control of salmonella cross-contamination between …

CONTROL OF Salmonella CROSS-CONTAMINATION BETWEEN GREEN, ROUND TOMATOES IN A MODEL FLUME SYSTEM

By

SCOTT GEREFFI

A THESIS PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2014

To my family and friends

4

ACKNOWLEDGMENTS

First and foremost, I would like to thank Dr. Keith Schneider, who gave me the

opportunity to first join his lab as a volunteer in January 2012 and as a graduate student

in August 2012. I could not have asked for a better major advisor and I am extremely

grateful for his guidance and financial assistance. I would also like to thank my

committee members, Dr. Michelle Danyluk and Dr. Steve Sargent, for their support of

this project.

I honestly could not have made it this far without the help of Susie Richardson.

From media preparation, to driving long distances to pick up tomatoes, to helping me

run experiments, her help has been priceless and I am eternally grateful for it. Thank

you to Dr. Aswathy Sreedharan, whose calm demeanor and sage advice kept me sane

in the craziest of times. A special shout out to Alina Balaguero, whose amazing sense of

humor made long days in the lab just a bit more bearable. Thank you to the rest of my

coworkers from the Aquatic Food Products Lab Building, including Dr. Marianne Fatica,

Samantha King Cekic, Kristina Underthun, Dr. You Li, Dr. Oleks Tokarskyy, Tyler

Austin, Shuang Wu, Ning Gao, Celia Lynch, Lei Fang, Amber Ginn, Evan Johnson, and

Sweeya Gopidi. These friends made graduate school an incredible experience.

Thank you to the USDA Specialty Crop Research Initiative (Grant 2011-51181-

30767), which provided funding for this research. Also, thank you to Pacific Tomato

Growers, DiMare Fresh, and Gadsden Tomatoes for their generous supply of tomatoes.

Finally, thank you to my family for their unwavering support.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 8

LIST OF FIGURES .......................................................................................................... 9

ABSTRACT ................................................................................................................... 10

CHAPTER

1 INTRODUCTION .................................................................................................... 12

2 LITERATURE REVIEW .......................................................................................... 14

Foodborne Disease ................................................................................................ 14 Fresh Produce Safety ............................................................................................. 15

Water Disinfection ................................................................................................... 20 Hypochlorous Acid (HOCl) ............................................................................... 21 Other Sanitizers: Peroxyacetic Acid (PAA) and Chlorine Dioxide (ClO2) .......... 27

Research Hypothesis and Objectives ..................................................................... 28

3 MATERIALS AND METHODS ................................................................................ 30

Bacterial Strains ...................................................................................................... 30 Inoculum Preparation .............................................................................................. 30 Determination of Salmonella Concentration in TSB/rif80 over 14 h at 37 °C s ....... 31

Tomato Inoculation ................................................................................................. 31 Recovery and Enumeration .................................................................................... 32

Model Flume System Preparation ........................................................................... 32 Sodium Hypochlorite (NaOCl) .......................................................................... 33

Peroxyacetic Acid (PAA) .................................................................................. 33 Water Quality Measurements ........................................................................... 34

Cross-contamination Studies .................................................................................. 35 Transfer of Salmonella from Inoculated to Uninoculated Tomatoes in a

Model Flume System Containing Free Chlorine under Clean or Organic Loading Conditions ....................................................................................... 35

Transfer of Salmonella from Inoculated Tomatoes to the Water of a Model Flume System Containing Free Chlorine or Peroxyacetic Acid under Clean or Organic Loading Conditions ........................................................... 35

Cross-contamination Study with Enrichment Examining Transfer of Low Levels of Salmonella from Inoculated Tomatoes to Uninoculated Tomatoes in a Model Flume System Containing Free Chlorine under Clean or Organic Loading Conditions ........................................................... 36

6

4 RESULTS ............................................................................................................... 38

Determination of Salmonella Concentration in TSB/rif80 over 14 h at 37 °C .......... 38


Model Flume System Containing Free Chlorine under Clean and Organic Loading Conditions ....................................................................................... 38

Salmonella recovery from inoculated tomatoes ......................................... 39 Salmonella recovery from uninoculated/cross contaminated tomatoes ..... 39

Model flume system water chemistry ......................................................... 40 Transfer of Salmonella from Inoculated Tomatoes to the Water of a Model

Flume System Containing Free Chlorine or Peroxyacetic Acid under Clean or Organic Loading Conditions ........................................................... 41


Salmonella recovery from the model flume system water .......................... 43

Model flume system water chemistry ......................................................... 43

Cross-contamination Study with Enrichment Examining Transfer of Low Levels of Salmonella from Inoculated Tomatoes to Uninoculated Tomatoes in a Model Flume System Containing Free Chlorine under Clean or Organic Loading Conditions ........................................................... 44

5 DISCUSSION AND CONCLUSIONS ...................................................................... 63

Determination of Salmonella Concentration in TSB/rif80 over 14 h at 37 °C .......... 63


Model Flume System Containing Free Chlorine under Clean and Organic Loading Conditions ....................................................................................... 65


Model flume system water chemistry ......................................................... 69 Transfer of Salmonella from Inoculated Tomatoes to the Water of a Model

Flume System Containing Free Chlorine or Peroxyacetic Acid under Clean or Organic Loading Conditions ........................................................... 70

Salmonella recovery from inoculated tomatoes ......................................... 70

Salmonella recovery from uninoculated/cross contaminated tomatoes ..... 71

Salmonella recovery from the model flume system water .......................... 72

Model flume system water chemistry ......................................................... 73 Cross-contamination Study with Enrichment Examining Transfer of Low

Levels of Salmonella from Inoculated Tomatoes to Uninoculated Tomatoes in a Model Flume System Containing Free Chlorine under Clean or Organic Loading Conditions ........................................................... 73

Conclusions and Suggestions for Further Research ............................................... 75

7

LIST OF REFERENCES ............................................................................................... 77

BIOGRAPHICAL SKETCH ............................................................................................ 84

8

LIST OF TABLES

Table page 4-1 Concentration of Salmonella serovars in TSB/rif200 over 14 h at 37 °C ............ 46

4-2 Salmonella recovery (log10 CFU/tomato) from inoculated tomato surfaces after treatment alongside uninoculated tomatoes in a model flume system containing FC under organic loading conditions ................................................. 48

4-3 Salmonella recovery (log10 CFU/tomato) from uninoculated tomato surfaces after treatment alongside inoculated tomatoes in a model flume system containing FC under organic loading conditions ................................................. 50

4-4 Initial and final sanitizer concentration, initial and final pH, initial COD, and TDS measured before and after those model flume system experiments summarized in Tables 4-2 and 4-3 ..................................................................... 52

4-5 NaOCl necessary to adjust appropriate FC levels and citric acid necessary to adjust the pH model flume system in Tables 4-2 and 4-3 ............................... 53

4-6 Salmonella recovery (log10 CFU/tomato) from inoculated tomato surfaces after treatment alongside uninoculated tomatoes in a model flume system containing FC or PAA under organic loading conditions ..................................... 54

4-7 Salmonella recovery (log10 CFU/tomato) from uninoculated tomato surfaces after treatment alongside inoculated tomatoes in a model flume system containing FC or PAA under organic loading conditions ..................................... 56

4-8 Salmonella recovery (log10 CFU/mL) from the water of a model flume system containing FC or PAA under organic loading conditions ..................................... 58

4-9 Initial and final FC or PAA concentration, initial and final pH, initial COD, initial TDS, and initial and final ORP measured before and after those model flume system experiments summarized in Tables 4-6, 4-7, and 4-8................... 60

4-10 Volumes of NaOCl and citric acid necessary to adjust appropriate FC levels and pH to 6.5, or PAA solution necessary to adjust PAA levels in the model flume system in those experiments summarized in Tables 4-6, 4-7, and 4-8 ..... 61

4-11 Positive Salmonella detection from inoculated and uninoculated tomatoes treated in a model flume system containing FC under organic loading conditions. .......................................................................................................... 62

9

LIST OF FIGURES

Figure page 4-1 Concentration of Salmonella serovars in TSB/rif200 over 14 h at 37 °C ............ 47

4-2 Salmonella recovery (log10 CFU/tomato) from inoculated tomato surfaces after treatment alongside uninoculated tomatoes in a model flume system containing FC under organic loading conditions ................................................. 49

4-3 Salmonella recovery (log10 CFU/tomato) from uninoculated tomato surfaces after treatment alongside inoculated tomatoes in a model flume system containing FC under organic loading conditions ................................................. 51

4-4 Salmonella recovery (log10 CFU/tomato) from inoculated tomato surfaces after treatment alongside uninoculated tomatoes in a model flume system containing FC or PAA under organic loading conditions with water testing ........ 55

4-5 Salmonella recovery (log10 CFU/tomato) from uninoculated tomato surfaces after treatment alongside inoculated tomatoes in a model flume system containing FC or PAA under organic loading conditions with water testing ........ 57

4-6 Salmonella recovery (log10 CFU/mL) from the water of a model flume system containing FC or PAA under organic loading conditions ..................................... 59

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the

Requirements for the Degree Master of Science

CONTROL OF Salmonella CROSS-CONTAMINATION BETWEEN GREEN, ROUND TOMATOES IN A MODEL FLUME SYSTEM

By

Scott Gereffi

August 2014

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

Tomato Best Management Practices (T-BMPs) require Florida packers to treat

tomatoes in a flume system containing at least 150 ppm free chlorine or other approved

sanitizers. However, there is a lack of research that examines the ability of these

sanitizers to prevent the transfer of pathogens from contaminated to uncontaminated

tomatoes, particularly under realistic packinghouse conditions. The goal of this research

was to assess the minimum levels of sanitizer needed to prevent Salmonella cross-

contamination between tomatoes in a model flume system under clean and organically

loaded conditions.

Inoculated tomatoes (~8.3 log10 CFU/tomato after drying) were treated alongside

uninoculated tomatoes in a model flume system containing 0, 10, or 25 ppm free

chlorine (FC) under organic loading conditions of 0, 500, or 4000 ppm chemical oxygen

demand (COD). In the absence of FC, uninoculated tomatoes were highly contaminated

(~5 log10 CFU/tomato) by 15 s. No contamination was detectable (<2 log10 CFU/tomato)

on uninoculated tomatoes when FC was present, except with 10 ppm FC under

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4000 ppm COD. This suggests failure of 10 ppm FC as a sanitizer under very high

organic loading conditions.

The model flume system water column was tested for contamination when

inoculated tomatoes were treated alongside uninoculated tomatoes in 25 ppm FC,

80 ppm peroxyacetic acid (PAA), or DI water. Salmonella was only detectable

(>1 log10 CFU/mL) in those water samples containing 80 ppm PAA at 2 s, but not at 30

or 60 s. No Salmonella was detected in water samples containing FC at any time point.

Finally, uninoculated whole tomatoes were enriched in TSB/rif80 after treatment

in 25 ppm FC under organic loading conditions of 0 or 500 ppm COD to detect

Salmonella as low as 1 CFU/tomato. One out of nine tomatoes was positive for

Salmonella at 15 s with 25 ppm FC in clean water. One out of 18 tomatoes was positive

at 15 s and 2/18 tomatoes were positive at 60 s with 25 ppm FC in 500 ppm COD.

Results from these studies suggest that currently required minimum free chlorine

concentrations in packinghouse flume systems may exceed necessity. Based on these

findings, 25 ppm FC may be adequate provided that this level is properly maintained,

that COD does not exceed 500 ppm, and that tomatoes are treated for at least 120 s.

The ability of packers to use less sanitizer could reduce associated chemical costs,

disposal costs, and mitigate negative environmental impact.

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CHAPTER 1 INTRODUCTION

Approximately 93,600 acres of fresh market tomatoes were harvested in 2013 in

the US, worth over $1.1 billion (USDA 2014). More fresh market tomato acreage was

harvested in Florida (34000 acres worth over $455 million) than any other state in 2013,

including California (28,000 acres worth over $304 million) (USDA 2014). These

tomatoes are distributed to millions of consumers both domestically and abroad. If

contamination by pathogens is not controlled, many will potentially fall ill. Between 1998

and 2011, tomatoes were confirmed as the source of 20 Salmonella outbreaks, nine of

which were multistate (CDC 2011). Any outbreak involving tomatoes can prove

financially devastating for the industry. In 2008, a Salmonella Saintpaul outbreak

thought to originate from tomatoes (the source was later identified as serrano and

jalapeño peppers) cost the Florida tomato industry an estimated $100 million (Taylor

and others 2010). Thus, it is important to keep this important economic commodity safe.

Upon arrival at the packinghouse, harvested tomatoes are typically unloaded

from containers into a large water-filled receiving tank. The primary purpose of this

dump tank/flume system is to cushion the fall of the tomatoes and transport the newly

received fruits onto the packing line for sorting, waxing, and packing. This procedure

also doubles as a wash step to assist in the removal of dirt and any other extraneous

material from the tomato surface. Some tomatoes may be contaminated with a

pathogen upon introduction to the flume system. The use and management of sanitizers

in the flume system are paramount, as improperly sanitized water can serve as a

vehicle for the transfer of bacteria from contaminated to uncontaminated tomatoes

(Lopez-Galvez and others 2009). Tomato Best Management Practices (T-BMPs)

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currently require Florida tomato packers to treat the fruits in a flume system that

contains at least 150 ppm free chlorine at pH 6.5 to 7.5, or other approved sanitizers

such as peroxyacetic acid (PAA) or chlorine dioxide (ClO2), for a maximum of 2 min

(FDACS 2007). While the ability of these sanitizers to disinfect tomato surfaces has

been evaluated sufficiently (Wei and others 1995; Beuchat and Ryu 1997; Yuk and

others 2005; Felkey and others 2006; Iturriaga and Escartin 2010), the ability of these

sanitizers to prevent cross-contamination between tomatoes is not adequately known to

establish a reliable metric. It is possible that current industry standards for sanitizer use

exceed necessity. Additionally, the effect of organic matter on sanitizing operations has

not been evaluated sufficiently. Soil, leaves, wax, and other organic material brought in

on tomato surfaces can accumulate in the flume system. This material can react with

the sanitizers, rendering them less effective (Nou and Luo 2010). Adjustment of

sanitizer levels may therefore be necessary under organic loading conditions to

maintain prevention of cross contamination.

The goal of this research was to assess the minimum levels of sanitizer

necessary to prevent Salmonella cross-contamination between tomatoes in a model

flume system with and without organic loading conditions. The ability of packers to use

less sanitizer in flume systems could significantly lower associated costs. Further, lower

sanitizer use could mitigate the negative environmental impact of these operations.

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CHAPTER 2 LITERATURE REVIEW

Foodborne Disease

Scallan and others (2011a, 2011b) estimate that each year there are

approximately 47.8 million cases of foodborne illness in the US. Approximately 9.4

million of these cases are attributed to known pathogens whereas approximately 38.4

million are of unspecified etiologic origin. These diseases are caused by viruses,

bacteria, parasites, toxins, metals, and prions (Mead and others 1999). Estimating the

number of foodborne illnesses is complicated since most cases are self-limiting and

therefore go unreported and undiagnosed. However, many foodborne diseases are

severe enough to warrant hospitalization and some may ultimately lead to death.

