IMPACT OF SELECTED UNIT OPERATIONS ON THE SPREAD OF ESCHERICHIA COLI
O157:H7 DURING PILOT-SCALE PRODUCTION OF FRESH-CUT LETTUCE
By
Lin Ren

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Food Science – Master of Science
2014

ABSTRACT
IMPACT OF SELECTED UNIT OPERATIONS ON THE SPREAD OF ESCHERICHIA COLI
O157:H7 DURING PILOT-SCALE PRODUCTION OF FRESH-CUT LETTUCE
By
Lin Ren
In response to continued foodborne outbreaks and recalls, two separate studies were
conducted to assess the importance of various unit operations in spreading E. coli O157:H7
during production of fresh-cut leafy greens. Initially, E. coli O157:H7 transfer from a
contaminated shredder and conveyor belt to previously uncontaminated iceberg lettuce, wash
water, and the equipment was quantified during pilot-plant production of fresh-cut lettuce using
9.1 kg of dip-inoculated radicchio (106 CFU/g, colored surrogate for iceberg lettuce) to
contaminate a pilot-plant-scale shredder or conveyor. After processing 90.7 kg of previously
uncontaminated lettuce, average E. coli O157:H7 populations on the shredder and conveyor belt
decreased from 3.7 to 0.9 and from 4.1 to 2.5 log CFU/100 cm2, respectively. E. coli O157:H7
levels in twenty 4.5 kg batches of lettuce collected after exiting the contaminated shredder and
conveyor also decreased significantly (P < 0.05) from 2.7 to 0.9 and from 1.8 to 0.9 log CFU/g
during processing. Thereafter, E. coli O157:H7 transfer from a single inoculated lettuce leaf (6.9
log CFU/g) to wash water was assessed at different water velocities in a custom-made pipe
system. Shedding of E. coli O157:H7 from lettuce was significantly greater at a laminar flow rate
of 0.01 m/s (5.1 log CFU) (P < 0.05) compared to turbulent flow at 0.07 m/s (3.8 log CFU) and
lower laminar flow at 0.005 m/s (3.2 log CFU) with numbers of E. coli O157:H7 peaking in the
water after 10, 4, 4, and 3 s at flow rates of 0.005, 0.01, 0.04, and 0.540 m/s, respectively. All of
these findings will be useful in the development of bacterial transfer models for current and
future risk assessments.

To my mother Guizhi Fu, my father Xiao Ren, my husband Bo Song, and my daughter Meeya
Song
谨以此献给我亲爱的父母，我的爱人和我可爱的女儿

iii

ACKNOWLEDGEMENTS

First and foremost, I would like to express my deepest appreciation to my advisor Dr.
Elliot Ryser for bringing me to food safety area. I really cherish the time being in Ryser’s Lab. It
is my great pleasure to work for and learn from him. Without his trust, support, understanding
and guidance, I would not be where I am today. I would also like to thank Dr. Bradley Marks for
guiding and supporting me through my tough time in research. I would thank Dr. John Linz for
being on my committee and giving me constructive suggestions.
I would like to thank my previous lab mate Gordon Davidson for teaching me the lab
rules and how to work efficiently. I would like to specially thank Hamoud Alnughaymishi,
Andrew Scollon for their friendships. I am really thankful that Guiomar D Posada- Izquierdo
came and help me with my big processing experiment in my first intensive semester. It is a
pleasure to work with Beatriz Mazon. I would also like to thank my all labmates, present and
past for their help, sharing and cares.
Last but not least, I would not thank my parents Guizhi Fu, Xiao Ren and my husband Bo
Song enough for their love and support, without them, I would not have time to finish my
research and thesis. I would give my special thanks to my adorable six-month-old daughter
Meeya Song, her birth let me know what the most beautiful moment in the world is. She taught
me the truth of life and being happy.

Lin Ren

iv

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................................ vii
LIST OF FIGURES ....................................................................................................................... ix
KEY TO SYMBOLS AND ABBREVIATIONS .......................................................................... xi
INTRODUCTION .......................................................................................................................... 1
CHAPTER 1: Review of Pertinent Literature ................................................................................ 5
1.1 Fresh-cut production and consumption ............................................................................ 6
1.2 Fresh-cut safety issues and related outbreaks .................................................................. 8
1.3 Escherichia coli O157:H7 .............................................................................................. 10
1.4 Lettuce contamination during pre-harvest and post-harvest handling ........................... 11
1.4.1 Pre-harvest .................................................................................................................... 11
1.4.2 At-harvest ..................................................................................................................... 12
1.4.3 Post-harvest .................................................................................................................. 13
1.5 Contamination during post-harvest processing .............................................................. 14
1.5.1 Shredding ...................................................................................................................... 14
1.5.2 Conveying ..................................................................................................................... 15
1.5.3 Flume washing .............................................................................................................. 16
1.5.4 Dewatering and drying ................................................................................................. 17
1.6 Industry washing ............................................................................................................ 17
1.6.1 Industry washing system .............................................................................................. 18
1.6.2 Problems in flume washing .......................................................................................... 19
1.7 Washing conditions ........................................................................................................ 21
1.7.1 Flow rates ..................................................................................................................... 21
1.7.2. Flow regime and Reynolds number ............................................................................. 22
1.7.3 Precious studies ............................................................................................................ 23
1.7.3.1 Wash flow rate and shear force ............................................................................. 23
1.7.3.2 Washing time ........................................................................................................ 25
1.7.3.2 Bacterial binding ability, attachment to surfaces, and the surrounding environment
........................................................................................................................................... 25
1.8
Overall goals. ................................................................................................................. 27
CHAPTER 2: Spread of Escherichia coli O157:H7 from a Contaminated Shredder or Conveyor
Belt to Fresh-Cut Iceberg Lettuce during Simulated Commercial Production ............................. 28
2.1 OBJECTIVE........................................................................................................................ 29
2.2 MATERIALS AND METHODS ........................................................................................ 30
2.2.1 Overall experimental design. ........................................................................................ 30
2.2.2 Leafy greens. ................................................................................................................ 30
2.2.3 Bacterial strains. ........................................................................................................... 30
2.2.4 Inoculum preparation. ................................................................................................... 31
v

2.2.5 Radicchio inoculation. .................................................................................................. 31
2.2.6 Processing line. ............................................................................................................. 31
2.2.7 Lettuce processing. ....................................................................................................... 32
2.2.8 Sample collection. ........................................................................................................ 32
2.2.9 Microbiological analyses. ............................................................................................. 33
2.2.10 Statistical analysis....................................................................................................... 33
2.3 RESULTS............................................................................................................................ 37
2.3.1. Radicchio. .................................................................................................................... 37
2.3.2. Iceberg lettuce.............................................................................................................. 40
2.3.3. Contaminated shredder/ conveyor. .............................................................................. 42
2.3.4. Water. .......................................................................................................................... 44
2.4 DISCUSSION ..................................................................................................................... 46
CHAPTER 3: Effect of Water Velocity on Escherichia coli O157:H7 Transfer from Inoculated
Lettuce to Wash Water in a Custom-made Pipe System .............................................................. 49
3.1 OBJECTIVE........................................................................................................................ 50
3.2 MATERIALS AND METHODS ........................................................................................ 51
3.2.1 Experimental design. .................................................................................................... 51
3.2.2 Leafy greens. ................................................................................................................ 51
3.2.3 Bacterial strains. ........................................................................................................... 51
3.2.4 Inoculum preparation. ................................................................................................... 51
3.2.5 Lettuce inoculation. ...................................................................................................... 51
3.2.6 Custom-made pipe system. ........................................................................................... 52
3.2.7 Flow rates. .................................................................................................................... 52
3.2.8 Processing. .................................................................................................................... 52
3.2.9 Sample collection. ........................................................................................................ 55
3.2.10 Microbiological analyses. ........................................................................................... 55
3.2.11 Percentage of E. coli O157:H7 cells recovered. ......................................................... 55
3.2.12 Statistical analysis....................................................................................................... 55
3.3 RESULTS............................................................................................................................ 56
3.3.1 Percentage of E. coli O157:H7 cells recovered. ........................................................... 56
3.3.2 Wash of inoculated lettuce. .......................................................................................... 58
3.3.2.1 Lettuce................................................................................................................... 58
3.3.2.2 Water samples. ...................................................................................................... 60
3.4 DISCUSSION ..................................................................................................................... 62
CONCLUSIONS AND FUTURE RECOMMENDATIONS ....................................................... 66
APPENDIX ................................................................................................................................... 69
REFERENCES ............................................................................................................................. 82

vi

LIST OF TABLES

Table 1.1: Leafy greens associated E. coli outbreaks from 1999 to 2013. Information based on
Foodborne Outbreak Online Database (CDC). The October 2013 outbreak are based on CDC …9
Table 2.1: Mean (±SD) weight (g) of radicchio and mean (±SD) E. coli O157:H7 populations on
equipment surfaces after processing (log CFU/cm2) (n=3). NA stands for not applicable. ......... 39
Table 3.1: Flow properties. ........................................................................................................... 52
Table AI.1: E. coli O157:H7 populations (log CFU/g) on previously uninoculated lettuce
collected (over time along processing) after contacting contaminated shredder. ......................... 70
Table AI.2: E. coli O157:H7 populations (log CFU/g) on previously uninoculated lettuce
collected (over time along processing) after contacting contaminated conveyor. ........................ 71
Table AI.3: E. coli O157:H7 populations (log CFU/g) on previously uninoculated lettuce
collected from each of the 20 collection baskets after washing and shaker table dewatering while
the shredder was contaminated. .................................................................................................... 72
Table AI.4: E. coli O157:H7 populations (log CFU/g) on previously uninoculated lettuce
collected from each of the 20 collection baskets after washing and shaker table dewatering while
the conveyor was contaminated. ................................................................................................... 73
Table AI.5: E. coli O157:H7 (log CFU/g) populations on previously uninoculated lettuce
collected (over time along processing) for each centrifugal drying batch while the shredder was
contaminated. ................................................................................................................................ 74
Table AI.6: E. coli O157:H7 (log CFU/g) populations on previously uninoculated lettuce
collected (over time along processing) for each centrifugal drying batch while the conveyor was
contaminated. ................................................................................................................................ 75
Table AI.7: E. coli O157:H7 populations (log CFU/cm2) on equipment surfaces while the
shredder was contaminated. * representing half of the LOD was used to calculate mean log value
for the surface samples yielded no E. coli O157:H7 colonies on the plates. ................................ 76
Table AI.8: E. coli O157:H7 populations (log CFU/cm2) on equipment surfaces while the
conveyor was contaminated. * representing half of the LOD was used to calculate mean log
value for the surface samples yielded no E. coli O157:H7 colonies on the plates. NA stand for
not applicable ................................................................................................................................ 77
Table AI.9: E. coli O157:H7 (log CFU/ml) populations in water samples for each centrifugal
drying batch while the shredder was contaminated. ..................................................................... 78

vii

Table AI.10: E. coli O157:H7 (log CFU/ml) populations in water samples for each centrifugal
drying batch while the conveyor was contaminated. .................................................................... 79
Table AI.11: Weight of radicchio (g) retrieved from each of the 20 collection baskets after
washing and shaker table dewatering while the shredder was contaminated. .............................. 80
Table AI.12: Weight of radicchio (g) retrieved from each of the 20 collection baskets after
washing and shaker table dewatering while the conveyor was contaminated. ............................. 81

viii

LIST OF FIGURES

Figure 1.1: Consumption of lettuce by type in United States from 1985 to 2013.. ........................ 7
Figure 1.2: Estimated value-added produce sales in select US retailers estimated by Cook from
various sources. ............................................................................................................................... 7
Figure 2.1: Sampling locations on the shredder. Locations with number of 1,3,5,7,9,11, and 13
were sampled before 90.7 kg of iceberg lettuce was processed. Locations with number 2, 4, 6, 8,
10, 12, and 14 were sampled after 90.7 kg of iceberg lettuce was processed……………………34
Figure 2.2: Sampling locations on the conveyor. All sampling locations were sampled after 90.7
of iceberg lettuce was processed when the shredder was contaminated. While the conveyor was
contaminated, locations with number of 15, 17, 19, and 21 were sampled before 90.7 kg of
iceberg lettuce was processed. Locations with number 16, 18, 20 and 22 were sampled after 90.7
kg of iceberg lettuce was processed. ............................................................................................. 35
Figure 2.3: Sampling locations on the flume tank, shaker table and centrifugal dryer. ............... 36
Figure 2.4: Mean (±SE) weight of radicchio retrieved after shaker table dewatering while the
shredder is contaminated (n=3). Mean weights from baskets without common letters are
significantly different (P < 0.05). .................................................................................................. 38
Figure 2.5: Mean (±SE) weight of radicchio retrieved after shaker table dewatering while the
conveyor is contaminated (n=3). Mean weights from baskets without common letters are
significantly different (P < 0.05). .................................................................................................. 38
Figure 2.6: Mean (±SE) E. coli O157:H7 populations on iceberg lettuce after contacting the
contaminated shredder and conveyor (n=3). Means without common letters for the shredder/
conveyor data sets are significantly different (P < 0.05). ............................................................. 41
Figure 2.7: Mean (±SE) E. coli O157:H7 populations on iceberg lettuce collected from each
collection basket after shaker table dewatering (n=3), while the shredder / conveyor was
contaminated. Means without common letters for the shredder/ conveyor data sets are
significantly different (P < 0.05). .................................................................................................. 41
Figure 2.8: Mean (±SE) E. coli O157:H7 populations on the shredder and conveyor (after
processing 9.1 kg of inoculated radicchio) before and after processing 90.7 kg of iceberg lettuce.
Means without common letters are significantly different (P < 0.05). ......................................... 43
Figure 2.9: Mean (±SE) E. coli O157:H7 populations in centrifugation water when the shredder
and conveyor were initially contaminated. Mean E. coli O157:H7 populations with same letter
are not significantly different (P > 0.05). ...................................................................................... 45

ix

Figure 3.1: Custom-made pipe system. Point ‘a’ is the inoculation port in the water study and the
lettuce insertion port in the lettuce study. Point ‘b’ is the water sample spout. Valve ‘c’ is an eye
valve for controlling water flow rates…………………………………………………….……...54
Figure 3.2: Mean E. coli O157:H7 populations in water samples collected during washing of
lettuce at four different flow rates. Half the limit of detection was used to calculate the mean log
value when a 500 ml water sample yielded no colonies by membrane filtration. ........................ 57
Figure 3.3: Mean (±SD) E. coli O157:H7 populations shed from inoculated lettuce into the water
based on the populations before and after washing. Different water flow rates with same letter
are not significantly different (P > 0.05). ...................................................................................... 59
Figure 3.4: Mean log E. coli O157:H7 populations in water samples collected during washing of
lettuce at four different flow rates. Half the limit of detection was used to calculate the mean log
value when a 500 ml water sample yielded no colonies by membrane filtration. ........................ 61
Figure 3.5: Mean (±SD) adjusted log E. coli O157:H7 populations shed from inoculated lettuce
pieces to wash water. Means of the different water flow rates with different letter are
significantly different (P < 0.05). .................................................................................................. 61

x

KEY TO SYMBOLS AND ABBREVIATIONS

ANOVA Analysis of Variance
CDC Centers for Disease Control and Prevention
CFU Colony forming unit(s)
CIP Clean in place
CSPI Center for Science in the Public Interest
d days(s)
EHEC Enterohemorrhagic Escherichia coli
FDA Food and Drug Administration
g grams(s)
GAPs Good Agricultural Practices
GFP Green fluorescent protein
h hour(s)
HUS Hemolytic uremic syndrome
L liter(s)
LOD limit of detection
min minute(s)
ml milliliter(s)
PBS Phosphate Buffered Saline
ppm Parts per million
PVC Polyvinyl chloride
Re Reynold’s number

xi

s second(s)
TSA-YE Trypticase Soy Agar with 0.6 % Yeast Extract
TSB-YE Trypticase Soy Broth with 0.6 % Yeast Extract
US United States of America
USDA United States Department of Agriculture
μl microliter(s)

xii

INTRODUCTION

1

Changes in eating habits are now leading to increased human consumption of fresh
vegetables. Although considered healthy and nutritious, leafy greens can also be a source of
bacterial foodborne pathogens. In 2009, leafy greens were ranked as the riskiest food by the
Food and Drug Administration, responsible for 363 outbreaks and 13,568 reported cases of
illness (Klein et al., 2009). In 2011, another risk ranking study concluded that enterohemorrhagic
Escherichia coli (EHEC) in leafy greens ranked first among the 53 pathogen-produce commodity
pairs considered, based on scenario and sensitivity analyses (Anderson et al., 2011). Between
2000 and 2007, lettuce contaminated with E. coli O157:H7 was responsible for 10 outbreaks and
242 illnesses (Anonymous, 2011). According to the 2012 annual report from CDC, among the 29
E. coli outbreaks, which caused 500 illnesses and 98 hospitalizations, 3 were associated with
contaminated lettuce consumption (CDC, 2012).
Cross-contamination can occur at any point during the farm-factory-fork continuum. At
the farm, lettuce can become contaminated with E. coli O157:H7 from domestic and wild
animals shedding the organism, as well as from contaminated irrigation water and soil (Solomon
et al., 2003; Oliveira et al., 2012).
Workers can further spread pathogens to large quantities of produce during harvesting
and subsequent handling. Although Good Agricultural Practices (GAPs) have been constantly
emphasized by FDA (FDA, 2006), pathogen contamination can still occur with improper storage
or transportation leading to pathogen growth and further spread in packing facilities.
The number of E. coli O157 leafy green-related outbreaks and recalls has risen sharply
during the past 10 years. In 2006, one nationwide E. coli O157:H7 outbreak was linked to
bagged baby spinach which resulted in 205 illnesses, 103 hospitalizations, and 3 deaths. In the
same year, E. coli O157:H7-contaminated lettuce was responsible for two additional outbreaks,

2

which sickened 105 people (CSPI, 2008). In 2010, a multistate outbreak of E. coli O145
infections was linked to shredded lettuce from a single processing facility (CDC, 2011). In a
Salmonella outbreak reported in Australia, shredded lettuce was contaminated by cutting wheel
of a shredder in a processing facility (Stafford et al., 2002).
Buchholz et al. (2012a) previously assessed the number of E. coli O157:H7 cells
transferred from equipment surfaces to lettuce during processing in a small-scale production line
with sanitizer-free water. In this work, 90.8 kg of previously uninoculated lettuce was
contaminated with E. coli O157:H7 from the processing equipment, which was initially
contaminated by processing inoculated lettuce containing E. coli O157:H7 at 106 and 104 log
CFU/g. Even at the lowest initial contamination level of 102 log CFU/g, 0.1 and 0.3% of the
original E. coli O157:H7 inoculum spread to previously uninoculated iceberg lettuce after
processing (Buchholz et al., 2012a). Improper post-harvest handling increases the potential for
cross-contamination during washing (Wachtel and Charkowski, 2002). Commercial processing,
especially washing, further facilitates the spread of foodborne pathogens to large volumes of
leafy greens (CSPI, 2008). Given the above outbreaks, a better understanding of pathogen
transfer during commercial flume washing is warranted. Several laboratory-scale studies
assessing the efficacy of various sanitizers have shown 1-3 log reductions of foodborne
pathogens on lettuce (Beuchat et al., 2004; Gil et al., 2009; Parish et al., 2003). In our previous
work, E. coli O157:H7 transfer from inoculated iceberg lettuce to wash water was quantified
during simulated commercial processing, with a 1 log reduction observed (Buchholz et al.,
2012b). However, commercial washing is a complicated process, with the extent to which
bacterial removal are impacted by a range of biological and physical factors. Lelièvre et al.
(2002) found that the removal of Bacillus cereus spores from a clean-in-place line was strongly

3

dependent on the hydrodynamic conditions during washing. In another study, removal of E. coli
O157:H7 from pre-inoculated apples and cantaloupe during washing increased with the water
flow velocity and agitation rate (Wang et al., 2007). However, the three flow velocities that were
assessed all fell in the laminar flow range, which is much lower than often seen in commercial
practice.
Consequently, the objectives of this research were to:
1. Quantify the spread of E. coli O157:H7 from a product contaminated shredder or

conveyor to subsequent product, other equipment surfaces, and the water during
simulated commercial production of fresh-cut iceberg lettuce.
2. Assess the impact of laminar, transition, and turbulent flow on E. coli O157:H7

transfer from lettuce to water during washing.

