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Microbiological Evaluation of Mexican Salsa

Permanent Link: http://ufdc.ufl.edu/UFE0022686/00001

Material Information

Title: Microbiological Evaluation of Mexican Salsa
Physical Description: 1 online resource (94 p.)
Language: english
Creator: Melazzini, Wendy
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: evaluation, foodborne, mexican, microbiological, pathogens, salsa
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Although Mexican food has become a mainstream in the US culture, little information about the safety of this food it is available. This study found that in the past 16 years (1990-2006) Mexican food was commonly related to foodborne outbreaks. Major bacteria reported in Mexican foodborne outbreaks were Salmonella spp., Clostridium spp., Shigella spp. and Staphylococcus. Most common location of the outbreaks reported for Mexican food was restaurants followed by private home. The results in this study, although limited by a small sample size and the evaluation of samples in only one city, indicated that salsa purchased from commercial restaurant have overall poor microbiological quality. Despite its low pH, restaurant-salsa samples if contaminated may present a good media for Salmonella spp. and Staphylococcus aureus survival during typical storage and consumption time. This information may be useful in the developing of safety material for the training and management of Mexican style food establishments.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Wendy Melazzini.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Simonne, Amarat H.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022686:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022686/00001

Material Information

Title: Microbiological Evaluation of Mexican Salsa
Physical Description: 1 online resource (94 p.)
Language: english
Creator: Melazzini, Wendy
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: evaluation, foodborne, mexican, microbiological, pathogens, salsa
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Although Mexican food has become a mainstream in the US culture, little information about the safety of this food it is available. This study found that in the past 16 years (1990-2006) Mexican food was commonly related to foodborne outbreaks. Major bacteria reported in Mexican foodborne outbreaks were Salmonella spp., Clostridium spp., Shigella spp. and Staphylococcus. Most common location of the outbreaks reported for Mexican food was restaurants followed by private home. The results in this study, although limited by a small sample size and the evaluation of samples in only one city, indicated that salsa purchased from commercial restaurant have overall poor microbiological quality. Despite its low pH, restaurant-salsa samples if contaminated may present a good media for Salmonella spp. and Staphylococcus aureus survival during typical storage and consumption time. This information may be useful in the developing of safety material for the training and management of Mexican style food establishments.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Wendy Melazzini.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Simonne, Amarat H.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022686:00001


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1 MICROBIOLOGICAL EVALUATI ON OF MEXICAN SALSA By WENDY FRANCO MELAZZINI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Wendy Franco Melazzini

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3 To our dreams (For two brighter futures)

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4 ACKNOWLEDGMENTS I sincerely want to express my gratitude towards my major advisor, Dr. A marat H. Simonne, who helped me to complete this study with her valuable advice, guidance and support. I extend special gratitude to my committee members (Dr. Maurice R. Marshall and Dr. Samuel R. Farrah) for their suggestions, advice and hel p. My deep appreciation also goes to Wei-Yea Hsu for her technical support, advice, time, gene rosity, and support in the development of this project. I express my most deep gratitude and love to my beloved husband Alfo, who always believed in me. His patience, encourage and love ga ve me the strength to complete this program and to look for a better future for our family. I would also like to thank my family (Milka, Jose Luis, Mauri, Patty, and little Ulri ch) for their love and support. My family and friends have been a constant source of encouragement and wi se advice for which I am very grateful. I would like to thank Nuria, Paula, Aleks, Ju an Manuel, Thabile, Claudia P., Leann, Mike, Cesar and Claudia F., who gave me their friends hip and support throughout this adventure. And to all my fellow classmates at the University of Florida a big thanks a nd mission accomplished! I would also like to thank the pr ofessors and staff at Food Science Department who in one way or another welcome me in the program and have left their contribution in this beautiful experience. Finally, I would like to thank the Fulbright Scholarship Program throughout its officers and staff members both here in th e United States as well in Bolivia for believe in me and gave me the opportunity to be a graduate student at the University of Florida. This research was partially funded by the USDA-CSREES-N IFSI (contract number 2006-5110-03597).

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 ABSTRACT...................................................................................................................................11 CHAPTER 1 INTRODUCTION ..................................................................................................................13 2 LITERATURE REVIEW.......................................................................................................16 Ethnic Food Safety Concerns.................................................................................................16 Mexican Food.........................................................................................................................20 Mexican Salsa.........................................................................................................................22 Mexican Food Safety Trends 1990-2000...............................................................................25 Selected Study Microorganisms.............................................................................................27 Salmonella spp.................................................................................................................29 Staphylococcus aureus ....................................................................................................31 3 MATERIALS AND METHODS........................................................................................... 35 Assessing Mexican Food Safety Trends................................................................................. 35 Microbiological Quality of Commercial Salsa....................................................................... 35 Sampling of Restaurants..................................................................................................35 Salsa Samples.................................................................................................................. 36 Sample Preparation..........................................................................................................36 Aerobic Plate Count and Total Coliform Count.............................................................. 36 Salmonella Determination............................................................................................... 37 Staphylococcus aureus Determination............................................................................ 37 Screening of Methicillin Resistant Staphylococcus aureus (MRSA).............................. 39 Microbiological Challenge Study ...........................................................................................39 Salsa Samples.................................................................................................................. 39 Bacterial Cultures Preparation......................................................................................... 40 Sample Inoculation..........................................................................................................41 Sample Storage................................................................................................................41 Staphylococcus aureus Challenge Study ......................................................................... 42 Salmonella Challenge Study ............................................................................................42 Statistical Analysis........................................................................................................... .......43

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6 4 RESULTS AND DISCUSSION............................................................................................. 45 Assessing Mexican Food Safety Trends................................................................................. 45 Microbiological Quality of Commercial Salsa....................................................................... 50 Temperature and pH Profiles........................................................................................... 50 Microbiological Quality.................................................................................................. 51 Total aerobic count.................................................................................................53 Total coliform count............................................................................................... 54 Salmonella spp.........................................................................................................55 Staphylococcus aureus .............................................................................................56 Methicillin resistant Staphylococus aureus (MRSA) screening............................... 57 Microbiological Challenge Study........................................................................................... 58 Staphylococcus aureus Study..........................................................................................59 Salmonella Study............................................................................................................. 61 5 SUMMARY AND CONCLUSION....................................................................................... 64 APPENDIX A PATHOGE NS DISTRIBUTION IN MEXICAN FOODBORNE ILLNESS OUTBREAKS (1990-2006) REPORTED BY THE CDC..................................................... 67 B MICROBIOLOGICAL QUALITY OF COMM ERCIAL M EXICAN SALSA (FL OW DIAGRAM)............................................................................................................................68 C SAMPLE PREPARATION FOR MICROBI OLOGICAL CHALLENGE STUDY (FLOW DIAGRAM).............................................................................................................. 70 D MICROBIOLOGICAL CHAL LENGE ST UDY IN COMMERCIAL MEXICAN SALSA (FLOW DIAGRAM)................................................................................................. 71 E COMMERCIAL SALSA SAMPLES.....................................................................................73 F GUIDELINES FOR THE MICROBIOLOGICA L QUALITY OF SOME READYTOEAT FOODS AT POINT OF SALE...................................................................................... 74 G MICROBIOLOGICAL QUALITY OF COMM ERCIAL MEXICAN SALSA: R AW DATA.....................................................................................................................................76 H MICROBIOLOGICAL QUALITY OF COMMERCIAL MEXICAN SA LSA: STATISTICAL ANALYSIS..................................................................................................78 I Staphylococcus aureus MICROBIOLOGICAL CHALLENGE STUDY: RAW DATA...... 79 J Staphylococcus aureus MICROBIOLOGICAL CHAL LENGE STUDY: STATISTICAL ANALYSIS..................................................................................................82 K Salmonella MICROBIOLOGICAL CHALLENGE STUDY: RAW DATA......................... 83

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7 L Salmonella MICROBIOLOGICAL CHALLENGE STUDY: STATISTICAL ANALYSIS ....................................................................................................................... ......85 LIST OF REFERENCES...............................................................................................................86 BIOGRAPHICAL SKETCH.........................................................................................................94

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8 LIST OF TABLES Table page 2-1 Most common types of food service es tablishments reported by surveyed food inspectors..................................................................................................................... ......194-1 Temperature and pH profiles of commer cial red salsa by type of restaurant....................514-2 Temperature and pH profiles of comm ercial red salsa by type of salsa............................ 514-3 Microbiological quality of Me xican salsa by restaurant types.......................................... 554-4 Microbiological quality of Mexican salsa by salsa type.................................................... 554-5 Staphylococcus aureus strains isolated from commercial salsa........................................ 574-6 Survival of Staphylococcus aureus in salsa samples during storage at 20 2C............... 614-7 Survival of Staphylococcus aureus in salsa samples during storage at 4 2C................. 614-8 Presence or absence of Salmonella spp. on inoculated salsa samples stored at 20 2C.....................................................................................................................................624-9 Presence or absence of Salmonella spp. on inoculated salsa samp les stored at 4 2C....62A-1 Pathogens distribution in Mexican foodbor ne illness outbreaks reported by the CDC (1990-2006)........................................................................................................................67F-1 Guidelines for the microbiological quality of some ready-to-eat foods at point of sale.... 75G-1 Microbiological quality of commercial Mexi can salsa: raw data...................................... 76H-1 Microbiological quality of commercial Mexican sals a: statistical analysis....................... 78I-1 Staphylococcus aureus microbiological challe nge study: raw data................................... 79J-1 Staphylococcus aureus microbiological challenge st udy: statistical analysis...................82K-1 Salmonella microbiological challe nge study: raw data..................................................... 83L-1 Salmonella microbiological challenge st udy: statistical analysis......................................85

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9 LIST OF FIGURES Figure page 2-1 Ethnic food practice concerns............................................................................................ 202-2 Green and red salsa....................................................................................................... ....222-3 Guacamole.................................................................................................................. .......232-4 Fresh salsa................................................................................................................ ..........232-5 Number of outbreaks per year as related to top thre Ethnic foods..................................... 262-6 Breakdown of etiology in all Ethnic foods........................................................................ 263-1 Petrifilm staph express plates......................................................................................... 383-2 Rapid Chek SELECTTM for Salmonella .........................................................................434-1 Total number of foodborne illness outbr eaks in Mexican food outbreaks in the US (1990-2006)........................................................................................................................464-2 Number of foodborne illn ess outbreaks associated with Mexican food (1990 2006).... 474-3 Most prevalent known e tiology involved in Mexican food outbreaks (1990-2006).........484-4 Location of foodborne illness outbreaks associated with Mexican foods due to known etiology from (1990-2006)..................................................................................... 494-5 Mexican food vehicles reported in foodborne illness outbreaks (1990-2006)..................494-6 The number of acceptable and unsatisfactory samples according to scientific criteria to ensure safe food............................................................................................................ .524-7 Disk diffusion method, inhi bition zone diameters for positive, negative control and isolated Staphylococcus aureus .........................................................................................584-8 Staphylococcus aureus survival in salsa samples storag e at room temperature (20C).... 594-9 Staphylococcus aureus survival in salsa samples storag e at refrigeration temperatures (4C)...................................................................................................................................59B-1 Flow diagram for commer cial Mexican salsa microbiological quality evaluation............ 69C-1 Sample preparation for the microbiological challenge study............................................ 70D-1 Flow diagrams for microbiological chal lenge study in commercial Mexican salsa.......... 72

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10 E-1 Commercial salsa samples: fr esh salsa (A) and red salsa (B)............................................ 73

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11 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MICROBIOLOGICAL EVALUATI ON OF MEXICAN SALSA By Wendy Franco Melazzini December 2008 Chair: Amarat H. Simonne Major: Food Science and Human Nutrition Annually, an average of 941 foodborne outbreaks we re reported in the US and more than 50% of these outbreaks were asso ciated with food service establishments including restaurants. As a result, in recent years consumption of food away from home was associated with an increased risk of gastrointestinal diseases. Ch anges in consumer habits (US population is eating out side the home more frequently) as well as the increase of the US ethnic population, Americans are more familiar with taste and flavors from foreign lands, often called ethnic food or ethnic cuisine.Mexican st yle food is one of the most popul ar ethnic foods, and along with Italian and Asian food, become mainstream foods in the US. Based on the CDC outbreaks data from 1990 to 2006 we found Mexican style food is often implicated in the foodborne outbreaks (560 out breaks; 18,581 cases). Of these 345 outbreaks (8,222 cases) were positively linked to a specific pathogen, 215 (10,359 cases) were attributed to unknown etiology. Pathogens often found in Mexican style food were Salmonella spp. (34%), Clostridium spp. (24%), Shigella spp. (6%), Staphylococcus (5%), and E. coli (5%). Foods commonly associated in the outbreaks were multiple food products (22%) followed by tacos (18%), chili (9%), and salsa (9 %). Majority of the outbreaks occurred in restaurant or delicatessens (46%) followed by private home (16%), workplace (8%), and school (6%).

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12 Because salsa is one of the most common side dish in the Mexican style foods, we examined microbiological qua lity (APC, total coliform, Staphylococcus aureus and Salmonella ) of commercial red and fresh sals a from 8 restaurants in Gainesv ille, FL twice during a six month period. Although, salsa samples were of poor micr obiological quality (h igh bacterial counts), none were above potentially hazardous limits. In general, red salsa had better microbiological quality (lower counts) than fresh salsa, but none of the samples tested positive for Salmonella. Salsa samples collected from locally owned restau rants tend to have higher bacterial counts than those from chain restaurants. Temperatures fo r how salsa samples were sold ranged between 4.9 to 27.0C, although most of the samples were sold at less than 10C. Fresh salsa samples were typically sold at lower temper ature. Salsa samples pH profile was between 3.4 and 4.3, and there was not significant difference between fresh and red salsa pH. Based on the microbial challenge study, regardle ss of its acidic pH salsa is capable to support survival of Staphylococcus aureus and Salmonella spp. long enough to cause foodborne illness to consumers. Inoculated salsa samples stored at room temperature (20C) sustained Staphylococcus aureus and Salmonella spp. survival for up to 24 h. Although Staphylococcus aureus decreased over time, the pathogen load was still high [2.15 log CFU/g when salsa inoculated at low levels (2.20 l og CFU/g), and 3.56 log CFU/g when inoculated at high levels (3.20 log CFU/g)]. Samples stored at refrigeration temperatures (4C) support Staphylococcus aureus survival for up to 7 days and Salmonella spp. for up to 3 days. We conclude that salsa if contaminated presents a potential health hazard. The biggest concern about salsa is that its usually served in combination with cooked i ngredients, which may increase chances for food contamination and bacterial growth.