Scallan and others (2011a) estimates that foodborne illnesses caused by 31 major

pathogens lead to 55,961 hospitalizations and 1,351 deaths annually. Similarly, Painter

and others (2013) estimates that for each year between 1998 and 2008, approximately

57,462 cases of foodborne illness of known etiologic origin resulted in hospitalization

while 1,451 resulted in death. Meanwhile, foodborne illnesses of unknown etiologic

origin are estimated to result in 71,878 hospitalizations and 1,686 deaths annually in the

US (Scallan 2011b). Surveillance of foodborne disease is further complicated

considering that many pathogens transmitted through food are also transmitted through

water or from person to person, which may mask foodborne transmission (Mead and

others 1999). Additionally, some foodborne illnesses are caused by agents that have

not yet been identified (Mead and others 1999). These diseases impart a tremendous

cost to society in terms of medical bills, lost wages and productivity, and illness-related

mortality. In 2011, the average foodborne illness in the US was estimated to have cost

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between $1,068 and $1,626 (Scharff 2012). This amounted to a total estimated cost of

$51.0 to $77.7 billion in 2011.

A foodborne disease outbreak is defined by the Centers for Disease Control and

Prevention (CDC) as the occurrence of two or more cases of a similar illness as a result

of eating a common food (CDC 2013). Leafy vegetables, dairy, and fruits and nuts are

associated with the highest number of outbreak-associated illnesses; dairy, leafy

vegetables, and poultry are associated with the highest number of hospitalizations; and

poultry, dairy, and vine-stalk vegetables are associated with the highest number of

deaths (Painter and others 2013). Between 2009 and 2010, 1,527 foodborne disease

outbreaks were reported in the US (CDC 2013). Norovirus was responsible for 42% of

these outbreaks while non-typhoidal Salmonella was responsible for nearly 30%. There

has been an increase in the number of cases of foodborne disease and foodborne

disease outbreaks over the past few decades. This increase can be attributed to a

variety of factors including larger-scale production and wider distribution of food,

globalization of the food supply, higher frequency of eating outside the home, evolution

of new pathogens, a growing population of susceptible consumers, and simply better

detection protocols (Nyachuba 2010).

Fresh Produce Safety

Fresh produce is defined as those fruits and vegetables that are sold to

consumers in an unprocessed form (e.g., no thermal treatment, freezing, dehydration,

etc.) (FDA 1998). Because fresh produce is often consumed raw and without a kill step

such as cooking, it can serve as a vehicle for the transmission of human pathogens

(Sivapalasingam and others 2004). The consumption of fresh produce has increased

over the past few decades as consumers have become increasingly aware of the

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associated health benefits. This increase in consumption has been paralleled by an

increase in produce-borne disease outbreaks. In the 1970s, approximately 0.7% of

foodborne disease outbreaks were associated with produce; this increased to 6% by the

1990s (Sivapalasingam and others 2004). For each year between 1998 and 2008, over

4.4 million cases of foodborne illness (45.9% of all foodborne illnesses) were associated

with fresh produce consumption. Of these, 21,885 resulted in hospitalization while 333

resulted in death (Painter and others 2013). The increases in fresh produce

consumption and disease risk have therefore placed an increased burden on the fresh

produce industry to provide consumers with safe products.

Produce items frequently associated with outbreaks include tomatoes, leafy

greens, melons, sprouts, and berries (Brackett 1999; Sivapalasingam and others 2004).

The most concerning pathogens that have been associated with produce include E. coli,

Salmonella spp., Shigella spp., Vibrio cholerae, and Listeria monocytogenes (Brackett

1999). Two recent examples of dangerous fresh produce-borne disease outbreaks

include an E. coli O104:H4 outbreak linked to sprouts that caused 3,816 illnesses and

54 deaths in Germany in 2011 (Frank and others 2011) and a Listeria monocytogenes

outbreak linked to cantaloupes that caused 32 deaths in the US in the same year (FDA

2011).

Fresh produce can become contaminated by a pathogen at any step of the

production process. Contact with improperly composted manure or fecal matter from

animals or humans, poor hygiene practices by field workers, contaminated water, soil,

and insect vectors are all potential sources of contamination (Brandl 2006). Fresh

produce can also be contaminated during transport, in retail and foodservice

17

establishments, and in the home (Harris and others 2003). As more demand has been

placed on the agriculture sector, crops are forced next to animal production areas,

which greatly increases the likelihood of contamination from animals (Lynch and others

2009). Water used for applying pesticides, hydrocooling, and irrigating crops may also

serve as a contamination source. Moreover, water used to transport and wash produce

in packinghouses can lead to the transfer of pathogens from contaminated items to non-

contaminated items. For this reason, it is vital that dump tank, flume system, and wash

water contain effective sanitizing agents that prevent cross-contamination (Lopez-

Galvez and others 2009).

Once produce becomes contaminated with a pathogen, it is nearly impossible to

completely remove it aside from cooking or irradiation (Parish and others 2003);

therefore, prevention of the initial contamination is the best strategy. Good Agricultural

Practices (GAPs) are general guidelines set by the FDA that reduce the risk of microbial

contamination of fresh produce if implemented properly (FDA 1998). GAPs have their

origin in the Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and

Vegetables published in 1998 by the FDA. The following general areas are emphasized:

protection from fecal contamination, prevention of handling by infected workers,

management of cold storage and supply chain, and maintenance of water quality (FDA

1998; Lynch and others 2009). The 2011 Food Safety and Modernization Act requires

all food producers to put into place a preventative food safety program for which GAPs

are typically a prerequisite (PL 2011). However, Tomato GAPs (T-GAPs) and Tomato

Best Management Practices (T-BMPs) have been required for Floridian tomato

producers and packers since 2008. These practices have been designed to increase

18

tomato safety at all levels of production. It is mandated that tomatoes are to be treated

in a flume system that contains at least 150 ppm free chlorine and has a pH of 6.5 to 7.5

for no more than 2 min (FDACS 2007). Water temperature must also be maintained

5 °C above the tomato pulp temperature to prevent internalization of water and

pathogens. Other EPA-approved sanitizers such chlorine dioxide (ClO2) and

peroxyacetic acid (PAA) may be used in flume systems provided that they achieve a

3 log10-unit reduction of Salmonella or Salmonella-like organisms on tomato surfaces.

Similar to T-GAPs and T-BMPs is the California Leafy Greens Marketing Agreement

(CA-LGMA), which is a mandatory audit program that certifies that California leafy

greens producers and packers are implementing food safety practices developed by

university and industry scientists, food safety experts, farmers, shippers and processors

(LGMA 2013).

Fresh produce items can contain spoilage organisms and plant pathogens.

These organisms include fungi such as Botrytis, Rhizopus, and Geotrichum, and

bacteria such as Pseudomonas, Xanthomonas, and Pectobacterium (Erwinia)

carotovorum (Narayanasamy 2006). Though these decay organisms alone are

harmless to humans, some can cause rot that may make the produce more susceptible

to human pathogen colonization (Wells and Butterfield 1997). It is therefore important

that flume system sanitizers can adequately prevent cross-contamination of these

microorganisms in addition to human pathogens.

Salmonella and Tomatoes

Though the efforts of the tomato industry have been successful in minimizing

tomato-borne disease outbreaks, a risk still exists. In the past, Salmonella outbreaks

associated with tomatoes were particularly common (CDC 2002, 2005, 2007;

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Cummings and others 2001; Greene and others 2008; Gupta and others 2007; Hedberg

and others 1999). Salmonella are Gram-negative, rod-shaped facultative anaerobes

belonging to the family Enterbacteriaceae (D’Aoust and Maurer 2007). Salmonella

infections are most commonly transmitted through food, leading to enterocolitis that

appears eight to 72 h after infection. Common symptoms are self-limiting diarrhea and

abdominal pain, though more serious or deadly issues may arise (D’Aoust and Maurer

2007). Salmonella invades intestinal cells via a Type III secretion system and causes an

influx of calcium ions into the intestinal tract. Salmonella can also produce an

enterotoxin and cytotoxin that can lead to apoptosis and cause diarrhea. The ingestion

of as low as 10-100 cells can cause illness (D'Aoust and Maurer 2007); thus it is vital to

prevent any Salmonella contamination of food. Salmonella infections cause more than

one million cases of foodborne disease, 19,336 hospitalizations, and 378 deaths in the

US annually (Scallan and others 2011a).

Typically, fresh market tomatoes are harvested from the field when they are

green for better shelf life and less bruising (Kader 1986). They are harvested by hand

into small buckets and dumped into larger field bins or specialized truck containers

(called gondolas). The trucks are sent to packinghouses and the tomatoes are unloaded

into a water-filled receiving tank before washing, waxing, and packaging. This initial

dump tank step minimizes impact damage to the fruits and aids in the removal of dirt

and other debris (Vigneault and others 2000). The tomatoes then flow from the dump

tank via a flume system onto the packaging line. At this point, if some tomatoes are

contaminated with pathogens, they have the potential to cross contaminate other

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uncontaminated product in the absence of proper sanitizer use (Lopez-Galvez and

others 2009).

A smaller number of tomato packing operations utilize a dry dump transfer

method, avoiding the complications associated with managing a large flume system.

With dry dumping, tomatoes are unloaded directly onto padded, sloped ramps or

conveyor belts to decrease fruit injury (Kitinoja and Kader 2002). Additionally, some

packing operations utilize an overhead spray brush and roller (OSBR) system, which

combines the overhead spraying of tomatoes with sanitizer with mechanical brushing by

roller brushes (Pao and other 2009). These systems are much less common than the

traditional dump tanks/flume systems found in most packinghouses.

Water Disinfection

The goal of aqueous disinfection in dump tanks and flume systems is to

inactivate pathogenic or spoilage bacteria, fungi, viruses, cysts, and other

microorganisms that may be present in the flume system water, minimizing the

probability of transfer of these microorganisms to uncontaminated produce items

(Lopez-Galvez and others 2009; Rana and others 2010). An increased use of sanitizer

allows for the continued recirculation of processing water, reducing the amount of

wastewater generated and reducing environmental impact (Olmez and Kretzschmar

2009). Conversely, an increase in the amount of sanitizer used has the disadvantage of

potential formation of toxic compounds in the presence of organic matter (Gil and others

2009), and thus a negative environmental impact as a result of disposal.

In laboratory-based studies, differing experimental conditions make it difficult to

compare sanitizer efficacies. Experiments with a dip inoculation procedure have shown

the greatest reduction in microorganisms compared to a spot or spray inoculation

21

procedure (Lang and others 2004). A longer time between inoculation and washing

decrease the efficacy of the wash treatment (Sapers 2001). Moreover, most laboratory-

based sanitizer studies typically do not take into consideration commercial processing

conditions (Allende and others 2007; Beuchat and others 2004; Sapers 2001; Stopforth

and others 2008). Wash water quality degrades quickly as soil, leaves, debris, and

microorganisms associated with the produce accumulate and lead to high levels of

organic loading (Allende and others 2008). When clean water is used in the assessment

of different sanitizers, the results obtained might not be indicative of what happens in a

real-world packinghouse. Many others factors make comparing sanitizer studies more

difficult. These include pH, temperature, sanitizer concentration, produce/water ratio,

rinsing of produce items after treatment, multiple washings, choice of microbial strain,

inoculation levels, detection and enumeration media, type of produce, etc. (Gil and

others 2009). A study by Zhou and others (2013) investigated the changes in wash

water quality and sanitizer concentration during routine operations of three large tomato

packinghouses in Florida. Water quality declined continuously throughout the day, as

measured by COD and turbidity. COD levels reached from 400-750 ppm COD at the

end of operation, depending on the length of the shift. As a result, free chlorine levels

varied considerably, requiring continuous adjustment of sanitizer concentration (Zhou

and others 2013).

Hypochlorous Acid (HOCl)

HOCl is the most common sanitizer used in tomato packinghouses (Suslow

1997). It is a potent antimicrobial and is relatively inexpensive. Sodium hypochlorite

(NaClO) is the most common source of HOCl in small-scale operations. Liquid solutions

are used most often since the solid form can react with water and generate potentially

22

harmful chlorine gas. Calcium hypochlorite, Ca(ClO)2, is another common source of

HOCl and is typically available as a granulated powder, compressed tablets, or large

slow-release tablets. Chlorine gas (Cl2) is the cheapest source of HOCl; however, it is

very cumbersome with regard to worker safety standards. Chlorine gas is sometimes

used in very large-scale operations and requires automated injection systems with in-

line pH monitoring (Suslow 1997).

Chlorine concentration can be expressed in two ways: total chlorine and free

chlorine. Total chlorine is the total amount of chlorine present in solution, which includes

both free and combined forms. Free chlorine refers to the sum of HOCl and hypochlorite

ions (OCl-), both of which are formed when any of the aforementioned chlorine sources

are added to water. Though the exact mechanism is not fully understood, HOCl is

believed to kill microbes by reacting with and destroying intracellular proteins, inhibiting

DNA synthesis, and disrupting membrane activity (McDonnell and Russell 1999). At

high pH levels, HOCl will dissociate to form OCl-, which is comparatively ineffective

(Suslow 1997). Between pH 4.5 and 5.5, the percentages of HOCl and OCl- are 100%

and 0%, respectively (Suslow 1997). At such low pH levels, however, potentially harmful

Cl2 may be produced. It is therefore recommended to maintain the pH of the water

system between 6.5 and 7.5 to avoid the generation of Cl2 and to minimize corrosion of

packinghouse equipment while still maintaining chlorine efficacy (Suslow 1997). The

percentages of HOCl and OCl- at pH 6.5 are 95% and 5%, respectively; at pH 7.5 they

are 50% and 50%, respectively.

There are several methods utilized to monitor the oxidant capacity of an aqueous

solution. Individual water samples can be taken, monitoring for sanitizer residual. While

23

accurate, the procedures involved can be time consuming and expensive. Continuous

monitoring of the disinfection potential in chlorinated water systems can be measured

by the oxidation-reduction potential (ORP). ORP monitoring systems provide continuous

real-time measurements and may have alarm systems that immediately notify the

operator when parameters become out of range (Suslow 2000). ORP monitors can be

coupled with chlorine injection systems as well as pH control injections to optimize

performance. A chlorinated water system with an ORP value of 650 to 700 mV has

been shown to kill spoilage organisms as well as E. coli and Salmonella within a few

seconds (Suslow 2000). ORP, however, may not necessarily have a linear relationship

with sanitizer concentration (Rana and others 2010; Zhou and others 2013), thus

making ORP monitoring systems difficult to calibrate and their readouts highly variable.

Chlorine-based sanitizers have a variety of advantages and disadvantages.

These sanitizers will react with organic matter, such as leaves, soil, and plant material in

the presence of oxygen. Not only will these reactions bind free chlorine and render the

sanitizer less effective (Suslow 1997), but they will lead to the formation of carcinogenic

by-products like trihalomethanes, haloacetic acids, haloketones, and chloropicrin (Gil

and others 2009). It is thus extremely important to change chlorinated water frequently,

filter out organic matter and debris, and maintain appropriate free chlorine

concentrations. Pre-washing produce transport bins and gondolas, as well as rinsing

very dirty produce prior to water system introduction can prolong the life of active

chlorinated water. The effect of organic matter on chlorine efficacy was demonstrated

by Nou and Luo (2010). In the study, inoculated whole-leaf lettuce was shown to have a

lesser level of cross-contamination after 60 s in chlorinated water (3.3% subsamples,

24

average MPN ≤ -0.3 log10 CFU/g) than cut lettuce (96.7% subsamples, average MPN

0.6 log10 CFU/g). Cell disruption in the cut lettuce likely led to a discharge of organic

matter into the water column, decreasing sanitizer efficacy. Another notable

disadvantage of chlorine-based sanitizers is their strong odor that may cause worker

discomfort (Suslow 1997). Furthermore, used chlorinated water must be disposed of

properly due to the potential formation of the aforementioned harmful compounds and

associated environmental discharge regulations. Chlorine sanitizers can also be highly

corrosive, especially at low pH, which can lead to the eventual breakdown of

packinghouse equipment. Finally, it should be noted that chlorine treatment might not

completely eradicate microorganisms from tomato surfaces, but rather, only reduce their

numbers (Beuchat and Ryu 1997; Felkey and others 2006; Iturriaga and Escartin 2010;

Wei and others 1995; Yuk and others 2005). Reduction is further inhibited on tomatoes

when microbes are located in surface wounds (Felkey and others 2006). As with any

sanitizer, chlorine-based sanitizers have no effect on microorganisms which have

infiltrated the tomato surface (Zhuang and others 1995). Despite these drawbacks, it is

generally accepted that the benefits of chlorine-based sanitizers, including efficacy and

ease of use, outweigh the risks (Suslow 1997).