4

CHAPTER 1:

Review of Pertinent Literature

5

1.1 Fresh-cut production and consumption
Lettuce is one of the most popular leafy green vegetables. Due to many perceived health
benefits such as reduction of chronic diseases, hypertension, ischemic stroke, and cancer (Hung
et al., 2004; López-Gálvez et al., 2010), consumption of leafy greens continues to increase.
According to Cook (2012), per capita consumption of head lettuce in United States was 12.5
pounds in 2013 , with romaine and leaf lettuce being 7.7 and 3.6 pounds, respectively
(USDA/ERS, 2014) (Figure 1.1). As shown in Figure 1.2, bagged fresh-cut salads comprised the
largest portion of produce sales in 2013 - approximate $3 billion in selected US retailers. In
2010, 200 million pounds of head lettuce and 300 pounds of lettuce were imported, while the
total exported reached 535 million pounds (USDA/ERS, 2011). Due to the significant economic
and nutritional value, lettuce is grown year- round in California and Arizona as well as in
Colorado, New Mexico, Washington, New Jersey, New York, Ohio, and Florida. Approximately
98 percent of commercial domestic output of lettuce is from California and Arizona.

6

Romaine & leaf

Head lettuce

Consumption (Lbs per capita)

35
30
25
20
15
10

5
0
Year

Figure 1.1: Consumption of lettuce by type in United States from 1985 to 2013 (USDA/ERS,
2014).
Fresh-cut fruit ,
15.50%
$582.9M

$3B

$1.4B
Value-add
vegetable
27%

Bagged salads,
60.80%

Figure 1.2: Estimated value-added produce sales in select US retailers estimated by Cook from
various sources (Cook, 2012).

7

1.2 Fresh-cut safety issues and related outbreaks
Fresh vegetables are inherently prone to bacterial contamination from infected wild and
domestic animals, soil, contaminated irrigation water, soil amendments, sewage, air and poor
worker hygiene (Nguyen-the & Carlin 1994, Nou & Luo 2010). Processing of fresh-cut fruits
and vegetables introduce wounds and cuts to the tissues, which leads to an increased respiration
rate (Watada et al., 1996). Vegetables are prone to bacterial attack and invasion which can lead
to various types of spoilage depending on the product pH (Heard, 2002). Damaged cells at the
cut surfaces of vegetables increase the sugar supply and provide microbial pathogens an
opportunity to adapt and grow.
As Americans change their eating habit towards more healthy food, the incidence of
foodborne outbreaks related to fresh produce has continued to increase. Among the 25 major
monitored microbial pathogens, Salmonella, Escherichia coli O157:H7 and Listeria
monocytogenes are the bacterial foodborne pathogens of most concern in fresh produce (FDA,
2008). Escherichia coli O157:H7 and Salmonella enterica have been most frequently linked to
illnesses involving fresh produce (Sivapalasingam et al., 2004), and have received considerable
attention following two leafy green outbreaks in 2006 and 2007.
Leafy greens are leafy vegetables that include Romaine lettuce, green leaf lettuce, red
leafy lettuce, baby leafy lettuce, escarole, endive, spring mix, spinach, cabbage, kale, and chard
(Matthews, 2014). From 1998 to 2008, the number of annual cases of foodborne illnesses was
estimated at 9.6 million, among which leafy vegetable-related illnesses attributed to 22% of the
cases in United States (Painter et al., 2013). Overall, 57,000 of 9.6 million individuals were
hospitalized, with 14% of these hospitalizations caused by leafy vegetables. Leafy greens
accounted for 6% of the 1451 fatalities annually (Painter et al., 2013). The Food and Drug

8

Administration ranked leafy greens as the riskiest food in 2009, responsible for 363 outbreaks,
and 13,568 illnesses (Klein et al., 2009), with enterohemorrhagic E. coli having the greatest
impact on human health. E. coli outbreaks since 1999 associated with leafy green consumption
are listed in Table 1.1 (CDC, 2014).
Table 1.1: Leafy greens associated E. coli outbreaks from 1999 to 2013. Information based on
Foodborne Outbreak Online Database (CDC). The October 2013 outbreak are based on CDC
(CDC, 2013).
Date
Location
Pathogen
Illnesses Reported
Produce
Feb. 99

NE

E. coli O157:H7

65

Lettuce based salad

Jul. 99

OH

E. coli O157:H7

18

Coleslaw

Aug. 99

Multistate

E. coli O157:H7

14

Romaine lettuce

Oct. 99

OH

E. coli O157:H7

47

Salad

Oct. 99

Multistate

E. coli O157:H7

45

Romaine lettuce

Nov. 01

TX

E. coli O157:H7

20

Lettuce-based salads

Jul. 02

WA

E. coli O157:H7

55

Caesar salad

Nov. 02

Multistate

E. coli O157:H7

16

Iceberg lettuce

Sep. 03

CA

E. coli O157:H7

51

Lettuce-based salads

Oct. 03

CA

E. coli O157:H7

16

Spinach

Nov. 04

NJ

E. coli O157:H7

6

Lettuce

Sep. 05

Multistate

E. coli O157:H7

34

Lettuce, prepackaged

Oct. 05

Multistate

E. coli O157:H7

12

Lettuce, prepackaged

Aug. 06

Multistate

E. coli O157:H7

238 (5 deaths)

Spinach

Nov. 06

Multistate

E. coli O157:H7

77

Lettuce

Nov. 06

Multistate

E. coli O157:H7

80

Lettuce

Jun. 07

AL

E. coli O157:H7

26

Lettuce-based salad

May 08

WA

E. coli O157:H7

10

Spinach

Aug. 08

Multistate

E. coli O157:H7

13

Spinach

Apr. 09

Multistate

E. coli O157:H7

16

Lettuce

Sep. 09

Multistate

E. coli O157:H7

22

Lettuce

Apr. 10

Multistate

E. coli O145

31

Romaine lettuce

9

Table 1.1 (cont’d)
Date
Location

Pathogen

Illnesses Reported

Produce

Oct. 11

MN

E. coli O157:H7

2

Romaine lettuce

Oct. 11

Multistate

E. coli O157:H7

26

Lettuce

Oct. 11

Multistate

E. coli O157:H7

60

Romaine lettuce

Apr. 12

CA

E. coli O157:H7

12

Lettuce

May. 12

GA

E. coli O157:H7

2

Iceberg lettuce

Jun. 12

Multistate

E. coli O157:H7

52

Romaine lettuce

Sep. 12

PA

E. coli O157:H7

9

Romaine lettuce

Oct. 12

Multistate

E. coli O157:H7

33

Prepackaged leafy greens

Oct. 12

Multistate

E. coli O145

16

Lettuce

Nov. 12

WA

E. coli O157:H7

4

Spinach

Nov. 12

MA

E. coli O157:H7

8

Leaf lettuce

Oct. 13

Multistate

E. coli O157:H7

33

Salad

E. coli O157:H7 outbreaks have continued to occur with the majority of the producerelated outbreaks associated with lettuce and spinach. In August 2006, a multistate E. coli
O157:H7 outbreak which caused 238 illnesses and 5 deaths was linked to consumption of bagged
spinach. FDA traced the spinach back to a company in California’s Salinas Valley. Four spinach
fields were possible origins of contamination with the same outbreak strain isolated from cattle
and a wild boar in nearby fields.
The E. coli O145 outbreak in April 2010 was related to the consumption of shredded
Romaine lettuce from a single processing facility (CDC, 2011). Therefore, the outbreaks
indicated that the microbial hazards present during pre-harvest and harvest cannot be completely
eliminated by post-harvest handling or processing.
1.3 Escherichia coli O157:H7

10

Escherichia coli is a Gram- negative, facultatively anaerobic rod-shaped bacteria which is
commonly found in the intestinal tract of humans and animals. Most strains are not harmful,
however, Escherichia coli serotype O157:H7 (expressing the O-antigen 157 and the H-antigen 7)
belongs to the group of pathogenic enterohemorrhagic E. coli (EHEC). EHEC are virulent due to
their ability to produce shiga- like toxins, adherence factors and enterohemolysin. E. coli
O157:H7 produces fimbriae that aid in attachment and colonization of intestinal mucosal cells
(Ashkenazi et al., 1992). Clinical symptoms of E. coli O157:H7 infections vary from non-bloody
diarrhea to hemorrhagic colitis and hemolytic uremic syndrome (HUS) which is the leading
cause of acute renal failure in children (Proulx et al., 2001).
E. coli O157:H7 was first recognized as a foodborne pathogen in 1982 when under
cooked hamburgers were linked to a large outbreak. Cattle are known as a main reservoir for E.
coli O157:H7, and can shed the pathogen in feces at level as high as 106 CFU/g (Chase-Topping
et al., 2007). However, some other species such as sheep were also reported to shed E. coli
O157:H7 at similar levels (Ogden et al., 2005). E. coli O157:H7 can survive for long periods of
time at a pH as low as 3.4 (Benjamin & Datta. 1995) in apple cider (Zhao et al., 1993) and
mayonnaise (Raghubeer et al., 1995). Furthermore, the low infectious dose of 10 - 100 cells
(FDA 2012) is problematic when fresh produce is consumed raw without further processing.
1.4 Lettuce contamination during pre-harvest and post-harvest handling
1.4.1 Pre-harvest
Before harvest, foodborne pathogens can harbor and persist in contaminated irrigation
water (Erickson et al., 2010; Islam et al., 2005; Solomon et al., 2002), sewage, soil (Ma et al.,
2014), dust, insects (Hancock et al., 1998; Rahn et al., 1997), animals (Doane et al., 2007),
animal feces (Kudva et al., 1998; Wang et al., 1996), and improperly treated soil amendments

11

(Islam et al., 2005; Jensen et al., 2013; Johannessen et al., 2005). Field workers can further
contaminate the lettuce during harvesting. Pathogens can attach to the leaf surfaces and/or
internalize into the lettuce tissue or be up taken by the roots from soil or irrigation water into
internal tissues. Pathogens tend to attach to the stomata, lenticels or sites of biological or
physical damage on the lettuce surface. Survival, contamination and internalization are affected
by many factors such as humidity, temperature, health and maturity of the plant (Kroupitski et
al., 2009; Pu et al., 2009), and motility of the pathogen (Kroupitski et al., 2009).
In most cases, foodborne pathogens are difficult to distinguish from microorganisms on
produce. The microbial populations on lettuce may not be sufficient to cause decay but may be
enough to cause human infection due to the low infectious dose of pathogens. Therefore,
perfection in appearance is not a guarantee for microbial safety. Increased outbreaks of E. coli
O157:H7 in recent years have prompted significant efforts towards the development and
implementation of improved Good Agricultural Practices (GAPs) to minimize pathogen
contamination in the field.
1.4.2 At-harvest
Lettuce is harvested as soon as the plants reach acceptable size and firmness (Salunkge
and Kadam, 1998). In most cases, lettuce is field packed and usually cut with a long-handled
sharp knife. Heads can be wrapped or bagged in water vapor permeable plastic film (Salunkge
and Kadam, 1998) by the cutter or packer. Cross-contamination might occur due to these
harvesting activities. Taormina reported that a coring knife contaminated by soil containing 2.72
and 1.67 log CFU/g E. coli O157:H7 further contaminated all 10 subsequent lettuce heads by
cutting and coring. Prolonged time for post-inoculation of knife did not decrease the likelihood
of lettuce being contaminated (Taormina et al., 2009). More cuts and damage introduced to

12

lettuce during harvest facilitates the survival, internalization and growth of pathogens, and make
the control and disinfection of the product during post-harvest handling even difficult.
1.4.3 Post-harvest
Soon after harvest, lettuce is usually transported from the field to a cooling warehouse to
be pre-cooled to -1 °C at 98%-100% relative humidity (Salunkge and Kadam, 1998). This precooling step is the most commonly used treatment to improve quality and prolong the shelf life
of lettuce. However, Li et al. (2008) reported that vacuum cooling had a significant impact on E.
coli O157:H7 cell infiltration into lettuce tissue. With vacuuming, a significantly larger portion
of cells were internalized in the lettuce tissue than that for the control group. Internalized
bacterial cells are unlikely to be removed during washing at fresh-cut processing facilities
(Aruscavage et al., 2006).
After pre-cooling, lettuce is transported in refrigerated trucks to maintain quality on the
way to the grocery store. After being sent to stores, lettuce is generally not stored for long
periods at 2°C. However, precise temperature control during transportation between the
manufacture and retailer are challenging due to exchange with extreme outside temperatures
during produce loading and unloading. Koseki & Isobe (2005) reported that the temperature for
lettuce in the supply chain fluctuated between 3° and 15°C. In a study conducted by McKellar et
al. (2012), increased temperatures were also observed during transport although the temperature
remained below the minimal growth temperature for E. coli O157:H7. However, this study was
conducted in winter with greater temperature increases to be expected during the warmer
months. The impact of temperature on E. coli O157:H7 growth during storage has been assessed
in many studies (Chua et al., 2008; Luo et al., 2010; McEvoy et al., 2009; Oliveira et al., 2010).
If E. coli O157:H7 is able to survive during post-harvest handling and processing, it is likely to

13

recover and grow during refrigerated storage (Delaquis et al., 2002; Diaz & Hotchkiss, 1996;
Oliveira et al., 2010).
1.5 Contamination during post-harvest processing
Lettuce harvested for processing is placed in large bulk bins for transportation to the precooling or processing facility. Lettuce can be cored either in the field or at local processing
facility. At the processing facility where the temperature is generally between 0-5°C (Kitinoja
and Gorny, 1999), lettuce which is processed for salads is cut by shredder, washed in cold,
chlorine-containing water, centrifuged to remove the extra moisture and then packaged. The
equipment used in processing facilities varies, but mostly includes a shredder, conveyor, flume
tank, and dewatering centrifugal dryer. Any human foodborne pathogens contaminating lettuce
in the field or at harvest could be transferred to a large quantity of product during subsequent
processing. Outbreaks traced back to packaging facilities have been reported, therefore, Good
Manufacturing Practices (GMPs) play an important role in preventing the risk of foodborne
pathogens contamination during processing and packing.
1.5.1 Shredding
At the shredding step, lettuce passes through the shredder/cutter and is cut into certain
sizes according to commercial needs. Shredder surface/ blades can be contaminated by fieldcontaminated lettuce or even animal feces/contaminated soil particles carried by the lettuce. In
May of 2001, 41 cases of Salmonella Bovismorbificans infection were reported. The infections
were associated with consumption of shredded lettuce. An investigation was conducted and
suggested that the contamination was from cutting equipment in a processing plant (Stafford et
al., 2002). If the shredding equipment is contaminated and efficient cleaning and sanitation
cannot be implemented immediately, it is highly possible that pathogens persist on surfaces and

