2028

Journal of Food Protection, Vol. 72, No. 10, 2009, Pages 20282037
Copyright G, International Association for Food Protection

Lack of Internalization of Escherichia coli O157:H7 in Lettuce

(Lactuca sativa L.) after Leaf Surface and Soil Inoculation
GUODONG ZHANG,{ LI MA, LARRY R. BEUCHAT, MARILYN C. ERICKSON, VANESSA H. PHELAN,
MICHAEL P. DOYLE*

AND

Center for Food Safety, University of Georgia, 1109 Experiment Street, Griffin, Georgia 30223-1797, USA
MS 09-096: Received 27 February 2009/Accepted 12 June 2009

ABSTRACT
Survival and internalization characteristics of Escherichia coli O157:H7 in iceberg, romaine, and leaf lettuce after
inoculation of leaf surfaces and soil were determined. A five-strain mixture of E. coli O157:H7 in water and cow manure extract
was used as an inoculum for abaxial and adaxial sides of leaves at populations of 6 to 7 log and 4 log CFU per plant. The five
strains were individually inoculated into soil at populations of 3 and 6 log CFU/g. Soil, leaves, and roots were analyzed for the
presence and population of E. coli O157:H7. Ten (4.7%) of 212 samples of leaves inoculated on the adaxial side were positive for
E. coli O157:H7, whereas 38 (17.9%) of 212 samples inoculated on the abaxial side were positive. E. coli O157:H7 survived for
at least 25 days on leaf surfaces, with survival greater on the abaxial side of the leaves than on the adaxial side. All 212
rhizosphere samples and 424 surface-sanitized leaf and root samples from plants with inoculated leaves were negative for E. coli
O157:H7, regardless of plant age at the time of inoculation or the location on the leaf receiving the inoculum. The pathogen
survived in soil for at least 60 days. Five hundred ninety-eight (99.7%) of 600 surface-sanitized leaf and root samples from plants
grown in inoculated soil were negative for E. coli O157:H7. Internalization of E. coli O157:H7 in lettuce leaves and roots did not
occur, regardless of the type of lettuce, age of plants, or strain of E. coli O157:H7.

Microbial contamination and colonization of food and

feed crops through root systems has been reported (14, 8,
12, 14, 2123, 27). Contamination and potential internalization of Escherichia coli O157:H7 in leafy greens are of
great interest to the fresh produce industry. In situ
glucuronidase staining of spinach seedlings has revealed that
a generic strain of E. coli was internalized within root tissue
and, to a limited extent, hypocotyls (26). When inoculated
seeds were sown in soil microcosms and cultivated for 42
days, E. coli was recovered from the external surfaces of
spinach roots and leaves as well as from surface-sterilized
roots (internalized). When 20-day-old spinach seedlings
grown from uninoculated seeds were transplanted in soil
inoculated with E. coli, the bacterium became established on
the plant surface, but internalization into the inner root tissue
was restricted (26). After seedlings were transferred to a
hydroponic system containing E. coli, the bacterium was
recovered from surface-sanitized roots, indicating that it had
been internalized (26). E. coli O157:H7 has been reported to
be internalized in spinach seedlings grown hydroponically,
but was not recovered within the tissues of mature plants (12).
Using laser scanning confocal microscopy and epifluorescence microscopy, E. coli O157:H7 was observed in
internal tissues of lettuce seedlings (21). E. coli O157:H7
was not internalized into lettuce tissue grown hydroponi* Author for correspondence. Tel: 770-228-7284; Fax: 770-229-3216;
E-mail: mdoyle@uga.edu.
{ Present address: Center for Food Safety and Applied Nutrition, U.S. Food
and Drug Administration, 5100 Paint Branch Parkway, College Park, MD
20740, USA.