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13 CHAPTER 1 INTRODUCTION Foodborne diseases are important worldwide as a public health problem. Although it is difficult to obtain a true estimate of foodborne diseases, according to the World Health organization (WHO) 1.8 million children died fro m acute diarrheal diseases, especially in developing countries (WHO, 2007). Contaminated food contribu tes to 1.5 billion cases of diarrhea worldwide, resulting in more than 3 million of premature deaths, mostly in children (WHO, 2002 and 2008). In the United States (US), the Center for Disease Control and Prevention (CDC) estimates that 76 million cases of foodborne diseases occurs each year with 325,000 hospitalizations and 5,000 deaths (Mead et al., 1999). According to the Center of Science in the Public interest (CSPI) from 1990 to 2005, a total of 5,316 foodborne outbreaks occurred with 157,830 individual cases. Seaf ood, produce, poultry, beef, and eggs were responsible for 60% of all outbreaks and 55% of disease cases. Produce items, by far, were implicated in the largest number of outbr eaks and cases (713 out breaks, 34,049 cases), and accounted for 22% of the total outbreaks in the 2007 CSPI database (CSPI, 2007). In past decades, the US population has e xperienced a demographic change with an exponential growth of the ethnic population. Currently a ccording to the US Census Bureau, 15% of the US population are of Hisp anic origin, with a majority being Mexican-American, and the Hispanic population is expected to reach 24% of the total population by 2050 (US Census Bureau, 2008). The demographic change and the increased growth of the ethnic food sector including ethnic food restaurants have led Americans to tast e new flavors and become more familiar with foods from international lands. Furthermore, as the per capita income increases, especially

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14 among ethnic populations, there has been an in crease in budgets for food away from home (Stewart et al., 2004; Geisler, 2007). In recent years, Mexican cuisine has become on e of the mainstream ethnic foods in the US, along with Italian and Asian cuisin es. It is common to find tortill as, refried beans, quesadillas, and guacamole in general groceries stores, restau rants and specialty stores. A similar trend is found in the restaurant industr y; among the top 100 menu cate gories, Mexican is the second largest segment with 12.5% of retail sales (Sloan, 2003). Furthe rmore, according to Packaged Facts, a market research firm, 46% of people in the US households who participated in the survey ate Mexican food four or mo re times per month (Dwyers, 1998). However, little information on ethnic food safe ty is available. Simonne et al. (2004) reported the incidences of foodborne diseases associated with et hnic foods in the US using the CDC foodborne illness data fr om 1990-2000. There was an increas ing trend (3 to 10 %) in the number of the outbreaks associat ed with ethnic foods (Mexica n, Italian, and Asian) from 1990 to 2000. In descending order, Mexican (41%), Italia n (39%), and Asian (20 %) foods represent the most frequent sources of repor ted foodborne diseases outbreaks. The most frequent location for ethnic food related outbreaks were in descending order: restaurant s (43%), private homes (21%), schools (7%), and others (29%) (Simonne et al., 2004). Among Mexican foods implicated in the outbreaks it appeared that salsa was one of the common food item in those foods (Simonne et al., 2004), thus salsa was selected in this study as a target food for its microbiologi cal evaluation. The selection of salsa as study food was based upon three major criteria. First, sals a is used as a side item in ev ery Mexican dish, as well as its being often used fresh (unprocessed) as a dip fo r snacks. Second, three of the major ingredients (tomato, onion, and cilantro) of salsa were ofte n implicated in the foodborne outbreaks (FDA,

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15 2004); therefore salsa may represent a good type of food for simulating pathogen contamination. Finally, preparation of salsa requires excessive handling of each and every ingredient, which increases the probability for b acterial cross-contamination from food handlers and equipment. The objectives of this study ar e to: (a) determine most prev alent etiology (microorganism) and vehicle (prepared food) related with Me xican foodborne outbreaks from the CDC foodborne diseases data from 1990 to 2006, (b) screen selected microbial populatio n [total aerobic plate count (APC), fecal coliform, Staphylococcus aureus and Salmonella spp.] in salsa samples from chain and locally owned restaurant s, and (c) conduct microbial chal lenge studies in salsa at room (20C) and refrigerated (4C) temp eratures to simulate typical practices in food service settings. This information could be used in developing food safety guidelines a nd educational programs for ethnic food vendors in this market segment.

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16 CHAPTER 2 LITERATURE REVIEW Ethnic Food Safety Concerns In the US annually, an average of 941 f oodborne illn ess outbreaks were reported by the Center for Disease Control and Prevention (CDC) from 1990 to 2006 (CDC, 2008). More than 50% of those outbreaks were attributed to food service sectors (Olsen et al., 2000). Moreover, results from the CDC FoodNet case co ntrol studies indicated that consumption of food outside the home was related to an increased risk of gastrointestinal illness and with specific types of food borne pat hogens (Shiferaw et al., 2004). In restaurant-related outbreaks, the most co mmon risk factors co rrelated to increased occurrence of foodborne illness and outbreaks as reported by the Environmental Health Specialists Network (EHS-Net) are: handling of food by an infected person or carrier, barehand contact with food, improper temperature control of potentia l hazardous foods, and lack of proper training for food handl ers (Hedber et al., 2006). In the last few years demand for ethnic food has shown an exponential growth, and it is projected that the ethnic food market will reach $75 billion record sales in 2008, and 65% and 35% of which will correspond to the restaurant and supermarket sectors, respectively (Geisler, 2007). In fact, the market for ethnic food has grown to such an extent that Italian, Mexican and Asian cuisines have joined th e mainstream foods. According to the National Restaurant Association's latest consumer study, Ethnic Cuisines II those three cuisines have become integrated into the American culture that they are no longer considered ethnic (Mills, 2000). There are several factors that affect the ethnic food market incl uding: increases in Americans expenditures in food away from home, demographic changes that influence Americans tastes and preferences, and the fast growing restaurant i ndustry (Stewart, et al. 2004.

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17 Food consumption away from home in the US has increased significantly, and in 2006 total expenditures from food consumed out side the home was $529,100 million, representing a 6% increase from 2005; 73% of the expenditure we re related to restaurants (ERS, 2005). In the past decade there has been a significan t increase of ethnic populations in the US. The largest ethnic group is the Hispanic population (15.1% or 45.5 million) followed by Asian (5% or 15.2 million). Hispanic was the fastest-growing ethnic group with a 3.3 percent increase between 2006 and 2007, while Asian wa s the second fastest-growing ethnic group, with a 2.9 percent population increase during the same period (Bernstein, 2008). According to the 2000 US Census, among Hispanic minoritie s, Mexicans are by far the largest with 28.3 million Mexican-born people out of the count rys 45.5 million total Latinos (US Census Bureau, 2008; Geisler, 2007). The Hispanic popu lation is expected to grow 40% by 2010 (US Census Bureau, 2008). As a result of this demographic change, the US population has become familiar with foods from foreign lands. The most preferre d among ethnic cuisines include pasta with shrimp for Italian, quesadilla for Mexican and vegetable stir fried fo r Asian (Sloan, 2002; Roseman, 2006). The restaurant and food service sectors have also shown increasing expansion. Restaurant industry sales are expected to reach a record high of $558 billion in 2008, an increase of 4.4% from 2007 (National Restaura nt Association, 2008). Fu ll-service restaurants will show higher increases than fast food re staurants, rising 18% and 6% respectively by 2020 (Stewart, 2004). Ethnic food was the fastest growing sector in this industry (Sloan, 2002; NRA, 2005). Few examples of fast causal restaurants that offer ethnic cuisines are Chipotle, Taco Cabana, La Salsa, Qdoba, or Baja Fresh (S outhwestern, Mexican, & Tex-Mex), Starbucks

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18 (Italian espresso), Panera bread (Italian paninis), and P.F. Cha ngs (Chinese) as well as other smaller local ethnic eateries in suburban areas (Paschel, 2007). As ethnic food establishments become hi ghly prevalent across the United States, limited food safety information pertaining to th ese foods is available. Moreover, science based inspection requirements, resources, and tr aining materials that focus on ingredients, preparation methods, storage and potential haza rds are not available (Simonne et al., 2004). Mauer et al (2006) surveyed 341 food safety professionals to identify concerns they may have on ethnic food safety. Participants were food safety inspectors from local jurisdictions (69%) [cou nty, city, township and district ag encies] federal/state jurisdictions (21%), and others including tribal and military agencies, industry, and consultants (10%). Respondents reported a wide vari ety of ethnic foods being pr epared, stored, and served in food establishments in their jurisdictions (Table 2-1). Chinese and Mexican/Latin American food were found in 90% of the partic ipating jurisdictions. Although not considered as mainstream, other ethnic foods such as Thai, Southeast Asian, Near/Middle Eastern and Indian/Pakistani food establishments were f ound in more than 47% of the participating jurisdictions.

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19 Table 2-1. Most common types of food servic e establishments repo rted by surveyed food inspectors Type of food Percentage of respondents Chinese 90.09 Mexican/Latin American 88.29 Thai 57.66 Southeast Asian 51.95 Indian/Pakistani 50.75 Near/Middle Eastern 47.15 Polynesian 21.32 Ethiopian 18.02 Japanese 13.51 Korean 6.92 African 6.92 Eastern European 5.15 Native American 4.50 Greek 3.90 Caribbean 3.00 South American 1.80 Adapted from Mauer et al., 2006 with permission from the Journal of Environmental Health June 2006, a publication of the National Environmental Health Association ( www.neha.org) C opyright 2006 Although Asian and Mexican/Latin American food were the most commonly available ethnic foods, few of these foods were placed on the list of most concern by the participants of the survey. It is likely that the food safety concern is more centered on foods that these professionals are not familiar with rather than specific knowledge of hazards associated with those foods. However food safety trends show th at some new mainstream ethnic foods, such as Mexican food, have an increa sing trend in foodborne outbreaks reported in the US. This is an indicator that more control, regulation, re search, and even training for food handlers and food inspectors is needed (Simonne et al., 2004 ; Adachi et al., 2002; Campbell et al., 2001). Improper food storage temperat ure, cross-contamination, and worker hygiene/illness were reported by the respondents as the most impor tant risk factors cont ributing to bacterial

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20 contamination in food service establishments. The majority of food-handling concerns were on worker hygiene/illness and cross-contaminat ion (Mauer et al., 2006 ). These poor handling practices are the same food safety concerns fo r mainstream foods and consistent with the practices reported by the CDC as contributing to foodborne illness outbreaks (FDA, National Retail Food Team, 2004). Finally, language barrier and lack of food safety knowledge are other important contributing factors diminishing ethnic food safety. Language barrier does not only affect food establishments, but also the whole food industry which employs the highest proportion of foreign workers. To ensure proper implem entation of ethnic food safety practices, food safety professionals will need to have tool s for communication with non-native food service workers as well as culturally appropriate e ducational materials and inspection practices (Mauer et al., 2006). 0102030405060708090 Improper food temperature Cross-contamination Worker hygiene/illness Unapproved food source Vermin Language Response in % Figure 2-1. Ethnic food practice co ncerns (Adapted from Mauer et al., 2006 with permission from the Journal of Environmental Health June 2006, a publication of the National Environmental Health Association ( www.neha.org ) C opyright 2006) Mexican Food Hispanic American cuis ine is a complex mi x of native and importe d flavors and tastes due to the colonization. Thus, it is a mix between traditions, cultu re and habits of the old

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21 and new worlds. It is a blend of foods and preparation styles of the native people of Central and South America and the culinary traditions of the Europeans who discovered these lands (Sloan, 2000; Long-Solis and Vargas, 2005). Culinary experts regard Mexican food as th e most interesting and complex in Latin America because it has the deepest roots in the indigenous cuisine of mezzo-America (Sloan, 2000). After the arrival of the Spanish, the Mexica n diet changed forever. The combination of the local and those food products introduced by Spaniards initiated the blending of the indigenous foods with Spanish cuisine, changi ng nutritional value and taste in the Mexican cuisine (Long Solis and Vargas, 2005). Ancient and modern Mexican cuisine has th ree major ingredients: corn, beans, and squash. These three crops were originated a nd domesticated in Me xico during the pre Hispanic time. These ingredients constitute the pr incipal food in ancient Mexico as well as in the modern diet. The most common meals prep ared with corn are Atole, beverages, Huitlacoche, Tamales and Torti llas (FDA, Training CDC 2007). Vegetables and fruits, such as amaranth, a vocados, cactus paddles, chayote, chocolate, jicama, peppers, plantains, tomatoes and tomatillos, pineapple, papaya, and guava are also important part of the Mexican cu isine. However, meat, citrus fruits, rice, sugar, wheat and spices were introduced into the Mexican cuis ine by the Spaniards (Long Solis and Vargas, 2005). In general, Mexican food is known for being mild to very spicy, thanks to the use of chili pepper. Americanized Mexican restaurants, catered to non-Hispanic Americans, offer popular dishes such as ground beef tacos, enchiladas, burritos, tostadas, chili rellenos, quesadillas, rice and beans, while others, cater to people of Mexican and Central American heritage, offering more traditional version of foods such as buche (pork stomach), lengua

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22 (beef tongue), menudo (a stew made with beef intestines and hominy), and specialty Mexican drinks such as horchata and tama rindo (Long Solis and Vargas, 2005). Typical Mexican dishes often have a combin ation of cooked and fresh ingredients. A taco, for example, it is a corn tortilla folded and filled with a selection of cooked meat (pork, beef or chicken), fresh vegetables (e.g. toma toes, lettuce), dairy pr oducts (e.g. cheese, sour cream), refried beans, and salsa. Salsa itself is a mix of different ot her fresh ingredients. Mexican Salsa According to the American Heritage Dictionary, salsa, the Spanish (Mexican) word for sauce, is a spicy product containing c hopped, uncooked vegetables or fruits such as tomatoes, onions, chili peppers, cilantro, and other seasonings. Salsa is one of the most common side dishes used in Mexican cuisine. It is consumed with tacos, tamales, quesadillas, and steaks. Salsa is also commonly served in the US as salsa dip or with snacks such as nachos. Different types of salsa exists, however the most recognized in the US are: red sauce, green sauce, fresh sauce and guacamole (Adachi et al., 2002). Red salsa salsa roja is used as a condiment in Mexican and southwestern US cuisine, and usually is made with cooked tomatoes, chil i peppers, onions, garlic and fresh cilantro (Figure 2-2 right). However, red salsa used for dips may be prepared from fresh ingredients without a cooking step. Green salsa salsa verde is usually made with cooked tomatillos, chili peppers, onions, garlic and fresh cilantro (Figure 2-2 left). Figure 2-2. Green sals a (L) and red salsa (R)

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23 Guacamole is a sauce in which the main in gredient is avocado, seasoned with lime, onions, salt, chili peppers a nd cilantro (Figure 2-3). Figure 2-3. Guacamole Raw salsa, also known as pico de gallo, or salsa picada ("chopped sauce"), salsa mexicana ("Mexican sauce"), or salsa fresca ("fresh sauce"), is usua lly made with raw diced tomatoes, lime juice, chili peppers, onions, ci lantro leaves, and other coarsely chopped ingredients (Figure 2-4). Figure 2-4. Fresh salsa Holding times and temperatures, contaminated equipment, cross contamination, improper cooling, poor personal hygiene, and foods from unsafe sources are among food safety risk factors for salsa described by the Food and Drug Administration (FDA) (FDA, 2004). Salsa requires considerable handling duri ng preparation and it is usually served at slightly elevated temperatures after prepar ation, and therefore sus ceptible to bacterial contamination and growth. Handling of fresh pr oduce ingredients in the preparation of salsa increases the risk of bacterial contamination a nd growth. When intact plant materials (fresh

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24 produce) are chopped or sliced, th e natural exterior barrier are broken causing plant cellular fluids to leak out and provide nutrient for bacteria to grow. Therefore, if the bacteria are present or survive, they can grow and create potential opportunity to further spread (UFPA, 2001). Improper sanitation during the production/prepar ation of fresh ingredient for salsa as well other fresh produce ingredie nts increases the potential for contamination by pathogens. Furthermore, the amount of handling and pr oduct mixing when preparing salsa can offer additional opportunities for contamination or sp reading contamination to large volumes. The potential for bacteria to survive and grow is increased by the high moisture and nutrient content of fresh-cut vegetables, the absence of a lethal process (e.g., heat) during production to eliminate pathogens, and the potential for temperature abuse during processing, storage, and transportation (UFPA, 2001). Foodborne microbial pathogens associated with consumption of fresh vegetables include Cyclospora cayetanensis Escherichia coli O157:H7, hepatitis A virus, Listeria monocytogenes, Norovirus, Salmonella spp., and Shigella spp. (FDA, 2008). Campbell et al. (2001) analyzed salsa made in restaurants, and found a high correlation between infection with Salmonella Thompson and cilantro from fresh salsa; they also found that refrigeration can limit growth of Salmonella species on cilantro for at least 3 days and in freshly made salsa for at least 1 day. Despite the low pH in salsa, the authors found that Salmonella can grow in salsa when it was stored at room temperature (Campbell et al., 2001). While it is a popular notion that low pH protects against bact erial growth, Adachi et al. (2002) analyzed Mexican sauces for the presence of enteric pa thogens, and found that pH is an ineffective deterrent for E. coli growth, which could be due to adaptability of the E. coli to acidic conditions. In the same study the author s found that the temperature profile at which sauces were prepared and serv e plays an important role in the growth and contamination.

