These f actors may contribute to the microbi al survival and growth trends which differed in mango, papaya and pineapple, and include pH and enzyme composition (i.e. proteases). The pH of cut mangoes, papa yas and pineapples are ca. 4.2, 5.7 and 3.6, respectively. Papayas and pin eapples contain the powerful pr oteases papain and bromelian, respectively, while mangoes do not contain similar proteases (M ynott et al., 1999). E. coli O157:H7 has the potential to grow on temperature abused fresh cut mangos, and survive for the shelf life of fresh cut ma ngoes. Cut mangoes support the growth of E. coli O157:H7 at 23 2C and the survival of E. coli O157:H7 at 12 2 and 4 2C for the shelf life of cut mangoes at each temperat ure. The ideal temperature for E. coli O157:H7 growth is 37C; however, it can grow at lower te mperatures if the appropriate nutrients, water activity and pH conditions exist. The inability of E. coli O 157:H7 to grow on cut mangoes at 12 2 and 4 2C, maybe due to the combination of temperature and acidic pH. Although no growth was observed, E. coli O157:H7 populations survived the shelf life of the cut mangoes at concentrations high enough to cau se illness. At 23 2C, E. coli O157:H7 grew following inoculation at high or low popula tion levels. The pH of cut ma ngoes (ca. 4.2) is within the minimum pH range of documented E. coli O157:H7 growth, of 4.0-4.5 unl ess acid resistance is induced (Lin et al., 1996). Salmonella grew on fresh cut mangos held at 23 2C and 12 2C, and survived for the shelf life of refrigerated fresh cut mangoes (4 2C) regardless of initial population concentration. The ideal temperature for Salmonella growth is 37C; however, it can grow at lower temperatures if given the conditions are adequate. Salmonella is also able to adapt and grow at what would normally be considered en vironmental extremes, such pH (tomatoes; pH 4.0; Beuchat and Mann, 2008) and te mperature (chicken, 2C; Foster and Spector, 1995). The 110

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pH of cut mangoes (ca. 4.2) did not inhibit Salmonella growth at 23 2 and 12 2C at any population concentration. Refrigerated (4 2C) cut mangoes allowed for the long term survival of Salmonella approximately the same concentration of the initial inoculum, even when at 1 log CFU/g. The low temperature (4 2C) and pH (4.2) were able to inhibit Salmonella growth, but unable to kill the Salmonella cells. Both E. coli O157:H7 and Salmonella grew on fresh cut mangoes; however, Salmonella grew at a faster rate than E. coli O157:H7. It can be hypothesized that the faster rate at which Salmonella grew led to a more rapid depletion of nut rients and buildup of byproducts resulting in lower populations of Salmonella than E. coli O157:H7 at the end of the experiment. This hypothesis was generated from general principles of the standard bacterial growth curve (Cox, 2000). The low pH of mangoes (ca. 4.3) does not s eem to affect the growth or survival of E. coli O157:H7 or Salmonella Salmonella can grow in acidic substrates as low as pH 3.0 if it is able to induce either one of two acid tolerance res ponse systems (Foster and Spector, 1995). These two acid tolerance response systems can be activated in either log phase or stationary phase cells. One is an acid shock response where log phase cells are subjected to a low pH shift (ca. pH 4.0) and the second occurs where stationary cells gradually beco me acid tolerant. E. coli O157:H7 can grow in acidic substrates if it is able to induce one of three acid response systems. These three systems include acid induced glutamatedependent, arginine-dependent, and oxidative systems (Lin et al., 1996). The growth observed in cut mangoes held at 23 2C may be due to the pH of mangoes triggering an aci d tolerance response system in E. coli O157:H7 and Salmonella. The pH of mangoes may have act ed as an acid shock as cells would have been in the lag phase initially following th e inoculation. Growth increases of 2-3 log CFU/g where seen 111

