SALMONELLA TYPHIMURIUM LT2 TRANSFER AND REDISTRIBUTION ON BABY
SPINACH AND CILANTRO DURING PILOT-SCALE PROCESSING
By
Haley Smolinski

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Food Science – Master of Science
2017

ABSTRACT
SALMONELLA TYPHIMURIUM LT2 TRANSFER AND REDISTRIBUTION ON BABY
SPINACH AND CILANTRO DURING PILOT-SCALE PROCESSING
By
Haley Smolinski
Several recent outbreaks traced to baby spinach and cilantro have been hypothesized to
involve cross-contamination during washing and processing. Consequently, this study aimed to
assess the redistribution of Salmonella Typhimurium LT2 during pilot-scale production of freshcut baby spinach and cilantro. Four inoculated:uninoculated product weight ratios (0.5:100,
1:100, 5:100, and 10:100) and three different inoculation levels (103, 101, and 10-1 CFU/g) were
used with spot-inoculated red leaf lettuce serving as a colored surrogate for baby spinach and
cilantro washing. Sanitizer-free wash water was used for all trials and a chlorine-based sanitizer
was used at 60 ppm available chlorine only for the highest inoculation level (103 CFU/g) and the
three highest weight ratios (1:100, 5:100, and 10:100). Overall, initial inoculation level had a
greater impact on the amount of Salmonella-positive samples than the weight ratios examined for
both commodities. The number of positive samples concurrently decreased as the initial
inoculation level of the surrogate decreased. Within each inoculation level, no significant
differences (P > 0.05) were found among the four product ratios. This is the first study to assess
the spread of Salmonella from incoming product to baby spinach and cilantro during processing.
These results will provide important data for microbial risk assessments associated with leafy
greens.

Copyright by
HALEY SMOLINSKI
2017

To my parents, Susan and Chet
Thank you for always supporting my dreams

iv

ACKNOWLEDGEMENTS

I would like to take a moment to acknowledge specific individuals who contributed to the
accomplishment of this work. First, I want to thank my advisor, Dr. Elliot Ryser for this
invaluable opportunity and his mentorship in the food safety and microbiology field. He was a
great advocate and always took the time to discuss project directions. I would also like to thank
Dr. John Linz and Dr. Cornelius Barry for their time and support in serving on my thesis
committee.
Special thanks must be made to my lab mates – Ryann Gustafson, Siyi Wang, Lin Ren,
Hamoud Alnughaymishi and our undergraduate laboratory assistants – Natasha Sloniker,
Lindsey DeFrain, Angela Shermetaro, Leah Thayer, Brooke Desposato, and Melaina Smith.
Without the contributions from these individuals, completing this work would not have been
possible.
Last but definitely not least, thank you to my amazing friends and family for sacrificing
our time together so that I could chase my dream of earning a master’s degree and becoming a
mad food scientist. Thank you for being patient. Without your advice, unconditional support and
encouragement, I would not be where I am today.

v

TABLE OF CONTENTS

LIST OF TABLES ....................................................................................................................... viii
LIST OF FIGURES ....................................................................................................................... ix
KEY TO ABBREVIATIONS ..........................................................................................................x
INTRODUCTION ...........................................................................................................................1
CHAPTER 1: REVIEW OF PERTINENT LITERATURE ............................................................5
1.1 Economic burden of foodborne illness ..................................................................................6
1.2 Salmonella .............................................................................................................................7
1.2.1 Salmonella Typhimurium LT2 ........................................................................................9
1.3 Fresh produce consumption and produce outbreaks in the U.S. .........................................10
1.4 Baby spinach and cilantro outbreaks ...................................................................................12
1.5 Previous cross contamination studies on pathogen transfer and redistribution during
processing ...................................................................................................................................14
1.6 Inoculation methods .............................................................................................................21
1.7 Previous microbial studies on fresh produce and spot inoculation .....................................22
1.8 Contamination of produce in the field ..................................................................................23
1.8.1 Harvesting of baby spinach and cilantro .......................................................................28
1.9 Post-harvest processing ........................................................................................................29
1.9.1 Shredding and cutting ....................................................................................................30
1.9.2 Conveying ......................................................................................................................31
1.9.3 Flume washing ...............................................................................................................32
1.9.4 Dewatering and drying ..................................................................................................36
1.9.5 Baby spinach..................................................................................................................36
1.9.6 Cilantro ..........................................................................................................................37
1.10 Food Safety Modernization Act ........................................................................................38
1.11 Risk assessment tools .......................................................................................................38
1.12 Overall goals ......................................................................................................................39

vi

CHAPTER 2: SALMONELLA TYPHIMURIUM LT2 TRANSFER AND REDISTRIBUTION
ON BABY SPINACH AND CILANTRO DURING PILOT-SCALE PROCESSING ................41
2.1 Introduction .........................................................................................................................42
2.2 Materials and methods ........................................................................................................45
2.2.1 Overall experimental design ..........................................................................................45
2.2.2 Produce ..........................................................................................................................45
2.2.3 Bacterial strain ...............................................................................................................45
2.2.4 Surrogate inoculation .....................................................................................................46
2.2.5 Wash water and sanitizer ...............................................................................................46
2.2.6 Leafy green processing line ...........................................................................................47
2.2.7 Baby spinach and cilantro processing and sample collection ........................................47
2.2.8 Microbiological analysis................................................................................................50
2.2.9 Red leaf lettuce samples ................................................................................................50
2.2.10 Water samples..............................................................................................................50
2.2.11 Bagged baby spinach and cilantro ...............................................................................50
2.2.12 GeneQuence assay .......................................................................................................51
2.2.13 Statistical analysis........................................................................................................51
2.3 Results .................................................................................................................................51
2.3.1 Baby spinach..................................................................................................................54
2.3.2 Cilantro ..........................................................................................................................57
2.3.3 Red leaf lettuce ..............................................................................................................60
2.3.4 Wash water ....................................................................................................................61
2.3.5 Sanitizer implementation ...............................................................................................62
2.4 Discussion ...........................................................................................................................66
CHAPTER 3: CONCLUSIONS AND FUTURE RECOMMENDATIONS ................................73
APPENDIX ....................................................................................................................................76
BIBLIOGRAPHY ..........................................................................................................................85

vii

LIST OF TABLES

Table 2.1: Numbers and percentage of Salmonella-positive baby spinach samples (23 samples
per trial) at different inoculation levels and inoculated:uninoculated product ratios ...................55
Table 2.2: Numbers and percentage of Salmonella-positive samples for cilantro samples (~ 22
total samples per trial) with different inoculation levels and product ratios .................................58
Table 2.3: Comparison of processing trials with and without a chemical sanitizer for baby
spinach ..........................................................................................................................................64
Table 2.4: Comparison of processing trials with and without a chemical sanitizer for cilantro....65
Table AI.1: Salmonella populations (log CFU/g) on red leaf lettuce before washing baby
spinach. This data served as the control for the surrogate samples and ensured the targeted initial
populations were achieved .............................................................................................................79
Table AI.2: Salmonella populations (log CFU/g) on red leaf lettuce before washing cilantro. This
data served as the control for the surrogate samples and ensured the targeted initial populations
were achieved.................................................................................................................................80
Table AI.3: Salmonella populations (log CFU/g) on red leaf lettuce after washing baby spinach.
Note: LOD: < -0.04 log CFU/g .....................................................................................................81
Table AI.4: Salmonella populations (log CFU/g) on red leaf lettuce after washing cilantro.
Note: LOD: <-0.04 log CFU/g ......................................................................................................82
Table AI.5: Percentage of Salmonella populations lost from red leaf lettuce surrogate during
baby spinach processing ...............................................................................................................83
Table AI.6: Percentage of Salmonella populations lost from red leaf lettuce surrogate during
cilantro processing ........................................................................................................................84

viii

LIST OF FIGURES

Figure 1.1: Breakdown of the economic costs caused by a Salmonella-related illness ...................7
Figure 2.1: Flume tank and dewatering shaker table .....................................................................48
Figure 2.2: Collection bin for washed produce following 90 s of flume washing.........................48
Figure 2.3: Centrifugal dryer .........................................................................................................49
Figure 2.4: Bagged samples containing 225 g baby spinach/cilantro after removal of the
inoculated surrogate .......................................................................................................................49
Figure 2.5: Percentage of Salmonella-positive baby spinach samples after 90 s of flume washing
with and without a chemical sanitizer. Means with different capital letters are significantly
different (P < 0.05). .......................................................................................................................52
Figure 2.6: Percentage of Salmonella-positive cilantro samples after 90 s of flume washing with
and without a chemical sanitizer. Means with different capital letters are significantly different
(P < 0.05) .......................................................................................................................................53
Figure 2.7: Percentage (+/- SD) of Salmonella-positive baby spinach samples after processing in
sanitizer-free water at inoculated:uninoculated weight ratios of a.) 10:100, b.) 5:100, c.) 1:100,
and d.) 0.5:100. Means with different capital letters are significantly different (P < 0.05) ..........56
Figure 2.8: Percentage (+/- SD) of Salmonella-positive cilantro samples after processing in
sanitizer-free water at inoculated:uninoculated weight ratios of a.) 10:100, b.) 5:100, c.) 1:100,
d.) 0.5:100. Means with different capital letters are significantly different (P < 0.05).................59
Figure 2.9: Percentage of Salmonella-positive samples after 90 s of flume washing in water
containing 60 ppm available chlorine. Means with different capital letters are significantly
different (P < 0.05). .......................................................................................................................63

ix

KEY TO ABBREVIATIONS

ANOVA – Analysis of Variance
°C – degrees Celsius
CDC – Centers for Disease Control and Prevention
CFU – Colony Forming Unit(s)
cm – centimeter
ERS – Economic Research Service
FAO – Food and Agriculture Organization of the United Nations
FDA – U.S. Food and Drug Administration
FSMA – Food Safety Modernization Act
g – gram
GAPs – Good Agriculture Practices
GMPs – Good Manufacturing Practices
GN Broth – Gram negative broth
kg – kilogram
L – liter
LOD – Limit of Detection
min – minute
mL – milliliter
nm – nanometers
PBS – Phosphate Buffered Solution
ppm – parts per million
QPRAM – Quantitative Predictive Risk Assessment Model
h – hour
RTE – Ready-to-Eat
RH – Relative Humidity

x

rpm – revolutions per minute
RV Broth – Rappaport-Vassilidas Broth
s – second
SD – Standard Deviation
SSOPs – Standard Sanitation Operating Procedures
TSA-YE – Trypticase Soy Agar with 0.6% Yeast Extract
TSB-YE – Trypticase Soy Broth with 0.6% Yeast Extract
TT Broth – Tetrathionate Broth
µL – microliter
U.S. – United States
USDA – United States Department of Agriculture
WHO – World Health Organization

xi

INTRODUCTION

1

Fruits and vegetables comprise a principal part of the diet for overall health and wellness.
As per the USDA MyPlate guidelines, it is recommended that adults consume 1½ - 2 cups of
fruit and 2½ - 3 cups of vegetables per day (USDA 2017). Therefore, these foods are consumed
every day, multiple times per day. However, since many of these foods are ready-to-eat (RTE) or
minimally cooked, the opportunity for reducing bacterial or pathogenic loads that may be present
on these foods is significantly less than for foods that are thoroughly cooked prior to
consumption. Given the high year round consumer demand for fresh fruits and vegetables, the
importation of produce, some of which is invariably contaminated, has also increased
(Merriweather 2015). Between the years 1996-2008, produce has been linked to 82 foodborne
outbreaks, of which leafy greens have accounted for 34% of the total (FDA 2009). Pathogens
linked to outbreaks related to fresh produce include Escherichia coli O157:H7, Listeria
monocytogenes, Salmonella Typhimurium, Hepatitis A, Campylobacter jejuni, and Norovirus,
among others. Produce linked to multistate outbreaks include salads, leafy vegetables (romaine,
spinach, shredded lettuce), tomatoes, sprouts, carrots, onions, cucumber, berries (strawberry,
blueberry, raspberry), melons (cantaloupe, honeydew, watermelon), and grapes, among many
others (Callejon et al. 2015).
Although commonly associated with egg and poultry outbreaks, Salmonella also poses a
threat to the safety of fresh produce. Between 2004 and 2012, Salmonella was responsible for the
most multi-state produce-related outbreaks in the United States and was also the most common
pathogen associated with contamination of sprouts (Callejon et al. 2015). The economic burden
caused by Salmonella is $3.7 billion, which is 24% of the total annual burden faced by the
industry from foodborne outbreaks.

2

Produce is prone to contamination at any point in the farm to fork continuum. Cross
contamination is often the catalyst, amplifying the spread of contamination during pre- and postharvest practices. The initial contaminant load does not have to be high for significant cross
contamination to occur (Buchholz et al. 2012; Buchholz et al. 2014; Davidson et al. 2013).
Vehicles for transmission during pre-harvest and harvest include: irrigation water, soil, manure,
wild or domestic animals, field workers, harvesting equipment and even insects (FDA 1998;
FDA 2013; FDA 2009). During post-harvest, produce can become compromised through direct
contact with other contaminated produce and contamination can spread to previously
uncontaminated produce through the wash water, mechanical equipment, or plant workers (FDA
1998; FDA 2009; FDA 2015a). Consumers can also cross-contaminate products in the home
from cutting boards, knives, and hands that have been in contact with raw meats (Buchholz et al.
2011; FDA 2015a).
The Food and Drug Administration utilizes risk assessment tools in order to determine
preventative methods that can be applied to ensure the safe handling of produce throughout the
distribution chain (FDA 2015b). Specific tools used by the produce industry include FDA iRISK
and the Quantitative Produce Risk Assessment Model (QPRAM) (FDA 2015b). These tools are
invaluable to the FDA when determining areas of improvement for the safe handling and
distribution of fresh produce. The FDA is also utilizing these same risk assessment tools to
develop guidelines of the Food Safety Modernization Act (FDA 2016). Under this act, the
industry is taking a preventative as opposed to a reactive approach to foodborne outbreaks. In
order to establish better preventative controls for the fresh produce industry, the FDA is utilizing
scientific data and risk assessment when establishing new or revising previous guidelines (FDA
2016a).

3

In order to ensure safer production of fresh produce and reduce the number of related
foodborne outbreaks, data gaps need to be filled in the QPRAM and FDA iRISK tools. An
accurate account of cross contamination occurring when a contaminant is introduced during
flume washing of leafy greens and other produce does not yet exist. Therefore, the need for
quantifying pathogen redistribution during this specific step in production is pertinent.
Consequently, the objectives for this research study were to:

1. Quantify Salmonella Typhimurium LT2 transfer and redistribution when
realistically low initial inoculation levels and weight ratios of inoculated surrogate
are introduced to uninoculated baby spinach and cilantro during post-harvest
processing, in sanitizer-free wash water.
2. Assess the efficacy of a chlorine-based sanitizer (60 ppm available chlorine,
acidified to pH 6.5) when the highest inoculation level 103 CFU/g and 1:100,
5:100, and 10:100 weight ratios of inoculated surrogate to uninoculated baby
spinach and cilantro are used.

4

CHAPTER 1:
REVIEW OF PERTINENT LITERATURE

5

1.1 Economic burden of foodborne illness
It is estimated by the CDC that 48 million people in the United States are sickened
annually by foodborne illness but only 9.4 million (20%) of these cases result from identifiable
pathogens (Hoffman et al. 2015). Of the 9.4 million identifiable cases, the USDA Economic
Research Service estimated the annual economic burden at $15.5 billion, although due to
difficulty in identifying cases, this value could be much higher (Hoffman 2015). As of 2015, the
five pathogens responsible for 90% of the economic burden included Salmonella, Toxoplasma
gondii, Listeria monocytogenes, Norovirus, and Campylobacter (Hoffman et al. 2015). Of these
five, Salmonella is responsible for $3.7 billion (24%) in annual economic losses which is the
highest of the top 15 foodborne illness-causing pathogens (Hoffman et al. 2015). Salmonella also
ranked 6th with per case monetary cost of $1,896 (Roos 2010; Hoffman et al. 2015). The
monetary amount lost per year from pathogens is determined from the severity of annual cases,
loss of productivity from missed work, associated medical costs such as physician office visits,
emergency room visits, outpatient clinic visits, hospitalizations, and premature death (Figure 1.1)
(Roos 2010; Hoffman et al. 2015). The average amount of money lost by a grower from a
foodborne outbreak is > $100,000, usually due to litigation costs and/or lost profit (GuiterrezRodriguez 2015).

6

(Source: USDA, Economic Research Service)
Figure 1.1: Breakdown of the economic costs caused by a Salmonella-related illness.

1.2 Salmonella
The genus Salmonella is found in the family Enterobacteriaceae, which contains rodshaped bacteria that are Gram-negative, motile with flagella or non-motile, and facultatively
anaerobic (FDA 2012; Motarjemi 2013). Because Salmonella are Gram-negative, they are more
resistant to antibiotics and sanitizers than Gram-positive bacteria. This is primarily due to their
thin peptidoglycan layer, which is located between two thin membranes. The thin outer
membrane surrounding the peptidoglycan layer is impermeable and resists toxic materials that
could damage the cell (Mitchell 2015; Silhavy et al. 2010).

7

The two serotypes of Salmonella most commonly associated with foodborne illness in the
United States are S. Typhimurium S. Enteritidis (CDC 2014; WHO 2013; Lawley 2013;
Motarjemi 2013). Depending on the serotype, there are two primary illnesses caused by
Salmonella: non-typhoidal salmonellosis and typhoid fever. Non-typhoidal salmonellosis estimated at 1,027,561 illnesses annually, is most commonly associated with foodborne
outbreaks (Hoffman et al. 2015). The infectious dose of Salmonella is 10-100,000 cells but
depending on strain characteristics and health of the individual exposed, the dose can be as low
as one cell (FDA 2015b; FDA 2012; Fellows 2009). Symptoms of illness will appear 6 - 72
hours following exposure, last 1 - 2 days if acute or 4 - 7 days if greater exposure occurs.
Mortality in healthy humans is about 1%, but if elderly, immunocompromised, or infants are
exposed, mortality increases to 3.6% (FDA 2015b).
Salmonella grows optimally at 37°C, but can also grow in temperatures between 7 - 45°C
(Motarjemi 2013). This pathogen can survive but will not grow at refrigeration or freezing
temperatures. Salmonella can grow at a pH range of 4.5 - 9.5, with optimal growth occurring at
6.5 - 7.5. While unable to grow at a water activity value of < 0.93, Salmonella can survive in
many low moisture foods, such as spices (Motarjemi 2013).
Animals are common asymptomatic carriers of Salmonella with this organism residing in
the intestinal tract and feces (Hoffman et al. 2015; WHO 2013; FDA 2012). Sources of
Salmonella include the intestinal tracts of livestock (cattle, pigs, and chickens), wildlife (snails,
insects, birds, and rodents), and household pets (dogs, cats, turtles) (WHO 2013; Lawley 2013;
FDA 2012; Hoffman et al. 2015). Pond water sediment can also be a potential source of the
bacteria (FDA 2012). Any contact with Salmonella-contaminated water or animals/animal feces
and subsequent contact with food or a food contact surface will spread the bacteria leading to a

8

potential illness (Hoffman et al. 2015). Person-to-person transmission can also occur through the
fecal-oral route (FDA 2012).
1.2.1 Salmonella Typhimurium LT2. Salmonella Typhimurium LT2 belongs to
subspecies I of S. Enterica, which causes 99% of Salmonella infections in humans and is
naturally found in mammals and birds (Selander et al. 1996; Popoff 2000). Salmonella
Typhimurium LT2 was first isolated in the 1940s and is a primary strain for investigating cellular
and molecular biology in Salmonella (Neidhardt 1996). In a study dedicated to the genomic
sequencing of Salmonella Typhyimurium LT2, it was found that 11% of the S. Typhimurium
LT2 genes are missing from S. enterica serovar Typhi (S. Typhi), and 29% are missing from
Escherichia coli K12 (McClelland et al. 2001). Escherichia coli K12 is a member of the closest
known genus to Salmonella, which is why the aforementioned comparison is relevant (Perna et
al. 2001). Since S. Typhimurium LT2 shares 352 genes with of S. enterica, this strain is useful
for epidemiological, host specificity, and pathogenicity studies (Selander et al. 1996). Specific
regions of the genome encode for various virulence genes which are important in pathogenicity.
For Salmonella, these regions are referred to as Salmonella pathogenicity islands (SPIs) (Perna et
al. 2001; Ochman et al. 200). SPI of S. Typhimurium LT2 strain 55% similar to the SPI of S.
Enterica (McClelland et al. 2001). Another important virulence factor in enteric bacteria is the
RpoS sigma factor. Specific to Salmonella, RpoS controls expression of the Salmonella plasmid
virulence genes, which are necessary for systemic infection (Nickerson and Curtiss 1997).
However, the spv gene is not expressed in the LT2 strain since the RpoS sigma factor is absent
(Nickerson and Curtiss 1997). This characteristic, as well as the genomic sequence of LT2, is
what make this strain avirulent.