The maximum free chlorine concentration permitted in a flume system for fresh

produce is 200 ppm (CFR 2013b). Recommendations for chlorine concentrations in

packinghouse water systems are largely reported anecdotally. Florida T-BMPs mandate

that free chlorine levels in flume systems be at least 150 ppm at pH 6.5 to 7.5. As

previously mentioned, this level may exceed necessity for tomato packinghouse

operations. The minimum free chlorine concentration required to prevent cross-

25

contamination of fruits and vegetables in packinghouse water systems is unclear. Most

microbial inactivation models are based on the disinfection of clean, potable water

where free chlorine levels are maintained for several minutes or longer. In real-world

packinghouse water systems, however, a sanitizer must prevent the movement of

pathogens from sources to potential infection sites, which may occur in a matter of

seconds (Bartz and others 2001).

Several studies have examined the role of chlorine in preventing cross-

contamination within a flume system. A study by Vigneault and others (2013) addressed

cross-contamination of tomato decay organisms. When tomatoes were immersed in

water contaminated with P. carotovorum and Rhizopus stolonifer for 10 min at 20 °C, 17

of 20 tomatoes developed decay after 10 days of storage. When the study was

repeated with the contaminated water adjusted to 200 ppm free chlorine, only one

tomato developed decay. No decay occurred when the water was adjusted to 400 ppm

free chlorine (Vigneault and others 2000). Though decay organisms can negatively

affect tomato quality and potentially make tomatoes more susceptible to human

pathogen colonization (Wells and Butterfield 1997), they alone pose little risk to human

health. In a study by Rana and others (2010), uninoculated tomatoes were treated

alongside inoculated tomatoes (approximately 7.3 log10 CFU/tomato) in clean tap water

containing 5 and 30 ppm free chlorine. Only 1/18 uninoculated tomato samples under

both conditions was positive for Salmonella after enrichment, suggesting low levels of

Salmonella cross contamination. No uninoculated tomato samples were positive for

Salmonella after enrichment when treated alongside inoculated tomatoes in clean tap

water containing 100 or 200 ppm free chlorine (Rana and others 2010). When the study

26

was repeated using spent industry dump tank water, 11/18 uninoculated tomatoes were

positive for Salmonella after treatment alongside inoculated tomatoes in 5 ppm free

chlorine and 2/18 were positive after treatment in 30 ppm free chlorine (Rana and

others 2010). The results of the study emphasize that organic matter can negatively

affect chlorine efficacy. In the study, the level of organic loading in the spent industry

dump tank water was not quantified, making it difficult to apply a metric to which tomato

packers should adhere. Also, a single serovar (S. Typhimurium) rather than a more

representative cocktail, which minimizes variability associated with serovar survival, was

used in these experiments. Lastly, the level of cross-contaminated Salmonella was not

quantified and treatment time examined unrealistically high at 5 min. In a study by Shen

and others (2013), the effects of free chlorine concentration, contact time, and organic

load on the inactivation of Salmonella, E. coli O157:H7, and non-O157 Shiga toxin-

producing E. coli (STEC) were evaluated in suspension. Organic matter was simulated

by lettuce or tomato extract. When no simulated organic matter was present, pathogen

inactivation was a function of initial free chlorine concentration, exposure time, along

with the strain of pathogen. When organic matter was present, pathogen inactivation

was dependent on the residual chlorine concentration. A >4.5 log10 CFU/mL reduction

was found after exposure to 0.5 ppm free chlorine for 30 s, or to 1.0 ppm free chlorine

for 5 s (Shen and others 2013). These data suggest that very low levels of chlorine

(much lower than the industry standard) can inactivate human pathogens in dump

tanks. This is further supported by another study in which 0.4 ppm free chlorine was

required to completely eradicate E. coli O157:H7 in lettuce wash water (Yang and

others 2012). If using lower levels of chlorine is as effective as using higher levels of

27

chlorine, money can be saved on chemical costs and there may be less environmental

impact as a result of disposal. While shown to be efficacious in laboratory studies, the

use of extremely low concentrations of sanitizers in flume systems remains impractical

since shifts in organic loading can quickly deplete residual sanitizer, rendering the

produce items potentially unsafe.

Other Sanitizers: Peroxyacetic Acid (PAA) and Chlorine Dioxide (ClO2)

A non-chlorine-based sanitizer commonly used in tomato packinghouse water

systems is peroxyacetic acid (PAA). PAA is an aqueous mixture solution of acetic acid

and hydrogen peroxide (Dell'Erba and others 2004). It is very pH tolerant, produces few

toxic volatile compounds, and has a high tolerance against organic materials (Fatica

2009). However, PAA is considerably more expensive than other sanitizers. The

maximum allowable concentration of PAA in produce sanitizing water is 80 ppm (CFR

2013b). The antimicrobial mechanism of PAA is believed to be based on the generation

of reactive oxygen species, denaturation of proteins, increase of cell wall permeability,

disruption of cell membranes, and blockage of vital transport systems in

microorganisms (Vandekinderen and others 2009). The efficacy of PAA as a produce

sanitizer has been demonstrated by a number of studies (Yuk and others 2005; Ruiz-

Cruz and others 2007).

Chlorine dioxide (ClO2) is another effective sanitizer that is frequently utilized in

tomato packinghouses (Pao and others 2007; Wu and Kim 2007; Zhang and Farber

1996). This water-soluble gas is a very potent antimicrobial, even in the presence of

organic matter. Because of its volatility, however, this sanitizer must be used in a

completely sealed or very well-ventilated area (Fatica 2009). Moreover, it must be

generated on-site as its gaseous form is explosive (Gomez-Lopez and others 2009).

28

Compared to HOCl, this compound is much less pH-dependent and can be used at

more alkaline pH levels, which leads to less corrosion of stainless steel equipment. ClO2

is approved by the FDA for use in sanitizing wash water at a concentration of 3 ppm or

below (CFR 2013a). The exact antimicrobial mechanism of ClO2 is not known; however,

it is believed to be due to the loss of permeability control in the cell membrane (Berg

and others 1986).

Research Hypothesis and Objectives

The proper use of sanitizers in packinghouse dump tanks and flume systems is

vital for preventing cross-contamination between tomatoes. The conditions for use of

these sanitizers are not adequately known to establish reliable metrics. The effect of

organic matter on sanitizing operations has not been evaluated sufficiently and few

studies have examined the efficacy of chlorine in preventing the cross-contamination of

Salmonella between tomatoes in a model flume system.

The goal of this research was to study the minimum levels of sanitizer needed to

prevent Salmonella cross-contamination in a model flume system with and without

organic loading conditions. It is hypothesized that an increased presence of organic

matter will increase the level of sanitizer necessary to prevent Salmonella cross-

contamination between tomatoes.

Specifically, the objectives of this research were to:

1. Establish growth curves for rifampicin-resistant Salmonella enterica serovars: Montevideo, Anatum, Javiana, Braenderup, and Newport.

2. Determine Salmonella recovery from inoculated and uninoculated tomatoes treated together in a model flume system containing 0, 10, and 25 ppm free chlorine under organic loading conditions of 0, 500, and 4000 ppm chemical oxygen demand (COD).

29

3. Determine the extent of water contamination from inoculated tomatoes treated in a model flume system containing 0 or 25 ppm free chlorine or 80 ppm PAA under organic loading conditions of 0 or 500 ppm COD.

4. Using enrichment, determine if low levels of Salmonella transfer from inoculated to uninoculated tomatoes in a model flume system containing 25 ppm free chlorine under organic loading conditions of 0 or 500 ppm COD.

30

CHAPTER 3 MATERIALS AND METHODS

Bacterial Strains

The serovars of Salmonella enterica used for all studies were Montevideo

(human-tomato linked LJH0519 G4639), Anatum (K2669 CDC clinical isolate), Javiana

(ATCC BAA-1593), Braenderup (04E61556), and Newport (environmental, tomato

outbreak from Virginia). These rifampicin-resistant strains were obtained from Dr.

Danyluk of the University of Florida Citrus Research and Education Center. Cultures

were stored at -80 °C as 70% glycerol stocks.

Each strain was streaked for isolation on tryptic soy agar (TSA) (Difco, Sparks,

MD) and incubated at 37 °C. After 24 h, a single colony was transferred to 10 mL tryptic

soy broth (TSB) (Difco) supplemented with 200 ppm rifampicin (TSB/rif200) and

incubated at 37 °C. A 10,000 ppm rifampicin stock was made by dissolving 0.4 g

rifampicin (Fisher Scientific, Fair Lawn, NJ) in 40 mL methanol. The stock solution was

filtered (0.20 µm pore size, Fisher Scientific) and stored at 2 °C until use. After 24 h, a

sterile loop was used to transfer approximately 10 μL of culture to fresh TSB/rif200 and

incubated at 37 °C. An additional transfer was performed once more after 24 h for a

total of three culture transfers.

Inoculum Preparation

After the cultures underwent the three-day successive transfers previously

described, the five cultures were combined as a 50 mL cocktail and centrifuged at

4000 x g for 10 min. The combined culture was then washed by disposing of the

supernatant and suspending the pellet in 5 mL buffered peptone water (BPW) (Difco).

The culture was centrifuged and washed twice more. The resulting inoculum had a

31

concentration of approximately 10 log10 CFU/mL. TSB and TSA used in experiments

were supplemented with 80 ppm rifampicin (denoted TSB/rif80 or TSA/rif80,

respectively) to reduce stress on the bacteria yet still select for resistant

microorganisms. The inoculum was serially diluted and plated on TSA/rif80 to verify

concentration. All serial dilutions in experiments were performed 1:10 in BPW.

Determination of Salmonella Concentration in TSB/rif80 over 14 h at 37 °C s

Growth of the five Salmonella serovars was measured over a period of 14 h.

Broth cultures were prepared individually and underwent the successive three-day

transfers in TSB/rif200 as previously described. Cultures were serially diluted to

approximately 4 log10 CFU/mL. One mL of each diluted culture was transferred to

separate bottles containing 99 mL TSB/rif200. These cultures were incubated at 37 °C

for 14 h. Each hour beginning with hour 0, cultures were serially diluted and pour plated

into TSA/rif80. Plates were incubated at 37 °C for 48 h and then counted. The

experiment was performed in triplicate. Average log10 concentration at each hour was

calculated for the three cultures per Salmonella serovar. For each growth curve,

analysis of variance (ANOVA) and mean separation using Tukey’s HSD with p<0.05

were performed using SAS 9.3 (SAS Institute Inc., Cary, NC) to determine differences

between serovar concentration by hour.

Tomato Inoculation

Unwashed, unwaxed, mature, green, round tomatoes were obtained from various

packers across the state of Florida. A new inoculum was prepared as previously

described for each experiment. Tomatoes were inoculated in a circle around the

blossom scar with 10 spots of approximately 10 μL each for a total of 100 μL per

tomato, or approximately 9.0 log10 CFU/tomato. The tomatoes were allowed to dry for

32

2 h in a biosafety hood to promote attachment. Inoculated tomatoes were marked

conspicuously with black permanent marker prior to inoculation to aid visibility in the

mode flume system.

Recovery and Enumeration

To obtain bacterial counts, tomatoes were placed into separate sterile

Stomacher® bags (Fisher Scientific) containing 100 mL BPW supplemented with

0.1% sodium thiosulfate (Na2S2O3) (Fisher Scientific) (BPW/0.1% Na2S2O3) to inactivate

chlorine. Chlorine was inactivated in order to determine the effect of chlorine at specific

time points and to negate residual effects. Each tomato was shaken, rubbed, and

shaken again (30 s, 30 s, and 30 s, respectively) to dislodge any Salmonella cells from

the tomato surface. One mL of the liquid was serially diluted (1:10) and pour-plated into

TSA/rif80. Plates were incubated at 37 °C and counted after 48 h. Negative controls of

all media were plated. For each experiment, an uninoculated tomato was used as a

negative control and was plated to ensure no rifampicin-resistant organisms were found

on the tomatoes.

Model Flume System Preparation

A 30.5 x 68.6 x 19.0 cm3 recirculating water bath (Model 2866, Thermo Scientific,

Waltham, MA) was used as a model flume system in these studies. For each

experiment with no organic loading, the water bath was filled with 20 L deionized water

(University of Florida) followed by sanitizer adjustment to predetermined level. For each

experiment with organic loading, autoclaved Scotts® Premium Topsoil (The Scotts

Miracle-Grow Company, Marysville, OH) was added to 20 L of water and mixed

thoroughly. The mixture was then filtered through two layers of cheesecloth (Fiberweb,

London, UK) to remove large particulate matter. Chemical oxygen demand (COD) was

33

used to measure the organic matter content in the water (procedure described below).

Approximately 6 kg of soil were used in the creation of high organic loading

(4000 ppm COD) while approximately 0.8 kg of soil were used in the creation of

moderate organic loading (500 ppm COD). The filtered soil-water mixture was then

used to fill the water bath, followed by sanitizer adjustment. The temperature of the

model flume system was maintained at 30 °C for each experiment.

Sodium Hypochlorite (NaOCl)

Free chlorine levels were adjusted using a 5.7-6% NaOCl solution (Fisher

Scientific). The free chlorine sanitizer solutions were adjusted to pH 6.5 using a 50%

citric acid solution (Alcide Corp., Redmond, WA). Free chlorine levels were verified with

a Hach DR/890 colorimeter (Hach Co., Loveland, CO) using the Hach method 8091,

which utilizes an N, N-diethyl-p-phenylenediamine (DPD) sulfate indicator. The DPD

method uses AccuVac free chlorine ampules (Hach Co.) that form a pink color when

reacted with chlorine. The effective range for the ampules is 0 to 2.00 ppm free chlorine

thus necessitating a 1:100 dilution with DI water to measure concentration. Each

ampule was filled with the diluted solution and measured using the DR/890 colorimeter,

with the resulting free chlorine value multiplied by a factor of 100 to determine the

oxidant level present in the test solution. For those studies with organic loading, a

sample was centrifuged at 4000 x g for 30 s to remove any large particulate matter from

suspension prior to dilution.

Peroxyacetic Acid (PAA)

PAA concentration was adjusted using the commercially available Tsunami 100

solution (Ecolab, Inc., St. Paul, MN). PAA concentration was verified using a LaMotte

hydrogen peroxide and peracetic acid measurement kit (LaMotte Co., Chestertown,

34

MD). A 25 mL sample was removed from the model flume system. Twenty-five drops of

64% sulfuric acid, 3 drops of ferroin indicator (0.3% 1,10-phenanthroline), and 3 drops

of 20% potassium iodide solution were added to the sample. A 2.5% sodium thiosulfate

solution was used as a peracetic acid titrant. Each drop of titrant required to change the

solution from a cloudy brown-orange color to a clear orange color was equivalent to

6 ppm PAA.