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in cut mangoes for both E. coli O157:H7 and Salmonella within the first 24 h. After the 24 h period, E. coli O157:H7 and Salmonella populations stabilized and th en proceeded to decline, likely due to limited nutrients or a further drop in pH due to the acid produced as byproducts. Hsin-Yi and Chou (2001) found that E. coli O157:H7 survived in mango juice for 6 days when held at ambient temperature (25 C); however, did not observe any E. coli O157:H7 growth. This may be due to the lower pH (ca. 4) of the juice. E. coli O157:H7 and Salmonella populations stabilized in cut mangoes held at both 12 2 and 4 2C. The temperature of 12 2C similar to those observed in open case retail refrigerat ion units. The likelihood of cut mangoes held at similar temperatures is extremely hi gh. Previous research has shown E. coli O157:H7 can survive in mango juice when held at refrigerati on temperatures 6C and 7C for periods of up to 13 and 8 days, respectively (Leite et al., 2002; Hsin-Yi and Chou, 2001). The results presented here are similar to previous re search trends determined for E. coli O157:H7 survival on mangoes. Tomatoes and mangoes both have a pH betw een 4 and 4.5. The growth and survival trends for E. coli O157:H7 and Salmonella observed here on cut ma ngoes are comparible to those seen on tomatoes. Beuchat and Mann (2008) observed Salmonella growth in tomato pulp (ca. pH 4.5) at both 21 and 12C within 72 h. Eribo and Ashenafi (2003) found long term survival of E. coli O157:H7 (23 days) in tomato juice held at 4C. E. coli O157:H7 populations grew in tomato juice held at 25C for up to 20 days (Eribo and Ashenafi, 2003). In this study, E. coli O157:H7 rapidly grew on fresh cu t papayas at 23 2 and 12 2C, and survived for the shelf life of cut papayas held at refrigeration. The near neutral pH (5.7) and presence of appropriate nutrients created an excellent substrate for E. coli O157:H7 growth. E. coli O157:H7 populations reached maximums of ca. 7 log CFU/g at both 23 2 and 12 2C and remained stable at this level for up to 7 days. Over the 28 day storage period the only 112

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decline in E. coli O157:H7 populations on cut refrigerated (4 2C) papayas was seen during the initial air drying. Salmonella grew rapidly on fresh cut papayas at 23 2 and 12 2C, and survived for the shelf life of refrigerated fresh cut papayas (4 2C). Salmonella was inoculated on papayas at a high (5 log CFU/g), medium (3 log CFU/g) a nd low (1 log CFU/g) to evaluate the effect of inoculum level on behavior. All three le vels of starting inoculum reached high Salmonella population levels within 24 h. On cut papayas he ld at the refrigeration temperature (4 2C) Salmonella populations survived the entire 28 day st orage period regardless of initial starting concentration. No growth was observed at 4 2C; however, if temperature abuse were to happen significant Salmonella growth will likely occur. E.coli O157:H7 and Salmonella both grow rapidly on fresh cut papayas. Similar to mangoes, Salmonella grew more rapidly than E. coli O157:H7 on papayas and had lower population counts upon completion of the experiment. Various pathogens have been shown to surviv e and grow in fresh cut papayas, papaya juice and pulp. Shigella and Salmonella grew on sliced papayas held at ambient (20C) temperatures for 6 h. Castillo and Escartin (199 4) noted growth increases at 3 and 6 h. They hypothesized this growth would continue to occu r, as is demonstrated in this thesis. Salmonella on fresh cut papayas held at 23 2C reached th e carrying capacity at ca. 7.2 log CFU/g for all inoculum levels within 72 h. Both E. coli O157:H7 and Salmonella rapidly grew in papaya juice at ambient temperatures within 48 h (Mutake et al., 2005; Yigeremu et al., 2001). Similar pathogen carrying capacities (max. log CFU/g lo ad on fruit sample) were observed by Mutake (2005) and Yigeremu (2001) in papaya juice. Small growth increases were observed for E. coli O157:H7 and Salmonella in papaya juice held at refriger ation temperatures (Yigeremu et al., 113