9

1.3 Fresh produce consumption and produce outbreaks in the U.S.
Between 1970 and 2004, the per capita consumption of produce consistently increased,
likely due to the active promotion of fruits and vegetables as components of a healthy diet (FDA
2015c). Between 1982 and 1997, per capita consumption rose most dramatically with an increase
of 32% from 91.2 kg to 121.1 kg (FDA 2015c). From 1997-2004, the increase in consumption
slowed more gradually and eventually saw a 7% decline from 2009-2014 (Produce for Better
Health Foundation 2015). A 5% increase in per capita consumption is projected from 2015 to
2020 (Produce for Better Health Foundation 2015). This increase in consumption has also led to
more produce-related outbreaks of illness primarily because more food was consumed outside of
the home, such as at buffets and restaurants, with consumption of fresh, RTE produce items like
bagged lettuce and pre-sliced fruits also increasing (FDA 2015c). These factors, coupled with
increased globalization for the fresh produce trade is likely to increase consumer exposure to a
broad range of foodborne pathogens (FDA 2015c).
The FAO and WHO declared that RTE leafy green vegetables and fresh herbs present the
greatest concern for microbiological hazards (FAO and WHO 2008). This is due to the lack of a
kill step during processing and in the home to eliminate pathogens that may be present. Between
1996 and 2008, produce has been linked to 82 foodborne outbreaks, of which leafy greens have
accounted for 34% of the total (FDA 2009). Salmonella, E. coli O157:H7. Bacillus cereus, and
Yersinia enterocolitica have been linked to sprouts, mung beans, and alfalfa seed outbreaks
(CDC 2016). Other produce implicated in multistate outbreaks involving E .coli O157:H7
include strawberries (2001), RTE salads (2013), spring mix (2012), romaine lettuce (2011), and
spinach (2006). Produce implicated in multistate outbreaks involving L. monocytogenes include
frozen vegetables (2016), cantaloupes (2011), sprouts (2014), caramel apples (2015), and

10

strawberries (2011) (CDC 2016; Kniel 2014). Salmonella, L. monocytogenes, Campylobacter,
Norovirus and E. coli O157:H7 have been linked to several multi-state melon outbreaks (CDC
2016; Castillo et al. 2014; Chapman 2005). Bagged lettuce products have also been a major
source of outbreaks; spring mix lettuce and spinach blend contaminated with E. coli O157:H7
caused 33 cases of illness and 13 hospitalizations (CDC 2012, cited in Matthews 2014). In 2013
and 2012, bagged and RTE lettuce was linked to two E. coli O157:H7 outbreaks, respectively
(CDC 2016).
Outbreaks caused by Salmonella have involved all major food groups with fresh produce
now the leading contributor to this foodborne illness. Tomatoes have been most commonly
associated with Salmonella with 5,324 cases of illness in the U.S. and 35 outbreaks between
1990 and 2012 (Center for Science in the Public Interest Outbreak Database 2013; Wang et al
2013). Salmonella was also responsible for outbreaks involving grapes, cabbage, lettuce, sprouts,
herbs, leafy green salads, and coleslaw between 2000 and 2007 (UGA’s Center for Food Safety;
CDC 2016). A Salmonella Copenhagen DT 104b outbreak in Finland was linked to fieldcontaminated iceberg lettuce originating from Spain that caused 60 confirmed illnesses
(Eurosurveillance 2005). Imported cucumbers from Mexico presumably contaminated in the
field were responsible for three Salmonella outbreaks in 2013, 2014, and 2015 (CDC 2016). In
2006, S. Oranienburg was found in cut fruit salads distributed to 10 northeastern U.S. states and
one Canadian province in 2006 causing 41 illnesses (CDC 2007). This outbreak most likely
resulted from cross contamination during the cutting process of fruit (CDC 2007). Salmonella
was also responsible for outbreaks involving mangoes in 2012, cantaloupe in 2008 and 2011, and
sprouts in 2011, 2013, 2014, and 2015 (CDC 2016).

11

1.4 Baby spinach and cilantro outbreaks
Cilantro (Coriandrum sativum), which is botanically and anatomically closely related to
parsley, has a long stem with small feathered leaves (Foley et al 2003). Cilantro is grown and
harvested close to the ground, which increases the probability of pathogen contamination from
the soil or groundwater (FDA 2001). Specific to cilantro, Salmonella has caused 8 multi-state
outbreaks in the past twenty years and caused two Class I recalls in 2011 and 2012 after random
FDA and USDA samples were Salmonella-positive (CDC 2016; US Foods 2016). In 1999,
Salmonella Thompson contaminated cilantro caused 41 confirmed cases after patrons had visited
a restaurant in California (Campbell et al. 2001). Poor record keeping of the distributor used by
the restaurant prevented a confirmed source to be found, however cilantro may have been
originally contaminated in the field (Campbell et al. 2001).
As per the Produce & Imported Foods Safety Initiative of 1997, the FDA was required to
gather data on specific fresh produce commodities imported at the highest volumes (FDA 2003).
Among the samples analyzed, 9% (n=177) of random imported cilantro samples were positive
for Salmonella or Shigella (FDA 2003). As a follow up to this study, 3.3% of imported samples
(n=33) were Salmonella-positive the following year (FDA 2003). Due to the high levels of
contaminated imported cilantro and other commodities, the FDA initiated a domestic follow-up
study in 2001 in which 1.2% (n=81) of domestically grown cilantro samples were positive for
Salmonella (FDA 2003). From 2002-2009 the FDA randomly tested 2510 cilantro samples for
the presence of enterohemorragic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), and
Salmonella and found 0.64% (n=16) to be positive for the presence of one of these pathogens
(FDA 2015a). Of the commodities tested, the 0.64% positive rate for cilantro was the second
highest, with spinach producing the highest positive rate of 0.74%.

12

In 2012, 2013, 2014 and 2015, cilantro was also responsible for a multistate Cyclospora
outbreak, causing over 1,310 illnesses of the four outbreaks combined (CDC 2016; FDA 2016c).
Investigations into these outbreaks revealed that the cilantro was imported from Puebla, Mexico,
where poor maintenance of hand washing stations and poor worker hygiene was the source of the
outbreak (CDC 2016). In 2014, the U.S. imported 6,518,000 metric tons of fresh vegetables and
10,870,000 metric tons of fresh fruit. Mexico has been the leading exporter of fresh produce to
the United States since 1995 (USDA ERS 2016). In response to the recent contamination issues
with cilantro, the FDA has advised those who grow, harvest, sort, pack, or distribute cilantro to
be especially vigilant of areas in their supply chain that could be improved to reduce hazards and
improve safety (FDA 2016c).
From 1995-2006, 22 outbreaks have been associated with fresh-cut lettuce or spinach
(Wendel et al. 2009; Cooley et al. 2007; Khalil and Frank 2009). The most notorious multi-state
outbreak involving baby spinach occurred in 2006. Spinach originating from a farm in Salinas,
CA was contaminated with E. coli O157:H7 and sickened 199 people, causing 3 deaths
(Hoffman et al. 2015). This outbreak most likely resulted from fecal contamination from wild
pigs or water runoff from a nearby livestock operation (Edward et al. 2015). From 2002-2009,
the FDA randomly tested 4433 spinach samples for the presence of enterohemorragic E. coli
(EHEC), Shiga toxin-producing E. coli (STEC), and Salmonella and found 0.74% to be positive
for the presence of one of these pathogens, which was the highest positive rate of the
commodities tested (FDA 2015a). Random sampling of baby spinach from farms in Spain found
5.2% (n=38) of collected samples to be contaminated with Salmonella following harvest (GarciaVillanova Ruiz et al. 1987). Spinach originating from the Netherlands caused a widespread
Salmonella outbreak across Europe in 2007 with 354 reported cases (Eurosurveillance 2005).

13

Domestically, Salmonella-contaminated spinach was implicated in one multi-state outbreak in
the United States (CDC 2016). More recently in 2011 and 2015, flat leaf spinach originating
from suppliers in California was recalled due to suspected Salmonella contamination after
random sampling resulted in a Salmonella-positive sample (US Foods 2016).
1.5 Previous cross contamination studies on pathogen transfer and redistribution during
processing
Numerous studies have examined pathogen cross contamination of fresh produce and
redistribution to previously uncontaminated produce during subsequent washing, slicing and
dicing. In order to provide vital information for future risk assessments, washing inoculated
produce in sanitizer-free water has been studied to generate baseline data. These studies have
generally found that a localized amount of contaminated produce can spread to contaminate a
larger amount of produce, regardless of initial amount of contamination. However, the results
from these studies are impacted by many variables including water quality, sanitizer
concentration and contact time, decontamination treatment(s), population and physiological state
of target microorganism(s), method of detection, type of produce and surface characteristics, and
the time interval between initial contamination and washing (Gil et al. 2009). Together, these
factors account for the similarities and differences found in past studies looking into pathogen
cross contamination and different sanitizing practices.
A pilot-scale study conducted by Buchholz and others (2012) examined the amount of
cross contamination that would occur during simulated commercial washing of romaine and
iceberg lettuce in sanitizer-free wash water. Dip-inoculated lettuce (22.7 kg) containing 106, 104,
or 102 CFU/g E. coli O157:H7 was washed for 90 s. After washing, the water used was drained

14

from the system and the inoculated lettuce was removed from the process line. Next, 90.8 kg of
uninoculated lettuce was washed for 90s in the same manner as the inoculated lettuce. Results
indicated cross contamination occurred throughout the previously uninoculated 90.8 kg batch,
regardless of inoculation level. Following washing, uninoculated lettuce had E. coli O157:H7
counts of 2.9, 0.7, and -1.1 log CFU/g for the 106, 104, and 102 CFU/g inoculation levels,
respectively. Water samples collected after washing the inoculated lettuce had 1.9, 2.2, 0.3 log
CFU/mL at the 106, 103 and 102 inoculation levels, respectively. Water samples collected after
washing the uninoculated lettuce, had 0.9, 0.0, and 1.8 log CFU/mL E. coli O157:H7 at the 106,
103 and 102 inoculation levels, respectively. The cell counts in water samples after processing
both the inoculated and uninoculated lettuce demonstrate water can be a vehicle of cross
contamination well after initial localized contamination. The inoculated lettuce transferred E.
coli O157:H7 to all major equipment surfaces (shredder, conveyor, flume tank, dewatering
shaker, and centrifuge) and the wash water. Highest levels of cross contamination occurred
during shredding and conveying, regardless of inoculation level. This study demonstrated water
and equipment contact surfaces can contribute to significant pathogen cross contamination
during pilot-scale processing of lettuce.
Radicchio was used as a colored surrogate to track the spread of E. coli O157:H7
contaminated product to iceberg lettuce in a pilot-scale processing line consisting of a
mechanical shredder, conveyor, flume tank, dewatering shaker, and centrifugal dryer.
Uninoculated iceberg lettuce was processed, followed by 9.1 kg of dip-inoculated surrogate, and
then 90.7 kg uninoculated iceberg lettuce. Flume water was drained from the system following
surrogate washing and refilled prior to processing the uninoculated iceberg lettuce. Mean E. coli
O157:H7 populations on radicchio following processing were 5.0 ± 0.4 log CFU/g, showing

15

there was ~1 unit log reduction of E. coli O157:H7 from radicchio leaves. Iceberg lettuce had E.
coli O157:H7 populations ranging from -0.1 to 3.0 log CFU/g with an average of 1.2 log CFU/g.
Processing equipment had statistically similar E. coli O157:H7 populations, with populations of
2.4 ± 0.4 log CFU/100 cm2 on the mechanical shredder, 3.2 ± 0.7 log CFU/100 cm2 on the
conveyor belt, 2.3 ± 0.5 log CFU/100 cm2 on the flume tank, and 1.9 ± 0.4 log CFU/100 cm2 on
the dewatering shaker. Populations in the flume water samples ranged from 1.6 log CFU/mL to 2.0 log CFU/mL (limit of detection). The authors noted that the radicchio, as could be seen on
the equipment surfaces and comingled with iceberg lettuce, visually demonstrated the spread of
contaminated product during processing, further emphasizing the risk of cross contamination
during leafy green processing. This study demonstrates the spread of E. coli O157:H7
contamination during sanitizer-free washing of leafy greens.
A previous pilot-plant scale study performed by Davidson and others (2013) examined
the efficacy of five different commercial chemical sanitizers when 5.4 kg of iceberg lettuce
inoculated with 106 CFU/g E. coli O157:H7 was washed for 90 s. Wash treatments included
water alone (control), 50 ppm peroxyacetic acid, 50 ppm of mixed peracid, 50 ppm available
chlorine alone or treated with citric acid acidified to pH 6.5 or treated with T-128 (an acidifier).
Results showed that no sanitizer was significantly more effective than water alone (P > 0.05) at
reducing E. coli O157:H7 populations on the iceberg lettuce (Davidson et al. 2013). Using
peracid, E. coli O157:H7 populations were reduced the by 1.4 log CFU/g however this reduction
was not significantly different than using water alone, which reduced populations by 0.75 log
CFU/g (Davidson et al. 2013). All five sanitizers significantly ( P< 0.05) reduced E. coli
O157:H7 populations on the flume tank and shaker table compared to using water alone. Nine
water samples (50 mL each) were also taken in 10 s intervals during the 90 s washing cycle to

16

assess the spread of E. coli O157:H7 in wash water when different sanitizers were used. The
chlorine alone, chlorine plus citric acid, and chlorine plus T-128 were generally more effective
than the other two sanitizer treatments in reducing E. coli O157:H7 populations from wash water
with reductions of 3.79, 5.47, and 5.37 log CFU/mL, respectively (Davidson et al. 2013). The
authors noted that the organic load in the study was lower than what may be seen in industry and
the higher organic load in commercial processing conditions would further reduce the
effectiveness of commercial sanitizers (Davidson et al. 2013). Therefore, proper maintenance of
wash water and consistent replenishment of sanitizer concentrations in wash water is essential to
mitigate cross contamination.
The change in the free chlorine concentration of wash water prepared both with and
without an acidulant (T128), was examined by Luo and others (2012). Baby spinach was
inoculated with E. coli O157:H7 at 2 x 105 CFU/g and washed with large amounts of
uninoculated iceberg lettuce in the presence of a chlorine-based sanitizer. At free chlorine
concentrations < 1 mg/L, E. coli O157:H7 was recovered from the wash water and uninoculated
iceberg lettuce. However when free chlorine concentration was > 1 mg/L in the presence of
T128, cross contamination to uninoculated iceberg lettuce was reduced. The researchers
recommended the importance of consistent monitoring of wash water quality and concentration
of free chlorine as ways to mitigate cross contamination during washing (Luo et al. 2012).
In other work, iceberg lettuce was inoculated with E. coli O157:H7 at 106 CFU/g and
washed in either sanitizer-free wash water, water containing sodium hypochlorite, or water
containing chlorine dioxide as the sanitizing agent (Lopez-Galvez et al. 2009). When inoculated
iceberg lettuce was washed prior to uninoculated iceberg lettuce, cross contamination occurred to
the uninoculated lettuce. In the sanitizer-free trial, the uninoculated lettuce had E.coli O157:H7

17

populations up to 3.4 log CFU/g after washing and the inoculated lettuce experienced a 0.5 +/0.1 log reduction. When sanitizer was used, similar increased E. coli O157:H7 reductions were
seen for both inoculated and uninoculated lettuce compared to the sanitizer-free trials. The
sanitizers were effective in mitigating E. coli O157:H7 presence in the water as no E. coli
O157:H7 was detected (Lopez-Galvez et al. 2009). This finding suggests that sanitizers are
effective in mitigating cross contamination that may occur through wash water (Lopez-Galvez et
al. 2009).
In another study, Nou and Luo (2010) examined the extent of cross contamination
occurring when romaine lettuce was sanitized before versus after mechanical shredding. When
romaine lettuce leaves were spot inoculated with 6.3 CFU/g of E. coli O157:H7 and washed for
60 s in water containing 70 ppm available chlorine, lettuce sliced prior to washing experienced a
1.1 log decrease in E.coli O157:H7 populations. However, romaine lettuce sliced after washing
experienced a 1.9 log decrease in E.coli O157:H7 (Nou and Luo 2010). When lettuce was
washed before slicing, E.coli O157:H7 populations were 0.6-1.3 log lower compared to that seen
when lettuce was sliced prior to washing (Nou and Luo 2010). The same study also evaluated
the extent of cross contamination occurring when radicchio was inoculated with 3.5×10³ CFU/g
E. coli O157:H7 and washed with uninoculated romaine lettuce in chlorinated water for ~90 s.
Cross contamination decreased when lettuce was washed before slicing, as 1 out of 12 red leaf
samples and 2 out of 60 romaine lettuce samples tested positive for E. coli O157:H7 (Nou and
Luo 2010). When lettuce was washed after slicing, 12 out of 121 red leaf lettuce samples and 58
out of 60 romaine lettuce samples tested positive for E. coli O157:H7 (Nou and Luo 2012). The
increased log reductions achieved when lettuce was washed before shredding in both
assessments indicates the importance of adequate washing procedures prior to further leafy green

18

processing. Similar results were found in a study examining the same order of washing/slicing
during post-harvest processing of iceberg lettuce as the Nou and Luo (2010) study. A 0.79-0.80
log CFU/g improvement in reduction of E. coli O157:H7 was found when artificially inoculated
iceberg lettuce was washed before cutting as opposed to washed following cutting (PalmaSalgado et al. 2014).
The quantification and spread of Listeria monocytogenes to previously uncontaminated
product during mechanical dicing of celery and growth during storage was assessed by Kaminski
and others (2014). To quantify L. monocytogenes transfer during mechanical dicing of celery,
275 g of inoculated celery containing 5.6 log CFU/g Listeria monocytogenes was diced prior to
15 batches of 250 g uninoculated celery (Kaminski et al. 2014). The first (out of 15)
uninoculated celery batch diced after the inoculated batch had similar L. monocytogenes levels as
the contaminated celery of 5.2 +/- 0.1 log CFU/g. Batches two and three had significantly lower
populations than batch one, with batches 8-15 containing about 2.0 log CFU/g of Listeria. Also
assessed in this study was the spread of L. monocytogenes-contaminated celery by weight during
mechanical dicing using Swiss chard as the colored surrogate. Swiss chard was dip-inoculated to
achieve 6.8 ± 0.4 log CFU/g. After initial dicing, the populations of L. monocytogenes on Swiss
chard remained relatively unchanged at 6.6 ± 0.7 log CFU/g. Diced pieces of Swiss chard were
allowed to comingle with diced pieces from the 15 batches of subsequently diced uninoculated
celery. Swiss chard was removed from all diced uninoculated celery pieces. L. monocytogenes
was found throughout the 15 batches of uninoculated celery with batches one and two having the
highest counts of 4.7±0.3 log CFU/g and 3.9±0.2 log CFU/g, respectively, and the remaining
batches having significantly lower L. monocytogenes counts of 1.7 log CFU/g. The amount of
contaminated product spread during mechanical dicing was measured by analyzing the weight of

19

contaminated surrogate found in diced uninoculated celery batches. Dicing of 250 g batches of
celery and the Swiss chard surrogate was performed, followed by 15 250 g batches of
uninoculated celery. All visible pieces of Swiss chard were removed from each batch of celery
and weighed. Overall, 25.0% (62.5 g) of Swiss chard was retained in the first batch of diced
celery, which was significantly more than what was retrieved from the remaining batches of
celery, which contained an average of 0.25% (0.6 g) of the surrogate in batches 2 through 15.
The weight amount of surrogate remaining on equipment was 4.33% (10.8 g). Changes in
populations of L. monocytogenes were analyzed based on storage temperature of diced celery (4,
7, and 10°C) over a 7 day period. For the storage study, inoculated celery with 3.2 ± 0.3 log
CFU/g L. monocytogenes was diced and stored at 4, 7, or 10°C. During storage, L.
monocytogenes populations reached maximum populations of 3.3 ± 0.6, 3.8 ± 0.3, and 5.2 ± 0.7
log CFU/g for the 4, 7, and 10°C storage temperatures, respectively. This study demonstrates the
ability for pathogens to cross contaminate to previously uncontaminated product during
mechanical dicing, especially when produce is temperature abused. Therefore, it is imperative
proper mitigation efforts are used during mechanical dicing of fresh produce and produce is held
at proper refrigeration temperatures in order to best prevent potential foodborne outbreaks.
Spanish yellow onions were diced at the pilot scale level to assess cross contamination of
Listeria monocytogenes (Scollon 2014). Peeled onions (2.2 kg) were inoculated with L.
monocytogenes at 5.9 or 4.2 log CFU/50 g to represent high and low inoculation levels and were
diced using a pilot scale dicer followed by 10 batches of uninoculated onions, each weighing 2.2
kg. L. monocytogenes was found on all surfaces of the dicer following the dicing of the
inoculated batch and after dicing of tenth uninoculated batch (Scollon 2014). Samples of
uninoculated onions from the first, fifth, and tenth batches were then analyzed for Listeria

20

presence. At the high inoculation level, 4.6, 3.0, and 2.3 log CFU/50 g was found on
uninoculated onions, respectively. At the low inoculation level, at least one out of three of the
samples collected in triplicate yielded L. monocytogenes (Scollon 2014). The diced onions were
then washed with chlorine sanitizer at 80 ppm free chlorine. After washing with a sanitizer, L.
monocytogenes populations decreased 1.4 log on the onions, which was significantly greater
compared to washing in sanitizer-free water (Scollon 2014). This study demonstrated that if
proper sanitation is not maintained during mechanical dicing of onions, then high volumes of
contamination can spread to previously uncontaminated product, even with a sanitizing wash
step implemented later in the process flow.
1.6 Inoculation methods
Three primary inoculation methods have been used to artificially contaminate produce
samples: dip, spray, and spot. Each method is unique and can provide different information in
regards to pathogen recovery, attachment, and elimination during washing or storage. The
specific inoculation method selected for studies should mimic real world contamination
conditions as closely as possible. These conditions include: location of contamination on the
produce, time of contamination in the production chain (pre-harvest, harvest, post-harvest,
packing), and surface characteristics of the produce studied such as surface area and weight
(Annous et al. 2005; Beuchat et al. 2001; Gil et al. 2009).
Spot inoculation tends to yield more consistent starting populations than dip and spray
inoculation because a known number of CFUs is applied to the sample (Annous et al. 2005; Lang
et al. 2004). This method most closely replicates random fecal contamination from animals or
biological soil amendments (e.g. manure) in the field rather than exposure to contaminated

