2308

Journal of Food Protection, Vol. 72, No. 11, 2009, Pages 2308–2312
Copyright G, International Association for Food Protection

Transfer of Escherichia coli O157:H7 from Soil, Water, and

Manure Contaminated with Low Numbers of the Pathogen to
Lettuce Plants
GABRIEL MOOTIAN, WEN-HSUAN WU, AND KARL R. MATTHEWS*

Department of Food Science, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, 65 Dudley Road,

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New Brunswick, New Jersey 08901-8520, USA

MS 09-004: Received 7 January 2009/Accepted 10 April 2009

ABSTRACT
The sources of contamination of leafy greens remain unclear, but it is evident that contaminated water, soil amendments, and
wildlife likely contribute. The objective of the present study was to determine transfer of low numbers of Escherichia coli
O157:H7 from soil, manure-amended soil, and water to growing lettuce plants. Lettuce plants, young (12 days of age) or mature
(30 days of age), were grown in soil, manure-amended soil, or irrigated with water containing 101, 102, 103, or 104 CFU E. coli
O157:H7 per g or ml. Harvested plants were processed to determine whether E. coli O157:H7 was associated with the entire plant
or within internal locations. Young plants (12 days) were harvested at 1, 10, 20, and 30 days postexposure. No samples were
positive for E. coli O157:H7 after direct plating of serial dilutions. Enrichment of all samples from young plants exposed to
contaminated soil, manure-amended soil, and irrigation water demonstrated that approximately 21% (113 of 552) of plants were
positive for E. coli O157:H7. Approximately 30% (36 of 120) of the mature plants initially irrigated with or grown in
contaminated soil (including manure-amended soil) for 15 days were positive for E. coli O157:H7. Based on sterilization of
surface tissue, E. coli O157:H7 was in protected locations of lettuce tissue. The results suggest that lettuce exposed to, and grown
in the presence of, low numbers of E. coli O157:H7 may become contaminated and thus present a human health risk.