14

contamination may be spread to other product during continued production. Moore et al. (2003)
conducted a study to evaluate the ability of Salmonella Typhimurium and Campylobacter jejuni
to transfer from stainless steel surfaces to both wet and dry romaine lettuce through direct
contact. For S. Typhimurium, 66% of the population transferred from dry stainless steel to dry
lettuce after 60 minutes of direct contact. In contrast, 23% to 31% of the Salmonella population
transferred from dry stainless steel to wet lettuce after 120 minutes of direct contact. Transfer of
C. jejuni to dry and wet lettuce was similar, ranging from 16% to 38% and from 15% to 27%
after 80 minutes of contact, respectively. These results suggest that effective cleaning and
sanitizing protocols should be developed since bacteria can attach to equipment surfaces and
produce biofilms.
Although the temperature in processing facilities is low, it is still possible for bacteria to
form biofilms. Research by Rossi et al. (2013) showed that at 4°C, out of the 32 strains isolated
from milk and vegetables, 20 strains (62.5%) were able to form biofilms on stainless steel in 24
hours, and 28 and 27 strains formed biofilms after 48 and 72 hours, respectively. At 20°C, more
strains were found to form biofilms than at 4°C.
1.5.2 Conveying
After being shredded, lettuce is step-conveyed and then flume washed. Conveyor can be
present at many different points of the processing line and play a major role in transportation.
Conveyors used in the leafy green industry are mainly smooth belted with one seam. Various
materials can be used for conveyor belts such high-density polyethylene, polypropylene, and
acetyl (Buchholz et al., 2010). In one study by Allen et al. (2005) in Florida, the survival of
Salmonella on a packing line was evaluated. Five types of surface materials were assessed,
including stainless steel, polyvinyl chloride (PVC), sponge rollers, conveyor belts and an

15

unfinished oak surface. During simulated spring conditions with a temperature of 30°C and a
relative humidity of 80%, Salmonella were able to survive on these conveyors for 3 days while
in simulated fall/ winter conditions with temperature of 20°C and humidity of 60%, Salmonella
were able to survive on the same materials for 21 days.
Although conveyor belt surfaces are visually smooth, crevices and holes can be observed
by scanning electron microscopy (Mafu et al., 1990). These crevices and holes serve as
attachment sites for bacteria. Therefore, cleaning and sanitizing conveyors remains challenging.
Rossi et al. (2013) reported, if the sponges used to clean stainless steel and polyethylene surfaces
were contaminated with fecal coliforms at 5 log CFU, then transfer varied from 3.3 to 5.5 log
CFU/cm2 for stainless steel and from 3.5 to 5.6 log CFU/cm2 for polyethylene. More than 1 log
CFU/cm2 survived after 24 hours on both surfaces.
1.5.3 Flume washing
In commercial processing, shredded lettuce is washed in recirculating cold water with
sanitizer for 30 seconds to 2 minutes to remove soil and debris (Herdt and Feng, 2009).
Commercial sanitizers used in leafy green industry include chlorine-based sanitizers,
peroxyacetic acid and mixed peracid (Herdt and Feng, 2009), with chlorine used most
commonly. The purposes of the washing are to reduce the temperature of the produce, provide an
appealing appearance, prolong shelf life and reduce the microbial load, which are also critical for
microbial safety (Herdt and Feng, 2009). However, the efficacy of the most commonly used
chlorine-based sanitizer in commercial washing is questionable since antimicrobial activity is
affected by organic load. In leafy green processing, the organic load of wash water consists of
debris, cellular fluids released from cut leaves, soil particles, insects, and microbes (Herdt and
Feng, 2009). It accumulates and increases during washing (Nou and Luo, 2010). The decreased

16

efficacy of sanitizers make the wash water an ideal vehicle of transferring microorganisms to a
much larger volume of produce and increases the risk of cross-contamination.
1.5.4 Dewatering and drying
After flume washing, excess water must be removed before packing to maintain product
quality and optimal shelf life. Leave that are wet with microorganisms or pathogens increase the
possibility of microbial proliferation and encourage spoilage or even microbial hazards. Various
types of dewatering and drying equipment can be used such as shaker/ spinner, forced-air drying
tunnel, blower, or centrifugal dryer (Buchholz et al., 2010; Turatti, 2011; Krasaekoopt and
Bhandari, 2011). Intensity is the main factor that needs to be adjusted for various products.
Shredded iceberg and romaine lettuce is normally spin blow dried followed by centrifugal drying
or centrifugally dried alone. Other products that cannot withstand intensive drying such as
centrifugal drying can be treated with other drying processes such as spin drying or drying in
drying tunnels. If wash water is contaminated, it is highly possible that lettuce leaves with
contaminated water further spread the contamination to drying equipment. In the work of
Buchholz et al. (2012b), leafy greens previously inoculated with E. coli O157:H7 that were
washed in sanitizer-free water were able to transfer ~90% of the original inoculum to wash water
and contaminate the whole processing line. Centrifugal drying, followed by conveying and
shredding, picked up more E. coli O157:H7 from product than the flume tank and shaker table.
1.6 Industry washing
In the fresh produce industry, washing is a critical step to eliminate potential chemical,
physical and microbial hazards. Since fresh-cut leafy greens are consumed raw, washing is even
more crucial to assure the quality as well the safety of the product.

17

1.6.1 Industry washing system
Leafy greens are crisp and subject to bruising and damage, therefore, other washing aids
such as surface brushing can be used for apples and tomatoes but not leafy greens. Therefore, a
well-designed washing system and an effective washing process are needed.
Multiple washes is more effective than a single wash if the produce is washed with fresh
water (Gil et al., 2013). Normally, an optimal washing system consists of three tanks (Gil et al.,
2013), with each tank having its own specific function in washing. The first of these tanks, is
used as a pre-washing procedure and aims to eliminate soil particles or other debris attached to
produce and conveys the product to later washing steps. The significance of the first step
washing is maximum removal of organic matter that might hinder sanitizer efficacy and ensure
washing effectiveness. In most cases, the water flow rate in this tank is comparatively low. In
some cases, the washing system includes some pipes at the bottom that blow high volumes of air
to ensure that the produce is floating on the water surface (Simons and Sanguansri, 1997). It also
generates a strong jaccuzi effect that causes produce to move and tumble, creating the
mechanical action needed to loosen and remove dirt and debris (FSAI, 2001). This dirt and
debris floating on water surface or sinking to the bottom of the tank, is either removed by an
incorporate system (Turatti, 2011) or cleaned by release through a periodic draining system. The
organic load and microbial counts accumulate rapidly in this tank, therefore, proper water
management is necessary. Proper sanitizing agents and treatment conditions should be applied
and frequently monitored to ensure water quality.
The second tank is where sanitation/decontamination takes place. Wash water added with
sanitizing agent further reduces the microbial load and prevents cross-contamination (Herdt and
Feng, 2009; Luo et al., 2011). The type of tank varies, it could be a flume tank with/without

18

overhead jets or a submersion tank with/or without spray disinfection application (FDA, 2008;
FDA, 2008). Generally, the flow rate in the flume tank is much higher than in the dump tank.
Water in the flume tank is recirculated, organic matter accumulates in the water which consumes
sanitizer and decreases the ability of the sanitizer to minimize cross-contamination (Zhang et al.,
2009), thereby the sanitizer concentration needs to be monitored and adjusted frequently. In
some cases, the flume tank is equipped with overhead jets. In addition to shear forces generated
by liquid flow and turbulence, wash water ejected by these jets generates a strong force which
acts on the produce surface (Dezuniga et al., 1991) to prolong the contact time with the sanitizer.
A submersion tank filled with sanitizer can be used as another method for decontamination.
Residence time of produce and concentration of sanitizer in wash water are key factors for
microbial reduction (FSAI, 2001). Sometimes, a submersion tank is used as a prewash step, after
which a sanitizer spray is applied to the produce surface for decontamination.
The third tank is not always present in all cases, however, the main purpose of the last
washing step is to eliminate sanitizer residues on the produce, give the produce a final wash and
reduce the temperature of the produce. A tank used here should be filled with sanitizer-free water
with the quality of water maintained in good condition. In some cases, a sanitizer-free spray is
used instead (Herdt and Feng, 2009).
1.6.2 Problems in flume washing
Generally, in postharvest processing of fresh produce, washing is the only intervention
activity. It is a key procedure to reduce potential microbiological hazards. Any foodborne
pathogen that contacted and contaminated produce in the earlier stage of harvesting is supposed
to be eliminated.

19

To increase washing efficacy, several sanitizing agents are used in the fresh-cut
processing industry to reduce potential microbial hazards, including chlorine-based sanitizers,
peroxyacetic acid and mixed peracid (peroxyacetic acid, organic acids, hydrogen peroxide, acetic
acid) (Herdt and Feng, 2009), among which, chlorine is the most widely used ( López-Gálvez et
al. 2009) due to its ease of use and relatively low cost. Numerous researchers (López-Gálvez et
al., 2009; Parish et al., 2003; Pirovani et al., 2004, Singh et al., 2002; Gil et al., 2013) have
assessed the reduction of pathogens on lettuce using sanitizers. However, the organic load builds
up quickly in dump and immersion tanks during washing. In addition, chlorine will react with
organic and inorganic matter in the wash water, with the efficacy of chlorine deceasing rapidly as
the organic load increases (Gil et al., 2013). Davidson et al. (2013) evaluated the efficacy of
some other sanitizers. After washing a 5.4 kg batch of pre-inoculated lettuce containing E. coli
O157:H7 at 6 log CFU/g with water alone and water with peroxyacetic acid, the processing
water yielded 3.47 and 3.01 log CFU/ml E. coli O157:H7, respectively, after 90 s of washing.
Mixed peracid and peroxyacetic acid yielded similar results in terms of E. coli O157:H7
reduction. Hence, use of a sanitizer does not ensure a microbiologically safe final product. In
some European countries, including Germany, the Netherlands, Denmark, Switzerland and
Belgium, use of antimicrobial agents such as chlorine in during washing of fresh-cut produce
washing is forbidden (Artés & Allende 2005; Nguyen-the and Carlin, 1994).
Cleaning of the processing/washing equipment is another potential problem. Most freshcut processing plants are using clean-in-place (CIP) cleaning. CIP means that the interior
surfaces of pipes, vessels, process equipment, filters and associated fittings are cleaned without
disassembly. If a sanitizer is not functioning during washing due to the increased organic load
and solid content in water, fresh produce carrying foodborne pathogens has chance to

20

contaminate wash water, other produce and the processing equipment. Biofilms (Ryu and
Beuchat, 2005) can be formed by E. coli O157:H7 on stainless steel and protect microorganisms
from sanitizers.
In industry, washing system vary from plant to plant. It is hard to define a best washing
system due to the different intrinsic properties of fresh produce being washed and different
requirements in microbial reduction, product appearance and washing costs. The exact sanitizer
type, wash time, flow rate, pH and temperature etc. in washing conditions for optimal washing of
produce remain unknown.
1.7 Washing conditions
Washing, in particular, is a sanitizing practice, the efficiency of which is often affected
by different factors (Adams et al., 1989). Washing conditions vary depending on the processing
plant and produce characteristics. Both the chemical factors, such as the types (Beuchat et al.,
2004; Davidson et al., 2013; Keskinen and Annous, 2011; Keskinen et al., 2009; López-Gálvez
et al., 2009; Singh et al., 2002) and concentrations (Keskinen and Annous, 2011; Kim et al.,
2008; Luo et al., 2011; Barrera et al., 2012) of sanitizer, and physical factors, such as wash water
flow rate, water hardness, washing time (Barrera et al., 2012), pH (Akbas & Ölmez 2007; Park et
al., 2011; Yang et al. 2003), temperature (Baur et al., 2005; Martin-Diana et al., 2005), waterproduct ratio (Lu et al. 2007; FDA, 2008) and organic load can influence the effectiveness of
washing (FDA, 2008). In addition, surface roughness of the washed object, and adhesion force of
different bacteria are also considered as factors that affect washing efficiency.
1.7.1 Flow rates
In fluid dynamics, flow rate refers to the mass flow rates or volumetric flow rate. The
volumetric flow rate is the volume of fluid which passes per unit time, whereas mass flow rate is

21

the mass of a fluid which passes per unit time (Semat and Katz, 1958). Under steady-state
conditions, the mass and volumetric flow rates remain constant.
Flowmeters are used to measure flow rates. End-line and in-line flowmeters are the two
broad categories, with end-line flowmeter used at the outlet or discharge of the flow and in-line
flowmeter placed in line with the pipe, respectively (Cimbala, 2009). End-line flowmeter is also
called discharge flowmeter: it calculate the flow rate by measuring the time to fill up a container
of known volume. Positive-displacement flowmeter is a commonly used in-line flowmeter which
measures the volumetric flow rate by collecting a fixed volume of fluid and then counting the
time needed for collection. Various units are used depending on the application and industry, but
might include gallons per minute, liters per second or cubic meters per second.
Flow velocity is the distance liquid particles travel per unit of time (Semat and Katz,
1958). It mathematically describes the motion of a fluid.
1.7.2. Flow regime and Reynolds number
Three different flow regimes are recognized - laminar, transitional, and turbulent flow
(Singh and Heldman, 2014). Laminar flow occurs when a fluid travels smoothly or in parallel
layers. In contrast, turbulent flow occurs when a fluid undergoes irregular fluctuations with
lateral mixing. Between laminar and turbulent flow, there is an intermediate level with both
laminar and turbulent called transitional flow. A simple dye can be used to characterize the type
of flow. At low flow rates, the dye moves in a linear manner in the axial direction, which is
characteristic of laminar flow. As the flow rate increases to some intermediate level, the dye
begins to blur at a certain distance away from the injection point, which is due to some of the dye
moving in a radial direction. At high flow rates of turbulent flow, the dye blurs immediately at
the point of injection and spreads in a random manner along both the radial and axial directions.

22

The Reynolds number (Re) is a dimensionless number (Munson et al., 2009), obtained
from the ratio of the inertial resistance to viscous resistance for a flowing fluid (as shown in the
equation below). It is a critical value for quantitatively describing the characteristics of a fluid
flowing either in a pipe or on the surfaces of objects with different shapes. It can be used to
specifically identify how a given liquid would behave under selected flow conditions.
𝑁𝑅𝑒 =

̅𝐷
𝜌𝑢
𝜇

(Singh and Heldman, 2014)

In this equation, ρ is the liquid density, D is tube diameter, 𝑢̅ is the average fluid velocity,
and μ is the liquid viscosity.
In a pipe, laminar flow has a Reynolds number of 2100 or less. When the Reynolds
number is greater than 4000, the type of flow is turbulent. Any Reynolds number between 2100
and 4000 signifies a transitional flow rate (Singh and Heldman, 2014).
1.7.3 Precious studies
1.7.3.1 Wash flow rate and shear force
The prime goal of produce washing is to reduce the microbial load (Ukuku and Fett,
2002). Because bacteria usually attach to the surface of produce by binding or adhesion forces,
additional force, particular shear force, is needed to break these bonds and detach the cells.
Bacterial binding to the produce surface depends on the surface characteristics of both the
bacterium and the material surface. For example, in ligand/receptor mediated attachment, the
number of bonds is a function of ligand and receptor density (Hubble et al., 1996). The number
of effective bonds between bacteria and the substrate surface determines the amount of
additional shear stress needed to detach the organism. There is an optimum flow rate (shear
force) for bacterial attachment that reflects the balance between the rate of delivery and the force
acting on attached bacteria (An and Friedman, 1998; Liu and Tay, 2002). Generally, high shear
23

rates on produce surfaces during washing increase detachment of bacteria (Chang et al., 1991).
At higher flow rates, a higher shear force is applied to attached bacteria, and the potential to form
higher numbers of bonds is of much greater significance (Hubble et al., 1996). When bacteria are
exposed to a sanitizer, increased turbulence during washing may aid in breaking up bacterial
clumps, thus increasing accessibility of the sanitizer (Wang et al. 2007). However, in some cases,
increased shear force tends to enhance the attachment of bacteria and their binding surfaces.
Thomas et al. (2004) and Rangel et al. (2013) reported higher shear forces facilitated the binding
of E. coli O157:H7 from FimH (a lectin-like protein responsible for adhesion properties of type
1 fimbriae).
Flow rate is an important factor that affects the efficiency of washing. Most laboratory
studies have focused on laminar flow. Wang et al. (2007) assessed E. coli O157:H7 reductions
from the surfaces of cantaloupe rind and cut apples under different washing conditions. After 3min of washing in a flow-through chamber, E. coli O157:H7 populations on cantaloupe rind and
cut apples decreased 2.5 and 2.3 log CFU/cm2, respectively, as the flow velocity increased from
0.0 to 0.52, and 0.65 to 0.8 m/min. Populations of E. coli O157:H7 decreased as the flow
velocity increased in both chambers (Wang et al., 2007). However, contradictory results were
reported by (Huang et al., 2013). In their research, a comparison in L. monocytogenes reductions
was made between dipping and washing with tap water. At an initial inoculation level of 3 log
CFU/g, the reduction caused by washing under running tap water (2 L/min) was less than
immersing in water. However, the treatment times for dipping (5 min) and washing (1 min) were
different; thus, it was difficult to conclude that dipping achieve a better performance than
washing. In order to quantitatively investigate the relationship between flow rate and bacterial
detachment, a number of studies have been conducted (Huang et al., 2013; Katsikogianni and

24

Missirlis, 2010; Mbaye et al., 2013; Yoshihara et al., 2014). Almost all of them suggested that
bacteria reduction was dependent on flow rate, with higher flow rates resulting in greater
reductions.
1.7.3.2 Washing time
Simply prolonging the washing period offers no significant improvement in bacterial
removal, except for the initial stage of washing. Adams et al., (1989) observed a rapid drop in
numbers of bacteria during the first 5 min of washing. Increasing the wash time for fresh lettuce
with hypochlorite did not result in further microbial reductions, even though 60% of the original
free available chlorine remained after 30 min of washing. In addition, 20 min of produce
washing yielded similar results, with a 10 min extension yielding no further improvement
(Adams et al., 1989)
Similar results were observed by Wang et al. (2007) who assessed removal of E. coli
O157:H7 during washing of fruits. During the first 2 to 3 min, a relatively rapid reduction in E.
coli O157:H7 was observed. However, extending the wash time an additional 3 min did not
significantly increase bacteria removal at the different flow velocities (Wang et al., 2007).
At the beginning of washing, time is an important factor regarding the reduction of
bacterial population. (Frank, 2001) proposed that washing lettuce under running tap water
permitted a maximum of 1 log CFU/g reduction in approximately 30 s; however, 10 s of washing
resulted in only a 0.6 log CFU/g reduction, with no difference in reduction seen after 5 and 15
min.
1.7.3.2 Bacterial binding ability, attachment to surfaces, and the surrounding environment
The ability to attach and detach from surfaces is an intrinsic characteristic of
microorganisms. Bacterial attachment is the initial stage of pathogen infections, which allows