21

irrigation water, where dip inoculation would be more appropriate (Land et al 2004; Gil at al.
2009).
Dip inoculation involves submerging the sample into a liquid containing the inoculum.
Bacteria are suspended in this liquid, typically water or a buffer solution, with the entire surface
of the sample exposed to the inoculum. This method is used to imitate a larger contamination
event such as during irrigation, hydrocooling, or flume washing (Annous et al. 2005). The dip
method is not as precise as spot inoculation because the amount of inoculum on each piece of
produce varies due to the nature of the application (Lang et al. 2004; Gil et al. 2009).
Spray inoculation is most commonly used to imitate contamination occurring during
spray irrigation (Erickson 2010). This method involves suspending the inoculum in a liquid and
atomizing the cocktail through a spray bottle or similar instrument. The primary disadvantage of
using this method is the inoculum may not reach the sample surface and the delivered spray may
not contain the targeted inoculum (Lang et al. 2004).
1.7 Previous microbial studies on fresh produce and spot inoculation
A study performed by Koseki and others (2003) analyzed the effectiveness of two
sanitizing methods in reducing the pathogen load on spot and dip inoculated lettuce pieces. This
study was conducted in order to assess if there was a correlation between inoculation method and
sanitizer efficacy. Acidic electrolyzed water and 200 ppm free chlorine were used to decrease the
levels of E. coli O157:H7 and Salmonella spp. Ten inner and outer leaf lettuce pieces were either
spot or dip inoculated to contain 7.3-7.8 log CFU/g. Spot inoculating yielded greater log
reductions for the inner as compared to outer leaves, presumably because the cells were able to
penetrate into leaf stomata, decreasing their accessibility to the sanitizer. Dip inoculation resulted

22

in reductions of < 1 log CFU/g compared to the 2.5-2.7 CFU/g log reduction obtained using spot
inoculation (Koseki et al 2003). Sanitizer efficacy was impacted by the inoculation method and
location of inoculum on the leaves; however neither sanitizer was capable of completely
inactivating either pathogen from the lettuce.
Another study assessed the effect of inoculation method and inoculum drying time on
recovery of E. coli O157:H7, Salmonella, and Listeria following washing of lettuce and parsley
(Lang et al. 2004). Produce samples were either dip, spot, or spray inoculated and allowed to dry
using one of two drying methods: 2 h at 22°C (single) or 2 h at 22°C followed by 22 h at 4°C
(dual). Spot inoculation yielded the most consistent recovery rates for lettuce and parsley after
the dual drying method. Based on the consistency of results collected for spot inoculation
method and dual drying time, the authors recommended this method be used to determine the
efficacy of chlorine sanitizers in order to enhance the reproducibility of results.
1.8 Contamination of produce in the field
The most notable sources for pre-harvest contamination of fruits and vegetables include
fecal contaminated irrigation water from domestic or wild animals, water runoff from nearby
livestock operations, ground soil, inadequately composted manure, field workers, and cleanliness
of tools (Sapers and Doyle 2014;Gil et al. 2009).
Outbreaks involving Salmonella, E. coli O157:H7, Giardia, and Cryptosporidium on
fresh produce have been traced back to contaminated irrigation water (Gerba and Rock 2014;
Thurston et al. 2012; Duffey et al. 2005; Materon et al. 2007). Irrigation water contaminated with
E. coli O157:H7 was implicated in two multi-state outbreaks involving lettuce in 1995 and 1996
(Hillborn et al. 1999). Contributing factors to the contamination of irrigation water include feces

23

originating from waterfowl or wild animals, poor storm water drainage, recent rainfall, and
runoff from nearby livestock operation(s) (Gerba and Rock 2014). Contamination of produce
through irrigation is affected by the irrigation method, frequency of irrigation, surface
characteristics of the edible portion of the plant, and the location of the edible portion of the plant
in relation to distance from the soil (Gerba and Rock 2014). Edible produce grown near the soil
is more likely to become contaminated than tree or vine crops (Manshadi et. al 2013). Produce
surface characteristics can also influence the extent of contamination. For example, plants that
can retain water in crevices or leaves are more likely to become contaminated, whereas plants
having a smooth hydrophobic waxy surface are more resistant to water pooling and consequently
less likely to be contaminated (Gerba and Rock 2014).
Another factor that may influence the likelihood of contamination is the irrigation
method. The three methods of irrigation commonly practiced are drip, surface, and overhead
sprinkler irrigation (Brouwer et al. 2016; Gerba and Rock 2014). Irrigation frequency in relation
to time of harvest also becomes an issue if the irrigation water is contaminated due to enhanced
pathogen survival. One study conducted by Soloman and others (2003) found that lettuce
sprayed either once or intermittently with irrigation water inoculated with E.coli O157:H7 at 104
CFU/mL or 102 CFU/mL resulted in contaminated produce, regardless of inoculation level or
irrigation frequency (2003). The only time E. coli O157:H7 contamination was not seen at
harvest was when the lettuce was irrigated once with water containing 102 CFU/mL at the start of
the 30-day trial (Soloman et al. 2003). The 30-day time period between initial irrigation in
combination with the low contamination level was long enough for the pathogen to be removed
from the system. This therefore demonstrates that increased time between irrigation and harvest
can reduce the likelihood of contamination if the initial contamination load is low.

24

Animal manure is commonly used as a crop fertilizer, especially since it helps maintain
overall soil quality by recycling nutrients and organic matter (Arthurson et al. 2011). However, if
manure is not treated correctly prior to field application, pathogenic bacteria may become
introduced to the soil and become disseminated throughout the field (Arthurson et al. 2011).
Another threat to field contamination is if improperly treated manure or feces from a nearby
livestock field comes into contact with produce (Millner 2014). Reports have shown that
livestock and wild animal feces may naturally contain between 102 and 105 CFU/g E. coli and
between 102 and 107 CFU/g Salmonella spp. (Himathongkham et al., 1999). Depending on the
animal source, manure can contain between 102 and 107 CFU/g Salmonella spp. (Pell 1997). A
study performed by Soloman and others (2002) found that E. coli O157:H7 from contaminated
manure and irrigation water can internalize into lettuce through the root system and lead to
decreased efficacy of sanitizers in wash water during post-harvest processing. Contamination of
produce can also occur from nearby cattle, swine, poultry, and sheep farms. A study conducted
on a dairy farm in Virginia reported 4.7% (n=531) of fecal, feed, water, and various
environmental samples contained Salmonella isolates (Warnick et al. 2001). Of the isolates,
Salmonella Typhimurium was the serotype found most frequently in studied herds (Warnick et
al. 2001). The data from the aforementioned study may be indicative of the amount of
Salmonella isolates present on other farms, which could be problematic if these farms are located
near produce fields since runoff or dust containing a pathogenic load can be transferred between
areas. The manure of ruminants such as cattle and sheep are considered the main sources of
Salmonella and E. coli O157:H7 (Pell 1997). Studies have shown that Salmonella can survive in
manure for at least 60 days when held at 4°C and 20°C, respectively and for up to 19 days when
held at 37°C (Himathongkam et al. 1999). Other studies have demonstrated Salmonella can

25

persist in soil for over 300 days following high initial loads (~10⁶ CFU/g) of contaminated
manure application (Jones 1986; Baloda et al. 2001; Islam et al. 2004). The FDA has established
guidelines for growers on proper manure management and treatment such as using active or
passive heat treatments for manure, preventing wildlife from getting into livestock housing units
and water troughs, and utilizing GAPs will contribute keeping manure used in crop fertilization
safe (Millner 2014; FDA 1998).
Maintenance of soil conditions is also pertinent to overall safety and quality of produce.
However, the same conditions required to grow robust crops are also prime for pathogen survival
and persistence. In terms of soil, water permeability, nutrient content, and the level of native
microflora present can all influence the proliferation of pathogens (Millner 2014). An important
factor is the amount of nitrogen present in the soil, as the application of fertilizers can enhance
the survival of pathogens, such as E. coli O157:H7 (Gagliardi and Karns 2000). A risk
assessment study detected Salmonella in soil samples from produce-growing regions in
California and New York State at 2.0% and 2.6%, respectively (Strawn et al. 2013). The same
study also found that farm management and irrigation practices influence the presence of
pathogens. In produce-growing regions of New York State, 6.1% and 17.5% of fields (n=263)
and 11% and 30% of water samples (n=74) were positive for Salmonella and L. monocytogenes,
respectively. A majority of the water samples positive for pathogens were from non-irrigation
surface water such as nearby ponds, rivers, and ditches (Strawn et al. 2013). The study also
reported that the odds of a Salmonella-positive field increased when manure application had
been used within 12 months and the odds of L. monocytogenes-positive fields increased when
irrigation and wildlife presence both occurred within 3 days of each other (Strawn et al. 2013).

26

Other pre-harvest sources of pathogen contamination include insects, dust, pesticides,
field workers, and equipment (Sapers and Doyle 2014). Contaminated equipment may transfer
bacteria to produce at the time of harvest (Patel et al. 2011). Any bins used to transport fresh
produce following harvest must also be properly washed and sanitized. Pesticides are capable of
transferring human pathogens if the water used to prepare the pesticide was previously
contaminated (Gerba and Rock 2014). Salmonella was isolated from pesticide spray prepared
from contaminated irrigation water in Japan (Izumi et al. 2008). Field workers can easily transfer
human pathogens to produce if proper sanitation practices are not followed such as ready access
to hand washing and bathroom facilities. Outbreaks of Cyclospora were caused by field
contamination of cilantro in 2012, 2013, 2014, and 2015 due to poor maintenance of bathroom
and handwashing facilities for field workers in Mexico (FDA 2016c).
Insects, flies and rodents are capable of transferring human pathogens to produce in the
field (Leliveld and Holah 2014). A case study of filth flies near leafy green growing regions was
performed in the Salinas and Imperial Valleys of California. Reports found that 90% of the filth
flies collected in the Imperial Valley and < 1% of filth flies in the Salinas Valley were positive
for E. coli O157:H7 (Wayadande and Talley 2010). Also collected in the same study were
spinach leaves where flies had left regurgitation spots. Spinach leaves were analyzed twice, one
day and one week after the flies had landed on the leaf surface. One day after landing, E. coli
O157:H7 populations were extremely low however evidence of bacterial replication on the
surfaces was evident after one week (Wayadande and Talley 2010). The researchers concluded
that flies are capable of transmitting E. coli O157:H7 due to the pathogen’s ability to survive in
the gut of flies and colonize the spinach phyllosphere after fly regurgitation (Wayadande and
Talley 2010). In another study by Pace (2013), house and blow flies artificially inoculated with

27

E. coli O157:H7 or Salmonella Enterica reportedly contaminated all analyzed lettuce samples
after 10 and 30 seconds of fly contact to the plant surface. The findings from the aforementioned
studies indicate that flies are potential sources of contamination to produce in the field.
1.8.1 Harvesting of baby spinach and cilantro. During harvest, both cilantro and baby
spinach are mechanically cut from the root of the plant (Smith et al. 2016; FDA 2013; Gunes and
Dogu 2010). The blades of the mechanical harvester can serve as a source of contamination
(FDA 2013). Opportunity for contamination occurs due to the close proximity the harvester
blades are to the soil since soil and contaminated plant material can cross-contaminate the
harvester blades (Patel et al. 2011; FDA 2013). Bacteria that become attached to the blades can
form biofilms utilizing exudates released from cut spinach, or other produce (Patel 2013 et al.).
Once formed, biofilms harbor bacteria that are protected from chemical sanitizers (Patel et al.
2011). When harvester blades were inoculated with E. coli O157:H7 at 1 log CFU/blade and held
at dynamic temperatures of 30 and 20oC to mimic day and night temperatures in the field
respectively, populations of attached bacteria increased to 6.09 log CFU/blade after 24 h (Patel et
al. 2011). This highlights the ability of E. coli O157:H7 and other enteric pathogens to persist on
machinery in the field.
For large commercial scale harvesting, cilantro is mechanically cut either directly below
the soil surface or 1.5 - 2 inches above the crown of the plant (Smith et al. 2016). In smaller
operations, cilantro is manually cut using the same measurements (Smith et al. 2016). Once cut,
cilantro is either placed into bins and taken for further processing or bundled into small bunches
and packed into 10 lb. boxes in the field (Smith et al. 2016). Once harvested and placed into bins
or boxes, cilantro is taken to a holding area where it is cooled using direct application of ice or
cool air to remove field heat prior to distribution (Smith et al. 2016; FDA 2013).

28

At the time of harvest, spinach leaves are mechanically cut 1 inch above the soil using a
lawn mower-type machine and then collected into bins or totes in the field (Gunes and Dogu
2010). Although effective for harvesting, this machine can introduce microorganisms and
perhaps pathogens into the product due to contact between the machine and soil, especially if
manure is present in the soil (Buchholz et al. 2012). In order to prevent moisture loss and wilting
from occurring, most produce is harvested in the morning when the air temperature is cooler
(Gunes and Dogu 2010). To prevent leaves from becoming crisp and breaking, it is also good
practice to not harvest soon after a heavy rainfall. Once spinach leaves have been removed from
the root, it is collected into bins and taken for further processing.
1.9 Post-harvest processing
Post-harvest operations also pose a risk of cross contamination. Washing and fluming are
key post-harvest processing steps that can spread contaminants, even if initial levels are low
(Farrar and Guzewich 2014; Buchholz et al. 2012; Buchholz et al. 2014; Gil et al 2009; Luo et
al. 2012). Proper maintenance and monitoring of the water quality is vital to reducing the risk of
pathogens and cross contamination.
Produce will come in contact with various equipment surfaces during post-harvest
processing. The most commonly used food contact surface in the industry is stainless steel
because it is nonporous, easy to clean, and robust (Fellows 2009; Holah and Thorpe1990). Other
equipment surfaces may include conveyor belt materials made from different plastic polymers
(Buchholz et al. 2011). However, pathogens have the ability to attach to the surface of stainless
steel and other equipment surfaces and form biofilms. Biofilms represent an accumulation of
surface-attached microorganisms within a matrix of exopolymers that affords protection from the

29

external environment and allows for their survival (Costerton et al. 1999). A related study found
that Salmonella Enteritidis survived on a stainless steel surface for 4 days when the initial
contamination level was 107 CFU/100 cm2, for 24 h when initial contamination was 105
CFU/100 cm2, and for 1 h when initial contamination was 103 CFU/100 cm2 (Kusumanigrum
2003). These results highlight the importance of frequent equipment surface monitoring using
swab analysis, or other method, in order to monitor the occurrence of pathogenic loads and
eliminate them immediately. Studies dedicated to the mitigation of biofilms on equipment and
food contact surfaces strongly emphasized the importance of proper sanitation techniques.
1.9.1 Shredding and cutting. Shredding is a value added processing step in the industry
because it is not mandatory and primarily done for consumer convenience (Buchholz et al.
2011). An example of a value added product is bagged, shredded lettuce because the product is
ready to use by the consumer with no further preparation steps needed. During mechanical
shredding of leafy greens, a high speed, rotating blade reduces the product to small, consistently
sized pieces. The mechanical act of shredding breaks the natural protective surface of produce,
releases nutrient rich exudates, and increases the cut surface area exposed to the environment
(Brackett 1994; Allende et al. 2009). Under these conditions, sanitizer efficacy is significantly
reduced and the likelihood of pathogen transfer and cross contamination increases (Seo and
Frank 1999; Khalil and Frank 2010).
A study tracking the transfer of E. coli O157:H7 through a pilot-scale leafy green
processing line found the greatest amount of E. coli O157:H7 transfer occurred from inoculated
lettuce to the mechanical shredder (Buchholz et al. 2012; 2014). Poor cleaning and sanitizing of
the shredding equipment was implicated as the source of a Salmonella Bovismorbifcas phage
type 32 outbreak involving lettuce (Stafford et al. 2002). Fresh-cut cilantro leaves were also

30

found to be more susceptible to microbial contamination than whole cilantro leaves (Wang et al.
2004; Allende et al. 2009). In another study, researchers found that Salmonella Chester was more
difficult to eliminate from cut surfaces of green peppers (Liao and Cooke 2001; Allende et al
2009).
If proper holding temperatures are maintained following shredding, the quality and safety
of produce can be maintained. Some studies have shown that certain pathogens, such as E.coli
O157:H7 present on produce will not grow if held at a low temperature (< 4°C); however other
pathogens, such as L. monocytogenes are still capable of growing (Herdt and Fang. 2009;
Kaminski et al. 2013). For example, one study found a decline in E. coli O157:H7 populations on
shredded iceberg lettuce held at 5°C, but a 2 log CFU/g increase occurred when lettuce was held
at 15oC for 7 days, and a 3 log CFU/g increase after 14 days (Li et al. 2001). Another study
found a plateau in E. coli O157:H7 growth on cut iceberg lettuce when held at 4°C and 5°C, but
recorded a 1 log CFU/g increase after 2 h with a holding temperature of 10°C (Francis and
O’Beirne 2001).
1.9.2 Conveying. Various mechanical conveyor systems are used for transporting fruits
and vegetables during post-harvest processing. Two common types of conveyor belt designs are
interlocking and continuous (Buchholz et al. 2011). Common materials used to make conveyor
belts include high-density polyethylene, and polypropylene (Buchholz et al. 2011). Conveyor
belts are highly prone to microbial contamination, proliferation, and sources of cross
contamination (Matthews 2014; Buchholz et al. 2011). Due to less vulnerability to contamination
and ease of cleaning, continuous belts are most commonly used in the industry (Buchholz et al.
2011).

31

Studies have been performed to assess cross contamination when produce comes into
contact with conveyors. Buchholz and others (2012) quantified the transfer of E. coli O157:H7
from artificially contaminated baby spinach, iceberg and romaine lettuce to equipment during
small scale processing. E. coli O157:H7 transfer was greatest to the shredder and conveyor. The
authors explained that the exudates released from leafy greens after shredding increased the
amount of liquid and organic load present on these two pieces of equipment due to their close
proximity and allowed for the survival of E. coli O157:H7 (Buchholz et al 2012).
In other work, Allen and others (2005) showed that Salmonella Typhimurium can survive
on stainless steel, PVC, and wooden surfaces for up to 28 days at 80% relative humidity and 11
days at 60% relative humidity. Moore and others (2003) reported similar trends of Salmonella
survival on stainless steel equipment when surfaces were initially contaminated with 106
CFU/28mm2. Salmonella Typhimurium transferred to uncontaminated lettuce that had been
placed on the stainless steel surfaces 1-2 h after initial contaminated (Moore et al. 2003).
1.9.3 Flume washing. ‘Fluming’ is a term used to describe the movement of foods by
water in troughs and is advantageous because produce is cleaned while simultaneously being
transported to the next stage of processing (Fellows 2009). Flume washing is widely used in the
fresh produce industry; however other methods of commercial washing include dump tanks,
flatbed and U-bed brush washers, reel washers, and pressure washers (Sapers 2014). The
aforementioned washing operations can be used alone or in combination. Selection of the proper
wash treatment is based on characteristics unique to the particular product such as fragility, size,
shape, the type and levels of contaminants anticipated to be present (pesticide residue, field
debris, spoilage microorganisms), and the extent of decontamination desired (Sapers 2014;
Fellows 2009). An important function of washing fresh produce is to improve overall quality and

32

appearance by removing dirt, field debris, cooling, and minimize physiological changes (Herdt
and Fang 2009). However, the quality of the water needs to be adequately maintained in order to
ensure the washing step remains effective. A study analyzing the efficacy of sanitizers on
shredded lettuce and spinach against E. coli O157:H7 found a negligible log reduction in
pathogens and attested that high organic load and poor microbial quality of the water were the
most significant limiting factors in sanitizer efficacy (Barrera et al. 2012). The researchers also
found that the final microbial quality of spinach was most impacted by the initial microbial
quality of the incoming produce from the field (Barrera et al. 2012).
Although effective in improving cosmetic appearance and removing planktonic bacteria,
washing can be a primary vehicle for the spread of pathogens. Chemical sanitizers are often
added to wash water, however their primary purpose is to maintain the microbial quality of the
water rather than inactivate pathogens that may be present on the product (Gil et al. 2009).
Pathogen persistence on the hydrophobic surface of the plant phylloplane (leaves) limits the
ability for chlorinated sanitizers to eliminate pathogens, increasing the chance of an infectious
dose remaining on the plant at the time of consumption (Beuchat, 1992, Delaquis et al., 1999 and
Heaton and Jones, 2007; Allende et al. 2009). Other process conditions impacting sanitizer
efficacy include the ratio of fresh produce to wash water, physical features of the produce such
as crevices or cracks, timing between initial contamination and washing, contamination load,
concentration of sanitizer used, and contact time between produce and wash water (Gil et al.
2009). The ability of chlorine and other sanitizers to reduce planktonic bacteria and human
pathogens in the wash water significantly reduces the risk of foodborne illness, however due to
the low infectious dose of certain enteric pathogens (i.e. Salmonella, E. coli O157:H7), complete
safety cannot be assured (Sapers 2014). Maximum reductions of generally no more than 2-3 logs

33

have been routinely reported when inoculated produce has been subjected to sanitizer washing
(Gil et al. 2009; Beuchat et al., 2004; Gonzalez et al.,2004; Inatsu et al., 2005; Ukuku et al.,
2005; Allende et al., 2007;Gómez-López et al., 2007; Selma et al., 2008b). Other studies have
reported a consistent 2 log maximum reduction of enteric pathogens under conditions that most
closely mimicked contamination during commercial production (Brackett, 1987; Zhuang et al.,
1995; Beuchat et al., 1998; Garcia et al., 2003). Additional studies have reported a maximum 1-2
log reduction for human foodborne pathogens on inoculated samples that were washed and
subsequently held at refrigeration temperatures (Sapers et al., 1999; Wisniewsky et al., 2000;
Wright et al., 2000; Lukasik et al., 2003; Caldwell et al., 2003; Nascimento et al., 2003; Beuchat
et al., 2004; Oh et al., 2005; Yuk et al., 2005 and Yuk et al., 2006; Shiron et al., 2009;
Vandekinderen et al., 2009). The variance in log reductions achieved from past studies using
chemical sanitizers in wash water is due to the unique physical characteristics of produce, wash
time, initial pathogen load, and time of contamination in relation to wash time, thus making each
wash operation highly unique for each produce commodity. Washing fresh produce in sanitizerfree water typically reduces the microbial load ~1 log for tomatoes, baby spinach, iceberg
lettuce, and romaine lettuce (Wang and Ryser 2014, Sapers 2006; Buchholz 2012, Chang et al.
2012).
Chlorine-based sanitizers are most commonly used for commercial washing of fresh
produce due their low cost, versatility, and capacity to reduce microbial populations (Fellows
2009; Buchholz 2011; Sapers 2014; Suslow 2000). The efficacy of chlorine-based sanitizers
relies heavily upon water quality and process conditions. The water must have a low organic
load, low turbidity, have an optimal pH range of 6-7, and be at the appropriate temperature for
the produce being washed (Sapers 2014). In the industry, produce is typically washed for 1-2