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25 Samples prepared and kept at room temperat ure were more prone to have pathogens, and hygiene practices among handlers, such as mu ltiple workers handling, and serving patrons reused sauce, also play an important role in contamination (Adachi et al., 2002) Raghubeer et al. (2000) found that E. coli, Listeria monocytogenes and Enterotoxigenic Staphylococcus aureus survived in fresh salsa for extende d periods of storage at different temperatures. After one week storage at 4C E. coli populations decreased from 1.24 x 108 CFU/g to 3.6 x 107 CFU/g (1-log decrease); however at room temperature, no E. coli was recovered. The foodborne pathogen that survived the longest (60 days at refrigeration temperature and 1 week at room temperature) in fresh salsa was Listeria monocytogenes. As for Staphylococcus aureus the most significant decrease was shown after 1 month of refrigeration storage (9.7 x 106 to 4.3 CFU/g), but no recovery was made after one week of room temperature storage (Raghubeer et al., 2000). Mexican Food Safety Trends 1990-2000 Simonne et al. (2004) examined the CDC f oodborne illness data from 1990 to 2000 in order to analyze the ethnic food safety trends the US. The authors grouped ethnic foods into three major ethnic categories: Asian, Italian and Mexican. Ov erall, total foodborne illness outbreaks associated with ethnic foods rose fr om 3% in 1990 to 10% in 2000 (Figure 2-5). There were no specific trends in the total numbe r of cases for illness. The highest numbers of outbreaks occurred in restaurant s (43%), private homes (21% ), schools (7%), and others (29%). The top five states with the highest numbers of outbreaks were Florida (n=136), California (n=74), New York (n=42), Maryland (n=40), and Michigan (n=37).

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26 6 7 2 6 5 9 4 8 15 20 32 13 8 1111 1212 8 19 42 43 67 15 7 13 8 7 8 14 12 49 54 530 10 20 30 40 50 60 70 801990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 Outbreaks numberYear Asian Mexican Italian Figure 2-5. Number of outbreaks per year as related to top th re Ethnic foods (Adapted from Simonne et al., 2004) The majority of the ethnic foodborne illness outbreaks were due to unknown etiologies (61%) followed by Salmonella spp. (18%), Clostridium spp. (6%), Bacillus spp. (4%), Staphylococcus spp. (4%), and all others (7%) (Figure 2-6). The profiles of microorganism in the known etiology category were different fo r each of the ethnic food types. The most frequent sources of reported food borne outbrea ks in descending order are Mexican (41%), Italian (39%), and Asian f ood (20%) (Simone et al., 2004). Salmonella spp 17% Clostridium spp 6% Bacillus spp 6% Staphylococcus spp 4% Other 7% Unknown 60% Figure 2-6. Breakdown of etiology in all Ethnic foods (Adapted from Simonne et al., 2004)

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27 The major microorganisms found in th e outbreak of Mexican foods were Salmonella spp. (47%), Clostridium spp. (26%), Shigella spp. (10%), and Staphylococcus (5%) (Simonne et al., 2004). The study indicated th at Mexican dishes appear to have more virulent type of pathogens in comparison to other ethnic food categories such as Italian or Asian food. The paper concluded that the differences in microorganism profile f ound among the three ethnic food categories could be attri buted to ingredients, cooking and preparation, and how the foods were served. Tacos (25%), burritos (14%), chili (8%), salsa (8%), enchiladas, refried beans and nachos (6 %), guacamole (4%), and others (23%) were among the food vehicles in the outbreaks from Mexican cuisine (Simonne et al 2004). These prepared foods often contain more than three ingredients, and often those ingredients are mixtures of cooked meat and fresh vegetables and dairy products which are often implicated in foodborne outbreaks (Simonne et al., 2004). The most common location of the outbreaks were restaurants (43%), private homes (21%), schools (7%), multiple locations (7%), workplaces (4%), unknown (4%), and others (14%) (Simonne et al., 2004). According to the National Restaurant Association, about 48% of the US population income is spent on meals away from home in restaurants, delicatessens, cafeterias, and other food serv ice establishments (NRA, 2007). As US consumers are eating more food away from home, little informati on regarding safety and the role of food establishments is availa ble (Simonne et al., 2004). Selected Study Microorganisms According to the CSPI 2007 report, bacteria are the most common cause of foodborne outbreaks. Si xty one percent of the total outbreaks reported from 1990 to 2005 were linked to bacteria including Salmonella spp. (24%), Clostridium spp. (11%), Staphylococcus spp. (8%), E. coli (6%), and Campylobacter (3%) (CSPI, 2007).

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28 Produce and produce dishes accounted for a total of 713 foodborne illness outbreaks (34,049 illness cases). The most commonly iden tified vehicles in these outbreaks were vegetables (260 outbreaks with 13,133 illness ca ses), and produce dishes (343 outbreaks with 8,668 illness cases) (CSPI, 2007). Five commodity groups make up to 75% of produce outbreaks. Leafy greens accounted for 30% of the outbreaks, followed by tomatoes (17%), cantaloupe (13%), herbs (basil parsley and cilantro) (11%), and green onions (5%) (FDA, 2008). The common bacteria asso ciated with outbreaks were Salmonella spp. (18%) and E. coli (8%). Half of the produce-related outbreaks we re attributed to food from restaurants and other food establishments (CSPI, 2007). Multi-ingredient foods acc ounted for a total of 952 out breaks (26,891 illness cases). Lasagna, tacos and lo mein, three major ethnic foods, were reported as the most common multi-ingredient foods involved in foodborne outbreaks (247 outbreaks) (CSPI, 2008). Fifty percent of these outbreaks occu rred in food from food servic e establishments. The most common risks factors noticed in these outbrea ks were cross-contamination, under cooking, inadequate cooling and storage, and employee contamination. Salmonella spp. was the most common pathogen associated with multi-ingredient outbreaks illness (CSPI, 2007). Greig et al. (2007) analyzed a total of 816 outbreaks (80,682 illness cases) where restaurant food workers have been implicated in the spread of the f oodborne illness. Major pathogens implicated in th e outbreaks were enteric Salmonella (151), Staphylococcus aureus (53), and Shigella spp. (33). Multiple foods and multi-i ngredient foods were identified most frequently with outbreaks, perhaps because of more frequent hand cont act during preparation and serving. Salsa, due to its preparation and consumption practices, falls under these food categories. From all pathogens related to produce and multi-ingredient outbreaks, Salmonella spp. and Staphylococcus aureus were selected as the bacterial targets for this study (Greig et al., 2007).

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29 Salmonella spp. Salmonella is the second most common cause of foodborne illness (salmonellosis) in the US, and is respon sible for millions of cases of illness each year. Typical symptoms of salmonellosis are nausea, vomiting, abdominal cr amps, fever, mild di arrhea, and headache; these symptoms usually last 6-48 hours. Salmone lla outbreaks have been associated with the consumption of raw and undercooked eggs, unde rcooked poultry and meat, dairy products made with unpasteurized milk, shrimp, fresh produce, and unpasteur ized fruit juice (FDA, Bad Bug Book). Salmonella is a genus of rod-shaped Gram-negativ e enterobacteria that causes typhoid fever, paratyphoid fever, and othe r non-typhoid type salmonellosis. Salmonella species are motile and produce hydrogen sulfide; they cause mo re than 1.4 million infections in the US and several hundred deaths annually. Because of the low infective dose for Salmonella (15 to 20 cells/sample), detection methods are required to prove the presence of even one cell in a defined food sample. Several isolation and identification methods are available for Salmonella in food products. Traditional methods recommend the use of selec tive enrichment broths to enhance Salmonella growth by inhibiting the growth of non Salmonella populations (background flora). Conventional enrichment media used by the FDA and USDA/FSIS are Tetrathiona te broth (TT) or Rappaport-Vassiliadis medium (RV) (Andrew et al., 2003; USDA/FSIS, 2002). Broths containing selenite, brilliant green and malachite green were found to be accurate in the detection of Salmonella spp. (Busse, 2005). Additionally to enhance growth of Salmonella spp. by enrichment media, incubation temperatures also play an important role. Recovery of Salmonella spp. from food with low background flora is usually done at 35C, while high background flora food samples should be in cubated at 43C (Mores et al., 2005).

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30 In order to increase specificity for Salmonella detection, since 1990 a wide variety of chromogenic media based on a combination of Salmonella biochemical characteristics and needs have been developed. Examples of chromogenic media include Rambach (Rambach, 1990), SM ID (Poupart et al., 1991), CHRO Magar (Galliot et al ., 1999), ABC medium (Perry et al., 1999), Chromogenic Salmonella esterease agar (Cooke et al., 1999), and Rainbow Salmonella Agar (Manafi, 2000). Mores et al. (2005) compared different media for Salmonella detection in naturally contaminated poultry. The authors analyzed two hundred and twenty five samples using three enrichment broths [Tetrationate brilliant green broth (TT); Selenite-Cystine broth (SC); Rappaport Vassialidis broth (R V)], and five plating agars [Rambach Agar; CHROMagar Salmonella (CAS); SS Agar; Brillant Green Agar (BGA); and Xylose Lysine Desoxyxholate Agar (XLD)]. There was no significant difference in using TT and RV as enrichment broths. Mores et al. (2005)s finding contradicted with other studies which found RV more effective than TT (Beckers et al., 1986; Hammack et al. 1999; June et al., 1995; Pi etzsch et al., 1984; Vassiliadis, 1983). The authors also conclude that SC is a useful enrichment broth for low Salmonella contaminated foods. As for chromagenic me dia, the authors findings showed that CAS was the most effective plating medium followed by Rambach, XLD, SS, and BGA, respectively (Mores et al., 2005). A number of rapid methods for the detection of Salmonella in foods have been developed, including electrical techniques, immunoassays and nucleic acid probe analyses. However, the analysis depends on sensitivity of the detection system, and in many cases, a positive result can only be reached when bacteria concentration is 104 106 cells/g (Bennet et al., 1998; Blivet et al ., 1998; Lee et al., 1990) The polymerase chain reaction (PCR) is a sensitive, rapid technique for Salmonella identification, in which a few copies of target DNA can be amplified to a level detectable by

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31 gel electrophoresis. However, P CR sensitivity may be inhibited by several factors, including food components and sample preparation. The re moval of inhibitory s ubstances is a major step in the preparation of samples for PCR-ba sed detection of food pathogens (Lantz et al., 1994; Schen et al.,1998). Immuno magnetic separation (IMS) is a powerful tool to extract bacteria from food samples. Bacteria are sp ecifically separated from the specimen, resulting in a useful sample for PCR with little or no nons pecific DNA or interferi ng factors (Safark et al., 1995). Staphylococcus aureus Staphylococcus aureus contamination in food is an indicator of poor handling and hygiene practices in the processing, ma nufactur ing and/or preparing of food. Handlers and equipment, or utensils used by them, contribute directly or indirectly to cross-contamination. Infected food handlers, whether symptomatic or not, who dont follow safety practices, may indirectly contaminate food (hand-to food) (Panisello et al., 2000). Staphylococcal food poisoning is a condition caused by the ingestion of enterotoxins produced by some strains of Staphylococcus aureus. Less than 1.0 microgram of toxin in food will produce symptoms of staphylococcal intoxication. The most common symptoms are nausea, vomiting, retching, abdominal cramping, a nd prostration. Individuals may not always demonstrate all the symptoms associated with the illness. In more severe cases, headache, muscle cramping, and transient changes in bl ood pressure and pulse rate may occur (FDA, Bad Bug Book). When Staphylococcus aureus populations exceed 100,000/g, the toxin will reach intoxication levels. Th e event will occur once food is contaminated with the microorganism and the food is left in the temper ature danger zone (5C, 41 F 57C, 135F) for a long period of time (Food Code, 2005). Foods that are frequently associated with staphylococcal food poisoning include meat and meat products, poultry and egg products, sa lads, bakery products, sandwich fillings, and

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32 milk and dairy products. Foods that require cons iderable handling during preparation and that are kept at slightly elevated temperatures after preparation are fr equently involved in staphylococcal food poisoni ng (FDA, Bad Bug Book). Humans and animals are the primary reservoirs of Staphylocococci. Fifty percent or more of healthy individuals may have Staphylococcus aureus in the nasal passage, throat, and on hair and skin. Staphylococci also exist in air, dust, sewa ge, water, milk, food or food equipment, and environmental surfaces. Incidence of human infection with Staphylococcus aureus is even higher for those who are associated with or who come in contact with sick individuals and hospital environments. Selective medium in the detection of Staphylococcus aureus is Manitol Salt Agar (MAS); this media minimize growth of normal flora and allow poten tial pathogens to be recovered more easily from co mplex microflora samples. Recently CHROMagar Staph aureus was reported to be useful for differen tial and selective use with this microorganism isolates without additional te sting and with 100% specificity (Sharp and Searcy, 2006). AOAC has approved a Petrifilm method for the determination of Staphylococcus aureus the Petrifilm Staph Express Count pl ate. The method, an alternative for the traditional AOAC Baird-Parker plate count me thod, uses a modified chromogenic Baird Parker, selective for Staphylococcus aureus. When other colored colonies (black and green) are encountered in testing, the Petrifilm Staph express disk is used to identify Staphylococcus aureus from all suspect colonies. Silbernagel et al. (2003) studied the effectiveness of Petrifilm Staph Express Count plate against traditional Baird Parker plate count (BPA) method in selected types of food such as frozen lasagna, custard, frozen mixed vegetables, frozen hash-browns, and frozen batter-coate d mushrooms. The authors found that the mean log10 counts of Petrifilm Sta ph Express Count plate method and the repeatability and