25

bacteria to further grow and survive. Attachment is influenced by the physical/chemical
properties of the cell surface, contact surface, and the surrounding environment (Frank, 2001).
Capsule and fimbriae are two major functional structures on bacterial cell surface for attachment.
The capsule of a bacterial cell often contains acidic residues which that impart a negative charge
to the bacteria surface (Sutherland, 1985). This hydrophilic capsule prefers a hydrophilic surface
and prevents adhesion between bacteria and hydrophobic surfaces (Ofek and Doyle, 1994). The
amino acids in fimbriae contain numerous nonpolar side chains that impart hydrophobicity to the
structure (Bitton and Marshall, 1980; Frank, 2001). The FimH adhesin is a two-domain protein
located on the tip of type 1 fimbriae. The E. coli adhesin FimH has two highly conserved
cysteines that form a disulphide bond under its ligand-binding (Choudhury et al., 1999). In the
research of (Nilsson et al., 2007), FimH was found to bind to mannose-coated surfaces through
catch bonds, which persist longer under higher tensile force. Thus, fluid flow-induced tensile
forces can significantly enhance the binding strength of bacteria (Nilsson et al., 2007).
Surface properties of produce can also contribute to microbial adhesion. These properties
include surface roughness and hydrophobicity. Surface roughness influences bacterial attachment
and removal from a surface (Wang et al., 2009). Han (2000) suggested that E. coli O157:H7
preferentially attached to coarse or injured surfaces compared to uninjured surfaces of green
peppers. Hydrophobicity relates to the physical properties of the surface of produce, which tends
to be non-polar and repel water. However, the relationship between surface hydrophobicity and
surface roughness of fresh produce is still unknown, although some comparison studies have
been conducted to evaluate the influence of surface roughness. (Fernandes et al., 2014) proposed
that surface roughness was not a dominant factor for bacterial adhesion. Both green peppers
(average roughness (Ra) =13.0 ± 2.7 nm) and melons (Ra =33.5 ± 7.9 nm) showed bacterial

26

adhesion (7.3 and 7.0 log CFU/cm2 for E. coli and S. enterica Enteritidis, respectively). These
findings suggest that adhesion is a multifactorial process (Fernandes et al., 2014).
Bacterial attachment and detachment is also influenced by temperature (Jaglic et al.,
2011; Rajkowski and Fan, 2008; Shwartz et al., 2003) and pH (Lewis et al., 1989). Normally,
bacterial detachment increases with increasing temperature and pH. This can be explained by the
thermal and pH inactivation of bacterial pathogens (Dixon et al., 2012; Spinks et al., 2006).
1.8 Overall goals.
In commercial fresh-cut processing facilities, microbial contaminants including bacterial
pathogens can spread to large volumes of product during washing, particularly when chlorinebased sanitizers become less effective in the presence of high organic loads. On one Salmonella
Bovismorbificans outbreak in May of 2001 (Stafford et al., 2002), contamination spread from a
shredder to subsequent product being processed. Therefore, it is important to assess the potential
contribution of mechanical shredding and conveying as well as washing at different flow rates in
spreading E. coli O157:H7 during simulated commercial production of fresh-cut leafy greens.
Such work is critical for the development of predictive mathematical models for pathogen
transfer during production of fresh-cut produce.

27

CHAPTER 2:

Spread of Escherichia coli O157:H7 from a Contaminated Shredder or Conveyor Belt to
Fresh-Cut Iceberg Lettuce during Simulated Commercial Production

28

2.1 OBJECTIVE
The objectives of this study were to 1assess the transfer of E. coli O157:H7 from a
product-contaminated shredder and conveyor to iceberg lettuce, wash water, and equipment
surfaces during the production of fresh-cut iceberg lettuce

29

2.2 MATERIALS AND METHODS
2.2.1 Overall experimental design. A small-scale commercial leafy green processing
line consisting of a shredder, conveyor, flume tank, shaker table and centrifugal dryer was used
to assess E. coli O157:H7 transfer during production of fresh-cut iceberg lettuce. Uninoculated
lettuce (22.7 kg) was first shredded or shredded and conveyed after which 9.1 kg of radicchio
(previously dip-inoculated with E. coli O157:H7 at 106 CFU/g) was processed to contaminate the
shredder or conveyor belt. In order to separately quantify transfer from the shredder and
conveyor, the conveyor and shredder were then respectively cleaned and sanitized before
processing an additional 90.7 kg of uninoculated iceberg uninoculated lettuce. Within a 5 min
processing, 15 radicchio-free lettuce samples were collected immediately after exiting the
contaminated shredder or conveyor. These samples, along with additional lettuce, sanitizer-free
water and equipment surface samples collected throughout processing were then quantitatively
examined for E. coli O157:H7.
2.2.2 Leafy greens. Individually wrapped heads of iceberg (Lactuca sativa L.) (24 heads
per case) and radicchio (Cichorium intybus) (2.3 kg per case) lettuce were obtained from a local
wholesaler (Stan Setas Produce Co., Lansing, MI), stored in a 4oC walk-in cooler, and used
within 2 days of delivery. All heads were hand-cored immediately before use.
2.2.3 Bacterial strains. Four non-toxigenic (stx1- and stx2-), green fluorescent protein
(GFP)-labeled, ampicillin-resistant strains of E. coli O157: H7 (6980-2, 6982-2, ATCC 43888,
and CV2b7) previously obtained from Dr. Michael Doyle (Center for Food Safety, University of
Georgia, Griffin, GA) were stored in tryptic soy broth (Difco, BD, Sparks, MD) containing 0.6%
(w/vol) yeast extract (TSBYE; Difco, BD) and 10% (vol/vol) glycerol (Sigma Chemical Co., St.
Louis, MO) at -80oC until use. Working cultures were prepared by streaking each stock culture

30

onto plates of tryptic soy agar (Difco, BD) containing 0.6% (w/vol) yeast extract and 100 ppm of
ampicillin (ampicillin sodium salt, Sigma Life Science, St. Louis, MO) (TSAYE plus amp).
After 18 to 24 h of incubation at 37oC, a single colony was transferred to 9 ml of TSBYE
containing 100 ppm ampicillin (TSBYE plus amp) and incubated at 37oC for 24 h.
2.2.4 Inoculum preparation. From those 9 ml of culture, a 0.2 ml aliquot of each nontoxigenic E. coli O157: H7 was added to 200 ml of TSBYE plus amp and incubated at 37oC for
24 h. As determined previously by Buchholz et al. (2012a), the growth rates for these four E. coli
O157: H7 strains were similar. Therefore, the four strains were combined in equal volumes to
obtain an 800 ml cocktail, which was added to 80 L of tap water in a 121 L plastic container
(Rubbermmaid, Wooster, OH) to obtain a population of ~7 log CFU/ml.
2.2.5 Radicchio inoculation. After removing the outer leaves, the radicchio heads (9.1
kg) were cored with a sterile knife, placed in a centrifugation basket, immersed into the inoculum
(Preparation described in 2.2.4) for 15 min, and then drained at room temperature (22 oC) for 1 h
before being spun in a commercial dewatering centrifuge (described in 2.2.6) to eliminate any
residual inoculum from the lettuce head. Based on triplicate analysis of 25-g samples collected
from random layers, the radicchio contained an E. coli O157:H7 population of ~6 log CFU/g
before processing.
2.2.6 Processing line. A small-scale commercial leafy green processing line was used
consisting of a shredder (model TRS 2500 Urschel TranSlicer, Valparaiso, IN), step conveyor
(ThermoDrive, Mol Industries, Grand Rapids, MI), 3.3-m long stainless steel flume tank
(Heinzen Manufacturing, Inc., Gilroy, CA) with two overhead spray jets, stainless steel shaker
table operated by a 1 HP Baldor washdown duty motor (Baldor Electric Co., Fort Smith, AR), a
~1000 L capacity stainless steel water recirculation tank, and a 22.7 kg capacity centrifugal Spin

31

Dryer (Model SD50-LT, Heinzen Manufacturing, Inc.)
2.2.7 Lettuce processing. Initially, 22.7 kg of iceberg lettuce were shredded or shredded
and conveyed to prime the processing line, followed by 9.1 kg of radicchio containing E. coli
O157:H7 at 106 CFU/g to contaminate the shredder or shredder and conveyor, respectively. In
the contaminated conveyor study, the shredder were sanitized, rinsed thoroughly and air dried to
assess transfer exclusively from the conveyor. Thereafter, iceberg lettuce (90.7 kg) was
shredded, conveyed to the flume tank, washed for 90 s in sanitizer-free water, and dewatered
using the shaker table. After collecting 20 5.5-kg samples of dewatered lettuce and removing the
shreds of radicchio by visual inspection, every four baskets of lettuce were combined and spun in
the centrifugal dryer for 80 s.
2.2.8 Sample collection. Three iceberg lettuce samples were collected before processing
as the control. Fifteen radicchio-free iceberg lettuce samples were also collected immediately
after contacting the pre-contaminated shredder/conveyer during processing, with 20 and 4
radicchio-free iceberg lettuce samples collected after shaker table dewatering and centrifugal
drying, respectively. Three 50-ml water samples were collected from the water return spout
above the recirculation tank at the beginning, middle, and end of processing. During centrifugal
drying, five water samples (50 ml each) were collected from the centrifuge drain. The equipment
surface sampling locations (100 cm2 each) as previously described by Buchholz et al (2012) were
shown in Figure 2.1, 2.2, and 2.3. The 1-ply composite tissue method was used for surface
sampling as described by Vorst et al. (2004). In the contaminated shredder study, 7 equipment
surface samples (Figure 2.1) were collected before and after processing the 90.7 kg of iceberg
lettuce, with 8, 11, 9, and 8 surface samples respectively collected from the conveyor belt
(Figure 2.2), flume tank (Figure 2.3), shaker table (Figure 2.3), and centrifugal dryer (Figure 2.3)

32

immediately after processing.
2.2.9 Microbiological analyses. All 25-g lettuce samples were added to 100ml sterile
PBS, homogenized in a stomacher (Stomacher 400 Circulator, Seward, Worthington, UK) at 260
rpm for 1 min and then quantitatively assayed for E. coli O157:H7 by plating appropriate
dilutions on TSAYE with ampicillin. As described previously, the lettuce and equipment surface
samples were either directly plated on TSAYE with amp or concentrated by membrane filtration,
with a limit of detection (LOD) of 0.2 CFU/g and 2 CFU/100cm2, respectively. Water samples
were concentrated by membrane filtration, with a LOD of 0.01 CFU/ml. All plates were
incubated at 37oC for 24 h, and observed for green fluorescing colonies of E. coli O157:H7 under
ultraviolet light (365 nm, Black-Ray, Ultra-violet Product Inc. San Gabriel, CA).
2.2.10 Statistical analysis. E. coli O157:H7 counts from triplicate experiments were
converted to log CFU/ml, log CFU/g or log CFU/100cm2, averaged and then subjected to an
analysis of variance (ANOVA) using JMP 11.0 (SAS Institute Inc., Cary, NC). Values equaling
half the limit of detection were used for samples yielded no E. coli O157:H7 counts. The TukeyKramer HSD test was used with P values of ≤ 0.05 considered significantly different.

33

Figure 2.1: Sampling locations on the shredder. Locations with number of 1,3,5,7,9,11, and 13
were sampled before 90.7 kg of iceberg lettuce was processed. Locations with number 2, 4, 6, 8,
10, 12, and 14 were sampled after 90.7 kg of iceberg lettuce was processed

34

Figure 2.2: Sampling locations on the conveyor. All sampling locations were sampled after 90.7
of iceberg lettuce was processed when the shredder was contaminated. While the conveyor was
contaminated, locations with number of 15, 17, 19, and 21 were sampled before 90.7 kg of
iceberg lettuce was processed. Locations with number 16, 18, 20 and 22 were sampled after 90.7
kg of iceberg lettuce was processed.

35

Figure 2.3: Sampling locations on the flume tank, shaker table and centrifugal dryer.

36

2.3 RESULTS
2.3.1. Radicchio. Before processing, radicchio was inoculated to contain E. coli
O157:H7 at 6.1 ± 0.1 log CFU/g. After processing 90.7 kg of iceberg lettuce, pieces of radicchio
pieces were retrieved from all surfaces of the shredder, conveyor, and shaker table. The weight
of radicchio recovered from each small basket of iceberg lettuce after shaker table dewatering
decreased (P < 0.05) from 13.9 to 0.2 g and from 9.7 to 0.1 g when the shredder and conveyor
were contaminated, respectively (Figures 2.4, 2.5, 2.6 and 2.7). E. coli O157:H7 populations on
the radicchio decreased from 6 to 5.4 log CFU/g after processing 90.7 kg of iceberg lettuce. The
amounts of radicchio retrieved from the shredder, conveyor, flume tank, shaker table and
dewatering centrifuge are shown in Table 2.1.

37

25
A

Weight (g)

20
15
10

B

B

B

5

B

B
B
B

B
B

B

B
B

B

B

B

B

B

B

B

0
1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20
Basket

Figure 2.4: Mean (±SE) weight of radicchio retrieved after shaker table dewatering while the
shredder is contaminated (n=3). Mean weights from baskets without common letters are
significantly different (P < 0.05).
14

A

12

Weight (g)

10
8
AB

6

B

4
AB

2

AB

B

B

B

B
B

B

B

0
1

2

3

4

5

6

7

8

B

B

B

B

B

B

B

B

9 10 11 12 13 14 15 16 17 18 19 20
Basket

Figure 2.5: Mean (±SE) weight of radicchio retrieved after shaker table dewatering while the
conveyor is contaminated (n=3). Mean weights from baskets without common letters are
significantly different (P < 0.05).

38

Table 2.1: Mean (±SD) weight (g) of radicchio and mean (±SD) E. coli O157:H7 populations on
equipment surfaces after processing (log CFU/cm2) (n=3). NA stands for not applicable.
Contaminated shredder

Contaminated conveyor

E. coli populations on
Radicchio

equipment

E. coli populations on
Radicchio

(log CFU/cm2)

equipment
(log CFU/cm2)

Weight(g)

Mean(±SD)

SE

Weight(g)

Mean(±SD)

SE

Shredder

15.6± 7.4

0.9± 1.8

0.7

NA

NA

NA

Conveyor

3.7± 6.0

1.4± 0.7

0.1

6.5± 0.9

2.5± 0.0

0.0

Flume tank

5.7± 9.1

2.1± 0.7

0.4

2.0± 1.7

1.1± 0.3

0.2

1.2± 0.8

1.6± 0.2

0.1

3.8± 3.6

1.4± 0.2

0.1

NA

1.1± 1.0

0.6

NA

0.1± 0.4

0.2

Shaker
table
Centrifuge

39

2.3.2. Iceberg lettuce. After contacting the contaminated shredder or conveyor, all 15
radicchio-free samples of iceberg lettuce contained E. coli O157:H7, with populations of 2.7 to
0.9 log CFU/g for the shredder and 1.8 to 0.9 log CFU/g for the conveyor. When the shredder
was contaminated, E. coli O157:H7 populations decreased significantly with time (P < 0.05). In
contrast, iceberg lettuce collected after contacting the contaminated conveyor belt was only
randomly contaminated, with no significant differences seen between samples (P > 0.05) (Figure
2.6). When the shredder was contaminated, E. coli O157:H7 populations on iceberg lettuce
decreased ~ 0.6 log CFU/g after flume washing and shaker table dewatering. Significant
differences (P < 0.05) in E. coli O157:H7 populations were only seen for the first ten washed
iceberg lettuce samples. No significant differences in E. coli O157:H7 populations were seen
when the conveyor was contaminated (P > 0.05) (Figure 2.7). Statistically similar E. coli
O157:H7 populations (P > 0.05) were recovered from iceberg lettuce after centrifugal drying,
while the shredder or conveyor contaminated.

40

E. coli O157:H7 population (log CFU/g)

Contaminated shredder

contaminated conveyor

3.5
3.0

A

AB
ABC

ABCD

2.5

ABCD

ABCD
A
A

2.0

A
A ABCD ABCD

A

A

A

1.5

ABCD
A
ABCD

A
BCD
A

A

A
ABCD

A
CD

A
BCD
D

1.0

A

0.5
0.0
1

2

3

4

5

6

7
8
9
Lettuce (No.)

10

11

12

13

14

15

E. coli O157:H7 population (log CFU/g)

Figure 2.6: Mean (±SE) E. coli O157:H7 populations on iceberg lettuce after contacting the
contaminated shredder and conveyor (n=3). Means without common letters for the shredder/
conveyor data sets are significantly different (P < 0.05).