34

minutes in water containing 50-200 ppm of free chlorine (Sapers 2014). Although highly
effective at removing planktonic bacteria, chlorine sanitizers are less effective against bacteria
that are attached or internalized in produce (Sapers 2014).
Other popular sanitizers used for commercial washing of fresh produce are chlorine
dioxide, ozone, and peroxyacetic acid (Buchholz 2011). While these sanitizers have all proven to
be effective in reducing pathogen cross-contamination from the wash water, reduction of
pathogens on the produce during washing has remained problematic, regardless of the sanitizer
used. Chlorine dioxide and peroxyacetic acid are more expensive than chlorine but have a greater
oxidation capacity and thus are more resilient to high organic loads in wash water (Buchholz et
al. 2011). Chlorine dioxide can be used at 3 ppm to sanitize fresh produce, provided that the
product receives a potable water rinse thereafter (Sapers 2014; 21 CFR 173.300). Studies
assessing the efficacy of chlorine dioxide have generally shown pathogen reductions on produce
of 1 to 3 logs after treatment with maximum reductions of 3 to 5 logs reported for tomatoes,
peaches, and fresh cut cabbage and carrots (Sy et al. 2005; Sapers 2014). Rodgers and others
(2004) achieved a ~5.6 log reduction for L. monocytogenes and E. coli O157:H7 on inoculated
apples, lettuce, strawberries, and cantaloupe (2004). Although these reductions are high, the
lengthy exposure times used remain problematic for the industry.
Ozone is effective against a broad range of microorganisms in wash and flume water and
can be used at low concentrations. However, its ability to reduce bacteria and pathogens on
produce is again dependent on the location of the organism. Ozone was ineffective in reducing E.
coli O157:H7 and L. monocytogenes on inoculated alfalfa seeds and the stem and calyx areas of
apples due to internalization of the pathogens (Sapers 2014). According to Rodgers and others
(2004), ozone (3 ppm) and chlorine dioxide were equally effective in achieving a ~5.6 log

35

reduction of E. coli O157:H7 and L. monocytogenes on dip-inoculated fresh produce however,
these treatment conditions would not be practical in a commercial setting.
1.9.4 Dewatering and drying. In order to maintain produce quality and shelf life, drying
operations are used to remove excess water from plant surfaces. Excess water can sustain the
growth of microorganisms and stress the plant, both of which can lead to a shorter shelf life and
diminished quality. Dewatering screens are commonly used to remove water following flume
washing (Fellows 2009). Other types of drying equipment include shaker tables, air blowers, and
centrifugal dryers (Buchholz et al. 2011). Similar to the selection of washing equipment, the
drying method must be tailored to the physical sensitivity and limitations of the specific product.
For example, due to their fragility, tomatoes are dried using both sponge rollers and air blowers
in order to minimize physical damage and prevent skin breakage (Suslow 2004; Buchholz et al.
2011). In contrast, iceberg and romaine lettuce can withstand dewatering and centrifugal dryers.
Centrifugal drying can expel up to 30% of the microbial population during drying making this
process ideal for microbial testing (Buchholz et al. 2012).
1.9.5 Baby spinach. Spinach can be consumed either fresh, frozen, canned, or
dehydrated (Gunes and Dogu 2010). After harvest, spinach is subjected to: pre trimming and
packaging, transportation or storage, trimming, sorting, grading, and washing (Gunes and Dogu
2010). The flume washing step can be performed with floating, immersion, rotary or high
pressure sprays and must be done with the proper sanitizers. Washing spinach is a crucial step
for reducing spoilage and pathogenic microorganisms. Chlorine-based disinfectants are most
commonly used for the washing of spinach (Gunes and Dogu 2010). After washing, spinach is
then further processing, if needed.

36

For fresh and fresh-cut RTE spinach, the minimal processing operations include washing
with a sanitizer, gentle drying, sorting, packaging, and storage (Gunes and Dogu 2010). The
most common forms of minimally processed spinach available on the market are whole leaf and
baby spinach. Physical damage will increase the naturally high respiration rate of spinach and
lead to a shorter shelf-life. Efforts to optimize spinach quality and control respiration include
reducing surface water through dewatering following rinsing, modified atmosphere packaging of
fresh cut spinach, and proper temperature control of packaged spinach during storage and
transportation (Gunes and Dogu 2010). The recommended conditions for holding spinach during
storage and transportation are 0°C with a relative humidity of 95-98% (Gunes and Dogu 2010).
1.9.6 Cilantro. The post-harvest processing steps taken for cilantro vary depending on
the capabilities of the growers, handlers, and packing houses. Cilantro can be packed in the field
or taken to a processing plant or packaging facility (FDA 2013; Smith et al. 2016). Immediately
after arriving at one of these destinations, cilantro must be cooled to maintain quality and shelflife. Cooling is achieved either by direct ice application or hydrocooling. The packing facility has
the option of washing cilantro; however, it is not required (FDA 2013). If washed, approved
sanitizers must be used and the water quality must be well maintained in order to minimize
bacteria and pathogens. Fresh-cut cilantro is most commonly sanitized with chlorine-based
sanitizers (Allende et al. 2009). The water used to make ice for cooling cilantro must meet USEPA standards and must contain an approved disinfectant in order to reduce microbial loads
present (FDA 2013). If cilantro is hydrocooled, the water must also meet US-EPA standards and
contain an approved disinfectant in order to maintain the microbial quality of the water (FDA
2013). Once cooled using the ice or hydro method, cilantro must then be held at 4°C during
storage and distribution (FDA 2013).

37

1.10 Food Safety Modernization Act
The FDA Food Safety Modernization Act (FSMA) was signed into law in 2011 and
refocuses the way in which the FDA responds to foodborne outbreaks. Under FSMA, the FDA
will take a preventative approach to foodborne outbreaks by utilizing scientific data as a
foundation of creating new and updating current regulations (FDA 2016b). A paramount
mandate for the produce industry was the establishment of science based minimum standards for
the growing, harvesting, packing, and holding of produce in order to minimize contamination
that may lead to serious health consequences or death (FDA 2016b). FSMA outlines
specifications for agriculture water quality and testing. The water intended for hand washing,
food contact surfaces, direct contact with produce before and after harvest, and contact with
sprouts is subject to water quality standards and consistent testing. There are also new guidelines
established for the use of raw manure, growing of sprouts and monitoring of domestic and wild
animal activity on farms, as well as new specifications for equipment, tools, buildings, and
sanitation practices (FDA 2016b; Gutierrez-Rodriguez 2015).
1.11 Risk assessment tools
The FDA has developed several risk assessment tools in order to estimate risks associated
with microbial and chemical contaminants in different foods. The data generated from these tools
are being used by the FDA to determine regulations based on scientific evidence. Ultimately,
these risk assessment tools will help make the food supply safer and integrate more preventative
measures. The two risk assessment tools of greatest value to the fresh produce industry are the
Food and Drug Administration’s i-RISK and Quantitative Produce Risk Assessment Model
(QPRAM). FDA i-RISK is a web-based tool used to compare and rank public health risks

38

associated with contaminants and different food combinations (FDA 2015c). The FDA uses the
data generated from this tool to prioritize high risk foods and allocate necessary funds to reduce
the associated risk (FDA 2015c). FDA i-RISK can also be used to assess the efficacy of various
preventative measures throughout the farm to fork continuum to mitigate risks (FDA 2015c).
This tool can be used to determine the risk associated with one hazard in multiple foods, multiple
hazards in one food, and multiple hazards in multiple foods. QPRAM, a risk assessment tool
used specifically for the fresh produce industry, predicts and characterizes risk associated with
the handling of fresh produce on the farm, during processing and at the point of consumption
(FDA 2015c). The data gathered from this tool can be used to predict and prevent future
outbreaks by determining places in the farm to fork continuum that are at highest risk for
contamination (FDA 2015c). Once these locations are identified, the proper preventative actions
can be implemented accordingly.
1.12 Overall goals
Due to the high numbers of recalls and outbreaks traced back to fresh produce, including
leafy greens and culinary herbs, the microbial safety of these products has come into question.
Large data gaps currently exist in the ability to accurately characterize produce crosscontamination during processing. A principal step in produce processing is washing, which has
the potential to either reduce or concurrently spread contamination to previously uncontaminated
produce; however the extent to which this may occur is not well understood.
The primary goals of the present cross contamination study were 1.) to quantify
Salmonella Typhimurium transfer and redistribution when realistically low initial inoculation
levels and weight ratios of inoculated surrogate were introduced to uninoculated baby spinach

39

and cilantro during post-harvest processing, in sanitizer-free wash water and 2.) assess the
efficacy of a chlorine-based sanitizer (60 ppm, acidified to pH 6.5) when the highest inoculation
level 103 CFU/g and 1:100, 5:100, and 10:100 weight ratios of inoculated surrogate to
uninoculated baby spinach and cilantro are used. Information gathered from this study will
support related cross contamination studies and help to fill data needs for future FDA risk
assessment tools.

40

CHAPTER 2:
SALMONELLA TYPHIMURIUM LT2 TRANSFER AND REDISTRIBUTION ON BABY
SPINACH AND CILANTRO DURING PILOT-SCALE PROCESSING

41

2.1 Introduction
Among food categories, leafy green vegetables and fresh herbs present the greatest
concern for microbiological hazards (FAO and WHO 2008). Between 2004 and 2013,
consumption of fresh produce led to over 643 outbreaks and sickened 20,456 individuals (CSPI
2015). These numbers surpassed all other outbreaks and illnesses occurring during this same
time period, including commodities such as meat, poultry and seafood.
Produce can become contaminated at any point in the farm to fork continuum. Specific
pre-harvest areas of major concern identified by the Food and Drug Administration are
agricultural water, soil amendments (i.e. manure), water runoff from nearby livestock and animal
operations, wild and domestic animal fecal contamination, insects, poor field worker health and
hygiene, and cleanliness of harvesting equipment, tools and buildings (FDA 2013). Poor field
worker hygiene and fecal contamination from wild animals and nearby livestock operations were
singled out as possible sources of cilantro and spinach outbreaks, respectively (FDA 2016;
Atwill et al. 2015).
Manure and soil are prone to pathogen contamination due to the nutrient rich environment
naturally present in these materials (Millner 2014). Pathogens can easily transfer from
contaminated manure or soil to produce through direct contact, irrigation water, and harvesting
techniques (Millner et al. 2014). Studies assessing the prevalence of pathogens in manure and
feces found Salmonella spp. in 17/287 sheep, 273/4977 cattle, 44/600 swine, and 12/67 poultry,
samples taken on farms where manure originated (Fedorka-Cray et al. 1998; Pao et al. 2005;
Callaway et al. 2010; Hutchison et al. 2004). Manure can contain initial loads of Salmonella spp.
between 102 and 107 CFU/g (Pell 1997). A risk assessment study reported that 2.0% and 2.6% of
soil samples were positive for Salmonella spp. from produce-growing regions of California and

42

New York State, respectively (Strawn et al. 2013). Fecal contamination is further complicated
by the ability of enteric pathogens (i.e. Salmonella) to attach, internalize, and form biofilms on
cilantro and spinach (Berger et al. 2009; Brandl and Mandrel 2002). The FDA advises that
growers take proper precautions when treating manure prior to field application and to also be
aware of contamination sources beyond the field including cattle and poultry operations. If these
guidelines are not followed, likelihood of produce field contamination increases significantly
(FDA 2016).
Surveys conducted by the FDA found 9% (n=177) of imported cilantro positive for
Salmonella or Shigella and 1.2% (n=85) of domestic cilantro positive for Salmonella (FDA
2003). As a follow up to this study, 3.3% of imported samples (n=33) were Salmonella-positive
(FDA 2003). From 2002-2009 the FDA randomly tested 2510 cilantro samples for the presence
of enterohemorragic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), and Salmonella and
found 0.64% (n=16) to be positive for the presence of one of these pathogens (FDA 2015a). Of
the commodities tested, the 0.64% positive rate for cilantro was the second highest, with spinach
producing the highest positive rate with 0.74% (n= 4433). Specific to cilantro, Salmonella has
caused 8 multi-state outbreaks in the past 20 years and two Class I recalls in 2011 and 2012 after
random FDA and USDA samples were Salmonella-positive (CDC 2016; US Foods 2017).
Random sampling of baby spinach from farms in Spain found 5.2% (n=38) of collected samples
to be contaminated with Salmonella following harvest (Garcia-Villanova Ruiz et al. 1987). In
2005, Salmonella Typhimurium contaminated spinach caused 60 reported illnesses in Finland
(Eurosurveillance 2005). In 2007, Salmonella java contaminated spinach caused 354 illnesses
across Europe in 2007 (Denny et al. 2007). Domestically, spinach has been linked to one multi-

43

state Salmonella outbreak and two Class I recalls in 2011 and 2015 (CDC 2016). Both recalls
were due to a random field sample of spinach testing positive for Salmonella.
Previous pilot-scale processing studies in our laboratory have shown that a surrogate
inoculated with 106, 104, and 102 CFU/g E. coli O157:H7 can contaminate large quantities of
uncontaminated iceberg lettuce as well as various equipment surfaces including a shredder,
conveyor, flume tank, shaker table, and dewatering centrifuge during washing in sanitizer-free
water (Buchholz et al. 2012; Buchholz et al. 2014. This data demonstrated that even small,
localized areas of contamination can spread to contaminate larger quantities of produce and
equipment. However, the initial inoculation levels and weight ratios of inoculated to
uninoculated product used in the aforementioned study as well as other cross contamination
studies were worst case, highly localized levels of contamination. As highly localized
contamination becomes disseminated over a wider area, subsequently lower numbers of bacteria
can contaminate irrigation water, equipment, or other areas of the field (Beuchat et al. 2010;
Ibekwe et al. 2004). In order to more accurately mimic pathogen redistribution in the field, low
initial inoculation levels were selected for this study (103, 101, and 10-1 CFU/g).
The objectives of the present cross contamination study were to 1.) quantify Salmonella
Typhimurium transfer and redistribution when realistically low initial inoculation levels and
weight ratios of inoculated surrogate were introduced to uninoculated baby spinach and cilantro
during post-harvest processing, in sanitizer-free wash water and 2.) assess the efficacy of a
chlorine-based sanitizer (60 ppm, acidified to pH 6.5) when the highest inoculation level 103
CFU/g and 1:100, 5:100, and 10:100 weight ratios of inoculated surrogate to uninoculated baby
spinach and cilantro were used. Information gathered from this study will support related cross
contamination studies and help to fill data needs for future FDA risk assessment tools.

44

2.2 Materials and methods
2.2.1 Overall experimental design. The spread of Salmonella during pilot-scale
processing of baby spinach and cilantro was assessed using four inoculated:uninoculated product
ratios (0.5:100, 1:100, 5:100, 10;100) and three inoculation levels. Red leaf lettuce (103, 101, 10-1
CFU/g) served as the colored surrogate and was spot inoculated 18-24 h before processing to
reach the targeted inoculation level. In each trial, product was flume-washed in sanitizer-free
water for 90 s. In order to better mimic industry practice, 60 ppm available chlorine (acidified to
pH 6.5) was used in wash water only at the 103 CFU/g inoculation level and 1:100, 5:100, and
10:100 weight ratios. All experiments were conducted in triplicate.
2.2.2 Produce. Baby spinach and cilantro were purchased from a local wholesaler (Stan
Setas Produce Co. LLC, Lansing, MI). Red leaf lettuce was purchased from a local retail
supermarket. All produce was stored in a walk in cooler at 4°C and produce was used within 3
days of delivery. Prior to use, any damaged leaves were discarded. When preparing the red leaf
lettuce, only the red portion from the inner leaves was used for inoculation.
2.2.3 Bacterial strain. To ensure worker safety during pilot-scale processing, avirulent
Salmonella Typhimurium LT2 was used (acquired from Dr. Michelle Danyluk University of
Florida, Gainesville), with this strain previously shown to exhibit similar attachment and growth
characteristics to other Salmonella strains implicated in fresh produce outbreaks (Wang and
Ryser 2014). This strain was maintained at -80°C in Trypticase Soy Broth containing 0.6%
(wt/vol) yeast extract (TSBYE; BD, Sparks, MD) and 10% (v/v) glycerol prior to use.
The working culture was prepared by streaking the frozen stock culture onto Trypticase
Soy Agar containing 0.6% (w/v) yeast extract (TSAYE; BD, Sparks, MS) and incubating for 24
h at 37°C. A single isolated colony was then subjected to two successive transfers in 9 Ml (24

45

h/37°C) of TSBYE. Thereafter, the culture containing ~109 CFU/mL was appropriately diluted
in sterile phosphate buffer solution (8.5 g of NaCl per liter, 1.44 g of Na2HPO4 per liter, and 0.24
g of KH2PO4 per liter) for red leaf lettuce inoculation.
2.2.4 Surrogate inoculation. Twenty-four hours before processing, red leaf lettuce was
aseptically cut with a knife into ~5 x 5 cm pieces (~1 g) to obtain the targeted
inoculated:uninoculated weight ratios of 0.5:100, 1:100, 5:100, and 10:100. The appropriate
number of leaves was placed in a biosafety chamber, spot-inoculated at multiple locations with
50 µL of S. Typhimurium LT2 at 103, 101, and 10-1 CFU/g, dried for 15 minutes, transferred to
sterile plastic containers (PLA NatureWorks NE) and held at 4°C for 18-24 h. Following
overnight storage and before processing, a 25 g sample of the inoculated red leaf lettuce was
analyzed by direct plating on XLT4 agar (Neogen Corp., Lansing, MI) to determine the starting
population of S. Typhimurium LT2.
2.2.5 Wash water and sanitizer Sanitizer-free filtered tap water at 10°C was used for
washing at the three inoculation levels and four inoculated:uninoculated produce ratios. In
addition, efficacy of a chlorine based sanitizer (XY-12, Ecolab, St. Paul, MN) (60 ppm of
available chlorine) was also assessed, but only at weight ratios of 1:100, 5:100, and 10:100 at the
highest inoculation level (103 CFU/g). Water containing the sanitizer was acidified to pH 6.5
with citric acid (Sigma-Aldrich, St. Louis, MO) and measured with a pH probe (pHTestr 30,
Oakton, Vernon Hills, IL). The level of available chlorine was measured using a rapid test kit
(Ecolab, St. Paul, MN).
Wash water (before and after processing) and expelled centrifuge water samples were
collected (~50 mL) and direct plated on XLT4 agar to determine Salmonella counts for each
trial. Water samples collected for the sanitizer trials were treated with 0.5% sodium thiosulfate

46

(Fisher Science Education, Hanover, IL) immediately following collection in order to neutralize
the sanitizer.
2.2.6 Leafy green processing line. The pilot-scale processing line consisted of a 3.6 mlong stainless steel flume tank, shaker table, dewatering centrifuge, and a non-refrigerated
recirculation tank (1000 L capacity). The flume tank (Heinzen Manufacturing, Inc., Gilroy, CA)
was equipped with two overhead spray jets. A 4.1-m long, 10 cm diameter hard plastic discharge
hose and centrifugal pump (model XB754FHA, Sterling Electric, Inc., Irvine, CA) circulated the
wash water at 15 L/sec through the flume tank. A stainless steel screen at the end of the flume
tank retained the product for the desired washing time. Immediately beyond the screen was a
stainless steel shaker table screen operated by a 1 HP Baldor wash-down duty motor (Baldor
Electric Co., Ft. Smith, AR) at 1760 RPM which was used for partial dewatering. Water removed
from the shaker table flowed back into the recirculation tank. After shaker table dewatering, the
produce was centrifugally dried using a 22.7 kg capacity Spin Dryer (model SD50-LT, Heinzen
Manufacturing, Inc.) with three internally timed spin cycles totaling 60 s.
2.2.7 Baby spinach and cilantro processing and sample collection. For processing, ~5
kg (± 0.5 kg) of uninoculated baby spinach or cilantro was added to the flume tank containing
~300 L of filtered sanitizer-free tap water (10°C) followed by addition of the required amount
inoculated red leaf lettuce. The combined batch of inoculated and uninoculated product was then
washed for 90 s in the flume tank. After 90 s, the batch was dewatered on the mechanical shaker
for ~15 s and collected in a perforated plastic bin and centrifugally dried for 60 s. After all
previously inoculated red leaf lettuce was manually removed, 25 g of washed and processed red
leaf lettuce was placed in a Whirl-PakTM bag for quantitative and qualitative analysis. The

47

remaining 5 kg of baby spinach or cilantro was collected into ~20-24 Whirl-PakTM bags, each
containing 225 g of produce.

Figure 2.1: Flume tank and dewatering shaker table.

Figure 2.2: Collection bin for washed produce following 90 s of flume washing.

48

Figure 2.3: Centrifugal dryer.

