Fate of Escherichia Coli O157

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Title:
Fate of Escherichia Coli O157 H7 and Salmonella Spp. on Whole Strawberries of Two Maturities under Different Storage Conditions
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1 online resource (108 p.)
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english
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Nguyen, Thao
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University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Danyluk, Michelle D.
Committee Members:
Sargent, Steven A
Schneider, Keith R
Welt, Bruce A

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Subjects / Keywords:
atmosphere -- bruised -- coli -- escherichia -- maturity -- modified -- salmonella -- strawberries
Food Science and Human Nutrition -- Dissertations, Academic -- UF
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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

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Abstract:
Strawberries are harvested at or near full ripe maturity for superior eating quality. These fruit tend to be less firm and more susceptible to bruising during harvest and transport, which may increase risks of foodborne pathogen proliferation. The objective of this research was to determine the fate of Escherichia coli O157:H7 and Salmonella spp. on bruised and intact surfaces of whole strawberries at shipping (2°C) and retail display (15.5°C) temperatures under different storage conditions.  Strawberries used in these experiments were either purchased from a supermarket or freshly harvested.  Store bought and freshly harvested strawberries (full and ¾ red maturity) were spot inoculated with either 6 or 7 log CFU/ml of inoculum.  Strawberries were transferred to whirl-pak bags and incubated at 2 ± 2°C and 15.5 ± 2°C.  Strawberries subjected to modified atmospheres were further transferred to campy bags and sealed in with an initial atmosphere of ca. 10% CO2 and 5% O2.  Strawberries stored at 2 ± 2°C and 15.5 ± 2°C were sampled at 0, 2, 5, and 24 h and on day 0, 1, 3, and 7, respectively.  After stomaching, samples were enumerated on nonselective and selective media, and populations were recorded as log CFU/berry.  Enrichments were conducted when populations fell below detection limits.  Over 24 h at 2 ± 2°C, E. coli O157:H7 populations declined by ca. 0.8-1.3 log CFU/berry; Salmonella populations declined by 0.9-1.2 log CFU/berry, except the ¾ red bruised samples - populations remained stable.  Over 7 days at 15.5 ± 2°C, E. coli O157:H7 and Salmonella populations declined by ca. 1.1-1.8 log and 1.4-2.4 log CFU/berry, respectively.  No significant differences, under all conditions, were observed in E. coli O157:H7 or Salmonella population declines between store bought or freshly harvested strawberries; freshly harvested ¾ and full red strawberries, except on day 3 at 15.5°C – Salmonella populations on ¾ (intact) strawberries were ca. 1.5 log less than on full red (bruised) strawberries; and strawberries stored under ambient and modified atmosphere conditions.  This research indicates that E. coli O157:H7 and Salmonella spp. do not grow on the surface of bruised or intact strawberries at both temperatures.
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In the series University of Florida Digital Collections.
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Includes vita.
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Description based on online resource; title from PDF title page.
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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 Thao Nguyen.
Thesis:
Thesis (M.S.)--University of Florida, 2012.
Local:
Adviser: Danyluk, Michelle D.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-08-31

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UFE0044767:00001


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1 FATE OF ESCHERICHIA COLI O157:H7 AND SALMONELLA SPP. ON WHOLE STRAWBERR IES OF TWO MATURITIES UNDER DIFFERENT STORAGE CONDITIONS By THAO PHUONG NGUYEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Thao Phuong Nguyen

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3 To my Mom and Dad, who may not have always agreed with my decisions but have always supported me through every s tep in this journey. To Travis, who has always given me the courage and inspiration to achieve this milestone.

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4 ACKNOWLEDGMENTS I would like to express my sincere appreciation and gratitude to my advisor and mentor, Dr. Michelle Danyluk. I am grate ful to Dr. Danyluk for believing in my abilities and giving me the amazing opportunity to be a research graduate assistant. Dr. Danyluk ceases to amaze me with her depth and wealth of knowledge as well as her dedication and enthusiasm for education and re search. I am thankful for the open door policy that she has always had, and she has provided with so much guidance and has molded me into the researcher I am today. I could not imagine going through this academic journey with anyone else. I would like to thank my supervisory committee members, Dr. Steve Sargent, Dr. Keith Schneider, and Dr. Bruce Welt. I would like to thank Dr. Sargent for opening up my eyes to agriculture in Florida the tour was one of the most fulfilling experiences in my academic ca such a fun topic I still remember your presentation from Product Development on how tiny microorganisms are compared to a nail! I would like to thank Dr. Welt for teaching me the fundamentals of packaging science and his willingness to provide me with the equipment needed to finish my research. I thank all of you for your time, as well as your insights on how to approach my research. Lastly, I am extremely grateful to everyone in the lab for their patience in assisting me in every imaginable way possible with my research: Lorrie Friedrich, Angela Valadez, Rachel McEgan, Pardeepinder Braur, Zeynal Toplacengiz, Gwen Lundy, Luis Martinez, and Brian Buzzie. I owe you guys the world f or your moral support and expertise!

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 2 LI TERATURE REVIEW ................................ ................................ .......................... 18 Strawberry ................................ ................................ ................................ .............. 18 Origin ................................ ................................ ................................ ................ 18 Nutritional Quality ................................ ................................ ............................. 18 Production ................................ ................................ ................................ ........ 19 Major geographical areas of the world ................................ ....................... 19 United States ................................ ................................ ............................. 20 US strawberry varieties ................................ ................................ .............. 21 Field and Postharvest Handling ................................ ................................ ........ 23 Harvesting ................................ ................................ ................................ .. 23 Cooling and temperature management ................................ ...................... 24 Shipping ................................ ................................ ................................ ..... 25 Controlled and modified atmosphere ................................ ......................... 26 Microbial safety ................................ ................................ .......................... 27 Fresh Produce ................................ ................................ ................................ ........ 28 Pathogenic Microorganisms of Concern ................................ ........................... 28 Sources of Contamination ................................ ................................ ................ 29 Field and Production ................................ ................................ ........................ 29 Postharvest Operations ................................ ................................ .................... 30 Prevention ................................ ................................ ................................ ........ 31 Outbreaks Associated with Strawberries ................................ .......................... 33 Prevalence of Pathogens on Strawberries ................................ ....................... 33 Escherichia coli O157:H7 ................................ ................................ ................. 34 Salmonella spp. ................................ ................................ ................................ 36 Listeria monocytogenes ................................ ................................ .................... 36 Pathogenic Escherichia coli ................................ ................................ .................... 38 Enterohaemorrhagic Escherichia coli ................................ ............................... 39 Physiology ................................ ................................ ................................ ........ 40 Cultural Isolation ................................ ................................ ............................... 40 Significance in Produce Outbreaks ................................ ................................ .. 42 Salmonella spp ................................ ................................ ................................ ...... 43

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6 Physiology ................................ ................................ ................................ ........ 45 Cultural Isolation ................................ ................................ ............................... 46 Significance in Produce Outbreaks ................................ ................................ .. 47 Efficacy of Decontaminants to Red uce E. coli O157:H7 and Salmonella ............... 48 Aqueous Solutions ................................ ................................ ........................... 48 Gaseous Agents ................................ ................................ ............................... 50 3 MATERIALS AND METHODS ................................ ................................ ................ 52 Strawberries ................................ ................................ ................................ ............ 52 Strawberry Bruising ................................ ................................ ................................ 52 Modified Atmosphere Packaging ................................ ................................ ............ 53 Bacterial Strains ................................ ................................ ................................ ...... 54 Inoculum Preparation ................................ ................................ .............................. 54 Strawberry Inoculation ................................ ................................ ............................ 55 Storage Conditions ................................ ................................ ................................ 56 Enumeration of Pathogens ................................ ................................ ..................... 56 Enrichment ................................ ................................ ................................ .............. 57 Escherichia coli O157:H7 ................................ ................................ ................. 57 Salmonella spp ................................ ................................ ................................ 57 Normalizing Data and Statistics ................................ ................................ .............. 58 4 RESULTS ................................ ................................ ................................ ............... 60 Pathogen Enumeration ................................ ................................ ........................... 60 Composition of Modified Atmosphere ................................ ................................ ..... 60 Fate of E. coli O157:H7 on Store Bought Strawberries ................................ ........... 61 Fate of E. coli O157:H7 on Freshly Harvested Strawberries ................................ ... 62 Fate of E. coli O157:H7 on Strawberries Stored under Modified Atmosphere ........ 63 Comparison of E. coli O157:H7 Behavior on Strawberries with Various Treatments ................................ ................................ ................................ .......... 64 Fate of Salmonella on Store Bought Strawberries ................................ .................. 65 Fate of Salmonella on Freshly Harvested Strawberries ................................ .......... 65 Fate of Salmonella on Strawberries Stored under Modified Atmosphere ............... 67 Comparison of Salmonella Behavior on Strawberries with Various Treatments ..... 67 Comparisons between E. coli O157:H7 and Salmonella Behavior on Strawberries ................................ ................................ ................................ ........ 68 5 DISCUSSION ................................ ................................ ................................ ......... 82 6 CONCLUSIONS AND FUTURE WORK ................................ ................................ 93 LIST OF REFERENCES ................................ ................................ ............................... 96 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 108

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7 LIST OF TABLES Table page 4 1 Change in carbon dioxide (CO 2 ) and o xygen (O 2 ) composition in the headspace of campy bags following incubation at 2 2C for up to 24 h. .......... 69 4 2 Change in carbon dioxide (CO 2 ) and oxygen (O 2 ) composition in the headspace of camp y bags following incubation at 15.5 2C for up to 7 days. ................................ ................................ ................................ ................... 69 4 3 Fate of E. coli O157:H7 populations on bruised and intact surfaces of store bought strawberries enumerated on TSAR and SMACR following incubation at 2 2C for up to 24 h. ................................ ................................ .................... 69 4 4 Fate of E. coli O157:H7 populations on bruised and intact surfaces of store bought strawberries enumerated on TSAR and SMACR following in cubation at 15.5 2C for up to 7 days. ................................ ................................ ............ 70 4 5 Fate of E. coli O157:H7 populations, inoculated with ca. 10 7 CFU/ml, on bruised and intact surfaces of freshly harvested full and red strawber ries enumerated on TSAR and SMACR following incubation at 2 2C ................... 70 4 6 Fate of E. coli O157:H7 populations, inoculated with ca. 10 7 CFU/ml, on bruised and intact surfaces of freshly harveste d full and red strawberries enumerated on TSAR and SMACR following incubation at 15.5 2C. ............. 71 4 7 Fate of E. coli O157:H7 populations, inoculated with ca. 10 7 CFU/ml, on bruised and intact surfaces of store bought strawberries stored under modified atmosphere storage (ca. 10% CO 2 5% O 2 ) at 2 2C. ....................... 71 4 8 Fate of E. coli O157:H7 populations, inoculated with ca. 10 7 CFU/ml, on bruised and intact surfaces of store bought strawberries stored under modified atmosphere storage (ca. 10% CO 2 5% O 2 ) at 15.5 2C. .................. 72 4 9 Fate of Salmonella spp. populations on bruised and intact surfaces of store bought strawberries enumerated on TSAR and BSAR following incubation at 2 2C for up to 24 h. ................................ ................................ ........................ 72 4 10 Fate of Salmonella spp. populations on bruised and intact surfa ces of store bought strawberries enumerated on TSAR and BSAR following incubation at 15.5 2C for up to 7 days. ................................ ................................ ................ 73 4 11 Fate of Salmonella spp. populations, inoculated with ca. 10 7 CFU/ml, o n bruised and intact surfaces of freshly harvested full and red strawberries enumerated on TSAR and BSAR. ................................ ................................ ...... 73

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8 4 12 Fate of Salmonella spp. populations, inoculated with ca. 10 7 CFU/ml, on brui sed and intact surfaces of freshly harvested full and red strawberries enumerated on TSAR and BSAR following incubation. ................................ ...... 74 4 13 Fate of Salmonella spp. populations, inoculated with ca. 10 7 CFU/ml, on bruised and intact surfaces of store bought strawberries stored under modified atmosphere storage (ca. 10% CO 2 5% O 2 ) at 2 2C. ....................... 74 4 14 Fate of Salmonella spp. populations, inocu lated with ca. 10 7 CFU/ml, on bruised and intact surfaces of store bought strawberries stored under modified atmosphere storage (ca. 10% CO 2 5% O 2 ) at 15.5 2C ................... 75

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9 LIST OF FIGURES Figure page 4 1 Behavior of E. coli O157:H7 on bruised (closed symbols) or intact (open symbols) surfaces of store bought (square symbols) or freshly harvested (triangle symbols) strawberries over 24 h of storage at 2 2C ......................... 76 4 2 Behavior of E. coli O157:H7 on bruised (closed symbols) or intact (open symbols) surfaces of store bought (square symbols) or freshly harvested (triangle symbols) strawberries over 7 d ays of storage at 15.5 2C ................. 76 4 3 Behavior of E. coli O157:H7 on bruised (closed symbols) or intact (open symbols) surfaces of freshly harvested full red (square symbols) or red (triangle symbols) strawberries over 24 h of storage at 2 2C. ........................ 77 4 4 Behavior of E. coli O157:H7 on bruised (closed symbols) or intact (open symbols) surfaces of freshly harvested full red (square sy mbols) or red (triangle symbols) strawberries over 7 days of storage at 15.5 2C. ................ 77 4 5 Behavior of E. coli O157:H7 on bruised (closed symbols) or intact (open symbols) surfaces of strawb erries stored under regular air storage (square symbols) or modified atmosphere storage (triangle symbols) over 24 h. ............ 78 4 6 Behavior of E. coli O157:H7 on bruised (closed symbols) or intact ( open symbols) surfaces of strawberries stored under regular air storage (square symbols) or modified atmosphere storage (triangle symbols) over 7 days. ........ 78 4 7 Behavior of Salmonella spp. on br uised (closed symbols) or intact (open symbols) surfaces of store bought (square symbols) or freshly harvested (triangle symbols) strawberries over 24 h of storage at 2 2C. ........................ 79 4 8 Behavior of Salmonella spp. on bruised (closed symbols) or intact (open symbols) surfaces of store bought (square symbols) or freshly harvested (triangle symbols) strawberries over 7 days ................................ ....................... 79 4 9 Behavi or of Salmonella spp. on bruised (closed symbols) or intact (open symbols) surfaces of freshly harvested full red (square symbols) or red (triangle symbols) strawberries over 24 h. ................................ .......................... 80 4 10 B ehavior of Salmonella spp. on bruised (closed symbols) or intact (open symbols) surfaces of freshly harvested full red (square symbols) or red (triangle symbols) strawberries over 7 days ................................ ....................... 80 4 11 Behavior of Salmonella spp. on bruised (closed symbols) or intact (open symbols) surfaces of strawberries stored under regular air storage (square symbols) or modified atmosphere storage (triangle symbols) ............................ 81

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10 4 12 Behavior of Salmonella spp. on bruised (closed symbols) or intact (open symbols) surfaces of strawberries stored under regular air storage (square symbols) or modified atmosphere storage (triangle symbols). ............................ 81

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science FATE OF ESCHERICHIA COLI O157:H7 AND SALMONELLA SPP. ON WHOLE ST RAWBERRIES OF TWO MATURITIES UNDER DIFFERENT STORAGE CONDITIONS By Thao Phuong Nguyen August 2012 Chair: Michelle Danyluk Major: Food Science and Human Nutrition Strawberries are harvested at or near full ripe maturity for superior eating quality. These fruit tend to be less firm and more susceptible to bruising during harvest and transport, which may increase risks of foodborne pathogen proliferation. The objective of this research was to determine the fate of Escherichia coli O157:H7 and Salmonella spp on bruised and intact surfaces of whole strawberries at shipping (2C) and retail display (15.5C) temperatures under different storage conditions. Strawberries used in these experiments were either purchased from a supermarket or freshly harvested. St ore bought and freshly harvested strawberries ( full and red maturity) were spot inoculated with either 6 or 7 log CFU/ml of inoculum. Strawberries were transferred to whirl pak bags and incubated at 2 2C and 15.5 2C. Strawberries subjected to mod ified atmospheres were further transferred to campy bags and sealed in with an initial atmosphere of ca. 10% CO 2 and 5% O 2 Strawberries stored at 2 2C and 15.5 2C were sampled at 0, 2, 5, and 24 h and on day 0, 1, 3, and 7, respectively. After sto maching, samples were enumerated on nonselective and selective media, and populations were recorded as log CFU/berry. Enrichments were conducted when

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12 populations fell below detection limits. Over 24 h at 2 2C, E. coli O157:H7 populations declined by c a. 0.8 1.3 log CFU/berry; Salmonella populations declined by 0.9 1.2 log CFU/berry, except the red bruised samples populations remained stable. Over 7 days at 15.5 2C, E. coli O157:H7 and Salmonella populations declined by ca. 1.1 1.8 log and 1.4 2 .4 log CFU/berry, respectively. No significant differences, under all conditions, were observed in E. coli O157:H7 or Salmonella population declines between store bought or freshly harvested strawberries; freshly harvested and full red strawberries, exc ept on day 3 at 15.5C Salmonella populations on (intact) strawberries were ca. 1.5 log less than on full red (bruised) strawberries; and strawberries stored under ambient and modified atmosphere conditions. This research indicates that E. coli O157:H 7 and Salmonella spp. do not grow on the surface of bruised or intact strawberries at both temperatures.