cally but was found at considerable densities in surfacesanitized leaves of lettuce grown in soil (6). However, the
pathogen was not detected in the edible parts of lettuce
grown in manure-amended soil (7). In another study, E. coli
O157:H7 was observed to be internalized by lettuce
seedlings but was not recovered from internal tissues of
mature plants (12). When manure or irrigation water
containing E. coli and E. coli O157:H7 was applied to soil
in which lettuce was grown, roots and edible parts of the
plant were positive for these bacteria (11, 21, 22, 25). Others
have reported that E. coli O157:H7 was not detected in roots
and leaves of lettuce grown in inoculated soil, indicating
that transmission of the pathogen from soil to leaves did not
occur through uptake or by splashing of soil or water (13,
14). Most of these studies used one lettuce cultivar, and all
but one study used only one strain of E. coli O157:H7.
The objective of the research reported here was to
evaluate several strains of E. coli O157:H7 for their ability
to persist on and internalize tissues of lettuce plants, and to
investigate the susceptibility of different types of lettuce to
internalization of E. coli O157:H7. The presence of E. coli
O157:H7 on and in roots and leaves of lettuce grown in
inoculated soil was also investigated.
MATERIALS AND METHODS
Bacterial strains and culture conditions. Strains of E. coli
O157:H7 and culture methods used in this study were the same as
those described in a previous report (28). Briefly, five strains of E.
coli O157:H7 were used: ATCC 43888 (human feces), EO122
(bovine isolate), K3995 (spinach isolate), K4492 (lettuce, clinical

J. Food Prot., Vol. 72, No. 10

LACK OF INTERNALIZATION OF E. COLI O157:H7 IN LETTUCE

isolate), and F4546 (alfalfa sprout outbreak isolate). These strains

were labeled with a green fluorescent protein (gfp) gene on the
pGFPuv plasmid (Clontech, Palo Alto, CA). All strains were
grown at 37uC for 24 h on tryptic soy agar (TSA; Difco, Becton
Dickinson, Sparks, MD) or in tryptic soy broth (TSB) supplemented with ampicillin (amp; Roche Diagnostics, Indianapolis, IN)
at a concentration of 100 mg/ml (TSA-amp and TSB-amp,
respectively). Ampicillin (100 mg/ml) was added to TSA and
TSB to maintain the pGFPuv plasmid, which contains not only a
gfp gene, but also a gene that encodes ampicillin resistance in E.
coli O157:H7. Colonies formed by GFP-labeled strains were
evident by viewing them under a 396-nm wavelength UV lamp.
All strains of E. coli O157:H7 were transferred to TSB-amp
three times at 24-h intervals preceding their use as inocula. Equal
volumes of 16- to 18-h cultures of each strain were combined to
create a five-strain mixture. Cells from the mixture were
centrifuged at 5,000 | g for 10 min and resuspended in sterile
deionized water. The suspension was diluted in sterile deionized
water to give desired population densities of 108 and 106 CFU/ml.
Populations were determined by serially diluted samples in sterile
0.1% peptone and surface plating on TSA-amp, and incubating
plates at 37uC for 24 h before counting colonies.
Conditions for growing lettuce. Iceberg lettuce (Great Lakes
no. 118), romaine lettuce (Parris Island Cos), and leaf lettuce
(Black Seeded Simpson) (Lactuca sativa L.) seeds were purchased
from a retail store in Griffin, GA. All seeds were distributed by
Ferry-Morse Seed Company, Fulton, KY.
Seeds were sown on the surface of sandy soil (9 cm deep) in a
plastic container (40.1 cm wide, 67.3 cm long, and 16.5 cm high) in
an environmental chamber (2.8 m wide, 2.8 m long, and 2.0 m
high). A tablespoon (approximately 15 g) of fertilizer (20-27-5,
N-P-K, United Industry Co., St. Louis, MO) was added to each
container and thoroughly mixed into the soil immediately before
sowing the seeds. Soil was then saturated with water, and seeds
were broadcast on the surface. Sandy soil was deposited (1 cm
deep) on top of the seeds. The containers were placed in an
environmental chamber set at 23uC during the day and 7uC at
night, with a 12-h photoperiod. The light intensity was at 600 to
700 mmol/m2/s. Plants were monitored daily. Water was applied
every other day when the soil surface started to dry and crack.
Lettuce seedlings with four or five leaves were transplanted to 15cm (diameter and height) pots (classic 200, Nursery Supplies, Inc.,
Fairless Hills, PA) containing sandy soil (Tifton, GA). These pots
were placed on capillary mats in shallow trays. Seedlings were
watered at 2- to 3-day intervals, depending on soil moisture
conditions. Plants were watered when the soil surface began to dry
and crack. Soil and plants accessed water from the capillary mat
through a hole in the bottom of the pot.
Preparation of cow manure extracts. Deionized water
(400 ml) was added to 100 g of fresh cow manure that had been
obtained from a local dairy, stored at 220uC, and thawed at 4uC.
The slurry was mixed and then autoclaved at 121uC for 30 min.
The sterilized slurry was centrifuged at 3,000 | g for 10 min. The
supernatant (manure extract) was deposited in sterile bottles and
stored at 220uC until used.
Leaf inoculation trials. In the first leaf inoculation study,
iceberg lettuce (Great Lakes no. 118) was grown at temperatures of
23uC during the day and 7uC at night, with a 12-h photoperiod.
Five-strain mixtures of GFP-labeled E. coli O157:H7 in deionized
water and cow manure extract were used as inocula for leaves.
Inoculum (20 ml) was pipetted onto two leaves per plant (about 5