Figure 2.4: Bagged samples containing 225 g baby spinach/cilantro after removal of the
inoculated surrogate.
49

2.2.8 Microbiological analysis. All water and red leaf lettuce samples before and after
processing, and bagged baby spinach and cilantro samples were assessed for Salmonella
Typhimurium LT2 using direct plating or GeneQuence Assay (Neogen Corp., Lansing, MI).
2.2.9 Red leaf lettuce samples. The two 25-g red leaf lettuce samples were collected
before and after processing, placed into individual Whirl-Pak™ bags containing 50 mL of
phosphate buffer solution, homogenized in a stomacher (Stomacher 400 Circulator, Seward,
Worthington, UK) for 2 min at 260 rpm and then plated in duplicate with or without membrane
filtration on XLT4 Agar. Another red leaf lettuce sample collected after processing was assessed
for presence of S. Typhimurium LT2 using the GeneQuence assay analysis.
2.2.10 Water samples. The 50 mL water samples collected before processing, after 90 s
of washing, and during centrifugal drying were subjected to membrane filtration and plated on
XLT4 agar. The plated samples were then incubated for 24 h at 37°C. All colonies resembling
Salmonella were counted.
2.2.11 Bagged baby spinach and cilantro. Each 225 g sample of baby spinach and
cilantro was enriched in 450 mL of sterile Lactose Broth (LB) and incubated for 24 h at 35°C.
Thereafter, 1 mL aliquot and 0.1 mL aliquot of the LB enrichment was transferred to 10 mL of
Tetrathionate (TT) Broth and Rappaport-Vassiliadis (RV) Broth, respectively and incubated for
18 h at 42°C. After incubation, 1 mL aliquots from each enrichment were transferred to 10 mL of
sterile Gram Negative (GN) broth and incubated for 6 h at 35°C. All GN enrichments were then
examined for presence/absence of Salmonella Typhimurium using the GeneQuence assay
(Neogen Corp., Lansing, MI). The red leaf lettuce sample collected after processing was
similarly enriched and examined using the GeneQuence assay.

50

2.2.12 GeneQuence assay. A 200 µL aliquot of GN enrichment from the previously
enriched TT and RV samples was transferred to the same sterile test tube after which 100 µL of
the GeneQuence lysis reagent was added. After 5 minutes of heating in a 65°C water bath, 150
µL of the sample was then transferred to a microwell. Thereafter, the GeneQuence testing
protocol for Salmonella was followed as outlined by the test kit manufacturer, with the results
read at 450 nm on a Stat Fax 4200 plate strip reader. Values > 0.10 were recorded as positive,
whereas readings < 0.09 were recorded as negative for Salmonella.
2.2.13 Statistical analysis. Colony counts obtained from direct plating were used to
determine the percentage of Salmonella CFUs transferred from the red leaf lettuce to the
uninoculated baby spinach or cilantro during processing. GeneQuence results were used to
determine the number of Salmonella-positive samples for each batch of baby spinach and
cilantro processed. Analysis of variance (ANOVA) using JMP 12.2 software (SAS Institute Inc.,
Cary, NC) was used to analyze data collected from the triplicate experiments. When P values
were ≤ 0.05, the Tukey—Kramer HSD test was used to determine statistical significance.
2.3 Results
Both baby spinach and cilantro yielded statistically similar (P > 0.05) percentages of
Salmonella-positive samples. The inoculated:uninoculated weight ratios did not significantly (P
> 0.05) affect the percentage of positive samples (Figures 2.5 and 2.6). The initial inoculation
level significantly (P < 0.05) impacted the number of positive samples with the 10⁻¹ CFU/g
inoculation level yielding significantly fewer Salmonella-positive samples compared to the two
higher inoculation levels, regardless of weight ratio (Figure 2.5 and 2.6). When the flume water
contained 60 ppm available chlorine (acidified to pH 6.5), greater Salmonella reductions were
seen for baby spinach and cilantro compared to washing without a sanitizer (Figures 2.5 and 2.6).

51

10:100

5:100

1:100

0.5:100

120
A

A

A

% Positive Samples

100

A

AB AB

AB

B

B

B

80

60
C
40
D

20

D

D

D
0
10³ CFU/g

10³ CFU/g with 60 ppm
Sanitizer in water

10¹ CFU/g

10⁻¹ CFU/g

Figure 2.5: Percentage of Salmonella-positive baby spinach samples after 90 s of flume washing with and without a chemical
sanitizer. Means with different capital letters are significantly different (P < 0.05).

52

10:100

5:100

1:100

0.5:100

120
A

A

A

A

AB AB

B

AB

B

% Positive Samples

100

80
C

60
CD

40

D
D

20

D

D

0
10³ CFU/g

10³ CFU/g with 60 ppm
Sanitizer in water

10¹ CFU/g

10⁻¹ CFU/g

Figure 2.6: Percentage of Salmonella-positive cilantro samples after 90 s of flume washing with and without a chemical sanitizer.
Means with different capital letters are significantly different (P < 0.05).

53

2.3.1 Baby spinach. As the initial inoculation level decreased, the percentage of positive
baby spinach samples also decreased, regardless of the inoculated:uninoculated product ratio.
Significantly fewer (P < 0.05) positive samples were observed at 10-1 CFU/g compared to the 101
and 103 CFU/g inoculation levels. All samples analyzed at the 103 CFU/g inoculation level were
positive for Salmonella at the four weight ratios tested. At the 101 CFU/g inoculation level:
100% (± 0%), 80.6% (± 14.9%), 84.1% (± 16.8%), 68.1% (± 33.6%) of the samples yielded
Salmonella at the 10:100, 5:100, 1:100, and 0.5:100 inoculated:uninoculated ratios, respectively
(Table 2.1). For the 10-1 CFU/g inoculation level, Salmonella-positive samples were: 11.6% (±
2.1%), 4.3% (± 3.5%), 11.6% (± 10.3%), 0% (± 0%) for the 10:100, 5:100, 1:100, and 0.5:100
inoculated:uninoculated ratios, respectively (Table 2.1). No significant differences were found
between inoculated:uninoculated ratios (P > 0.05) at the same inoculation level. Sanitizer helped
to reduce the percentage of positive samples to comparable numbers collected in the sanitizerfree 10¹ CFU/g inoculation trials for the 10:100 and 5:100 ratios; however this decrease was not
significant (Figure 2.5). When the ratio was reduced to 1:100 during the sanitizer trials; fewer
samples were positive (P < 0.05) compared to the sanitizer free 103 CFU/g trials (Figure 2.5).
Spread of Salmonella was again generally greater at the 103 and 101 CFU/g inoculation levels
compared to the 10-1 CFU/g level at each inoculated:uninoculated product ratio (Figure 2.7).

54

Table 2.1: Numbers and percentage of Salmonella-positive baby spinach samples (23 samples
per trial) at different inoculation levels and inoculated:uninoculated product ratios.

Inoculation
Level
(CFU/g)

103

Sanitizer
103

101

10-1

Inoculated:uninocul
ated Ratio

Avg. Percent
Positive Samples

Positive/Total Samples

Rep 1

Rep 2

Rep 3

Mean (± SD)

10:100

23/23

23/23

23/23

100% (± 0%)

5:100

23/23

23/23

23/23

100% (± 0%)

1:100

23/23

23/23

23/23

100% (± 0%)

0.5:100
10:100

23/23
19/23

23/23
22/23

23/23
22/23

100% (± 0%)
91.3% (± 7.5%)

5:100

23/23

23/23

23/23

100% (± 0%)

1:100

10/23

10/23

8/23

40.6% (± 5.0%)

10:100

23/23

23/23

23/23

100% (± 0%)

5:100

23/23

14/22

18/23

80.6% (± 14.9%)

1:100

21/23

23/23

14/23

84.1% (± 16.8%)

0.5:100

5/23

19/23

23/23

68.1% (± 33.5%)

10:100

3/23

3/23

2/23

11.6% (± 2.1%)

5:100

0/23

2/23

1/23

4.3% (± 3.5%)

1:100

6/23

1/23

1/23

11.6% (± 10.3%)

0.5:100

0/23

0/23

0/23

0% (± 0%)

55

120

120
a.)

A

A
Positive Samples (%)

Positive Samples (%)

A
100
80
60
40

B

20
0

40

20

B

10³

10¯¹
d.)

A

A

100
80
60

40
B
20

0
Baby spinach 1:100
10¹

10¹

120

c.)
A
Positive Samples (%)

Positive Samples (%)

60

10¯¹

120

10³

80

0
Baby spinach 5:100

Baby spinach 10:100
10³ 10¹

A

b.)

A

100

100
80
60

40
20
B

0
Baby spinach 0.5:100
10¯¹

10³

10¹

10¯¹

Figure 2.7: Percentage (+/- SD) of Salmonella-positive baby spinach samples after processing in
sanitizer-free water at inoculated:uninoculated weight ratios of a.) 10:100, b.) 5:100, c.) 1:100,
and d.) 0.5:100. Means with different capital letters are significantly different (P < 0.05).

56

2.3.2 Cilantro. Similar to results obtained in the baby spinach trials, the number of
Salmonella-positive cilantro samples decreased as the initial inoculation level decreased.
Significantly fewer (P < 0.05) Salmonella-positive samples were seen at the10-1 CFU/g
inoculation level compared to the two higher inoculation levels (Figure 2.6). Sanitizer use
decreased the percentage of positive samples to comparable numbers collected at the 101 CFU/g
inoculation level for the 10:100 and 5:100 ratios; however this decrease was not significant (P >
0.05) (Figure 2.6). Similar reductions (P < 0.05) were attained at the 101 CFU/g inoculation level
at the same weight ratio when no sanitizer was used. These values were comparable to those
attained in the baby spinach trials. At 103 CFU/g, all samples tested positive for Salmonella at
each weight ratio. As expected, the number of positive samples decreased as the initial
inoculation level decreased. At the 101 CFU/g inoculation level, 92.1% (± 11.2%), 100% (± 0%),
87.9% (± 17.1%), and 37.1% (± 22.1%) of the samples tested positive for Salmonella at the
10:100, 5:100, 1:100, and 0.5:100 inoculated:uninoculated ratios, respectively. At the 10-1 CFU/g
inoculation level, 10.6% (± 7.7%), 15.3% (± 11.8%), 4.8% (± 6.7%), 4.8% (± 3.9%) of the
samples tested positive for Salmonella at the 10:100, 5:100, 1:100, and 0.5:100
inoculated:uninoculated weight ratios, respectively (Table 2.2). Spread of Salmonella was again
generally greater at the 103 and 101 CFU/g inoculation levels compared to the 10-1 CFU/g level at
each inoculated:uninoculated product ratio (Figure 2.8). However, unlike for baby spinach,
significantly less spread of Salmonella occurred for the 101 and 10-1 CFU/g inoculation levels
and the 0.5:100 inoculated:uninoculated product ratio compared to the 103 CFU/g inoculation
level at the same weight ratio.

57

Table 2.2: Numbers and percentage of Salmonella-positive samples for cilantro samples (~ 22
total samples per trial) with different inoculation levels and product ratios.
Inoculation
Level
(CFU/Leaf)

103

Sanitizer
103

101

10-1

Inoculated:uninoculated
Ratio

Positive/Total Samples

Avg. Percent
Positive Samples

Rep 1

Rep 2

Rep 3

Mean (± SD)

10:100

22/22

22/22

21/21

100% (± 0%)

5:100

22/22

22/22

20/20

100% (± 0%)

1:100

22/22

22/22

21/21

100% (± 0%)

0.5:100
10:100

22/22
21/21

22/22
21/21

21/21
16/21

100% (± 0%)
92.0% (± 13.7%)

5:100

17/20

20/20

20/20

95.0% (± 8.7%)

1:100

7/22

8/21

5/21

31.2% (± 7.2%)

10:100

22/22

22/22

16/21

92.1% (± 11.2%)

5:100

21/21

22/22

21/21

100% (± 0%)

1:100

22/22

21/22

14/22

87.9% (± 17.1%)

0.5:100

14/22

8/21

2/21

37.1% (± 22.1%)

10:100

4/22

0/22

3/22

10.6% (± 7.7%)

5:100

1/20

7/22

2/22

15.3% (± 11.8%)

1:100

3/21

0/21

0/22

4.8% (± 6.7%)

0.5:100

1/21

2/21

0/21

4.8% (± 3.0%)

58

a.)

120
A

A

100

Positive Samples (%)

Positive Samples (%)

A
80
60
40

B

20

Cilantro 10:100
10³

10¹

80
60
40

B

20

Cilantro 5:100
10³

10¯¹
c.)

120

10¹

10¯¹
d.)

120

A

A

A

100

Positive Samples (%)

Positive Samples (%)

A

100

0

0

80
60
40
B

20
0

Cilantro 1:100
10³

b.)

120

100
80
B
60
40
20

B

0
10¹

Cilantro 0.5:100
10³ 10¹

10¯¹

10¯¹

Figure 2.8: Percentage (+/- SD) of Salmonella-positive cilantro samples after processing in
sanitizer-free water at inoculated:uninoculated weight ratios of a.) 10:100, b.) 5:100, c.) 1:100,
d.) 0.5:100. Means with different capital letters are significantly different (P < 0.05).

59

2.3.3 Red leaf lettuce. Salmonella populations on red leaf lettuce decreased after 90 s of
flume washing. In the baby spinach trials, populations decreased an average of 1.5, 1.0, and 1.8
log CFU/g for the 103, 101, and 10-1 CFU/g inoculation levels, respectively. Using the
GeneQuence assay, all red leaf lettuce samples were Salmonella-positive after washing.
Additionally, during the baby spinach trials, red leaf lettuce inoculated with 103 CFU/g
Salmonella experienced population decreases of 3.6 to 1.7, 3.2 to 2.1, 3.2 to 1.4, and 3.3 to 2.3
log CFU/g at the 10:100, 5:100, 1:100, and 0.5:100 weight ratios, respectively. At the 101 CFU/g
inoculation level, Salmonella populations decreased from 1.6 to 0.3, 1.4 to 0.2, 1.5 to 0.4, and
1.4 to 0.3 log CFU/g for the 10:100, 5:100, 1:100, and 0.5:100 weight ratios, respectively. At the
10-1 CFU/g inoculation level, Salmonella populations decreased from -0.37 to below limit of
detection (0.04 CFU/g) , -0.4 log CFU/g to below limit of detection (0.04 CFU/g), -0.17 to -0.04
log CFU/g, and -0.4 to below limit of detection for the 10:100, 5:100, 1:100, and 0.5:100 weight
ratios, respectively (Tables AI.1 and AI.3). At the 103 CFU/g inoculation level, 97.1, 88.5, 94.2,
89.4%, of the Salmonella population was shed from the red leaf lettuce during washing at the
10:100, 5:100, 1:100, and 0.5:100 weight ratios, respectively. At the 101 CFU/g inoculation
level, 94.0, 92.6, 91.6, and 93.4% Salmonella was shed, whereas at the 10-1 CFU/g inoculation
level, 94.8, 100.0, 98.27, and 97.0% was shed at the 10:100, 5:100, 1:100, and 0.5:100 weight
ratios, respectively (Table AI.5).
For the cilantro trials, average Salmonella reductions on red leaf lettuce were 1.4, 1.4,
and 1.6 log CFU/g for the 103, 101, and 10-1 CFU/g inoculation levels, respectively. Based on
GeneQuence analysis, all red leaf lettuce samples were Salmonella-positive after washing. Red
leaf lettuce inoculated at 103 CFU/g experienced Salmonella reductions of 3.4 to 1.7, 3.2 to 1.3,
3.3 to 2.2, and 3.1 to 2.1 log CFU/g at the 10:100, 5:100, 1:100, and 0.5:100 weight ratios,

60

respectively. At the 101 CFU/g inoculation level, Salmonella populations decreased from 1.4 to 0.3 log CFU/g, 1.7 to 0.3 log CFU/g, 1.4 to 0.5 CFU/g, and 1.4 to -0.4 log CFU/g for the 10:100,
5:100, 1:100, and 0.5:100 weight ratios, respectively. At the 10-1 CFU/g inoculation level,
numbers of Salmonella decreased from -0.6 log CFU/g to below limit of detection (0.04 CFU/g),
-0.3 to below limit of detection, 0.17 to below limit of detection, and -0.2 log CFU/g -0.04 log
CFU/g for the 10:100, 5:100, 1:100, and 0.5:100 weight ratios, respectively (Tables AI.2 and
AI.4). The average percentage of the Salmonella population shed from red leaf lettuce during 90s of flume washing for the 103 CFU/g level was 94.8, 96.4, 93.1, and 94.6% for the respective
weight ratios of 10:100, 5:100, 1:100, and 0.5:100. At the 101 CFU/g and 10-1 CFU/g inoculation
levels, 97.9, 95.1, 95.6, 95.6%, and 100.0, 94.4, 82.2, and 84.0% of the Salmonella population
was lost for the weight ratios of 10:100, 5:100, 1:100, and 0.5:100, respectively (Table AI.6).
2.3.4 Wash water. At the time of processing, three 50 mL water samples were collected
before washing (control), after 90 s of washing, and during centrifugal drying. All water samples
taken before processing were negative for Salmonella. Based on the experimental design, a total
of twelve water samples were taken at each inoculation level studied (4 weight ratios conducted
in triplicate =12 trials per level). Overall, 0/12, 1/12, and 0/12 water samples after baby spinach
washing and 0/12, 0/12, and 1/12 water samples after cilantro washing yielded detectable counts
of Salmonella at the 103, 101, and 10-1 CFU/g inoculation levels, respectively. Salmonella
populations detected in the water following 90 s of washing ranged from 1.0 log CFU/100 mL to
below limit of detection (0.3 log CFU/100 mL) for both baby spinach and cilantro. When water
samples were analyzed following centrifugal drying, a total of 1/12, 2/12, 0/12 baby spinach
water samples and 10/12, 3/12, and 1/12 cilantro water samples had detectable counts of
Salmonella at the 103, 101, and 10-1 CFU/g inoculation levels, respectively. Salmonella

61

populations ranged from 0.7 log CFU/mL to 1.0 log CFU/100 mL for baby spinach and 1.0 log
CFU/100 mL to 1.6 log CFU/100 mL for cilantro.
2.3.5 Sanitizer implementation. A chlorine-based sanitizer was added to wash water,
acidified to pH 6.5 with citric acid, and concentrated to have 60 ppm available chlorine before
washing the surrogate inoculated at 103 Salmonella CFU/g with uninoculated baby spinach or
cilantro. Three weight ratios were analyzed: 1:100 5:100, and 10:100.When sanitizer was used,
Salmonella populations decreased by an additional log on red leaf lettuce compared to sanitizerfree trials (Tables 2.3 and 2.4). None of the 50 mL water samples collected yielded Salmonella
by direct plating. Among the three weight ratios studied, the 1:00 ratio produced significantly (P
< 0.05) fewer positive samples compared to the two higher ratios (Figure 2.9). Comparing the
number of bagged samples positive for Salmonella between the sanitizer-free trials and those
where sanitizer was used, significant reductions (P < 0.05) were seen at the 1:100 ratio (Figures
2.5 and 2.6).

62

Baby Spinach

Cilantro

120
A

Positive Samples (%)

100

A

A

A

80

60
B
B

40

20

0
10:100

5:100

1:100

Figure 2.9: Percentage of Salmonella-positive samples after 90 s of flume washing in water
containing 60 ppm available chlorine. Means with different capital letters are significantly
different (P < 0.05).