Water Quality Measurements

Total dissolved solids (TDS) content was verified using an Oakton EcoTestr TDS

Low pocket TDS tester (Oakton Instruments, Vernon Hills, IL). Oxidation-reduction

potential (ORP) was verified using a Hanna Combo pH and ORP meter (Hanna

Instruments USA, Smithfield, RI). Chemical oxygen demand (COD) was verified using

an AQUAFast II Orion AQ2040 COD photometer (Thermo). Prior to the addition of

sanitizer, a 2 mL water sample was removed from the water bath and placed into an

Orion CODH00 medium range COD test vial (Thermo). The vial was then placed in an

Orbeco-Hellige TR125 thermoreactor (Orbeco-Hellige Inc., Sarasota, FL) and digested

at 150 °C for 2 h. Each vial contains a highly acidic solution of potassium dichromate

(K2Cr2O7) of known concentration. The dichromate ion (Cr2O72-) reacts with any organic

matter and is reduced, forming Cr3+, which produces a green color in solution. The vial

was removed and allowed to cool to room temperature before placement in the

photometer for reading.

35

Cross-contamination Studies

Transfer of Salmonella from Inoculated to Uninoculated Tomatoes in a Model Flume System Containing Free Chlorine under Clean or Organic Loading Conditions

The sanitizer concentrations and organic loading levels selected for this study

were 0, 10, 25 ppm free chlorine (FC) and 0, 500, or 4000 ppm COD, respectively.

Twelve uninoculated tomatoes were added to the water bath containing the appropriate

organic loading level and sanitizer concentration. Three uninoculated tomatoes were

removed as 0 s controls. Three inoculated tomatoes were also taken as 0 s controls.

Nine inoculated tomatoes were added to the water bath. At 15, 60, and 120 s, three

inoculated tomatoes and three uninoculated tomatoes were removed and immediately

placed into separate Stomacher® bags containing 100 mL BPW/0.1% Na2S2O3. Any

Salmonella present on the tomato surfaces were recovered and enumerated as

previously described. ANOVA and mean separation using Tukey’s HSD with p<0.05

were performed using SAS 9.3 (SAS Institute Inc., Cary, NC) to determine differences

between Salmonella recovery by treatment and by time.

Transfer of Salmonella from Inoculated Tomatoes to the Water of a Model Flume System Containing Free Chlorine or Peroxyacetic Acid under Clean or Organic Loading Conditions

The sanitizer concentrations and organic loading levels selected for this study

were 0 or 25 ppm FC or 80 ppm PAA and 0, 500, or 4000 ppm COD, respectively.

Twelve uninoculated tomatoes were added to the water bath containing the appropriate

organic loading level and sanitizer concentration. Three uninoculated tomatoes were

removed as 0 s controls. Three inoculated tomatoes were also taken as 0 s controls.

Nine inoculated tomatoes were added to the water bath. At 30 and 60 s, three

inoculated tomatoes and three uninoculated tomatoes were removed and immediately

36

placed into separate Stomacher® bags containing 100 mL BPW/0.1% Na2S2O3. Any

Salmonella present on the tomato surfaces was enumerated as previously described.

Three 1 mL water samples were also removed at 2, 30, and 60 s and placed into

test tubes containing 9 mL BPW/0.1% Na2S2O3. Three 1 mL water samples were

removed prior to the addition of inoculated tomatoes as 0 s controls. A 1 mL sample

was removed from each tube and pour plated into TSA/rif80. The plates were incubated

at 37 °C and counted after 48 h to enumerate any Salmonella present in the water.

ANOVA and mean separation using Tukey’s HSD with p<0.05 were performed using

SAS 9.3 (SAS Institute Inc., Cary, NC) to determine differences between Salmonella

recovery by treatment and by time.

Cross-contamination Study with Enrichment Examining Transfer of Low Levels of Salmonella from Inoculated Tomatoes to Uninoculated Tomatoes in a Model Flume System Containing Free Chlorine under Clean or Organic Loading Conditions

The sanitizer concentration and organic loading levels selected for this study

were 25 ppm FC and 0 or 500 ppm COD, respectively. Twelve uninoculated tomatoes

were added to the water bath containing the appropriate organic loading level and

sanitizer concentration. Three of uninoculated tomatoes were removed as 0 s controls.

Three inoculated tomatoes were also taken as 0 s controls. Nine inoculated tomatoes

were added to the water bath. At 15, 60, and 120 s, three inoculated tomatoes and

three uninoculated tomatoes were removed and immediately placed into separate

Stomacher® bags containing 100 mL TSB/rif80 supplemented with 0.1% Na2S2O3

(TSB/rif80/0.1% Na2S2O3). Any Salmonella on the tomato surfaces were recovered as

previously described. The bags were then incubated at 37 °C for 48 h to enrich any

Salmonella present. An inoculation loop was used to streak a portion of the enriched

37

liquid onto TSA/rif80 to confirm for the presence of Salmonella. The TSA/rif80 was

incubated at 37 °C and examined for growth after 48 h.

38

CHAPTER 4 RESULTS

Determination of Salmonella Concentration in TSB/rif80 over 14 h at 37 °C

Growth of five rifampicin-resistant Salmonella serovars (Montevideo, Anatum,

Javiana, Braenderup, and Newport) was measured over a period of 14 h in TSB/rif200

at 37 °C. The concentration (log10 CFU/mL) of each serovar was determined from

triplicate experiments each hour, beginning with hour 0 (Table 4-1 and Figure 4-1).

Average initial concentration at hour 0 was 2.12 log10 CFU/mL for all serovars.

Significant differences in concentration were present between serovars from hours 0

through 12 (p<0.05). Initially, S Braenderup and S. Newport grew more slowly than

S. Montevideo, S. Anatum, and S. Javiana. However by hour 13, no significant

differences (p>0.05) in concentration were observed between serovars. Average final

concentration at hour 14 was 8.95 log10 CFU/mL.


Transfer of Salmonella from Inoculated to Uninoculated Tomatoes in a Model Flume System Containing Free Chlorine under Clean and Organic Loading Conditions

This study was conducted to assess the ability of free chlorine (FC) to prevent

cross-contamination of Salmonella between tomatoes in a model flume system with

clean water and under organic loading conditions. Dried, inoculated tomatoes were

treated alongside uninoculated tomatoes in a recirculating water bath (model flume

system) containing 0, 10, or 25 ppm free chlorine (FC) under organic loading levels of 0,

500, or 4000 ppm chemical oxygen demand (COD) for 0, 15, 60, or 120 s. Tomatoes

were rinsed in 100 mL BPW/0.1% Na2S2O3, resulting in a limit of detection of

0 log10 CFU/mL (1 CFU/mL) of rinsate or 2 log10 CFU/tomato (100 CFU/tomato).

39

However, the statistical limit of detection is 3.4 log10 CFU/tomato when considering the

acceptable range for countable colonies on plates (25-250 CFU). To convert from

log10 CFU/mL to log10 CFU/tomato, log10 CFU/mL data were adjusted by a factor of

2 log10 units. All treatments were designed as a ratio of COD to FC. Statistical analysis

looked for significance between time and COD/FC concentration.

Salmonella recovery from inoculated tomatoes

Salmonella recovery data from inoculated tomatoes are summarized in Table 4-2

and Figure 4-2. The mean inoculation for positive control samples was

8.29 log10 CFU/tomato after 2 h drying. No significant differences (p>0.05) were

observed in Salmonella recovery for untreated, inoculated tomatoes (t = 0 s).

At 60 and 120 s, differences in recoveries from inoculated tomatoes were more

pronounced than at 15 s. Recoveries from inoculated tomatoes treated in the absence

of free chlorine were significantly higher (p<0.05) than from those treated in 10 or

25 ppm FC with a difference of >4.80 log10 CFU/tomato, except under organic loading

conditions of 4000 ppm COD. Recoveries from inoculated tomatoes treated in 4000/10

ppm FC/COD were not significantly different (p>0.05) from those treated in the absence

of FC at 15, 60, and 120 s.

For all individual treatment conditions, significant reductions in Salmonella

populations were observed after 15 s treatment. Reductions of approximately 5

log10 CFU/tomato were achieved after 60 s treatment in the presence of 10 or 25 ppm

FC, except under organic loading conditions of 4000 ppm COD.

Salmonella recovery from uninoculated/cross contaminated tomatoes

Salmonella recovery data from uninoculated/cross contaminated tomatoes are

summarized in Table 4-3 and Figure 4-3. Salmonella was only detectable from

40

uninoculated tomatoes when treated alongside inoculated tomatoes in 0/0, 500/0,

4000/0, and 4000/10 ppm COD/FC for ≥15 s, at which cross contaminated Salmonella

levels reached >5 log10 CFU/tomato. At 0 s, no Salmonella was detectable

(<2.00 log10 CFU/tomato) on uninoculated tomatoes. Salmonella was undetectable at all

time points from uninoculated tomatoes treated in 0/10, 0/25, 500/10, 500/25, and

4000/25 ppm COD/FC.

Model flume system water chemistry

Water chemistry data for the initial cross-contamination study are summarized in

Table 4-4. Initial and final FC concentration, initial and final pH, initial COD, and initial

TDS were measured for each experiment. FC concentration tended to decrease more

between initial and final measurements in those experiments with organic loading

compared to those with no organic loading. For example, initial and final FC

concentrations were 11 and 11 ppm, respectively, in experiments with 0/10 COD/FC;

while initial and final FC concentrations were 12 and 2 ppm, respectively, in

experiments with 4000/10 COD/FC

The average volumes of 5.65-6.0% sodium hypochlorite (NaOCl) necessary to

adjust FC levels and 50% citric acid necessary to adjust the pH to 6.5 are summarized

in Table 4-5. Much higher volumes of NaOCl are required to reach the desired free

chlorine levels in those systems under organic loading. Approximately 4.7 mL of NaOCl

was required to adjust the model flume system water to 10 ppm FC under no organic

loading conditions while approximately 319.7 mL were required to adjust the model

flume system water under the organic loading condition of 4000 ppm COD. In turn, the

same generally appears to hold true for the required amount of citric acid necessary to

adjust the pH to around 6.5. For example, approximately 0.5 mL citric acid was required

41

to adjust the model flume system water to 6.5 under no organic loading, while 8.7 mL

was required under the organic loading condition of 4000 ppm COD.


Similar to the initial cross contamination study, this study examined the ability of

various chemical sanitizers to prevent Salmonella cross-contamination between

tomatoes in a model flume containing water under varying organic load levels. Unlike

the initial cross-contamination study, this study had the addition of Salmonella

enumeration in the flume water.

Tomatoes were inoculated with approximately 9 log10 CFU/tomato and allowed to

dry for 2 h in a biosafety hood at room temperature. Dried, inoculated tomatoes were

then treated alongside uninoculated tomatoes in a model flume containing 25 ppm FC,

80 ppm peroxyacetic acid (PAA), or deionized water under organic loading conditions of

0 or 500 ppm COD for 0, 30, or 60 s. Tomatoes were rinsed in 100 mL

BPW/0.1% Na2S2O3, resulting in a limit of detection of 0 log10 CFU/mL (1 CFU/mL) of

rinsate or 2 log10 CFU/tomato (100 CFU/tomato). However, the statistical limit of

detection is 3.4 log10 CFU/tomato when considering the acceptable range for countable

colonies on plates (25-250 CFU). To convert from log10 CFU/mL to log10 CFU/tomato,

log10 CFU/mL data were adjusted by a factor of 2 log10 units.

Additionally, water samples were removed at 0, 2, 30, and 60 s to enumerate any

Salmonella present. Water samples were diluted in 9 mL BPW/0.1% Na2S2O3, resulting

in a limit of detection of 1 log10 CFU/mL. However, the statistical limit of detection is

2.4 log10 CFU/tomato when considering the acceptable range for countable colonies on

plates (25-250 CFU).

42


Salmonella recovery data from inoculated tomatoes are summarized in Table 4-6

and Figure 4-4. The mean inoculation for positive control samples was

8.01 log10 CFU/tomato after 2 h drying. No significant differences (p>0.05) were

observed in Salmonella recovery for any untreated, inoculated tomatoes (t = 0 s).

At 30 and 60 s, recoveries from inoculated tomatoes treated in

0/0 ppm COD/sanitizer were significantly higher (p<0.05), with a difference of >2.69

log10 CFU/tomato, than from those treated in 0/25 ppm COD/FC, 0/80 ppm COD/PAA,

500/25 ppm COD/FC, and 500/80 ppm COD/PAA. Recoveries from inoculated

tomatoes treated in 0/25 ppm COD/FC, 0/80 ppm COD/PAA, 500/25 ppm COD/FC, and

500/80 ppm COD/PAA were not significantly different from each other at 30 s (p>0.05).

At 60 s, recoveries from inoculated tomatoes treated in 0/25 ppm COD/FC, 500/25 ppm

COD/FC, and 500/80 ppm COD/PAA were not significantly different from each other

(p>0.05) while Salmonella populations dropped below the limit of detection on

inoculated tomatoes treated in 0/80 ppm COD/PAA.

At 30 and 60 s, recovery from inoculated tomatoes treated in the absence of

sanitizer were >6.00 log10 CFU/tomato and were significantly higher than from those

treated in all other conditions (p<0.05). Reductions in Salmonella population were >4.35

log10 CFU/tomato when either 25 ppm FC or 80 ppm PAA were present under clean

water or organically loaded water at 500 ppm COD.



summarized in Table 4-7 and Figure 4-5. Salmonella was only detectable from

uninoculated tomatoes when treated alongside inoculated tomatoes in 0/0 ppm

43

COD/sanitizer for ≥30 s at slightly less than 5.00 log10 CFU/tomato. At 0 s, no

Salmonella was detectable (<2.00 log10 CFU/tomato) on uninoculated tomatoes.

Salmonella was undetectable at all time points from uninoculated tomatoes treated in

0/25 ppm COD/FC, 500/25 ppm COD/FC, 0/80 ppm COD/PAA, and 500/80 ppm

COD/PAA.

Salmonella recovery from the model flume system water

Salmonella recovery data from the model flume system water samples are

summarized in Table 4-8 and Figure 4-6. Because water samples (1 mL) were diluted in

9 mL BPW/0.1% Na2S2O3, the limit of detection was 10 CFU/mL (1 log10 CFU/mL).

Plate count data were therefore adjusted by a factor of 1.00 log10 units. Salmonella was

detectable in 0/0 ppm COD/sanitizer at ≥2 s at approximately 3.5 log10 CFU/mL and

reached nearly 5.00 log10 CFU/mL by 30 s. Salmonella was detectable in

0/80 ppm COD/PAA and 500/80 ppm COD/PAA at 2 s at slightly above 2.00 log10

CFU/mL, but not at 30 or 60 s. Salmonella was undetectable (<1.00 log10 CFU/mL) at all

time points in 0/25 and 500/25 ppm COD/FC.


Water chemistry data for the second cross-contamination study are summarized

in Table 4-9. Initial and final sanitizer concentration, initial and final pH, initial COD,

initial TDS, and initial and final ORP were measured. As in the initial cross-

contamination study, FC concentration tended to decrease more between initial and

final measurements in those studies with organic loading compared to those with no

organic loading. PAA concentration also decreased slightly between initial and final

measurements. The pH varied considerably in all experiments. Initial and final ORP

values ranged from 827 to 838 mV in those experiments with 25 ppm FC. ORP values

44

were considerably lower in those studies with 80 ppm PAA (~470 mV) and 0 ppm

sanitizer (~580 mV).