2029

droplets per leaf). Caution was used to prevent runoff of the

inoculum or contamination of adjacent leaves. Inoculated leaves
were marked with a permanent ink marker. These leaves were
inoculated on abaxial or adaxial sides (one side only for each plant)
at populations of 6 to 7 log CFU per plant on days 3, 30, and 60
after transplanting four- to five-leaf seedlings to pots, and analyzed
for E. coli O157:H7 19 and 54; 3, 7, and 25; and 3 and 7 days after
inoculation, respectively. At each sampling time, soil in which
lettuce plants were grown, rhizosphere (root surfaces, including
soil attached to roots), macerated roots after surface sterilization,
leaf surface, and macerated leaves after surface sterilization were
analyzed for the pathogen.
In another leaf inoculation study, iceberg lettuce plants were
inoculated with E. coli O157:H7 at a population of 4 log CFU per
plant 7 days after transplanting seedlings to pots. Inoculation
procedures were the same as described above. Leaves, rhizosphere,
roots, and soil were analyzed for E. coli O157:H7 15, 26, 35, and
44 days after inoculation.
Soil inoculation. Iceberg, romaine, and leaf lettuces were
grown in sandy soil in an environmental chamber at temperatures
of 23uC during the day (12 h) and 7uC at night (12 h). The light
intensity was at 600 to 700 mmol/m2/s. Lettuce seedlings were
transplanted in the inoculated soil when they had four or five true
leaves. Soil was inoculated with single strains of five GFP-labeled
E. coli O157:H7 at a population of 6 log CFU/g of soil at the time
of transplanting seedlings. Using a polypropylene, conical 15-ml
screw-cap test tube (Sarstedt, Inc., Newton, NC), two holes 6-cm
deep were made in the top of the soil at two opposite sides of each
pot (ca. 5 cm from the plant stem). E. coli O157:H7 suspension
(15 ml, 8 log CFU/ml) was added via the two holes in each pot to
result in a population of ca. 6 log CFU/g of soil. Leaves, soil,
rhizosphere, and roots of plants were analyzed for E. coli O157:H7
17, 45, and 60 days after seedlings were transplanted.
A second soil inoculation experiment was done by using E.
coli O157:H7 at 3 log CFU/g of soil. Leaves, soil, rhizosphere, and
roots were analyzed for E. coli O157:H7 26 and 60 days after
seedlings were transplanted in inoculated soil. The protocol for the
remainder of the experiment was the same as for the trial with
inoculation of 6 log CFU/g of soil as described above.
Microbiological analysis. Soil (10 g) was combined with
45 ml of sterile 0.1% peptone water in a Whirl-Pak bag (Nasco,
Fort Atkinson, WI). Samples were pummeled at regular speed for
1 min with a Stomacher 400 laboratory blender (Seward, London,
UK). For enumeration of E. coli O157:H7 on leaf surfaces, 25 to
35 g of leaves (depending on leaf size) were mixed with an
appropriate amount of sterile 0.1% peptone water (1:4.5
[leaf:peptone water]) and agitated vigorously by hand for 1 min.
The solution was assayed for E. coli O157:H7 by enumeration and
enrichment culture. Leaves were transferred to a new Whirl-Pak
bag and surface sanitized by using a procedure described by Zhang
et al. (28), and then macerated with a mortar and pestle. An
appropriate amount of 0.1% peptone water (1:4.5 [leaf:peptone
water]) was added to the macerated leaves and thoroughly mixed
manually (agitated vigorously by hand for 1 min). The homogenate was assayed for E. coli O157:H7 by enumeration and
enrichment culture. A similar protocol was used in analysis for E.
coli O157:H7 on root surfaces (rhizosphere counts) and macerated,
surface-sanitized roots. The weight of root samples was 13.6
6.8 g.
Portions (0.25 ml in quadruplicate and 0.1 ml in duplicate) of
undiluted homogenates and duplicate samples (0.1 ml) of homogenates serially diluted in 0.1% peptone water were surface plated

2030

ZHANG ET AL.