63

Table 2.3: Comparison of processing trials with and without a chemical sanitizer for baby spinach.
Sanitizer Free
Inoculation Level
Inoculated:uninoculated ratio
% Positive samples
% CFU loss on red leaf lettuce after processing
Average log reduction after processing (log CFU/g)
Salmonella populations in wash water (log CFU/100 mL)
Salmonella populations in centrifugation water (log CFU/100
mL)

60 ppm Sanitizer
10³

10:100

10:100

5:100

100% (± 0%)

91.3% (± 7.5%)

100% (± 0%)

97.1% (± 1.5%)

99.8% (± 0.1%)

91.7% (± 6.7%)

1.7 (± 0.5)
< 0.3

2.7 (± 0.5)
< 0.3

2.4 (± 0.8)
< 0.3

1:100
40.6%
(± 5.0%)
99.3
(± 4.0%)
2.1
(± 0.1)
< 0.3

< 0.3

< 0.3

< 0.3

< 0.3

64

Table 2.4: Comparison of processing trials with and without a chemical sanitizer for cilantro.
Sanitizer Free
Inoculation Level
Inoculated:uninoculated ratio
% Positive samples
% CFU loss on red Leaf lettuce after processing
Average log reduction after processing (log CFU/g)
Salmonella populations in wash water (log CFU/100
mL)
Salmonella populations in centrifugation water (log
CFU/100 mL)

60 ppm Sanitizer

10³
10:100
10:100
5:100
100% (± 0%) 92.1% (± 13.8%) 95.0% (± 8.7%)
94.8% (±
5.8%)
99.5% (± 0.3%)
97.8% (± 3.7%)
1.7 (± 0.5)
2.7 (± 0.5)
2.6 (± 0.8)

1:100
31.2% (± 7.2%)
95.3 (± 4.7%)
2.3 (± 0.2)

< 0.3

< 0.3

< 0.3

< 0.3

1.6

< 0.3

< 0.3

< 0.3

65

2.4 Discussion
This study assessed the impact of inoculum size and the amount of inoculated product
being processed on the spread of Salmonella to previously uncontaminated product during pilotscale washing in sanitizer-free water. Spot, rather than dip or spray inoculation was selected to
artificially contaminate red leaf lettuce due to a greater ability to achieve the targeted inoculation
level (Lang et al. 2004). This technique closely mimics random fecal contamination from
animals or biological soil amendments (e.g. manure) in the field rather than exposure to
contaminated irrigation water, where dip inoculation would be more appropriate (Land et al
2004; Gil at al. 2009). Although enteric pathogen contamination from fecal material is
hypothesized to be highly localized in the field, bacterial pathogens are capable of spreading to
previously uncontaminated produce during and after harvest (Williams et al. 2008). Past cross
contamination studies have demonstrated that initial inoculation levels of E. coli O157:H7
ranging from 107 to102 CFU/g can spread to uncontaminated produce during pilot-scale washing,
regardless of sanitizer use (Buchholz et al. 2012; Buchholz et al. 2014; Davidson et al. 2013;
Nou and Luo 2010). However, the initial inoculation levels used in these past studies represented
worst-case scenario and highly localized field contamination such as direct contact with
contaminated manure. Although often highly localized, these same contaminants can be
introduced into irrigation water or equipment and can be subsequently spread over wider field
areas (Beuchat et al. 2010; Ibekwe et al. 2004). In order to more accurately mimic low level field
contamination, which is most commonly presumed, initial inoculation levels of 103, 101, and 10-1
CFU/g were selected for this study.
Previous studies by our lab group have shown that an inoculated surrogate containing
106, 104, or 102 CFU/g E. coli O157:H7 can transfer 1.51 to 2.90 log CFU/g to uninoculated

66

lettuce during pilot-scale processing without the use of a chemical sanitizer. In in similar work
by our group using a chemical sanitizer, initial levels of E. coli O157:H7 106 CFU/g decreased
~1.5 to 2.5 log CFU/g following 90 s washing. The extent of pathogen transfer to uninoculated
lettuce and log reductions seen in these previous studies are similar to the initial inoculation
levels used in the present study. The inoculation levels of the present study can also represent
potential secondary and tertiary contamination occurring during post-harvest washing as bacteria
can subsequently spread through flume water in lower numbers following an initial large
contaminant exposure.
By necessity, most of the baby spinach and cilantro trials were conducted using sanitizerfree wash water in order to quantify the spread of Salmonella to previously uncontaminated
product. Other studies have used sanitizer-free water for pilot-scale studies in order to generate
baseline data for the number of pathogens transferred during this operation (Buchholz et al.
2012; Buchholz et al. 2014; Davidson 2013; Nuo and Luo 2010). This baseline data can then be
extrapolated and applied to predictive models for risk assessments (Pérez-Rodríguez et al. 2011).
The present study aimed to generate baseline data that can be applied to future risk assessments
for fresh-cut leafy greens.
When red leaf lettuce was inoculated to contain 103, 101 10-1 CFU/g, significant
differences in the number of positive samples were observed between inoculation levels. The
different weight ratios used did not produce significant differences in Salmonella-positive
samples. Therefore, the inoculation level has a greater impact on spread of cross contamination
than the amount of contaminated product. A similar trend was seen in a related pilot-scale study
from our laboratory by Buchholz and others (2012) where sanitizer-free wash water was used to
process lettuce inoculated to contain different levels of E. coli O157:H7. In their study, 22.7 kg

67

of dip inoculated lettuce containing 106, 104, or 102 CFU/g E. coli O157:H7 was processed
without a chemical sanitizer through a shredder, conveyor, flume tank (90 s washing), shaker
table, and centrifugal dryer. Immediately thereafter, 90.8 kg uninoculated lettuce was processed
in the same manner. Results from the 102 CFU/g inoculation level revealed transfer of E. coli
O157:H7 to 85% of the 90.8 kg uninoculated lettuce (Buchholz et al. 2012). At the two higher
inoculation levels, complete transfer of E. coli O157:H7 occurred to the entire 90.8 kg
uninoculated lettuce batch (Buchholz et al. 2012). The decrease in transfer seen at the lowest
inoculation level compared to the increased transfer at the two higher inoculation levels supports
the present findings where fewer Salmonella-positive samples were obtained as the initial
inoculation level decreased. Therefore, the extent of cross contamination is more highly
dependent on the level of contamination in the product rather than the amount of product
processed
Using sanitizer-free wash water, Salmonella populations on red leaf lettuce decreased 1.5,
1.0, and 1.8 log CFU/g for the baby spinach and 1.4, 1.4, and 1.6 log CFU/g for cilantro trials at
the 103, 101, 10-1 CFU/g inoculation levels, respectively. In other pilot-scale studies, a ~1 log
CFU/g reduction was achieved after inoculated radicchio, iceberg, and romaine lettuce was
washed for 90 s in sanitizer-free water (Buchholz et al. 2012; Buchholz et al. 2014; Davidson et
al. 2013; Luo and Nou et al. 2012).
Salmonella was generally not detected in samples of sanitizer-free wash water or spent
centrifugation water collected after 90 s of processing. When compared to the aforementioned
study by Buchholz et al. (2012), 90% of the E. coli O157:H7 population transferred to sanitizerfree wash water after 90 s washing of 22.7 kg inoculated lettuce at 106, 104, and 102 CFU/g
inoculation levels (Buchholz et al. 2012). Furthermore, E. coli O157:H7 was quantifiable in their

68

50 mL water samples at the following levels: 1.9, 2.2, -0.3 log CFU/g for iceberg lettuce trials
and 3.0, 1.2, 0.0 log CFU/g for romaine lettuce trials at the high, medium, and low inoculation
levels, respectively. At the lowest inoculation level (102 CFU/g), a -0.3 and 0.0 log CFU/g count
was recorded for iceberg and romaine, respectively (Buchholz et al. 2012). These low counts at
the 102 CFU/g inoculation level along with the high ratio of contaminated to uncontaminated
product processed (1:4 ratio) help explain the low detection rates for Salmonella in wash water
samples from the present study. Our weight ratios and inoculation levels were much lower and
therefore more difficult to detect due to the small water samples taken (50 mL). Buchholz et al.
(2012) also reported that centrifugal drying can expel up to 30% of the microbial population
present on the lettuce with such spent centrifugation water best suited for pathogen testing.
Although, Salmonella counts were limited following centrifugal drying in the present study,
these counts were still higher than those seen in the wash water samples after processing.
Sanitizers used in commercial flume water will typically reduce microbial populations on
the washed product by 90-99% (Burnett et al. 2004, Keskinen et al. 2009, Sapers 2001;
Weissinger et al. 2000; Zhang et al. 2009). However, chlorine-based sanitizers are sensitive to
changes in pH, organic load, and temperature, therefore consistent monitoring of wash water is
paramount to ensure safety and quality of the produce (Davidson et al. 2013; Gil et al. 2009). If a
sanitizer had been used for the lower inoculation levels in the present study, few Salmonella cells
likely would have remained after processing.
Chlorine-based sanitizers are most commonly used in industry due to their relatively low
cost and low negative impact on product quality (Fellows 2009; Buchholz 2011; Sapers 2014;
Suslow 2000). They are used at concentrations of 50 - 200 ppm, and exposure time of 1-2
minutes, and are typically treated with an acidifying agent to lower the water pH to 6.0~7.0

69

(Davidson et al. 2013; Beuchat et al. 1998; Sapers 2014). This same protocol was followed in the
present study as wash water containing 60 ppm available chlorine was acidified to pH 6.5 using
citric acid for a 90 s exposure time. Although unable in completely remove Salmonella from red
leaf lettuce when the 103 CFU/g inoculation level and 10:100, 5:100, 1:100 weight ratios were
used, cross contamination during washing was reduced, indicating marginal success of the
sanitizer. The primary purpose of a sanitizer in wash water is to maintain the microbial quality of
the water and prevent widespread cross contamination of pathogens that may be present (Gil et
al. 2009; Lopez-Galvez et al. 2009; Davidson et al; 2013). In the present study, sanitizer use
decreased Salmonella levels below the limit of detection (0.3 log CFU/100 mL) in both the wash
water and centrifugation water with greater log reductions seen on red leaf lettuce compared to
the sanitizer-free trials. In the present study, a 2.7 log reduction in Salmonella on red leaf lettuce
was achieved. A previous pilot-plant scale study performed by our group reported a 0.88 log
CFU/g reduction of E. coli O157:H7 when 5.4 kg of iceberg lettuce inoculated with 5.93 log
CFU/g was washed for 90 s with chlorine-based sanitizer (50 ppm available chlorine) acidified
with citric acid (Davidson et al. 2013). In another study, spinach inoculated with 2×105 CFU/g
was washed in two different wash treatments, a chlorine-based sanitizer with citric acid and a
chlorine-based sanitizer with T-128, both treatments showed log reductions of 0.8-0.9 CFU/g
following ~90 s of washing (Luo and Nou et al. 2012). Other pilot-scale and laboratory studies
have reported a maximum reductions of 1-3 logs for E .coli O157:H7 and Salmonella spp.
following washing of inoculated produce in the presence of a sanitizer and under practical
conditions (Luo and Nou et al. 2012).
The limited ability of sanitizers to achieve complete removal of Salmonella can be
attributed to attachment of the pathogen to the red leaf lettuce surface. Similar findings were

70

obtained in a separate study where the use of chemical sanitizers decreased but did not
completely eliminate Salmonella Typhimurium or E.coli O157:H7 from parsley and spinach,
respectively (Lapidot et al. 2006; Barrera et al. 2010). The ability of Salmonella to form biofilms
on plant surfaces after 24 h of storage at 4°C also leads to protection against disinfection
(Lapidot et al. 2006). Such pathogens become more difficult to eliminate using chlorine-based
sanitizers when internalized in plant tissue with the hydrophobic surface characteristics of fresh
produce also decreasing the effectiveness of aqueous hypochlorite solutions (Adams et al. 1989;
Pirovani et al. 2004). At best, sanitizers are capable of reducing pathogens a maximum of 3 logs
under ideal commercial processing conditions (Gil et al. 2009; Beuchat et al., 2004; Gonzalez et
al.,2004; Inatsu et al., 2005; Ukuku et al., 2005; Allende et al., 2007;Gómez-López et al., 2007;
Selma et al., 2008b). Therefore, the sanitizer used in this study was limited in preventing
Salmonella cross contamination during washing simply due to the contaminant load and
attachment to red leaf lettuce.
In summary, this study shows that the magnitude of transfer and redistribution of
Salmonella during pilot-scale washing of leafy greens in sanitizer-free water is influenced by
inoculum size, rather than the amount of inoculated product. Such cross contamination can still
occur even if the initial contamination level is low, as the 10-1 CFU/g inoculation level produced
Salmonella-positive samples. Use of a chlorine-based sanitizer led to greater Salmonella
reductions on washed red leaf lettuce and also reduced the number of Salmonella-positive
samples compared to trials where sanitizer was not used, indicating its presence in wash water
made a positive difference. Therefore, proper use of an approved sanitizer is recommended to
minimize microbial cross contamination during washing. These results will be essential in future
risk assessment strategies as this study is the first of our knowledge to analyze Salmonella

71

transfer to baby spinach and cilantro in sanitizer-free water using realistically low levels of initial
contamination.

72

CHAPTER 3:
CONCLUSIONS AND FUTURE RECOMMENDATIONS

73

Fresh produce has been implicated in numerous outbreaks, with leafy greens, culinary
herbs, and sprouts being particularly problematic. Salmonella is the leading cause of multi-state
produce outbreaks and has the third highest economic burden to the food industry and consumer.
Through the Food Safety Modernization Act, the FDA has taken a more proactive approach to
preventing foodborne outbreaks through various risk assessment strategies. Given the threat that
pathogen contamination poses to the fresh produce industry, the need for transparency on the
degree of cross contamination that may occur during commercial processing of leafy greens is
pertinent to risk assessments.
The present study aimed to fill critical data gaps identified by the FDA in regard to the
cross contamination and redistribution of Salmonella Typhimurium LT2 during pilot-scale
processing of baby spinach and cilantro. One objective was to quantify Salmonella Typhimurium
LT2 transfer and redistribution during production of fresh-cut baby spinach and cilantro in
sanitizer-free wash water. Three inoculation levels (103, 101, and 10-1 CFU/g) and four different
weight ratios of surrogate to uninoculated produce were used to assess the extent of cross
contamination that would occur during 90 s of flume washing. Overall, as the initial inoculation
level of surrogate decreased, cross contamination decreased. When analyzing the weight ratios,
there was no significant difference in cross contamination.
In an effort to best mimic real-world scenarios, low inoculation levels of Salmonella
Typhimurium and low inoculated:uninoculated product weight ratios were used to represent
contamination occurring at pre-harvest. Based on the data collected, decreased contamination of
uninoculated produce occurred when the initial contamination load was low following washing
and drying, indicating a lower food safety risk. Although contamination occurred at the lowest
inoculation level and weight ratio, there was a significant decrease in the percentage of

74

Salmonella-positive samples revealing that the risk can be minimized by utilizing sanitizers in
the water during processing.
The second objective evaluated the efficacy of a chlorine-based sanitizer used at 60 ppm
available chlorine, acidified to pH 6.5 under the same conditions as specified above. This was
done in order to imitate industry practices, as 50-200 ppm chlorine (acidified to pH 6.0-7.0) can
be used in produce wash water. The sanitizer was limited in its ability to completely eliminate
Salmonella from the system; however it was able to reduce the spread of contamination during
washing compared to the sanitizer-free trials.
Future studies should focus on work that adds value to risk assessments and can
strengthen food safety at pre-and post-harvest points of fresh produce processing. At pre-harvest,
more data is needed on field contamination and subsequent spread of pathogens through the
field. This data will provide invaluable information that can be applied to future cross
contamination studies where more accurate initial contamination levels can be used. At postharvest, one potential avenue is to assess the spread of contamination occurring to equipment
surfaces such as the shredder, conveyor, flume tank, dewatering shaker, collection bins, and
centrifuge during sanitizer-free processing of inoculated product containing initial pathogen
levels of 103, 101, and 10-1 CFU/g. When applied to the data gathered in the present study, such
findings will lead an improved understanding of the spread of cross contamination to surfaces
when low initial inoculation levels are used, thus providing added information to further
strengthen future risk assessment studies.

75

APPENDIX

76

Chapter 10, Volume 1
Microbiology of Fresh and Processed Vegetables
Haley Smolinski and Elliot T. Ryser

Abstract
Produce is vulnerable to being implicated in foodborne outbreaks due its ready to eat nature and
lack of cooking step prior to consumption. There are multiple routes during pre- and post-harvest
in which produce can become initially contaminated with pathogens. Also, due to the
increasingly globalized market of the fresh produce industry, international transport of produce
can potentially increase the chances of contamination due to a longer distribution chain to reach
the end consumer. Specific strategies have been implemented in order to preserve produce
quality and enhance shelf life including hydrocooling, washing, refrigeration, freezing, and
utilization of active or modified packaging. Although these techniques are beneficial, they can
also directly lead to produce contamination or cross contamination.
Introduction
An increasing number of foodborne outbreaks traced to fresh fruits and vegetables are partially
attributed to production, processing, and consumption patterns. In the United States, the
progression from locally grown produce to centralized production has led to numerous multistate
and nationwide outbreaks of foodborne illnesses. Additionally, consumer demand for year-round
availability of fresh produce has made the industry increasingly globalized. In many cases, fruits
and vegetables are grown on centralized large-scale farms in locations that specialize in a
specific product. A few examples from the United States include the production of baby spinach
in California and Arizona, tomatoes in Florida and New Jersey, blueberries in Michigan and New
Jersey, and mushrooms in Pennsylvania. Under these conditions, one contamination incident at a
large centralized grower or processor could quickly lead to a multistate outbreak with near
catastrophic consequences for the industry, as was seen in several recent outbreaks involving
baby spinach and tomatoes. By definition, fresh fruits and vegetables do not typically undergo
any treatment other than washing for the reduction and/or elimination of potentially hazardous
microorganisms. Use of chemical sanitizers in wash water is common practice throughout the
industry, however the primary function of these sanitizers is to maintain the microbial quality of
the water and improve overall cosmetic appearance. Unfortunately, most commercial sanitizers
used for washing fresh produce can only reduce the microbial levels by 99% to 99.9% at best,
which still makes these products potential vehicles for the transmission of such microbial
pathogens as Salmonella, Escherichia coli O157:H7, Cryptosporidium, Hepatitis A, and
Norovirus.

Handbook of Vegetables and Vegetable Processing. Edited by N. K. Sinha. © 2011 Blackwell Publishing Ltd.

77

SPREAD OF ESCHERICHIA COLI O157:H7 DURING FLUME WASHING AND DRYING
OF FRESH-CUT ROMAINE LETTUCE

By
Siyi Wang, Haley Smolinski, Elliot T. Ryser

Abstract
The microbiological safety of leafy greens remains a concern as evidenced from recent
outbreaks. This study assessed the spread of E. coli O157:H7 during washing and drying of fresh
cut romaine lettuce. Radicchio was spot-inoculated at 10-1, 101 and 103 CFU/g and mixed with
uninoculated romaine lettuce to obtain 5 kg batches with inoculated vs uninoculated ratios of
0.5:100, 1:100, 5:100 and 10:100. After 90 s of sanitizer-free flume washing followed by shaker
table and centrifugal dryer, the radicchio was removed and the lettuce was divided into 225 g
samples to test presence/absence of E. coli O157:H7 using GeneQuence assay. Based on
triplicate trials, lower inoculation levels led to decreased E. coli O157:H7 transfer to romaine
lettuce (P < 0.05). All lettuce samples yielded E. coli O157:H7 when radicchio was inoculated at
103 CFU/g. At 101 CFU/g, the percentage of positive samples decreased from 96.8% to 93.7%,
81.0% and 63.5% while at 10-1 CFU/g, 22.2%, 6.3%, 4.8% and 6.3% were positive at 10:100,
5:100, 1:100 and 0.5:100 ratios. Within each inoculation level, there was no significant
difference (P > 0.05) among four product ratios. These findings will provide important data for
improving exposure assessment in risk assessments for leafy greens.

78

Table AI.1: Salmonella populations (log CFU/g) on red leaf lettuce before washing baby
spinach. This data served as the control for the surrogate samples and ensured the targeted initial
populations were achieved.

Inoculation
Level
(CFU/g)

103

103
Sanitizer

101

10-1

Inoculated:Uninoculated
Ratio
10:100
5:100
1:100
0.5:100
10:100
5:100
1:100
10:100
5:100
1:100
0.5:100
10:100
5:100
1:100
0.5:100

Salmonella populations before
washing - control (log CFU/g)
Rep 1
3.90
3.20
3.20
3.40
3.30
3.10
3.30
1.90
1.50
1.90
1.50
-0.20
-0.30
0.10
-0.20

79

Rep 2
3.30
3.10
3.30
3.30
3.70
3.10
3.30
1.50
1.10
1.20
1.50
-0.30
-0.80
-0.80
-0.40

Rep 3
3.70
3.40
3.20
3.20
3.70
3.10
3.30
1.30
1.60
1.40
1.20
-0.60
-0.10
0.20
-0.60

Avg. populations
Mean (± SD)
3.63 (± 0.31)
3.23 (± 0.15)
3.23 (± 0.06)
3.30 (± 0.10)
3.57 (± 0.23)
3.10 (± 0.0)
3.30 (± 0.0)
1.57 (± 0.31)
1.40 (± 0.26)
1.50 (± 0.36)
1.40 (± 0.17)
-0.37 (± 0.21)
-0.4 (± 0.36)
-0.17 (± 0.55)
-0.4 (± 0.20)

Table AI.2: Salmonella populations (log CFU/g) on red leaf lettuce before washing cilantro. This
data served as the control for the surrogate samples and ensured the targeted initial populations
were achieved.
Inoculation
Level
(CFU/g)

103

103
Sanitizer

101

10-1

Inoculated:Uninoculated
Ratio
10:100
5:100
1:100
0.5:100
10:100
5:100
1:100
10:100
5:100
1:100
0.5:100
10:100
5:100
1:100
0.5:100

Salmonella populations before
washing - control (log CFU/g)
Rep 1
3.20
3.30
3.30
3.10
3.10
3.20
3.30
1.40
1.60
1.10
1.10
-0.70
-0.30
0.20
-0.20

80

Rep 2
3.50
3.10
3.20
3.20
3.20
3.30
3.20
1.40
1.60
1.90
1.40
-0.80
-0.20
-0.10
-0.10

Rep 3
3.50
3.20
3.30
3.10
3.10
3.20
3.20
1.30
1.90
1.10
1.70
-0.20
-0.40
0.40
-0.30

Avg. populations
Mean (± SD)
3.40 (± 0.17)
3.20 (± 0.10)
3.27 (± 0.06)
3.13 (± 0.06)
3.13 (± 0.06)
3.23 (± 0.06)
3.23 (± 0.06)
1.37 (± 0.06)
1.70 (± 0.17)
1.37 (± 0.46)
1.40 (± 0.3)
-0.57 (± 0.32)
-0.30 (± 0.10)
0.17 (± 0.25)
-0.20 (± 0.10)

Table AI.3: Salmonella populations (log CFU/g) on red leaf lettuce after washing baby spinach.
Note: LOD: < -0.04 log CFU/g.
Inoculation
Level
(CFU/g)

103

103
Sanitizer

101

10-1

Inoculated:Uninoculated
Ratio
10:100
5:100
1:100
0.5:100
10:100
5:100
1:100
10:100
5:100
1:100
0.5:100
10:100
5:100
1:100
0.5:100

Salmonella populations after
washing (log CFU/g)
Rep 1
2.10
1.70
2.20
2.40
0.60
1.20
1.30
0.30
0.30
0.60
0.20
<-0.04
<-0.04
-0.04
<-0.04

81

Rep 2
1.30
2.30
0.20
2.30
1.60
1.90
1.20
0.40
0.10
0.20
0.50
-0.04
<-0.04
<-0.04
<-0.04

Rep 3
1.80
2.20
1.90
2.10
0.90
2.20
1.10
0.20
0.30
0.40
0.10
<-0.04
<-0.04
<-0.04
<-0.04

Avg. populations
Mean (± SD)
1.73 (± 0.40)
2.07 (± 0.32)
1.43 (± 1.08)
2.27 (± 0.15)
1.03 (± 0.51)
1.77 (± 0.51)
1.20 (± 0.1)
0.3 (± 0.10)
0.23 (± 0.11)
0.40 (± 0.20)
0.27 (± 0.21)
<-0.04 (± 0.01)
<-0.04 (± 0.0)
-0.04 (± 0.01)
<-0.04 (± 0.0)

Table AI.4: Salmonella populations (log CFU/g) on red leaf lettuce after washing cilantro.
Note: LOD: <-0.04 log CFU/g.
Inoculation
Level
(CFU/g)