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2001; Mutake et al., 2005). No growth increase s were observed in fresh cut papayas held at refrigeration. This difference may be due to th e substrate matrices (i.e. pH or composition). Penteado et al. (2004) observed Salmonella growth in papaya pulp at 10, 20 and 30C. Growth rates in papaya pulp at 10 and 20C were sim ilar to the rates observed here at 12 and 23C, respectively. The trends observed for pathogen fate on cut papayas are comparable to other fruit commodities, such as melons, with similar intr insic factors. Delrosario and Beuchat (1995) observed E. coli O157:H7 growth at 25C and survival at 5C on fresh cut cubes of cantaloupe and watermelon during 34 h storage. Go lden et al. (1993) observed rapid Salmonella growth in cut cantaloupe, water and honeydew melons when stored at 25C for 24 h. Salmonella populations were stable in the same cut melons stored at 5C for the 24 h (Golden et al., 1993). Additionally, Penteado and Leitao (2004) found Salmonella growth in melon and watermelon pulp stored at 30, 20 and 10C within 24 h. E. coli O157:H7 did not grow and had limited surv ival on fresh cut pine apples held at 23 2, 12 2 and 4 2C. The pH of pineapples is ca. 3.6, below the minimum pH limits for E. coli O157:H7. Pineapples also have a high perc entage of unfermentable fiber, which may decrease the availability of nutrients for E. coli O157:H7 growth (Mutake et al., 2005). The survival of E. coli O157:H7 on fresh cut pineapple is affected by temperature. E. coli O157:H7 survives longer on fresh cut pineapples at refrig eration temperatures (4 2C) than at ambient temperatures (23 2C). The slower rate of E. coli O157:H7 decline at lower temperatures maybe due to to the overall reduced metabolism of the organism at cooler temperatures. At 23 2C, the low level inoculum (3 log CFU/g) of E. coli O157:H7 reached undetectable levels the 114

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most rapidly proving additional evidence that the length of E. coli O157:H7 survival on fresh cut pineapples is influenced by temperature. No growth and limited survival of Salmonella was seen on fresh cut pineapples held at 23 2, 12 2 and 4 2C at all inoculum levels. Salmonella does not normally grow below a pH of 4.0 (Foster and Spector, 1995). The survival of Salmonella like that of E. coli O157:H7 appears to be influenced by temperature. Salmonella survives longer on fresh cut pineapples at refrigeration temperatures (4 2C) than at the other higher temperatures (23 2 and 12 2C). The same trends were observed with Salmonella as in E. coli O157:H7 on fresh cut pineapples. A small scale study was conducted using acid adapted Salmonella on cut pineapples held at 23 2C. There were no significant differences between the acid adapte d and non acid adapted Salmonella on fresh cut pineapples. The acid adapted Salmonella did not grow or exhibit a ny significant improvement in survival, thus it is unlikely pH alone accounts for the rapid decline in pathogen populations. Beuchat and Mann (2008) determined that the fate of Salmonella in tomato pulp tissue (pH ca. 4.2) was unaffected by predisposing Salmonella cells to acid. Salmonella inoculated into tomato pulp tissue was able to grow re gardless of acid adaptation, simila r to results shown here where no differences in Salmonella survival were observed following acid adaptation. Other factors, such the protease bromelain, maybe involved in the decline of E. coli O157:H7 and Salmonella populations in fresh cut pineapples; howev er, more research is needed. Neither E. coli O157:H7 or Salmonella grew on fresh cut pineapples. E. coli O157:H7 was able to survive longer than Salmonella in fresh cut pineapples at all temperatures. The pH of cut pineapples is below either minimum pH standard for E. coli O157 or Salmonella (Lin et al., 1996; Foster and Spector, 1995). 115