J. Food Prot., Vol. 72, No. 10

TABLE 1. Occurrence of Escherichia coli O157:H7 on iceberg lettuce inoculated 3 days after transplanting seedlings to pots a
Time between inoculation
and analysis (days)

19

Leaf side
inoculated

Adaxial
Abaxial
Control

54

Adaxial
Abaxial
Control

Inoculum carrier

Inoculated
leaf surfacesb

Uninoculated
leaf surfacesc

Water
Cow manure extract
Water
Cow manure extract
Not inoculated

0
0
2
2
d

0
0
1
0
0

Water
Cow manure extract
Water
Cow manure extract
Not inoculated

0
0
0
0

0
0
0
0
0

Occurrence ~ number of positive samples/3 samples analyzed.

Leaves were inoculated with E. coli O157:H7 at 6 to 7 log CFU per plant. Soil in pots was not inoculated.
c
Surfaces of uninoculated leaves from inoculated and control (uninoculated) plants.
d
, no data.
b

on TSA-amp. The remaining homogenate was incubated at 35uC

for 3 h to facilitate resuscitation of injured cells, combined with an
equal volume of double-strength TSB-amp, enriched at 37uC for
21 h, and streaked in duplicate on TSA-amp. Direct enumeration
plates and enrichment culture plates were incubated at 37uC for
24 h before colonies were viewed under UV illumination. Colonies
presumptive for E. coli O157:H7 were counted, and cells from
random colonies were subjected to confirmation tests using the E.
coli O157 latex agglutination assay (Remel, Lenexa, KS).
Statistical analysis. Data were analyzed with the general
linear models procedure of SAS (version 9.1.3, SAS Institute, Inc.,
Cary, NC) at a ~ 0.05. Duncans multiple range tests were used
to determine significant (a ~ 0.05) differences between mean
values.

RESULTS
In both leaf inoculation experiments, regardless of
inoculation level, leaf side inoculated, lettuce age when
inoculated, sampling time after inoculation, or inoculum
carrier, all soil samples in which lettuce plants were grown,
macerated leaves (uninoculated leaves from inoculated and
uninoculated plants) that were surface sanitized, rhizosphere
(unwashed root surfaces, including attached soil), and
macerated roots (from inoculated and uninoculated plants)
that were surface sanitized, were negative for E. coli
O157:H7 after enrichment culture. Data are not shown in
tables.
E. coli O157:H7 on and in inoculated leaves. In the
first leaf inoculation trial, with initial populations of E. coli
O157:H7 at 6 to 7 log CFU per plant, the pathogen survived
longer on the abaxial side of the iceberg lettuce leaves than
on the adaxial side of the leaves (Table 1). For leaves
inoculated with E. coli O157:H7 3 days after transplanting
seedlings, all samples of leaves inoculated on the adaxial
side were negative for the pathogen 19 days postinoculation.
However, four of six samples of leaves inoculated on the
abaxial side were positive.
Five of 18 samples of leaves from plants inoculated on
the adaxial side were positive for E. coli O157:H7, whereas

14 of 18 leaf samples from plants inoculated on the abaxial

side were positive 3, 7, and 25 days postinoculation
(Table 2). Twenty-five days after inoculation of leaves (30
days posttransplanting), 2 of 12 samples of inoculated
leaves were positive for E. coli O157:H7.
For iceberg lettuce leaves inoculated 60 days after
transplanting, 1 of 12 leaf samples from plants inoculated on
the adaxial side was positive, whereas 6 of 12 samples from
plants inoculated on the abaxial side were positive
(Table 3). Fifty-four days after inoculation of leaves (3
days posttransplanting), E. coli O157:H7 was not detected
(Table 1).
In most cases, E. coli O157:H7 populations on the
adaxial side of inoculated iceberg lettuce leaves were
significantly lower than those populations on the abaxial
side (Table 4). Considering all seven sampling times, E. coli
O157:H7 was detected only at one sampling time (3 days
after inoculating plants that had been transplanted for 30
days) at populations of 1.23 and 0.85 log CFU per sample of
leaves inoculated on the adaxial side with suspensions of E.
coli O157:H7 in water and cow manure extract, respectively
(Table 4). Populations ranging from 0.57 to 3.65 log CFU per
sample of leaves inoculated on the abaxial were recovered on
six of seven sampling times.
In the second leaf inoculation trial using 4 log CFU per
plant, E. coli O157:H7 was detected by enrichment culture
of inoculated leaves 15 days postinoculation but not 26, 35,
or 44 days postinoculation (Table 5). E. coli O157:H7 was
detected at populations of 0.93 and 0.66 log CFU per leaf
sample from plants inoculated on the abaxial side of leaves
15 days postinoculation with water and cow manure extract
suspension, respectively (Table 6). E. coli O157:H7 was not
detected on leaves inoculated on the adaxial side.
E. coli O157:H7 on and in uninoculated leaves and
roots of inoculated plants. Among 212 uninoculated leaf
samples from the same plants with inoculated leaves, the
surfaces of four of them were positive for E. coli O157:H7
before surface sanitizing. All 212 root samples were
negative for E. coli O157:H7 before surface sanitizing.