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33 reproducibility variances of that method were si milar to those of trad itional BPA method, and they did not find any statistical di fference between the two methods. Methicillin-resistant Staphylococcus aureus (MRSA) is a resistant variation of the common bacterium Staphylococcus aureus. It is a bacterium respons ible for difficult-to-treat infections in humans. It may also be referred to as multiple-resistant Staphylococcus aureus or oxacillin-resistant Staphylococcus aureus (ORSA). The organism is often sub-categorized as Community-Associated MRSA (CA-MRSA) or Hospital-Associated MRSA (HA-MRSA) depending upon the circumstances of acquiring the disease. Base d on current data both CAMRSA and HA-MRSA are two distinct strains of the bacteria l species (Okuma et al., 2002). Antimicrobial resistance is an important public health concern worldwide. The development of resistance both in human and an imal bacterial pathogens has been associated with the extensive therapeutic use of antimicr obials or with their administration as growth promoters in animal foods (Normanno, 2005). MRSA has an evolved ability to survive treatment with beta-lactam antibiotics, including penicillin, methicilli n, and cephalosporins (Foster, 1996). It is especially troublesome in hospital-associated infections Patients with open wounds, invasive devices, and weakened immune systems are at a greate r risk for MRSA infection than the general public. Hospital staff who does not follow prope r sanitary procedures may transfer the bacteria from patient to patie nt. MRSA infection has been reported in the United States for over 30 years. Initially, MRSA infections were primarily a problem of hospitals and nursing homes. By 1997, 50% of health-care-acquired Staphylococcus aureus isolates in the United States were methicillin resistant (Lowy, 1998) In the early 1980s, cases of communityacquired MRSA were reported primarily in pers ons with a history of drug use and other highrisk patients (Saravolatz, 1982), but more recently, the community-acquired MRSA has been

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34 described in both adults and children who did not have extens ive exposure to hospitals or other apparent risk fact ors (Gorak et al., 1999). The first report in the US of a community -acquired outbreak of acute gastroenteritis caused by MRSA was reported in 2002 in pork ba rbeque and coleslaw (Jones et al., 2002). The MRSA strains were isolated from ill persons, the implicated foods, and food handlers (asymptomatic carrier) who prepared the food pr oducts at a convenience market (Jones et al., 2002). Internationally speaking there are few studi es relating the transmission of MRSA by contaminated food. However in every case, contamination was notice from a sick foodhandler to food products. Moreover, the food products related in the outbreaks were repeatedly food of animal origin, including but not limiting to dairy products, meat and meat products (Normanno et al., 2005, Lee, 2003, Kluytmans et al., 1995). Although food handlers are usually the main source of food contamination in food poisoning outbreaks, equipment and environm ental surfaces can also be sources of contamination with MRSA. Several phenotype methods for the dete ction of methicillin resistance in Staphylococcus aureus isolates have been developed, incl uding oxacillin agar screen test, cefotoxin test and disk diffusion test or commercial automate d tests such as MRSA latex agglutination test and Vitek 2 system (GPS-SA card). As these methods in some cases are not sufficient sensitive or specific, PCR detection of the mecA gene could be applied (Normanno, 2005; Lee, 2003; Gorak, 1999).

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35 CHAPTER 3 MATERIALS AND METHODS Assessing Mexican Food Safety Trends The ethnic foodborne illness data from Si monne et al. (2004) from 1990 to 2000 was further expanded through 2006, using the Center for Control and Prevention (CDC) outbreak surveillance database (2001-2006) (CDC, 2008). Ethnic foods were grouped as previously described by Simonne et al. ( 2004) into three major categorie s: Mexican, Asian, and Italian foods. Whenever possible, food items impli cated in outbreaks were verified with the Websters New World Dictionary of Culinary Ar ts (Labensky et al., 1997) to determine its origin before they were assi gned to one of the categories. Mexican food was also verified with the book Food Culture in Mexico (Long-Soli and Vargas, 2005). Combined data for Mexican food (1990-2006) were processed and ranked based on number of outbreaks, etiology, outbreak location, and outbreak vehicle. Microbiological Quality of Commercial Salsa Sampling of Restaurants According to the Florida Restaurant a nd Loading Association 21 restaurants in Gainesville, FL are considered as Mexican or Mexican style operations and am ong those, 38% were categorized as locally owned restaurants, while the remaining 62% were categorized as chain type rest aurants (Florida Restaurant a nd Loading Association, 2008). A chain restaurant is defined as a set of related restaurants with the same brand-name in many different locations either under shared cor porate ownership or franchising agreements. Typical chain restaurants are built to specific standards and offer standard menus set by parent companies; these may include fast f ood restaurants as well as midand up-scale establishments. Locally owned restaurants, as op posed to chain restaurants, do not have more than two stores in the same area, and ther efore, do not have a nationwide brand-name reputation. Its management and menu offerings are established under local owner standards.

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36 A total of 8 restaurants were sampled fo r this study. Half of them were chain restaurants including fast food and middle scale restaurants. The remaining half was locally owned restaurants. A total of 48 samples were co llected from these restaurants. Samples were subcategorized according to salsa type (fresh or red salsa) and visually characterized for ingredients. Salsa Samples Red sauce Salsa roja and fresh salsa Pico de gallo samples used in this study were purchased from both locally owned and chain re s taurants in Gainesville, FL. Salsa samples were typically sold in variab le size plastic containers ranging from 25 to 600 g capacity. Temperature of salsa samples was measured at the point of sale using Alarm Thermometer with Probe (Fisher Scientific Laboratory, P ittsburgh, PA) and pH was measured upon arrival at the research laboratory us ing a pH meter (MP230 Meter To ledo, Austria). Samples were transported in a cooler to the research laboratory at the Univer sity of Florida within 20 min, and they were stored at 4C on the same day of purchase (within 6 hours). Sample Preparation Twenty-five grams of each salsa sample were placed aseptically into a sterile 400-m l stomacher bag (Seward Scientific, London, UK) c ontaining 225 ml of 0.1 % peptone water to make a tenfold dilution for all tests except Salmonella For Salmonella 225 ml of lactose broth were used (Difco, Becton Dickinson, Sparks MD). Further serial dilution was made as required using 1 ml of the homogenate in 9 ml of 0.1% peptone water (AOAC method # 2001.05; AOAC-RI# 020502). Aerobic Plate Count and Total Coliform Count After stomac hing, the homogenate was further serially diluted in 0.1% peptone water and then inoculated on PetrifilmTM Aerobic Count plates (3M Microbiology, St. Paul, MN) and E. coli Count plates (3M Microbiology) in duplicates for enum eration of total aerobic

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37 plate count and total E. coli counts, respectively. The manu facturer instructions were followed and results were reported as col ony forming units (CFU/g) (3M Microbiology). Salmonella Determination AOAC-RI# 020502 CHROMagarTM Salmonella (Difco, Becton Dickinson) was used. Twenty-five grams of sample placed in a ster ile 400-ml stomacher bag containing 225 ml of lactose broth was homogenized in the stomacher (Seward) for 1 min at normal speed and the homogenate was incubated at 37C for 18 h. Af ter the incubation, 1 ml aliquot of sample was transferred to 10 ml of selenite cys tine broth (SC; Difco, Becton Dickinson) and RappaportVassiliadis broth (RV; Difco, Becton Dickson) in duplicates, and incubated at 37C for 24 h. After overnight incubation, 3 mm loopful (about 10 l) was taken from each selective enrichment broth a nd streaked onto xylose lysine desoxycholate agar (XLD; Difco, Becton Dickinson) and CHROMagarTM Salmonella (Difco, Becton Dickson) in triplicate. Plates were incubated at 37 C for 24 h. Black colored colonies on XLD agar and mauve colored colonies on CHROMagarTM Salmonella were presumptively identified as Salmonella spp. All presumptive Salmonella colonies were confirmed as Salmonella by the Salmonella latex agglutination test (Oxoid, Basingstoke, UK). Staphylococcus aureus Determination AOAC m ethod # 2001.05 was followed for Staphylococcus aureus challenge study (Silbernagel, et al., 2003). Twenty-five gram s of sample were placed in a sterile 400-ml stomacher bag containing 225 ml of 0.1% peptone water, and homogenized in the stomacher (Seward) for 1 min at normal speed. Serial dilu tions in 0.1% peptone water were done, and an aliquot (1 ml) of serial diluen ts was inoculated on PetrifilmTM Staph Express Count (3M Microbiology) following the manufacturer instruc tions. Plates were incubated at 37C for 24 h. Red-violet colonies were recorded as Staphylococcus aureus (Figure 3-1-A) When colony colors other than red-violet were pres ent (black, green or blue), a PetrifilmTM Staph Express

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38 disk (3M Microbiology) was used, and the sample were incubated for an additional incubation period at 37C for 1-3 h (Figure 3-1B). From this, colonies showing a pink zone around red, blue or colorless center were r ecorded as Staphylococcus aureus. Three presumptive Staphylococcus aureus colonies from each positive sample were picked, streaked on tryptic soy agar (TSA; Difco, Bect on Dickinson), and incubated at 37C for 24 h. After the overnight incubation, cultures were resuspended in tryptic soy broth (TSB; Difco, Becton Dickinson) containing 30% sterile gl ycerol solution, and st ored at -80C for subsequent coagulase agglutination test. (A) (B) Figure 3-1. Petrifilm staph express plat es. (A) Red-violet and green colonies on Petrifilm Staph Express plate. (B) Pink zones around presumptive Staphylococcus aureus colonies, colored by Petrifilm Staph Express Disk. Presumptive Staphylococcus aureus colonies were confirmed by coagulase agglutination test. Frozen cultu res were first streaked on TSA and incubated at 37C for 24 h. One colony was inoculated in brain heart infu sion broth (BHI; Difco, Becton Dickinson) and incubated at 37C for 24 h. After the incuba tion, 0.5 ml of reconstituted rabbit plasma (Remel, Lenexa, KS) was added to a sterile tube containing 0.2 ml of the overnight cultures

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39 in BHI and reincubated for up to 6 h at 37C. Any degree of clot formation within 6 h was recorded as positive for Staphylococcus aureus (Sperber, 1975). Screening of Methicillin Resistant Staphylococcus aureus (MRSA) The agar disk diffusion method was perform ed to screen the methicillin resistant strains of Staphylococcus aureus (MRSA) following the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS, 2000). A Staphylococcus aureus strain (ATCC 12600-U) and a MRSA strain (ATCC 33591) were used as negative and positive controls, respectively. Both strains we re purchased from the American Type Culture Collection (ATCC, Manassas, VA). Each Staphylococcus aureus strain was enumerated in 5 ml Muller-Hinton broth (MHB, Difco, Becton Di ckson) at 37C for 3 h, and the culture was adjusted to turbidity equivalent to 0.5 McFarl and to achieve a concentration of approximately 1.5x108 CFU/ml. Bacterial suspension was spread onto Muller-Hinton agar (MHA, Difco, Becton Dickson) and manitol salt agar (MSA; Di fco, Becton Dickson) in duplicates by sterile cotton swabs. Oxacillin disks (BBL, Diagnostic System, Sparks, MD) (at 1 g per disc) were applied onto each of the inoculated plate in triplicates, After inc ubation at 37C for 24 h inhibition zones were measured and recorded in millimeters of diameters. The size of the inhibition zones on MHA and MSA plates 10 and 16 mm, respectively, were defined as resistant. A flow diagram of microbiological quality of sa lsa evaluation can be found in Appendix B. Microbiological Challenge Study Salsa Samples Red salsa Salsa roja s amples were purchased from a commercial restaurant in Gainesville, FL. Salsa samples in large plastic containers (1000 g/container) were transported in a cooler to the laboratory with in 20 min. Immediately after arrival, samples from three large plastic containers were thor oughly mixed in a sterile 5000-ml container.

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40 Individual portions of 25 0.2 g of salsa mixtures were aseptically transferred to 150-ml individual sterile graduated c ontainers with lids (Fisher Scientific, Pittsburgh, PA), and each container was considered as one sample. The in dividual samples were then inoculated with Salmonella and Staphylococcus aureus cocktail suspensions for microbiological challenge study; respectively. Both micr obial challenge studies for Staphylococcus aureus and Salmonella spp. were completed sepa rately in triplicates. A flow diagram of microbiological qualit y of microbiological challenge sample preparation can be found in Appendix C. Bacterial Cultures Preparation Three Staphylococcus aureus strains (ATCC 29247, ATCC 12600-U and ATCC 35548) and three Salmonella serotypes Enteritidis, Typhimuri um, and Thompson (ATCC 8391) were used as inocula for the Staphylococcus aureus and Salmonella microbiological challenge studies, respectively. Staphylococcus aureus strains (ATCC 29247, ATCC 12600U and ATCC 35548) and Salmonella serotype Thompson (ATCC 8391) were purchased from the American Type Culture Collect ion (ATCC, Manassas VA), while Salmonella serotypes Enteritidis and Typhimurium were obtained from Dr. Tun-Shi Huang (Auburn University, Auburn, AL). Bacterial cultures were stored at 80C in st erile 30% glycerol solution. Working cultures were maintained on TSA (D ifco, Becton Dickinson, Sparks, MD) at room temperature and transferred to fresh TSA plates once a week. One day prior to the experiment, bacter ial cultures were streaked onto TSA and incubated at 37C for 18-24 h. Five isolated colonies of each overnight culture were transferred to a 50-ml centrifuge tube (Corning Costar Inc., Corning, NY) containing 30 ml of sterile TSB (Difco, Becton Dickinson) and incubated at 37C for approximately 3 h. After incubation when there is visible turbid ity, each bacterial cultu re was harvested by centrifugation at 2,000 x g for 15 min (Centra CL3, Thermo Electron Corporation, UK) and

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41 washed once with 0.1% peptone water (Dif co, Becton Dickinson). The cell pellet was resuspended in 0.1% peptone water and the cell density was adjusted to achieve a final cell concentration of approximately 1.5 x 108 CFU/ml (Hsu et al., 1998, 2006). Equal volumes of each Staphylococcus aureus strain or Salmonella serotype suspension were combined, and dilutions were prepared with 0.1% peptone water. Appropriate final cell diluents were used as cocktails for salsa inoculation, depending on the inoculation level, 155 and 1,555 cells/g as low and high inoculation levels for Staphylococcus aureus challenge study, and 15-20 cells/sample for Salmonella study. TSA plate spreading technique was used to count cells of the cocktail prepared for sample inoculation. Sample Inoculation Salsa samples were inoculated using spot i noculation technique as described by Hsu et al. (2006) and the FDA (2001). Aliquots of a ppropriately diluted cocktails prepared as previous des cribed were inoculated into each salsa sample container (25 g/container) at 5 locations for even distribution of bacterial inoculum. Low (155 cells/g) and high (1,550 cells/g) inoculation levels were used for Staphylococcus aureus study, while only one inoculation level (15 20 cells/sample) was used for Salmonella Fifteen to 20 Salmonella cells/g is determined as the infective dose for this microorganism (FDA, Bad Bug Book). After inoculation, half of the s amples at each inoculation level were stored at room temperature (20 2C) (30 samples for Staphylococcus aureus and 15 for Salmonella studies, respectively) and the other half at refriger ated temperature (4 2C) [Appendix C, D]. Original populations of Salmonella, Staphylococcus aureus and total aerobic counts were determined on three un-inoculated sample s at each microbiological challenge study. Sample Storage Population (log CFU/g) of Staphylococcus aureus and presence of Salmonella spp. in salsa samples were evaluated over periods of 0, 2, 4, 6, and 24 h for room t emperature