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Previous research on E. coli O157:H7 and Salmonella in cut pineapples has also found no growth and limited survival. A two day study by Nazuka et al. (2004) concluded fresh cut pineapples when stored at 4, 10 and 20C to be a poor substrate for growth of both E. coli O157:H7 and Salmonella Fresh cut pineapples remained approximately the starting inoculum (4.5 log CFU/g) for the 2 day duration (Nazuka et al., 2004). Th ese results are similar to the results presented here where no decreases in E. coli O157:H7 or Salmonella populations were observed until day 3. The shelf life of fresh cut pineapples (ca. 10 days for 4C; ca. 5 days for 10C) stored at 4 and 10C is longer than 2 days so the Nazuka et al. (2004) study. Studies in pineapple juice by Mutake et al. (2005) and Yigeremu et al. (2001) determined that E. coli O157:H7 and generic E. coli, respectively were unable to grow in this matix. Mutake et al. (2005) observed E. coli O157:H7 populations survived in pinea pple juice for 3 days when held at both ambient (20-25C) and refrigeration (4C) temperatures. Yigeremu et al. (2001) observed a similar trend for E. coli in pineapple juice held at the sa me temperatures, and populations to decline in refrigeration pineapple juice as well (Yigeremu et al., 2001). However, this same study also observed Salmonella to grow in pineapple juice held at 37C within 24 h (Yigeremu et al., 2001). Unless pineapple juice was subjected to extreme temperature abuse this temperature condition, 37C, is unlikely. While, the mediums ar e not the same (fresh cut vs. juice), these studies support the finding th at pineapple is a poor substrate for growth of E. coli O157:H7 and Salmonella at 4, 12 and 23C. Research in other high ac id commodities, such as oranges, determined E. coli O157:H7 and Salmonella growth in peeled oranges within 24 h at 24C (Pao et al., 1998). However, after 24 h a decline in both E. coli O157:H7 and Salmonella populations were observed. E. coli O157:H7 and Salmonella populations survived for the 14 days on peeled oranges at 4C, but 116

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declined continually dur ing this storage period (Pao et al ., 1998). The initial growth of pathogens at 24C varies from pineapple result s discussed in this th esis, which observed no E. coli O157:H7 or Salmonella growth in cut pineapple at any stor age temperature. This difference could be due to the fact that while peeled orange s are high in acid, they ha ve an intact membrane unlike cut pineapples. Overall, like our observations in pineapple, E. coli O157:H7 and Salmonella populations continued to dec line in peeled oranges duri ng storage regardless of the difference in intact versus broken membrane on the fruit surface. Long term survival of E. coli O157:H7 and Salmonella was observed on frozen (-20C) cut mangoes, papayas and pineapples. At this temperature, E. coli O157:H7 and Salmonella limit metabolic function in order to prevent deat h. Initial decreases were observed for both E. coli O157:H7 and Salmonella populations, prior to st abilizing. If temperature abuse or thawing under improper conditions were to occur, E. coli O157:H7 and Salmonella would likely be able to grow. Previous work has shown E. coli O157:H7 in mango juice co uld survive for up to 2 weeks when held at freezing temperatures (10 C) (Leite et al., 2002). Similar studies were conducted on frozen pin eapple juice and found both E. coli O157:H7 and Salmonella to exhibit long term survival. Leite et al. (200 2) and Oyarzabal et al. (2003) observed E. coli O157:H7 and Salmonella populations to remain in frozen pinea pple juice for up to the duration of the experiments: 2 and 4 weeks, respectively. In conclusion, E. coli O157:H7 and Salmonella displayed similar behavior on cut mangoes, papayas and pineapples. This is most likely due to the fact that E. coli O157:H7 and Salmonella are both enteric pathogens a nd share similar survival and growth characteristics. Alternatively, mango, papaya and pi neapple all displayed different survival and growth trends. 117

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For instance, if mangoes are temperatur e abused (23 2C) they have the E. coli O157:H7 and Salmonella for pathogen growth; however, temperature a bused (23 2C) pineapples only have limited E. coli O157:H7 and Salmonella survival. E. coli O157:H7 and Salmonella grow very well on papayas even in the absence of temp erature abuse (23 2C). This research demonstrates the potential for E. coli O157:H7 and Salmonella survival and grow th in mangoes, papayas and pineapples and the importance on ha rvest and postharvest practices. High-quality sanitation is required to prevent contamination of pathogens on ra w commodities like cut tropical fruits. Packinghouses and fresh cut facilities sh ould implement safety and sanitation programs. The ultimate goal of these programs is to ensure a safe, raw product entering the facility and eliminate possible contamination within the faci lity. This thesis proves the potential for E. coli O157:H7 and Salmonella to survive and grow in cut mangoes, papayas and pineapples held at major storage conditions. 118