3.5

Contaminated shredder

Contaminated conveyor

3
2.5
2

A

AB
A

ABC
A

1.5

A

ABC
A BC
A
A BC

A
BC

A BC
A
BC

BC
A

C
CA

1

C

A

A CA
CA C

C
A

C
C

A

A

A C

A

0.5
0
1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20
Basket

Figure 2.7: Mean (±SE) E. coli O157:H7 populations on iceberg lettuce collected from each
collection basket after shaker table dewatering (n=3), while the shredder / conveyor was
contaminated. Means without common letters for the shredder/ conveyor data sets are
significantly different (P < 0.05).
41

2.3.3. Contaminated shredder/ conveyor. When the shredder surface was contaminated
to contain 3.7 log E. coli O157:H7 CFU/cm2 by processing inoculated radicchio, 0.9 log
CFU/cm3 remained on the shredder after processing 90.7 kg of iceberg lettuce (P < 0.05), with
99.8% transferred from the conveyor to the lettuce, wash water and product contact surfaces of
the processing line. Similarly, when the conveyor initially contained 4.1 log CFU/cm2, 2.5 log
CFU/cm2 remained after conveying 90.7 kg iceberg lettuce (P < 0.05) (Figure 2.8), with only
97.5% subsequently transferred during processing. After processing, all equipment surfaces
examined beyond the initially contaminated shredder and conveyor yielded E. coli O157:H7
(Table 2.1).

42

E. coli O157:H7 population (log CFU/cm2)

5
A

4.5

Shredder

A

Conveyor

4
3.5
3

B

2.5
2
B

1.5
1
0.5
0
Before

After

Figure 2.8: Mean (±SE) E. coli O157:H7 populations on the shredder and conveyor (after
processing 9.1 kg of inoculated radicchio) before and after processing 90.7 kg of iceberg lettuce.
Means without common letters are significantly different (P < 0.05).

43

2.3.4. Water. E. coli O157:H7-free wash water was immediately contaminated from the
lettuce during flume washing. No significant differences were observed for the water samples
collected from the recirculation spout at the beginning, mid-point, and end of processing (P >
0.05) when either the shredder or conveyor were initially contaminated, with E. coli O157:H7
populations of 1.4 and 0.6 log CFU/ml, respectively. Similar results were observed for the water
samples collected from the centrifuge drain for both contamination scenarios (P > 0.05) (Figure
2.9).

44

Contaminated shredder

E. coli O157:H7 population (log CFU/ml)

2.5

Contaminated conveyor

A

A
A
A

A

2

A

A

A

A

A

1.5

1

0.5

0
1

2

3

4

5

Lettuce

Figure 2.9: Mean (±SE) E. coli O157:H7 populations in centrifugation water when the shredder
and conveyor were initially contaminated. Mean E. coli O157:H7 populations with same letter
are not significantly different (P > 0.05).

45

2.4 DISCUSSION
An unusually high E. coli O157:H7 inoculum level (106 log CFU/g) was used to
contaminate both the shredder and conveyor in order to generate quantifiable results for
subsequent mathematical modeling. However, in field-grown lettuce, exposure to feces from
“super-shedder” cattle and sheep (Chase-Topping et al., 2007; Ogden et al. 2005) or highly
contaminated irrigation water remains plausible.
Radicchio was used as a colored surrogate for iceberg lettuce as in previous work
(Buchholz et al., 2014), which allowed the physical separation of inoculated and previously
uncontaminated product. Over 50% of the radicchio processed was recovered from the first three
collection baskets for washed iceberg lettuce. The higher populations of E. coli O157:H7
observed in radicchio-free lettuce from these same three baskets also supports pathogen transfer
via direct radicchio/lettuce contact during removal of the radicchio prior to analysis. Thereafter,
the decrease in E. coli O157:H7 populations on radicchio-free iceberg lettuce in subsequent
baskets paralleled the decrease in the radicchio/iceberg lettuce ratio. Taormina et al. (2009) also
observed a decrease in E. coli O157:H7 populations during hand-coring of lettuce.
When the initial contamination level was ~4 log CFU/cm2 on shredder and conveyor, E.
coli O157:H7 populations decreased to ~1 log CFU/cm2 on the shredder as compared to ~2.5 log
CFU/cm2 on the conveyor after processing 90.7 kg of previously uncontaminated lettuce. Over
99.8% of the initial E. coli O157:H7 population was transferred from the contaminated shredder
to previously uncontaminated lettuce, wash water, and equipment surfaces. Using the same
processing line, Buchholz et al. (2012) also reported higher E. coli O157:H7 populations on the
conveyor belt compared to the shredder, flume tank, shaker table, and dewatering centrifuge.
This observation is supported by several studies showing greater bacterial adherence to

46

polyurethane as compared to stainless steel (Sinde & Carballo 2000; López-Gálvez et al., 2009).
When the conveyor was contaminated by conveying previously inoculated radicchio, the
conveyor belt was contaminated at certain areas, where the radicchio had contacted. E. coli
O157:H7 recovery from sequentially collected lettuce samples was inconsistent, with no
discernable pattern, suggesting that the conveyor belt was also non-uniformly contaminated.
During flume washing, E. coli O157:H7 population on the lettuce decreased significantly,
with the numbers of E. coli O157:H7 becoming homogenized mid-way through processing. No
significant difference was found after the tenth lettuce sample when either the shredder or
conveyor was initially contaminated, indicating that flume washing soon led to uniform
distribution of E. coli O157:H7 in the water. Furthermore, similar E. coli O157:H7 populations
were recovered from the centrifugation water for each batch of product.
Although chemical sanitizers are routinely used in commercial flume washing systems to
minimize contamination from the water (López-Gálvez et al., 2009), the efficacy of chlorinebased sanitizer efficacy decreases significantly at higher organic load (Buchholz et al., 2012b). In
addition, some of the lettuce - contaminated or otherwise - will tend to linger in the processing
line during continued operation and gradually spread to subsequent product, as shown in this
study using radicchio.
Given the ability of mechanical conveyors to continually shed bacteria and contaminated
product over time, their use in commercial operations should be minimized with lettuce directly
shredded into the flume washing system to minimize the chance of cross-contamination. With a
great ability of the shredder to spread contamination and implication of a lettuce shredder in the
previously outbreak of salmonellosis in Australia (Stafford et al., 2002), introduction of a
continuous sanitizer spray within the shredder may be useful in minimizing product build-up and

47

decreasing the subsequent spread of microbial contaminants. However, appropriate use of
chemical sanitizers during flume washing along with effective pre- and post-operative cleaning
and sanitation programs remain critically important for maintaining the safety of fresh-cut
produce.

48

CHAPTER 3:

Effect of Water Velocity on Escherichia coli O157:H7 Transfer from Inoculated Lettuce to
Wash Water in a Custom-made Pipe System

49

3.1 OBJECTIVE
The objective of this study was to assess the impact of laminar, transition, and turbulent
flow on E. coli O157:H7 transfer from lettuce to water during washing.

50

3.2 MATERIALS AND METHODS
3.2.1 Experimental design. The spread of E. coli O157:H7 from inoculated pieces of
iceberg lettuce to wash water under different flow rates was assessed by horizontally inserting 5
x 5 cm pieces of iceberg lettuce dip-inoculated to contain E. coli O157:H7 at 6.7± 0.4 log CFU/g
into a continuously flowing stream of water pumped through a 10-cm diameter custom-made
pipe at 0.005, 0.01, 0.04, and 0.07 m/s. Water and lettuce samples were collected at 1-, 5-, or 10s intervals for 2 or 5 min and quantitatively essayed for E. coli O157:H7 by plating appropriate
dilutions with/without membrane filtration on TSAYE with ampicillin. The ability of the
custom-made pipe and sampling method to recover bacterial populations from the water was
measured and calibrated by adding 1 ml of a 4-strain non-toxigenic E. coli O157:H7 cocktail
(~7.6 log CFU/ml) using the respective flow rates and the same recovery method. Data from
triplicate experiments were assessed for significance using the Tukey-Kramer HSD test.
3.2.2 Leafy greens. Individually wrapped heads of iceberg lettuce (Lactuca sativa L.)
were purchased from a local grocery store (Meijer, Okemos, MI), stored in a 4oC walk-in cooler,
and used within 2 days of purchase.
3.2.3 Bacterial strains. Identical to 2.2.3.
3.2.4 Inoculum preparation. A 25 μl aliquot of each non-toxigenic E. coli O157: H7
strain was added to 25 ml of TSBYE plus amp and incubated at 37oC for 24 h. Since the growth
rates for the four E. coli O157: H7 strains were previously shown to be similar (Buchholz et al.,
2012b), these strains were combined in equal volumes to obtain a 100 ml E. coli O157:H7
cocktail, and then added to 200 ml of tap water (~15oC) to make 300 ml of inoculum containing
~8.1 log CFU/ml.
3.2.5 Lettuce inoculation. Iceberg lettuce heads were hand-cored, with the leaves

51

removed and cut into 5 x 5 cm pieces with a sterilized knife. After immersing in the inoculum for
15 min (preparation described in 3.2.4) followed by 15 min of drying in a bio-safety hood at
room temperature, the leaf pieces contained E. coli O157:H7 at 6.7± 0.4 log CFU/g based on
direct plating (n=3).
3.2.6 Custom-made pipe system. A custom-made pipe system consisted of a 10-cm
diameter PVC pipe (Figure 3.1), with an inlet and an outlet on the ends, an inoculum/lettuce
insertion port on the top, and a water collecting spout on the bottom. An eye valve preceding the
inlet was used to obtain different water flow rates. A centrifugal pump (B633, Magnetek,
Menomonee Falls, WI) was connected by a flexible hose to pump tap water (~15oC) through the
pipe from a ~1000 L capacity stainless steel tank.
3.2.7 Flow rates. Four different flow listed below (Table 3.1) were achieved by adjusting
the valve and then verified by the time required to fill a 3.78 L container, with these times being
96, 50, 12, and 7 s, respectively.
Table 3.1: Flow properties.
Flow velocity (m/s) Volumetric flow rate (L/s)

Flow regime

Reynolds number (Re)

0.005

0.039

Laminar

506

0.01

0.076

Laminar

1012

0.04

0.315

Transitional

3846

0.07

0.549

Turbulent

7085

3.2.8 Processing. To calibrate the pipe system and sampling method, 1 ml of the E. coli
O157:H7 cocktail containing ~7.6 ± 0.3 CFU /ml was rapidly pipetted at point ‘a’ into water
flowing at 0.005, 0.01, 0.04 or 0.07 m/s (Figure 3.1). To evaluate the impact of flow rate on the
transfer of E. coli O157:H7 from lettuce, a 5 x 5 cm piece of iceberg lettuce dip-inoculated to
contain E. coli O157:H7 at 6.7± 0.4 log CFU/g were secured to a thin-wired clamp (4.2 x 4.2
cm) and inserted at point ‘a’ (shown in Figure 3.1) into water flowing at the same rates. Based on
52

results from preliminary experiments, wash times of 5, 2, 2 and 2 minutes were used for flow
rates of 0.005, 0.01, 0.04 m/s and 0.07 m/s, respectively. Differences in E. coli O157:H7 transfer
were accordingly related to differences in the flow rate and flow regime.

53

c

a

b
a

Figure 3.1: Custom-made pipe system. Point ‘a’ is the inoculation port in the water study and the
lettuce insertion port in the lettuce study. Point ‘b’ is the water sample spout. Valve ‘c’ is an eye
valve for controlling water flow rates.

54

3.2.9 Sample collection. Water samples (500 ml each) were analyzed for E. coli
O157:H7 by membrane filtration, which yielded a lower limit of detection (LOD) of 0.002
CFU/ml. Water samples (50 or 500 ml) were collected in Whirl-PakTM bags (Nasco, Fort
Atkinson, WI) from the sampling spout at 1-, 5-, or 10-s intervals for 2 min (flow rates of 0.01,
0.04, and 0.07 m/s) or 5 min (flow rates of 0.005 m/s) with collection starting either 5 s before
inserting inoculum or lettuce (flow rates of 0.07 m/s) or at time 0 (flow rates of 0.005, 0.01, and
0.04 m/s). After each experiment, the inoculated lettuce piece was removed and added to a
Whirl-PakTM bag (Nasco, Fort Atkinson, WI) containing 25 ml of Phosphate Buffered Saline
(PBS) for further analysis.
3.2.10 Microbiological analyses. All lettuce samples were homogenized in a stomacher
(Stomacher 400 Circulator, Seward, Worthington, UK) at 260 rpm for 1 min and then
quantitatively essayed for E. coli O157:H7 by plating appropriate dilutions on TSAYE plus
ampicillin. Water samples were concentrated by membrane filtration on TSAYE with amp
plates, with limits of detection (LOD) of 0.02 and 0.002 CFU/ml for the 50 and 500 ml samples,
respectively. All plates were incubated at 37oC for 24 h and then observed for green fluorescing
colonies under an ultraviolet light (365 nm, Black-Ray, Ultra-violet Product Inc. San Gabriel,
CA), which were counted as E. coli O157:H7.
3.2.11 Percentage of E. coli O157:H7 cells recovered. The percentage of E. coli
O157:H7 cells recovered and the adjusted E. coli O157:H7 populations during wash of
inoculated lettuce at the four different flow rates were determined as follows:
% 𝐸. 𝑐𝑜𝑙𝑖 O157: H7 population recovered =

𝐸.𝑐𝑜𝑙𝑖 O157:H7 population recovered to water
𝐸.𝑐𝑜𝑙𝑖 O157:H7 population released to water

Adjusted 𝐸. 𝑐𝑜𝑙𝑖 O157: H7 population recovered =

𝐸.𝑐𝑜𝑙𝑖 O157:H7 population recovered in water
% 𝐸.𝑐𝑜𝑙𝑖 O157:H7 population recovered

3.2.12 Statistical analysis. Identical to 2.2.10.
55

× 100%

3.3 RESULTS
3.3.1 Percentage of E. coli O157:H7 cells recovered. When a 1 ml aliquot of E. coli
O157:H7 (7.6 ± 0.3 CFU/ml) was pipetted into water flowing at 0.005 m/s, water samples
collected during 5 to 180 s yielded the organism. In contrast, at the higher flow rates of 0.01 and
0.04 m/s, E. coli O157:H7 was only recovered from samples collected during 5 to 30 s and 3 to
15 s, respectively. At the highest flow rate of 0.07 m/s, E. coli O157:H7 was only observed
during 2 to 10 s. E. coli O157:H7 populations peaked in the water after 20, 10, 5, and 3 s at 3.2,
3.5, 3.3, and 4.1 log CFU/ml for flow rates of 0.005, 0.01, 0.04, and 0.07 m/s, respectively
(Figure 3.2). E. coli O157:H7 persisted in the water for 175, 25, 12, and 8 s at flow rates of
0.005, 0.01, 0.04, and 0.07 m/s, respectively. Cumulative E. coli O157:H7 populations after
processing were 6.7, 6.0, 6.3, and 6.3 log CFU for flow rates of 0.005, 0.01, 0.04, and 0.07 m/s,
respectively. Percent recovery decreased from 98 to 92, 88 to 84% as the water flow rate
increased.

56

0.07 m/s

0.04 m/s

0.01 m/s

0.005 m/s

E. coli O157:H7 (Log CFU/ml)

5
4
3
2
1
0
-5
-1

15

35

55

75

95 115 135 155 175 195 215 235 255 275 295

-2
-3
-4

Time (seconds)
Figure 3.2: Mean E. coli O157:H7 populations in water samples collected during washing of
lettuce at four different flow rates. Half the limit of detection was used to calculate the mean log
value when a 500 ml water sample yielded no colonies by membrane filtration.

57

3.3.2 Wash of inoculated lettuce.
3.3.2.1 Lettuce. After washing iceberg lettuce pieces (initially containing E. coli
O157:H7 populations of 6.7± 0.4 log CFU/g) at flow rates of 0.005, 0.01, 0.04, and 0.07 m/s,
6.1, 6.2, 6.3, and 6.1 log CFU were shed into the water, respectively (Figure 3.3), none of which
were significantly different. Shedding of E. coli O157:H7 into the water ceased after 2 min at
flow rates of 0.01, 0.04, and 0.07 m/s, and after 5 min at a flow rate of 0.005 m/s.

58

0.07

0.04

0.01

0.005

Flow rate (m/s)
Figure 3.3: Mean (±SD) E. coli O157:H7 populations shed from inoculated lettuce into the water
based on the populations before and after washing. Different water flow rates with same letter
are not significantly different (P > 0.05).

59

3.3.2.2 Water samples. At the lowest flow rate of 0.005 m/s, E. coli O157:H7 was found
in water samples collected after 3 to 180 s. In contrast, at the next two higher flow rates of 0.01
and 0.04 m/s, water samples collected from 5 to 60 s and from 3 to 15 s yielded E. coli O157:H7,
respectively (see Figure 3.4). At the highest flow rate of 0.07 m/s, E. coli O157:H7 was only
observed in water samples collected after 3 to 5 s. E. coli O157:H7 persistence in the water
decreased from 177 to 57, 12, and 2 s at the flow rates of 0.005, 0.01, 0.04, and 0.07 m/s,
respectively. E. coli O157:H7 populations peaked in water samples at 10, 4, 4, and 3s at 0.7, 2.3,
2.1, and 0.7 log CFU/ml for flow rates of 0.005, 0.01, 0.04, and 0.07 m/s, respectively. The total
populations of E. coli O157:H7 recovered from water samples collected after processing were
3.2 ± 0.2, 5.1 ± 0.2, 4.6 ± 0.8, and 3.8 ± 0.0 log CFU for flow rates of 0.005, 0.01, 0.04, and 0.07
m/s, respectively. E. coli O157:H7 populations in the water were significantly higher (P < 0.05)
at a flow rate of 0.01 m/s (5.1 log CFU) compared to 0.07 m/s (3.8 log CFU) and 0.005 m/s (3.2
log CFU) (Figure 3.5). The adjusted E. coli O157:H7 populations shed from lettuce during
washing (using the equations in 3.2.11) were 3.3 ± 0.2, 5.5 ± 0.2, 5.3 ± 0.8, and 4.1 ± 0.0 log
CFU, respectively, which were significantly affected by flow rates (P < 0.05). For laminar and
transition flow, the higher the flow rate, the more transfer to the water from lettuce. At the higher
flow rates (0.540 and 0.04 m/s), significantly less transfer (P < 0.05) was seen after 10 s, whereas
at the lower flow rates (0.01 and 0.005 m/s), significantly less transfer (P < 0.05) was seen after
4 s.