The average volumes of 5.65-6.0% NaOCl and 50% citric acid necessary to

adjust FC levels as well as the volumes of Tsunami 100 PAA solution necessary to

adjust PAA levels are summarized in Table 4-9. As in the initial cross-contamination

study, much higher volumes of NaOCl are required to reach the desired FC levels in

those experiments with moderate organic loading as opposed to no organic loading.

Conversely, the volumes of Tsunami 100 PAA required to adjust to the appropriate PAA

levels were similar in experiments with and without organic loading. For example,

approximately 25.8 and 25.0 mL Tsunami 100 PAA were required to adjust the model

flume system water to 80 ppm PAA under no organic loading and the organic loading

condition of 500 ppm COD.


Similar to the first two cross-contamination studies, tomatoes were inoculated

with approximately 9 log10 CFU/tomato and allowed to dry for 2 h in a biosafety hood at

room temperature (ca. 25 ºC). Dried, inoculated tomatoes were then treated alongside

uninoculated tomatoes in a recirculating water bath (model flume system) containing 0,

10, or 25 ppm FC under organic loading conditions of 0, 500, or 4000 ppm COD for 0,

15, 60, or 120 s. After treatment, the tomatoes were removed and placed into separate

Stomacher® bags containing 100 mL TSB/rif80/0.1% Na2S2O3. The tomatoes were then

incubated at 37 °C for 48 h to enrich any Salmonella present. Presumptive positive as

45

well as negative samples were streaked onto TSA/rif80 for confirmation. The recovery

data for these studies are summarized in Table 4-11.

After treatment in 0/0 COD/FC, all inoculated tomatoes at 0, 15, 60, and 120 s

were positive for Salmonella while only 1 out of 9 uninoculated tomatoes was positive at

15 s. After treatment in 500/25 COD/FC, all inoculated tomatoes at 0, 15, and 60 s were

positive for Salmonella while 7 out of 9 tomatoes were positive for Salmonella at 120 s.

For uninoculated tomatoes, 1 out of 9 was positive for Salmonella at 15 s while 2 out of

9 were positive for Salmonella after 60 s. No uninoculated tomatoes, however, were

positive for Salmonella after 120 s.

46

Table 4-1. Concentration of Salmonella serovars in TSB/rif200 over 14 h at 37 °C

Salmonella concentration (log10 CFU/mL)a

Hour Montevideo Anatum Javiana Braenderup Newport

0 2.33 ± 0.04 a 2.13 ± 0.07 ab 2.29 ± 0.07 a 2.02 ± 0.05 ab 1.85 ± 0.08 b

1 2.41 ± 0.10 a 2.23 ± 0.11 ab 2.36 ± 0.03 a 2.07 ± 0.07 ab 1.94 ± 0.05 b

2 2.70 ± 0.15 a 2.66 ± 0.23 ab 2.84 ± 0.17 a 2.27 ± 0.07 bc 2.24 ± 0.22 c

3 3.17 ± 0.24 a 3.29 ± 0.29 a 3.42 ± 0.29 a 2.72 ± 0.22 b 2.73 ± 0.28 b

4 3.81 ± 0.29 a 4.00 ± 0.28 a 4.04 ± 0.30 a 3.26 ± 0.28 b 3.41 ± 0.41 b

5 4.45 ± 0.28 a 4.53 ± 0.37 a 4.56 ± 0.35 a 3.89 ± 0.33 b 4.00 ± 0.49 b

6 5.09 ± 0.39 a 5.24 ± 0.43 a 5.24 ± 0.46 a 4.51 ± 0.38 b 4.67 ± 0.43 b

7 5.74 ± 0.37 ab 5.93 ± 0.50 a 5.88 ± 0.41 a 5.19 ± 0.43 c 5.37 ± 0.38 bc

8 6.29 ± 0.41 ab 6.64 ± 0.37 a 6.62 ± 0.34 a 5.82 ± 0.34 c 6.03 ± 0.29 bc

9 7.05 ± 0.35 ab 7.32 ± 0.31 a 7.19 ± 0.21 a 6.60 ± 0.40 c 6.73 ± 0.20 bc

10 7.59 ± 0.28 ab 7.89 ± 0.37 a 7.86 ± 0.25 a 7.07 ± 0.41 c 7.28 ± 0.19 bc

11 8.11 ± 0.27 ab 8.50 ± 0.34 a 8.39 ± 0.21 a 7.74 ± 0.37 b 7.89 ± 0.23 b

12 8.55 ± 0.23 abc 8.90 ± 0.20 a 8.78 ± 0.03 ab 8.36 ± 0.22 c 8.42 ± 0.22 bc

13 8.88 ± 0.10 a 9.06 ± 0.14 a 9.03 ± 0.05 a 8.75 ± 0.19 a 8.81 ± 0.03 a

14 8.92 ± 0.07 a 8.99 ± 0.01 a 9.01 ± 0.04 a 8.99 ± 0.07 a 8.85 ± 0.03 a aValues are mean ± standard deviation of triplicate experiments (n=3). Means with same letter in the same row (abc) are not statistically different (p>0.05).

47

Figure 4-1. Concentration of Salmonella serovars over 14 h at 37 °C

Error bars represent standard deviation of triplicate experiments (n=3)

48

Table 4-2. Salmonella recovery (log10 CFU/tomato) from inoculated tomato surfaces after treatment alongside uninoculated tomatoes in a model flume system containing 0, 10, or 25 ppm free chlorine (FC) under organic loading conditions of 0, 500, or 4000 ppm chemical oxygen demand (COD)

Salmonella recovery (log10 CFU/tomato) from tomatoesa

0 ppm FC 10 ppm FC 25 ppm FC

Immersion Time (s) 0 ppm COD

0 8.25 ± 0.16 a, x 8.42 ± 0.10 a, x 8.49 ± 0.11 a, x 15 6.74 ± 0.53 abc, y 5.31 ± 1.31 de, y 5.14 ± 1.80 e, y 60 6.40 ± 0.24 a, y 2.78 ± 0.85 b, z 2.52 ± 0.56 b, z

120 6.01 ± 0.24 a, y 2.26 ± 0.60 bc, z 2.17 ± 0.40 c, z

500 ppm COD

0 8.14 ± 0.25 a, x 7.93 ± 0.18 a, x 8.43 ± 0.13 a, x 15 7.03 ± 0.54 ab, y 6.17 ± 0.89 bcd, y 6.08 ± 1.46 cde, y 60 6.41 ± 0.41 a, y 3.17 ± 0.61 b, z 3.38 ± 0.34 b, z

120 6.42 ± 0.28 a, y 2.72 ± 0.60 bc, z 3.14 ± 0.68 b, z

4000 ppm COD

0 8.31 ± 0.07 a, x 8.33 ± 0.11 a, x 8.34 ± 0.14 a, x 15 7.17 ± 0.32 a, y 7.32 ± 0.29 a, y 6.44 ± 0.84 abc, y 60 6.68 ± 0.14 a, y 6.62 ± 0.17 a, yz 3.31 ± 0.94 b, z

120 6.76 ± 0.28 a, y 6.29 ± 0.21 a, z 3.00 ± 1.01 bc, z aValues are mean ± standard deviation of triplicate experiments of 3 tomatoes each adjusted for the 2 log10 CFU/tomato loss from rinsing tomatoes in 100 mL BPW/0.1% Na2S2O3 (n=9). Means with same letter in the same row (abcde) or in the same column (xyz) are not statistically different (p>0.05).

49

Figure 4-2. Salmonella recovery (log10 CFU/tomato) from inoculated tomato surfaces after treatment alongside uninoculated tomatoes in a model flume system containing 0, 10, or 25 ppm free chlorine (FC) under organic loading conditions of 0, 500, or 4000 ppm chemical oxygen demand (COD)

Error bars represent standard deviation of triplicate experiments of three tomatoes each (n=9)

50

Table 4-3. Salmonella recovery (log10 CFU/tomato) from uninoculated tomato surfaces after treatment alongside inoculated tomatoes in a model flume system containing 0, 10, or 25 ppm free chlorine (FC) under organic loading conditions of 0, 500, or 4000 ppm chemical oxygen demand (COD)




0 <2.00 ± 0.00 a, y <2.00 ± 0.00 a, x <2.00 ± 0.00 a, x 15 5.14 ± 0.21 b, x <2.00 ± 0.00 c, x <2.00 ± 0.00 c, x 60 5.03 ± 0.18 b, x <2.00 ± 0.00 c, x <2.00 ± 0.00 c, x

120 5.05 ± 0.11 b, x <2.00 ± 0.00 c, x <2.00 ± 0.00 c, x

500 ppm COD

0 <2.00 ± 0.00 a, y <2.00 ± 0.00 a, x <2.00 ± 0.00 a, x 15 5.11 ± 0.35 b, x <2.00 ± 0.00 c, x <2.00 ± 0.00 c, x 60 5.09 ± 0.30 b, x <2.00 ± 0.00 c, x <2.00 ± 0.00 c, x

120 5.14 ± 0.30 b, x <2.00 ± 0.00 c, x <2.00 ± 0.00 c, x

4000 ppm COD

0 <2.00 ± 0.00 a, y <2.00 ± 0.00 a, z <2.00 ± 0.00 a, x 15 5.42 ± 0.21 a, x 5.28 ± 0.12 ab, x <2.00 ± 0.00 c, x 60 5.35 ± 0.16 a, x 5.09 ± 0.12 b, y <2.00 ± 0.00 c, x

120 5.40 ± 0.25 a, x 5.05 ± 0.15 b, y <2.00 ± 0.00 c, x aValues are mean ± standard deviation of triplicate experiments of 3 tomatoes each adjusted for the 2 log10 CFU/tomato loss from rinsing tomatoes in 100 mL BPW/0.1% Na2S2O3 (n=9). Means with same letter in the same row (abc) or in the same column (xyz) are not statistically different (p>0.05).

51

Figure 4-3. Salmonella recovery (log10 CFU/tomato) from uninoculated tomato surfaces after treatment alongside inoculated tomatoes in a model flume system containing 0, 10, or 25 ppm free chlorine (FC) under organic loading conditions of 0, 500, or 4000 ppm chemical oxygen demand (COD)

Error bars represent standard deviation of triplicate experiments of 3 tomatoes each (n=9) Recovery data for 0/10 COD/FC, 0/25 COD/FC, 500/10 COD/FC, 500/25 COD/FC, and 4000/25 COD/FC are below the detection limit of 2.00 log10 CFU/tomato

52

Table 4-4. Initial and final sanitizer concentration, initial and final pH, initial chemical oxygen demand (COD), and initial total dissolved solids (TDS) measured before and after those model flume system experiments summarized in Tables 4-2 and 4-3


0 ppm COD

Initial Final Initial Final Initial Final

FC (ppm)a b b 11± 1 11 ± 1 27± 1 25 ± 2

pHa 6.54 ± 0.04 8.20 ± 0.19 6.50 ± 0.01 6.61 ± 0.21 6.51 ± 0.01 6.39 ± 0.09

COD (ppm)a b b b b b b

TDS (ppm)a b b b b b b

500 ppm COD

FC (ppm)a b b 12 ± 3 2 ± 3 30 ± 2 22 ± 2

pHa 6.51 ± 0.02 6.47 ± 0.03 6.46 ± 0.02 6.37 ± 0.01 6.50 ± 0.02 6.32 ± 0.13

COD (ppm)a 568 ± 64 b 588 ± 38 b 501 ± 36 b

TDS (ppm)a 113 ± 6 b 97± 6 b 110 ± 0 b

4000 ppm COD

FC (ppm)a b b 12 ± 2 4 ± 1 29 ± 1 23 ± 2

pHa 6.51 ± 0.01 5.93 ± 0.07 6.50 ± 0.03 6.27 ± 0.23 6.49 ± 0.01 6.30 ± 0.27

COD (ppm)a 3680 ± 274 b 4158 ± 184 b 3817 ± 472 b

TDS (ppm)a 200 ± 0 b 200 ± 10 b 203 ± 6 b

aValues are mean ± standard deviation of measurements from triplicate experiments (n=3). bNot measured.

53

Table 4-5. Volumes of 5.7-6.0% sodium hypochlorite (NaOCl) necessary to adjust appropriate free chlorine (FC) levels and 50% citric acid necessary to adjust the pH to 6.5 in the model flume system in those experiments summarized in Tables 4-2 and 4-3


0 ppm COD

Volume of 5.65-6% NaOCl (mL)a

b 4.7 ± 0.1 10.1 ± 0.4

Volume of 50% citric acid

(mL)a

b 0.5 ± 0.0 1.4 ± 0.1

500 ppm COD


b 38.3 ± 2.9 98.3 ± 2.9


(mL)a

b 2.3 ± 0.6 1.2 ± 0.3

4000 ppm COD


b 319.7 ± 41.4 458.3 ± 77.7


(mL)a

b 8.7 ± 0.6 21.9 ± 6.2

aValues are mean ± standard deviation of volumes added from triplicate experiments (n=3). bNone added or not applicable.

54

Table 4-6. Salmonella recovery (log10 CFU/tomato) from inoculated tomato surfaces after treatment alongside uninoculated tomatoes in a model flume system containing 0, 10, or 25 ppm free chlorine (FC) or 80 ppm peroxyacetic acid (PAA) under organic loading conditions of 0, 500, or 4000 ppm chemical oxygen demand (COD) with water testing


0 ppm sanitizer 25 ppm FC 80 ppm PAA

0 ppm COD

Immersion Time (s)

0 8.09 ± 0.22 a, x 8.07 ± 0.25 a, x 8.09 ± 0.20 a, x 30 6.33 ± 0.41 a, y 2.78 ± 1.04 b, y 3.02 ± 1.44 b, y 60 6.38 ± 0.33 a, y 2.59 ± 0.85 bc, y <2.00 ± 0.00 c, z

500 ppm COD

0 b 7.99 ± 0.14 a, x 7.83 ± 0.18 a, x 30 b 3.64 ± 1.07 b, y 3.19 ± 1.00 b, y 60 b 3.15 ± 1.04 b, y 2.30 ± 0.66 bc, z

aValues are mean ± standard deviation of triplicate experiments of 3 tomatoes each adjusted for the 2 log10 CFU/tomato loss from rinsing tomatoes in 100 mL BPW/0.1% Na2S2O3 (n=9). bNot tested. Means with same letter in the same row (abc) or in the same column (xyz) are not statistically different (p>0.05).

55

Figure 4-4. Salmonella recovery (log10 CFU/tomato) from inoculated tomato surfaces after treatment alongside uninoculated tomatoes in a model flume system containing 0, 10, or 25 ppm free chlorine (FC) or 80 ppm peroxyacetic acid (PAA) under organic loading conditions of 0, 500, or 4000 ppm chemical oxygen demand (COD) with water testing

Error bars represent standard deviation of triplicate experiments of 3 tomatoes each (n=9)

56

Table 4-7. Salmonella recovery (log10 CFU/tomato) from uninoculated tomato surfaces after treatment alongside inoculated tomatoes in a model flume system containing 0, 10, or 25 ppm free chlorine (FC) or 80 ppm peroxyacetic acid (PAA) under organic loading conditions of 0, 500, or 4000 ppm chemical oxygen demand (COD) with water testing




0 <2.00 ± 0.00 a, z <2.00 ± 0.00 a, x <2.00 ± 0.00 a, x 30 4.92 ± 0.18 a, x <2.00 ± 0.00 b, x <2.00 ± 0.00 b, x 60 4.72 ± 0.25 a, y <2.00 ± 0.00 b, x <2.00 ± 0.00 b, x

500 ppm COD

0 b <2.00 ± 0.00 a, x <2.00 ± 0.00 a, x 30 b <2.00 ± 0.00 b, x <2.00 ± 0.00 b, x 60 b <2.00 ± 0.00 b, x <2.00 ± 0.00 b, x

aValues are mean ± standard deviation of triplicate experiments of 3 tomatoes each adjusted for the 2 log10 CFU/mL loss from rinsing tomatoes in 100 mL BPW/0.1% Na2S2O3 (n=9). bNot tested. Means with same letter in the same row (ab) or in the same column (xyz) are not statistically different (p>0.05).