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CHAPTER 6 FUTURE WORK Tropical fruits are a common commodity in st ores and food service across the United States. As the marketplace continues to demand mo re tropical fruits, the need continues to be met by importing tropical fruits The potential for pathogen growth and long term survival on tropical fruits has been demonstrated by the re sults in this thesis; however, much remains unknown. Governments need to work together to formulate and implement global food safety strategies. In the meantime, more work is n eeded on a variety of safety issues concerning tropical fruits. One of the main issues that requires furt her examination are the routes of pathogen transmission and sources of cross contamination in tropical fruits. The initial source of pathogen contamination should be investig ated, in addition to risks associated with various modes of pathogen transmission. Postharv est handling practices in mangoes (i.e. hot water dips) have directly contributed to two large Salmonella outbreaks associated with mango consumption due to of pathogen internalization. However, initial sources of Salmonella contamination are less concrete. Field studies could be conducted to determine wher e pathogen contamination may occur. Possible sources of initial contamination include soil, air, animals, water or worker personal hygiene. Understandi ng the degree of fruit entering the postharvest environment already contaminated and understanding how cross contamination occurs down the continuum from farm to fork will be the key for risk managers. An additional topic that requires further ex amination is the behavior of pathogens on whole tropical fruit surf aces. Few studies have evaluated th e survival of human pathogens on the surface of tropical fruits. Tropical fruit surfac es are extremely variable, ranging from the rough exterior of a pineapple, the fuzzy exterior of a mamey, and the smooth exterior of a mango. 119

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Studies investigating the modes of pathogen attachment for each different tropical fruit and lengths of time various pathogens can survive on the surface should be identified. Additionally, work with varying sanitizers to investigate th e degree of pathogen removal from the surface of tropical fruits remains to be evaluated. For example, before mangoes enter the postharvest hot water dip to eliminate the Caribbean Fruit Fly, th ey could be dipped in a sanitizer to remove potential surface contamination. The goal in this pre-treatment would be to eliminate surface pathogen contamination so internal ization does not occur upon transfer to the cool water after the hot water dip. The work presented in this thesis sought to open the door to understanding pathogen behavior on the fruit flesh. It demonstrated the potential for E. coli O157:H7 and Salmonella to grow and survive on mangoes, papayas and pineapples for the duration of their shelf lives and in some cases much longer. Temperature a buse is critical to the prevention of E. coli O157:H7 and Salmonella proliferation, was observed by the growth of these pathogens on mango and papayas at room temperature. None of th e temperatures evaluated prevented E. coli O157:H7 and Salmonella survival. Even E. coli O157:H7 or Salmonella survived for the duration of the industry recommended shelf lives on fresh cut pineapples held at 23 2, 12 2 and 4 2C. These results further demonstrat e the food safety risks that ma ybe associated with fresh cut tropical fruits. This research only evaluated the behavior of E. coli O157 and Salmonella on fresh and frozen cut tropical fruits; howev er, other pathogens such as Shigella have been associated with tropical fruit outbreaks, for which no research exists. Listeria monocytogenes is a serious pathogen of concern in refrigera tion foods. Little to no research exists on the fate of this pathogen in various refrigerated fresh cut tropical fruits. Additionally, more tropical fruit remain 120

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121 to be investigated in regards to their ab ility to support the grow th or survival for E. coli O157:H7 and Salmonella Acerola and aai are two tr opical fruits found in a wi de variety of new healthy juice blends and no studies have been conducted concerning pathogen behavior in the matrixes of these emerging tropical fruits. Every tropical fruit, as demonstrated by mangoes, papayas and pineapples, has different characteristics. However, the risks associated with pathogen growth and survival on tropical fruits is the common thread. Fresh cut comm odities offer no barrier between pathogens and consumers. There is no kill step to eliminate pathogen contamination. Preventive and safety measures are needed during the entire process from harvest to consumer storage to ensure a safe product. More research on the above menti oned areas will aid in the formulation and implementation of preventive and safety measures thus creating a wide variety of fresh cut tropical fruits for consumers to enjoy in their diets.