Foodborne illness outbreaks linked to the consumption
of contaminated leafy greens continues not only in the
United States, but also throughout the world (8). The most
common etiologic agents associated with these outbreaks
are Escherichia coli O157:H7 and Salmonella. Many of
these outbreaks have been linked to lettuce. For example, in
Sweden, a total of 135 cases, including 11 cases of
hemolytic uremic syndrome, were linked to the consump-
tion of locally produced lettuce that was contaminated with
E. coli O157. Water samples from a stream used for
irrigation were positive for the outbreak strain, as were cattle
at a farm upstream of the irrigation point (12). Contaminated
water, soil amendments, flooding of fields, and wildlife are
all likely sources of contamination of lettuce (3). A limited
number of studies have addressed the interaction of E. coli
O157:H7 (or for that matter, other enteric pathogens) with
growing lettuce plants; most studies have used seedlings
(that may not be representative of mature plants) or mature
plants (10, 15, 16). A major criticism of studies addressing
E. coli O157:H7 interaction (external and internal) with
lettuce plants and tissue is the use of challenge populations
that were unrealistically high. Populations of E. coli
O157:H7, if present in the environment, are likely extremely
low (5, 11). High-challenge inocula of E. coli O157:H7
were used in many studies, since the goal was to determine
* Author for correspondence. Tel: 732-932-9611; Fax: 732-932-6776;
E-mail: matthews@aesop.rutgers.edu.
the location and route of contamination through tracking of
the microbe in the rhizosphere and the phyllosphere (13).
The ability of E. coli O157:H7 to invade the internal
tissue of lettuce plants has been reported (13, 15, 16).
Researchers have suggested that E. coli O157:H7 can be
taken up by the roots and transported to the edible portion of
a lettuce plant (7, 15, 16). Solomon et al. (15) reported the
internalization of E. coli O157:H7 into edible tissue of
lettuce, detected by laser scanning confocal microscopy,
through root-associated uptake of the pathogen. After
surface sanitizing for 10 min, internalized cells were
detected in plants exposed to 108 CFU E. coli O157:H7,
but not to 104 CFU E. coli O157:H7. Other researchers
report contradictory outcomes such as association with root
tissue, but not within edible tissue (9, 10). A number of
variables including plant age, soil type, growth conditions
(e.g., hydroponics versus soil), method of exposure (seed or
plant), and lettuce variety may influence whether enteric
pathogens will enter a plant through the root system and
migrate to the phyllosphere (6, 13).
Given the lack of conclusive data concerning the
internalization of enteric foodborne pathogens, whether
through the roots, stomata, or wounds in the edible tissue of
growing plants, the cultivation of crops in soils containing
low populations of E. coli O157:H7 should be considered a
potential human health risk Indeed, low numbers of bacteria
associated with the phyllosphere are capable of persisting
for extended periods, and the infectious dose for E. coli
O157:H7 may be less than 100 cells; therefore, exposure of
J. Food Prot., Vol. 72, No. 11
growing crops to any level of the pathogen is unacceptable.
Solomon et al. (14) demonstrated the persistence of E. coli
O157:H7 on lettuce for more than 20 days under laboratory
conditions. The lettuce plants were 30 days old when
initially exposed to E. coli O157:H7–contaminated irriga-
tion water. The interaction of E. coli O157:H7 with plants
may be influenced by the age of the plant. A recent study
demonstrated that the population of E. coli O157:H7 was
consistently greater on young (inner) leaves as compared
with older, middle leaves of lettuce (4).
The objective of this study was to determine whether
the exposure of the rhizosphere and phyllosphere of lettuce
plants to low populations of E. coli O157:H7 would result in
detectable levels of the pathogen associated with the
phyllosphere. In this study, plants were grown either in soil
or manure-amended soil containing 101, 102, 103, or
104 CFU E. coli O157:H7/g, or surface irrigated once with
water containing 101, 102, 103, or 104 CFU E. coli O157:H7
per ml. Two groups of plants were used: 12-day-old (young)
and 30-day-old (old) plants were followed for 30 and 15
days postexposure, respectively.
MATERIALS AND METHODS
Bacteria. E. coli O157:H7 strain ATCC 43895 (isolated from
ground beef) was used in this study. The isolated was used in
previous studies (14, 15). The strain was transformed to express
green fluorescent protein (GFP) by using the plasmid pGFP
(Clontech, Inc., Palo Alto, CA), as described previously (15).
Working cultures were maintained at 4uC on tryptic soy agar
(TSA; Difco, Becton Dickinson, Sparks, MD) plates containing
100 mg/ml ampicillin (Amp; Sigma, St. Louis, MO) (TSA-Amp).
Lettuce plant cultivation. Seeds of green ice leaf lettuce
(Lactuca sativa L.) were purchased from W. Atlee Burpee Co.
(Warminster, PA). Plants were grown to 12 days of age in plugs,
and then transplanted to larger pots as required. For experiments
requiring plants 30 days of age and older, plants were grown in 3-
in.-diameter pots containing a 1:1 mixture of Canadian peat moss
and agricultural-grade vermiculite. Plants were watered daily and