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13 CHAPTER 1 INTRODUCTION Fruit and vegetable consumption is a growing trend in the US evidenced by the increase in produce production in comparison to the last 4 decade s (USDA, 2011b) The implications of overall health benefits and its role in reducing the incidence of c ancer have all been driving forces behind this increase in consumption (NAS, 1982) Along with increased health benefits, demands for fresh fruits and vegetables have also changed over the decades as more fruits become available year round due to improved agricultural practices and improved transportation of imports. Due to the increased availability of imported produce, larger arrays of various fresh fruits and vegetables have also enticed American consumers to eat more fresh produce (USDA, 2012b) Strawberr y is a major global crop with billions in tons of production worldwide (USDA, 2010) The US leads the world in strawberry production with production values estimated to be $2.4 billion in 2011 (USDA, 2010) California is the largest producer of strawberries in the US, capturing 89% of th e US strawberry market in 2009. Florida follows California in strawberry production, however has notably less of the market share, with only 8.5% of the US strawberry production (USDA, 2010) Strawberry consumption in the US has grown favorably over the last two decades and is one of the top five most consumed fruits. In 2009, 3.9 kg of strawberries were consumed per capita, with over 80% consumed as fresh fruit (USDA, 2011a) The year round supply of strawberries has contributed immensely to this growing popularity for fresh straw berries, with Florida being the major producer for strawberries grown in the winter (USDA, 2011a) The production of strawberries sold as a fresh fruit accounted for 81%

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14 of the total strawberries produced in 2010; notably, virtually produc tion is destined for the fresh market (USDA, 2011a) As the popularity of fresh fruits and vegetables grow s the concerns for foodborne illness due to consumption of these ready to eat commodities need to be further investigated. According to the CDC (2011c) there are approximately 1,000 reported dis ease outbreaks, an estimated 48 million illnesses, 128,000 hospitalizations, and 3,000 deaths caused by consumption of contaminated food annually. Scallan et al. (2011) estimates that 59% (5.5 million) of foodborne illnesses are caused by viruses, 39% (3.6 million) caused by bacteria, and 2% (0.2 million) caused by parasites. Additionally, norovirus, nontyphoidal Salmonella C perfringens and Campylobacter spp. are the primary causative pathogens in most foodborne illnesses, in that order, respectively (Scallan et al., 2011) Norovirus and Salmonella are the primary pathogens in outbreaks and illnesses caused by both fruits and vegetables (DeWaal, 2007) These overwhelming numbers have prompted a great deal of research to further investigate foodborne pathogen be havior on various produce commodities such as lettuce, carrots, jalapenos, strawberries, zucchini, mangoes, papayas, and pineapples (Abdulraouf et al., 1993; Castro Rosas et al., 2011; Castro Rosas et al., 2010; Fles sa et al., 2005; Knudsen et al., 2001; Strawn and Danyluk, 2010a; Strawn and Danyluk, 2010b) Possible sources of contamination exist at all steps during production and harvesting to process and handling. Preharvest contamination sources include soil, ir rigation water, inadequate composted manure, air, wild and domestic animals, human handling, and water used for pesticides or growth hormones applications (FDA, 2011a) Postharvest contamination sources include human handling (workers and consumers),

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15 wash and cooling water, harvesting equipment, transport containers, improperly cl eaned transport vehicles, unclean storage facilities, and cross contamination (FDA, 2011a) Preharvest contamination can be highly detrimental to society as crops will likely cross contaminate through the whole postharvest handling chain, increasing risks of foodborne illness to consumers (Gorny, 2006) Risk assessments for specific commodities need to analyzed and appropriate actions must be enforced to ensure risks for pre and post harvest contamination are minimized. For small fruits, the CDC (2011a) reported several confirmed outbreaks implicating raspberries and blackberries contaminated with Cyclospora cayetanensis which, in total, caused 109 illnesses and resulted in three ho spitalizations. In 1999, there was a confirmed Cyclospora outbreak in Florida implicating raspberries, blackberries, and strawberries, which caused 94 illnesses and resulted in one hospitalization (CDC, 2011a) Another three outbreaks (Texas, 1998; Massachusetts, 2000; Florida, 2007) involving 40 illnesses and two hospitalizations due to a viral infection resulting in Hepatitis A were linked to strawberries (CDC, 2011a) Strawberries, again, were implicated in another three outbreaks involving norovirus, which resulted in 113 illnesses (CDC, 2011a) In 2003, there was an outbreak in California caused by strawberries infected with Salmonella which caused 13 illnesses and two hospitalizations (CDC, 2011a) More recently, a reported outbreak in Oregon implicating strawberries contaminated with E. coli O157:H7 caused 14 illnesses and one death (FDA, 2011b) With novel packaging technologies on the rise, applications using modified atmosphere packaging, in addition to proper post harvest management and techniques,

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16 are being utilized to prolong shelf life of fresh produce commodities (Ozkaya et al., 2009) To prolong or enhance the qu ality of fruits and vegetables, atmosphere surrounding the commodity is often adjusted to specific concentrations of selected gases such as oxygen, carbon dioxide, carbon monoxide, ethylene, ozone, etc. (Adaskaveg et al., 2002) Though extension of shelf life is desirable, favorable conditions for bacterial growth may arise and become a problem. While quality attributes c an be heightened and extended with the right balance of carbon dioxide, oxygen, and nitrogen, the extended shelf life may allow bacteria to proliferate to numbers of which can cause illness. Most importantly, the food product can still appear edible. Res earch pertaining to this idea is very limited, though this is a valid concern. If modified atmosphere can be successfully applied to extend the shelf life of the produce commodity, this concern should be further investigated. Strawberries are non clima cteric fruits, therefore no sharp increase in ripening is observed after the strawberries are harvested from the parent plant (Kader, 2002) Organoleptic qualities such as flavor, sweetness, and acidity are important to the f ully ripen on the parent plant (Moing et al., 2001) However, strawberry firmness decreases as ripening progresses, which make these s pongy fruit highly susceptible to bruising during harvest and transport. Additionally, strawberries have different responses to bruising at low and high temperatures. Ferreira et al. (2009) inflicted two types of forces onto strawberries, including impact and compression. Interestingly, strawberries inflicted with compression forces were more resistant to bruising at low temperatures, and the opposite occurred w hen strawberries were inflicted with impact

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17 forces, where higher temperatures made strawberries more resistant to bruising. Bruising is highly likely to occur during harvesting and handling, regardless of temperature, and risks involving pathogen prolifer ation on bruised strawberries need to be further investigated. This research focuses on the behavior of two well known foodborne pathogens, E. coli O157:H7 and Salmonella on bruised and intact strawberries (1) purchased from retail supermarkets; (2) fres hly harvested and of 2 maturities (full red and red color); and (3) stored under modified atmospheric conditions. The objectives were further classified and include E. coli O157:H7 and Salmonella population comparisons between: (1) full red, sto re bough t and freshly harvested strawberries; (2) full red and red, freshly harvested strawberries; and (3) full red strawberries stored under ambient and modified atmosphere conditions. All of these objectives were evaluated under storage at 2 2C (simulatio n of shipping temperature) and 15.5 2C (simulation of retail display temperature ). Ultimately, this research can help agriculturalists make decisions on whether there are any increased risks of pathogen proliferation if strawberries are picked at a ful ly ripened stage, with or without the occurrence of bruising. This research can also provide insights on whether modified atmosphere packaging can heighten risks associated with pathogen behavior.

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18 CHAPTER 2 LITERATURE REVIEW Strawberry Origin Strawberri es are perennials belonging to the family Rosaceae and are members of the genus Fragaria (Mitcham, 2012) Th ey have been documented in literature since 23 79 AD, though cultivation of these wild berries did not begin until the early 1300s (Darrow, 1966) In the 1300s, Europeans began to plant strawberries for ornamental purposes rather than consumption. In the 1700s, strawberries were planted in North America and i ts earliest varieties were introduced however, it was not until the 1800s that strawberries were commercially cultivated in the US (Darrow, 1966) S trawberry cultivation began to expand in the US during the 1860s, due to a break through new variety of strawberry (Darrow, 1966) These newly transformed larger strawberries, named the Wilson varieties, began to replac e the early Virginian varieties, revolutionizing and expanding the strawberry industry in the US to more than 100,000 acres of production and allowing strawberries to bec o me a major crop of the Americas (Darrow, 1966) Nutritional Quality Strawberries are highly sought after due to their vibrant colors, fresh t aste, and excellent nutritional quality. The a mainly due to increased amounts of anthocyanin during development (Nunes et al., 2006) one of the many compoun ds in strawberries that contributes to its antioxidant quality (Lopez et al., 2010) The two predominant anthocyanins in strawberries are pelargonidin 3 gluco side (80%) and cyanidin 3 glucoside (Bakker et al., 1994) The

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19 flavor profile of a strawberry var ies depending on the balance of sugars and acids at time of consumption (Shaw, 1990) and the ratio of the two can differ depending on strawberry v ariety, maturity of harvest, storage temperatures as well as other postharvest conditions (Montero et al., 1996; Nunes et al., 2006) Sucrose, glucose, and fructose comprise over 99% of the total sugars in fully r ipened strawberries (Makinen and Soderling, 1980; Sturm et al., 2003; Wrolstad and Shallenberger, 1981) T race amounts of sorbitol, xylitol, and xylose can also be found in full red strawberries (Makinen et al., 1980) As the strawberry ripens the percentage of sucrose typically declines while the amount of glucose and fructose increases almost proportionally (Montero et al., 1996) The principal organic acids in strawberries are citric and malic acid, where citric acid accounts for approximately 90% of the total acids (Montero et al., 1996; Sturm et al., 2003) Ascorbic acid is also found in strawberries and increase s during strawberry development (Montero et al., 1996) Ascorbic acid contributes to the antioxidant cap acity of strawberries and has beneficial effects on health. Alt hough these are the main constituents of strawberries, many other nutritionally sound compounds, such as flavonols and other phenolics (Olsson et al., 2004) as well as small amounts of additional vitamins and minerals (USDA, 2012a) can also be fo und in strawberries Production Major geographical areas of the world With 18,415,541 tons of strawberry production from 1990 2010, the US is the world leading producer of strawberries (FAO, 2012; USDA, 2010) Following the US with 5,879,313 and 4,198,500 tons of production in th e same time frame are Spain and Japan, respectively (FAO, 2012; USDA, 2010) Poland, Italy, Republic of Korea,

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20 Russian Federation, Mexico, Turkey, and Germany were also among st the top ten strawberry producers in the world between 1990 and 2010 (FAO, 2012; USDA, 2010) In addition to being the leading strawberry producer in the world, the US is the second largest exporter of strawberries w ith 1,199,872 tons of export from 1990 2007 (USDA, 2010) Spain led the world export industry from 1990 2007 with 3,242,251 tons export ed Although Italy was a major exporter from 1990 2007, with 729,385 tons of exports (USDA, 2010) strawberry production began to steadily decline from 1 995 2004. By 2004, strawberry production in Italy decreased by approximately 74% (USDA, 2010) due to high labor costs and international competition (USDA, 2012c) Mexico was the third largest expo rter of strawberries in 2003, 2005, 2006, and 2007, with increases in export of 23% and 36% from 2003 to 2005 and 2005 to 2006, respectively (USDA, 2010) Mexico exports roughly 98% of its fresh strawberr ies to the US, making the US its largest export market (USDA, 2012c) United States California leads the US in strawberry production, producing about 89% of the domestic crop in 2009 (USDA, 2010) The harvested acreage was approximately 39,800, produc ing roughly 2. 5 billion pounds of strawberries (USDA, 2010) strawberry production regions are located mainly along the central and southern coast and can be categorized into five different regions: (1) Central Coast Region, Watsonville, Salinas, Gilroy, Aromas, and adjace nt areas; (2) Santa Maria Region, coastal regions of Santa Cruz, Santa Clara, Monterey, San Luis Obispo, and northern Santa Barbara counties; (3) Oxnard Plain Region, Oxnard and Ventura County; (4) South Coast Region, Ventura, Orange, Los Angeles, San Dieg o, and western Riverside counties; and (5) Interior Valley Regions, interior valleys such as central San Joaquin

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21 Valley. Though there are five regions of strawberry production in California, the Santa Maria region covers nearly two thirds of the productio n acreage (UCDavis, 1999) Florida is the second largest strawberry producer in the US and has tremendously acreage was 8,800, which yielded nearly 238 million pounds of stra wberries and accounted for approximately 8.5% of the total US production that year (USDA, 2010) production can also be found in near by counties including Pasco, Polk, and Manatee County. Strawberries are also produced in South Florida, specifically in Collier and Dade County (Bertelson, 1995) Oregon and North Carolina follow California and Florida in notable US strawberry production and are the third and fourth largest producers, respectively. Oregon and North Carolina produced 0.75% and 0.70% of the 2009 US strawberry yield (USDA, 2010) In 2009, Oregon had a harvested acreage of 1700 and produced about 21 million pounds of strawberries (USDA, 2010) In the same year, North Car olina had a harvested acreage of 1500 and produced 19.5 mil lion pounds of strawberries (USDA, 2010) US strawberry v arie ties There are numerous varieties of strawberries planted in the US, and most of these cultivars are developed from breeding programs in California and Florida (Chandler and Legard, 2012) Although they are bred to fit the needs of reg ion specific climates in the US, mainly the coastal California area and west central Florida area, these cultivars are adopted and planted all over the world (Chandler et al., 2012; Santos et al., 2007) The general goal of all breeding programs is to produce strawberries that are aesthetically

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22 pleasing, with prerequisites of fruits which are greater than 10g (average weight of approximately 20g), have bright red color firm texture, and strawberry flavor and that are free of d efects such as cracks or splits. Ease of harvest is also a factor that breeders consider (Chandler et al., 2012) Although these breeding programs essentially have these general goals, they start to branch off into different direction s when taking into consideration time of production as well as disease and pests related to the region of interest (Chandler et al., 2012) Because California and Florida are the leading producers of US strawberries, only varieties from these two states will be discussed. California strawberries are harvested almost year round, beginning in late winter (January) with strawberries from the southern region until late fall (November) with strawberries from the northern region (Kader, 2002) Peak harvesting for fresh market strawberries occurs i n April (Bertelson, 1995) Cultivars planted in California inc lude (1) Diamante (introduced in 1997); (2) Aromas (introduced in 1997); (3) Gaviota (introduced in 1997); (4) Camarosa (introduced in 1993); (5) Seascape (introduced in 1991); (5) Oso Grande (introduced in 1987); (7) Chandler (introduced in 1983); (8) Sel va (introduced in 1983); (9) Parker (introduced in 1983); and (10) Pajaro (introduced in 1979) (Chandler et al., 2012) New varieties patented by the University of California include: (1) Monterey; (2) Palomar; (3) Portola; and (4) San Andreas (UCDavis, 2012) Though there are many varietie s, most are grown and harvested in certain time periods and are usually region specific (Bertelson, 1995) Florida strawberry production occurs predominantly during the winter season, beginning in November and lasting through until April, with peak harvesting in March

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23 (Bertelson, 1995) Notably, virtually fresh markets (Boriss et al., 2006) Florida cultivars include: (1) Florida Radiance (introduced in 2008); (2) Florida Elyana (introduced in 2008); (3) Winter Dawn (introduced in 2005); (4) Rubygem (introduced in 2003); (5) Carmine (introduced in 2002); (5) Straw berry Festival (introduced in 2000); (6) Earl i brite (introduced in 2000); (7) Rosa Linda (introduced in 1996); (8) Sweet Charlie (introduced in 1992); (9) Florida Belle (introduced in 1975); (10) Dover (introduced in 1979); and (11) Florida Ninety (introdu ced in 1952) (Whitaker, 2 010) Field and Postharvest Handling Harvesting Strawberries are highly perishable fruit and must be properly handled during and after harvest to maintain consumer acceptability as well as ensuring an extended shelf life. Although postharvest management is a critical factor in maximizing quality and shelf life, attention and care taken while harvesting is also important. Factors to consider during harvest include: (1) maturity of fruit picked, which is typically measured by pickers observing the amount of red color on the surface of berries ; fruits must have at least red color to be picked, however, picking of overripe fruit should also be avoided; (2) amount of force used when picking the berry to ensure no physical injury is inflicted and no discolor ation takes place; berries should be loosely handled in the palm; and (3) strawberry selection or grading to make certain berries are free from defects, such as disease and injury. Berries are generally packed on the field directly into containers, either the commonly used 1 lb. thermoformed plastic clamshell containers or propylene mesh baskets (Mitcham and Mitchell, 2002)

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24 Cooling and temperature m anagement Because strawberries have a high respiration rate (12 18 mg CO 2 /kg h at 0C), it is critical to precool strawberries promptly to remove residual field heat to minimize early onset of deterioration. Strawberry respiration rates double or even qua druple with a 10 C increase in temperature from 0C (Talbot and Chau, 1991) It is recommended that strawberries are precooled to 0C as close to 1 h after h arvest as possible, and this temperature should be maintained throughout the postharvest handling chain (Talbot and Chau, 1991) In Florida it is suggested that industry follow the 7/8 cooling time to assess the rate of precooling. The 7/8 cooling time requires that 7/8 of the difference between the pulp temperature and cooling temperature be removed before berries can be stored in cold rooms (at 0C) or sh ipped off to its destination market (Talbot and Chau, 1991) It allows more control in maximizing cooling efficiency due to berries with lower pulp tempera tures not need ing as much precooling time as berries with higher pulp temperatures. Not only does this recommendation provide for better efficiency but also maintains quality by avoiding excessive dehydration of the berry (Talbot and Chau, 1991) Nunes et al. (1995) found that when strawberrie s were delayed fo r up to 8 h at 30C prior to cooling, water loss increased to 16.7% compared to 9.9% water loss when precooled immediately. This water loss reduced the quality of the strawberry appearance; s trawberries were shriveled with a dull color on the exterior. Furthermore, greater decreases in firmness, soluble solids content (SSC), ascorbic acid levels, and sugar levels were found in strawberries with delayed cooling (Nunes et al., 1995) Precooling of strawberries is typically done by forced air cooling which rapidly cool strawberries by moving cold air through the pallets. The key is that the cold air comes in contact with the st rawberries, facilitating faster cooling. To prevent extensive

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25 water loss, cool air should be humidified to 90% or greater (Ferreira et al., 2006) There are two types of forced air cooling: Forced air Tunnel and Cold Wall. Forced air Tunnel is the simpler and more cost effective design of the two, in which rows of pallets are placed on both sides of an exhaust fan. This creates a tunnel in between the rows and air is forced through this tunnel and ultimately through the vents of the pallets. The Cold Wall method differs in that air plenums are placed in front of exhaust fans and both air plenum and exhaust fans are permanent fixtures along that wal l. Pallets are placed in front of the plenum openings, which allow for even distribution of cold air. This method also allows one pallet or even just half of a pallet to be cooled at once, rather than having to have two rows of pallets (for the tunnel ef fect) to begin the cooling process (Talbot and Chau, 1991; Thompson et al., 2002) Shipping Strawberries destined for remote markets spend most of their shelf life in transport vehicles ( truck s or airplane s) It is critical that loading docks for trucks have refrigeration and sealed loading doors so strawberries stay cool dur ing loading. Pallets should be stacked towards the front and center of the truck to minimize transport damage and to safeguard strawberries from abused temperature emitted along the wall from outside heat. To reduce transport damage due to road vibration al and impact effects trailers should be equipped with air suspension. Dunnage blocks are placed along the wall of the truck and between pallets to stabilize the load and to ensure that strawberries have sufficient air flow to keep them cool. Temperatur e inside transport vehicle s should be kept near 1C, and, freezing point of strawberry must be considered (approximately 0.8C, depending on the levels of soluble solids). The same precautions and conditions apply to air freight, and care should be taken as to not