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42 storage, and at 0, 1, 3, 5 and 7 da ys for refrigerated storage. Each 25-g salsa in a container was processed as one sample, and all experiment was done in triplicates. Staphylococcus aureus Challenge Study AOAC method # 2001.05 used and described in the Microbiological Quality of Commercial Salsa section was followed fo r the enumeration and identification of Staphylococcus aureus in the challenge study (AOAC, 2001). Salmonella Challenge Study Inoculated samples were analyzed by both AOAC-RI# 020502 CHROMagarTM Salmonella (Becton Dickson) and AOAC-RI# 030301Rapid Chek SELECTTM for Salmonella (Strategic Diagnostics Inc., Newar k, DE) in order to compare the media performance. For AOAC-RI# 020502 CHROMagarTM Salmonella each sample was processed according to the procedures men tioned in the Microbiological Quality of Commercial Salsa section. For AOAC-RI# 030301 Rapid Chek SELECTTM for Salmonella the individual sample was placed in a sterile stomacher bag (Seward) containing 225 ml of Rapid Enrichment Media for Salmonella (Strategic Diagnostics Inc.), and was homogenized in a stomacher (Seward) for 1 min at normal speed. Homogenate samples in stomacher bags were incubated at 42C for 24 h. After incubation, an 1 ml aliquot was tran sferred to 10 ml of tetrathionate broth ( TT, Difco, Becton Dickinson), and wa s incubated at 42C for 24 h. After incubation, approximately 0.5 ml of sample TT broth was transferre d to a plastic tube (provided with the kit), one kit te st strip was inserted into each tube and the strip was left to develop color for 10 min. A single red line (c ontrol line) on the stri p was recorded as a negative result while two red lines on the strip we re recorded as a positive result (Figure 3-2). Tubes with positive readings were streaked onto XLD agar, in duplicate, and incubated at 37C for 24 h. Black colonies in XLD agar were recorded as presumptive Salmonella and

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43 subsequently were confirmed as Salmonella by the Salmonella latex agglutination test (Oxoid, Basingstoke, UK). (A) (B) Figure 3-2. Rapid Chek SELECTTM for Salmonella. (A) Negative (one line colored control line). (B) Positive (two lines colored) Statistical Analysis For Microbiological Quality of Commerci al Salsa study, each experime nt was replicated two times and triplicate salsa samples were analyzed at each selected restaurant, with a total sample number of 48 (three salsa samples, eight different restaurants, and two trials). For each Microbiological Challenge Study, each experiment was replicated two times and triplicate salsa samples were used at each time point, with a to tal sample number of 63 per Staphylococcus aureus study, including control sample (five time points at room temperature, five at refrigeration temperature, two inoculation levels, and three samples for each time point, and two trials). For Salmonella study, 33 samples including control samples (five time points at room temperature, five at refrigeration temperature, one inoculation level and three samples for each time point) were analyzed. Results from each time point were

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44 used to evaluate survival of Staphylococcus aureus and Salmonella. Overall storage effect was determined by using results at each time point. Statistical analysis of the response (log CF U/g) for both Microbiological Quality of Commercial Salsa and Micr obiological Challenge Study was analyzed at significance level of = 0.05 by the general linear model (Proc GLM) (SAS system for Windows 9, SAS Institute, Cary, NC). Means separations we re done by Duncans multiple range test.

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45 CHAPTER 4 RESULTS AND DISCUSSION Assessing Mexican Food Safety Trends Foodborne outbreaks reported by Simonne et al. (2004) and the CDC from 1990 2006 were categorized following Simonne et al. (2004) m ethodology. Mexican foodborne outbreaks were analyzed and ranked based on number of outbreak s, etiology, outbreak location and outbreak vehicle. Overall, a total of 560 outb reaks (18,581 cases) related to Mexican food were reported from 1990 to 2006; out of the total outbr eaks 62% were due to unknown etiology (345 outbreaks, 8,222 cases), and 38% were due to specific know pathogens (215 outbreaks; 10,359 cases). Among the 38% for the know n pathogens, the majority were Salmonella spp. (34%), Clostridium spp. (24%), Shigella spp. (6%), Staphylococcus (5%), and Escherichia coli (5%) (Appendix A). Foods often associated with the outbreaks were multiple foods (22%), followed by tacos (18%), chili (9%) and salsa (9%). Overall, there was an increase in numbers of foodborne illness cases associated with Mexican food (relative to the total number of foodborne outbreaks reported for the period of study) from 2% in 1990 to 3% in 2006 for both combined known and unknown etiology categories. In some years (2000, 2002, 2003, and 2005) there seems to be a higher percentage (5% on average) in the number of outbreaks a ssociated with Mexican foods as compared to the total numbers the foodborne illness. While the total numbe r of outbreaks did not follow any specific trend, there is an increasing trend of incidenc es of foodborne outbreaks in Mexican food (Figure 4-1). In 1998, the CDC enhanced its outbreak surv eillance network causing an increase in the total number of foodborne illness outbreaks reported to the network, which also impacted the numbers of foodborne illness outbr eaks associated with Mexican foods.

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46 138111111128184243675569495652352% 3% 2% 2% 2% 1% 2% 3%3% 5% 4% 5% 5% 4% 5% 2% 3%0 10 20 30 40 50 60 70 801990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 Total outbreaks numbe r 0% 1% 2% 3% 4% 5% 6% % Total outbreaks % Figure 4-1. Total number of foodborne illness outbreaks in Mexican food outbreaks in the US (1990-2006) In general, during the first 10 years of the study period, unknown outbreaks were more frequently reported, while star ting in 2000, outbreaks reported were positively linked to a specific pathogen (Figure 4-2). Based on this study, the major genera of b acteria causing foodborne illness in Mexican foods were Salmonella (35%), Clostridium (24%), Shigella (6%), and Staphylococcus (5%) (Figure 4-3). According to the CDC, Salmonella, E. coli O157:H7, and Listeria monocytogenes are among the top five bacteria that can cause severe diseases (CDC, 2008), and these bacteria are often implicated in illn esses associated with Mexican foods. Norovirus (formerly called Norwalk virus) (13%) was also an important known etiology in the outbreaks relates to Mexican food (Appendix A).

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47 8 33 9 3 8 5 9 13 14 27 11 21 15 21 19 26 55 8 2 8 4 3 9 2929 40 44 48 34 35 33 9 0 10 20 30 40 50 60 19901991199219931994199519961997199819992000200120022003200420052006 YearOutbreaks number Known Unknown Figure 4-2. Number of foodborne illness outbreaks associated with Mexican food (1990 2006) Based on the 10 year data from Simonne et al (2004) and the current findings in this study (16 year data), there seems to be a consis tent pattern of foodborne illness and profile of microorganisms associated with Mexican foods. Salmonella, Clostridium, Shigella and Staphylococcus were the most commonly found pathogens in outbreaks related to Mexican foods; however, prevalence (%) for each bacteria were higher in Simonne et al (2004) than those found in this study. Among the outbreaks with known etiology, the highest number occurred in restaurants or delicatessens (47%), followed by private home (16%), schools (6%), workplace (8%), prisons (3%), unknown, church or temples and multiple locations (2% each), and other locations (15%) [Figure 4-4]. Other locations category includes hotels, conferences, camps, festivals, fairs, and picnics. The numbers found in this study are comparable to the information reported by the CDC and other studies (Shiferaw et al., 2004).

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48 According to the CDC from 1990 to 2006, an average of 941 foodborne illness outbreaks were reported, and 50% were attribut ed to the food service sectors (CDC, 2008; Olsen et al., 2000). Moreover, results from th e CDC FoodNet case control studies, indicated that consumption of food outside the home was related with an increased risk of gastrointestinal illness and w ith specific types of foodborne pathogens (Shiferaw et al., 2004). Clostridium 24% Norovirus 13% Salmonella 35% Shigella 6% Staphylococcus 5% Other 17% Figure 4-3. Most prevalent known etiology i nvolved in Mexican food outbreaks (1990-2006) Among top food items, tacos (18%), chili (9%) salsa (9%), refried beans (8%), burritos (7%), and guacamole (4%) were implicated in the outbreak with known etiology (Figure 4-5). Other food items such as enchiladas, Carnitas, Tortilla and Fajitas (3% each) were also implicated in outbreaks, but less frequently. Multiple food items (22%) and others (8%) are also often associated with outbreaks in Me xican foods during the study period (1990-2006). These findings are also consistent with thos e reported in the 2007 Outbreak Alert by the Center for Science in the Public Interest (CSP I). In the CSPI report a total of 952 outbreaks (26,891 illness cases) from 1990 to 2005 were attributed to the multi-ingredient foods. In

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49 descending order the most common identified vehicles were multi-ingredient dishes, including lasagna, tacos and lo mein (26%), multi-ingredient sa lads (24%), foods, including rice, stuffing and pasta dishes (21%), sandwiches (15%), and sauces (7%). The most common risk factors noticed in th ese outbreaks were cross-contamination, inadequate cooking, inadequate cooling and storage, and contamination by workers (CSPI, 2007). Restaurant or delicatessen 47% Prison 3% Multiple locations 2% Unknown 2% Church or temple 2% Private home 16% Other 15% Workplace 7% School 6% Figure 4-4. Location of foodborne illness outbreaks associated with Mexican foods due to known etiology from (1990-2006) Multiple 23% Tacos 18% Chili 9% Salsa 9% Refried beans 8% Other 8% Guacamole 4% Enchiladas 3% Carnitas 3% Fajitas 3% Tortilla 3% Nachos 1% Carne asada 1% Burrito 7% Figure 4-5. Mexican food vehicles reporte d in foodborne illne ss outbreaks (1990-2006)

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50 Microbiological Quality of Commercial Salsa Temperature and pH Profiles Temperature profiles for fresh salsa at sale ranged between 4.9 and 21C. Fresh salsa samples (62.5%) were sold at temperatures less than 10C, while the remaining 37.5% of salsa were sold at temperatures higher than 14C. Red salsa temperature profiles ranged between 5.4 and 27C, and 62.5% of red salsa samp les were sold at temperatures less than 10C, while the remaining 37.5% of samples were at temperatures higher than 18C (Table 41 and 4-2). Although service practices diffe r among restaurant types, the majority of restaurants kept and sold salsa under refrigerated conditions (Table 4-1 and 4-2). It was observed that although the fast food restaurants sampled in this study kept their salsa in ice-refrigerated displays, their temperature profiles were hi gher than those found in midscale chain and locally owned restaurants where salsa was usuall y kept in the restaura nt kitchen and brought to the table once ordered. One part icular restaurant ke pt its red salsa under room temperature conditions. This salsa showed ch aracteristics similar to Tabas co sauce. When waiter was asked about the characteristics of salsa, he mentioned that the salsa was usually bought in bulk at retail centers, and was not a fresh home-made salsa. According to the FDA-Food Code (2005) recommendations, food products should be kept colder than 5C (41F) or hotter than 57C (135F). The temperatures between 5 and 57C are defined as the danger zone. Keep ing potentially hazardous food products (TCS for time/temperature control for safety food, as in the 2005 Food Code) under the danger zone increases the risk of rapid bacterial grow th. Temperatures at which salsa samples were kept fall under this temperature range (tempera ture danger zone), whic h indicates a potential hazard. Due to serving practice and consumption of salsa, it is common to have salsa displayed or served at room temperatures. The Food Code (2005) recommends, when food is

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51 removed from a cold holding temperature cont rol (5C or less) it could sit at room temperature (up to 21C) for up to 6 h, after th at time it should be thrown out. Potentially hazardous foods (like cut vegetables, meats, dair y, and fish.) should never be eaten if they have been sitting out for more than 6 h (FDA-Food Code 2005, 2005). In this study we did not determine if restaurants follo w these recommendations or not. Table 4-1. Temperature and pH profiles of co mmercial red salsa by type of restaurant Temperature (C) pH Restaurant type Average Range Average Range Fast food 19.4 A 5.4 27.0 3.8 A 3.4 4.2 Chain Midscale 13.4 B 4.9 21.2 4.0 A 3.7 4.3 Locally owned 14.0 C 5.4 27.0 3.9 A 3.6 4.3 Averages followed by different letters in row are different from each other by Duncans multiple range test ( = 0.05) Table 4-2. Temperature and pH profiles of commercial red salsa by type of salsa Temperature (C) pH Salsa type Average Range Average Range Red 14.9 A 5.4 27.0 3.8 A 3.6 4.1 Fresh 11.4 B 4.9 21.2 3.9 A 3.4 4.3 Averages followed by different letters in row are different from each other by Duncans multiple range test ( = 0.05) The pH of salsa samples ranged from 3.3 to 4.3 showing acidic pH profiles (Table 4-1 and 4-2). There was no significant difference in the pH of fresh and red salsa; however red salsa seemed to have less acidic pH values th an fresh salsa. While the pH values of salsa sampled in this study fall in the category of f oods previously documented as not favorable for pathogen growth, in recent years several foodbo rne illness outbreaks were associated with food having low pH (< 4.6) (Raghubeer et al., 2000). Many studies have demonstrated that some bacteria such as E. coli and Salmonella spp. can adapt and su rvive under acidic conditions (Conner et al., 1996 and Smith, 2003). Microbiological Quality Overall, salsa samples collected from lo cally owned and chain restaurants had poor microbiological quality. Although 87.5% of th e samples tested were ranked under

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52 unsatisfactory conditions according to the Scie ntific Criteria to Ensure Safe Food (2003), none of them were classified above potentially hazardous limits. Overall, red salsa samples had better microbiological quality than fresh salsa. Seventy five percent of red salsa samples (18 samples) were ranked under acceptable condi tions, while only 21% (5 samples) of fresh salsa samples were ranked under th at category (Figure 4-6). 18 5 33 6 5 21 210 5 10 15 20 25RedFreshLocally ownedChainNumber of Samples Acceptable Unsatisfactory Figure 4-6. The number of acceptable and unsatisfactory samples according to scientific criteria to ensure safe food (2003) Salsa samples from locally owned restaura nts had higher bacteria l counts (aerobic counts, E. coli and Staphylococcus aureus ) than chain restaurants. These bacterial populations gave an idea of hygienic practices used in restaurant s. Perhaps chain restaurants have to follow more rigorous hygiene standa rds than locally owned restaurants. It appeared that higher counts of pathogens were found in fres h salsa than in red salsa. Preparation of fresh salsa may require exte nsive handling such as chopping of fresh ingredients including tomato, onions, cilantro and chili pe ppers, which brings more opportunities for mishandling and cross-contam ination from food handlers and other food contact surfaces such as utensils. On the othe r hand, red salsa is usually prepared with the same ingredients, but the prep aration includes cooking of toma to, which may reduce the risk of pathogen contamination, as cooking is considered a killing step. Additionally, some

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53 salsa ingredients such as tomatoes and cilantro have been implicated in many foodborne illnesses due to Salmonella and other microbes which increase the potential risk of salsa (CSPI, 2007; CDC, 2003; Campbell et al, 2001). Due to the lack of microbial standards fo r Mexican salsa in the US food regulations, international standards and speci fications for ready-to-eat vege table products were considered as parameters for the evaluation of salsa sa mples (Appendix F). This standard indicates criteria to classify food products, according to its microbial c ontent, under four categories: acceptable, satisfactory, unacceptable and potential hazardous. Total aerobic Count On average, salsa samples tested in this study contain a broad range of total aerobic count (1.23 6.03 log CFU/g). No statistically significant difference was found in total aerobic count between locally owne d and chain restaurants (p>0.005; =0.05). The average total aerobic counts of salsa samp les from the locally ow ned and chain restaurants were 3.53 and 3.76 log CFU/g, respectively (Table 4-3). Sa mples for both types of restaurants fall under satisfactory limits for ready-to-eat vegetables (< 4 log CFU/g). Statisti cal analysis of aerobic plate count by salsa type (Table 4-4) indicated a si gnificant difference between red and fresh salsa. Average total aerobi c counts for red and fresh salsa were 3.11 and 4.17 log CFU/g, respectively. Although fresh salsa samples had higher total aerobic coun ts, the values were under acceptable levels (4 to < 5 log CFU/g). Salsa samples stored at temperatures higher than 10C had higher total aerobic counts. Overall total aerobic counts found in salsa sa mples in this study (3.38 log CFU/g) are lower than those reported in fresh tomato-based salads by Kubhela et al. (2002) [ 5.9 log CFU/g] and Kokkinakis and Fragki adakis (2007) [6.91 log CFU/g].