2031

LACK OF INTERNALIZATION OF E. COLI O157:H7 IN LETTUCE

J. Food Prot., Vol. 72, No. 10

TABLE 2. Occurrence of Escherichia coli O157:H7 on iceberg lettuce inoculated 30 days after transplanting seedlings a
Time between inoculation
and analysis (days)

Leaf side
inoculated

Adaxial
Abaxial
Control

Adaxial
Abaxial
Control

25

Adaxial
Abaxial
Control

a
b

Inoculum carrier

Inoculated
leaf surfaces

Uninoculated
leaf surfaces

Water
Cow manure extract
Water
Cow manure extract
Not inoculated

3
1
3
3
b

0
0
1
0
0

Water
Cow manure extract
Water
Cow manure extract
Not inoculated

1
0
3
3

0
0
0
0
0

Water
Cow manure extract
Water
Cow manure extract
Not inoculated

0
0
1
1

0
0
0
0
0

Occurrence ~ number of positive samples/3 samples analyzed. See Table 1 footnotes for description of headings.
, no data.

In the first leaf inoculation trial using an inoculum of 6

log CFU per plant, regardless of the side of the leaf
inoculated, all uninoculated leaf samples (Tables 1 through
3) and surface-sanitized roots from the same plants
inoculated by leaf were negative for E. coli O157:H7. All
uninoculated leaf samples (Tables 5 and 6) and surfacesanitized roots were negative for E. coli O157:H7,
regardless of time elapsed (3, 30, or 60 days) between
transplanting seedlings and inoculation. Similar results were
obtained with a lower (4 log CFU per plant) inoculum.
Soil in which surface-inoculated plants was grown.
All 212 samples of soil in which inoculated plants were
grown were negative for E. coli O157:H7, regardless of the
time elapsed between inoculation and analysis. Controls
were also negative.
Surface contamination and internalization of E. coli
O157:H7 on and in leaves and roots of plants grown in
inoculated soil. All samples of surface-sanitized leaves

from plants grown in soil inoculated with E. coli O157:H7

at populations of 3 and 6 log CFU/g of soil were negative
for E. coli O157:H7, regardless of the time elapsed after
inoculation (Tables 7 and 8). At an initial population of 6
log CFU/g of soil, 49 of 60 leaf surface samples were
positive for E. coli O157:H7 17 days after inoculating
transplanted seedlings. E. coli O157:H7 was not detected on
the surface of 120 leaf samples 45 or 60 days after
inoculation (Table 7). At an initial inoculum population of 3
log CFU/g of soil, E. coli O157:H7 was detected on surfaces
of leaves from 120 samples 26 and 60 days after inoculation
(Table 8).
All but 2 of the 300 surface-sanitized macerated root
samples were negative for E. coli O157:H7 (Tables 7 and
8). At an inoculum population of 6 log CFU/g of soil, all 60
rhizosphere samples were positive for E. coli O157:H7 17
days after inoculation of soil (Table 7). Fifty-four and 28 of
the samples were positive for E. coli O157:H7 45 and 60
days, respectively, after inoculation. At an inoculum
population of 3 log CFU/g of soil, only 6 of 120 rhizosphere

TABLE 3. Occurrence of Escherichia coli O157:H7 on iceberg lettuce inoculated 60 days after transplanting seedlings a
Time between inoculation
and analysis (days)

Leaf side
inoculated

Adaxial
Abaxial
Control

Adaxial
Abaxial
Control

a
b

Inoculum carrier

Inoculated
leaf surfaces

Uninoculated
leaf surfaces

Water
Cow manure extract
Water
Cow manure extract
Not inoculated

0
0
2
0
b

0
0
1
0
0

Water
Cow manure extract
Water
Cow manure extract
Not inoculated

0
1
3
1

0
0
0
0
0

Occurrence ~ number of positive samples/3 samples analyzed. See Table 1 footnotes for description of headings.
, no data.