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26 leaving pallets out on tarmac, under direct sunlight, for prolonged amounts of time. Reflective covering should be placed over pallets, along with cold packs, to prevent warming while on tarmac (Mitcham et al., 2002) Controlled and modified a tmosphere Controlled and/or modified atmosphere to maintain optimal quality of strawberries during storage and transport have been extensively studied (Caner and Aday, 2009; Caner et al., 2008; Garcia et al., 1998; Holcroft and Kader, 1999; Nunes et al., 2002; Ozkaya et al., 2009; Sanz et al., 2000) To prolong or enhance the quality of fruits, atmosphere surround ing the commodity is often adjusted to specific concentrations of selected gases such as oxygen, carbon dioxide, carbon monoxide, ethylene, ozone, etc (Adaskaveg et al., 2002) When these atmospheres are closely monitored for specific concentrations of a selected mix of gases, it is called a controlled atmosphere (CA). Controlled atmospheres are typically used with bulk storage of commodities (Sandhya, 2010) Modified atmosphere storage is defined as when the initial gas composition surrounding the food product is changed to specific concentrations, however no monitoring of the atmosphere takes place therea fter (Zagory and Kader, 1988) If the film of the package is tailored to the respiration rate of the produce commodity, an equilibrium modified atmosphere (EMA) will be established soon thereafter, which will slow down respiration, thus deterioration rates (Sandhya, 2010) For strawberries, an atmosphere of 5 10% oxygen (O 2 ) and 12 20% carbon dioxide (CO 2 ) is beneficial (Adaskaveg et al., 2002) Generally, atmospheres with reduced levels of oxygen an d increased levels of carbon dioxide (up to a certain extent) can slow down respiration rates, thus increasing shelf life (Zagory and Kader, 1998) However, care must be taken so that oxygen levels do not get too low where anaerobic respiration will take

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27 place (can cause development of off flavo rs and aromas) and carbon dioxide levels do not get too high, which can have negative physiological effects on the commodity (Zagory et al., 1998) Strawberries are often shipped with a modified atmosphere of increased carbon dioxide levels (12 15%) to slow down respiration as well as decrease ri sks of decay (gray mold) spreading. This practice is highly recommended if berries have water collected on the surface after harvest, due to moist and foggy weather, where there is increased risk of mold development. In practice, pallets are covered with plastic bags and sealed off at the bottom of the pallet liner. A hole is pierced by a nozzle that first pulls a vacuum then injects carbon dioxide inside the plastic bag. The hole is then covered by tape so that carbon dioxide does not escape. This mech anism does prevent cold air from reaching the strawberries once the plastic bags are taped off, therefore, strawberries should be properly cooled to prevent condensation from forming on the plastic bag. Generally, treatment with modified atmosphere is don e right before loading strawberries onto the truck (Mitcham et al., 2002) Microbial s afety Extended shelf life of produce commodities due to modified at mosphere packaging has raised concerns of whether it presents increased risks of biological hazards. Since shelf life is extended, the produce may still appear edible, however, human pathogens may have the advantage to proliferate over the extended time p eriod and cause illness (Phillips, 1996) Very limited research has been conducted to determine pathogen behavior on fresh fruits and vege tables packaged in modified atmospheres. S trawberries packaged in an EMA (5% CO 2 3% O 2 balance N 2 ) and in a high oxygen atmosphere (HOA, 95% O 2 5% N 2 ) had an extension in shelf life from 3 to 5 days (for

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28 both systems); however, the extended shelf life of raspberries went from 3 to 5 days and 3 to 7 days in an EMA and HOA system, respectively, when stored at 7C (Siro et al., 2006) Escherichia coli and Listeria monocytogen es did not grow but c ould survive over the 5 days on strawberries. E scherichia coli, Salmonella and L. monocytogenes also did not grow, but survived through the extended shelf life from both modified atmosphere packaging systems on raspberries. In othe r studies, L. monocytogenes was able to survive on strawberries for up to 7 days at 4C (Flessa et al., 2005) ; both E. coli O157:H7 and Salmonella were able to surviv e on strawberries for up to 7 days at 5C (Knudsen et al., 2001) From the evidence suggested by (Siro et al., 2006) no increased risks were found when shelf life of strawberries was e xtended. Fresh Produce Typically, fresh fruits and vegetables are consumed in their natural state without a pose a risk of foodborne illness to consumers. Immense precautionary steps must be ta ken from field to fork to ensure these fresh products pose little to no foodborne illness risk to consumers. The responsibility of the field to fork concept belongs to each individual involved in production all the way through to consumption; this include s growers, processors, shippers, distributors, retailers, and consumers (Gorny, 2006) Pathogenic Microorganisms of Concern Pathogenic microorganisms of concern in fresh produce include Salmonella enterotoxig enic and enterohemorrhagic E. coli, Shigella spp. Listeria monocytogenes, Yersinia enterocolitica, Staphylococcus aureus Aeromonoas species spore forming pathogenic bacteria ( Bacilleus cereus Clostridium botulinum and Clostridium perfringens ), viruses (Hepatitis A and norovirus ), and protozoan parasites

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29 ( Cryptosporidium parvum Cyclospora cayetanensis and Giardia lamblia ) (FDA, 2011a) Norovirus and Salmonella are the primary pathogens in outbreaks and illnesses caused by both fruits and vegetables (DeWaal, 2007) Sources of Contamination Fruits and vegetables are commonly grown in open fields which are nonsterile environments. Growers have little control over environmental conditions and wildlife occurrences that may aid in facilitation of biological contamination. After harvesting, raw commodities are introduced to a variety of steps, such as culling, washing, waxing, cutting, and/or packaging, which can introduce new sources of contamination. To decrease risks associated with biological contamination, special attention should be directed towards pre and post harvest handling operations. Field and Production Great care must be taken during production, especially in the field, to ensure risks of contamination with human pathogens are greatly minimized. Consequences of preharvest contamination can be highly detrimental to society due to the increased possibility of cross contamination through the whole postharvest handling chain (Gorny, 2006) A variety of factors, including soil, feces, and irrigation water, can be source s of preharvest produc e contamination. In a two year microbiological survey of fresh produce grown on conventional, semiorganic, and organic farms, Mukherjee et al. (2006) found evidence of E. coli on leafy greens, lettuce, and cabbages. Out of 2029 samples, 8% had detectable levels of E. coli contamination. Leafy greens, lettuce, and cabbages are grown close to the ground, and a likely source of contam ination could have been soil. Ishii et al. (2006) found that E. coli could reach populations of 5 log CFU/g soil when climate conditions were above 30C and were able to survive over a

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30 month when climate conditions were above 25C. Factors that can influence growth and survival of E. coli in soil include soil temperature, moisture, n utrients, and occurrence of other microorganisms found in the soil (Ishii et al., 2010) Control of wild animals in open environments is almost an impossible task, thus animal feces could be a source of contamination. Kudva et al. (1998) found that E. coli O157:H7 was able to survive in ovine manure for up to 21 months under natural environmental conditions. Additionally, E. coli O157:H7 was able to survive for up to 49 and 56 days in bovine species at 37 C and 22C, respectively (Wang et al., 1996) E. coli was able to survive longer at the lower temperature because there was less water loss (Wang et al., 1996) As far as irrigation water is concerned, laboratory studies showed that recovery of up to 5 log CFU/g of E. coli O157:H7 on lettuce plants were attained when plants were irrigated with water contaminated with 4 log CFU/ml of E. coli O157:H7 (Solomon et al., 2003) Th ese studies indicate that human pathogens are able to survive long term in open, natural environments, and these agricultural environments need to be managed and controlled carefully to reduce risks of pathogen contamination. Postharvest Operations After h arvesting, there are many points of contamination, whether the commodity will be shipped off to destination markets or further processed inside processing plants. C ontamination can occur by ill human hygiene, dirty transport and/or final packaging contain ers, unclean processing equipment, contaminated wash water, unclean storage facilities, and improperly cleaned transport vehicles (FDA, 2011a) It is common practice that field workers, as well as processing plant workers, often wear gloves, hairnets, and smocks to prevent risks of cross contamination. Although this may be a good safety precaution, it is also important to train employees of good hygiene regimens

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31 and safe food handling practices (Gorny, 2006) Processing equipment, as well as transport containers, should be kept clean and free from organic material such as dirt, stems, leaves, etc. When possible, plastic containers should be used instead of wood, due to the mere aspect that plastic can be sanitized effectively whereas wood is porous, thus practically impossible to dis infect (Gorny, 2006) Wash water coming into direct contact with fresh produce must be treated with an appropriate, approved level of disinfectant so as to not contaminate the commodity as well as aid in disi nfecting the produce (Gorny, 2006) Cold storage rooms must be kept in top notch working conditions to prevent any possibility of condensation, from any equipment or the ceiling, dripping onto raw commodities It is important to note that refrigerated storage with inefficient cooling capacity can easily facilitate formation of condensation on walls, ceilings, and equipment as well as fog and mist, thus equipment needs to be checked and maintained regularly (Gorny, 2006) Transport vehicles should be cleaned and sanitized before every new load. Unless appropriate and adequate cleaning has taken place, trucks previously used to transport livestock, animal products, or toxic materials should not be used to transport fresh produce (Gorny, 2006) If all of these steps are taken, risks for postharvest cross contamination are greatly minimized. Prevention Most produce items are generally eaten without any further thermal treatment, thus, prevention of the produce commodity becoming contaminated at any point in the harvest and process handling chain is crucial. C ritical points which elevate potential risks for contamination include: in the field, initial point of processing, and during consumer preparation in the kitchen (Lynch et al., 20 09) The water that comes in contact with the produce item at any time should be free of biological contaminants.

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32 This includes, but is not limited to: (1) water used to apply pesticides, (2) irrigation water, and (3) postharvest precooling water (Lynch et al., 2009) Harvesting bins should be cleaned and sanitized daily to reduce risks of cross contamination (Yoon et al., 2010) When applicable, culling of damaged fruits should be implemented. Damaged fruits may be contaminated and can allow for better survival of microbes, thus spreading of contamination can occur if it is not removed from the group of healthy fruits (Keller, 200 6) Conveyor belts and packaging tables need to be cleaned and sanitized according to standard operating procedures to avoid cross contamination and formation of bacterial biofilms (Yoon et al., 2010) Management of proper cooling temperatures through the distribution chain is also important to ensure favorable climates and atmospheres do not arise, which can allow for proliferation and amplification of biological contaminants (Lynch et al., 2009) The FDA has generated guidance o Minimize Microbial Food Safety Hazards of Fresh cut Hazard Analysis Critical Control Point (HACCP) has not yet been mandated for fresh produce handling, implementation o f HACCP systems, along with prerequisite programs (ie. standard operating procedures [ SOPs ] good agricultural practices [ GAPs ] good manufacturing practices [ GMPs ] and sanitation standard operating procedures [ SSOPs ] ) can be a step in the right direction to minimize the risks associated with biological contamination of fresh produce (Hurst, 2006) Consumer awareness and education for safe handling practices of fresh produce should also be a focus.

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33 Outbreaks Associated with Strawberries Strawberries have been implicated as a vehicle in almost a dozen outbreaks and associat ed with over 300 illnesses and resulted in one death. In 1999, there was a confirmed Cyclospora outbreak in Florida implicating raspberries, blackberries, and strawberries, which caused 94 illnesses and resulted in one hospitalization (CDC, 2011a) Another three outbreaks (Texas, 1998; Massachusetts, 2000; Florida, 2007) involving 40 illnesses and two hospitalizations due to a viral infection resulting in Hepatitis A were also linked to strawberries (CDC, 2011a) From 1998 to 2007, strawberries were implicated as vehicles in four outbreaks involving norovirus, which resulted in ov er 150 illnesses (CDC, 2011a) In 2003, there was an outbreak in California caused by strawberries infected with Salmonella which caused 13 illnesses and two hospita lizations (CDC, 2011a) Many of these foodborne illnesses occurred from consumption of the berries in a home, school, or banquet setting (CDC, 2011a) which likely indicates berries were contaminated in a postharvest setting. However, in 2011, an outbreak implicating strawberries from a farm in Oregon prompted a recall for fresh st rawberries; these strawberries were contaminated preharvest with E. coli O157:H7, which resulted in 15 illnesses and one death (FDA, 2011b) Prevalence of Pathogens on Strawberries Though strawberries are not linked to a significant amount of outbreaks, its structural characteristic and growing conditions contribute to its r anking as a high risk commodity for biological contamination (FDA, 2001 ) Strawberries are grown in close proximity to the ground, which can be a potential source of contamination with human pathogens that may be present in the soil. Strawberries have nonsmooth, irregular surfaces, which may provide suitable hiding places for pathogens, making it more

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34 difficult to decontaminate, if contaminated, by just washing alone. A survey was conducted by the FDA in 1999 to determine the prevalence of E. coli O157:H7, Salmonella and Shigella on high risk, high volume imported produce items. Strawberries, among seven other produce items, were chosen for evaluation and contributed to 0.1% of the 4.4% positive for bacterial contamination. A sample of strawberries was found to be contaminated with Salmonella ; however, none were found to be contaminated with E. coli O157:H7, and Shigella was not tested for strawberries (FDA, 2001) Though strawberries represented a low percentage of samples contaminated, this survey elucidates that strawberries can provide a suitable environment for pathogens. Thus, research on the persistence of pathogens on strawberries is important to allow understanding of their behavior on the fruit and to assess the risks associated with contaminated strawberries. Escherichia coli O157:H7 Escherichia coli O157:H7 outbreaks, though first linked to ground beef, are becoming increasing ly linked to fresh produce (Kaper et al., 2004) The primary reservoir for E. coli O157:H7 is thought to be the intestinal tracts of cattle (Kaper et al., 2004) thus, manure and soil can be direct paths of contamination for strawberries. If contamination occurs, studies have shown th at E. coli O157:H7 can persist on strawberries. Knudsen et al. (2001) reported that populations of E. coli O157:H7 on cut strawberries stored at 5C showed no decline over 7 days, thus, indicating E. coli O15 7:H7 could survive on the open flesh of strawberries. Populations declined by approximately 2 log cycles when inoculated on the surface of intact whole berries over 7 days under refrigerated storage (5C), and lower counts on selective media indicated cel ls were injured. Yu et al. (2001) also reported significant differences between

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35 reductions of E. coli O157:H7 populations over 2 4 h at 23C when injected inside the fruit versus surface inoculation, with the latter manifesting in greater decreases. Even though the pH of strawberries is low, the ability of E. coli O157:H7 to survive on the open flesh rather than the surface probabl y has to do with the increased availability of the nutrients and water activity. Han and Linton (2004a) reported that E. coli O157:H7 was able to surv ive over 3 days at pH 3.6 in strawberry juice at 4C but not at 37C; significant cell injury had occurred during the first 2 h of incubation at 37C. Since the optimum growth temperature for E. coli O157:H7 is 37C, this seems contradicting. However, Co nner and Kotrola ( 1995) found that the pathogen did not grow or survive in solutions of citric or malic acid (primary acids in strawberries) at pH 4.0; therefore, the type of organic acid, coupled with other treatments such as temperature, can also be a factor in the inhib ition of E. coli O157:H7 in acidic environments. In the case of freezing temperatures ( 20C), Knudsen et al. (2001) reported similar behavior of E. coli O157:H7 to L. monocytogenes (Flessa et al., 2005) in which the presence of sucrose supported better survival. However, the same study also reported that the strain of E. coli O157:H7 used to inoculate the strawberry wa persistence during storage at 20C; one strain of E. coli O157:H7 exhibited a larger decline over 30 days without the protection of sucrose versus the other. Furthermore, cells frozen without sucrose resulted in m ore injury than cells frozen with sucrose. Strain differences were also found to be a factor of E. coli O157:H7 behavior at 23C, where one strain was found to show minor increases over 24 h inside and on the surface of strawberry fruit and the other decr eased under both conditions (Yu et al.,

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36 2001) At abusive storage temperatures for strawberries (24C), E. coli O157:H7 was able to s urvive over 2 days on cut and whole berries (Knudsen et al., 2001) Salmonella spp. Salmonella has been the pathogen of concern in many produce outbreaks, including papayas, cantaloupes, alfalfa sprouts, tom atoes, and jalapeno peppers (CDC, 2012) Strawberries have not been linked to any multistate foodborne outbreaks involving Salmonella and there is limited research on Salmonella behavior on strawberries. Knudsen et al. (2001) reported that Salmonella was able to survive over 48 h at 24C on the surface of whole and cut strawberries, with no significant declines in population and no occurrences of significant cell injury. However, at refrigerated temperatures (5C), there were reported significant differences between survival of Salmonella on the surface of whole and cut berries, where the latter exhibited smaller decreases over 7 days (Knudsen et al., 2001) These results are similar to the findings with the behavior of E. coli O157:H7 on the cut surface of strawberries (Knudsen et al., 2001; Yu et al., 2001) Listeria monocytogenes Listeria monocytogenes can commonly be found on raw vegetables and can survive for up to 12 years in plant materials (Beuchat, 1996a) Though listeriosis has not been linked to consumption of strawberries, a study conducted on fresh produce sold in Norway retail markets found strawberries to harbor L. monocytogenes (Johannessen et al., 2002) As mentioned earlier, strawberries are grown in close association to the soil, and this growing practice increases their susceptibility to pathogens such as L. monocytogenes which is often isolated from soil (Beuchat, 1996a) A study conducted by Flessa et al. (2005) found that L. monocytogenes was

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37 able to survive for at least one week on fresh whole and cut strawberries stored at refrigerated storage temperatures ( 4C) and survived better on cut strawberries. At freezing temperatures of 20C, populations of L. monocytogenes showed no decline over approximately 1 month on strawberries frozen with 20% sucrose; however, when strawberries were not frozen with sucrose, a significant decline of 1.2 log CFU/sample was observed (Flessa et al., 2005) This data indicates that the presence of sucrose favorably affects the survival of L monocytogenes on frozen strawberries. In the same study, Flessa et al. (2005) reported that contamination levels of L. monocytogenes also play a role in its behav ior on strawberries stored at 24C. Strawberries contaminated at a higher level (7.5 log CFU/sample) rather than a lower level (5.6 log CFU/sample) showed smaller decreases in pathogen population on the surface of intact whole strawberries over 2 days. T hough reasoning for the accelerated decline at the lower level of inoculation was not discussed, microbial load of lower inoculum levels is more probable in natural environments and should be considered during assessments of microbial risks (Flessa et al., 2005) The effect of pH coupled with temperature can also affect the behavior of L. monocytogenes on strawberry products. Han and Linton (2004a) found that L. monocytogenes was able to survive without becoming injured in strawberry juice (pH 3.6) at 4C over 3 days; however, over 3 days at 37C, populations fell below the detection limit (<1 CFU/ml), and cells were found to be injured after 2 h at 37C. These studies indicate that L. monocytogenes can survive over a range of temperatures and even under extreme pH conditions and should be considered in risk assess ments with strawberry cultural and handling practices.