60

3

0.07 m/s

0.04 m/s

0.001 m/s

0.005 m/s

E. coli O157:H7 (Log CFU/ml)

2
1

0
-5
-1

15

35

55

75

95

115 135 155 175 195 215 235 255 275 295

-2
-3
-4

Time (seconds)

Figure 3.4: Mean log E. coli O157:H7 populations in water samples collected during washing of
lettuce at four different flow rates. Half the limit of detection was used to calculate the mean log
value when a 500 ml water sample yielded no colonies by membrane filtration.

Adjusted total E. coli O157:H7
population in water (Log CFU)

A
6

A

5
B
4

C
3
2
1
0

Figure 3.5: Mean (±SD) adjusted log E. coli O157:H7 populations shed from inoculated lettuce
pieces to wash water. Means of the different water flow rates with different letter are
significantly different (P < 0.05).

61

3.4 DISCUSSION
The flow rates achieved using the present pipe system more closely resembled those seen
for commercial dump tanks and water flow conveyors than commercial leafy green flume
washing systems (~3.6 m/s). However, various conditions of laminar, transitional, and turbulent
flows were obtained with their impacts on bacterial transfer investigated for the first time during
washing.
Dip-inoculation was used to ensure uniform distribution of E. coli O157:H7 on the lettuce
pieces (Lang et al., 2004). Although E. coli O157:H7 could potentially internalize into lettuce
through the cut edges (Erickson, 2012) during inoculation, such internalized cells would not
affect the transfer results since homogenization by stomaching recovers very few internalized
cells (Davidson et al., 2013).
When 1 ml of inoculated water was pipetted to the continuously flowing stream of water
in the pipe, E. coli O157:H7 persistence in the water increased as the flow rate decreased. This
observation is in accordance with the high velocity of rapidly moving water (Semat and Katz,
1958), which validates the custom-made pipe system and sampling method. Similarly, during the
washing of inoculated lettuce, a rapid initial increase in numbers of E. coli O157:H7 was seen at
all flow rates. However, this initial increase was of short duration with E. coli O157:H7
populations decreasing in the water after 10 s. The time used to reached thresholds for initial
detection increased with the decrease of flow rates, which can be explained by classic fluid
mechanics since higher flow rates generate higher shear forces (Semat and Katz, 1958).
Eventually, the E. coli O157:H7 populations reached a steady state, below limit of detection of
0.002 CFU/ml (half the LOD of -3 log CFU/ml was used to calculate the mean log), regardless
of the flow rate, which is consistent with other reports (Adams et al., 1989; Doménech et al.,

62

2013; Ukuku and Fett 2002). The declining numbers of E. coli O157:H7 shed into the water are
also related to the flow rate, with a higher flow rate more rapidly decreasing the residual
population of the E. coli O157:H7 on the lettuce surface as also observed by Wang et al. (2007).
Consequently, washing at a higher low rate leads to more efficient removal of surface bacteria.
Based on the populations before and after washing, total E. coli O157:H7 populations
shed from the inoculated lettuce into water increased with flow rate within the laminar and
transitional flow regimes. The population decreased to the lowest level at a flow rate of 0.07 m/s
in the turbulent regime. However, these data were too scattered to confirm this finding.
Alternatively, the total numbers of E. coli O157:H7 shed in the wash water can be calculated. In
the lettuce study, total E. coli O157:H7 reductions were obtained by adjusting the populations
recovered from the water samples with the recovery rates for the four different flow rates in the
water study (as explained in 3.2.11). Again, bi-phasic removal was observed during washing.
Because of physical limitations in the experimental design, the possibility of multi-phase (more
than one phase) E. coli O157:H7 reductions at different flow rate cannot be ruled out.
During laminar flow (0.005 and 0.01 m/s), bacterial transfer increased with the increase
of flow rate, since shear force increases with velocity. The wall shear force at the lettuce surface
is likely partly responsible for shedding of E. coli O157:H7 into the water. In support of these
observations, Wang et al. (2007) also reported more effective bacterial transfer from cantaloupe
rinds to water at higher shear forces. However, neither transitional nor turbulent flow was
assessed in their study. From our observations, bacterial transfer to the water was lowest at the
highest flow rate of 0.07 m/s (turbulent flow); therefore, bacterial detachment is not always
increased with the increase of flow rate/shear force. Normally, a higher flow rate leads to an
increase in shear force which can affect bacterial adhesion to a surface (Rusconi et al., 2014).

63

This shear stress-enhanced adhesion of E. coli O157:H7 to surfaces is not simply due to fluid
transport or an increased number of bonds formed, but is consistent with the formation of catch
bonds that strengthen as the force increases (Forero et al., 2004; Nilsson et al., 2006; Thomas et
al., 2004; Thomas et al., 2006).
Typically, at a low shear force, bacterial cells will pass over a surface unhindered.
However, higher shear forces tend to enhance surface binding (Rangel et al., 2013; Thomas et
al., 2004) with FimH (a lectin-like protein responsible for adhesion properties of type 1
fimbriae) expressed by E. coli O157:H7 (Thomas et al., 2004). FimH has two functional states
corresponding to weak and strong bonds. At a low shear force, most FimH proteins that interact
with the substrate are in the unstressed equilibrium state. These weak bonds can be easily
broken, allowing the bacterial cell to detach from the surface. Under such conditions, the rate of
bacterial removal is fairly high. As the shear force increases, more bacteria tend to roll forward
instead of detach. In the “rolling” state, a faster flow rate could rotate and carry bacteria forward
faster, with the weak bonds still leading to rapid rolling (Chang et al., 2000). As the shear force
increases further, mechanical force fosters another change in FimH, in which the interdomain
linker chain is disassociated from the lectin domain to form a strong long-lasting bond. At this
point, most bacterial cells would have been immobilized on the surface leading to decreased
removal (Thomas et al., 2004). Since lettuce is a non-anti-FimH material, the surface interactions
between lettuce and FimH need to be considered when assessing E. coli transfer. Due to physical
limitations regarding the power of the pump and the feasibility of water sampling, higher flow
rates could not be achieved. However, given a sufficiently strong shear force to break the bonds
between FimH and the lettuce surface; increased removal of E. coli O157:H7 might be possible
in the regime of turbulent flow.

64

In conclusion, this study assessed the relationship between bacterial removal rates and
water velocity. Overall, flow rate significantly impacted the rate of bacterial transfer from lettuce
to water. Transfer of E. coli O157:H7 from lettuce to wash water increased with flow rate within
the laminar flow regime due to the increased shear force; however, when the flow rate entered
the turbulent flow regime, the greater shear force may have facilitated adhesion of E. coli
O157:H7 with the lettuce, decreasing transfer to the water.

65

CONCLUSIONS AND FUTURE RECOMMENDATIONS

66

Fresh produce is now responsible for more than half of all cases of foodborne illness with
fresh-cut leafy greens posing a major public health concern as evidence by recent recalls and
outbreaks. Although sanitizers are commonly used during production of fresh-cut produce, their
antimicrobial efficacy is difficult to maintain due to the rapid built up of organic material in the wash
water. Therefore, the presence of sanitizers cannot guarantee effective washing.
The first objective of this research was to quantify the spread of E. coli O157:H7 from a

product-contaminated shredder or conveyor to subsequent product, other equipment surfaces,
and the water during simulated commercial production of fresh-cut iceberg lettuce. The results
indicated that while the shredder was contaminated by previously inoculated radicchio, 99.8% of
the E. coli O157:H7 was spread to the wash water and other equipment surfaces. After
processing 90.7 kg of previously uninoculated iceberg lettuce, 2.5 log CFU/cm2 remained on the
conveyor while the conveyor belt was initially contaminated to contain ~4 log CFU/cm2 E. coli
O157:H7. Considering the ability of the shredder to spread E. coli O157:H7 during processing, a
continuous sanitizer spray should be introduced within the shredder to minimize the spread of
microbial contaminants. The use of mechanical conveyors after shredding should be eliminated
or minimized due to their ability to transfer bacteria over a long period of time. Therefore
shredding lettuce directly into the flume washing system is strongly recommended.
Being the only post-harvest microbial reduction step for, washing remains critical for
enhancing end-product safety. The second objective of this research assessed the impact of
laminar, transition, and turbulent flow on E. coli O157:H7 transfer from lettuce to water during
washing. Persistence of E. coli O157:H7 in the wash water increased with the decrease in flow
rate. The time needed for E. coli O157:H7 to reach the detection threshold decreased with
increasing flow rate. Within the laminar regime, removal of E. coli O157:H7 from inoculated
lettuce increased with the increase in flow rate, however, at the highest flow rate achieved by the
67

custom-made pipe system, E. coli O157:H7 removal was lowest compared to the lower flow
rates. In order to better understand the impact of flow rate on E. coli O157:H7 transfer during
washing of lettuce, at least two additional flow rates within the turbulent regime should be
included in the future research. This additional work would reveal if an increase in flow rate
leads to less removal of E. coli O157:H7 from inoculated lettuce during washing. Additionally,
the product-to-water ratio should be considered since this is another critical factor affecting
washing efficacy. A much greater product-to-water ratio should be obtained to mimic
commercial washing conditions.

68

APPENDIX

69

Table AI. 1: E. coli O157:H7 populations (log CFU/g) on previously uninoculated lettuce
collected (over time along processing) after contacting contaminated shredder.
E. coli O157:H7 populations (log CFU/g)
Lettuce
sample (No.)

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1

2.5

2.4

3.2

2.7

0.4

2

1.8

3.0

2.9

2.6

0.7

3

1.8

2.8

1.7

2.1

0.6

4

2.0

2.4

2.2

2.2

0.2

5

2.3

2.7

2.1

2.3

0.3

6

1.7

2.3

2.5

2.2

0.4

7

1.8

2.3

1.2

1.8

0.6

8

1.6

1.2

1.7

1.5

0.3

9

1.7

1.4

0.9

1.3

0.4

10

1.4

0.9

1.3

1.2

0.3

11

2.8

1.2

1.7

1.9

0.8

12

1.4

1.2

1.5

1.4

0.2

13

1.7

0.2

1.0

0.9

0.7

14

1.7

0.9

0.9

1.2

0.5

15

0.8

0.6

1.2

0.9

0.3

70

Table AI. 2: E. coli O157:H7 populations (log CFU/g) on previously uninoculated lettuce
collected (over time along processing) after contacting contaminated conveyor.
E. coli O157:H7 populations (log CFU/g)

Lettuce
sample (No.)

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1

1.4

1.4

1.5

1.4

0.1

2

0.6

2.0

1.1

1.2

0.7

3

1.6

1.3

1.1

1.3

0.2

4

0.6

2.8

0.9

1.4

1.2

5

1.5

2.1

1.7

1.8

0.3

6

1.9

1.8

1.3

1.7

0.3

7

1.2

2.2

0.6

1.3

0.8

8

1.2

2.2

0.9

1.4

0.7

9

0.6

1.3

1.4

1.1

0.4

10

0.6

1.2

1.5

1.1

0.4

11

2.7

1.4

1.2

1.8

0.8

12

0.9

1.9

0.8

1.2

0.6

13

0.6

1.8

1.2

1.2

0.6

14

1.2

1.6

-0.1

0.9

0.9

15

1.3

0.9

0.6

0.9

0.3

71

Table AI. 3: E. coli O157:H7 populations (log CFU/g) on previously uninoculated lettuce
collected from each of the 20 collection baskets after washing and shaker table dewatering while
the shredder was contaminated.
E. coli O157:H7 populations (log CFU/g)
Basket
(No.)

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1

2.1

2.0

2.2

2.1

0.1

2

1.7

2.3

1.6

1.9

0.4

3

1.2

2.0

1.2

1.5

0.4

4

1.4

1.7

1.2

1.4

0.3

5

1.5

1.5

0.9

1.3

0.3

6

1.0

1.4

1.1

1.2

0.2

7

1.0

1.1

1.2

1.1

0.1

8

1.0

1.2

1.2

1.1

0.1

9

1.0

1.4

0.9

1.1

0.3

10

0.9

1.2

1.8

1.3

0.5

11

1.0

1.0

1.1

1.0

0.1

12

1.1

0.9

0.7

0.9

0.2

13

1.0

1.4

0.9

1.1

0.3

14

1.0

0.9

1.4

1.1

0.2

15

0.9

1.1

1.1

1.0

0.1

16

1.2

1.1

0.8

1.0

0.2

17

1.4

1.0

0.8

1.1

0.3

18

1.4

1.2

0.7

1.1

0.4

19

1.3

0.9

0.8

1.0

0.2

20

0.9

1.1

0.9

1.0

0.2

72

Table AI. 4: E. coli O157:H7 populations (log CFU/g) on previously uninoculated lettuce
collected from each of the 20 collection baskets after washing and shaker table dewatering while
the conveyor was contaminated.
E. coli O157:H7 populations (log CFU/g)
Basket
(No.)

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1

1.0

1.4

1.7

1.4

0.3

2

0.4

1.4

1.4

1.1

0.6

3

0.7

1.5

1.2

1.1

0.4

4

1.3

1.1

1.4

1.2

0.1

5

1.3

0.9

0.6

1.0

0.4

6

0.9

1.6

1.2

1.3

0.3

7

0.7

1.5

1.0

1.1

0.4

8

0.9

1.2

1.2

1.1

0.2

9

0.8

1.1

0.8

0.9

0.2

10

1.6

0.9

1.2

1.2

0.4

11

1.1

1.1

1.0

1.1

0.1

12

0.8

1.0

1.2

1.0

0.2

13

0.5

0.9

1.1

0.8

0.3

14

0.6

0.9

1.0

0.8

0.2

15

0.9

0.9

1.2

1.0

0.2

16

0.7

0.9

0.8

0.8

0.1

17

1.1

0.9

0.9

1.0

0.1

18

0.6

0.9

0.9

0.8

0.2

19

0.6

1.0

0.8

0.8

0.2

20

0.7

0.7

0.5

0.7

0.1

73

Table AI. 5: E. coli O157:H7 (log CFU/g) populations on previously uninoculated lettuce
collected (over time along processing) for each centrifugal drying batch while the shredder was
contaminated.
E. coli O157:H7 populations (log CFU/g)
Lettuce sample
(No.)

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1

1.4

2.4

1.3

1.7

0.6

2

0.7

1.2

0.9

1.0

0.2

3

0.7

1.3

1.4

1.1

0.4

4

0.7

1.0

0.6

0.8

0.2

5

0.8

1.8

1.3

1.3

0.5

74

Table AI. 6: E. coli O157:H7 (log CFU/g) populations on previously uninoculated lettuce
collected (over time along processing) for each centrifugal drying batch while the conveyor was
contaminated.
E. coli O157:H7 populations (log CFU/g)
Lettuce
sample (No.)

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1

0.9

1.4

1.2

1.2

0.3

2

1.2

0.2

1.0

0.8

0.5

3

0.5

1.3

0.9

0.9

0.4

4

1.0

2.0

1.6

1.5

0.5

5

0.9

0.8

0.7

0.8

0.1

75

Table AI. 7: E. coli O157:H7 populations (log CFU/cm2) on equipment surfaces while the
shredder was contaminated. * representing half of the LOD was used to calculate mean log
value for the surface samples yielded no E. coli O157:H7 colonies on the plates.
E. coli O157:H7 populations (log CFU/cm2)
Surface sample (No.)/
Surface of equipment

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1

4.7

3.7

3.6

4.0

0.6

2

0.0*

0.0*

0.0*

0.0

0.0

3

1.3

4.4

2.4

2.7

1.6

4

0.0*

0.0*

0.0*

0.0

0.0

5

2.4

4.0

3.5

3.3

0.8

6

0.0*

0.0*

0.3

0.1

0.2

7

3.6

1.3

4.0

3.0

1.5

8

0.0*

0.0*

1.6

0.5

0.9

9

3.0

4.2

2.8

3.3

0.7

10

3.3

0.3

0.0*

1.2

1.8

11

2.3

5.0

4.8

4.1

1.5

12

4.1

5.0

4.9

4.6

0.5

13

5.8

5.5

5.8

5.7

0.2

14

0.0*

0.3

0.0*

0.1

0.2

15

0.0*

0.3

1.7

0.7

0.9

16

0.0*

1.2

1.8

1.0

0.9

17

0.3

0.3

3.2

1.3

1.6

18

0.3

2.5

1.9

1.6

1.1

19

2.4

2.8

1.8

2.3

0.5

20

2.1

2.2

0.8

1.7

0.8

21

0.0*

2.9

1.3

1.4

1.4

22

0.0*

3.3

1.8

1.7

1.6

Flume tank

3.0

1.9

1.5

2.1

0.7

Shaker table

1.8

1.5

1.5

1.6

0.2

centrifuge

1.4

1.9

0.1

1.1

0.9

76

Table AI. 8: E. coli O157:H7 populations (log CFU/cm2) on equipment surfaces while the
conveyor was contaminated. * representing half of the LOD was used to calculate mean log
value for the surface samples yielded no E. coli O157:H7 colonies on the plates. NA stand for
not applicable
E. coli O157:H7 populations (log CFU/cm2)
Surface sample (No.)/
Surface of equipment

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1-14

NA

NA

NA

NA

NA

15

4.9

4.0

4.0

4.3

0.5

16

2.9

2.8

2.6

2.8

0.2

17

3.9

5.0

5.5

4.8

0.8

18

2.4

2.8

2.7

2.7

0.2

19

4.9

4.1

3.7

4.2

0.6

20

2.8

1.9

2.3

2.3

0.5

21

2.6

3.7

3.2

3.2

0.6

22

2.1

2.5

2.3

2.3

0.2

Flume tank

1.0

1.4

0.9

1.1

0.3

Shaker table

1.5

1.2

1.4

1.4

0.2

centrifuge

1.4

0.8

0.8

1.0

0.3

77

Table AI. 9: E. coli O157:H7 (log CFU/ml) populations in water samples for each centrifugal
drying batch while the shredder was contaminated.
E. coli O157:H7 populations (log CFU/ml)
Water sample
(No.)