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Hedberg, C. W., Angulo, F. J., White, K. E., La ngkop, C. W., Schell, W. L., Stobierski, M. G., Schuchat, A., Besser, J. M., Dietrich, S., He lsel, L., Griffin, P. M., McFarland, J. W., Osterholm, M. T., 1999. Outbreaks of salm onellosis associated with eating uncooked tomatoes: implications for public hea lth. Epidemiol. Infect. 122, 385-389. Huang, H-Y., Chang, C-K., Tso, T. K., Huang, JJ., Chang, W-W., Tsai, Y-C., 2004. Antioxidant activities of various fruits and vegetables produ ced in Taiwan. Intl. J. Food Sci. Nutr. 55, 423-429. Huang, S., Huang, K., 2007. Increased U.S. imports of fresh fruit and ve getables. Washington, D.C.: U.S. Dept. Agric. Available at: http://www.ers.usda.gov/Public ations/fts/2007/08Aug/fts32801/ Accessed 20 June, 2009. Huhtanen, C. N., 1975. Use of pH gradient pl ates for increasing the acid tolerance of Salmonellae. J. Applied Microbiol. 29, 309-312. Hutin, Y. J., Pool, V., Cramer, E. H., Nainan, O. V., Weth, J., Williams, I. T., Goldstein, S. T., Gensheimer, K. F., Bell, B. P., Shapiro, C. N., Alter, M. J., Margolis, H. S., 1999. A multistate foodborne outbreak of hepatitis A. National Hepatitis A Investigation Team. N. Engl. J. Med. 340, 595-602. Hoge, C. W., Bodhidatta, L., T ungtaem, C., Echeverria, P., 1995. Emergence of nalidixic acid resistant Shigella dysenteriae type 1 in Thailand: An outbreak associated with consumption of coconut milk desse rt. Intl. J. Epidemiol. 24, 1228-1232. Hsin-Yi, C., Chou, C., 2001. Acid adaption a nd temperature effect on the survival of E. coli O157:H7 in acidic fruit juice and lactic fermen ted milk product. Intl. J. Food Microbiol. 70, 189-195. Iturriaga, M. H., Arvizu-Medrano, S. M., Escartin, E. F., 2002. Behavior of Listeria monocytogenes in avocado pulp and processed guacamole. J. Food Prot. 65, 1745-1749. Jimenez-Escrig, A., Rincon, M., Pulido, R., Sa ura-Calixto, F., 2001. Guava fruit (Psidium guajava L.) as a new source of antioxidant dietary fiber. J. Agric. Food Chem. 49, 54895493. Kader, A. A., 2002. The importance of fruit and nut s in human nutrition and health. In: Knee, M. (Ed.) Fruit quality and its biological basis. Press LLC, Florida, pp. 7-8. Katz, D. J., Cruz, M. A., Trepka, M. J., Suarez, J. A., Fiorella, P. D., Hammond, R. M., 2002. An Outbreak of typhoid fever in Florida associated with an imported frozen fruit. J. Infect. Dis. 186, 234-239. Knudsen, D. M., Yamamoto, S. A., Harris, L. J., 2001. Survival of Salmonella spp. and Escherichia coli O157:H7 on Fresh and Frozen Strawberries. J. Food Prot. 64, 14831488. 127

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133 BIOGRAPHICAL SKETCH Laura Kathryn Strawn was born in Long Beac h, California to John and Kathy Strawn. She has two younger brothers Matt and Zach St rawn. Laura attended the University of California, Davis from 2003-2007 where she graduate d with a B.S. degree in food science with an emphasis in microbiology. Following her gra duation she started her M.S. degree under the instruction of Dr. Michelle Danyluk. At the University of Florida, Laura studied food microbiology and safety. Future plans include starting her PhD program in the fall (2009) at Cornell University, traveling to Fiji and pursuing a career in academia or government.