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38 Pathogenic Escherichia coli Initially named Bacterium coli Eschericha coli was discovered in the late 1800s by a Bavarian pediatrician named Theodor Escherich. Though he described the bacteria to be a natural colonizer in the human gut of healthy individuals, he also had an inclination that they were somehow linked in causing intestinal and urinary tract infections. Early developments of serotyping schemes in the 1930s and 1940s provided evidence tha t only certain strains of E. coli were pathogenic and others were harmless in healthy individuals (Kaper, 2005) In terms of intestinal disease causing E. coli currently there are six known pathotypes: (1) enteropathogenic E. coli (EPEC); (2) enterohaemorrhagic E. coli (EHEC); (3) enterotoxigenic E. coli (ETEC); (4) enteroaggregative E. coli (EAEC); (5) enteroinvasive E. coli (EIEC); and (6) diffusely adherent E. coli (DAEC) (Kaper et al., 2004) Escherichia coli is a gram negative, non spore forming, rod shaped (1.1 1.5 m x 2.0 6.0 m) microorganism and is part of the Enterobacteri aceae family (Fratamico and Smith, 2006) It is facultatively anaerobic and typically motile by a peritrichous flagella, meaning it has flagellates all around the cell (Fratamico et al., 2006) Its biochemical characteristics include: oxidase negative; catalase positive; ability to ferment glucose, lactose, D mannitol, D galactosidase p ositive. Most strains are indole and methyl red positive and Voges Proskauer and citrate negative (Fratamico et al., 2006) Isolates of E. coli can be distinguished by three major surface antigens ( serotyping): the O (lipopolysaccharide), H (flagellar), and K (capsular) antigens. E. coli strains causing diarrheal disease need only be serotyped by its O and H antigens. Presently, a total of 173 O antigens, 56 H antigens, and 103 K antigens have been identified (Meng et al., 2007)

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39 Enterohaemorrhagic Escherichia coli EHEC is primarily found in the intes tinal tract of cattle and was first recognized as a human pathogen in 1982 (Kaper et al., 2004) Though serogroups O111 and O26 of EHEC are also capable of causing illness (prevalent in other countries) (Kaper et al., 2004) this pathotype is dominantly associated with serogroup O157 in the US (Meng et al., 2007) The first reported E. coli O157:H7 outbreak in 1982 was linked to consumption of undercooked grou nd beef. Though ground beef was still reported to be the most prominent vehicle in E. coli O157:H7 foodborne outbreaks (41%) from 1982 2002, raw produce accounted for 21% of the E. coli O157:H7 outbreaks in the same time frame (Rangel et al., 2005) Symptoms of infection with E. coli O157:H7 include nonbloody diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic uremic syndrome (HUS) (Kaper et al., 2004) Though it can range from 2 to 12 days, there is a typical incubation period of up to 3 days after ingestion of the contaminated food. The onset of symptoms classically originates with nonbloody diarrhea and severe abdominal cramping. Following the second or third day, illness may progre ss to hemorrhagic colitis and can last for up to 10 days. Symptoms typically persist for about one week, and then will usually resolve itself; however, 6% of patients will progress to HUS (Meng et al., 2007) Children aged 10 years or younger have a higher probability (15%) of progressing to HUS (Tar r et al., 2005) E. coli O157:H7 infections have a case fatality rate of 1% (Meng et al., 2007) Evidence suggests th at the infectious dose for E. coli O157:H7 is low. Two outbreaks involving frozen ground beef patties and salami were reported to having only 0 to 15 isolates per gram (Meng et al., 2007) Additionally, the fact that EHEC infection

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40 can be spread via water (recreational and drinking) and human to human contact also elucidates that the infectious dose for disease is low (Meng et al., 2007) Physiology Escherichia coli O157:H7, though they share some similar physiological properties to non O157 strains (Jacobsen et al., 2009) also possess characteristics that distinguishes it from non O157 strains, such as an inability to ferment sorbitol rapidly (within 24h), an inability to successively grow at temperatures above 44.5C in E. coli broth, and an i glucuronidase (Meng et al., 2007) E scher i chia coli O157:H7 grows optimally at 37C, however it can grow and survive in temperatures ranging from 7C to 50C (WHO, 2012). The growth phase ha s a significant impact on the heat resistance of E. coli O157:H7; cells in the late stationary phase had greater heat resistant than log phase cells at 55C and 60C (Kaur et al., 1998) The optimum pH f or E. coli O157:H7 growth is 7.0, with minimum pH between 4.0 and 4.5, depending on other growth factors (Meng et al., 2007) Escherichia coli O157:H7 has been known to survive and cause infection in food matrixes with low pH, such as apple cider and mayonnaise (Besser et al., 1999; Zhao and Doyle, 1994) Escherichia coli O 157:H7 was found to survive for up to approximately one month in apple cider (pH 3.6 4.0) stored at 8C and for up to 3 days at 25C (Zhao et al., 1993) Additionally, E. coli O157:H7 was able t o survive in mayonnaise (pH 3.6 3.9) for up to approximately 2 months at 5C and 21 days at 20C (Zhao et al., 1994) Cultural Isolation Foods contaminated with E. coli O157:H7 may not contain detectable levels of the pathogen, therefore, steps must be taken to increase levels to that of detection. Development of rapid tests utilizing real time PCR is extremely beneficial and can be

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41 used to rule out negative samples within 24 h; however, presumptive positive samples still need be confirmed via cultural isolation (Feng et al., 2011) Thus, the method for cultu ral isolation will be discussed. The first step involves enriching food samples either using EHEC enrichment broth (EEB) or modified Buffered Peptone Water with pyruvate (mBPWp). The latter is now (BAM) due to its sensitivity in detection (<1 CFU/g in foods) as well as its ability to suppress growth of normal microflora and non targeted competitors (Feng et al., 2011) After overnight streake d onto selective agar plates to isolate single colonies. Examples of selective plates for E. coli O157:H7 include Sorbitol MacConkey agar (SMAC), SMAC modified with cefixime and postassium tellurite (CT SMAC), SMAC modified with cefixime and rhamnose (CR SMAC), and Rainbow agar O157 (Fratamico et al., 2006) Since E. coli O157:H7 does not possess the ability to ferment sorbitol within 24 h (Meng et al., 2007) colonies appear colorless on SMAC. Inclusion of reagents added to modify SMAC is thought to enhance the isolation of E. coli O157:H7 by reducing growth of oth er sorbitol fermenting and nonsorbitol fermenting microbes, however, not affecting growth of E. coli O157:H7. For example, rhamnose can be fermented by nonsorbitol fermenting pathogens but not E. coli O157:H7; cefixime inhibits growth of nonsorbitol ferme nting microbes at concentrations that are not inhibitory to E. coli (O'Brien and Kaper, 1998) On Rainbow agar, colonies appear as black or blue black colonies. After overnight incubation on selective media, portions of typical E. coli O157:H7 colonies are selected for latex agglutination of the O157 antige n. Positively screened colonies are then

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42 streaked onto tryptic soy agar supplemented with yeast extract (TSAYE), where a ColiComplete (CC) disc is placed onto the heaviest st r eak area of the plate. The CC disc is used as a chromogenic assay for galactopy ranosidase (positive for coliforms) and a fluorogenic assay for glucuronidase (negative for E. coli O157:H7). Following overnight incubation of the TSAYE plates, E. coli O157:H7 colonies will produce a blue color around the CC disc, however should not flu oresce under a fluorescent light. The last step involves an isolate confirmation test, where positive colonies will be subjected to a commercial antisera test confirming the presence of the O157 and H7 antigen (Feng et al., 2011) The FDA BAM suggests using the RIM E. coli O157:H7 Latex Test for the final confirmation step (Remel, Lenexa, KS). Significance in Produce Outbreaks From 1982 to 2002, there were 350 reported outbreaks associated with E. coli O157:H7, and 52% of these outbreaks were transmitted by consumption of a food item pred ominantly from a community setting (food item not isolated to a single venue or event) (Rangel et al., 2005) Additionally, 38 (21%) of the 183 foodborne outbreaks were associated with consumption of a pro duce item. Implicated produce items include lettuce (34%), apple cider or apple juice (18%), salad (16%), coleslaw (11%), melons (11%), sprouts (8%), and grapes (3%). The setting for the produce associated outbreaks primarily occurred in restaurants, in which approximately 50% were caused by cross contamination during food handling and preparation (Rangel et al., 2005) In 2006, a multistate outbreak linking fresh spinach to E. coli O157:H7 contamination resulted in 199 people becoming infected. Of the 199 people, 102 were hospitalized, 31 developed HUS and three deaths were confirmed (CDC, 2006) Another outbreak linking romaine lettuce as the vehicle occurred in 2011, where contamination was

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43 suspected to have occurred before it reached its retail destination. Sixty persons were reportedly infected, howeve r, information was only obtained for 45 individuals; of the 45, 30 were hospitalized and two developed HUS (CDC, 2011b) Salmonella spp Salmonella are gram negative, facultatively anaerobic, oxidase negative, catalase positive glucose fermenters that can cause serious gastrointestinal infec tions in humans. These typically motile, rod shaped bacilli are of the Enterobacteriaceae family (D'Aoust and Maurer, 2007) Nomenclature of the Salmonella taxonomy has been quite controversial over the years (Tindall et al., 2005) ; nonetheless, present day nomenclature identifies the genus Salmonella as divided into two species: Salmonella enterica and Salmonella bongori Salmonella enterica is further divided into six subspecies : S. enterica subsp. enterica S. enterica subsp. salamae S. enterica subsp. arizonae S. enterica subsp. diarizonae S. enterica subsp. houtenae and S. enterica susp. Indica Additionally, serova rs contained in each subspecies total to over 2600 (Guibourdenche et al., 2010) Salmonella species are classified and identified into serovars according to the Kauffman White scheme and are uniquely identified by their combination of surface antigens, which is known as the antigenic formula. The 3 surface antigens that make up the antigenic formula include the O antigen (somatic antigen), H antigen, and Vi antigen. Salmonella are first serogrouped by their O antigen, which is related to the lipopolysacchari de of the bacterial cell wall. They are then further classified into serovars by their H antigen and Vi antigen, which is related to the flagella and the bacterial capsule, respectively (Robinson, 2000) A list of serovars and its antigenic formula can be found in the White Kauff man Le Minor scheme, which

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44 is updated by the WHO Collaborating Center for Reference and Research on Salmonella annually (Guibourdenche et al., 2010) Salmonellosis has been the most commonly reported foodborne illness, and infection rates from 1996 2010 were a bout 17.6 illnesses per 100,000 persons. An estimated 43% of hospitalizations and deaths from bacterial foodborne illnesses occurred due to infections with nontyphoidal Salmonella (CDC, 2011c) Transmission of the disease to humans is usually through contaminated foods of animal origin, predominantl y poultry, meat, and eggs (D'Aoust et al., 2007) There are two common human diseases associated with Salmonella : typhoid (enteric fever) and non typhoid salmonellosis. Salmonella serovars associated with typhoid fever are onl y adapted to humans and higher primates and include Salmonella enterica serovars Typhi and Paratyphi A, B, and C (Santos et al., 2001) Typhoid fever is a systemic disease and rarely occurs in the U.S (Mead et al., 1999) The incubation period for typhoid fever is 8 28 days, with the onset of symptoms resulting in fever, diarrhea, and abdominal pain. Infection can spread systemically after 7 days of symptoms, and the case fatality rate ranges from 1 to 10% for developed and undevelope d countries, respectively (D 'A oust, 1991) N on typhoid salmonellosis is much more common in the US and ranks as one of the top two foodborne illnesses, behind illness caused by Campylobacter spp. (M ead et al., 1999) Infection with non typhoid strains of Salmonella is less serious and typically results in entercolitis; illness is usually self limiting and will last no more than 5 days. The incubation period is 8 72 h and symptoms include diarrh ea, abdominal pain, and fever (short duration, < 48 h) (Mead et al., 1999)

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45 Infections with Salmonella follow a dose response model, and evidence suggests the number of bacterial cells required to cause illness can be very low. Factors influencing the dose for infections include: strain virulence; human host condition (age and health), food matrix (fatty foods may protect against gastric acids), and portal of entry (aerosols or orally) (Blaser and Newman, 1982) Physiolog y Salmonella continues to be a threat to the food industry, due to its ability to grow at extreme low and high temperatures. There has been an abundance of studies conducted to gain knowledge on how to control or inhibit the growth of Salmonella Doing s o largely relates to a number of variables, such as temperature, pH, and water activity. Salmonella can be found within the gastrointestinal tracts of warm blooded animals; therefore, their optimum growth temperature is 37 C (D 'Aoust et al., 2007) Though Salmonella can be found growing at temperatures in the range of 2 54 C, growth of Salmonella below 7 C and above 48 C has only been observed on laboratory media and in mutant strains, respectively (Robinson, 2000) Salmonella pose a risk to the pr oduce industry largely due to the fact that it is capable of growing at refrigerated and sometimes freezing temperatures. Additionally, cells can increase their potential to survive and grow at refrigerated temperatures when preconditioned to these lower temperatures (Airoldi and Zottola, 1988) The optimum pH to enhance Salmonella growth is 6.5 7.5, however, extremes of low (pH 4.05) and high (pH 9.5) can also support growth depending on the constituents of the food system (Robinson, 2000) Factors such as presence of salts and volatile fatty acids can inhibit Salmonella growth. Optimal water activity for Salmonella is different in foods than in laboratory

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46 applications. Salm onella can grow in water activity of 0.93 in food systems and 0.945 0.999 in laboratory media (Robinson, 2000) Cultural Isolation Isolation of Salmonella includes many crucial steps because Salmonella spp. are not always at levels capable of being detected in food. Salmonella tes ts can take up to five days for results; however, rapid test kits have been developed to increase efficiency. Rapid test kits are only used to rule out negative samples, and if positive, will require further testing to confirm the presence of the Salmonel la spp. The first step in isolating and confirming positive samples is the pre enrichment broth. This step is done to revive injured cells back to health and does not inhibit other bacteria that may be present from growing (Doyle and Cliver, 1990) Examples of pre enrichment broths are lactose, Universal pre enrichment, and trypticase soy (Andrews et al., 2011) Samples are incubated in enrichment broths for 24 h and then specific volumes are transferred to selective enrichment broths, which allow Salmonell a growth and inhibits growth of other bacteria. Commonly used selective enrichment broths are Selenite Cysteine (SC) and Tetrathionate (TT) broth; however, SC has largely been replaced with Rappaport Vassiliadis (RV) broth, due to its versatile use in foo ds with high and low levels of competitive microflora (Andrews et al., 2011) After overnight incubation in selective enri chment broths, inoculum is then streaked onto selective agar plates (Andrews et al., 2011) This selective plating allows for the isolation of Salmonella as single colonies and inhibition of non Salmonella bacteria from growing (Robinson, 2000) Selective plates include bismuth sulfite (BS) agar, xylose lysine desoyxcholate (XLD) agar, and Hektoen enteric (HE) agar. Colonies on BS agar appear brown, gray, or black with or without a metallic sheen; colonies on the XLD agar appear pink with or without black

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47 centers (colonies may appear completely black); and colonies on the HE agar appear blue green to blue with or without black centers (colonies may a ppear completely black) (Andrews et al., 2011) The fourth step is done to identify Salmonella colonies by observance of i ts biochemical reactions (Doyle et al., 1990) Typical Salmonella colonies from selective plates are picked, then streaked and stabbed into agar slant s. Slants used for this step include triple sugar iron (TSI) agar and lysine iron (LI) agar. A red slant (alkaline) and yellow butt (acid) indicates positive results for TSI, and a purple butt (alkaline) in LIA slants indicate positive results. A distin ct yellow butt (acidic) in LIA slants indicates negative results. Both media can also appear black, which indicates hydrogen sulfide (H 2 S) was produced (Andrews et al., 2011) These two are often used in conjunction with each other to confirm positive results (Robinson, 2000) The last step serologically identifies isolates that were screened positive from the previous step by agglutination assays (Doyle et al., 1990) Significance in Produce Outbreaks In the time periods of 1973 1987 and 1988 1992, reported outbreaks involving consumption of fres h produce had doubled, and outbreaks with known etiologic agents were primarily linked to Salmonella (Buck et al., 2003) From 1990 2005, Salmonella was found to be the causative agent for 18% of all produce outbreaks, accounting for 21% and 28% of vegetable and fruit outbreaks, respectiv ely (DeWaal, 2007) Salmonella has been linked to a wide array of fruits and vegetables since the 1950s including artichokes, cabbage, carrots, cauliflower, celery, eggplant, green onions, herbs ( basil and cilantro ) lettuce, mangoes, melons (cantaloupe, honeydew and wat ermelon), mushrooms, peppers (red chili and Serrano), potatoes, sprouts ( alfalfa and mung ),strawberries and tomatoes (Buck et al., 2003; Hanning et al., 2009) Since