57

Figure 4-5. Salmonella recovery (log10 CFU/tomato) from uninoculated tomato surfaces after treatment alongside inoculated tomatoes in a model flume system containing 0, 10, or 25 ppm free chlorine (FC) or 80 ppm peroxyacetic acid (PAA) under organic loading conditions of 0, 500, or 4000 ppm chemical oxygen demand (COD) with water testing

Error bars represent standard deviation of triplicate experiments of 3 tomatoes each (n=9) Recovery data for 0/25 COD/FC, 0/80 COD/PAA, 500/25 COD/FC, and 500/80 COD/PAA are below the detection limit of 2.00 log10 CFU/tomato

58

Table 4-8. Salmonella recovery (log10 CFU/mL) from the water of a model flume system containing 0, 10, or 25 ppm free chlorine (FC) or 80 ppm peroxyacetic acid (PAA) under organic loading conditions of 0, 500, or 4000 ppm chemical oxygen demand (COD) in which inoculated tomatoes were treated alongside uninoculated tomatoes

Salmonella recovery (log10 CFU/mL) from watera



0 <1.00 ± 0.00 a, z <1.00 ± 0.00 a, x <1.00 ± 0.00 a, y 2 3.49 ± 1.46 a, y <1.00 ± 0.00 c, x 2.14 ± 1.02 b, x 30 4.81 ± 0.20 a, x <1.00 ± 0.00 b, x <1.00 ± 0.00 b, y 60 4.76 ± 0.14 a, x <1.00 ± 0.00 b, x <1.00 ± 0.00 b, y

500 ppm COD

0 b <1.00 ± 0.00 a, x <1.00 ± 0.00 a, y 2 b <1.00 ± 0.00 c, x 2.26 ± 0.93 b, x 30 b <1.00 ± 0.00 b, x <1.00 ± 0.00 b, y 60 b <1.00 ± 0.00 b, x <1.00 ± 0.00 b, y

aValues are mean ± standard deviation of triplicate experiments of three 1 mL water samples each adjusted by 1 log10 CFU/mL for dilution in 9 mL BPW/0.1% Na2S2O3 (n=9). bNot tested. Means with same letter in the same row (abc) or in the same column (xyz) are not statistically different (p>0.05).

59

Figure 4-6. Salmonella recovery (log10 CFU/mL) from the water of a model flume system containing 0, 10, or 25 ppm free chlorine (FC) or 80 ppm peroxyacetic acid (PAA) under organic loading conditions of 0, 500, or 4000 ppm chemical oxygen demand (COD) in which inoculated tomatoes were treated alongside uninoculated tomatoes

Error bars represent standard deviation of triplicate experiments of 3 water samples each (n=9) Recovery data for 0/25 COD/FC and 500/25 COD/FC are below the detection limit of 1.00 log10 CFU/mL

60

Table 4-9. Initial and final free chlorine (FC) or peroxyacetic acid (PAA) concentration, initial and final pH, initial chemical oxygen demand (COD), initial total dissolved solids (TDS), and initial and final oxidation reduction potential (ORP) measured before and after those model flume system experiments summarized in Tables 4-6, 4-7, and 4-8


0 ppm COD

Initial Final Initial Final Initial Final

Measured FC or PAA (ppm)a b b 27 ± 2 24 ± 2 78 ± 0 76 ± 7

Measured pHa 6.52 ± 0.01 5.78 ± 0.58 6.47 ± 0.03 6.16 ± 0.40 3.57 ± 0.02 3.27 ± 0.21

COD (ppm)a b b b b b b

TDS (ppm)a

b b b b b b

ORP (mV)a 574 ± 157 591 ± 45 827 ± 17 836 ± 15 474 ± 5 464 ± 12

500 ppm COD

Measured FC or PAA (ppm)a 27 ± 2 22 ± 1 78 ± 0 74 ± 3

Measured pHa 6.49 ± 0.03 5.88 ± 0.46 3.75 ± 0.05 3.43 ± 0.36

COD (ppm)a 570 ± 71 b 488 ± 69 b

TDS (ppm)a

97 ± 6 b 100 ± 10 b

ORP (mV)a 840 ± 16 838 ± 27 476 ± 6 469 ± 3

aValues are mean ± standard deviation of triplicate experiments (n=3). bNot measured.

61

Table 4-10. Volumes of 5.7-6.0% sodium hypochlorite (NaOCl) and 50% citric acid necessary to adjust appropriate free chlorine (FC) levels and pH to 6.5, or Tsunami 100 peroxyacetic acid (PAA) solution necessary to adjust peroxyacetic acid (PAA) levels in the model flume system in those experiments summarized in Tables 4-6, 4-7, and 4-8

0 ppm FC 25 ppm FC 80 ppm PAA

0 ppm COD


b 10.0 ± 0.0 b

Volume of 50% citric acid (mL)a

0.0 ± 0.1 1.1 ± 0.1 b

Volume of Tsunami 100 PAA (mL)a b b 25.8 ± 1.4

500 ppm COD


b 95.0 ± 5.0 b

Volume of 50% citric acid (mL)a

b 1.6 ± 0.5 b

Volume of Tsunami 100 PAA (mL)a b b 25.0 ± 0.0

aValues are mean ± standard deviation of volumes added from triplicate experiments (n=3). bNone added or not applicable.

62

Table 4-11. Positive Salmonella detection from inoculated and uninoculated tomatoes treated in a model flume system containing 25 ppm free chlorine (FC) under organic loading conditions of 0 or 500 ppm chemical oxygen demand (COD) and subsequently enriched in TSB/rif80/0.1% Na2S2O3 and confirmed on TSA/rif80.

Positive Salmonella recovery from tomatoesa

Inoculated Tomatoes

0 ppm COD 500 ppm COD

Immersion Time (s) 25 ppm FC 25 ppm FC

0 9 9 15 9 9 60 9 9

120 9 7

Uninoculated Tomatoes

0 ppm COD 500 ppm COD

25 ppm FC 25 ppm FC

0 0 0 15 1 1 60 0 2

120 0 0 aValues are number of tomatoes positive for rifampicin-resistant Salmonella growth from triplicate experiments of 3 tomatoes each (n=9).

63

CHAPTER 5 DISCUSSION AND CONCLUSIONS

Between 1998 and 2011, tomatoes were confirmed as the source of 20

Salmonella outbreaks in the US (CDC 2011). While Good Agricultural Practices (GAPs)

and other intervention measures aid in minimizing the risk of these outbreaks,

contamination can still occur. Improper sanitizer use in a packinghouse flume system

may allow for the transfer of pathogens from contaminated to uncontaminated tomatoes

(Lopez-Galvez and others 2009). Few studies have addressed the ability of sanitizers to

prevent cross-contamination of human pathogens between tomatoes under realistic

packinghouse conditions. Dirt, leaves, wax, tomato exudate, and other organic debris

that accumulate in recirculating water systems can negatively affect sanitizer efficacy

(Allende and others 2007; Gil and others 2009). The overall goal of this research was to

assess the minimum levels of sanitizer necessary to prevent the cross-contamination of

Salmonella between intact, green, round tomatoes in a model flume system with clean

and organically loaded water.

Determination of Salmonella Concentration in TSB/rif80 over 14 h at 37 °C

A five-serovar cocktail of rifampicin-resistant Salmonella enterica was used as

the tomato inoculum for all experiments. The serovars included Montevideo, Anatum,

Javiana, Braenderup, and Newport. These strains were selected due to their

association with past multistate tomato outbreaks (Hedberg and others 1999; CDC

2005; CDC 2007). A growth curve study was conducted to confirm that each of the five

serovars reached stationary phase at the same concentration after the same incubation

period, ensuring that all serovars in the combined cocktail would be equivalent in

concentration. The use of a cocktail reduces variability in recovery associated with

64

differences in serovar survival on tomatoes during treatment. Growth of each serovar

was measured individually over 14 h (Table 4-1 and Figure 4-1). Growth for all serovars

began with a lag phase from hours 0 to 2, followed by a logarithmic growth phase from

hours 0 to 12. In the stationary phase, the rate of cell proliferation is equivalent to the

rate of cell death; therefore, live cell concentration plateaus (Zwietering and others

1990). In this study, significant differences in cell concentration were present until hour

13, at which point cell concentration leveled off and stationary phase likely began. At

hours 13 and 14, no significant differences in concentration were present between

serovars (p>0.05). The average concentration of the five serovars at hour 14 was

8.95 log10 CFU/mL.


Three cross-contamination studies were performed to assess the ability of

sanitizers in a model flume system to prevent the transfer of Salmonella from inoculated

to uninoculated tomatoes in clean and organically loaded water. Transfer was

determined by quantifying Salmonella recovered from uninoculated tomatoes after

flume treatment. Reduction of Salmonella on inoculated tomatoes was also measured.

In the second cross-contamination study, levels of Salmonella released into the water

from contaminated tomatoes were determined. In the final study, Salmonella was not

enumerated; rather, presence or absence was determined by enriching the entire

tomato in 100 mL of TSB/rif80. This method was employed to increase sensitivity, as

the survival of a single, viable Salmonella bacterium could be detected.

A study by Zhou and others (2013) measured dump tank water quality

parameters over the course of one day in three separate Florida tomato packinghouses.

Organic matter content in the water was quantified by chemical oxygen demand (COD).

65

COD began near 100 ppm in each production shift, and ranged from 400 to 750 ppm by

the end of production depending on the length of operation (Zhou and others 2013). A

COD of 500 ppm was selected in this study to represent typical packinghouse organic

loading conditions, while 4000 ppm COD was selected to represent a worst-case

scenario.

Transfer of Salmonella from Inoculated to Uninoculated Tomatoes in a Model Flume System Containing Free Chlorine under Clean and Organic Loading Conditions

This study was conducted to assess the ability of free chlorine (FC) to prevent

cross-contamination of Salmonella between tomatoes in clean and organically loaded

water. Reduction of Salmonella population on tomato surfaces and transfer to

uninoculated tomatoes were determined.


Recovery data from inoculated tomatoes are summarized in Table 4-2 and

Figure 4-2. Untreated, inoculated tomatoes had an average recovery of

8.29 log10 CFU/tomato after the 2 h drying period in the biosafety hood. This is lower

than the theoretical inoculation level of 9 log10 CFU/tomato. The 2 h drying period was

intended to promote Salmonella attachment on the tomato surfaces. Desiccation as a

result of exposure to airflow likely led to a small decrease in Salmonella population (Wei

and others 1995). It is also possible that some Salmonella failed to dislodge into the

recovery medium. This difference between Salmonella recovery from inoculated, dried

tomato surfaces and theoretical inoculation levels is similar to a study by Chang (2011),

in which an average difference of approximately 0.73 log10-units was observed between

a Salmonella cocktail inoculum and inoculated tomatoes dried for 2 h.

66

Salmonella recoveries from inoculated tomatoes were generally higher in those

experiments where FC was absent. One exception was inoculated tomatoes treated in

4000/10 COD/FC, where recoveries after 60 and 120 s were not significantly different

from those in all other experiments with 0 ppm FC (p>0.05). This result suggests that

FC fails as a sanitizer at 10 ppm under the very high organic loading conditions of

4000 ppm COD. This could be explained by FC reacting preferentially with organic

matter over bacterial cells (CDC 2006). Moreover, FC decay influenced by the presence

of high levels of organic matter may have altered reaction kinetics, exacerbating the

decline in efficacy.

Significantly lower Salmonella recoveries (p<0.05) were obtained from inoculated

tomatoes treated 0/10 COD/FC, 0/25 COD/FC, 500/10 COD/FC, 500/25 COD/FC, and

4000/25 COD/FC (not in 4000/10 COD/FC) than from those treated in the absence of

FC. Reductions under these conditions at ≥60 s exceeded the minimum 3.00 log10-units

reduction of Salmonella populations required for tomato packinghouse sanitizing

operations by T-BMPs regulations (FDACS 2007). These data suggest that maintaining

FC concentrations as low as 25 ppm may be adequate for industrial tomato sanitation

practices under current T-BMPs requirements (≥3 log10-unit reduction of Salmonella or

like bacteria on tomato surfaces), provided that organic loading levels in the water do

not exceed 4000 ppm COD. Such high COD levels are unlikely to occur if dump tank

and flume system water is changed daily (Zhou and others 20013). These results,

however, do not address the potential for cross contamination.

Recoveries from inoculated tomatoes treated in 0/10 COD/FC, 0/25 COD/FC,

500/10 COD/FC, 500/25 COD/FC, and 4000/25 COD/FC declined much more sharply

67

over time than recoveries from inoculated tomatoes treated in 0/0 COD/FC, 500/0

COD/FC, 4000/0 COD/FC, and 4000/10 COD/FC. Compared to untreated, inoculated

tomatoes (t = 0 s), significant reductions occurred on inoculated tomatoes after ≥15 s

treatment in 0/0 COD/FC, 500/0 COD/FC, 4000/0 COD/FC, and 4000/10 COD/FC

(p<0.05). These reductions were likely due to mechanical removal as a result of tomato

agitation in the model flume system (Chang 2011). Reductions on inoculated tomatoes

after treatment in 0/10 COD/FC, 0/25 COD/FC, 500/10 COD/FC, 500/25 COD/FC, and

4000/25 COD/FC can be partially attributed to mechanical removal by agitation in the

water. However, the presence of FC appears to have greatly aided in further reduction.

In a study by Felkey and others (2006), tomatoes inoculated with Salmonella were

treated in a model flume system containing 50 ppm FC. Salmonella reductions were

1.24, 1.47, 3.96, and 4.61 log10-units after 15, 30, 60, and 120 s, respectively (Felkey

and others 2006). These reductions are considerably lower than those achieved in the

present study, despite the use of higher FC concentrations. The use of different

serovars in the Felkey and others (2006) study may potentially explain these

differences. Additionally, it is possible that more tomato agitation was achieved in the

present study, thereby increasing Salmonella detachment. Zhuang and others (1995)

examined the fate of S. Montevideo on the surfaces of tomatoes treated in FC. A

reduction of approximately 1.50 log10-units was achieved after 2 min treatment in

320 ppm FC (Zhuang and others 1995). Again, this reduction is considerably lower than

what was observed in the present study, despite substantially higher FC concentrations.

A number of factors can potentially explain this difference, including differing inoculation

methods (dip inoculation vs. spot inoculation), treatment methods (sanitizer dip vs.

68

model flume system treatment), enumeration media (bismuth sulfite agar vs. TSA/rif80),

and serovar(s) used (S. Montevideo alone vs. a five-serovar rifampicin-resistant

cocktail).



summarized in Table 4-3 and Figure 4-3. Salmonella was undetectable from

uninoculated tomatoes after treatment alongside inoculated tomatoes in 0/10 COD/FC,

0/25 COD/FC, 500/10 COD/FC, 500/25 COD/FC, and 4000/25 COD/FC. Salmonella

recoveries from uninoculated tomatoes after treatment alongside inoculated tomatoes in

0/0 COD/FC, 500/0 COD/FC, 4000/0 COD/FC, and 4000/10 COD/FC reached over

5 log10 CFU/tomato after ≥15 s treatment. In a study by Rana and others (2010),

uninoculated tomatoes were treated alongside tomatoes inoculated with S.