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54 Total coliform Count Total coliform ranged from 1.00 to 4.73 log CFU/g in 93.7% of samples (n = 45/48). Locally owned restaurants had significantly lower coliform (2.43 log CFU/g) than chain restaurants (3.23 log CFU/g) tested in this study. Total coliform for both salsa types were significantly different; on average total coliform of fresh salsa were hi gher than those of red salsa (3.13 and 2.42 log CFU/g, respectively). Total coliform counts in salsa (2.46 log CFU/g) in this study are lower than those reported by Kokkinakis and Fragkiadakis. (2007) [5.64 log CFU/g]. Six samples (13.3%) from both locally owned and chain restaurants tested positive for E. coli with counts values ranging from 2.60 to 3.18 log CFU. Half of the samples, positive for E. coli, were from locally owned restaurant, and the other half were from chain restaurants (Table 4-3). Samples from the locally owned restaurants had higher E. coli populations than those samples from the chain restaurants (3.05 and 2.69 log CFU/g, respectively), but both numbers fall under unsatisfactory limits. Only fresh salsa samples tested positive for E. coli and levels of this bacteria fall under unsatisfactory limits (Table 44). The presence of fecal coliform indicates in adequate hygienic-sanitary conditions in the processing and preparation of food products Counts higher than 2 log CFU/g for E. coli represent an unsatisfactory level. In this case, it is impossible to indentify if the contamination comes from the restaurants or from the raw materials. These groups of microorganisms are common in raw vegetables and are not necessarily associated with fecal contamination; the majority of the genera ar e part of the endogenous microflora of the product (Brackett and Sp littstoesser, 2001). E. coli values found in this study are comparable with those reported for tomato-based salad prepared in restaurants, hospitals and the military (Kokkinakis and Fragkiadakis, 2007;

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55 Ayc cek et al., 2004). In those two studies, the E. coli populations were higher than 2 log CFU/g, indicating a poor micr obiological quality. Table 4-3. Microbiological qu ality of Mexican salsa by re staurant types (Averages) Microbiological parameter Locally own Restaurants Chain Restaurant APC (log CFU/g) (n=48) 3.53 A 3.76 A Total coliform (log CFU/g) (n=45) 2.43 A 3.23 B E. coli (log CFU/g) (n=6) 3.05 A 2.69 B Staphylococcus aureus (log CFU/g) (n=21) 2.44 A 1.79 B Salmonella spp. (n=48) Lower than minimum detection level Lower than minimum detection level Averages followed by different letters in line are different from each other by Duncans multiple range test ( = 0.05) Table 4-4. Microbiological quality of Mexican salsa by salsa type (Averages) Microbiological parameter Red salsa Fresh salsa APC (log CFU/g) (n=48) 3.11 A 4.17 B Total coliform (log CFU/g) (n=45) 2.42 A 3.13 B E. coli (log CFU/g) (n=6) Absent 2.87 Staphylococcus aureus (log CFU/g) (n=21) 2.44 A 1.92 B Salmonella spp. (n=48) Lower than minimum detection level Lower than minimum detection level Averages followed by different letters in a row are different from each other by Duncans multiple range test ( = 0.05) Salmonella spp. None of the salsa samples evaluated contained Salmonella spp. The results are in accordance with safety criteria standards, which establish no Salmonella spp. should be present in 25g of ready-to-eat vegetable food products (Scientif ic Criteria to Ensure Safe Food, 2003). Compared to minimally processed vegetables and ready-to-eat sa lads studied by Sagoo et al. (2003) and Froder et al. (2007) salsa samples had better microbiological quality when analyzing for the presence of Salmonella spp. Froder et al (2007) reported the presence of Salmonella in 1 (4.8%) of 21 mixed salads analyz ed, while Sagoo et al. (2003) reported isolation of Salmonella from 5 (0.12%) of 3,852 ready-to -eat salads analyzed.

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56 Staphylococcus aureus Staphylococcus aureus was present in 21 samples of sa lsa. Of those 9 samples (37.5%) were from l ocally owned restaurants and 12 samp les (50%) were from chain restaurants. On an average, Staphylococcus aureus populations in locally owned and chain restaurants samples were 2.44 log CFU/g and 1.79 log CFU/g, respectively; these were statistically different (Table 4-3). Red salsa samples tested in this study had a significantly higher Staphylococcus aureus population than those tested in fresh salsa. Average counts for Staphylococcus aureus in red and fresh salsa were 2.44 and 1.92 lo g CFU/g, respectively (Table 4-4). Staphylococcus aureus was present in 6 red salsa sample s, and in 15 fresh salsa samples. Staphylococcus aureus levels in red salsa samples from locally owned restaurants tested in this study fell under unsatisfactory levels (2 to 4 log CFU/g), while the Staphylococcus aureus populations in fresh salsa samples from chain restaurants fall under acceptable limits (1.3 to < 2 log CFU/g). The presence of Staphylococcus aureus has no correlation to the types of salsa, but rather the presence of this microorganism is st rongly related to unsanitary hygienic conditions and improper handling practices. Staphylococcus aureus isolated from salsa samples is an indicator of poor hygiene practices at restaurants. In general, food handlers are the main source of this organism; however, equipment and environmental surfaces can also be sources of contamination with Staphylococcus aureus, when they were previously touch by infected handlers or workers (Panisel lo et al., 2000). Counts of Staphylococcus aureus found in some salsa samples fall under the unsatisfactory catego ry for ready-to-eat vegetable products, but they do not fall on potentially hazardous category.

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57 The incidence of Staphylococcus aureus found in this study is higher than those published by Ayc cek et al. (2004), who showed out of 70 salad samples studied, 8 (11.4%) tested positive for coagulase staphylococci. Methicillin resistant Staphylococus aureus (MRSA) screening Twenty one Staphylococcus aureus isolates from salsa samples were tested by the disk diffusion me thod on MHA and MSA. Inhibition zone diameters for each disk, in triplicate, were measured, and compared against the National Committee for Clinical Laboratory Standards (NCCL) size of inhibition zone on MHA and MSA plates. Values 10 and 16 mm, respectively were defined as resistant. All isol ates presented inhibiti on zones greater than 10 mm, and therefore all of 21 Staphylococcus aureus strains were determined as methicillinsusceptible (Table 4-5). Average inhibition z one diameters for the MRSA and MSSA control strains were 27.8 mm and 0 mm in MHA and 23.6 mm and 0 mm in MSA, respectively. A graphic comparison between positive and negative control and isolated strain inhibition zones could be observed in Figure 4-7. Table 4-5. Staphylococcus aureus strains isolated from commercial salsa Average inhibition zone diameter (mm) Sample MSA1 MHA1 Fresh salsa 19.2 19.4 Red salsa 16.4 19.3 1 Represents average diameter replicates tested in two trials Methicillin resistant Staphylococcus aureus infections are a global health issue due to the severity of the illness. In the past, MR SA infections were mainly hospital acquired infections (HA-MRSA), but in recent years, the incidence of community acquired infections (CA-MRSA) have increased in industrialized countries (N ormanno et al, 2005). Some of these CA-MRSA infections have been related to food (Kluytmans et al, 1995; Jones et al., 2002, Normanno et al., 2005). The role of contaminated food in the spread of the infections is still unclear because limited information is available (Normanno et al., 2005). Foods of

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58 animal origin such as milk and meat are the most common implicated foods, but so far fruit or vegetable have not been implicated in MRSA outbreaks (Normanno et al., 2005, Jones et al., 2002, Kluytmans et al., 1995). Isolated strain Positive Control Negative Control (A) Isolated strain Positive Control Negative Control (B) Figure 4-7. Disk diffusion method, inhibition zone diameters for positive, negative control and isolated Staphylococcus aureus (A) Inhibition zone di ameters in MullerHinton Agar (MHA), (B) Inhibition zone diameters in Manito Salt Agar (MSA) Microbiological Challenge Study Red salsa was selected to be used in the m icrobiological challenge study for two reasons. First, preliminary work revealed that red salsa samples had overall lower microbial counts (See Table 4-4) than fresh salsa samples. Total aerobic counts and total coliform in red salsa are lower than those found in fresh salsa. E. coli was absent in red salsa samples. Although overall Staphylococcus aureus populations in red salsa ar e higher than those found in fresh salsa, the restaurant chain from which red salsa samples were purchased showed the lowest populations of this bacteria. Second, it is common to consume red salsa with snacks (nachos and chips), and red salsa and nacho ch ips are often served as free appetizers in Mexican style restaurants while customers are waiting for their main course.

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59 Total aerobic counts (APC), Staphylococcus aureus and Salmonella spp. were determined on control samples (un-inoculated sa mples) for the microbial challenge studies. On average aerobic plate counts were 2.21 log CF U/g, and none of the control samples tested positive for Staphylococcus aureus or Salmonella spp. Staphylococcus aureus Study A decline in Staphylococcus aureus population was observed for samp les stored at both room (20C) and refrigerated (4C) temperatures and at both hi gh (1,550 cells/g) and low (155 cells/g) inoculation levels (p<0.0001) (Figures 4-8, 4-9 and Tables 4-6 and 4-7) 1.5 2 2.5 3 3.5 4 4.5 024624logCFU/gTime in hours Low inoculation High inoculation Figure 4-8. Staphylococcus aureus survival in salsa samples storage at room temperature (20C) 1 1.5 2 2.5 3 3.5 4 4.5 01357logCFU/gTime in days Low inoculation High inoculation Figure 4-9. Staphylococcus aureus survival in salsa samples storage at refrigeration temperatures (4C)

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60 Staphylococcus aureus populations in salsa samples remained high during the first 6 h at room temperature for both i noculation levels. The decrease began to occur after 6 h, and the most significant decreased was observed at 24 h of storage. However, the decrease was of no practical purpose because the final Staphylococcus aureus populations were 2.15 and 3.56 log CFU/g for both low and high inoculation levels respectively. These fi nal levels were still high and therefore placed the samples under unsa tisfactory microbiological quality (2 to 4 log CFU/g). According to the FDA Food Code 2005, when ready-to-eat food products are taken out of refrigeration and kept at room temperature, and when the room temperature does not exceed 21C (70F) during that period of time, th e food may remain for up to 6 h. After that time, the food product is not safe for consump tion. Salsa samples which were kept at room temperature for an extended period of time (> 6 h) presented a poor appearance and started to have strong decomposition smell and eventua lly will not be appealing for consumption. However, before the 6 h of storage and alt hough the appearance of salsa samples were still good, if salsa was contaminated with Staphylococcus aureus, the remaining bacterial content is still high representing some microbial risk. Staphylococcus aureus populations remained practically constant in refrigerated samples (at 4C) during the first two days of storage, although the bacterial population slightly started to decline after day 3 of storage, and reached the greatest reduction after 7 days of storage for both high and low inoculat ion levels (1.67 log CFU/g and 1.44 log CFU/g, respectively). Nevertheless, the final bacteria populations remained within the acceptable microbial quality category (1.3 to 2 log CFU/g). The fact that the microbial load remained high during the first 3 days of storage suggests that Staphylococcus aureus will persist in salsa once it is contaminated.

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61 Table 4-6. Survival of Staphylococcus aureus in salsa samples during storage at 20 2C (log CFU/g) Inoculation level Time (h) Low High 0 3.23 a 4.18 a 2 3.25 a 4.24 ab 4 3.19 ab 4.15 ab 6 3.14 ab 4.08 b 24 2.15 b 3.56 c Averages followed by different letters in columns are different from each ot her by Duncans multiple range test ( = 0.05) Table 4-7. Survival of Staphylococcus aureus in salsa samples during storage at 4 2C (log CFU/g) Inoculation level Time (days) Low High 0 3.23 a 4.18 a 1 3.20 a 4.20 a 3 2.46 b 3.17 b 5 1.95 c 2.19 c 7 1.44 d 1.69 d Averages followed by different letters in columns are different from each ot her by Duncans multiple range test ( = 0.05) The results of Staphylococcus aureus challenge study reveals that salsa can be a potential health hazard if it is contaminated. Several studies have s hown that there is a significantly longer survival of pathogens in acidic foods stored at 4C compared with storage at 20 to 23C (Erickson and Jenkins 1991, Gomex-Lucia et al., 1987, Miller and Kaspar, 1994, and Raghubeer et al., 1995, Raghubeer et al., 2000). Salmonella Study The Salmonella microbiological challenge study indica tes that refriger ation can support survival of Salmonella spp. for at least 3 days. Despite the low pH, salsa will support bacterial growth when stored at room temperature for up 24 h. Salmonella survival was done in a side by side study, using two different types of selective media and different incubation temperatures. XLD Agar and CHROMagarTM Salmonella media were used with SC and RV enrich ment broths and incubated at 37 2C following AOAC-RI # 020502. Rapid Check SELECTTM for Salmonella was used with TT broth and incubated at 42 2C following AOAC-RI# 03030. Both methods were tested

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62 under one inoculation level (15 to 20 cell/sample) and two different storage temperatures, room temperature (20 2C) and refrigeration (4 2C). The bacterial population of samples was evaluated at different time points throughout the study. While using XLD agar and CHROMagarTM fail to detect the presence of inoculated Salmonella under all test conditions, the Rapid Chek SELECTTM showed positive results (Tables 4-8 and 4-9). Salmonella tested positive at all times in samples stored at room temperature from hour 0 to hour 24. While in refrigeration, Salmonella was present for the first three days, and thereafter, no Salmonella was found. Table 4-8. Presence or absence1 of Salmonella spp. on inoculated salsa samples stored at 20 2C Time (h) CHROMagar XLD Rapid Check SELECTTM 0 Absent Absent Present 2 Absent Absent Present 4 Absent Absent Present 6 Absent Absent Present 24 Absent Absent Present 1 Absent means less than minimum detection level Table 4-9. Presence or absence1 of Salmonella spp. on inoculated salsa samples stored at 4 2C Time (days) CHROMagar XLD Rapid Check SELECTTM 0 Absent Absent Present 1 Absent Absent Present 3 Absent Absent Present 5 Absent Absent Absent 7 Absent Absent Absent 1 Absent means less than minimum detection level Level of sensibility for each method may be due to different enrichment broths, and incubation temperatures. Another important factor to mention is that Rapid Chek SELECTTM uses a selective pre-enrichme nt broth, which may enhance Salmonella growth by eliminating other background flora by the antibi otics in the media, while the CHROMagar method uses Lactose broth which is a unive rsal broth that enhances growth of Salmonella spp. as well as other background flora. A lthough several studies have shown that

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63 CHROMagar is a very effective selective medi a, even more than XLD agar (Mores et al., 2005, Maddocks et al., 2002), in th is study, it failed to detect Salmonella presence in inoculated salsa samples. Microbiological challenge studies results de monstrated that despite the high acidity levels typically found in salsa, Salmonella can persist during typical storage and consumption time. Furthermore, salsa will support Salmonella survival when stored under refrigeration for longer periods of time. These findings are c onsistent with those found by Campbell et al (2001) in unprocessed salsa samples. These results reinforce the concept that on ce salsa is contaminated by pathogens such as Salmonella and Staphylococcus aureus they may survive long enough to cause foodborne illness to consumers. However, survival characteristics of different pathogens in similar foods that are stored in similar conditions may be diff erent, and therefore each type of food must be tested separately to assess the risk using the pathogens of interest to public health.