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48 2006, there have been 10 multistate outbreaks implicating produce infected with Salmonella which led to hospitalization of at least 394 persons and possibly caused 2 deaths. These produce items include tomatoes (2006); cantaloupes (2008 and 2011); raw produce (Serrano and jalapeno peppers 2008); a lfalfa sprouts (2009, 2010 [2], 2011); frozen mamey fruit pulp (2010); and imported papayas (2011) (CDC, 2012) The April 2008 outbreak involving the consumption of multiple raw produce items was the largest foodborne produce outbreak observed in the US in the past decade (CDC, 2008) Early investigations of the outbreak proposed illnesses were linked to eating raw t omatoes; however, later studies indicated illnesses were associated with items commonly eaten with Mexican style cuisine, namely jalapeno peppers. The outbreak strain, Salmonella serotype Saintpaul, was finally isolated in July of 2008 from jalapeno and S errano peppers that were grown and packed in Mexico (CDC, 2008) ; source of contamination was contaminated irrigation water (Hanni ng et al., 2009) The prevalence of Salmonella in fresh produce is significant, and Salmonella can grow and cause infection in a variety of produce items. Prevent ative plans and programs, as well as consumer awareness, should be focal points in reducing Salmonella outbreaks associated with produce. Efficacy of Decontaminants to Reduce E. coli O157:H7 and Salmonella Aqueous Solutions The use of sodium chlorite at a concentration of 50 200 ppm of free chlorine has been utilized as an industry standard to decontaminate fruits and vegetables for a long time (Beuchat, 1996b) However, the concern of c arcinogen effects associated with chlorine by products has led the industry to explore new alternatives to ensure safety of fresh eaten produce (Lukasik et al., 2003) There have been a number of studies to test

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49 several poss ible new options for decontamination of strawberries, such as use of detergents, washes, sanitizers, and disinfectants. Detergents include Tween 80 (polysorbate 80) and sodium lauryl sulfate (SLS); accessible household products include vinegar (10%), Fit (over the counter produce wash), Healthy Harvest (over the counter produce wash) and sodium chloride (NaCl, 2%); chemical disinfectants include Alcide (100 or 200 ppm of acidified sodium chlorite), cetylpyridinium chloride (CPC, 0.1%), and hydrogen peroxid e (H 2 O 2 0.5%). Raiden et al. (2003) studied the efficacy of Tween 80, SLS, and water for use with strawberries. Results showed that Tween 80 and water, at temperatures of both 22C and 40C, were both effective, removing on average 4.22 0.10 log CFU/ml of Salmonella populations on strawberries ; SLS was extremely less effective at both temperatures. Strawberries treated with 50 to 300 ppm of free chlorine reduced Salm onella and E. coli O157:H7 populations by over 90% (initial concentration: 10 6 10 7 CFU/berry); treatment with over 100 ppm were not statistically different than using 50 ppm of free chlorine (Lukasik et al., 2003). Alcide (100 and 200 ppm), H 2 O 2 (0.5%), and CPC (0.1%) all had >97% (initial concentration: 10 6 10 7 CFU/berry) reduction of Salmonella and E. coli O157:H7 populations on strawberries and were more effective than the free chlorine washes (Lukasik et al., 2003) Ten percent vinegar and 2.0% NaCl was able to give a Salmonella reduction of >90% (initial concentration: 10 6 10 7 CFU/berry) on strawberries, but were less effective in reducing E. coli O157:H7 populations (Lukasik et al., 2 003) Fit was the most effective at reducing both pathogens on strawberries, providing over 99% (initial concentration: 10 6 10 7 CFU/berry) reduction. In comparison, significantly smaller

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50 reductions (3 7%) in pathogen counts were observed with the othe r over the counter produce wash, Healthy Harvest (Lukasik et al., 2003) Gaseous Agents Gaseous agents such as chlorine dioxide (ClO 2 ) and ozone (O 3 ) gas are being introduced into the produce scene as decontaminant alternat ives to conventional sanitizers (Bialka and Demirci, 2007a) Although ozone has been used since the late 19th century as a purifier in bottled water (Graham, 1997) it was not until 2001 that it was approved to be used as a treatment with raw commodities ( Federal Regi ster, 2001) O zone, which decays into oxygen, does not leave behind residues, as does chlorine (Bialka and Demirci, 2007b) Studies have shown that both of th ese gaseous agents are means to significantly reduce the microbial population on strawberries without drastically compromising organoleptic properties. According to Bialka et al., ( 2007b) pressurized ozone (83 kPa) administered for 64 min reduced Salmonella and E. coli O157:H7 populations on strawberries by 2.2 log CFU/g and 2.3 log CFU/g, respectively. Treatment of non pressurized, continuous application of ozo ne for 64 min resulted in a smaller reduction of 0.9 log CFU/g and 1.8 log CFU/g for Salmonella and E. coli O157:H7, respectively (Bialka et al., 2007b) Increas ing pressure may help gases get into inaccessible crevices of fruits, which may explain why pressurized ozone was more effective at reducing both pathogens on strawberries. The efficacy of aqueous ozone has also been studied to reduce Salmonella and E. co li O157:H7 populations on strawberries. T he reduction of both pathogens is directly proportional to exposure time with aqueous ozone treatment, the longer the exposure to aqueous ozone, the higher the reduction. At 64 min, treatment with 8.9 mg/l of aque ous ozone

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51 led to reductions of 2.9 and 3.3 log CFU/g in populations of E. coli O157:H7 and Salmonella respectively (Bialka et al., 2007a) Gaseous ClO 2 has shown to be very capable of reducing Salmonella count on strawberries. Greater than 5 log CFU of E. coli O157:H7 per strawberry was observed with batch (30 min of exposure to 4.0 mg/l ClO 2 ) and continuous (3.0 mg/l ClO 2 for 10 min ) treatments with chlorine dioxide ga s (Han et al., 2004b) Sy et al. (2005) found that 8.0 mg/l of ClO 2 (released over a period of 120 min ) was able to reduce Salmonella populations by 3.76 log CFU/g of strawberry, when inoculated on the skin, and by 4.41 log CFU/g of strawberry, when inoculated on the stem scar. There were no significant differences in reduction of Salmonella between the two inoculation sites. Yuk et al. (2006) also found no significant dif ferences in reduction of Salmonella populations when inoculated at the stem scar and surface; however, when inoculated at a puncture site on the strawberry, ClO 2 was not as effective in reducing Salmonella populations. In a study conducted by Mahmoud et a l. (2007) treat ment of strawberries with 5 mg/l of ClO 2 for 10 min reduced the population of Salmonella by 4.3 log CFU/berry and extended its shelf life from 8 to 16 days. Though reduction of E. coli O157:H7 and Salmonella populations seem promising with gaseous ClO 2 re gimens, only aqueous ClO 2 is approved for use as an antimicrobial for fruits and vegetables (Federal Register, 1996)

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52 CHAPTER 3 MATERIALS AND METHODS Strawberries Strawberries were ei ther purchased from a local store near Lake Alfred, FL or freshly harvested from a farm in Dover, FL. Purchased strawberries were stored up to one day at 4 2C before use and were either of California or Florida origin. Freshly harvested strawberries w ere picked up from Dover, FL (BBI Produce, Inc.) on the day of harvest and stored up to 4 h before use. Only strawberries free of defects were used, and freshly harvested strawberries were additionally sorted of two maturities: full red and red. Strawbe rry Bruising A 2.54 cm diameter PVC pipe (Lowes; Winter Haven, FL) was cut to a length of 20 cm. A 32.6 g steel ball (20 mm diameter) was selected to impact the strawberry. This PVC pipe height and steel ball weight and diameter were chosen to match the highest energy of impact needed to cause strawberry bruising due to impact forces (Ferreira et al., 2009) The energy of impact was calculated using the formula E = mgh [where m is the mass of the steel ball (32.6 g); g = acceleration constant (9.82 m/s 2 ; h is the vertical height (20 cm)]. The highest energy of impact in the study was 0.075 J, and our energy of impact was 0.064 J. This created the highest level of bruising without breaking the skin of the strawberry. The PVC pipe was positioned directly above the strawberry, and the steel ball was dropped through the PVC pipe, directly impacting the strawberry. A new, sterile steel ball was used to bruise eac h strawberry within one repetition of the experiment.

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53 Modified Atmosphere Packaging A campy gas system (Gibson Laboratories, LLC; Lexington, KY) was used in all modified atmosphere packaging experiments. A campy gas cylinder, with a gas composition of 9.9 8% carbon dioxide and 4.98% oxygen (balance of nitrogen), was used to create the modified atmosphere. Whirl pak bags (Nasco Whirl Pak, Fort Atkinson, WI; 207 ml) containing strawberry samples were placed into special campy bags (Gibson Laboratories, LLC; Lexington, KY; 22.9x30.5 cm) with a TempTale (TempTale 4, Sensitech Inc., Beverly, MA) to measure temperature and relative humidity throughout storage. Bags were flushed once with the gas from the campy gas cylinder then inflated to capacity and sealed. Sealed bags were placed into incubators until sampling. The carbon dioxide and oxygen composition in the headspace of the campy bags containing strawberry samples were measured, in separate experiments, using a Mocon headspace analyzer (Pac Check 650, Mi nneapolis, MN). Strawberries were spot inoculated with 20 l of 0.1%peptone water (Difco, Becton Dickinson) and allowed to dry at room temperature for 1 h under a bio safety cabinet. After drying, strawberries were transferred into whirl pak bags, placed into campy bags and incubated at either 2 2C or 15.5 2C to simulate shipping and retail display temperatures, respectively. The headspace composition of samples stored at 2 2C and 15.5 2C were measured at 0, 5, and 24 h and on day 0, 1, 3, 5 and 7, respectively (n=3). A sticky nickel septum was placed onto the outer surface of campy bags (to prevent air from escaping the bag during sampling ), and the syringe was inserted through the septum and into the bag to sample the air.

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54 Bacterial Stra ins Five rifampicin resistant (80 g/ml) E. coli O157:H7 strains were used in each experiment. Isolation sources of strains include: Odwalla juice (MDD 20; strain #223), c antaloupe outbreak (MDD 326; LJH 1214; clinical isolate), l ettuce outbreak (MDD 18; LJH 1153; human feces), a lfalfa sprout outbreak (MDD 19; F 4546; human feces), and s pinach outbreak (MDD 327; human feces ). Five rifampicin resistant (80 g/ml) Salmonella serovars were used in each experiment. Sources of serovars include: Salmonella Ago na (MDD 17; LJH 0517; a lfalfa sprouts), Salmonella Poona (MDD 237; LJH 0631; c antaloupe outbreak, human isolate), Salmonella Montevideo (MDD 22; LJH 0519; t omato linked, human isolate), Salmonella Newport (MDD 314; t omato outbreak, environmental isolate), and Salmonella St. Paul (MDD 32; LJH 1262; j alapeno, product isolate). A stock solution of rifampicin was prepared by adding 1 g of rifampicin to 20 ml of methanol and solution was then filter sterilized (Nalgene [0.20 m pore size], Rochester, NY). To pr epare media with a final concentration of 80 g/ml of rifampicin, 1.6 ml of stock solution was added to 1 L of media. Before rifampicin was added, media was either boiled or autoclaved (120C for 15 min ), then tempered in a 45C water bath for approximate ly 1 h Inoculum Preparation Bacterial strains, stored at 80C in glycerol bead stock cryogenic vials, from the Danyluk laboratory culture collection were thawed at room temperature for ca. 15 min and streaked onto tryptic soy agar plates supplemented wit h rifampicin (TSAR; Difco, Becton Dickinson, Sparks, MD) 4 days prior to experimentation. Plates were incubated for 18 2 h at 35 2C, and one isolated colony was transferred into 10 ml of fresh

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55 tryptic soy broth supplemented with rifampicin (TSBR; Dif co, Becton Dickinson). Test tubes of TSBR were incubated for 24 h at 35 2C. Two subsequent 24 h transfers (incubated at 352C) were made using a 10 l inoculum loop into 10 ml of fresh TSBR. Cultures were collected by centrifugation (Allegra X 12, B eckman Coulter, Fullerton, CA) at 3000 rpm for 10 min. Cells were washed by first removing the supernatant, suspending the cell pellet in 10 ml of 0.1% peptone water (Difco, Becton Dickinson), vortexing, and centrifugation. Cells were washed twice and su spended in 5 ml of 0.1% peptone water. Serial dilutions were carried out in 0.1% peptone water (9 ml) to produce an inoculum concentration of 10 6 or 10 7 CFU/ml. Each bacterial strain was added in equal volume (1 ml) to produce its respective cocktail. I noculum cocktails were stored on ice up to 2 h prior to experiments. Inoculum concentrations were confirmed by enumeration on selective and nonselective media. Initial experiments with store bought strawberries were carried out using an inoculum concentra tion of 10 6 CFU/ml. After 1 h dry under a bio safety cabinet, initial bacterial populations were inconsistent, and sometimes undetectable by standard plating methodologies. Subsequent experiments were performed with a higher inoculum concentration of 10 7 CFU/ml. Experiments with store bought strawberries were carried out using 10 6 CFU/ml (n=3) and 10 7 CFU/ml (n=3). All replicates (n=6) from the harvested strawberries experiments and modified atmosphere packaging experiments were carried out using an ino culum concentration of 10 7 CFU/ml. Strawberry Inoculation Twenty microliters of either E. coli O157:H7 or Salmonella cocktails were spot inoculated (8 10 drops) onto either the bruised or intact surface of the strawberry. Inoculum was air dried for 1 h under a biosafety cabinet.

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56 Storage Conditions Strawberries were stored at either 2 2C or 15.5 2C to simulate shipping and retail display temperature, respectively. Strawberries stored at 2C were sampled at 0, 2, 5, and 24 h. Strawberries stored at 15.5C were sampled on day 0, 1, 3, and 7. Enumeration of Pathogens To homogenize samples, filtered whirl pak bags (270 ml) containing strawberry samples were filled with 30 ml of 0.1 M Phosphate Buffered Saline (PBS; 0.02 M NaH 2 PO 4 0.02 M Na 2 HPO 4 ; Fish er; pH 7.2) and placed in a stomacher (BAGMIXER 400, Interscience Laboratories Inc., Weymouth, MA; 8 strokes/sec) for 2 min. PBS was used to increase the pH of the sample homogenization for better recovery of pathogens (Flessa et al., 2005; Knudsen et al., 2001) After stomaching, serial dilutions were made in 0.1% peptone water (9 ml) and surface plated in duplicates (0.1 ml) onto selective and nonselective media supplemented with rifampicin. Sorbitol MacConkey agar supplemented with rifampicin (SMACR; 80 g/ml; Difco, Becton Dickinson) was used as the selective media for E. coli O157:H7. Bismuth sulfite agar supplemented with rifampicin (BSAR; 80 g/ml; Difco, Becton Dickinson) was used as the selective media f or Salmonella TSAR was used as the nonselective media for both E. coli O157:H7 and Salmonella To increase the sensitivity of the experiment, 1 ml of the lowest dilution was spread over four plates (0.25 ml per plate) to increase the limit of detection t o 30 CFU/strawberry (1.5 log CFU/strawberry). Control samples were also plated on selective and nonselective media. TSAR plates were incubated at 352C for 24 h, and SMACR and BSAR plates were incubated at 352C for 48 h. Following respective incubat ion times, colonies were

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57 hand counted, and E. coli O157:H7 and Salmonella populations were expressed as log CFU/strawberry. Enrichment When counts fell below the detection limit (1.5 log CFU/strawberry), enrichments for E. coli O157:H7 and Salmonella were performed. Escherichia coli O157:H7 enrichments were conducted following protocols from a nut study conducted in the Danyluk lab ( personal communication ). Salmonella enrichments were conducted following protocols from the US Food and Drug Administration B acteriological Analytical Manual (BAM) (Andrews et al., 2011) Escherichia coli O157:H7 For E. coli O157:H7 enrichment, equ al volume of (30 ml) of double strength modified buffered peptone water with pyruvate (mBPWp; Acumedia, Neogen Corporation, Lansing, MI) was added to the sample and incubated at 35 2C for 5 h. Following 5 h incubation, stock solution of rifampicin was added to standard concentration (96 l of rif to 60 ml; 80 g/ml) and incubated again at 42 2C for 18 24 h. After overnight incubation, the enrichment was streaked onto SMACR and incubated for 24 h at 35 2C. After 24 h, if typical colonies were pre sent, enrichment was recorded as positive, and populations were recorded as 1.5 log CFU/strawberry (limit of detection). Salmonella spp For Salmonella enrichment, equal volume (30 ml) of double strength lactose broth (Difco, Becton Dickinson) was added to the sample and incubated at 35 2C for 24 h. After overnight incubation, 1 ml of enrichment was added to 9 ml of tetrathionate broth (TT; Difco, Becton Dickinson), and 0.1 ml of enrichment was added to 9.9 ml of

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58 Rappaport Vassiliadis R10 (RV; Difco, Be cton Dickinson). TT broth and RV broth tubes were incubated at 352C (24 h) and 422C (48 h), respectively. Following incubation, a 10 l loopful, from each broth, was streaked onto BSAR, xylose lysine desoxycholate agar supplemented with rifampicin (X LDR; 80 g/ml; Difco, Becton Dickinson), and Hektoen enteric agar supplemented with rifampicin (HER; 80 g/ml; Difco, Becton Dickinson). XLDR and HER were incubated at 352C for 24 h, and BSAR was incubated at 352C for 48 h. Following incubation, if t ypical colonies were present, portions of those colonies were picked and stabbed and streaked into triple sugar iron (TSI; Difco, Becton Dickinson) and lysine iron agar (LIA; Difco, Becton Dickinson) slants. Slants were incubated at 352C for 24 h. Posi tive confirmation of Salmonella on TSI slants appears to have a yellow butt and red slant (with or without H 2 S gas formation [black]). There is no change in color (remains purple) for positive confirmation of Salmonella on LIA slants (can have H 2 S gas for mation [black]). When enrichments were positive, populations were recorded as 1.5 log CFU/strawberry (limit of detection). Normalizing Data and Statistics Data from experiments with store bought strawberries were normalized to negate the effects of using two different inoculum levels. Data was normalized using the expression log N/N o where N is bacterial counts at each sampling time, other than time 0, and N o is initial bacterial counts at time 0, on strawberries. Results obtained were averaged (duplicat e plates) or summed (quadruplicate plates) to obtain final counts. Comparisons in differences among populations (log CFU/strawberry) were made between selective an d nonselective media, bruised and intact strawberries, harvested and store bought strawberri es, full red and red strawberries, modified and ambient atmosphere packaging, between each time point,

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59 and between E. coli O157:H7 and Salmonella populations for both temperatures (2C and 15.5C) at each time point. Data were statistically analyzed usi ng analysis of variance (ANOVA; JMP, software version 9.0.2; SAS Institute Inc., Cary, NC, USA.), performed to see which groups were different. Differences were consider ed significant when 0.05.