2032

ZHANG ET AL.

J. Food Prot., Vol. 72, No. 10

TABLE 4. Escherichia coli O157:H7 on inoculated iceberg lettuce leaves a

Inoculated on the adaxial side
(log CFU/plant sample)

Time (days) between:

Transplanting
and inoculation

Inoculation
and analysis

19
54

3
7
25

3
7

30

60
a
b
c

Inoculated on the abaxial side

(log CFU/plant sample)

Water

Cow manure extract

NDb,c
ND

A
A

1.23 1.08
ND
A ND
B

ND
ND

0.85 1.47
ND
A ND

ND
ND

B
B

ND
ND

Water
A
A

A
A
A

Cow manure extract

1.53 1.33
ND

3.65 0.12
2.03 0.35
ND

1.88 1.64
1.80 0.17

A
A

ND
ND
3.09 0.06
1.76 1.53
0.57 0.98
ND
0.67 1.15

Leaves of plants were inoculated with 6 log CFU E. coli O157:H7 per plant by using water and cow manure extract as carriers.
Values in same row that are preceded by the same letter are not significantly (a ~ 0.05) different.
ND, not detected. Limit of detection was 1 log CFU per plant.

samples (26 and 60 days after inoculating soil) were positive

for E. coli O157:H7 (Table 8).
Persistence of E. coli O157:H7 in soil. All samples
from soil inoculated at a population of 6 log CFU/g were
positive for E. coli O157:H7 17 days after inoculation
(Table 7). There were 54 and 45 of 60 samples positive for
E. coli O157:H7 for 45 and 60 days, respectively, after
inoculation. At a population of 3 log CFU/g of soil, all soil
samples were positive for E. coli O157:H7 in the 26-day
postinoculation samples. Only 16 of 60 were positive at 60
days (Table 8).

DISCUSSION
E. coli O157:H7 inoculated on the surface of lettuce
leaves survived for at least 25 days. The pathogen was not
detected on leaves 26 and 35 days after inoculation. This
finding indicates that if irrigation water or surface runoff
water contains E. coli O157:H7, lettuce harvested within 25
days of application of these waters may be contaminated.
Solomon et al. (20) reported that E. coli O157:H7 persisted
on leaf surfaces of 9 of 11 plants for 20 days after spray
irrigation with contaminated water. In another study, E. coli
O157:H7 survived 30 days on lettuce plants after spray
irrigation, and repeated irrigation with contaminated water

TABLE 5. Occurrence of Escherichia coli O157:H7 on iceberg lettuce leaves inoculated 30 days after transplanting seedlings a
Time between inoculation
and analysis (days)

15

Leaf side
inoculated

Adaxial
Abaxial
Control

26

Adaxial
Abaxial
Control

35

Adaxial
Abaxial
Control

44

Adaxial
Abaxial
Control

Inoculum carrier

Inoculated leaf
surfaces

Uninoculated
leaf surfaces

Water
Cow manure extract
Water
Cow manure extract
Not inoculated

1
3
7
7
b

0
0
1
0
0

Water
Cow manure extract
Water
Cow manure extract
Not inoculated

0
0
0
0

0
0
0
0
0

Water
Cow manure extract
Water
Cow manure extract
Not inoculated

0
0
0
0

0
0
0
0
0

Water
Cow manure extract
Water
Cow manure extract
Not inoculated

0
0
0
0

0
0
0
0
0

Occurrence ~ number of positive samples/8 samples analyzed. Leaves were inoculated with E. coli O157:H7 at 4 log CFU per plant. Soil
in pots was not inoculated.
, no data.