Typhimurium (7.4 log10 CFU/tomato) for 5 min in a model flume system containing clean

tap water. Under this condition, Salmonella recovery from uninoculated tomatoes after

washing was approximately 3.0 log10 CFU/tomato in the absence of FC (Rana and

others 2010). The extent of cross-contamination is lower than what was observed in the

present study; however, this can be attributed to lower inoculation levels and a higher

model flume system volume utilized the Rana and others (2010) study. In the present

study, FC at 10 ppm under very high organic loading conditions (4000 ppm COD) failed

to prevent cross contamination. As previously discussed, this is likely explained by FC

preferentially reacting with organic matter over Salmonella (CDC 2006). These results

emphasize that in the absence of proper sanitation, cross-contamination can occur in

15 s. Additional studies should be conducted to examine the potential for cross-

contamination in <15 s, though sampling logistics make this difficult. These results

69

suggest that FC levels as low as 25 ppm in a packinghouse flume system are adequate

to prevent Salmonella cross-contamination at levels ≥2 log10 CFU/tomato, provided that

organic loading conditions do not exceed 4000 ppm COD. To determine if these lower

levels of FC could completely prevent cross contamination, the third cross-

contamination study was performed to address contamination levels as low as

1 CFU/tomato.


Table 4-4 summarizes water chemistry parameters for each of the first cross-

contamination study experiments. It is important to note that FC levels were less stable

in those experiments where organic loading conditions were present. For example,

initial and final FC concentrations in studies with 0/10 COD/FC were both 11 ppm. Initial

and final FC concentrations in studies with 4000/10 COD/FC were 12 and 4 ppm,

respectively. Thus, the rate of FC decay increased with increasing COD. This

underlines the importance of frequent monitoring of sanitizer levels in flume systems,

especially when high levels of organic matter are present, which can lead to the rapid

depletion of FC (Gil and others 2009). Initial COD measurements are summarized, as

well. In the Zhou and others (2013) study, dump tank organic matter content increased

significantly throughout the day and reached between 400 and 750 ppm COD by the

end of operation. As a result of organic matter accumulation, FC levels (indicated by

ORP) were unstable and needed to be frequently adjusted throughout the day. This

climb in COD was strongly correlated with an increase in TDS (r=0.841) as well as an

increase in turbidity (r=0.635). Considering the length of time required for COD analysis

(~2.5 h), the authors considered TDS and turbidity more practical indicators of organic

loading for a tomato packinghouse operator (Zhou and others 2013). In the present

70

study, the volumes of NaOCl and citric acid required to adjust the appropriate sanitizer

conditions increased greatly with increased organic loading (Table 4-5). For example,

the volumes of NaOCl and citric acid required to adjust the model flume system to

25 ppm FC and pH 6.5 in clean water were approximately 10 and 1.4 mL, respectively.

Conversely, the volumes of NaOCl and citric acid required to adjust the flume system to

25 ppm free chlorine and pH 6.5 under 4000 ppm COD were approximately 458 and

22 mL, respectively. This enormous difference emphasizes that organic matter can

bind/deplete the sanitizer, which requires the packer to add more sanitizer as organic

matter accumulates in the flume system (Suslow 1997).


This study was primarily conducted to assess the ability of sanitizers under

organic loading conditions to inactivate any Salmonella dislodged from inoculated

tomato surfaces and released into the model flume system water. In addition to

Salmonella survival in the water column, survival on inoculated tomatoes and transfer to

uninoculated tomatoes were determined, as well. This study also examined the efficacy

of peroxyacetic acid (PAA) as compared to FC. PAA is frequently utilized in Florida

packinghouses and increasingly looked at as a move environmentally-friendly

alternative to chlorine-based sanitizers.


Recovery data from inoculated tomatoes are summarized in Table 4-6 and

Figure 4-4. Average recoveries from inoculated tomatoes after treatment in 0/0 COD/FC

for 30 and 60 s were significantly higher than from those treated in 0/25 COD/FC, 0/80

COD/PAA, 500/25 COD/FC, and 500/80 COD/PAA (p<0.05). These results are similar

71

to what was observed in the initial cross-contamination study. Mechanical action alone

likely significantly reduced Salmonella populations on inoculated tomato surfaces after

≥30 s (p<0.05). The presence of sanitizer, however, appears to have had a large effect

in terms of further reduction. In a study by Yuk and others (2005), Salmonella

populations on inoculated tomato surfaces were reduced by over 5-log10-units after

treatment in a recirculating water bath containing 87 ppm PAA at 35 °C for 1 min (Yuk

and others 2005). The results from the Yuk (2005) study parallel the results observed in

the present study. From the initial cross-contamination study, it is known that

10 ppm FC fails as a sanitizer under very high organic loading conditions of 4000 ppm

COD. Additional studies should be conducted to assess the efficacy of 80 ppm PAA as

a tomato surface disinfectant under organic loading conditions of >500 ppm COD.


Recovery data from uninoculated tomatoes are summarized in Table 4-7 and

Figure 4-5. Salmonella was only detectable from uninoculated tomatoes treated

alongside inoculated tomatoes in 0 ppm COD/0 ppm. Similar to the initial cross-

contamination study, Salmonella levels reached near 5 log10 CFU/tomato on

uninoculated tomatoes under this treatment. FC levels of 10 and 25 ppm as well as

80 ppm PAA appear to prevent cross-contamination at levels ≥2 log10 CFU/tomato

under clean conditions and organically loaded conditions of 500 ppm COD. Additional

studies should be conducted to assess the ability of 80 ppm PAA to prevent Salmonella

cross-contamination between tomatoes under organic loading conditions >500 ppm

COD.

72

Salmonella recovery from the model flume system water

Recovery data from the model flume system water are summarized in Table 4-8

and Figure 4-6. For those experiments with 0/0 COD/sanitizer, Salmonella levels in the

water reached 4.81 and 4.76 log10 CFU/mL after 30 and 60 s, respectively. Salmonella

was recoverable at 3.49 log10 CFU/mL only ~2 s after the introduction of inoculated

tomatoes. This suggests that cross-contamination between tomatoes can potentially

occur in 2 s in the absence of proper sanitation. Salmonella was undetectable in the

model flume system water in those experiments with 0/25 COD/FC or 500/25 COD/FC.

Salmonella was recoverable at 2 s from the water in those studies with 0/80 COD/PAA

and 500/80 COD/PAA; however, not at 30 or 60 s. These results suggest that FC

inactivates Salmonella in water nearly instantaneously whereas PAA requires more than

2 s but fewer than 30 s. These results agree with a study by Shen and others (2013),

which showed a >4.5 log10 CFU/mL reduction of Salmonella, E. coli O157:H7, and

non-O157 Shiga toxin-producing E. coli (STEC) in suspension after exposure to

0.5 ppm FC for 30 s, or to 1.0 ppm free chlorine for 5 s. In the Rana and others (2010)

study, clean tap water in a model flume system containing no FC reached

approximately 3.68 log10 CFU/mL 5 min after the introduction of inoculated tomatoes

(Rana and others 2010). This is lower than the water contamination levels observed in

the present study with no FC; however, higher inoculation levels and a smaller model

flume system volume in the present study likely led to this difference. In the Rana and

others (2010) study, Salmonella was undetectable in clean tap water when FC

concentrations were between 5 and 200 ppm, and in organically loaded, spent industry

dump tank water when FC concentrations were between 5 and 100 ppm (Rana and

others 2010).

73

Additional studies should be conducted to find the minimum treatment time

required for 80 ppm PAA to inactivate Salmonella in the model flume system water.

Further, additional studies should be conducted with enriched water samples of larger

volumes to extend the detection limit below 1.00 log10 CFU/mL.


As in the initial cross-contamination study, the presence of organic matter

appears to have led to more rapid depletion of free chlorine (Table 4-9). Levels of PAA

also tended to decrease, further illustrating the importance of frequent sanitizer

monitoring. In experiments with 25 ppm FC, ORP levels exceeded range (650-700 mV)

necessary to kill E. coli and Salmonella (Suslow 2000). However, in experiments with

80 ppm PAA, these levels were not achieved, suggesting that ORP is not a good

indicator of PAA efficacy.


This study was conducted to assess the ability of FC to prevent the cross-

contamination of Salmonella between tomatoes at levels between 0 and

2 log10 CFU/tomato. Detection data from these experiments are summarized in

Table 4-11. One out of 9 uninoculated tomatoes treated alongside inoculated tomatoes

in 0/25 COD/FC was positive for Salmonella after 15 s, while no uninoculated tomatoes

were positive for Salmonella after 60 or 120 s under this treatment. One out of nine

uninoculated tomatoes treated alongside inoculated tomatoes in 500/25 COD/FC was

positive for Salmonella after 15 s while two out of nine were positive for Salmonella after

60 s treatment under this treatment. No uninoculated tomatoes were positive for

74

Salmonella after treatment for 120 s. These results suggest that low levels of

Salmonella (0-2 log10 CFU/tomato) can occur in the presence of 25 ppm FC with and

without organic loading. However, it appears that a minimum of 15-30 s (with no organic

loading) or 60-120 s (with 500 ppm COD) was required for 25 ppm FC to completely

inactivate Salmonella that were dislodged from inoculated tomatoes. In the Rana and

others (2010) study, uninoculated tomatoes were treated alongside inoculated tomatoes

in clean tap water containing 5 and 30 ppm FC. Under each treatment, only 1/18

uninoculated tomato samples was positive for Salmonella after enrichment, suggesting

low levels of Salmonella cross contamination. In the study, no uninoculated tomatoes

were positive for Salmonella after enrichment when treated alongside inoculated

tomatoes in clean tap water containing 100 or 200 ppm FC (Rana and others 2010).

However, when the study was repeated using spent industry dump tank water, 11/18

uninoculated tomatoes were positive for Salmonella after treatment in 5 ppm FC and

2/18 were positive after treatment in 30 ppm FC (Rana and others 2010). These results

are similar to what was observed in the present study, although the organic loading

level in the spent industry dump tank water was not quantified, making it difficult to

directly compare results to the present study. However, it is clear that organic loading

does indeed negatively impact the ability of FC to prevent Salmonella cross-

contamination between tomatoes in a flume system.

The results of the present study suggest that tomatoes should be treated in a

flume system for ≥120 s with FC levels of ≥25 ppm to completely prevent cross-

contamination of Salmonella provided that COD levels do not exceed 500 ppm.

Additional cross-contamination studies with enrichment should be conducted to see if

75

this treatment is adequate under organic loading conditions of >500 ppm COD.

Presently, T-BMPs regulations allow for a maximum flume system treatment of 2 min to

minimize potential pathogen internalization (FDACS 2007). Therefore, more studies with

enrichment should be conducted to examine the ability of FC at concentrations above

25 ppm under organic loading conditions to prevent cross-contamination in under 120 s.

Conclusions and Suggestions for Further Research

The results from these studies have potentially far-reaching effects with regard to

tomato packinghouse sanitation practices. Based on these findings, maintaining FC

levels in dump tanks and flume systems at a minimum of 25 ppm may be adequate to

completely prevent cross-contamination of Salmonella provided that a) this

concentration is properly maintained, b) that the organic loading level of the flume water

does not rise above 500 ppm COD, and c) that tomatoes are treated for a minimum of

120 s. The ability of packers to use lower levels of sanitizer could save money on

chemistry costs and reduce the environmental impact associated with spent water

disposal.

Future studies should involve enrichment to address lower levels of cross

contamination. FC concentrations ≥25 ppm with levels of organic loading between

500 and 4000 ppm COD should be examined to a) find the minimum concentration

required to completely prevent Salmonella cross-contamination under higher organic

loading conditions, and b) to potentially lessen the required FC contact time. Other

common packinghouse sanitizers such as PAA and chlorine dioxide (ClO2) should also

be examined in greater detail. A suggestion for future work is to examine Salmonella

survival on cross contaminated tomatoes 24 h after flume treatment to more closely

mimic post-wash storage of packed tomatoes prior to consumption. Additionally, the

76

effect of tomato waxing following Salmonella cross contamination could be examined.

While the present studies show that these lower levels of free chlorine can inactivate

Salmonella, the same may not necessarily apply to other human pathogens or to plant

pathogens that can cause tomato quality issues. Further studies should be conducted to

address the control of cross-contamination of other microorganisms.

77

LIST OF REFERENCES Allende A, Martinez B, Selma V, Gil M, Suarez J, Rodriguez A. 2007. Growth and

bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing Listeria monocytogenes in vitro and in fresh-cut lettuce. Food Microbiology 24(7-8):759-66.

Allende A, Selma M, Lopez-Galvez F, Villaescusa R, Gil M. 2008. Impact of wash water quality on sensory and microbial quality, including Escherichia coli cross-contamination, of fresh-cut escarole. Journal of Food Protection 71(12):2514-8.

Bartz J, Eayre C, Mahovic M, Concelmo D, Brecht J, Sargent S. 2001. Chlorine concentration and the inoculation of tomato fruit in packinghouse dump tanks. Plant Disease 85(8):885-9.

Berg J, Roberts P, Matin A. 1986. Effect of chlorine dioxide on selected membrane functions of Ecscherichia coli. Journal of Applied Bacteriology 60(3):213-20.

Beuchat L, Adler B, Lang M. 2004. Efficacy of chlorine and a peroxyacetic acid sanitizer in killing Listeria monocytogenes on iceberg and Romaine lettuce using simulated commercial processing conditions. Journal of Food Protection 67(6):1238-42.

Beuchat LR. 1996. Pathogenic microorganisms associated with fresh produce. Journal of Food Protection 59(2):204-16.

Beuchat LR, Ryu J. 1997. Produce handling and processing practices. Emerging Infectious Diseases 3(4):459-65.

Beuchat LR. 2002. Ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. Microbes and Infections 4:413-23.

Brandl M. 2006. Fitness of human enteric pathogens on plants and implications for food safety. Annual Review of Phytopathology 44:367-92.

Brackett RE. 1999. Incidience, contributing factors, and control of bacterial pathogens in produce. Postharvest Biol Tec 15:305-11.

[CDC] Centers for Disease Control and Prevention. 2002. Outbreak of Salmonella

serotype Javiana infections. MMWR 51(31):683-684.

[CDC] Centers for Disease Control and Prevention. 2005. Outbreaks of Salmonella infections associated with eating Roma tomatoes - United States and Canada, 2004. MMWR 54(13):325-328.

[CDC] Centers for Disease Control and Prevention. 2006. Safe Water Systems (SWS) Publications – Disinfectant. Available from: http://www.cdc.gov/safewater/publications_pages/pubs_disinfectant.htm. Accessed 22 May 2014.

http://www.cdc.gov/safewater/publications_pages/pubs_disinfectant.htm

78

[CDC] Centers for Disease Control and Prevention. 2007. Multistate outbreaks of Salmonella infections associated with raw tomatoes eaten in restaurants - United States, 2005-2006. MMWR 56(35):909-11.

[CDC] Centers for Disease Control and Prevention. 2011. Foodborne outbreak online database. Available from: http://wwwn.cdc.gov/foodborneoutbreaks/default.aspx. Accessed 20 May 2014.

[CDC] Centers for Disease Control and Prevention. 2013. Surveillance for foodborne disease outbreaks - United States, 2009-2010. MMWR 62(03):41-47.