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64 CHAPTER 5 SUMMARY AND CONCLUSION This study found that Mexican food is of ten susceptible to foodborne pathogen contamination. Among t he bacterium etiology, th e most common microorganisms related to Mexican food outbreaks (1990-2006) were Salmonella (34%), Clostridium (24%), Shigella (6%) and Staphylococcus (5%). Forty six percent of the know outbreaks were related to food consumed at restaurants. The top four vehicle of contamination, the top four foods related to outbreaks were tacos (18%) followed by chili (9%), salsa (9%), and refried beans (8%). Salsa is an important side dish in Mexican cuisine, and it is usually served with main dishes (among others steaks, taco s and tamales) as well as a dip for snacks. The results in this study, although limited by a small sample size and th e evaluation of samples in only one city, indicated that overall salsa purchased from co mmercial restaurants has poor microbiological quality. Total aerobic counts and total coliform ranged from 1.23 to 6.03 log CFU/g and 1.00 to 4.73 log CFU/g, respectively. E. coli populations were found in only six fresh salsa samples, while the pathogen was not detected in red salsa samples. Staphylococcus aureus populations were found in 21 samples. Although Staphylococcus aureus populations were more prevalent in fresh salsa samples (12 samples), red salsa samples had the highest population counts (2.44 log CFU/g). None of the Staphylococcus aureus isolates were resistant to antibiotics. No Salmonella spp. was detected in tested samples. Overall samples collected from locally owned restaurants had poorer microbiological quality than those collected from chain restaurants. Although service practices differ among restau rants, overall salsa samples were kept and sold under the temperature danger zone (5C, 41 F 57C, 135F). Red salsa average temperature at point of sale was 14.9C while th e average temperature of fresh salsa at point of sale was 11.4C. The ideal storage temperature for perishable food products recommended by the Food Code (2005) is 5C (41F). In fa st food restaurants salsa was kept under ice-

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65 refrigerated displays, and these samples showed the highest temperature profiles. While in midscale chain and locally owned restaurants, sals a was usually kept in the restaurant kitchen and brought to the table once or dered, these samples showed c ooler temperature profiles. The microbiological challenge study tested two pathogens, Salmonella spp. and Staphylococcus aureus. Selection of bacterial targets was done on the basis of previous Mexican food safety data, which indicated Salmonella spp. as the most common pathogen found in Mexican food outbreaks. Furthermore, salsa ingredients (tomato, cilantro and onions) were related in recent years to Salmonella foodborne outbreaks (CDC, 2008; CSPI, 2007; Adachi et al. 2002). Staphylococcus aureus was selected because of its importance as an indicator of food handli ng and preparation practices. The study demonstrated that despite the hi gh acidity levels typically found in salsa these pathogens can persist during typical consumption time. Of greater interest, salsa will allow bacteria survival when stored at refrige ration for longer periods of time. Salmonella spp. were present in all inoc ulated salsa samples stored at room temperature, while Staphylococcus aureus populations decreased after 24 h, a nd all bacterial levels remained high the first 6 h when stored at room temperature. Under refrigeration Salmonella spp. can survive in salsa for at least 3 days, and Staphylococcus aureus populations remain considerably high for the same period of time. Several studies have shown that there is a signi ficantly longer survival of pathogens in acidic foods stored at 4C compared with storag e at 20 to 23C (Erickson and Jenkins, 1991, Gomex-Lucia et al., 1987, Miller and Kaspar 1994, and Raghubeer et al., 1995, Raghubeer et al., 2000). Contaminated salsa presents a potential health hazard. The biggest concern with salsa is that it is usually served in combination with warm cooked ingredients, which increases the

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66 risk of rapid bacterial growth. Moreover, food sa fety practices are not we ll identified for this type of cuisine and there are no specific guidelines for service and handlers practices. The evaluation of food safety for ethnic food in the US is a starting point to address the increasing number of outbreaks related with this type of food. The chal lenge for food safety governmental agencies should be to establis h specific guidelines and standards based on current food safety trends.

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67 APPENDIX A PATHOGENS DISTRIBUTION IN MEXI CAN FOODBORNE ILLNESS OUTBREAKS REPORTED BY THE CDC (1990-2006) Table A-1. Pathogens distribut ion in Mexican foodborne illness outbreaks reported by the CDC (1990-2006) Etiology Outbreak numb er % Salmonella 74 34% S. enteriditis 29 13% S. heidelberg 10 5% S. typhimurium 10 5% Other Salmonella types 25 12% Clostridium 52 24% C. botulinum 1 0% C. perfringens 51 24% Norovirus 28 13% Shigella 13 6% S. sonnei 8 4% Other Shigella types 5 2% Staphylococcus 11 5% Staphylococcus aureus 9 4% Staphylococcus aureus (enterotoxin type A) 2 1% Escherichia 10 5% E. coli O157:H7 8 4% Enterotoxigenic E. coli 2 1% Bacillus cereus 4 2% Campylobacter jejuni 4 2% Listeria monocytogenes 4 2% Norwalk* 4 2% Hepatitis A 2 1% Other and multiple 9 4% Total 215 100% *Norwalk virus it is currently called Norovirus

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68 APPENDIX B MICROBIOLOGICAL QUALITY OF COMM ERCIAL MEXICAN SA LSA (FLOW DIAGRAM)

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69 Figure B-1. Flow diagram for co mmercial Mexican salsa microbi ological quality evaluation Collect Salsa samples Homogenize 25 g of sample in 225 ml Lactose Broth RappaportVassiliadis b roth selenite cystine broth XLD Agar and ChromA g a r Black-colored colonies on XLD and mauve-colored colonies on ChromAgar are presumptively i de ntifi ed as Salmonella spp. Incubate 24 h at 37 C Confirmation Test by Latex Agglutinationtest Incubate 24 h at 37 C Incubate 24 h at 37 C Measure tem p erature at p oint of sale and p H at laborator y Homogenize 25 g of sample in 225 ml 0.1% Pe p tone wate r Serial dilute salsa (1:10 v/v) with 0.1% Pe p tone wate r Plate on 3M Petrifilm: APCplates Count, E. coli, Staphylococcus au r eus Incubate 24 h at 37 C Read results (CFU/g) Test presumptive Staphylococcus aureus strains by Coagulase agg l u tin a ti o n t est MRSA determination of positive Staphylococcus aureus strains by disk d iff us i o n m et h od

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70 APPENDIX C SAMPLE PREPARATION FOR MICROBIOL OGICAL CHALLENGE STUDY (FLOW DIAGRAM) Figure C-1. Sa mple preparation for the microbiological challenge study 1000 g 1000 g 1000 g Red salsa samples from commercial restaurant sold in individual containers Mixed samples in sterile container (5000 ml) Transportation to laboratory facility in cooler, 20 min Transfer aseptically 25 g to 150 ml sterile containers with lid 15 containers for storage at room temperature (20C) 15 containers for storage at refrigeration (4C) 15 containers for storage at room temperature (20C) 15 containers for storage at refrigeration (4C) Sta p h y lococcus aureus Salmonella Sam p le Inoculation Low: 155 cells/ g Hi g h: 1 550 cells/ g 15 20 cells/sam p le 15 containers for storage at room temperature (20C) 15 containers for storage at refrigeration (4C) Continue in Appendix D

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71 APPENDIX D MICROBIOLOGICAL CHALLENGE STUDY IN COMMERCIAL MEXICAN SA LSA (FLOW DIAGRAM)

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72 Figure D-1. Flow diagrams for microbiological challenge study in commercial Mexican salsa Bacterial cocktail preparation Homogenize 25 g of sample in 225 ml Lactose Broth Rappaport Vassiliadis selenite cystine b roth XLD Agar and CHROMa g ar Black-colored colonies on XLD and mauve-colored colonies on CHROMagar are presumptively identified as Salmonella s pp Incubate 24 h at 37 C Confirmation Test by Latex agglutination test Incubate 24 h at 37 C Incubate 24 h at 37 C Sample inoculation 15 to 20 cells/sample Homogenize 25 g of sample in 225 ml 0.1% Pe p tone wate r Serial dilute salsa (1:10 v/v) with 0.1% Pe p tone wate r Plate on 3M Staphylococcus Incubate 24 h at 37 C Read results (CFU/g) Test presumptive Staphylococcus aureus strains by Coa g ulase Sample inoculation High: 1,550 cells/g Low: 155 cells/g Salmonella Sta p h y lococcus aureus From diagram C: When stored at 20C at 0, 2, 4, 6 and 24 h When stored at 4C at 0, 1, 3, 5 and 7 day Homogenize 25 g of sample in 225 ml Enrichment Ra p id Check Incubate 24 h at 42 C Tetrathionate broth Incubate 24 h at 42 C Rapid Check SELECT Stri p s test XLD Agar Incubate 24 h at 42 C

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73 APPENDIX E COMMERCIAL SALSA SAMPLES (A) (B) Figure E-1. Commercial salsa samples: fresh salsa (A) and red salsa (B)

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74 APPENDIX F GUIDELINES FOR THE MICRO BIOLOGICAL QUALITY OF S OME READY-TO-EAT FOODS AT POINT OF SALE

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75Table F-1. Guidelines for the microbiological qualit y of some ready-to-eat foods at point of sale Microbiological quality (CFU/g unless otherwise stated) Food categorya Criterion Satisfactory Acceptable Unsatisfactory Unacceptable/Potentially hazard Aerobic colony count (30C/48h) A <103 103 to <104 104 N/Ab B <104 104 to < 105 105 N/A C <105 10 to < 106 106 N/A D <106 10 to < 107 107 N/A E N/A N/A N/A N/A Indicator organismsc A E Enterobacteriaceae < 100 100 to < 104 104 N/A A E Escherichia coli (total) < 20 20 to < 100 100 N/A A E Listeria spp. (total) < 20 20 to < 100 100 N/A Pathogens A E Salmonella spp. Not detected in 25 g Detected in 25 g A E Campylobacter spp. Not detected in 25 g Detected in 25 g A E E. coli O157 and other verocytotoxin producing E. coli Not detected in 25 g Detected in 25 g A E Vibrio cholerae Not detected in 25 g Detected in 25 g A E Vibrio parahaemolyticusd < 20 20 to < 100 100 to < 103 A E L. monocytogenes < 20 20 to < 100 N/A A E Staphylococcus aureus < 20 20 to < 100 100 to < 104 A E Clostridium perfringens < 20 20 to < 100 100 to < 104 A E Bacillus cereus and other pathogenic Bacillus spp. < 103 103 to < 104 100 to < 106 a Category A includes beef burgers, meat pies pork pies, sausages rolls, scorch eggs, raw pickled fish, mousse/desserts; Catego ry B includes meat meals, poultry cakes and pastries (without dairy cream), quiche, mayonnaise, vegetables, ice cr eam, ready-to-eat meals; Category C in cludes sliced beef, sliced pork, slic ed poultry, crustaceans, fish, cakes and pastries (with dairy cream), dried fruits and vegetables, and rice. Categor y D includes sliced ham, mollusks, and other shellfish, prepared mixed salads; Ca tegory E includes raw ham, smoked sausages, cheesecake, fermented foods, fresh fruits an d vegetables, cheese, yogurt. b N/A = not available. c On occasion some stra ins may be pathogenic. d Relevant to seafood only Adapted from: Scientific criteria to ensure safe food. 2003. Institute of Medicine, National Research Council. National Academ y Press. Washington, DC.

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76 APPENDIX G MICROBIOLOGICAL QUALITY OF COMMERCI AL MEXICAN SA LSA: RAW DATA Table G-1. Microbiological quality of commercial Me xican salsa: raw data Trial Rep Salsa type Restaurant type Tem (C) pH APC Staphylococcus aureus Total Coliform Salmonella 1 S1 Red Local 10.1 3.95 2.82 2.77 1.40 1 S2 Red Local 10.2 3.84 2.87 2.52 2.10 1 S3 Red Local 10 3.81 2.86 2.65 2.15 1 S1 Red Chain 7.3 4.11 2.73 1 S2 Red Chain 7.3 3.74 2.70 1 S3 Red Chain 7.3 3.75 2.72 1 S1 Red Local 12.4 3.59 3.48 2.38 3.08 1 S2 Red Local 12.2 3.58 3.47 2.13 2.98 1 S3 Red Local 11.9 3.57 3.50 2.20 3.10 1 S1 Red Local 26.4 3.65 2.89 2.51 1 S2 Red Local 26.7 3.62 2.81 1.93 1 S3 Red Local 27 3.62 2.15 2.08 1 S1 Pico Chain 6.4 4.12 1.30 1.85 1 S2 Pico Chain 6.5 4.15 1.32 2.06 1 S3 Pico Chain 6.2 4.07 1.23 1.90 1 S1 Pico Chain 4.9 4.14 6.30 2.61 4.36 1 S2 Pico Chain 4.9 4.08 4.95 2.04 4.11 1 S3 Pico Chain 4.9 4.26 6.10 2.80 4.27 1 S1 Pico Local 10.3 4.06 4.77 2.40 3.51 1 S2 Pico Local 10.2 4.09 6.00 2.48 3.45 1 S3 Pico Local 10.3 4.06 4.98 2.46 3.68 1 S1 Pico Chain 10.2 3.67 3.59 2.97 1 S2 Pico Chain 10 3.39 3.43 2.72 1 S3 Pico Chain 10.4 3.62 3.10 2.80 2 S1 Red Local 5.8 3.97 2.82 1.00 2 S2 Red Local 5.7 3.94 2.87 1.54 2 S3 Red Local 5.4 3.92 2.86 1.65 2 S1 Red Chain 19.7 3.99 5.35 2.81 2 S2 Red Chain 20.4 4.00 3.60 3.40 2 S3 Red Chain 21.2 4.00 3.36 2.60 2 S1 Red Local 11.6 3.96 3.81 3.13 2 S2 Red Local 12 3.94 3.48 3.10 2 S3 Red Local 11.9 3.94 3.65 3.04 2 S1 Red Local 25 3.89 2.40 1.98 2 S2 Red Local 25.1 3.89 2.54 2.48 2 S3 Red Local 24.8 3.89 3.00 2.78 2 S1 Pico Chain 14 4.15 4.27 1.00 2.81 2 S2 Pico Chain 14.5 4.12 4.23 1.00 3.02 2 S3 Pico Chain 14.1 4.17 4.24 1.00 2.88 2 S1 Pico Chain 20.8 4.01 4.35 1.81 2.90 2 S2 Pico Chain 21.2 3.98 3.85 1.60 3.16 2 S3 Pico Chain 21.2 3.96 3.88 1.48 3.16

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77 Table G-1. Continued 2 S1 Pico Local 10.3 4.19 4.35 1.48 2 S2 Pico Local 10.5 4.21 5.20 2.04 2 S3 Pico Local 10.2 4.28 5.04 2.10 2 S1 Pico Chain 14.1 3.53 5.11 2.16 4.73 2 S2 Pico Chain 13.8 3.56 4.23 1.95 4.67 2 S3 Pico Chain 14.5 3.56 4.27 2.04 4.58