fertilized as needed with Pete’s General Purpose 15-15-15 fertilizer
(Peter’s Professional, Allentown, PA) in the Rutgers University
floriculture greenhouse. Plants were grown in a 12-h photoperiod
at 21uC during the day and 15uC at night. Plants were watered
when the soil surface started to dry. Water was applied directly to
soil by using a 25-ml pipette. Green ice lettuce reaches maturity
between 40 and 50 days of growth. Age of plants in this study
refers to time after emergence. Twelve-day-old plants typically had
four to five leaves.
Inoculum preparation and challenge of 12-day-old
lettuce plants. E. coli O157:H7 was cultured in tryptic soy broth
(TSB; Difco, Becton Dickinson) and Amp (TSB-Amp) at 37uC for
24 h. To prepare the inoculum, cells were harvested by
centrifugation (3,500 | g for 10 min) and resuspended in distilled
water to achieve approximately 109 CFU/ml. Soil, irrigation water,
and manure were contaminated with E. coli O157:H7. In brief, for
soil, E. coli O157:H7 was added to achieve levels of approximately
101, 102, 103, and 104 CFU/g. Cow manure obtained from the
Rutgers University farm was inoculated to achieve E. coli
O157:H7 levels of approximately 5 | 101, 5 | 102, 5 | 103,
and 5 | 104 CFU/g. The inoculated samples (soil or manure) were
mixed vigorously to ensure even distribution of the pathogen.
TRANSFER OF E. COLI O157:H7 TO LETTUCE PLANTS 2309
Water for irrigation was prepared by adding E. coli O157:H7 to
achieve levels of 101, 102, 103, and 104 CFU/ml. Bacterial levels in
soil, manure, and water were confirmed by plating dilutions on
TSA-Amp. On the day of challenge, plants were moved from the
greenhouse to our laboratory. Each experiment included 12-day-
old plants that were separated into four groups of six plants per
group. Experiments using contaminated soil, manure, and
irrigation water were conducted independently. An uninoculated
control group was included.
Plugs containing a single 12-day-old plant were planted in 3-
in. pots containing soil or manure-amended soil (1 part manure to 4
parts soil) having E. coli O157:H7 at levels of 101, 102, 103, and
104 CFU/g. In experiments with E. coli O157:H7–contaminated
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water; a 20-ml volume of water was applied directly to the soil of
plants in 3-in. pots. Soil was irrigated only once with the
contaminated water. For all experiments, the outer leaves were
not prevented from touching the soil, to closely model in-field
conditions. Experiments were conducted twice.
Challenge of 30-day-old plants. Thirty-day-old lettuce
plants grown as described above were transplanted to soil,
manure-amended soil, or irrigated with water contaminated with
E. coli O157:H7. E. coli O157:H7 was at levels of 101, 102, 103,
and 104 CFU/g or ml. In the surface-irrigation experiments, the soil
in each pot was irrigated with 200 ml of water containing E. coli
O157:H7 at levels stated previously. The inoculum was applied
carefully to prevent splashing of the inoculum onto the edible
portion of the lettuce plant. Soil was irrigated only once with the
contaminated water. When the soil surface started to dry, plants
were watered as with pathogen-free water. Each group contained
six plants. An untreated control group was included. Experiments
were conducted twice.
Microbiological analysis. Plants that were 12 days old at the
initiation of the study were harvested 1, 10, 20, and 30 days
postchallenge. Plants that were 30 days old at initiation of the study
were harvested 1 and 15 days postchallenge. The plants were cut
from the root systems approximately 1 cm above the soil surface to
minimize surface contamination of the edible portion of the plant
through contact with the planting mixture. Six plants per treatment
were collected and divided into two sets of three plants. One set of
three plants from each treatment group were surface disinfected as
previously described (15). In brief, plants were dipped in 80%
ethanol for 5 s, which was followed by immersion in 0.1% HgCl2
(wt/vol) for 5 min. The plants or lettuce leaves were washed twice
in sterile water and shaken to remove excess water. The remaining
set of three plants were immersed in sterile water for 5 min,
washed twice in sterile water, and then shaken to remove excess
water. This was done to mirror the procedure used for surface
sanitizing. The above method was conducted in a Biosafety Level
2 cabinet. All leaves on a plant were aseptically removed, weighed,
and placed into sterile Whirl-Pak bags (Nasco, Fort Atkinson, WI)
containing TSB-Amp at a volume twofold greater than the weight
of the lettuce. The samples were homogenized either by hand or in
a stomacher. A 100-ml volume was surface plated on TSA-Amp.
Plates were incubated at 37uC for 24 h, and then illuminated with
UV light, and GFP-expressing colonies were enumerated. The
remaining sample homogenate was also incubated at 37uC for
24 h, and 100-ml of the enriched sample spread plated on TSA-
Amp only when no E. coli O157:H7 colonies were present after
direct plating of the homogenate. Plates were illuminated with UV
light, and GFP-expressing colonies identified.
Composite soil samples (10 g) were collected on each sample
day for all treatments. Soil samples were dispensed into sterile
2310 MOOTIAN ET AL. J. Food Prot., Vol. 72, No. 11