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60 CHAPTER 4 RESULTS Pathogen Enumeration All tables include data for nonselective and selective media at all temperatures and treatments and are expressed as population counts in log CFU/berry. Populations recovered on nonselective (TSAR) and sel ective media (SMACR for E. coli O157:H7 and BSAR for Salmonella ) were not significantly different for all experiments, except for some modified atmosphere experiments ( see below ). Selective media results will only be discussed for modified atmosphere expe riments, and nonselective media results will be presented for all other experiments. Under modified atmosphere storage at 2 2C and 15.5 2C, the average relative humidity inside campy bags containing the samples was 62% 1.5 and 83% 1.7, respectiv ely (data not shown). Composition of Modified Atmosphere Initial modified atmospheric conditions for strawberries held at 2 2C were 9.5% CO 2 and 5.9% O 2 (Table 4 1). Carbon dioxide concentrations changed slightly by 5 and 24 h to 8.8% and 9.1%, respec tively (Table 4 1). Oxygen concentrations did not deviate from initial concentrations, dropping only to 5.7% and 5.4% by 5 and 24 h (Table 4 1). Initial modified atmospheric conditions for strawberries held at 15.5 2C were 9.5% CO 2 and 5.7% O 2 (Table 4 2). Carbon dioxide concentrations increased steadily over 7 days, with the largest increase of 8% on day 3 and day 5 (Table 4 2). By day 7, carbon dioxide concentration was at its highest 35% (Table 4 2). Oxygen concentrations dropped by 1.3% on day 1 of storage at 15.5 2C, and on day 3 and thereafter, no oxygen (0%) was detected in the headspace of all campy bags (n=3, Table 4 2).

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61 Fate of E. coli O157:H7 on Store Bought Strawberries Escherichia coli O157:H7 populations on bruised strawberries sign ificantly declined ( P Table 4 3 ) during 24 h of storage at 2 2C. Populations on intact strawberry surfaces remained stable through 24 h of storage at 2 2C, with less than a 1 log CFU/berry decrease ( P ; Table 4 3) Escherichia coli O157:H7 populations on bruised and intact strawberries over the course of storage at 2 2C were not significantly different ( P inoculated with an inoculum concentration of ca. 10 6 CFU/ml fell below the limit of detection ( Table 4 3) Enrichment was not conducted; populations were averaged in at the limit of detection. No samples fell below the limit of detection when an inoculum concentration of 10 7 CFU/ml was used (Table 4 3 ) Populatio ns of E. coli O157:H7 on bruised and intact strawberries decreased significantly ( P ( Table 4 4 ). By day 7, populations had decreased by 1.5 log CFU/berry on bruised samples and 1.6 log CFU/berry on intact samples ( P significant ( P 7 on bruised strawberries, but were significant by day 3 on intact strawberries. No significant differences ( P E. coli O157:H7 populations exist between bruised and intact treatments on each sampling day on strawberries held at 15.5 2C. Wh en inoculated with an inoculum concentration of ca. 10 6 CFU/ml (n=3; Table 4 4 ), populations fell below the limit of detection (1.5 log CFU/berry) on day 1 (1/3 for bruised strawberries), day 3 (1/3 for intact strawberries), and day 7 (2/3 for bruised stra wberries; 1/3 for intact strawberries). These samples were not enriched; populations were averaged into results at the limit of detection.

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62 When the inoculum concentration was increased to 10 7 CFU/ml, no samples fell below the limit of detection (Table 4 4 ). Fate of E. coli O157:H7 on Freshly Harvested Strawberries Populations of E. coli O157:H7 on full red and red, bruised and intact strawberries decreased significantly ( P Table 4 5 ) Populations on all samples, under all conditions, remained relatively stable through the first 5 h of storage ( P and decreased by approximately 1 log CFU/berry by 24 h ( P 5 ) There were no significant differences ( P E. coli O157:H7 population decli nes between bruised and intact treatments or between full red and red strawberries at each sampling point ( P Table 4 5 ) Populations on full red and red strawberries did not fall below the limit of detection (Table 4 5 ) Escherichi a coli O157:H7 populations on full red bruised and intact strawberries decreased significantly ( P (Table 4 6 ) Populations on full red, bruised strawberries remained stable through the first 3 days, then decrea sed by 1.8 log CFU/berry by day 7 ( P ; Table 4 6 ). Significant population decreases on full red intact strawberries were seen by day 3 ( P 4 6 ) by 1.2 log CFU/berry; population levels remained unchanged through day 7. E scherichia coli O157:H7 populations on red, bruised strawberries held at 15.5 2C remained unchanged through the first 3 days of storage, then significantly decreased ( P Table 4 6 ) There were n o significant declines in populat ions ( P (Table 4 6 ) No significant differences in E. coli O157:H7 populations ( P between full red and red strawberries (under all conditions) for each sampling day

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63 when held at 15.5 2C ( Table 4 6 ) There were n o significant differences ( P E. coli O157:H7 populations between bruised and intact treatments for full red and red strawberries on each sampling day (Table 4 6 ) On day 3, two full red bruised samples (n =6) fell below the limit of detection (Table 4 6 ) Following enrichment, both samples were positive for E. coli O157:H7 presence and populations were averaged at the limit of detection. On day 7, one intact red sample (n=6) fell below the limit of dete ction ( Table 4 6 ) Upon enrichment, the sample was positive for E. coli O157:H7 and populations were averaged in at the limit of detection. Fate of E. coli O157:H7 on Strawberries Stored under Modified Atmosphere Escherichia coli O157:H7 populations on br uised and intact strawberries stored under a modified atmosphere at 2 2C remained relatively stable through the first 5 h of storage ( P 7 ) Both bruised and intact treatments had slight, but insignificant ( P ions between 2 to 5 h, but populations declined ( P ) by 24 h of storage by 0.7 and 1.1 log CFU/berry for bruised and intact treatments, respectively (Table 4 7 ). No significant differences ( P population declines on bruised or intact straw berries were seen at each sampling point ( Table 4 7 ) Significant differences ( P Table 4 7 ) in populations were not observed between nonselective and selective media for strawberries stored under modified atmosphere at 2 2C, and populations nev er fell below the limit of detection. Escherichia coli O157:H7 populations ( P and intact strawberries stored under modified atmosphere at 15.5 2C over 7 days ( Table 4 8 ) Populations remained stable ( P from day 0 to day 3 for both

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64 bruised and intact treatments, and decreased by 1.2 and 1.1 log CFU/berry for bruised and intact treatments, respectively, by day 7 ( P Table 4 8 ) In some instances, significant differences ( P E. coli O157:H7 populations enumerated on nonselective and selective media were seen when strawberries were held at 15.5 2C ( Table 4 8 ) On i ntact strawberries at day 3, populations recovered from TSAR were 4.2 log CFU/berry, while recovery on SMACR yielded significa ntly lower population levels of 2.9 log CFU/berry (Table 4 8 ) Following 7 days on bruised strawberries, population recovery on TSAR of 3.8 log CFU/berry was significantly higher than population recovery of 2.7 log CFU/berry on SMACR (Table 4 8 ) S ignifi cant differences ( P bruised and intact strawberries for each sampling day (Table 4 8 ) Under no conditions did p opulations fall below the limit of detection; no enrichments were conducted. Compariso n of E. coli O157:H7 Behavior on Strawberries with Various Treatments Comparing E. coli O157:H7 population declines on store bought and harvested full red strawberries, no significant differences ( P time when stored o ver 24 h at 2 2C and 7 days at 15.5 2C (Figure 4 1 and 4 2 ). No significant differences ( P E. coli O157:H7 population declines between freshly harvested, full red and red strawberries, were observed at each sampling time during storage at 2 2C and 15.5 2C (Figure 4 3 and 4 4). Between strawberries stored under unmodified and modified atmosphere at 2 2C and 15.5 2C no significant differences ( P E. coli O157:H7 population declines were observed (Figure 4 5 and 4 6 ).

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65 Fate of Salmonella on Store Bought Strawberries For store bought strawberries stored at 2 2C, Salmonella populations decreased significantly ( P treatments over 24 h ( Table 4 9 ) Salmonella popu lations on bruised strawberries declined significantly ( P through 24 h ( P ( P Table 4 9 ) N o differences in population levels between bruised and intact strawberries occurred at each sampling point ( P Table 4 9 ). Salmonella populations on all samples did not fall below the limit of detection (n=3; Ta ble 4 9 ). For store bought strawberri es stored at 15.5 2C, Salmonella populations decreased significantly ( P Table 4 10 ) by 1.9 and 1.6 log CFU/berry on bruised and intact strawberries, respectively, by day 7. No differences in population declines were seen between bruised and int act treatments on any sampling day ( P Table 4 10 ). All bruised samples (n=3) and one intact sample (n=3) fell below the limit of detection when inoculated with an inoculum concentration of 10 6 CFU/ml (Table 4 1 0 ) These samples were not e nriched; the limit of detection was used to average these samples. No samples inoculated with an inoculum concentration of 10 7 CFU/ml fell below the limit of detection (Table 4 1 0 ) Fate of Salmonella on Freshly Harvested Strawberries Over 24 h of storage 2 2C significant declines in Salmonella populations ( P 0.05) of 0.9, 1.1, and 1.1 log CFU/berry were observed for freshly harvested, full red bruised, full red intact, and red intact samples, respectively( Table 4 1 1 ) Populations remained stable on red bruised samples, with less than a 0.5 log CFU/berry decrease

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66 in population over 24 h of storage ( 0.05; Table 4 1 1 ) No significant declines ( P 0.05) in populations were observed between bruised and intact samples or between full red and red samples for each sampling point ( Table 4 11 ). Population s did not fall below the limit of detection for full red or red strawberries at any sampling point ( Table 4 11 ). On full red and red, freshly harvested strawberries, Salmonella populations significant ly declined ( P e 7 days of storage at 15.5 2C (Table 4 12 ) Populations on full red bruised and intact strawberries decreased by approximately 2 log CFU/berry by day 7 (Table 4 1 2 ) Small and insignificant ( P 0.05) increases in Salmonella population occurred betwe en days 1 and 3 for full red bruised samples (Table 4 1 2 ) Salmonella populations on red, bruised and intact strawberries declined significantly ( P by day 3 (Table 4 12 ) On day 7, populations on br uised samples decreased by 2.4 log CFU/berry ( P insignificant ( P (Table 4 12 ) No significant differences ( P n bruised and intact treatments at each sampling day; however, on day 3, samples (intact) had lower Salmonella populations ( P log CFU/berry (Table 4 1 2 ) Populations in one sample (n=6) of the red bruised treatment and two samples (n=6) of the red intact treatment fell below the limit of detection on day 7 (Table 4 12 ) Upon enrichment, the samples were positive for Salmonella and the limit of detection was used for averaging.

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67 Fate of Salmo nella on Strawberries Stored under Modified Atmosphere Salmonella populations on bruised and intact strawberries stored under a modified atmosphere at 2 2C have slightly different survival trends over 24 h (Table 4 13 ) On bruised strawberries, Salmone lla populations declined significantly ( P h of storage by 1.1 log CFU/berry and remained unchanged thereafter ( Table 4 13 ) On intact strawberries, Salmonella populations declined significantly ( P storage, by 0.9 log C FU/berry, and remained relatively stable thereafter ( Table 4 13 ) However, populations on intact strawberries did not significantly decline over 24 h ( P 0.05; Table 4 13 ) S ignificant differences ( P bruised and intact treatments at each sampling period, and samples did not fall below the limit of detection over the 24 h of storage (Table 4 13 ). Salmonella populations on bruised and intact strawberries stored under a modified atmosphere declined significantly ( P over 7 days of storage at 15.5 2C (Table 4 14 ) Both bruised and intact samples had significant population declines of 1.4 and 1.9 CFU/berry, respectively, by day 3 (Table 4 14 ). On day 7, bruised samples had dropped in populations by 2.3 log CFU/berr y, however, intact treatments had a slight, but insignificant ( P Table 4 14 ) Significant population differences ( P treatments at each sampling day, and samples did not fall below the limit of detection over the 7 days of storag e (Table 4 14 ). Comparison of Salmonella Behavior on Strawberries with Various Treatments Comparing Salmonella population declines on strawberries between store bought and harvested full red strawberries, no significant differences ( P were observed at each sampling time when stored over 24 h at 2 2C and 7 days at 15.5 2C

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68 (Figure 4 7 and 4 8). No significant differences ( P in Salmonella population declines are seen when comparing freshly harvested, full red and red strawberries at ea ch sampling time, when strawberries were held at 2 2C (Figure 4 9). Salmonella populations on red strawberries (intact) had significantly larger declines ( P than full red (bruised) strawberries on day 3, by 2.3 log CFU/berry, when held at 15. 5 2C (Figure 4 10). Between strawberries stored under ambient and modified atmosphere, no significant differences ( P Salmonella population declines were observed when strawberries were held at 2 2C or 15.5 2C (Figure 4 11 and 4 12). Co mparisons between E. coli O157:H7 and Salmonella Behavior on Strawberries Population declines of E. coli O157:H7 and Salmonella on freshly harvested strawberries and modified atmosphere experiments were significantly different under some instances. In all cases, Salmonella populations were significantly lower than E. coli O157:H7 populations. Salmonella populations on freshly harvested red intact strawberries were significantly lower ( P ; 2.2 log CFU/berry; Table 4 12 ) than E. coli O157:H7 populations (Table 4 6 ) on full red, bruised strawberries, following three days of storage at 15.5 2 2C. For strawberries held under modified atmosphere storage for 3 days at 15.5 2C, Salmo nella populations (Table 4 14 ) were significantly lower ( P E. coli O157:H7 populations,(Table 4 8 ) by 1.2 and 1.7 log CFU/berry on bruised and intact surfaces, respectively. Following 5 h of storage at 2 2C under modified atmosphere condit ions Salmonella populations (Table 4 13 ) on bruised strawberries were significantly lower ( P E. coli O157:H7 populations (Table 4 7 ) by 1.3 log CFU/berry.

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69 Table 4 1. Change in carbon dioxide (CO 2 ) and oxygen (O 2 ) composition in the headspac e of campy bags following incubation at 2 2C for up to 24 h. Time (hour) CO 2 O 2 0 1 9.5 0.3 5.9 0.4 5 8.8 0.1 5.7 0.1 24 9.1 0.1 5.4 0.3 1 Values represent m ean standard deviation of triplicate samples from 1 replication (n=3), expressed as % composition of gas. Table 4 2. Change in c arbon dioxide (CO 2 ) and oxygen (O 2 ) composition in the headspace of campy bags following incubation at 15.5 2C for up to 7 days. Time (day) CO 2 O 2 0 1 9.5 0.1 5.7 0 1 13 0.3 1.3 0.3 3 21 1. 0 0.0 0 5 29 2.4 0.0 0 7 35 0.7 0.0 0 1 Values represent m ean standard deviation of triplicate samples from 1 replication (n=3), expressed as % composition of gas. Table 4 3. Fate of E. coli O157:H7 populations on bruised and intact surfac e s of store bought strawberries enumerated on TSAR and SMACR following incubation at 2 2C for up to 24 h Inoculum Time Bruised Intact (CFU/ml) (hour) TSAR SMACR TSAR SMACR 10 6 0 1 3.30.2 3.20.1 3.00.6 2.60.6 2 3.30.7 2.90.6 3.10.2 2.70.5 5 2.80.4 2.50.7 3.00.4 2.30.3 24 2.30.7 1.90.7 <2.31.5 a <2.21.3 b 10 7 0 1 5.20.0 4.50.0 4.90.2 4.30.2 2 4.50.3 4.00.3 4.50.2 3.60.8 5 4.21.1 3.71.3 4.70.1 4.50.1 24 3.50.5 2.80.8 4.10.3 3.60.4 1 Values represent the mean plu s or minus standard deviation of triplicate samples from 1 replication (n=3), expressed as log CFU/berry. a n=3, one rep licate below limit of detection b n=3, two replicates below limit of detection

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70 Table 4 4. Fate of E. coli O157:H7 populations o n bruised and intact surface s of store bought strawberries enumerated on TSAR and SMACR following incubation at 15.5 2C for up to 7 days. Inoculum Time Bruised Intact (CFU/ml) ( day ) TSAR SMACR TSAR SMACR 10 6 0 1 4.10.1 3.60.2 4.10.2 3.30.2 1 <2.71.1 a <2 .00.7 a 3.00.3 1.90.4 3 3.10.4 1.70.3 <2.61.1 a <1.80.6 a 7 <1.80.6 b <1.50.0 b <2.21.3 a <2.21.2 a 10 7 0 1 5.30.1 4.60.0 5.10.3 4.00.4 1 5.00.4 4.20.7 4.70.1 3.50.3 3 4.50.2 3.00.9 4.00.5 2.41.2 7 4.61.4 4.21.7 2.80.4 1.60.3 a 1 Values represent the mean plus or minus standard deviation of triplicate samples from 1 replication (n=3), expressed as log CFU/berry. a n=3, one replicate below limit of detection b n=3, two replicates below limit of detection Table 4 5. Fate of E. c oli O157:H7 populations, inoculated with ca. 10 7 CFU/ml on bruised and intact surface s of freshly harvested full and red strawberries enumerated on TSAR and SMACR following incubation at 2 2C for up to 24 h. Maturity Time Bruised Intact (hour) TS AR SMACR TSAR SMACR Full Red 0 1 4.50.5 4.00.5 4.30.5 3.50.5 2 4.00.6 3.20.6 4.20.3 3.40.4 5 3.90.9 3.11.1 3.80.2 2.70.4 24 3.40.7 2.70.8 3.30.8 2.40.6 Red 0 1 4.40.6 3.80.6 4.20.3 3.50.3 2 3.70.2 2.90.4 4.00.1 3.20.1 5 3.60.3 2.60.4 3.90.3 3.10.4 24 3.40.9 2.51.1 3.10.6 2.20.8 1 Values represent the mean plus or minus standard deviation of triplicate samples from each of 2 replications (n=6), expressed as log CFU/berry.