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78 APPENDIX H MICROBIOLOGICAL QUALITY OF COMMERCI AL MEXICAN SA LSA: STATISTICAL ANALYSIS Table H-1. Microbiological qual ity of commercial Mexican sa lsa: statistical analysis Source DF Squares Mean Square F Value Pr > F Model 6 23.25 3.87 3.7 0.005 Error 41 42.95 1.05 Corrected Total 66.19 Source DF Type III SS Mean Square F Value Pr > F Trial 1 2.39 2.39 2.28 0.1389 Rep 2 0.41 0.21 0.20 0.8223 Type 1 14.16 14.16 13.52 0.0007 Rest 1 1.40 1.40 1.33 0.2548 Type*Rest 1 5.63 5.63 5.38 0.0255

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79 APPENDIX I STAPHYLOCOCCUS AUREUS MICROBIOLOGICAL CHALLEN GE STUDY: RAW DATA Table I-1. Staphylococcus aureus m icrobiological challe nge study: raw data Trial Rep Temperature Inoculation] Storage time log CFU/g 1 S1 Room Low 0 h 3.114 1 S2 Room Low 0 h 3.267 1 S3 Room Low 0 h 3.230 1 S1 Room Low 2 h 3.255 1 S2 Room Low 2 h 3.190 1 S3 Room Low 2 h 3.255 1 S1 Room Low 4 h 3.176 1 S2 Room Low 4 h 3.217 1 S3 Room Low 4 h 3.161 1 S1 Room Low 6 h 3.021 1 S2 Room Low 6 h 3.176 1 S3 Room Low 6 h 3.190 1 S1 Room Low 24 h 2.243 1 S2 Room Low 24 h 2.130 1 S3 Room Low 24 h 2.041 1 S1 Room High 0 h 4.230 1 S2 Room High 0 h 4.130 1 S3 Room High 0 h 4.230 1 S1 Room High 2 h 4.279 1 S2 Room High 2 h 4.190 1 S3 Room High 2 h 4.130 1 S1 Room High 4 h 4.114 1 S2 Room High 4 h 4.146 1 S3 Room High 4 h 4.204 1 S1 Room High 6 h 4.161 1 S2 Room High 6 h 4.114 1 S3 Room High 6 h 4.097 1 S1 Room High 24 h 3.525 1 S2 Room High 24 h 3.562 1 S3 Room High 24 h 3.544 1 S1 RefrigerationLow day 0 3.114 1 S2 RefrigerationLow day 0 3.267 1 S3 RefrigerationLow day 0 3.230 1 S1 RefrigerationLow day 1 3.061 1 S2 RefrigerationLow day 1 3.079 1 S3 RefrigerationLow day 1 3.161 1 S1 RefrigerationLow day 3 2.477 1 S2 RefrigerationLow day 3 2.301 1 S3 RefrigerationLow day 3 2.544 1 S1 RefrigerationLow day 5 1.929 1 S2 RefrigerationLow day 5 1.813 1 S3 RefrigerationLow day 5 1.978 1 S1 RefrigerationLow day 7 1.301 1 S2 RefrigerationLow day 7 1.602

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80 Table I-1. Continued 1 S3 RefrigerationLow day 7 1.398 1 S1 RefrigerationHigh day 0 4.230 1 S2 RefrigerationHigh day 0 4.130 1 S3 RefrigerationHigh day 0 4.230 1 S1 RefrigerationHigh day 1 4.079 1 S2 RefrigerationHigh day 1 4.267 1 S3 RefrigerationHigh day 1 4.301 1 S1 RefrigerationHigh day 3 3.061 1 S2 RefrigerationHigh day 3 3.190 1 S3 RefrigerationHigh day 3 3.267 1 S1 RefrigerationHigh day 5 2.176 1 S2 RefrigerationHigh day 5 2.267 1 S3 RefrigerationHigh day 5 2.267 1 S1 RefrigerationHigh day 7 1.740 1 S2 RefrigerationHigh day 7 1.875 1 S3 RefrigerationHigh day 7 1.699 2 S1 Room Low 0 h 3.290 2 S2 Room Low 0 h 3.322 2 S3 Room Low 0 h 3.146 2 S1 Room Low 2 h 3.204 2 S2 Room Low 2 h 3.342 2 S3 Room Low 2 h 3.243 2 S1 Room Low 4 h 3.061 2 S2 Room Low 4 h 3.301 2 S3 Room Low 4 h 3.217 2 S1 Room Low 6 h 3.204 2 S2 Room Low 6 h 3.061 2 S3 Room Low 6 h 3.204 2 S1 Room Low 24 h 2.097 2 S2 Room Low 24 h 2.176 2 S3 Room Low 24 h 2.190 2 S1 Room High 0 h 4.267 2 S2 Room High 0 h 3.875 2 S3 Room High 0 h 4.371 2 S1 Room High 2 h 4.243 2 S2 Room High 2 h 4.301 2 S3 Room High 2 h 4.301 2 S1 Room High 4 h 4.312 2 S2 Room High 4 h 4.021 2 S3 Room High 4 h 4.130 2 S1 Room High 6 h 3.778 2 S2 Room High 6 h 4.079 2 S3 Room High 6 h 4.230 2 S1 Room High 24 h 3.602 2 S2 Room High 24 h 3.667 2 S3 Room High 24 h 3.484 2 S1 RefrigerationLow day 0 3.290 2 S2 RefrigerationLow day 0 3.322 2 S3 RefrigerationLow day 0 3.146

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81 Table I-1. Continued 2 S1 RefrigerationLow day 1 3.290 2 S2 RefrigerationLow day 1 3.407 2 S3 RefrigerationLow day 1 3.230 2 S1 RefrigerationLow day 3 2.544 2 S2 RefrigerationLow day 3 2.301 2 S3 RefrigerationLow day 3 2.602 2 S1 RefrigerationLow day 5 2.000 2 S2 RefrigerationLow day 5 2.000 2 S3 RefrigerationLow day 5 1.978 2 S1 RefrigerationLow day 7 1.301 2 S2 RefrigerationLow day 7 1.544 2 S3 RefrigerationLow day 7 1.477 2 S1 RefrigerationHigh day 0 4.267 2 S2 RefrigerationHigh day 0 3.875 2 S3 RefrigerationHigh day 0 4.371 2 S1 RefrigerationHigh day 1 4.204 2 S2 RefrigerationHigh day 1 4.301 2 S3 RefrigerationHigh day 1 4.079 2 S1 RefrigerationHigh day 3 3.279 2 S2 RefrigerationHigh day 3 3.114 2 S3 RefrigerationHigh day 3 3.114 2 S1 RefrigerationHigh day 5 2.190 2 S2 RefrigerationHigh day 5 2.146 2 S3 RefrigerationHigh day 5 2.097 2 S1 RefrigerationHigh day 7 1.301 2 S2 RefrigerationHigh day 7 1.740 2 S3 RefrigerationHigh day 7 1.778

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82 APPENDIX J STAPHYLOCOCCUS AUREUS MICROBIOLOGICAL CHALLENGE STUDY: STATISTICA L ANALYSIS Table J-1. Staphylococcus aureus microbiological challenge st udy: statistical analysis Source DF Sum of Squares Mean Square F Value Pr > F Model 15 79.09 5.27 37.35 <0.01 Error 104 14.68 0.14 Corrected Total 119 93.77 Source DF Type III SS Mean Square F Value Pr > F Trial 1 0.00 0.00 0.03 0.87 Rep 2 0.03 0.01 0.10 0.90 Temp 1 16.66 16.66 118.06 <.0001 Inolevel 1 21.29 21.29 150.80 <.0001 Time 4 39.11 9.78 69.27 <.0001 Trial*Inolevel 1 0.05 0.05 0.37 0.55 Inolevel*Time 4 0.61 0.15 1.08 0.37 Temp*Inolevel 1 1.33 1.33 9.44 0.00

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83 APPENDIX K SALMONELLA MICROBIOLOGICAL CHALLE NGE S TUDY: RAW DATA Table K-1. Salmonella microbiological challenge study: raw data Trial Rep Temperature Time CHROMagarKit 1 S1 Room 0 h Absent Present 1 S2 Room 0 h Absent Present 1 S3 Room 0 h Absent Present 1 S1 Room 2 h Absent Present 1 S2 Room 2 h Absent Present 1 S3 Room 2 h Absent Present 1 S1 Room 4 h Absent Present 1 S2 Room 4 h Absent Present 1 S3 Room 4 h Absent Present 1 S1 Room 6 h Absent Present 1 S2 Room 6 h Absent Present 1 S3 Room 6 h Absent Present 1 S1 Room 24 h Absent Present 1 S2 Room 24 h Absent Present 1 S3 Room 24 h Absent Present 1 S1 Refrigeration day 0 Absent Present 1 S2 Refrigeration day 0 Absent Present 1 S3 Refrigeration day 0 Absent Present 1 S1 Refrigeration day 1 Absent Present 1 S2 Refrigeration day 1 Absent Present 1 S3 Refrigeration day 1 Absent Present 1 S1 Refrigeration day 3 Absent Present 1 S2 Refrigeration day 3 Absent Present 1 S3 Refrigeration day 3 Absent Present 1 S1 Refrigeration day 5 Absent Absent 1 S2 Refrigeration day 5 Absent Absent 1 S3 Refrigeration day 5 Absent Absent 1 S1 Refrigeration day 7 Absent Absent 1 S2 Refrigeration day 7 Absent Absent 1 S3 Refrigeration day 7 Absent Absent 2 S1 Room 0 h Absent Present 2 S2 Room 0 h Absent Present 2 S3 Room 0 h Absent Present 2 S1 Room 2 h Absent Present 2 S2 Room 2 h Absent Present 2 S3 Room 2 h Absent Present 2 S1 Room 4 h Absent Present 2 S2 Room 4 h Absent Present 2 S3 Room 4 h Absent Present 2 S1 Room 6 h Absent Present 2 S2 Room 6 h Absent Present 2 S3 Room 6 h Absent Present 2 S1 Room 24 h Absent Present 2 S2 Room 24 h Absent Present 2 S3 Room 24 h Absent Present

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84 Table K-1. Continued 2 S1 Refrigeration day 0 Absent Present 2 S2 Refrigeration day 0 Absent Present 2 S3 Refrigeration day 0 Absent Present 2 S1 Refrigeration day 1 Absent Present 2 S2 Refrigeration day 1 Absent Present 2 S3 Refrigeration day 1 Absent Present 2 S1 Refrigeration day 3 Absent Present 2 S2 Refrigeration day 3 Absent Present 2 S3 Refrigeration day 3 Absent Present 2 S1 Refrigeration day 5 Absent Absent 2 S2 Refrigeration day 5 Absent Absent 2 S3 Refrigeration day 5 Absent Absent 2 S1 Refrigeration day 7 Absent Absent 2 S2 Refrigeration day 7 Absent Absent 2 S3 Refrigeration day 7 Absent Absent

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85 APPENDIX L SALMONELLA MICROBIOLOGICAL CHALL ENGE STU DY: STATISTICAL ANALYSIS Table L-1. Salmonella microbiological challenge st udy: statistical analysis Source DF Sum of Squares Mean Square F Value Pr > F Model 8 6 0.75 10.63 <0.01 Error 51 3.6 0.071 Corrected Total 59 9.6 Source DF Type III SS Mean Square F Value Pr > F Trial 1 0 0 0 1 Time 4 3.6 0.9 12.75 <.0001 Temp 1 2.4 2.4 34 <.0001 Rep 2 0 0 0 1

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86 LIST OF REFERENCES 1. Adachi, J.A., J.J. Mathewson, Z. Jiang, C.D. Ericsson, and H.L. DuPont. 2002. Enteric Pathogens in Mexican Sauces of Popular Restaurants in Guadalajara, Mexico, and Houston, Texas. Ann Intern Med. 136:884-887. 2. American Heritage Dicctionary. 4th Edition. 2000. Houghton Mifflin Company, Boston, MA. 3. Andrews, W.H. and T.S. Hammack. 2003. Salmonella Chapter 5, rev. April 2003. In FDA Bacteriological analytical manual, 8th ed., Rev. A. AOAC International, Gaithersburg, MD. 4. Ayc cek, H., B. Sarimehmetoglu, S. Cakiroglu. 2004. Assessment of the microbiological quality of meals sampled at the meal serv ing units of a military hospital in Ankara, Turkey. Food Control. 15: 379. 5. Beckers, H.J., D. Roberts, O. Price, R.R. Beremer, R. Peter.1986. Evaluation of reference material for the detection of Salmonella Int. J. Food Microbiol. 3: 287-298. 6. Bennet, A.R., D. Greenwood, C. Tennant, J.G. Banks, R.P. Betts. 1998. Rapid and definitive detection of Salmonella in foods by PCR. Lett Appl Microbiol. 26:437. 7. Berstein, R. 2007. Minority population tops 100 million. US Census Bureau, News Room. Available at http://www.census.gov/PressRelease/www/releases/arc hives/population/010048.html Accessed May 24, 2008. 8. Blivet, D., G. Salvat, F. Humbert, P. Co lin.1998. Development of a new culture medium for the rapid detection of Salmonella by indirect conductance measurement. J. Appl Microbiol. 84:399. 9. Brackett, R. E., and D.F. Splittstoesse r. 2001. Fruits and vegetables, p. 515 520. In F.P. Downes and K. Ito (ed.). Compendium of methods for the microbial examination of foods, 4th edition. American Public Hea lth Association. Washington, DC. 10. Busse, M. 1995. Media for Salmonella Inter. J. Food Microbiol. 26: 117-131. 11. Campbell, J.V., J. Mohle-Boetani, R. Repor ter, S. Abbott, J. Farrar, M. Brandl, R. Mandrell, and S.B.Werner. 2001. An Outbreak of Salmonella Serotype Thompson Associated with Fresh Cilantro. J. Infect Dis. 183:984. 12. Center for Disease Control and Prev ention. Foodborne outbreak response and surveillance unit. 2003.U.S. f oodborne disease outbreaks, annual listing 1990 2003. Available at http://www.cdc.gov/foodbornoutbreaks/us_outb.htm Accessed November 25, 2006. 13. Center for Science in the Public Interest. 2007. Outbreak Alert. Available at http://www.cspinet.org/foodsafety/ou tbreak_alert.pdf. Accessed by May 24 2008.

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94 BIOGRAPHICAL SKETCH Wendy Franco Melazzini was born in 1977, in La Paz, Bolivia. After her high school graduation in 1995, she attended Universidad Nues tra Senora de La Paz (Our Lady of La Paz University) and graduated with a degree in food engineering in 2002. From 2002 to 2004 she was a research assistant at th e same university wh ile she was studying industrial engineering as her second academic degree. She graduated in June 2004. After gradua tion, she joined the faculty at the Engineer ing department at Our Lady of La Paz University. She taught two undergraduate courses as well organized and partic ipated actively in ma ny projects related to food quality, food safety and food security. In 2006 she was granted with the Fulbright Scholarship to pursue graduate studies at Univ ersity of Florida under the supervision of Dr. Amarat H. Simonne. In August 2008, she earned dual Master of Science degrees in food science and in sgribusiness from University of Florid a. After graduation she will join Dr. Simonne research laboratory to do her academic training. In further years she plans to continue her studies toward a doctoral degree.