Chang A. 2011. Evaluation of overhead spray-applied sanitizers for the reduction of Salmonella on tomato surfaces. [MS thesis]. Gainesville, FL: Univ. of Florida.

[CFR] Code of Federal Regulations. 2013a. Chlorine dioxide. Title 21, Part 173.300. Washington, DC: US Food and Drug Administration, Office of the Federal Register.

[CFR] Code of Federal Regulations. 2013b. Sanitizing solutions. Title 21, Part 178.1010 Washington, DC: US Food and Drug Administration, Office of the Federal Register.

Cummings K, Barrett E, Mohle-Boetani J, Brooks J, Farrar J, Hunt T, Fiore A, Komatsu K, Werner S, Slutsker L. 2001. A multistate outbreak of Salmonella enterica serotype Baildon associated with domestic raw tomatoes. Emerging Infectious Diseases 7(6):1046-8.

D'Aoust J, Maurer J. 2007. Salmonella Species.In: Doyle MP, Beuchat, LR. Food Microbiology: Fundamentals and Frontiers. Washington, DC: ASM Press. p 187-236.

Dell'Erba A, Falsanisi D, Liberti L, Notarnicola M, Santoro D. 2004. Disinfecting behaviour of peracetic acid for municipal wastewater reuse. Desalination 168:435-42.

Fatica M. 2009. The use of chlorination and alternative sanitizers in the produce industry. Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 4(052):1-10.

Felkey K, Archer D, Bartz J, Goodrich R, Schneider K. 2006. Chlorine disinfection of tomato surface wounds contaminated with Salmonella spp. Horticulture Technology 16(2):253-6.

[FDA] Food and Drug Administration. 1998. Guide to minimize microbial food safety hazards for fresh fruits and vegetables. Washington, D.C.: US Department of Health and Human Services, FDA. Available from: http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/ProduceandPlanProducts/UCM064574. Accessed 20 May 2014.

http://wwwn.cdc.gov/foodborneoutbreaks/default.aspx

http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/ProduceandPlanProducts/UCM064574

http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/ProduceandPlanProducts/UCM064574

79

[FDA] Food and Drug Administration. 2011. FDA confirms Listeria monocytogenes on Jensen Farms’ Rocky Ford-brand cantaloupes. Available from: http://www.fda.gov/newsevents/newsroom/pressannouncements/ucm272527.htmAccessed 20 May 2014.

[FDACS] Florida Department of Agriculture and Consumer Services. 2007. Tomato best practices manual. Tallahassee, FL: Florida Department of Agriculture and Consumer Services, Division of Food Safety. Available from: http://www.doacs.state.fl.us/fs/TomatoBestPractices.pdf. Accessed 21 May 2014.

Frank C, Werber D, Cramer JP, Askar M, Faber M, an der Heiden M, Bernard H, Fruth A, Prager R, Spode A, Wadl M, Zoufaly A, Jordan S, Kemper MJ, Follin P, Muller L, King LA, Rosner B, Buchholz U, Stark K, Krause G. 2011. Epidemic profile of shiga-toxin-producing Escherichia coli O104:H4 outbreak in Germany. New Engl J Med 365:1771-80.

Gil M, Selma M, Lopez-Galvez F, Allende A. 2009. Fresh-cut product sanitation and wash water disinfection: problems and solutions. International Journal of Food Microbiology 134(1-2):37-45.

Gomez-Lopez V, Rajkovic A, Ragaert P, Smigic N, Devlieghere F. 2009. Chlorine dioxide for minimally processed produce preservation: A review. Trends in Food Science & Technology 20(1):17-26.

Greene S, Daly E, Talbot E, Demma L, Holzbauer S, Patel N, Hill T, Walderhaug M, Hoekstra R, Lynch M, Painter J. 2008. Recurrent multistate outbreak of Salmonella Newport associated with tomatoes from contaminated fields, 2005. Epidemiology and Infection 136(2):157-65.

Gupta S, Nalluswami K, Snider C, Perch M, Balasegaram M, Burmeister D, Lockett J, Sandt C, Hoekstra R, Montgomery S. 2007. Outbreak of Salmonella Braenderup infections associated with Roma tomatoes, northeastern United States, 2004: a useful method for subtyping exposures in field investigations. Epidemiology and Infection 135(7):1165-73.

Harris L, Farber J, Beuchat L, Parish M, Suslow T, Garrett E, Busta F. 2003. Outbreaks associated with fresh produce: incidence, growth, and survival of pathogens in fresh and fresh-cut produce. Comprehensive Reviews in Food Science and Food Safety 2(s1):78-141.

Hedberg C, Angulo F, White K, Langkop C, Schell W, Stobierski M, Schuchat A, Besser J, Dietrich S, Helsel L, Griffin P, McFarland J, Osterholm M, Team I. 1999. Outbreaks of salmonellosis associated with eating uncooked tomatoes: implications for public health. Epidemiology and Infection 122(3):385-93.

Iturriaga M, Escartin E. 2010. Changes in the effectiveness of chlorine treatments during colonization of Salmonella Montevideo on tomatoes. Journal of Food Safety 30(2):300-6.

http://www.fda.gov/newsevents/newsroom/pressannouncements/ucm272527.htm

http://www.fda.gov/newsevents/newsroom/pressannouncements/ucm272527.htm

http://www.doacs.state.fl.us/fs/TomatoBestPractices.pdf

80

Kader AA. 1986. Effects of postharvest handling procedures on tomato quality. Acta Hort. (ISHS) 190:209-22.

Kitinoja L, Kader AA. 2002. Small-scale postharvest handling practices: A manual for horticultural crops. Davis, CA: University of California, Davis Postharvest Technology Research and Information Center. Available from: http://www.fao.org/docrep/009/ae075e/ae075e00.htm. Accessed 20 May 2014.

Lang MM, Harris LJ, Beuchat LR. 2004. Evaluation of inoculation method and inoculum drying time for their effects on survival and efficiecny of recovery of Escherichia coli O157:H7, Salmonella and Listeria monocytogenes inoculated on the surfaces of tomatoes. Journal of Food Protection 57:732-741.

[LGMA] Leafy Greens Marketing Agreement. 2013. Commodity specific food safety guidelines for the production and harvest of lettuce and leafy greens. Sacramento, CA: California Leafy Green Handler Marketing Board. Available from: http://www.caleafygreens.ca.gov/. Accessed 14 April 2014.

Lopez-Galvez F, Allende A, Selma MV, Gil MI. 2009. Prevention of Escherichia coli cross-contamination by different commercial sanitizers during washing of fresh-cut lettuce. International Journal of Food Microbiology 133(1-2):167-71.

Lynch M, Tauxe R, Hedberg C. 2009. The growing burden of foodborne outbreaks due to contaminated fresh produce: risks and opportunities. Epidemiology and Infection 137(3):307-15.

Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV. 1999. Food-related illness and death in the United States. Emerg Infec Dis 5:607-25

McDonnell G, Russell A. 1999. Antiseptics and disinfectants: activity, action, and resistance. Clinical Microbiology Reviews 12(1):147-79.

Narayanasamy P. 2006. Ecology of postharvest microbial pathogens. In: Narayanasamy P. Postharvest pathogens and disease management. Hoboken, NJ: John Wiley & Sons, Inc. p 79-116.

Nou X, Luo Y. 2010. Whole-leaf wash improves chlorine efficacy for microbial reduction and prevents pathogen cross-contamination during fresh-cut lettuce processing. Journal of Food Science 75(5):M283-90.

Nyachuba DG. 2010. Foodborne illness: Is it on the rise? Nutrition Reviews 68(5):257-69.

Olmez H, Kretzschmar U. 2009. Potential alternative disinfection methods for organic fresh-cut industry for minimizing water consumption and environmental impact. LWT-Food Science and Technology 42(3):686-93.

Painter J, Hoekstra R, Ayers T, Tauxe R, Braden C, Angulo F, Griffin P. 2013. Attribution of foodborne illnesses, hospitalizations, and deaths to food

http://www.fao.org/docrep/009/ae075e/ae075e00.htm

http://www.caleafygreens.ca.gov/sites/default/files/California%20LGMA%20metrics%2008%2026%2013%20%20Final.pdf

81

commodities by using outbreak data, United States, 1998-2008. Emerg Infect Dis 19(3):407-15.

Pao S, Kelsey DF, Khalid M, Ettinger M. 2007. Using aqueous chlorine dioxide to prevent contamination of tomatoes with Salmonella enterica and Erwinia carotovora during fruit washing. Journal of Food Protection 70(3):629-34.

Pao S, Kelsey DF, Long W. 2009. Spray washing of tomatoes with chlorine dioxide to minimize Salmonella on inoculated fruit surfaces and cross-contamination from revolving brushes. J Food Prot 72:2448-52.

Parish M, Beuchat L, Suslow T, Harris L, Garrett E, Farber J, Busta F. 2003. Methods to reduce/eliminate pathogens from fresh and fresh-cut produce. Comprehensive Reviews in Food Science and Food Safety 2(s1):161-73.

[PL] Public Law. 2011. FDA Food Safety Modernization Act. Pub L No. 111-353, 124 Stat 3885.

Rana S, Paris B, Reineke KM, Stewart D, Schlesser, Tortorello M, Fu TJ. 2010. Factors affecting Salmonella cross-contamination during postharvest washing of tomatoes. Available from: http://www.iit.edu/ifsh/resources_and_tools/pdfs/2010poster_iafp_rana_salmonellatomatoes.pdf. Accessed 15 May 2014.

Ruiz-Cruz S, Acedo-Felix E, Diaz-Cinco M, Islas-Osuna M, Gonzalez-Aguilar G. 2007. Efficacy of sanitizers in reducing Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes populations on fresh-cut carrots. Food Control 18(11):1383-90.

Robbs PG, Bartz JA, Brecht JK, Sargent SA. 1995. Oxidation-reduction potential of chlorine solutions and their toxicity to Erwinia carotovora subsp. carotovora and Geotrichum candidum. Plant Disease 79(2):158-62.

Sapers G. 2001. Efficacy of washing and sanitizing methods for disinfection of fresh fruit and vegetable products. Food Technology and Biotechnology 39(4):305-11.

Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, Griffin PM. 2011a. Foodborne illness acquired in the United States--major pathogens. Emerg Infect Dis 17(1):7-15.

Scallan E, Griffin PM, Angulo FJ, Tauxe RV, Hoekstra RM. 2011b. Foodborne illness acquired in the United States – unspecified agents. Emerg Infect Dis 17(1):16-22.

Scharff RL. 2012. Economic Burden from Health Losses Due to Foodborne Illness in the United States. Journal of Food Protection 75(1):123-31.

Shen CL, Luo YG, Nou XW, Wang Q, Millner P. 2013. Dynamic effects of free chlorine concentration, organic load, and exposure time on the inactivation of Salmonella, Escherichia coli O157:H7, and non-O157 Shiga toxin-producing E. coli. Journal of Food Protection 76(3):386-93.

http://www.sciencedirect.com/science?_ob=RedirectURL&_method=externObjLink&_locator=url&_cdi=271165&_issn=09639969&_origin=article&_zone=art_page&_plusSign=%2B&_targetURL=http%253A%252F%252Fwww.iit.edu%252Fifsh%252Fresources_and_tools%252Fpdfs%252F2010poster_iafp_rana_salmonellatomatoes.pdf

http://www.sciencedirect.com/science?_ob=RedirectURL&_method=externObjLink&_locator=url&_cdi=271165&_issn=09639969&_origin=article&_zone=art_page&_plusSign=%2B&_targetURL=http%253A%252F%252Fwww.iit.edu%252Fifsh%252Fresources_and_tools%252Fpdfs%252F2010poster_iafp_rana_salmonellatomatoes.pdf

82

Sivapalasingam S, Friedman C, Cohen L, Tauxe R. 2004. Fresh produce: a growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. Journal of Food Protection 67(10):2342-53.

Stopforth J, Mai T, Kottapalli B, Samadpour M. 2008. Effect of acidified sodium chlorite, chlorine, and acidic electrolyzed water on Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes inoculated onto leafy greens. Journal of Food Protection 71(3):625-8.

Suslow T. 1997. Postharvest chlorination: basic properties and key points for effective disinfection. Davis, CA: Regents of the University of California. Available from: http://anrcatalog.ucdavis.edu/pdf/8003.pdf. Accessed 20 February 2014.

Suslow T. 2000. Chlorination in the production and postharvest handling of fresh fruits and vegetables. In: Suslow. Fruit and Vegetable Processing. p 2-15.

Taylor E, Kastner J, Renter D. 2010. Challenges involved in the Salmonella Saintpaul outbreak and lessons learned. Journal of Public Health Management Practices 16:221-31.

[USDA] United States Department of Agriculture. 2014. Vegetables 2013 Summary. p 42-3.

Vandekinderen I, Devlieghere F, De Meulenaer B, Ragaert P, Van Camp J. 2009. Optimization and evaluation of a decontamination step with peroxyacetic acid for fresh-cut produce. Food Microbiology 26(8):882-8.

Vigneault C, Bartz J, Sargent S. 2000. Postharvest decay risk associated with hydrocooling tomatoes. Plant Disease 84(12):1314-8.

Wei C, Huang T, Kim J, Lin W, Tamplin M, Bartz J. 1995. Growth and survival of Salmonella Montevideo on tomatoes and disinfection with chlorinated water. Journal of Food Protection 58(8):829-36.

Wells J, Butterfield J. 1997. Salmonella contamination associated with bacterial soft rot of fresh fruits and vegetables in the marketplace. Plant Disease 81:867-72.

Wu V, Kim B. 2007. Effect of a simple chlorine dioxide method for controlling five foodborne pathogens, yeasts, and molds on blueberries. Food Microbiology 24(7-8):794-800.

Yang Y, Luo YG, Millner P, Shelton D, Nou XW. 2012. Enhanced chlorine efficacy against bacterial pathogens in wash solution with high organic loads. Journal of Food Processing and Preservation 36(6):560-6.

Yuk H, Bartz J, Schneider K. 2005. Effectiveness of individual or combined sanitizer treatments for inactivating Salmonella spp. on smooth surface, stem scar, and wounds of tomatoes. Journal of Food Science 70(9):M409-14.

http://anrcatalog.ucdavis.edu/pdf/8003.pdf

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Zhang S, Farber J. 1996. The effects of various disinfectants against Listeria monocytogenes on fresh-cut vegetables. Food Microbiology 13(4):311-21.

Zhou B, Luo Y, Turner E, Wang Q, Schneider K. 2013. Evaluation of current industry practices for maintaining tomato dump tank water quality during packinghouse operations. Journal of Food Processing and Preservation. doi: 10.1111/jfpp.12200.

Zhuang R, Beuchat L, Angulo F. 1995. Fate of Salmonella Montevideo on and in raw tomatoes as affected by temperature and treatment with chlorine. Applied and Environmental Microbiology 61(6):2127-31.

Zwietering MH, Jongenburger I, Rombouts FM, Van 't Riet K. 1990. Modeling of the bacterial growth curve. Applied and Environmental Microbiology 56(6):1875-81.

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BIOGRAPHICAL SKETCH

Scott Gereffi was born and raised in Fort Lauderdale, Florida. Scott earned a

bachelor’s degree in food science and human nutrition from the University of Florida in

2012 and joined Dr. Keith Schneider’s food safety lab as a graduate student shortly

thereafter. He plans to graduate with a master's degree in food science and human

nutrition in summer 2014 to pursue a career as a food scientist in industry. Scott’s

passion in life is travel. He also enjoys playing the violin and spending time with friends

and family.