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1

1.9

2.6

2.0

2.2

0.4

2

1.8

2.0

1.9

1.9

0.1

3

1.6

2.0

1.8

1.8

0.2

4

1.6

1.9

1.7

1.7

0.1

5

1.6

2.5

2.1

2.1

0.4

78

Table AI. 10: E. coli O157:H7 (log CFU/ml) populations in water samples for each centrifugal
drying batch while the conveyor was contaminated.
E. coli O157:H7 populations (log CFU/ml)
Water sample
(No.)

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1

1.4

1.9

2.4

1.9

0.5

2

1.7

1.9

2.0

1.9

0.2

3

1.6

1.7

2.0

1.8

0.2

4

1.3

1.9

1.8

1.7

0.3

5

1.6

1.6

1.9

1.7

0.2

79

Table AI. 11: Weight of radicchio (g) retrieved from each of the 20 collection baskets after
washing and shaker table dewatering while the shredder was contaminated.
Weight of radicchio (g)
Basket
(No.)

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1

4.2

28.3

9.3

13.9

12.7

2

5.8

9.4

3.9

6.4

2.8

3

2.4

11.1

5.3

6.3

4.4

4

2.2

5.5

4.5

4.1

1.7

5

0.5

5.3

5.1

3.6

2.7

6

0.3

3.2

2.4

2.0

1.5

7

0.9

1.6

1.1

1.2

0.4

8

1.1

0.6

0.8

0.8

0.3

9

0.7

0.3

5.9

2.3

3.1

10

0.3

0.5

0.4

0.4

0.1

11

0.5

0.2

0.2

0.3

0.2

12

0.0

1.2

2.6

1.3

1.3

13

0.1

2.4

1.2

1.2

1.2

14

0.1

1.2

0.4

0.6

0.6

15

0.1

0.2

0.0

0.1

0.1

16

0.2

0.6

1.2

0.7

0.5

17

0.0

0.8

0.1

0.3

0.4

18

0.0

0.9

0.5

0.5

0.4

19

1.1

0.4

0.2

0.6

0.5

20

0.1

0.3

0.2

0.2

0.1

80

Table AI. 12: Weight of radicchio (g) retrieved from each of the 20 collection baskets after
washing and shaker table dewatering while the conveyor was contaminated.
Weight of radicchio (g)
Basket
(No.)

Replicate 1

Replicate 2

Replicate 3

Mean

STDEV

1

13.5

3.0

12.7

9.7

5.8

2

2.6

3.1

7.3

4.3

2.6

3

1.1

1.3

3.2

1.9

1.2

4

2.2

1.2

1.1

1.5

0.6

5

1.2

1.7

1.4

1.4

0.3

6

1.8

0.6

1.6

1.3

0.6

7

1.6

1.4

0.6

1.2

0.5

8

0.7

1.4

0.8

1.0

0.4

9

1.4

0.4

0.3

0.7

0.6

10

0.4

0.2

0.2

0.3

0.1

11

0.5

0.2

0.2

0.3

0.2

12

5.9

0.3

0.2

2.1

3.3

13

0.4

0.2

0.2

0.3

0.1

14

0.4

0.3

0.2

0.3

0.1

15

0.3

0.1

0.2

0.2

0.1

16

0.3

0.1

3.0

1.1

1.6

17

0.2

0.1

0.4

0.2

0.2

18

0.3

0.2

0.2

0.2

0.1

19

0.1

0.1

0.2

0.1

0.1

20

0.1

0.2

0.1

0.1

0.1

81

REFERENCES

82

REFERENCES

Adams, M.R., Hartley, a. D., Cox, L.J., 1989. Factors affecting the efficacy of washing
procedures used in the production of prepared salads. Food Microbiol. 6, 69–77.
Akbas, M.Y., Ölmez, H., 2007. Inactivation of Escherichia coli and Listeria monocytogenes on
iceberg lettuce by dip wash treatments with organic acids. Lett. Appl. Microbiol. 44, 619–
624.
Allen, R.L., Warren, B.R., Archer, D.L., Sargent, S.A., Schneider, K.R., 2005. Survival of
Salmonella spp. on the surfaces of fresh tomatoes and selected packing line materials.
Horttechnology 15, 831–836.
Allende, A., Selma, M. V., López-Gálvez, F., Villaescusa, R., Gil, M.I., 2008. Role of
commercial sanitizers and washing systems on epiphytic microorganisms and sensory
quality of fresh-cut escarole and lettuce. Postharvest Biol. Technol. 49, 155–163.
An, Y.H., Friedman, R.J., 1998. Concise review of mechanisms of bacterial adhesion to
biomaterial surfaces. J. Biomed. Mater. Res. 43, 338–348.
Anderson, M., Jaykus, L.A., Beaulieu, S., Dennis, S., 2011. Pathogen-produce pair attribution
risk ranking tool to prioritize fresh produce commodity and pathogen combinations for
further evaluation (P3ARRT). Food Control 22, 1865–1872.
Anonymous, 2011. Illnesses in U.S. Attributed to Leafy Greens. Available at:
http://www.ugacfs.org/producesafety/Pages/Basics/USIllnesses.html (accessed 9.29.14).
Artés, F., Allende, A., 2005. Processing lines and alternative preservation techniques to prolong
the shelf-life of minimally fresh processed leafy vegetables. Eur. J. Hortic. Sci.
Artés, F., Gómez, P., Aguayo, E., Escalona, V., Artés-Hernández, F., 2009. Sustainable
sanitation techniques for keeping quality and safety of fresh-cut plant commodities.
Postharvest Biol. Technol.
Aruscavage, D., Lee, K., Miller, S., LeJeune, J.T., 2006. Interactions affecting the proliferation
and control of human pathogens on edible plants. J. Food Sci.
Ashkenazi, S., Larocco, M., Murray, B.E., Cleary, T.G., 1992. The adherence of verocytotoxinproducing Escherichia coli to rabbit intestinal cells. J. Med. Microbiol. 37, 304–309.
Barrera, M.J., Blenkinsop, R., Warriner, K., 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, 745–751.

83

Baur, S., Klaiber, R., Wei, H., Hammes, W.P., Carle, R., 2005. Effect of temperature and
chlorination of pre-washing water on shelf-life and physiological properties of ready-to-use
iceberg lettuce. Innov. Food Sci. Emerg. Technol. 6, 171–182.
Benjamin, M.M., Datta, A.R., 1995. Acid tolerance of enterohemorrhagic Escherichia coli. Appl.
Environ. Microbiol. 61, 1669–1672.
Beuchat, L.R., Adler, B.B., Lang, M.M., 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, 1238–1242.
Bitton, G., Marshall, K.C., 1980. Adsorption of microorganisms to surfaces. Wiley, New York.
Buchholz, A., Davidson, G., Ryser, E., 2010. Microbiology of Fresh and Processed Vegetables.
Veg. 159–181.
Buchholz, A.L., Davidson, G.R., Marks, B.P., Todd, E.C.D., Ryser, E.T., 2012a. 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.
Buchholz, A.L., Davidson, G.R., Marks, B.P., Todd, E.C.D., Ryser, E.T., 2012b. Quantitative
Transfer of Escherichia coli O157:H7 to equipment during small-scale production of freshcut leafy greens. J. Food Prot. 75, 1184–1197.
CDC, 2011. Investigation Update: Multistate Outbreak of Human E. coli O145 Infections Linked
to Shredded Romaine Lettuce from a Single Processing Facility. Available at:
http://www.cdc.gov/ecoli/2010/ecoli_o145/index.html?s_cid=ccu051010_006 (accessed
9.29.14).
CDC, 2012. Surveillance for Foodborne Disease Outbreaks United States, 2012. Available at:
http://www.cdc.gov/foodsafety/pdfs/foodborne-disease-outbreaks-annual-report-2012508c.pdf (accessed 9.24.14).
CDC, 2013. Multistate outbreak of shiga toxin-producing Escherichia coli O157:H7 infections
linked to ready-to-eat salads (final update). Available at:
http://www.cdc.gov/ecoli/2013/o157h7-11-13/index.html (accessed 10.27.14).
CDC, 2014. Foodborne outbreak online database. Available at:
http://wwwn.cdc.gov/foodborneoutbreaks/ (accessed 10.29.14).
Chang, H.T., Rittmann, B.E., Amar, D., Heim, R., Ehlinger, O., Lesty, Y., 1991. Biofilm
detachment mechanisms in a liquid-fluidized bed. Biotechnol. Bioeng. 38, 499–506.
Chang, K.C., Tees, D.F., Hammer, D.A., 2000. The state diagram for cell adhesion under flow:
leukocyte rolling and firm adhesion. Proc. Natl. Acad. Sci. U. S. A. 97, 11262–11267.

84

Chase-Topping, M.E., McKendrick, I.J., Pearce, M.C., MacDonald, P., Matthews, L., Halliday,
J., Allison, L., Fenlon, D., Low, J.C., Gunn, G., Woolhouse, M.E.J., 2007. Risk factors for
the presence of high-level shedders of Escherichia coli O157 on Scottish farms. J. Clin.
Microbiol. 45, 1594–1603.
Chia, T.W.R., Goulter, R.M., McMeekin, T., Dykes, G.A., Fegan, N., 2009. Attachment of
different Salmonella serovars to materials commonly used in a poultry processing plant.
Food Microbiol. 26, 853–859.
Choudhury, D., Thompson, A., Stojanoff, V., Langermann, S., Pinkner, J., Hultgren, S.J.,
Knight, S.D., 1999. X-ray structure of the FimC-FimH chaperone-adhesin complex from
uropathogenic Escherichia coli. Science 285, 1061–1066.
Chua, D., Goh, K., Saftner, R.A., Bhagwat, A.A., 2008. Fresh-cut lettuce in modified atmosphere
packages stored at improper temperatures supports enterohemorrhagic E. coli isolates to
survive gastric acid challenge. J. Food Sci. 73.
Cimbala, J.M. (Pennsylvania S.U., 2009. Volume and mass flow rate measurement. Available at:
http://www.mne.psu.edu/me345/lectures/Volume_flow_rate_measurement.pdf (accessed
9.27.14).
Cook, R., 2012. Trends in the marketing of fresh produce and fresh-cut/value-added produce.
Available at: http://ucanr.edu/datastoreFiles/234-2435.pdf (accessed 10.24.14).
CSPI, 2008. Outbreak alert! 2008. Available at:
http://www.cspinet.org/new/pdf/outbreak_alert_2008_report_final.pdf (accessed 9.24.14).
Davidson, G.R., Buchholz, A.L., Ryser, E.T., 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.
Delaquis, S., Stewart, S., Cazaux, S., Toivonen, P., 2002. Survival and growth of Listeria
monocytogenes and Escherichia coli O157:H7 in ready-to-eat iceberg lettuce washed in
warm chlorinated water. J. Food Prot. 65, 459–464.
Dezuniga, A., Anderson, M., Marshall, R., 1991. A model system for studying the penetration of
microorganisms into meat. OURNAL FOOD Prot. 54, 256–258.
Dixon, P.F., Smail, D.A., Algoët, M., Hastings, T.S., Bayley, A., Byrne, H., Dodge, M., Garden,
A., Joiner, C., Roberts, E., Verner-Jeffreys, D., Thompson, F., 2012. Studies on the effect of
temperature and pH on the inactivation of fish viral and bacterial pathogens. J. Fish Dis. 35,
51–64.
Doane, C.A., Pangloli, P., Richards, H.A., Mount, J.R., Golden, D.A., Draughon, F.A., 2007.
Occurrence of Escherichia coli O157:H7 in diverse farm environments. J. Food Prot. 70,
6–10.
85

Doménech, E., Botella, S., Ferrús, M.A., Escriche, I., 2013. The role of the consumer in the
reduction of Listeria monocytogenes in lettuces by washing at home. Food Control 29, 98–
102.
Erickson, M.C., 2012. Internalization of fresh produce by foodborne pathogens. Annu. Rev. Food
Sci. Technol.
Erickson, M.C., Webb, C.C., Diaz-Perez, J.C., Phatak, S.C., Silvoy, J.J., Davey, L., Payton, A.S.,
Liao, J., Ma, L., Doyle, M.P., 2010. Surface and internalized Escherichia coli O157:H7 on
field-grown spinach and lettuce treated with spray-contaminated irrigation water. J. Food
Prot. 73, 1023–1029.
FDA, 2006. Commodity specific food safety guidelines for the lettuce and leafy greens supply
chain. Available at:
http://www.fda.gov/downloads/Food/FoodSafety/ProductspecificInformation/ (accessed
9.29.14).
FDA, 2008. Guidance for Industry: Guide to Minimize Microbial Food Safety Hazards of Freshcut Fruits and Vegetables. Available at:
http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/
ProducePlantProducts/ucm064458.htm (accessed 10.24.14).
Fernandes, P.É., São José, J.F.B., Zerdas, E.R.M. a., Andrade, N.J., Fernandes, C.M., Silva,
L.D., 2014. Influence of the hydrophobicity and surface roughness of mangoes and
tomatoes on the adhesion of Salmonella enterica serovar Typhimurium and evaluation of
cleaning procedures using surfactin. Food Control 41, 21–26.
Food and Drug Administration, 2012. Bad bug book, Foodborne pathogenic microorganisms and
natural toxins handbook, Listeria monocytogenes.
Forero, M., Thomas, W.E., Bland, C., Nilsson, L.M., Sokurenko, E. V., Vogel, V., 2004. A
catch-bond based nanoadhesive sensitive to shear stress. Nano Lett. 4, 1593–1597.
Frank, J., 2001. Microbial attachment to food and food contact surfaces. Adv. Food Nutr. Res.
43, 319–370.
FSAI, F.S.A. of I., 2001. Code of practice for food safety in the fresh produce supply chain in
Ireland.
Gil, M.I., Selma, M. V, Suslow, T., Jacxsens, L., Uyttendaele, M., Allende, A., 2013. Pre- and
post-harvest preventive measures and untervention strategies to control microbial food
safety hazards of fresh leafy vegetables. Crit. Rev. Food Sci. Nutr. 37–41.
Gil, M.I., Selma, M. V., López-Gálvez, F., Allende, A., 2009. Fresh-cut product sanitation and
wash water disinfection: Problems and solutions. Int. J. Food Microbiol.

86

Han, Y., 2000. Inactivation of Escherichia coli O157:H7 on surface-uninjured and -injured green
pepper (Capsicum annuum L.) by chlorine dioxide gas as demonstrated by confocal laser
scanning microscopy. Food Microbiol. 17, 643–655.
Hancock, D.D., Besser, T.E., Rice, D.H., Ebel, E.D., Herriott, D.E., Carpenter, L. V, 1998.
Multiple sources of Escherichia coli O157 in feedlots and dairy farms in the northwestern
USA. Prev. Vet. Med. 35, 11–19.
Heard, G.M., 2002. Microbiology of fresh-cut produce, in: Lamikanra, O. (Ed.), Fresh-cut fruits
and vegetables: Science, Technology, and Market. pp. 188–236.
Herdt, J., Feng, H., 2009. Aqueous antimicrobial treatments to improve fresh and fresh-cut
produce safety, in: Fan, X., Niemira, B.A., Doona, C.J., Feeherry, F.E., Gravani, R.B.
(Eds.), Microbial Safety of Fresh Produce. pp. 169–190.
Huang, Z., Mclamore, E.S., Chuang, H.S., Zhang, W., Wereley, S., Leon, J.L.C., Banks, M.K.,
2013. Shear-induced detachment of biofilms from hollow fiber silicone membranes.
Biotechnol. Bioeng. 110, 525–534.
Hubble, J., Ming, F., Eisenthal, R., Whish, W., 1996. Progressive detachment of cells from
surfaces: a consequence of heterogeneous ligand populations or a multi-site binding
equilibrium. J. Theor. Biol. 182, 169–171.
Hung, H.-C., Joshipura, K.J., Jiang, R., Hu, F.B., Hunter, D., Smith-Warner, S.A., Colditz, G.A.,
Rosner, B., Spiegelman, D., Willett, W.C., 2004. Fruit and vegetable intake and risk of
major chronic disease. J. Natl. Cancer Inst. 96, 1577–1584.
Islam, M., Doyle, M.P., Phatak, S.C., Millner, P., Jiang, X., 2005. Survival of Escherichia coli
O157:H7 in soil and on carrots and onions grown in fields treated with contaminated
manure composts or irrigation water. Food Microbiol. 22, 63–70.
Jaglic, Z., Cervinkova, D., Michu, E., 2011. Effect of milk temperature and flow on the
adherence of Staphylococcus epidermidis to stainless steel in amounts capable of biofilm
formation. DAIRY Sci. Technol. 91, 361–372.
Jensen, A.N., Storm, C., Forslund, A., Baggesen, D.L., Dalsgaard, A., 2013. Escherichia coli
contamination of lettuce grown in soils amended with animal slurry. J. Food Prot. 76,
1137–1144.
Johannessen, G.S., Bengtsson, G.B., Heier, B.T., Bredholt, S., Wasteson, Y., Rørvik, L.M.,
2005. Potential uptake of Escherichia coli O157:H7 from organic manure into crisphead
lettuce. Appl. Environ. Microbiol. 71, 2221–2225.
Katsikogianni, M.G., Missirlis, Y.F., 2010. Interactions of bacteria with specific biomaterial
surface chemistries under flow conditions. Acta Biomater. 6, 1107–1118.