TABLE 1. Contamination of 12-day-old lettuce plants, based on sample enrichment after exposure to soil or manure-amended soil
contaminated with Escherichia coli O157:H7
Level of Escherichia coli O157:H7a:
Day(s)
Treatment Surface examined postexposure 101 CFU/g 102 CFU/g 103 CFU/g 104 CFU/g

Soil Total 1 NDb 0/6 3/6 6/6

10 ND 4/6 5/6 6/6
20 ND 1/6 0/6 1/6
30 ND 1/6 0/6 1/6
Internal 1 0/6 0/6 4/6 3/6
10 0/6 0/6 0/6 0/6
20 0/6 0/6 0/6 0/6

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30 0/6
Manure Total 1 4/6
10 0/6
20 1/6
30 0/6
Internal 1 2/6
10 0/6
20 0/6
30 0/6
a
Samples tested positive by enrichment in TSB-Amp, as determined by surface plating on TSA-Amp. Values are number of positive
plants/number of plants tested.
b
ND, not determined.
Whirl-Pak bags containing 10 ml of TSB-Amp. The sample was
homogenized with a stomacher, and a 100-ml volume was surface
plated on TSA-Amp. Plates and remaining sample homogenate
were incubated at 37uC for 24 h. Plates were illuminated with UV
light, and GFP-expressing colonies were enumerated. Plating from
the enrichment culture, incubation of the plates, and plate counts
were performed as described above.
RESULTS AND DISCUSSION
The outcome of research addressing the preharvest
persistence and location (internal or external) of human
pathogens on leafy greens and other crops is often
contradictory. The difficulty in detecting E. coli O157:H7
in the environment suggests that when present, the
populations are extremely low (5, 11). Researchers
investigating the interactions between food crops and
enteric foodborne pathogens use levels of a target pathogen
that are orders of magnitude greater than what would be
expected in the environment. We utilized low levels (101,
102, 103, and 104 CFU) of E. coli O157:H7 to investigate
contamination during growth of ca. 800 young and mature
lettuce plants. Table 1 summarizes the number of young
lettuce plants that were positive for E. coli O157:H7 after
being transplanted at 12 days of age into soil or manure-
amended soil contaminated with low levels of the pathogen.
Results indicate that approximately 22% of plants were
positive for E. coli O157:H7. Approximately 7% of surface-
sterilized plants were positive for the pathogen, suggesting
cells were protected at surface or subsurface locations.
There also exists the possibility that cells were in protected
locations on the leaf surface and as such, survived the
surface-sanitizing treatment. Similar results occurred in
plants irrigated with contaminated water (Table 2). All
control plants were negative for E. coli O157:H7.
0/6 0/6 0/6
4/6 4/6 4/6
4/6 5/6 0/6
1/6 0/6 4/6
0/6 0/6 0/6
2/6 3/6 6/6
0/6 0/6 0/6
0/6 0/6 0/6
0/6 0/6 0/6
The ability of enteric foodborne pathogens such as E.
coli O157:H7 to survive under environmental conditions is
largely unknown. Research suggests that E. coli O157:H7
can survive in soil, manure, and water for extended periods
(1). In studies investigating the association of human
pathogens with food crops, plants were grown in soil,
hydroponic systems, or gnotobiotic systems containing up
to 108 CFU/g of the target pathogen—levels far exceeding
those reportedly found in nature (2, 7, 10, 15, 16, 18). The
cultivars used and the age of plants tested were often
different precluding direct comparison of the studies.
However, the conclusion drawn from those studies was
that E. coli O157:H7 at high densities (108 CFU/g) became
established on roots and edible tissues of plants, whereas at
low densities (102 to 104 CFU/g), colonization was
comparatively low. E. coli counts in this study remained
below the level of detection even in seedlings transplanted
into soil containing 104 CFU E. coli O157:H7/g (Table 1).
At the end of the cultivation period, 30 days postexposure,
only 2 of 42 non–surface-sterilized samples were positive,
based on microbiological enrichment (Table 1). E. coli
O157:H7 was not recovered from surface-sterilized leaf
tissue. Mature plants, such as the 42-day-old plants, have a
well-developed secondary root system, which may increase
the likelihood of interaction with bacteria in the soil.
The ability of a microbe to interact with the root system
of a plant is likely influenced by a multitude of variables.
Enteric pathogens may enter the plant through the branch
root junctions and root tips (13). However, the ability of
bacteria associated with soil particles or components of
manure to interact with a plants root system may be limited.
In this study, microbiological analysis was not conducted on
root tissue. E. coli was recovered from surface-sterilized
root tissue, but not from within leaves, of 62-day-old
J. Food Prot., Vol. 72, No. 11 TRANSFER OF E. COLI O157:H7 TO LETTUCE PLANTS 2311

TABLE 2. Contamination of 12-day-old lettuce plants, based on sample enrichment after exposure to irrigation water contaminated with
Escherichia coli O157:H7
Level of Escherichia coli O157:H7a:
Day(s)
Treatment Surface examined postexposure 101 CFU/g 102 CFU/g 103 CFU/g 104 CFU/g

Water Total 1 0/6 0/6 1/6 3/6

10 3/6 5/6 4/6 3/6
20 0/6 1/6 0/6 0/6
30 6/6 1/6 0/6 1/6
Internal 1 0/6 0/6 0/6 0/6
10 1/6 1/6 0/6 0/6
20 0/6 4/6 0/6 0/6

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30 0/6 0/6 0/6 0/6
a
Samples tested positive by enrichment in TSB-Amp, as determined by surface plating on TSA-Amp. Values are number of positive
plants/number of plants tested.