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71 Table 4 6 Fate of E. coli O157:H7 popu lations, inoculated with ca. 10 7 CFU/ml, on bruised and intact surfaces of freshly harvested full and red strawberries enumerated on TSAR and SMACR following incubation at 15.5 2C for up to 7 days. Maturity Time Bruised Intact ( day ) TSAR SMACR TSA R SMACR Full Red 0 1 5.10.2 4.80.3 4.60.2 4.10.2 1 4.60.5 4.20.5 4.20.5 3.40.6 3 4.30.4 b 3.70.4 b 3.40.8 <2.61.1 a 7 3.31.2 <2.41.0 a 3.00.9 <1.90.7 a Red 0 1 4.90.3 4.60.5 4.50.3 4.00.4 1 4.00.9 3.30.8 3.91.1 <3.11.5 a 3 3.7 0.6 3.00.9 3.11.5 <2.61.3 a 7 3.41.4 <2.71.4 a <2.91.3 a <2.41.3 a 1 Values represent the mean plus or minus standard deviation of duplicate samples from each of 3 replications (n=6), expressed as log CFU/berry. a n=6, one replicate below limit of det ection b n=6, one replicate not sampled due to lab error Table 4 7 Fate of E. coli O157:H7 populations, inoculated with ca. 10 7 CFU/ml, on bruised and intact surfaces of store bought strawberries stored under modified atmosphere storage (ca. 10% CO 2 5% O 2 ) at 2 2C for up to 24 h and enumerated on TSAR and SMACR. Time Bruised Intact (hour) TSAR SMACR TSAR SMACR 0 1 5.0 0.2 4.5 0.3 4.8 0.1 4.1 0.4 2 4.7 0.5 4.2 0.6 4.2 0.5 3.4 0.7 5 4.7 0.3 4.2 0.4 4.4 0.4 3.5 0.8 24 4.2 0.6 3.7 0.8 3.7 0.5 2.9 0 .8 1 Values represent the mean plus or minus standard deviation of triplicate samples from each of 2 replications (n=6), expressed as log CFU/berry.

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72 Table 4 8. Fate of E. coli O157:H7 populations, inoculated with ca. 10 7 CFU/ml on bruised and intact sur faces of store bought strawberries stored under modified atmosphere storage (ca. 10% CO 2 5% O 2 ) at 15.5 2C for up to 7 days and enumerated on TSAR and SMACR. Time Bruised Intact (day) TSAR SMACR TSAR SMACR 0 1 5.0 0.4 4.5 0.5 4.8 0.3 4.2 0.2 1 4.8 0.3 4.1 0.3 4.4 0.4 3.2 0.7 3 4.9 0.2 4.0 0.4 4.2 0.2 2.9 0.5 7 3.8 0.6 2.6 1.0 3.7 1.0 2.7 1.0 1 Values represent the mean plus or minus standard deviation of triplicate samples from each of 2 replications (n=6), expressed as log CFU/berry. Ta ble 4 9 Fate of Salmonella spp. populations on bruised and intact surfaces of store bought strawberries enumerated on TSAR and BSAR following incubation at 2 2C for up to 24 h. Inoculum Time Bruised Intact (CFU/ml) (hour) TSAR BSAR TSAR BSAR 10 6 0 1 4.20. 8 4.1 1.0 3.90.1 3.5 0.2 2 3.20.5 2.9 0.6 3.60.6 3.4 0.7 5 3.21.1 2.8 1.5 3.60.6 2.6 0.5 24 2.90.4 2.4 0.1 2.40.6 <1.9 0.4 a 10 7 0 1 5.20.1 5.2 0.1 4.3 0.5 4.1 0.6 2 3.90.7 3.7 0.8 4.2 0.3 4.0 0.3 5 4.00.2 3.8 0.1 4.2 0.4 4.1 0.5 24 4. 10.9 3.8 1.1 3.5 0.9 3.3 1.1 1 Values represent the mean plus or minus standard deviation of triplicate samples from 1 replication (n=3), expressed as log CFU/berry. a n=3, one replicate below limit of detection

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73 Table 4 1 0 Fate of Salmonella spp. popul ations on bruised and intact surfaces of store bought strawberries enumerated on TSAR and BSAR following incubation at 15.5 2C for up to 7 days. Inoculum Time Bruised Intact (CFU/ml) ( day ) TSAR BSAR TSAR BSAR 10 6 0 1 3.50.1 3.20.1 3.10.8 3.00.7 1 2.90.4 2.90.4 2.30.5 <1.90.5 a 3 2.91.0 2.91.0 2.00.1 <1.70.2 a 7 <1.50.0 c <1.50.0 b <1.50.0 a <1.50.0 a 10 7 0 1 4.90.1 4.90.1 4.50.1 4.40.2 1 3.70.3 3.60.3 4.20.2 4.10.2 3 3.41.5 3.51.8 3.80.8 3.80.8 7 3.30.8 3.10.8 3.00 .3 2.80.4 1 Values represent the mean plus or minus standard deviation of triplicate samples from 1 replication (n=3), expressed as log CFU/berry. a n=3, one replicate below limit of detection b n=3, two replicates below limit of detection c n=3, three repl icates below limit of detection Table 4 11. Fate of Salmonella spp. populations, inoculated with ca. 10 7 CFU/ml, on bruised and intact surfaces of freshly harvested full and red strawberries enumerated on TSAR and BSAR following incubation at 2 2C for up to 24 h. Maturity Time Bruised Intact (hour) TSAR BSAR TSAR BSAR Full Red 0 1 4.60.3 4.30.8 4.20.3 3.60.8 2 3.90.6 3.21.2 3.60.5 3.01.0 5 3.90.3 3.30.6 3.50.3 2.80.5 24 3.70.9 <3.41.0 a 3.10.4 <2.60.6 a Red 0 1 4.10.2 3.7 0.6 4.00.2 3.40.4 2 4.20.5 3.90.7 3.40.4 <2.80.8 a 5 3.60.5 2.71.1 3.40.3 2.70.7 24 3.70.5 3.30.7 2.90.4 2.00.5 1 Values represent the mean plus or minus standard deviation of triplicate samples from each of 2 replications (n=6), express ed as log CFU/berry. a n=6, one replicate below limit of detection

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74 Table 4 1 2 Fate of Salmonella spp. populations, inoculated with ca. 10 7 CFU/ml, on bruised and intact surfaces of freshly harvested full and red strawberries enumerated on TSAR and BSAR following incubation at 15.5 2C for up to 7 days. Maturity Time Bruised Intact ( day ) TSAR BSAR TSAR BSAR Full Red 0 1 4.80.3 4.70.4 4.40.2 4.10.5 1 3.90.5 3.70.6 3.90.5 3.70.6 3 4.50.5 4.30.5 3.80.6 3.60.7 7 2.81.2 2.71.1 <2.41 .3 b <2.51.2 b Red 0 1 4.40.5 4.20.8 4.00.5 3.60.6 1 3.70.9 3.41.0 2.80.6 2.50.7 3 2.81.0 <2.61.2 a 2.10.8 <1.90.6 a 7 <2.00.4 a <1.70.3 a <2.61.3 c <2.21.2 c 1 Values represent the mean plus or minus standard deviation of duplicate sample s from each of 3 replications (n=6), expressed as log CFU/berry. a n=6, one replicate below limit of detection b n=6, two replicates below limit of detection c n=6, one replicate negative upon enrichment Table 4 13 Fate of Salmonella spp. populations, ino culated with ca. 10 7 CFU/ml on bruised and intact surfaces of store bought strawberries stored under modified atmosphere storage (ca. 10% CO 2 5% O 2 ) at 2 2C for up to 24 h and enumerated on TSAR and BSAR. Time Bruised Intact (hour) TSAR BSAR TSAR B SAR 0 1 4.8 0.6 4.8 0.6 4.0 0.3 3.70.3 2 3.7 0.7 3.60.6 3.6 0.5 3.20.7 5 3.9 0.5 3.80.5 3.1 1.0 2.90.8 24 3.8 0.8 3.60.9 3.1 0.8 2.71.0 a 1 Values represent the mean plus or minus standard deviation of triplicate samples from each of 2 replication s (n=6), expressed as log CFU/berry. a n=6, one replicate below limit of detection

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75 Table 4 14 Fate of Salmonella spp. populations, inoculated with ca. 10 7 CFU/ml, on bruised and intact surfaces of store bought strawberries stored under modified atmospher e storage (ca. 10% CO 2 5% O 2 ) at 15.5 2C for up to 7 days and enumerated on TSAR and BSAR. Time Bruised Intact (day) TSAR BSAR TSAR BSAR 0 1 5.10.3 5.00.2 4.40.5 4.20.7 1 4.20.4 4.10.4 3.40.7 2.51.0 3 3.70.3 3.50.5 2.40.7 2.30.7 7 2.8 1.0 2.71.2 a 2.80.7 2.70.9 a 1 Values represent the mean plus or minus standard deviation of triplicate samples from each of 2 replications (n=6), expressed as log CFU/berry a n=6, one replicate below limit of detection

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76 Figure 4 1. Behavior of E. col i O157:H7 on bruised (closed symbols) or intact (open symbols) surfaces of store bought (square symbols) or freshly harvested (triangle symbols) strawberries over 24 h of storage at 2 2C (n=6). Error bars represent standard deviations. Figure 4 2. Behavior of E. coli O157:H7 on bruised (closed symbols) or intact (open symbols) surfaces of store bought (square symbols) or freshly harvested (triangle symbols) strawberries over 7 days of storage at 15.5 2C (n=6). Error bars represent standard devi ations.

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77 Figure 4 3. Behavior of E. coli O157:H7 on bruised (closed symbols) or intact (open symbols) surfaces of freshly harvested full red (square symbols) or red (triangle symbols) strawberries over 24 h of storage at 2 2C (n=6). Error bars rep resent standard deviations. Figure 4 4. Behavior of E. coli O157:H7 on bruised (closed symbols) or intact (open symbols) surfaces of freshly harvested full red (square symbols) or red (triangle symbols) strawberries over 7 days of storage at 15.5 2C (n=6). Error bars represent standard deviations.

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78 Figure 4 5. Behavior of E. coli O157:H7 on bruised (closed symbols) or intact (open symbols) surfaces of strawberries stored under regular air storage (square symbols) or modified atmosphere storage (triangle symbols) over 24 h at 2 2C (n=6). Error bars represent standard deviations. Figure 4 6. Behavior of E. coli O157:H7 on bruised (closed symbols) or intact (open symbols) surfaces of strawberries stored under regular air storage (square s ymbols) or modified atmosphere storage (triangle symbols) over 7 days at 15.5 2C (n=6). Error bars represent standard deviations.

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79 Figure 4 7. Behavior of Salmonella spp. on bruised (closed symbols) or intact (open symbols) surfaces of store bought (square symbols) or freshly harvested (triangle symbols) strawberries over 24 h of storage at 2 2C (n=6). Error bars represent standard deviations. Figure 4 8. Behavior of Salmonella spp. on bruised (closed symbols) or intact (open symbols) surfac es of store bought (square symbols) or freshly harvested (triangle symbols) strawberries over 7 days of storage at 15.5 2C (n=6). Error bars represent standard deviations.

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80 Figure 4 9. Behavior of Salmonella spp. on bruised (closed symbols) or intac t (open symbols) surfaces of freshly harvested full red (square symbols) or red (triangle symbols) strawberries over 24 h of storage at 2 2C (n=6). Error bars represent standard deviations. Figure 4 10. Behavior of Salmonella spp. on bruised (cl osed symbols) or intact (open symbols) surfaces of freshly harvested full red (square symbols) or red (triangle symbols) strawberries over 7 days of storage at 15.5 2C (n=6). Error bars represent standard deviations.

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81 Figure 4 11. Behavior of Salm onella spp. on bruised (closed symbols) or intact (open symbols) surfaces of strawberries stored under regular air storage (square symbols) or modified atmosphere storage (triangle symbols) over 24 h at 2 2C (n=6). Error bars represent standard deviati ons. Figure 4 12. Behavior of Salmonella spp. on bruised (closed symbols) or intact (open symbols) surfaces of strawberries stored under regular air storage (square symbols) or modified atmosphere storage (triangle symbols) over 7 days at 15.5 2C ( n=6). Error bars represent standard deviations.

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82 CHAPTER 5 DISCUSSION The recent E. coli O157:H7 outbreak implicating strawberries as a vector has raised concerns on the safety of strawberries. The practice of growing strawberries in close proximity to soil is believed to increase risks associated with biological hazards, yet very little is known about the fate of pathogens on strawberries. The goal of this research was to evaluate biological risks associated with postharvest handling of strawberries, i ncluding pathogen behavior on strawberries of two maturity stages, displayed at retail shelf or shipping temperature, and as affected by modified atmosphere packaging. Strawberry firmness is known to decrease extensively during ripening, and degree of firm ness typically correlates with the maturity stage of the strawberry. At harvest, strawberries with half red and red color were reported to be 25 and 10%, respectively, firmer than full red strawberries (Nunes et a l., 2006) ; t hough red strawberries were slightly firmer than full red strawberries, evidence suggests that firmness remains relatively unchanged from the to full red stage (Menager et al., 2004) Though firmness can decrease during refrigerated storage (4C ; Nunes et al., 2002) Nunes et al. (2006) found that the firmness of 2 out of 3 varieties did not change significantly when harvested full red and stored at 1C up to 8 d ays. Thus we hypothesized that bruising, without breaking the epidermal surface of the strawberry, would not affect the behavior of E. coli O157:H7 or Salmonella on strawberries, regardless of whether they were store bought or freshly harvested at the r ed or full red stage of maturity.

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83 At 15.5 2C, E. coli O157:H7 and Salmonella populations declined under all conditions, regardless of bruising. Escherichia coli O157:H7 populations declined by 1.6 to 1.8 log CFU/berry on store bought (full red) and f reshly harvested and full red strawberries (bruised and intact) over 7 days, when held at 15.5 2C. Under the same conditions, Salmonella populations declined by 1.4 to 2.4 log CFU/berry on store bought (full red) and freshly harvested and full red strawberries, respectively. The declines we observed are comparable to those of Yu et al. (2001) where declines of up to 2.3 l og CFU/g were observed when E. coli O157:H7 was inoculated onto outer surfaces of strawberries and held for 3 days at 10C. The larger decline observed by Yu et al. (2001) over the shorter amount of time (3 vs 7 days) may be due to the lower storage temperature. Similar population decreases were also reported for E. coli O157 and Salmonella populations inoculated onto fresh cut pi neapples, where declines of 1.4 and 3.3 log CFU/g, respectively, were reported over 7 days, when held at 12C (Strawn and Danyluk, 2010a) Pineapples and strawberries are considered high acid fruits, with a pH of less than 4.0, which more than likely co ntributed to the inability for E. coli O157:H7 and Salmonella growth (Bassett and McClure, 2008) That E. coli O157:H7 and Salmonella were not able to grow on the fresh cut surface of pineapples, where moisture and nutrients are in abundance, elucidates that pH may be a critical contributing factor in its behavior on strawberries as well as pineapples. Conversely, other fruits and vegetables, with relatively low pH are good substrates for E. coli O157:H7 and Salmonella growth and survival. Tomatoes, wi th a pH profile of ca. 3.99 4.37 (Asplund and Nurmi, 1991) are well documented to support growth of Salmonella on cut surfaces when held at 12C (Ma et al., 2010) Escherichia coli O157:H 7

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84 survived, with relatively no change in population density, for up to 10 days on cut mangoes (pH 4.2) held at 12C; while Salmonella grew during the first 3 days, then slowly declined over 28 days (Strawn and Danyluk, 2010b) Escherichia coli O157:H7 and Salmonella inoculated on minimally peach plugs) and held at 10C grew after 3 days and survived for up to 14 days (Alegre et al., 2010b) Salmonella was not able to grow, but could survive, on the outer intact surface of tomatoes over 7 days at 12C (Ma et al., 2010) Similarly, Salmonella did not grow on the skin of grape tomatoes (pH 4.67) held at 14C for up to 14 days (Beucha t and Mann, 2008) Pathogen growth on fruits and vegetables with low pH is seen in some cases but not others, indicating there are limiting growth factors beyond pH for E. coli O157:H7 and Salmonella on surfaces of strawberries, held at abusive temperatu res. These factors may include surface characteristics, strawberry variety, nutritional compositional differences and variance from berry to berry. Differences between our results and those of others may also be attributed to pathogen strains, as well a s level of inoculum concentration used. Salmonella populations on freshly harvested intact red strawberries, on day 3 at 15.5 2C, were significantly lower than E. coli O157:H7 populations on freshly harvested bruised full red strawberries by 2.2 log C FU/berry. Similarly, Salmonella populations declined more quickly and by more than E. coli inoculated onto whole zucchini squash held at 25C and 3 5C (Castro Rosas et al., 2010) The differences in behavior of the two pathogens on strawberries could be due to different mechanisms of attachment to the strawberry, which is beyond the scope of this project. However, as differences in behavior b etween the two pathogens were not found consistently