87

Keskinen, L.A., Annous, B.A., 2011. Efficacy of adding detergents to sanitizer solutions for
inactivation of Escherichia coli O157:H7 on Romaine lettuce. Int. J. Food Microbiol.
Keskinen, L.A., Burke, A., Annous, B.A., 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.
Kim, Y.J., Lee, S.H., Park, J., Park, J., Chung, M., Kwon, K., Chung, K., Won, M., Song, K.
Bin, 2008. Inactivation of Escherichia coli O157:H7, Salmonella typhimurium, and Listeria
monocytogenes on stored iceberg lettuce by aqueous chlorine dioxide treatment. J. Food
Sci. 73.
Kitinoja, L., Gorny, J.R., 1999. Fresh-cut produce, in: Post-Harvest Technology for Small-Scale
Produce Marketers: Economic Opportunities, Quality and Food Safety. Davis: University of
California., pp. 12.1–12.15.
Klein, S., Witmer, J., Tian, A., DeWaal, C., 2009. The ten riskiest foods regulated by the US
Food and Drug Administration. Publ. Cent. Sci. ….
Koseki, S., Isobe, S., 2005. Prediction of pathogen growth on iceberg lettuce under real
temperature history during distribution from farm to table. Int. J. Food Microbiol. 104, 239–
248.
Krasaekoopt, W., Bhandari, B., 2011. Fresh-cut vegetables, in: Sinha, N.K. (Ed.), Handbook of
vegetables and vegetable processing. Blackwell Publishing Ltd, pp. 221–242.
Kroupitski, Y., Golberg, D., Belausov, E., Pinto, R., Swartzberg, D., Granot, D., Sela, S., 2009.
Internalization of Salmonella enterica in leaves is induced by light and involves chemotaxis
and penetration through open stomata. Appl. Environ. Microbiol. 75, 6076–6086.
Kudva, I.T., Blanch, K., Hovde, C.J., 1998. Analysis of Escherichia coli O157:H7 survival in
ovine or bovine manure and manure slurry. Appl. Environ. Microbiol. 64, 3166–3174.
Lang, M.M., Harris, L.J., Beuchat, L.R., 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 67, 1092–1103.
Lelièvre, C., Antonini, G., Faille, C., Bénézech, T., 2002. Cleaning-in-Place: Modelling of
cleaning kinetics of pipes soiled by Bacillus spores sssuming a process combining removal
and deposition. Food Bioprod. 80, 305–312.
Lewis, S.J., Gilmour, A., Johnston, D.E., 1989. Factors influencing the detachment of a polymerassociated Acinetobacter sp. from stainless steel. Int. J. Food Microbiol. 8, 155–164.

88

Li, H., Tajkarimi, M., Osburn, B.I., 2008. Impact of vacuum cooling on Escherichia coli
O157:H7 infiltration into lettuce tissue. Appl. Environ. Microbiol. 74, 3138–3142.
Liu, Y., Tay, J.H., 2002. The essential role of hydrodynamic shear force in the formation of
biofilm and granular sludge. Water Res.
López-Gálvez, F., Allende, A., Selma, M. V., Gil, M.I., 2009. Prevention of Escherichia coli
cross-contamination by different commercial sanitizers during washing of fresh-cut lettuce.
Int. J. Food Microbiol. 133, 167–171.
López-Gálvez, F., Allende, A., Truchado, P., Martínez-Sánchez, A., Tudela, J.A., Selma, M. V.,
Gil, M.I., 2010. Suitability of aqueous chlorine dioxide versus sodium hypochlorite as an
effective sanitizer for preserving quality of fresh-cut lettuce while avoiding by-product
formation. Postharvest Biol. Technol. 55, 53–60.
Lu, Z.X., Lu, F.X., Zhang, L.K., Bie, X.M., Zou, X.K., 2007. Predictive modeling and growth
models of aerobic mesophilic bacteria on fresh-cut lettuce by hypochlorite-washing. J. Food
Saf. 27, 157–168.
Luo, Y., He, Q., McEvoy, J.L., 2010. Effect of storage temperature and duration on the behavior
of Escherichia coli O157:H7 on packaged fresh-cut salad containing romaine and iceberg
lettuce. J. Food Sci. 75.
Luo, Y., Nou, X., Yang, Y., Alegre, I., Turner, E., Feng, H., Abadias, M., Conway, W., 2011.
Determination of free chlorine concentrations needed to prevent Escherichia coli O157:H7
cross-contamination during fresh-cut produce wash. J. Food Prot. 74, 352–358.
Ma, J., Mark Ibekwe, A., Crowley, D.E., Yang, C.H., 2014. Persistence of Escherichia coli O157
and non-O157 strains in agricultural soils. Sci. Total Environ. 490, 822–829.
Mafu, A., Roy, D., Goulet, J., Magny, P., 1990. Attachment of Listeria monocytogenes to
stainless steel, glass, polypropylene, and rubber surfaces after short contact times. J. Food
Prot. 742–746.
Martin-Diana, A.B., Rico, D., Barry-Ryan, C., Frias, J.M., Mulcahy, J., Henehan, G.T.M., 2005.
Calcium lactate washing treatments for salad-cut Iceberg lettuce: Effect of temperature and
concentration on quality retention parameters. Food Res. Int. 38, 729–740.
Matthews, K.R., 2014. The produce contamination problem, Second. ed, The Produce
Contamination Problem. Elsevier.
Mbaye, S., Séchet, P., Pignon, F., Martins, J.M.F., 2013. Influence of hydrodynamics on the
growth kinetics of glass-adhering Pseudomonas putida cells through a parallel plate flow
chamber. Biomicrofluidics 7, 54105.

89

McEvoy, J.L., Luo, Y., Conway, W., Zhou, B., Feng, H., 2009. Potential of Escherichia coli
O157:H7 to grow on field-cored lettuce as impacted by postharvest storage time and
temperature. Int. J. Food Microbiol. 128, 506–509.
McKellar, R.C., LeBlanc, D.I., Lu, J., Delaquis, P., 2012. O157:H7 behavior in fresh-cut lettuce
under dynamic temperature conditions during distribution from processing to retail.
Foodborne Pathog. Dis.
Moore, C.M., Sheldon, B.W., Jaykus, L.-A., 2003. Transfer of Salmonella and Campylobacter
from stainless steel to romaine lettuce. J. Food Prot. 66, 2231–2236.
Munson, B.R., Young, D.F., Okiishi, T.H., Huebsch, W.W., 2009. Fundamentals of fluid
mechanics, 6th ed. Don Fowley.
Nilsson, L.M., Thomas, W.E., Sokurenko, E. V., Vogel, V., 2006. Elevated shear stress protects
Escherichia coli cells adhering to surfaces via catch bonds from detachment by soluble
inhibitors. Appl. Environ. Microbiol. 72, 3005–3010.
Nilsson, L.M., Yakovenko, O., Tchesnokova, V., Thomas, W.E., Schembri, M.A., Vogel, V.,
Klemm, P., Sokurenko, E. V., 2007. The cysteine bond in the Escherichia coli FimH
adhesin is critical for adhesion under flow conditions. Mol. Microbiol. 65, 1158–1169.
Nou, X., Luo, Y., 2010. Whole-leaf wash improves chlorine efficacy for microbial reduction and
prevents pathogen cross-contamination during fresh-cut lettuce processing. J. Food Sci. 75.
Ofek, I., Doyle, R.J., 1994. Bacterial adhesion to cells and tissues. Chapman & Hall, New York.
Ogden, I.D., MacRae, M., Strachan, N.J.C., 2005. Concentration and prevalence of Escherichia
coli O157 in sheep faeces at pasture in Scotland. J. Appl. Microbiol. 98, 646–651.
Oliveira, M., Usall, J., Solsona, C., Alegre, I., Viñas, I., Abadias, M., 2010. Effects of packaging
type and storage temperature on the growth of foodborne pathogens on shredded “Romaine”
lettuce. Food Microbiol. 27, 375–380.
Oliveira, M., Viñas, I., Usall, J., Anguera, M., Abadias, M., 2012. Presence and survival of
Escherichia coli O157:H7 on lettuce leaves and in soil treated with contaminated compost
and irrigation water. Int. J. Food Microbiol. 156, 133–140.
Painter, J.A., Hoekstra, R.M., Ayers, T., Tauxe, R. V., Braden, C.R., Angulo, F.J., Griffin, P.M.,
2013. Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities
by using outbreak data, United States, 1998-2008. Emerg. Infect. Dis. 19, 407–415.
Parish, M.E., Beuchat, L.R., Suslow, T.V., Harris, L.J., Garrett, E.H., Farber, J.N., Busta, F.F.,
2003. Methods to reduce/eliminate pathogens from fresh and fresh-cut produce. Compr.
Rev. Food Sci. Food Saf. 2, 161–173.

90

Park, S.H., Choi, M.R., Park, J.W., Park, K.H., Chung, M.S., Ryu, S., Kang, D.H., 2011. Use of
organic acids to inactivate Escherichia coli O157: H7, Salmonella typhimurium, and
Listeria monocytogenes on organic fresh apples and lettuce. J. Food Sci. 76.
Pirovani, M., Piagentini, A., Güemes, D., Arkwright, S., 2004. Reduction of chlorine
concentration and microbial load during washing-disinfection of shredded lettuce. Int. J.
Food Sci. Technol. 39, 341–347.
Proulx, F., Seidman, E.G., Karpman, D., 2001. Pathogenesis of Shiga toxin-associated hemolytic
uremic syndrome. Pediatr. Res. 50, 163–171.
Pu, S., Beaulieu, J.C., Prinyawiwatkul, W., Ge, B., 2009. Effects of plant maturity and growth
media bacterial inoculum level on the surface contamination and internalization of
Escherichia coli O157:H7 in growing spinach leaves. J. Food Prot. 72, 2313–2320.
Raghubeer, E. V, Ke, J.S., Campbell, M.L., Meyer, R.S., 1995. Fate of Escherichia coli
O157:H7 and other coliforms in commercial mayonnaise and refrigerated salad dressing. J.
Food Prot. 58, 13–18.
Rahn, K., Renwick, S.A., Johnson, R.P., Wilson, J.B., Clarke, R.C., Alves, D., McEwen, S.,
Lior, H., Spika, J., 1997. Persistence of Escherichia coli O157:H7 in dairy cattle and the
dairy farm environment. Epidemiol. Infect. 119, 251–259.
Rajkowski, K.T., Fan, X., 2008. Microbial quality of fresh-cut iceberg lettuce washed in warm or
cold water and irradiated in a modified atmosphere package. J. Food Saf. 28, 248–260.
Rangel, D.E., Marín-Medina, N., Castro, J.E., González-Mancera, A., Forero-Shelton, M., 2013.
Observation of bacterial type I pili extension and contraction under fluid flow. PLoS One 8.
Rossi, E.M., Scapin, D., Tondo, E.C., 2013. Survival and transfer of microorganisms from
kitchen sponges to surfaces of stainless steel and polyethylene. J. Infect. Dev. Ctries. 7,
229–234.
Rusconi, R., Guasto, J.S., Stocker, R., 2014. Bacterial transport suppressed by fluid shear. Nat.
Phys. 10, 212–217.
Ryu, J.H., Beuchat, L.R., 2005. Biofilm formation by Escherichia coli O157:H7 on stainless
steel: Effect of exopolysaccharide and curli production on its resistance to chlorine. Appl.
Environ. Microbiol. 71, 247–254.
Salunkge, D.K., Kadam, S.S., 1998. Lettuce, in: Handbook of vegetable science and technology.
pp. 493–498.
Semat, H., Katz, R., 1958. Hydrodynamics (Fluid in motion), in: Physics. Rinehart & Company,
INC., NewYork, pp. 165–180.

91

Shwartz, H., Shtienberg, D., Vintal, H., 2003. The interacting effects of temperature, duration of
wetness and inoculum size on the infection of pear blossoms by Erwinia amylovora, the
causal agent of fire blight. PHYTOPARASITICA 31, 174–187.
Simons, L., Sanguansri, P., 1997. Advances in the washing of minimally processed vegetables.
Food Austrilia 49, 75–80.
Sinde, E., Carballo, J., 2000. Attachment of Salmonella spp. and Listeria monocytogenes to
stainless steel, rubber and polytetrafluorethylene: the. Food Microbiol. 17, 439–447.
Singh, N., Singh, R.K., Bhunia, A.K., Stroshine, R.L., 2002. Efficacy of chlorine dioxide, ozone,
and Thyme essential oil or a sequential washing in killing Escherichia coli O157:H7 on
lettuce and baby carrots. Leb. und-Technologie 35, 720–729.
Singh, R.P., Heldman, D.R., 2014. Fluid flow in food processing, in: Taylor, S.L. (Ed.),
Introduction to Food Engineering. Elsevier, pp. 65–209.
Sivapalasingam, S., Friedman, C.R., Cohen, L., Tauxe, R. V, 2004. Fresh produce: a growing
cause of outbreaks of foodborne illness in the United States, 1973 through 1997. J. Food
Prot. 67, 2342–2353.
Solomon, E.B., Pang, H.-J., Matthews, K.R., 2003. Persistence of Escherichia coli O157:H7 on
lettuce plants following spray irrigation with contaminated water. J. Food Prot. 66, 2198–
2202.
Solomon, E.B., Yaron, S., Matthews, K.R., 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.
Spinks, A.T., Dunstan, R.H., Harrison, T., Coombes, P., Kuczera, G., 2006. Thermal inactivation
of water-borne pathogenic and indicator bacteria at sub-boiling temperatures. Water Res.
40, 1326–1332.
Stafford, R.J., McCall, B.J., Neill, A.S., Leon, D.S., Dorricott, G.J., Towner, C.D., Micalizzi,
G.R., 2002. A statewide outbreak of Salmonella bovismorbificans phage type 32 infection
in Queensland. Commun. Dis. Intell. 26, 568–573.
Sutherland, I., 1985. Biosynthesis and composition of gram-negative bacterial extracellular and
wall polysaccharides. Annu. Rev. Microbiol.
Taormina, P.J., Beuchat, L.R., Erickson, M.C., Ma, L., Zhang, G., Doyle, M.P., 2009. Transfer
of Escherichia coli O157:H7 to iceberg lettuce via simulated field coring. J. Food Prot. 72,
465–472.

92

Thomas, W., Forero, M., Yakovenko, O., Nilsson, L., Vicini, P., Sokurenko, E., Vogel, V., 2006.
Catch-bond model derived from allostery explains force-activated bacterial adhesion.
Biophys. J. 90, 753–764.
Thomas, W.E., Nilsson, L.M., Forero, M., Sokurenko, E. V., Vogel, V., 2004. Shear-dependent
“stick-and-roll” adhesion of type 1 fimbriated Escherichia coli. Mol. Microbiol. 53, 1545–
1557.
Turatti, A., 2011. Process design, facility and equipment requirements, in: Advances in FreshCut Fruits and Vegetables Processing. pp. 339–361.
Ukuku, D.O., Fett, W., 2002. Behavior of Listeria monocytogenes inoculated on cantaloupe
surfaces and efficacy of washing treatments to reduce transfer from rind to fresh-cut pieces.
J. Food Prot. 65, 924–930.
USDA/ERS, 2011. U.S. Lettuce statistics. Available at:
http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1576
(accessed 10.24.14).
USDA/ERS, 2014. U.S. per capita use of fresh and processing vegetables, dry pulse crops, and
potatoes; cash receipts; U.S. vegetable trade. Available at: http://www.ers.usda.gov/dataproducts/vegetables-and-pulses-data/yearbook-tables.aspx (accessed 10.24.14).
Vorst, K.L., Todd, E.C.D., Rysert, E.T., 2004. Improved quantitative recovery of Listeria
monocytogenes from stainless steel surfaces using a one-ply composite tissue. J. Food Prot.
67, 2212–2217.
Wachtel, M.R., Charkowski, A.O., 2002. Cross-contamination of lettuce with Escherichia coli
O157:H7. J. Food Prot. 65, 465–470.
Wang, G., Zhao, T., Doyle, M.P., 1996. Fate of enterohemorrhagic Escherichia coli O157:H7 in
bovine feces. Appl. Environ. Microbiol. 62, 2567–2570.
Wang, H., Feng, H., Liang, W., Luo, Y., Malyarchuk, V., 2009. Effect of surface roughness on
retention and removal of Escherichia coli O157:H7 on surfaces of selected fruits. J. Food
Sci. 74.
Wang, H., Liang, W., Feng, H., Luo, Y., 2007. Modeling of the effect of washing solution flow
conditions on Escherichia coli O157:H7 population reduction on fruit surfaces. J. Food
Prot. 70, 2533–2540.
Watada, A.E., Ko, N.P., Minott, D.A., 1996. Factors affecting quality of fresh-cut horticultural
products. Postharvest Biol. Technol. 9, 115–125.
Yang, H., Swem, B.L., Li, Y., 2003. The effect of pH on inactivation of pathogenic bacteria on
fresh-cut lettuce by dipping treatment with electrolyzed water. J. Food Sci. 68, 1013–1017.
93

Yoshihara, A., Narahara, H., Kuriyama, Y., Toyoda, S., Tokumoto, H., Konishi, Y., Nomura, T.,
2014. Measurement of microbial adhesive forces with a parallel plate flow chamber. J.
Colloid Interface Sci. 432, 77–85.
Zhang, G., Ma, L., Phelan, V.H., Doyle, M.P., 2009. Efficacy of antimicrobial agents in lettuce
leaf processing water for control of Escherichia coli O157:H7. J. Food Prot. 72, 1392–
1397.
Zhao, T., Doyle, M.P., Besser, R.E., 1993. Fate of enterohemorrhagic Escherichia coli O157:H7
in apple cider with and without preservatives. Appl. Environ. Microbiol. 59, 2526–2530.

94