spinach plants cultivated in soil that was contaminated with
E. coli (18). However, microbiological enrichment of leaf
tissue samples was not conducted; low levels of the
pathogen may have been present and simply not detected.
Bacteria in surface irrigation water may initially have
an increased opportunity to interact with the root system as
the water and bacteria flows through the soil stratum. In the
present study, 12-day-old plants were transplanted to fresh
potting soil and subsequently surface irrigated with 20 ml of
water containing 101, 102, 103, or 104 CFU E. coli
O157:H7/ml. A total of eight non–surface-sterilized samples
(plants) collected 30 days postexposure to contaminated
irrigation water were positive for E. coli O157:H7
(Table 2); the pathogen was detected in all soil samples
after enrichment (data not shown). It is important to reiterate
that, in this study, leaves were not prevented from
contacting the soil, thereby eliminating root uptake as the
sole means by which leaf tissue could become contaminat-
ed. A clear correlation of pathogen level in soil or irrigation
water to plant tissue contamination was not evident
(Tables 1 through 4). Indeed, for several sample points, a
greater number of plants exposed to 101 as compared with
104 E. coli O157:H7 were microbiologically positive for the
pathogen. Approximately, 55, 14, and 9% of surface-
sterilized (5 min) tissue samples from young lettuce plants
exposed to 108, 106, and 104 CFU E. coli O157:H7,
respectively, were positive when harvested 9 days or earlier
postexposure (15).
There are notable morphological, developmental, and
anatomical differences in the root systems of plants as they
mature (17). These differences may influence the ability of
enteric microbes to interact with, enter plant roots, and
travel to the leaf tissue. In this study, 30-day-old plants were
transplanted into soil (soil alone or manure-amended soil)
contaminated with E. coli O157:H7. During transplanting,
care was taken to prevent damage to the root system of
plants. The pathogen persisted in the soil for the 15-day
experimental period. E. coli O157:H7 was recovered from
39% of all non–surface-sterilized and 11% of all surface-
sterilized samples of plants that had been cultivated for 15
days posttransplantation (Table 3). Nearly 63% of plants
(non–surface sterilized) that had been cultivated for 15 days
after irrigation with contaminated water were positive for E.
coli O157:H7 (Table 4), whereas only 13% of surface-
sterilized plants were positive for the target pathogen.
Regardless of the exposure scenario, E. coli O157:H7 was

TABLE 3. Contamination of 30-day-old lettuce plants, based on sample enrichment after exposure to soil or manure-amended soil
contaminated with Escherichia coli O157:H7
Level of Escherichia coli O157:H7a:
Day(s)
Treatment Surface examined postexposure 101 CFU/g 102 CFU/g 103 CFU/g 104 CFU/g

Soil Total 1 0/6 3/6 5/6 5/6

15 1/6 2/6 1/6 2/6
Internal 1 0/6 0/6 0/6 0/6
15 2/6 2/6 0/6 0/6
Manure Total 1 NDb ND 1/6 1/6
15 ND ND 4/6 4/6
Internal 1 ND ND 0/6 0/6
15 ND ND 0/6 0/6
a
Samples tested positive by enrichment in TSB-Amp, as determined by surface plating on TSA-Amp. Values are number of positive
plants/number of plants tested.
b
ND, not determined.
2312 MOOTIAN ET AL.
TABLE 4. Contamination of 30-day-old lettuce plants, based on sample enrichment after exposure to irrigation water contaminated with
Escherichia coli O157:H7
Day(s)
Treatment Surface examined postexposure 101 CFU/g
Water Total 1 1/6
15 5/6
Internal 1 0/6
15 1/6
a
Samples tested positive by enrichment in TSB-Amp, as determined by surface plating on TSA-Amp. Values are number of positive
plants/number of plants tested.
associated with leaf tissue such that it was protected from
the action of a stringent surface-sanitizing regime. Research
is needed to determine whether low numbers of surviving
bacteria associated with lettuce tissue may be capable of
growth during transport and storage, and thus present a
human health risk.
In this study, E. coli O157:H7 was only detected after
enrichment of samples, suggesting that the level of
contamination of lettuce tissue was extremely low even
though the pathogen persisted in the soil during the growth
period. A greater percentage (includes total and surface
sanitized samples) of mature plants as compared with young
plants were positive at harvest, although all plants were ca. 45
days of age at harvest. These results suggest that contamina-
tion of lettuce at times increasingly close to harvest may
increase the risk of the pathogen being present on the crop at
harvest. Future efforts must center on avoiding human
pathogen contamination of produce rather than focusing on
disinfection technologies with limited efficacy.
ACKNOWLEDGMENT
This work was supported with funds from the Western Institute for
Food Safety and Security.
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