103

103
Sanitizer

101

10-1

Inoculated:Uninoculated
Ratio

Salmonella populations after
washing (log CFU/g)
Rep 1

Rep 2

Rep 3

10:100

2.20

1.10

1.90

5:100

1.30

1.50

1.20

1:100

2.20

2.20

2.20

0.5:100

2.10

2.10

2.20

10:100

0.20

1.20

0.50

5:100

2.10

0.10

0.30

1:100

1.50

1.60

2.20

10:100

-0.60

-0.50

0.10

5:100

0.30

0.30

0.40

1:100

1.10

0.20

0.10

0.5:100

0.30

-0.70

-0.70

10:100

<-0.04

<-0.04

<-0.04

5:100

-0.04

-0.04

<-0.04

1:100

0.04

-0.04

<-0.04

0.5:100

<-0.04

-0.08

<-0.04

82

Avg. populations
Mean
(± SD)
1.73
(± 0.57)
1.33
(± 0.15)
2.20
(± 0.0)
2.13
(± 0.06)
0.63
(± 0.51)
0.83
(± 1.10)
1.77
(± 0.38)
-0.33
(± 0.38)
0.33
(± 0.06)
0.47
(± 0.55)
-0.37
(± 0.58)
<-0.04
(± 0.0)
<-0.04
(± 0.01)
<-0.04
(± 0.01)
-0.04
(± 0.03)

Table AI.5: Percentage of Salmonella populations lost from red leaf lettuce surrogate during
baby spinach processing.
Inoculation
Level
(CFU/g)

103

Inoculated:Uninoculated
Ratio

Percent lost from surrogate

Rep 1

Rep 2

Rep 3

10:100

95.61%

96.57%

99.24%

5:100

94.86%

74.10%

96.55%

1:100

95.65%

95%

92.02%

0.5:100

94.74%

86.98%

86.58%

10:100

99.90%

99.74%

99.67%

5:100

85.00%

91.82%

98.31%

1:100

98.81%

99.34%

99.60%

10:100

94.45%

90.87%

96.81%

5:100

94.14%

90.77%

92.90%

1:100

89.00%

92.08%

93.73%

0.5:100

94.17%

90.58%

95.56%

10:100

100%

84.29%

100%

3

10
Sanitizer

101

10-1

5:100

100.00% 100.00% 100.00%

1:100

98.37%

100.00%

96.43%

0.5:100

100.00%

90.90%

100.00%

83

Avg. percent lost
Mean
(± SD)
97.14%
(± 1.54%)
88.50%
(± 10.21%)
94.22%
(± 1.58%)
89.43%
(± 3.76%)
99.77%
(± 0.10%)
91.71%
(± 6.65%)
99.25%
(± 0.40%)
94.04%
(± 2.44%)
92.60%
(± 1.39%)
91.60%%
(± 1.96%)
93.44%
(± 2.10%)
94.76%
(± 7.41%)
100.00%
(± 0%)
98.27%
(± 1.46%)
96.97%
(± 4.29%)

Table AI.6: Percentage of Salmonella populations lost from red leaf lettuce surrogate during
cilantro processing.
Inoculation
Level
(CFU/g)

103

103
Sanitizer

101

10-1

Inoculated:Uninoculated
Ratio

Percent lost from surrogate

Rep 1

Rep 2

Rep 3

10:100

97.98%

99.73%

86.76%

5:100

98.62%

95.64%

94.78%

1:100

94.52%

92.42%

92.34%

0.5:100

99.16%

94.81%

89.79%

10:100

100.00%

100.00%

76.19%

5:100

99.88%

99.95%

93.46%

1:100

89.83%

97.60%

98.33%

10:100

96.40%

98.68%

98.50%

5:100

93.43%

95.00%

96.80%

1:100

98.60%

97.18%

91.03%

0.5:100

93.42%

91.41%

96.00%

10:100

100.00%

100.00% 100.00%

5:100

97.23%

100.00%

86.00%

1:100

80.00%

100.00%

66.70%

0.5:100

100.00%

84.00%

67.00%

84

Avg. percent lost
Mean
(± SD)
94.82%
± 5.75%)
96.35%
(± 1.65%)
93.09%
(± 1.00%)
94.59%
(± 3.83%)
92.06%
(± 11.22%)
97.76%
(± 3.73%)
93.25%
(± 4.70%)
97.86%
(± 1.03%)
95.08%
(± 1.38%)
95.60%
(± 3.30%)
95.94%
(± 2.04%)
100.00%
(± 0%)
94.41%
(± 6.05%)
82.23%
(± 13.69%)
83.66%
(± 13.47%)

BIBLIOGRAPHY

85

BIBLIOGRAPHY

Adams, M.R., A.D. Hartley, and L.J. Cox. 1989. Factors affecting the efficacy of washing
procedures used in the production of prepared salads. Food Microbiol. 6:69–77.
Allen, R.L., B.R. Warren, D.L. Archer, S.A. Sargent, and K.R. Schneider. 2005. Survival of
Salmonella spp. on the surfaces of fresh tomatoes and selected packing line materials. Hort
Technol. 15:831-6.
Allende, A., B. Martínez, M.V. Selma, M.I. Gil, J.E. Suárez, and A. Rodríguez. 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 Microbiol. 24:759–766.
Allende, A., J. McEvoy, Y. Tao, and Y. Luo. 2009. Antimicrobial effect of acidified sodium
chlorite, sodium chlorite, sodium hypochlorite, and citric acid on Escherichia coli O157:H7 and
natural microflora of fresh-cut cilantro. Food Control. 20:230-4.
Allende, A.J., and J. Monaghan. 2015. Irrigation water quality for leafy crops: a perspective of
risks and potential solutions. Int. J. Environ. Res. Public Health. 12:7457-7477.
Allende, A., M.V. Selma, F. Lopez-Galvez, R. Villaescusa, and M.I. Gil. 2008. Role of
commercial sanitizers and washing systems on epiphytic microorganisms and sensory quality of
fresh-cut escarole and lettuce. Postharv. Biol. and Tech. 49:155-63.
Annous, B.A., G.M. Sapers, D.M. Jones, and A. Burke. 2005. Improved recovery procedure for
evaluation of sanitizer efficacy in disinfecting contaminated cantaloupes. J. Food Sci. 70:242-7.
Annous, B. A., G. M. Sapers, A. M. Mattrazzo, and D. C. Riordan. 2001. Efficacy of washing
with a commercial flatbed brush washer, using conventional and experimental washing agents, in
reducing populations of Escherichia coli on artificially inoculated apples. J. Food Prot. 64:159163.
Arthurson, V., A. Sessitsch, and L. Jandurlund. 2011. Persistence and spread of Salmonella
enterica serovar Weltvreden in soil and spinach plants. FEMS Microbiol. Let. 314:64-74.
Babic, I., and A.E. Watada. 1996. Microbial populations of fresh-cut spinach leaves affected by
controlled atmospheres. Postharvest Biol. and Tech. 9:187-93.
Baloda, S.B., L. Christensen, and S. Trajcevska. 2001. Persistence of Salmonella enterica serovar
Typhimurium DT12 clone in a piggery and in agricultural soil amended with Salmonella
contaminated slurry. Appl. Environ. Microbiol. 67:2859–2862.
Barrera, M.J., R. Blenkinsop, and K. Warriner. 2012. The effect of different processing
parameters on the efficacy of commercial post-harvest washing of minimally processed spinach
and shredded lettuce. Food Control. 25:745-51.

86

Berger, C.N., R.K. Shaw, D.J. Brown, H. Mather, S. Clare, G. Dougan, M.J. Pallen, G. Frankel.
2009. Interaction of Salmonella enterica with basil and other salad leaves. J. Microbial. Ecol.
3:261-265.
Beuchat, L. R. 2002. Ecological factors influencing survival and growth of human pathogens on
raw fruits and vegetables. Microbiol. Infect. 4:413-423.
Beuchat, L. R. 1998. Surface decontamination of fruits and vegetables eaten raw: a review.
Available at: http://www.who.int.proxy2.cl.msu.edu/foodsafety/
publications/fs_management/en/surface_decoapdf. Accessed 28 October 2016.
Beuchat, L.R., B.B. Adler, and M.M. Lang. 2004. Efficacy of chlorine and a peroxyacetic acid
sanitizer in killing Listeria monocytogenes on iceberg and romaine lettuce using simulated
commercial processing conditions. J. Food Prot. 67:1238–42.
Beuchat, L.R., J.M. Farber, E. Garrett, L.J. Harris, M.E. Parish, T.V. Suslow, and F.F. Busta.
2001. Standardization of a method to determine the efficacy of sanitizers in inactivating human
pathogenic microorganisms on raw fruits and vegetables. J. Food Prot. 64:1079–84.
Beuchat, L.R., B.V. Nail, B.B. Adler, and M.R.S Clavero. 1998. Efficacy of spray application of
chlorinated water in killing pathogenic bacteria on raw apples, tomatoes and lettuce. J. Food
Prot. 61:1305–1311.
Buchholz, A.L., G.R. Davidson, and E.T. Ryser. 2011. Microbiology of Fresh and Processed
Vegetables, p. 159-181. In. N.K. Sinha, Y.H. Hui, E.O., Evranuz, M, Siddiq, and J. Ahmed (ed.),
Handbook of Vegetables and Vegetable Processing. Wiley-Blackwell, Ames Iowa.
Buchholz, A.L., G.R. Davidson, B.P., Marks, E.C.D. Todd, and E.T. Ryser. 2012. Transfer of
Escherichia coli O157:H7 from equipment surfaces to fresh-cut leafy greens during processing
in a model pilot-plant production line with sanitizer-free water. J. Food Prot. 75:1920-9.
Buchholz A.L., G.R. Davidson, B.P. Marks, E.C.D. Todd, and E.T. Ryser. 2014. Tracking an
Escherichia coli O157:H7-Contaminated batch of leafy greens through a pilot-scale fresh-cut
processing line. J. Food Prot. 77:1487-94.
Burnett, A. B., M. H. Iturriaga, E. F. Escartin, C. A. Pettigrew, and L R. Beuchat. 2004.
Influence of variations in methodology on populations of Listeria monocytogenes recovered
from lettuce treated with sanitizers. J. Food Prot. 67:742-750.
Brackett, R.E. 1994. Microbiological spoilage and pathogens in minimally processed refrigerated
fruits & vegetables, p. 269-312. In R.C. Wiley (ed.), Minimally processed refrigerated fruits and
vegetables, Chapman and Hall, New York.
Brandl, M.T., and R.E. Mandrell. 2002. Fitness of Salmonella enterica Thompson in the cilantro
phyllosphere. Appl. Environ. Microbiol. 68:3614-21.

87

Brouwer, C., K Prins, M. Kay, and M. Heibloem. 2016. Choosing an irrigation method. In. Food
and Agriculture Organization of the United Nations, Irrigation water management: irrigation
methods.
Buchanan, R. L., S. G. Edelson, R. L. Miller, and G. M. Sapers. 1999. Contamination of intact
apples after immersion in an aqueous environment containing Escherichia coli O157:H7. J. Food
Prot. 62:444-450.
Callejon, R.M., M.I. Rodriguez-Naranjo, C. Ubeda, R. Hornedo-Ortega, M.C. Garcia-Parrilla,
and A.M. Troncoso. 2015. Reported foodborne outbreaks due to fresh produce in the United
States and European Union: Trends and Causes. Foodborne Path. & Dis. 12:32-8.
Campbell, J.V., J. Mohle-Boetani, R. Reporter, S. Abbott, J. Farrar, M. Brandl, R. Mandrell, and
S.B. Werner. 2001. An Outbreak of Salmonella serotype Thompson associated with fresh
cilantro. J. Infect. Dis. 183:984-7.
Centers for Disease Control and Prevention (CDC). 2007. Salmonella Oranienburg infections
associated with fruit salad served in healthcare facilities - northeastern United States and Canada.
Available at: https://www.ncbi.nlm.nih.gov/pubmed/17914330. Accessed 30 November 2016.
Centers for Disease Control and Prevention (CDC). 2012. Multistate outbreak of shiga toxinproducing Escherichia coli O157:H7 infections linked to organic spinach and spring mix blend
(final update). Available at: http://www.cdc.gov.proxy2.cl.msu.edu/ecoli/2012/O157H7-1112/index.html. Accessed 01 November 2016.
Centers for Disease Control and Prevention (CDC). 2014. Foodborne Diseases Active
Surveillance Network (FoodNet): FoodNet Surveillance Report for 2012 (Final Report). Atlanta,
Georgia: U.S. Department of Health and Human Services.
Centers for Disease Control and Prevention (CDC). 2015. Reports of Selected Salmonella
Outbreak Investigations. Available at: http://www.cdc.gov/salmonella/outbreaks.html. Accessed
2 Sep 2016.
Centers for Disease Control and Prevention (CDC). 2016. Foodborne outbreak and reporting:
foodborne outbreak online database (FOOD Tool). Available at:
https://wwwn.cdc.gov/foodborneoutbreaks/. Accessed 4 March 2014.
Centers for Disease Control and Prevention (CDC). 2016. List of selected multistate foodborne
outbreak investigations. Centers for Disease Control and Prevention. Available at:
http://www.cdc.gov/foodsafety/outbreaks/multistate-outbreaks/outbreaks-list.html. Accessed 30
November 2016.
Center for Science in the Public Interest (CSPI). 2015. CSPI outbreak alert data: info on produce
outbreaks.https://cspinet.org/sites/default/files/attachment/outbreak-alert-2015.pdf. Accessed 18
February 2017.

88

Chang, A. S., and K. R. Schneider. 2012. Evaluation of overhead spray-applied sanitizers for the
reduction of Salmonella on tomato surfaces. J. Food Sci. 71:M65–M69.
Chimbombi, E., R, Moreira, E.M. Castell-Perez, A.F. Puerta-Gomez. 2013. Assessing
accumulation (growth and internal mobility) of Salmonella Typhimurium LT2 in fresh-cut
cantaloupe (Cucumis melo L.) for optimization of decontamination strategies. Food Control
32:574-81.
Cook, R. 2011. Tracking demographics and U.S. fruit and vegetable consumption patterns.
Department of Agriculture and Resource Economics University of California, Davis. Available
at:
https://arefiles.ucdavis.edu/uploads/filer_public/2014/05/19/blueprintseoeconsumptioncookfinalj
an2012figures.pdf. Accessed 20 Nov 2016.
Costerton, J.W., P.S. Stewart, and E.P. Greenberg. 1999. Bacterial biofilms: a common cause of
persistent infections. Sci. 284:1318-22.
Cooley, M., D. Carychao, L. Crawford-Miksza, M. T. Jay, C. Myers, C. Rose, C. Keys, J. Farrar,
and R. E. Mandrell. 2007. Incidence tracking of Escherichia coli O157:H7 in a major produce
production region in California. Plos one 2:e1159. doi: 10.1371/journal.pone.0001159.
Denney, J., J. Threlfall, S. Lofdahl, T. Westrell, C. Varela, B. Adak, N. Boxall, S. Ethelberg, M.
Torpdahl, M. Straetemans, W. van Pelt. 2007. Multinational Salmonella Paratyphi B variant Java
(Salmonella Java) outbreak August-December 2007. Available at:
http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=3332. Accessed 10 March 2017.
Erickson, M.C. 2012. Microbial Ecology, p 3-41. In. V.M. Gomez-Lopez (ed.). Decontamination
of Fresh and Minimally Processed Produce. Wiley-Blackwell, Ames, IA.
Erickson, M.C., C.C. Webb, J.C., Diaz-Perez, S.C. Phatak, and J.J. Silvoy. 2010. Surface and
internalized Escherichia coli O157:H7 on field-grown spinach and lettuce treated with spraycontaminated irrigation water. J. Food Prot. 73:1023-9.
Erickson, M.C., J. Liao, J.L. Cannon, and Y.R. Ortega. 2015. Contamination of knives and
graters by bacterial foodborne pathogens during slicing and grating of produce. Food Mirco.
52:138-45.
Eurosurveillance. 2005. A nationwide outbreak of multiresistant Salmonella Typhimurium var
Copenhagen DT104B infection in Finland due to contaminated lettuce from Spain, May 2005.
Available at: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=2734. Accessed 18
March 2017.
Elsevier. Available at: http://app.knovel.com/hotlink/toc/id:kpEFAGHN0D/encyclopedia-foodsguide/encyclopedia-foods-guide. Accessed 1 December 2016.

89

Davidson, G.R., A.M. Buchholz, and E.T. Ryser. 2013. Efficacy of commercial produce
sanitizers against nontoxigenic Escherichia coli O157:H7 during processing of iceberg lettuce in
a pilot-scale leafy green processing line. J. Food Prot. 76:1838-45.
Davidson, G.R., N.K. Kaminski, and E.T. Ryser. 2014. Impact of organic load on Escherichia
coli O157:H7 survival during pilot-scale processing of iceberg lettuce with acidified sodium
hypochlorite. J. Food Prot.77: 1669-81.
Delaquis, P.J., S. Stewart, P.M.A. Toivonen, and A.L. Moyls. 1999. Effect of warm, chlorinated
water on the microbial flora of shredded iceberg lettuce. Food Res. Int’l. 32:7-14.
Duffy, E.A., L.M. Lucia, J.M. Kells, A. Castillo, S.D. Pillai, and G.R. Acuff. 2005.
Concentrations of Escherichia coli and genetic diversity and antibiotic resistance profiling
Salmonella isolated from irrigation water, packing shed equipment, and fresh produce in Texas.
J. Food Prot. 68:70-9.
Food and Agriculture Organization of the United Nations and the World Health Organization
(FAO and WHO). 2008. Microbiological hazards in fresh leafy vegetables and herbs.
Department of Food Safety and Zoonoses and Foodborne Diseases, World Health Organization,
Geneva, Switzerland.
Farrar, F.J., and J. Guzewich. 2014. Identification of the source of contamination. p. 59-83. In.
K.M. Matthews, G.M. Sapers, and C.P. Gerba (ed.), The Produce Contamination Problem, 2nd
Ed. Elsevier, San Diego, CA.
Fellows, P.J. 2009. Raw material preparation, p. 99-124. In. P.J. Fellows (ed). Food processing
technology, 3rd Ed. Woodhead Publishing, Oxford, UK.
Foley, D., M. Euper, F. Caporaso, and A. Prakash. 2004. Irradiation and chlorination effectively
reduces Escherichia coli O157:H7 inoculated on cilantro (Coriandrum sativum) without
negatively affecting quality. J. Food Prot. 67:2092-98.
Francis, G.A., and D. O’Beirne. 2001. Effects of vegetable type, package, atmosphere and
storage temperature on growth and survival of Escherichia coli O157:H7 and Listeria
monocytogenes. J. Ind. Microbiol. Biotech. 27:111-6.
Garcia-Villanova Ruiz, B., R. Galvez-Vargas, and R. Garcia-Villanova.1987. Contamination on
fresh vegetables during cultivation and marketing. Int. J. Food Microbiol. 4:285-291.
Gerba, C.P., and C. Rock. 2014. Water Quality. In K.M. Matthews, G.M. Sapers, and C.P. Gerba
(ed.), The Produce Contamination Problem, 2nd Ed. Elsevier, San Diego, CA.
Gil, M.I., M.V. Selma, F. Lopez-Galvez, and A. Allende. 2009. Fresh-cut product sanitation and
wash water disinfection: Problems and solutions. Intl. J. Food Microbiol.134:37-45.

90

Gomez-Lopez, V.M., F. Devlieghere, P. Ragaert, and J. Debevere. 2007. Shelf-life extension of
minimally processed carrots by gaseous chlorine dioxide. Intl. J. Food Microbiol. 116:221–7.
Gonzalez, R.J., Y. Luo, S. Ruiz-Cruz, and J.L. McEvoy. 2004. Efficacy of sanitizers to inactivate
Escherichia coli O157:H7 on fresh-cut carrot shreds under simulated process water conditions. J.
Food Prot. 67:23375–80.
Gunes, G., and E. Dogu. 2010. Green leafy vegetables: Spinach and lettuce. p. 705-716. In. N.K.
Sinha, Y.H. Hui, E.O., Evranuz, M, Siddiq, and J. Ahmed (ed.), Handbook of Vegetables and
Vegetable Processing. Wiley-Blackwell, Ames Iowa.
Gutierrez-Rodrigez, E. 2015. Fresh produce safety before and after the food safety
modernization act (FSMA). NC State University Cooperative Extension. Available at:
http://www.nciom.org/wp-content/uploads/2015/05/NCIOM-Guest-Gutierrez-2015-Handout.pdf.
Accessed 26 Nov 2016.
Haute, S., F. Lopez-Galvez, V. L. Gomez, V. Ericksson, M. Devlieghere, A. Allende, and I.
Sampers. 2015. Methodology for modeling disinfection efficiency of fresh-cut leafy vegetables
wash water applied on peracetic acid combined with lactic acid. Intl. J. Food Microbiol.
208:102-13.
Heaton, J.C., and K. Jones. 2007. Microbial contamination of fruit and vegetables and the
behaviour of enteropathogens in the phyllosphere: a review. J. Appl. Microbiol. 104:613–26.
Herdt, J., and H. Fang. 2009. Aqueous antimicrobial treatments to improve fresh and fresh-cut
produce safety, p. 169-190. In. X. Fan, B.A. Niemira, C.J. Doona (ed.). Institute of Food
Technologists Series: microbial safety of fresh produce, 1st Ed. Wiley-Blackwell.
Hillborn, E.D., J.H. Mermin, P.A. Mshar, J.L. Hadler, A. Voetsch, Wojtkunski, C., Swartz, M.,
Mshar, R., Lambert-Fair, M.A., Farrar, J.A., Glynn, and M.K., Slutsker, L. 1999. A multistate
outbreak of Escherichia coli O157:H7 infections associated with consumption of mesclun
lettuce. Arch. Intern. Med. 159:1758–1764.
Himathongkam, S., S. Bahari, H. Riemann, D. Cliver. 1999. Survival of Escherichia coli
O157:H7 and Salmonella Typhimurium in cow manure and cow slurry. FEMS Microbiol. Lett.
178:251-257.
Hoffman, S., B. Maculloch, and M. Batz. 2015. Economic burden of major foodborne illnesses
acquired in the United States. United States Department of Agriculture.
Holah, J.T., and R.H. Thorpe. 1990. Cleanability in relation to bacterial retention on unused and
abraded domestic sink materials. J. Appl. Microbiol. 69:599-608.
Ibekwe, A. M., P. M. Watt, P. J. Shouse, and C. M. Grieve. 2004. Fate of Escherichia coli
O157:H7 in irrigation water on soils and plants as validated by culture method and real-time
PCR. Can. J. Microbiol. 50:1007-1014.