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85 throughout all experiments, significant differences seen at this time point might be due to differences in strawberry to strawberry characteristics (e.g. surface characteristics and degree of firmness). Also on day 3, Salmonella populations on red intact berries were lower than those on full red bruised berries by 1.5 log CFU/berry, providing further justification that the significant difference had more so to do with berry to berry variations. An alt ernative possibility may be a higher level of bruising occurred on these particular bruised strawberries (due to strawberry firmness variations), increasing moisture and nutrients availability, better supporting survival in both scenarios. At 2 2C, de clines were observed after 24 h under all experimental conditions. Experiments were not carried out longer than 1 day due to preliminary trials where population declines after 24 h of storage at 2 2C resulted in levels below the limit of detection (dat a not shown). No differences in declines between E. coli O157:H7 and Salmonella populations exist at each time point, under all conditions, regardless of bruising. Escherichia coli O157:H7 and Salmonella populations declined by ca. 0.8 1.3 log CFU/berry and 0.4 1.2 log CFU/berry, respectively, on bruised and intact surfaces of store bought (full red) and freshly harvested red and full red strawberries over 24 h when held at 2 2C. Similar results have been previously reported by Knudsen et al. (2001) where declines of 1 to 2 log CFU/sample were observed with both E. coli O157:H7 and Salmonella on outer surfaces of strawberry, when held at 5C over 7 days. Larger initial populations (exact data not shown ) were established at time 0 for these experiments, which can explain why survival occurred over the 7 days of storage. Flessa et al. (2005) established that popula tions of L. monocytogenes behaved differently on strawberries held at 24C when inoculated at low and high concentrations,

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86 with the latter showing slower declines. Das et al. (2006) also reported dose dependent differences in behavior of Salmonella Enteritidis on the surface o f cherry tomatoes. Escherichia coli O157:H7 declined by 1.3 to 1.8 log CFU/g on the surface of strawberries over 3 days at 5C, when initially inoculated at 4.3 log CFU/g (Yu et al., 2001) Cut pineapples stored at 4C supported survival of E. coli O157:H7 for 7 days; while Salmonella significantly declined by 1.1 log CFU/g after 3 days of storage at 4C (Strawn and Danyluk, 2010a) Results for E. coli O157:H7 on cut pineapples are quite different than what we observed on strawberries. Escherichia coli O157:H7 has been capable to survive in food matrices with low pH, as it has been implicated in outbreaks involving apple cider (Besser et al., 1993) and can survive in apple cider (pH 3.7 4.1), containing 0.1% sodium benzoate, for 21 days at 4C (Miller and Kaspar, 1994) Similar to population dynamics at 15.5C, E. coli O157:H7 and Salmonella are also able to survive, and som etimes grow, on produce with relatively acidic pH. Populations of E. coli O157:H7 inoculated at 5 log CFU/g were found to survive, with relatively no change, on cut mangoes and cut papayas over 28 days at 4C (Strawn and Danyluk, 2010b) Salmonella was able to grow on cut mangoes after 24 h of storage at 4C but declined after 3 days and survived for at least 28 days on cut papayas (Strawn and Danyluk, 2010b) Escherichia coli and Salmonella declined ca. 1 log CFU/plug over 14 days on minimal ly processed peaches stored at 5C (Alegre et al., 2010b) Salmonella was able to survive, with no significant deviations from initial populations of ca. 3 log units (whole log CFU/tomato; chopped log CFU/g), on the surface of whole and chopped tomatoes for 7 days when held at 4C (Ma et al., 2010) Beuchat and Mann (2008) also reported similar findings on chopped tomatoes stored at 4C over 10 days of storage.

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87 Pr oduce with higher pH than strawberries, including jalapeno pepper (3, 4, or 7C; Salmonella E. coli and E. coli O157:H7), Serrano pepper (3C; Salmonella and E. coli ), cilantro (4C; Salmonella ), zucchini squash (3 5C; E. coli and Salmonella ), and icebe rg lettuce (5C; E. coli O157:H7) do not support growth of Salmonella E. coli, or E. coli O157:H7 when inoculated onto intact produce surfaces and held at lower temperatures (Abdulraouf et al., 1993; Castro Rosas et al., 2011; Castro Rosas et al., 2010; Huff et al., 2012; Ma et al., 2010) Though growth is not commonly observed on intact surfaces of produce with low and near neutral pH, evidence does suggest, even at low pH, produce can support survival of pathogen s when inoculated onto flesh/pulp surfaces. E scherichia coli O157:H7 and Salmonella survived on strawberry surfaces, even at low temperatures, but did not grow. Though bruising of the strawberries did not influence E. coli O157:H7 and Salmonella behavior; different results have been documented for other commodities. Bruised McIntosh (pH 4.5) and Red Delicious (pH 5.5) apples were inoculated by injection of E. coli O157:H7 into bruised tissues then incubated at room temperature for 2 days. Pathogens grew in Red Delicious, but not in McIntosh; further investigations revealed that storage of McIntosh apples for 1 month at 4C before bruising and inoculation allowed populations of E. coli O157:H7 to reach those of Red Delicious by day 6 when stored at room te mperature. Bruised apple tissues were shown to have significantly higher pH than intact tissues (Ding man, 2000) Salmonella Montevideo, suspended in TSB and inoculated onto stem scars, survived better than when inoculated onto intact tomato skin over 7 day storage at 20 and 25C (Wei et al., 1995) Salmonella Montevideo suspended in distilled water and inoculated into punctured sites

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88 on full red and green tomatoes were able to either grow or remain unchanged over 24 h at 25C, with no differences observed between the two maturity levels (Wei et al., 1995) Injured jalapenos, with exposed pericarp tissue, did not support growth of E. coli O157:H7 or Salmonella at 7C for 14 days; however, L. monocytogenes initially declined but grew back to initial population levels. At 12C, injured jalapenos supported Salmonella and L. monocytogenes growth and survival of E. co li O157:H7 over 14 days (Huff et al., 2012) All of these studies examined the behavior of pathogens on exposed tissues of the produce at higher temperatures than tested in our experimental design, potentially explaining why enhanced growth and survival were observed in apples, tomatoes, and jalapenos. There are also other studies that indicate the ability of E. coli O157:H7, Salmonella and L. monocytogenes to survive on exposed tissue of strawberries (Flessa et al., 2005; Knudsen et al., 2001; Yu et al., 2001) Since our experiments did not bruise strawberries to a level where the epidermal surface was exposed to inner tissue, this could explain wh y only declines were observed for bruised strawberries. Modification of atmosphere surrounding produce commodities is utilized to prolong shelf life, as well as enhance quality attributes. However, careful consideration must be taken to ensure conditions inside the package do not become favorable for biological growth. Precautions should be considered to ensure respiration of the produce does not adversely affect quality attributes of the produce or enhance microbial growth. Abusive storage temperatures increase biological reactions, and high relative humidity can cause condensation to form on the surface of produce, promoting growth of mold or bacteria (Zagory et al., 1988)

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89 At 15.5C, no differences exist between E. coli O157:H7 and Salmonella behavior on strawberries stored under a modified atmosphere compared to ambient atmosphere storage. Escherichia coli O157:H7 and Salmonella populations decline d by ca. 1.1 1.2 log CFU/berry and 1.6 2.3 log CFU/berry, respectively, on bruised and intact strawberries stored under a modified atmosphere. Due to strawberry respiration, initial atmospheres of ca. 5.7%O 2 / 9.5%CO 2 changed to 0%O 2 / 35%CO 2 over 7 days of storage at 15.5 2C. Though respiration became anaerobic on day 3, the behavior of E. coli O157:H7 and Salmonella did not change when compared to storage under ambient conditions. Escherichia co li O157:H7 and Salmonella are facultative anaerobes and can survive and grow without the presence of oxygen. Though growth may be more favorable under aerobic conditions, survival under anaerobic conditions is not significantly altered from aerobic condit ions for E. coli O157:H7 and Salmonella on strawberries. Population differences in E. coli O157:H7 were noted during recovery on TSAR and SMACR following days 3 and 7 in modified atmosphere conditions. This may be an indication that anaerobic conditions stressed and injured E. coli O157:H7 cells. Selective media is a harsher environment when compared to nonselective media for bacteria to thrive, thus significant differences in populations between the two are often a good indicator that cells are stresse d. Han and Linton (2004a) have previously determined that E. coli O157:H7 becomes injured when stored at higher temperatures under acidic conditions. Injury can occur in a relatively short amount of time at higher temperatures. The low acidity, absence of oxygen, abusive temperature, and low nutrient and moisture availability could all have been contributing factors for the

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90 observed E. coli O157:H7 ce ll injury. In accordance with our data, Das et al. (2006) found no significant differences in behavior of Salmonella Enteritidis on cherry tomatoes stored under ambient and modified atmosphere conditions (6%O 2 /4%CO 2 ), when inoculated at 7.0 log CFU/tomato and held at either 7 or 22C. Differences in the behavior dynamics of E. coli O157:H7, Salmonella and L. innocua were not observed when inoculated onto minimally processed peaches and apples, regardless of storage under ambient or modified atmosphere conditions (Alegre et al., 2010a; Alegre et al., 2010b) At 2 2C, no differences between E. coli O157:H7 and Salmonella behavior under ambient and modified atmosphere conditions exist, regardless of bruising. Declines of ca. 0.8 1.1 log CFU/berry and 0.9 1.1 log CFU/berry are reported for E. coli O157:H7 and Salmonella respectively, on bruised and intact strawberries stored under modified atmosphere conditions. The initial atmosphere of 9.5%CO 2 / 5.9%O 2 remained relatively constant ov er 24 h of storage (9.1% CO 2 / 5 .4%O 2 ) at 2 2C. Because 2C is reasonably close to the recommended storage temperature for strawberries, no increased respiration rate was observed; hence, there was no change in atmosphere in the short amount of storage ti me. On sliced tomatoes stored under modified atmosphere (5%CO 2 /95%N 2 ), Salmonella Enteritidis populations declined during storage at 4C; survived, with little change, during storage at 10C; and behaved similarly to ambient conditions at both temperature s (Drosinos et al., 2000) Escherichia coli O157:H7 and Salmonella inoculate d onto peaches and apples declined over 14 days of storage at 5C under passive MA conditions (final atmosphere: peaches : 3.9%CO 2 / 18.5%O 2 ; apples : 2.8%CO 2 / 19.6%O 2 ), and no differences were observed

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91 between ambient and modified atmosphere conditions (Alegre et al., 2010a; Alegre et al., 2010b) Similar to our findings with freshly harvested strawberries, storage on day 3 at 15.5 2C under modified atmosphere, Salmonella populations were significantly lower than E. coli O157:H7 populations. Following 5 h of storage at 2 2C, Salmonella populations were significantly lower than E. coli O157:H7 populations. As there were no differences in E. coli O157:H7 and Salmonella behavior when stored under modified atmospher e and ambient conditions, these differences are likely due to berry to berry differences as previously discussed for the freshly harvested berries. Alt hough modification of the atmosphere did not affect the behavior of E. coli O157:H7 and Salmonella on st rawberries at both temperatures, there was an observed increase in overall aesthetic quality to the strawberries during modified atmosphere storage (data not shown). Strawberries appeared to have a more vibrant color and shine by the end of storage when h eld at 15.5 2C compared to strawberries stored under ambient conditions, which were dull brown by day 7 at 15.5 2C. Storage of strawberries under modified atmospheres has proven to be effective in maintaining overall quality of strawberries and exte nding its shelf life (Caner et al., 2008; Ozkaya et al., 2009; Siro et al., 2006) Visual comparisons between modified atmosphere packaging and ambient storage were not as obvious with strawberries stored at 2C, l ikely due to the short incubation period. However, as stated earlier, 2C is close to optimal temperature storage for strawberries; thus, visual changes are not expected to have changed extensively. Anaerobic respiration was seen during storage at 15.5C therefore off flavor development may have occurred due to formation and accumulation

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92 of ethanol and acetaldehyde (Kader, 2002) Larsen and Watkins (1995) described an accumulation of acetaldehyde, ethanol, and ethyl acetate in packaged strawberries stored under various modified atmosphere conditions. Additionally, ethyl acetate was found to be the highest correlated with off flavors in s trawberries stored under high carbon dioxide and low oxygen concentrations. Sensory analysis was not considered for our experiments since strawberries were inoculated with foodborne pathogens, but precautions to not induce anaerobic conditions should be c onsidered when modifying atmospheric conditions with strawberries.

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93 CHAPTER 6 CONCLUSIONS AND FUTURE WORK Strawberry firmness is known to decrease as ripening progresses; however, firmness stabilizes, with little to no change, from the to full red stage As hypothesized, strawberries that were bruised, without rupturing the skin, did not support the growth of Escherichia coli O157:H7 or Salmonella spp. at both low shipping temperature (2 2C) and abusive retail display temperature (15.5 2C). The a dditional storage time for strawberries purchased from local grocery stores did not compared with strawberries that were freshly harvested the same day. A modification o f atmosphere from ambient conditions did not support growth of E. coli O157:H7 or Salmonella spp. on bruised or intact strawberries, as predicted. This research reveals that strawberry surfaces are not suitable conditions for E. coli O157:H7 and Salmonel la spp. growth under all conditions tested. However, research to determine behavior of psychrotrohic bacteria and other spoilage organisms on bruised and intact strawberry surfaces could be conducted. Listeria monocytogenes is abundantly found in soil an d plant environment s thus risk of contamination, due to strawberry agricultural practices, is a reasonable concern. Additionally, L. monocytogenes grows at low temperatures, therefore, if contaminated with this psychrotroph, strawberries being shipped lo ng distances may potentially become favorable conditions for its growth. The behavior of L. monocytogenes on bruised and intact strawberries is unknown and could be explored. Strawberries exist in many shapes and sizes, and sometimes, differences from str awberry to strawberry of the same cultivar can be great. It is also reported that

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94 strawberries of different cultivars can have nutritional compositional differences, including amount of amino acids, organic acids, and soluble solids accumulation. These p hysical and biochemical differences may impact how pathogens behave on the surface of bruised and intact strawberries (e.g. attachment efficacy and nutrient bioavailability) and could be further investigated. If done, this follow up study should be a coll aborative effort to also examine the biochemical content of the strawberry cultivar being examined to further understand, if any significant differences are observed, which physical and chemical characteristics influence pathogenic behavior. Our bruising methodology was carefully conducted to ensure the bruise would not rupture the outer surface of the strawberry. Though studies have been conducted to examine the behavior of foodborne pathogenic microorganisms on the cut surface of strawberries, no resea rch has been conducted to examine their behavior on severely bruised strawberries, where juices are released, making nutrients and moisture available. Different degrees of bruising may make a difference as to how E. coli O157:H7 and Salmonella spp. behave on the surface of bruised strawberries. Postharvest strawberry losses due to bruising is a major concern for agriculturalists, therefore bruising severity and its effect on the dynamics of pathogen behavior is an important and relevant subject to explore With respect to modified atmosphere packaging, our methodology was simplified for practical purposes. Strawberries were packaged and examined individually, outside of its typical retail clamshell container. As we observed with our packaging system, str awberries can alter the atmosphere surrounding it due to its high respiration rate; therefore multiple strawberries packaged together may alter the environment much

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95 differently and more drastically. The behavior of E. coli O157:H7 and Salmonella spp. on s trawberries packaged together in a clamshell container with a controlled atmosphere environment, remains to be explored, to ensure only beneficial conditions for strawberries are maintained. This will portray a more realistic situation, and transfer studi es can also be conducted to examine the severity of transfer scenarios from one strawberry to another and how this may affect strawberry aesthetic properties. The recent E. coli O157:H7 strawberry outbreak in Oregon demonstrate s the potential for strawberr ies to be vectors of foodborne pathogens and how they can result in illness and death. This outbreak warrants the research of foodborne pathogen behavior on strawberries, which will allow us to further understand how they behave on strawberries and which conditions are favorable for their growth and survival. With this knowledge, suggestions can be made for risk analysis and prevention, which will allow government agencies to regulate the safe practice of growing and handling strawberries.

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98 CDC ( Cent ers for Disease Control and Prevention Cen t er), 2006. Update on multi state outbreak of E. coli O157:H7 infections from fresh spinach. Available at http://www.cdc.gov/foodborne/ecolispina ch/100606.htm Accessed on April 15, 2012. CDC ( Centers for Disease Control and Prevention Cen t er), 2008. Outbreak of Salmonella serotype Sainpaul infections associated with multiple raw produce items United States, 2008. Available at http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5734a1.htm Accessed on April 19, 2012. CDC ( Centers for Disease Control and Prevention Cen t er), 2011a. Foodborne Outbreak Online Database (FOOD). Available at http://wwwn.cdc.gov/foodborneoutbreaks/ Accessed on May 26, 2012. CDC ( Centers for Disease Control and Prevention Cen t er), 2011b. Investigation Announcement Multistate outbreak of E. coli O157:H7 i nfections linked to romaine lettuce. Available at http://www.cdc.gov/ecoli/2011/ecoliO157/romainelettuce/120711/index.html Accessed on April 15, 2012. CDC ( Centers f or Disease Control and Prevention Cen t er), 2011c. Vital signs: incidence and trends of infection with pathogens transmitted commonly through food foodborne diseases active surveillance network, 10 US Sites, 1996 2010. Available at http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6022a5.htm?s_cid=mm6022a5 _w Accessed on April 18, 2012. CDC ( Centers for Disease Control and Prevention Cen t er), 2012. Multistate foodborne outbrea ks. Available at http://www.cdc.gov/outbreaknet/outbreaks.html#salmonella Accessed on April 14, 2012. Chandler, C.K., Legard, D.E. 2012. Strawberry cultivars for annual production systems Available at http://strawberry.ifas.ufl.edu/strawberrycultivars.htm Accessed on March 28, 2012. Conner, D.E., Kotrola, J.S. 1995. Growth and survival of E scherichia coli O15 7:H7 under acidic conditions. Applied and Environmental Microbiology 61, 382 385. D'Aoust, J.Y., Maurer, J. 2007. Salmonella species. Third ed. ASM Press, Washington, D.C. D oust, J.Y. 1991. Pathogenicity of foodborne S almonella International Journal o f Food Microbiology 12, 17 40. Darrow, G.M. 1966. The Strawberry History, Breeding and Physiology. 1st ed. Holt, Rinehart and Winston, New York, NY.

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108 BIOGRAPHICAL SKE TCH Thao Nguyen was born in Houston, Texas to Ky Nguyen and Ngoi Tran. She has an older brother, Vuong Nguyen, and a younger sister, Yen Nguyen. Thao received her B.S. in f ood s cience and h uman n utrition, with an emphasis in f ood s cience, from the Univer sity of Florida in May 2010. In August 2010, she began her M.S. degree under the guidance of Dr. Michelle Danyluk, where she studied f ood s cience and h uman n utrition, with an emphasis in f ood m icrobiology. She also received a minor in p ackaging s cience w hile pursuing her M.S. degree. In the future, Thao wants to pursue a career in the industry with plans to become a food safety specialist.