91

Inatsu, Y., M.L. Bari, S. Kawasaki, K. Isshiki, and S. Kawamoto. 2005. Efficacy of acidified
sodium chlorite treatments in reducing Escherichia coli O157:H7 on Chinese cabbage. J. Food
Prot. 68:251–5.
Islam, M., M.P. Doyle, S.C. Phatak, P. Millner, and X. Jiang. 2004. Persistence of
enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in
fields treated with contaminated manure composts or irrigation water. J. Food Prot. 67:1365–
1370.
Iturriaga, M.H., E.F. Escarin, L.R. Beuchat, and R. Martinez-Peniche. 2003. Effect of inoculum
size, relative humidity, storage temperature, and ripening stage on the attachment of Salmonella
Montevideo to tomatoes and tomatillos. J. Food Prot. 66:1756-61.
Johannessen, G.S., R.B. Froseth, L. Solemdal, J. Jarp, Y. Wasteson, and L.M. Rorvik. 2004.
Influence of bovine manure as fertilizer on the bacteriological quality of organic Iceberg lettuce.
J. Appl. Microbiol. 96:787-94.
Jones, P.W. 1986. Sewage sludge as a vector of salmonellosis, p. 21-33. In. J.C. Block, A.H.
Haielaar, and P. L’Hermite (ed.), Epidemiological studies of risks associated with the
agricultural use of sewage sludge, Elsevier, London.
Kaminski, C.N., G.R. Davidson, and E.T. Ryser. 2014. Listeria monocytogenes transfer during
mechanical dicing of celery and growth during subsequent storage. J. Food Prot. 77:765-71.
Keskinen, L. A., A. Burke, and B. A. Annous. 2009. Efficacy of chlorine, acidic electrolyzed
water and aqueous chlorine dioxide solutions to decontaminate Escherichia coli O157:H7 from
lettuce leaves. Int. J. Food Microbiol. 132:134-140.
Khalil, R., and J.F. Frank. 2010. Behavior of Escherichia Coli O157:H7 on damaged Leaves of
spinach, lettuce, cilantro, and parsley stored at abusive temperatures. J. Food Prot. 73:212-20.
Kisluk, G., S. Yaron. Presence and Persistence of Salmonella enterica serotype Typhimurium in
the phyllosphere and rhizosphere of spray irrigated parsley. 2012. Appl. Env. Microbiol.
78:4030-4036.
Kondo, N., M. Murata, and K. Isshiki. 2006. Efficiency of sodium hypochlorite, fumaric acid
and mild heat in killing native microflora and E. coli O157:H7, Salmonella Typhimurium DT104
and Staphylococcus aureus attached to fresh-cut lettuce. J. Food Prot. 69:323–29.
Koseki, S., K. Yoshida, Y. Kamitani, and K. Itoh. 2003. Influence of inoculation method, spot
inoculation site, and inoculation size on the efficacy of acidic electrolyzed water against
pathogens on lettuce. J. Food Prot. 66:2010-2016.
Kusumaningrum, H.D., G. Riboldi, W.C. Hazeleger, and R.R. Beumer. 2003. Survival of
foodborne pathogens on stainless steel surfaces and cross-contamination to foods. Intl. J. Food
Microbiol. 85:227-236.

92

Lang, M.M., L.J. Harris, and L.R. Beuchat. 2004. Evaluation of inoculation method and
inoculum drying time for their effects on survival and efficiency of recovery of Escherichia coli
O157:H7, Salmonella and Listeria monocytogenes inoculated on the surfaces of tomatoes. J.
Food Prot.57:732–41
Lang, M.M., L.J. Harris, and L.R. Beuchat. 2004. Survival and recovery of Escherichia coli
O157:H7, Salmonella, and Listeria monocytogenes on lettuce and parsley as affected by method
of inoculation, time between inoculation and analysis, and treatment with chlorinated water. J.
Food Prot. 67(6):1092-1103.
Lapidot, A., U. Romling, and S. Yaron. 2006. Biofilm formation and the survival of Salmonella
Typhimurium on parsley. Intl. J. Food Microbiol. 109:229-33.
Lawley, R. 2013. Salmonella. Food safety watch. Available at:
http://www.foodsafetywatch.org/factsheets/salmonella/. Accessed 11 November 2016.
Li, Y., R. Brackett, J. Chen, and L. Beuchat. 2001. Survival and growth of Escherichia coli
O157:H7 inoculated onto cut lettuce before or after heating in chlorinated water, followed by
storage at 5 or 15C. J. Food Prot. 64:305-9.
Liao, C.H., and P.H. Cooke. 2001. Response to trisodium phosphate treatment of Salmonella
chester attached to fresh-cut green pepper slices. Can. J. Microbiol. 47:25–32.
Luo, Y., X. Nou, P. Millner, B. Zhou, C. Shen, Y. Yang, Y. Wu, Q. Wang, H. Feng, and D.
Shelton. 2012. A pilot plant scale evaluation of a new process aid for enhancing chlorine efficacy
against pathogen survival and cross-contamination during produce wash. Intl. J. Food Microbiol.
158:133-139.
Lopez-Galvez, F., M.I. Gil, M.V. Selma, and A. Allende. 2010. Cross-contamination of fresh-cut
lettuce after a short-term exposure during pre-washing cannot be controlled after subsequent
washing with chlorine dioxide or sodium hypochlorite. Food Microbiol. 27:199-204.
Lopez-Galvez, F., A. Allende, M.V. Selma, and M. Gil. 2009. Prevention of Escherichia coli
cross-contamination by different commercial sanitizers during washing of fresh-cut lettuce. Int.
J. Microbiol. 133:167-71.
Materon, L.A., M. Martinez-Garcia, and V. McDonald. 2007. Identification of sources of
microbial pathogens on cantaloupe rinds from pre-harvest to post-harvest operations. Wld. J.
Microbiol. Biotechnol. 32:1281–87.
Matthews, K.M. 2014. Leafy Vegetables, p. 187-206. In K.M. Matthews, G.M. Sapers, and C.P.
Gerba (ed.), The Produce Contamination Problem, 2nd Ed. Elsevier, San Diego, CA
McClelland, M. 2000. Comparison of the Escherichia coli K-12 genome with sampled genomes
of a Klebsiella pneumoniae and three Salmonella Enterica serovars, Typhimurium, Typhi and
Paratyphi. Nucleic Acids Res. 28, 4974-86.

93

Merriweather, S., T.C. Cloyd, and D.Gubernot. ‘‘Memorandum to the File 2011–2014 Produce
Related Outbreaks and Illnesses, May 2015.’’ Food and Drug Administration. Available at:
https://www.regulations.gov/document?D=FDA-2011-N-0921-18587. Accessed March 3, 2017.
Millner, P.D. 2014. Manure Management, p. 85-106. In K.M. Matthews, G.M. Sapers, and C.P.
Gerba (ed.), The Produce Contamination Problem, 2nd Ed. Elsevier, San Diego, CA.
Mitchell, E. 2015. Gram positive vs gram negative bacteria and the fight against HAIs. Cupron
Enhanced EOS Surfaces. Available at: http://blog.eoscu.com/blog/gram-positive-vs-grampositive. Accessed 23 November 2016.
Montville, R., and D. W. Schaffner. 2003. Inoculum size influences bacterial cross
contamination between surfaces. Appl. Environ. Microbiol. 69:7188-7193.
Moore, C.M., B.W. Sheldon, and L. Jaykus. 2003. Transfer of Salmonella and Campylobacter
from stainless steel to romaine lettuce. J. Food Prot. 12:2231-36.
Motarjemi, Y. 2013. Bacteria. p. 337-580 In. Y. Motarjemi, G.G. Moy, and E.C.D. Todd (ed.),
Encyclopedia of Food, Vol 1. Elsevier, San Diego, CA.
Neidhardt, F. C. 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM,
Washington DC.
Nickerson, C.A., R. Curtiss III. 1997. Role of sigma factor RpoS in intial stages of Salmonella
typhimurium infection. J. of Infection and Immunity 65: 1814-23.
Nou, X., and Y. Luo. 2010. Whole leaf wash improves chlorine efficacy for microbial reduction
and prevents pathogen cross-contamination during fresh cut lettuce processing. J. Food Sci.
75:283-90.
Ochman, H., J.G. Lawrence, E.A. Groisman. 2000. Lateral gene transfer and the nature of
bacterial innovation. Nature 405: 299-304.
Pace, R.C. 2013. Interaction of filth flies, plants and human pathogens. Oklahoma State
University, ProQuest/Dissertation.
Palma-Salgado, S., Pearlstein, A.J., Luo, Y., Park, H.K., and Feng, H. 2014. Whole-head
washing, prior to cutting, provides sanitization advantages for fresh-cut Iceberg lettuce (Latuca
sativa L.). Intl. J. Food Microbiol. 179(2): 18-23.
Patel, J., M. Sharma, P. Millner, T. Calaway, and M. Singh 2011. Inactivation of Escherichia
coli O157:H7 attached to spinach harvester blade using bacteriophage. Foodborne Path. Dis.
8:541-46.
Pell, N. Manure and microbes: public and animal health problem? J. Dairy Sci. 80:2673-2681.

94

Pérez-Rodríguez, F., D. Campos, E.T. Ryser, A.M. Buchholz, B.P. Marks, Z, Gonzalo, and
E.C.D. Todd. 2011. A mathematical risk model for Escherichia coli O157:H7 crosscontamination of lettuce during processing. Food Microbiol. 28:694-701.
Perna, N. T. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature
409, 529-533.
Pirovani, M., A. Piagentini, D. Güemes, and S. Arkwright. 2004. Reduction of chlorine
concentration and microbial load during washing-disinfection of shredded lettuce. Intl. J. Food
Sci. & Tech. 39:341–47.
Plym-Forshell, L. 1995. Survival of Salmonella and Ascaris suum eggs in a thermophilic biogas
plant. Acta. Vet. Scand. 36:79–85.
Popoff, M.Y., J. Bockemuhl, F.W. Brenner. 1999. Supplement 1999 to the Kauffmann-White
scheme. Res. Microbiol. 43: 893-6.
Produce for Better Health Foundation. 2015. State of the Plate: 2015 study on America’s
consumption of Fruits and Vegetables. Available at:
http://www.pbhfoundation.org/pdfs/about/res/pbh_res/State_of_the_Plate_2015_WEB_Bookmar
ked.pdf. Accessed 19 Nov 2016.
Roos, R. 2010. USDA estimates E.coli, Salmonella costs at $3.1 billion. Center for Infectious
Disease Research and Policy. Available from: http://www.cidrap.umn.edu/newsperspective/2010/05/usda-estimates-e-coli-salmonella-costs-31-billion. Accessed: 2016
November 1.
Ruiz-Cruz, S., E. Acedo-Felix, M. Diaz-Cinco, M.A. Islas-Osuna, and G.A. Gonzalez-Aguilar.
2007. Efficacy of sanitizers in reducing Escherichia coli O157:H7, Salmonella spp. and Listeria
monocytogenes populations on fresh-cut carrots. Food Control 18:1383-90.
Sapers, G.M. 2014. Disinfection of contaminated produce with conventional washing and
sanitizing technology, p. 389-431. In K.M. Matthews, G.M. Sapers, and C.P. Gerba (ed.), The
Produce Contamination Problem, 2nd Ed. Elsevier, San Diego, CA.
Sapers, G. M. 2001. Efficacy of washing and sanitizing methods for disinfection of fresh fruit
and vegetable products. Food Technol. Biotechnol. 39:305-311.
Sapers, G. M., and D. M. Jones. 2006. Improved sanitizing treatments for fresh tomatoes. J.
Food Sci. 71:M252–M256.
Selander, R.K., J. Li, K. Nelson. 1996. Escherichia coli and Salmonella. p. 2691-2707. In F.C.
Neidhardt (ed.), Cellular and Molecular Biology. ASM, Washington DC.

95

Selma, M.V., A.M. Ibañez, A. Allende, M. Cantwell, and T. Suslow. 2008. Effect of gaseous
ozone and hot water on microbial and sensory quality of cantaloupe and potential transference of
Escherichia coli O157:H7 during cutting. Food Microbiol. 25:162–68.
Seo, K. H., and J. F. Frank. 1999. Attachment of Escherichia coli O157:H7 to lettuce leaf surface
and bacterial viability in response to chlorine treatment. J. Food Prot. 62:3-9.
Scollon, A.M. 2014. Transfer and survival of Listeria monocytogenes during slicing, dicing, and
storage of onions. Pro Quest, Ann Arbor, MI.
Shirron, N., G. Kisluk, Y. Zelikovich, I. Eivin, E. Shimoni, and S. Yaron. 2009. A comparative
study assaying commonly used sanitizers for antimicrobial activity against indicator bacteria and
a Salmonella Typhimurium strain on fresh produce. J. Food Prot. 72:2413-17.
Silhavy T.J., D. Kahne, and S. Walker. 2010. The bacterial cell envelope. Cold Spring Harb.
Perspect. Biol. 2:a000414.
Smith, R., J. Bi, M. Cahn, M. Cantwell, O. Daugovish, S. Koike, E. Natwick, and E. Takele.
2015. Cilantro production in California. University of California Agriculture and Natural
Resources. Available at: http://anrcatalog.ucanr.edu/pdf/7236.pdf. Accessed 9 Sep 2016.
Soloman, E.B., H. Pang, and K.R. Matthews. 2003. Persistence of Escherichia coli O157:H7 on
lettuce plants following spray irrigation with contaminated water. J. Food Prot. 12:2198-2202.
Soloman, E.B., Y. Sima, and K.R. Matthews. 2002. Transmission of Escherichia coli O157:H7
from contaminated manure and irrigation water to lettuce plant tissue and its subsequent
internalization. Appl. Environ. Microbiology. 68:397-400.
Solomon, E. B., S. Yaron, and K.R. Matthews. 2002. Transmission of Escherichia coli O157:H7
from contaminated manure and irrigation water to lettuce plant tissue and its subsequent
internalization. Appl. Environ. Microbiol. 68:397–400.
Stafford, R., B. McCall, A. Neil. D. Leon, G. Dorricott., C. Towner, and G. Micalizzi. 2002. A
statewide outbreak of Salmonella Bovismorbificans phage type 32 infection in Queensland.
Comm. Dis. Intel. 26:588-73.
Strawn, L.K., Y.T. Grohn, S. Warchocki, R.W. Worobo, E.A. Bihn, and M. Weildmann. 2013.
Risk factors associated with Salmonella and Listeria monocytogenes contamination of produce
fields. Appl. Env. Microbiol. 79:7618-7627.
Suslow, T.V. 2004. Key points of control and management of microbial food safety: information
for producers, handlers, and processors of fresh market tomatoes. University of California,
Davis. Available at: http://anrcatalog.ucanr.edu/pdf/8150.pdf. Accessed 30 November 2016.
Thurston-Enriquez, J.A., P. Watt, S.C. Dowd, R. Enriquez, I.L. Pepper, and C.P. Gerba. 2002.
Detection of protozoan parasites and microsporidia in irrigation waters used for crop production.
J. Food Prot. 65:378–82.

96

Ukuku, D.O., M.L. Bari, S. Kawamoto, K. Isshiki. 2005. Use of hydrogen peroxide in
combination with nisin, sodium lactate and citric acid for reducing transfer of bacterial pathogens
from whole melon surfaces to fresh-cut pieces. Intl. J. Food Microbiol. 104:225–33.
University of Georgia Center for Produce Safety. 2011. Illnesses in U.S. attributed to leafy
greens. Available at: http://www.ugacfs.org/producesafety/pages/basics/USIllnesses.html.
Accessed 26 Nov 2016.
U.S. Department of Agriculture (USDA). 2017. USDA Choose MyPlate.gov. Available at:
https://www.choosemyplate.gov/MyPlate. Accessed 15 Feb 2017.
U.S. Food and Drug Administration (FDA). 1998. Guidance for industry: guide to minimize
microbiological food safety hazards for fresh fruits and vegetables. Available at:
https://www.fda.gov/regulatoryinformation/guidances/ucm064574.htm. Accessed 15 March
2017.
U.S. Food and Drug Administration (FDA). 2003. FDA survey of domestic fresh produce FY
2000/2001 field assignment. Available at:
http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/Prod
ucePlantProducts/ucm118306.htm. Accessed: 2016 November 3.
U.S. Food and Drug Administration (FDA). 2009. Draft guidance for industry: guide to
minimize microbial food safety hazards of leafy greens. U.S. Department of Health and Human
Services. Available at:
http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/Prod
ucePlantProducts/ucm174200.htm#ref1. Accessed 23 Nov 2016.
U.S. Food and Drug Administration (FDA). 2012. Bad bug book, foodborne pathogenic
microorganisms and natural toxins, 2nd Ed.
U.S. Food and Drug Administration (FDA). 2013. Commodity specific Food safety guidelines
for the production, harvest, post-harvest and processing unit operations of fresh culinary herbs.
Available at:
http://www.fda.gov/downloads/food/guidanceregulation/guidancedocumentsregulatoryinformati
on/produceplantproducts/ucm365142.pdf. Accessed 2016 November 12.
U.S. Food and Drug Administration (FDA). 2015a. Final qualitative risk assessment of risk to
public health from on-farm contamination of produce. Available at:
file:///C:/Users/Owner/Downloads/QAR-11.16.15_Waiver.pdf. Accessed 3 March 2017.
U.S. Food and Drug Administration (FDA). 2015b. Risk analysis at FDA: food safety. U.S.
Department of Health and Human Services. Available at:
http://www.fda.gov/Food/FoodScienceResearch/RiskSafetyAssessment/ucm243439.htm.
Accessed 15 Nov 2016.

97

U.S. Food and Drug Administration (FDA). 2015c. Safe practices for food processes Analysis
and evaluation of preventive control measures for the control and reduction/elimination of
microbial hazards on fresh and fresh-cut produce: Chapter IV, outbreaks associated with fresh
and fresh-cut produce: incidence, growth, and survival of pathogens in fresh and fresh-cut
produce. U.S. Department of Health and Human Services. Available at:
http://www.fda.gov/food/foodscienceresearch/safepracticesforfoodprocesses/ucm091265.htm.
Accessed 2 Nov 2016.
U.S. Food and Drug Administration (FDA). 2016a. FSMA final rule on produce safety.
Available at: http://www.fda.gov/Food/GuidanceRegulation/FSMA/ucm334114.htm. Accessed
18 Nov 2016.
U.S. Food and Drug Administration (FDA). 2016b. Produce safety standards. Available at:
http://www.fda.gov/Food/GuidanceRegulation/FSMA/ucm304045.htm. Accessed 18 Sep 2016.
US Foods, Inc. 2016. Recall Notices. Available at: https://www.usfoods.com/recall-notices.html.
Accessed 3 Sep 2016.
Wang, H., and E.T. Ryser. 2014. Salmonella Transfer during pilot plant scale washing and roller
conveying of tomatoes. J. Food Prot. 77:380-87.
Wang, H., H. Feng, and Y. Luo. 2004. Microbial reduction and storage quality of fresh-cut
cilantro washed with acidic electrolyzed water and aqueous ozone. Food Res. Intl. 37:949–56.
Warnick, L.D., L.M. Crofton, K.D. Pelzer, M.J. Hawkins. 2001. Risk factors for clinical
salmonellosis in Virginia, USA cattle herds. Prev. Vet. Med. 49:259-275.
Wayadande, A. J. Talley. 2010. Fly Reservoirs of E. coli O157:H7 and their role in
contamination of leafy greens. UC Davis Center for Produce Safety: California Leafy Greens
Research Program. Available at:
http://calgreens.org/control/uploads/1Wayadande_CPS_CLGRP_Final_Report_4-30-10.pdf.
Accessed 15 March 2017.
Wendel, A. M., D. H. Johnson, U. Sharapov, J. Grant, J.R. Archer, T. Monson, C. Koschmann,
and J. P. Davis. 2009. Multistate outbreak of Escherichia coli O157:H7 infection associated with
consumption of packaged spinach, August to September 2006: the Wisconsin investigation. Clin.
Inf. Dis. 48:1079-86.
Weissinger, W. R., W. Chantarapanont, and L. R. Beuchat. 2000. Survival and growth of
Salmonella baildon in shredded lettuce and diced tomatoes, and effectiveness of chlorinated
water as a sanitizer. Int. J. Food Microbiol. 62:123-131.
Williams, A. P., H. Gordon, D. L. Jones, N. J. Strachan, L. M. Avery, and K. Killham. 2008.
Leaching of bioluminescent Escherichia coli O157:H7 from sheep and cattle feces during
simulated rainstorm events. Int. J. Food Microbiol. 105:1452-1460.

98

World Health Organization. 2013. Salmonella (non-typhoidal). Available at:
http://www.who.int/mediacentre/factsheets/fs139/en/. Accessed 2016 5 November.
Zhang S, and J.M. Farber. 1996. The effects of various disinfectants against Listeria
monocytogenes on fresh-cut vegetables. Food Micro. 13:311-21.
Zhang, G, L. V. Ma, H. Phelan and M. P. Doyle. 2009. Efficacy of antimicrobial agents in
lettuce leaf processing water for control of Escherichia coli O157:H7. J. Food Prot. 72:13921397.

99