LACK OF INTERNALIZATION OF E. COLI O157:H7 IN LETTUCE

J. Food Prot., Vol. 72, No. 10

2033

TABLE 6. Escherichia coli O157:H7 on inoculated iceberg lettuce leaves inoculated 7 days after transplanting seedlings a
Inoculated on the adaxial side
(log CFU/plant sample)
Time between inoculation
and analysis (days)

15
26
35
44
a
b
c

Water

NDb,c
A ND
A ND
A ND
B

Inoculated on the abaxial side

(log CFU/plant sample)

Cow manure extract

ND
ND
A ND
A ND
B

Water
A

0.93 0.63
A ND
A ND
A ND

Cow manure extract

A

0.66 0.56
A ND
A ND
A ND

Leaves of plants were inoculated with 4 log CFU E. coli O157:H7 per plant by using water and cow manure extract as carriers.
Values in same row that are preceded by the same letter are not significantly (a ~ 0.05) different.
ND, not detected. Limit of detection was 1 log CFU per plant.

increased the pathogen level on the plant (19). However, in

our study, E. coli O157:H7 survived in soil for 60 days
(Table 7). Since E. coli O157:H7 in irrigation water would
contaminate soil and lettuce plants before or during
harvesting, use of contaminated irrigation water should be
avoided.
E. coli O157:H7 survived longer on the abaxial side of
the lettuce leaves than on the adaxial side. The adaxial side
of the leaf faces the sunlight during daytime hours and tends
to be drier than the abaxial side. The abaxial side of the leaf
is exposed to indirect light and may be at a lower
temperature. The environment on the abaxial side of leaves
would be more favorable than that on the adaxial side in
terms of preserving viability of E. coli O157:H7. We are not
aware of previous studies comparing the survival of E. coli
O157:H7 on the two sides of lettuce leaves.
Irrigation water containing cow manure may prolong
the survival of E. coli O157:H7 on lettuce leaves because of
the nutrients it provides. E. coli O157:H7 suspensions with
and without manure extract were inoculated on lettuce
leaves to compare the effect of contamination with manure.
Combining the results of the two leaf trials, 26 of 106 leaf
samples inoculated with water as the E. coli O157:H7
carrier were positive for E. coli O157:H7, and 22 of 106 leaf
samples inoculated with cow manure extract as the E. coli
O157:H7 carrier were positive (Tables 1 through 3 and 5).
In 22 direct comparisons of enumeration data between leaf
samples inoculated with water and cow manure extract as
inoculum carriers, significant differences were observed in
only two cases (Tables 4 and 6). Therefore, it is concluded
that cow manure extract did not substantially enhance the
survival or colonization of E. coli O157:H7 on lettuce
leaves.
Survival of E. coli O157:H7 on lettuce leaf surfaces
was largely unaffected by the inoculum carrier, i.e., water
versus manure extract. Perhaps factors such as light
intensity, moisture, and temperature have more influence
on the survival of E. coli O157:H7 on leaf surfaces than
does cow manure extract. Nutrients in manure extract may
not have been concentrated enough to affect E. coli
O157:H7 survival. E. coli O157:H7 has been reported to
survive on lettuce plants after irrigation with contaminated
water (1921, 23); however, to our knowledge, there are no
published studies comparing the effect of water contami-

nated with and without manure extract on the survival of E.

coli O157:H7 on lettuce leaves. Monior and Lindow (17)
reported that cell aggregates formed by resident bacterial
populations on bean plants (Phaseolus vulgaris) can either
enhance or hinder the survival of migrant bacteria,
depending on the nature of the interactions between those
species. Leaf surface sites differed in their ability to
influence the survival of migrant bacterial cells, and the
fate of these cells depended on the nature of the leaf surface
on which they deposited, and the amount of nutrients and
water available at that site.
Four of 212 leaf surface samples of uninoculated leaves
from leaf-inoculated plants were positive for E. coli
O157:H7. This was probably due to cross-contamination
during inoculation or subsequent handling of leaves. As for
the soil inoculation trial in which the inoculation level was
106 CFU/g of soil, all leaf surface samples were negative for
E. coli O157:H7 for 45 and 60 days after inoculations;
however, 49 of 60 leaf surface samples were positive 17 days
postinoculation (Table 7). All leaf surface samples from the
trial with an inoculation level of 103 CFU/g of soil were
negative for E. coli O157:H7. When plants were sampled at
17 days postinoculation, we used leaves which had contact
with soil in order to obtain enough samples. Samples at 26
days postinoculation and older did not contain leaves that had
been in contact with soil (Table 8). E. coli O157:H7 may
have contaminated bottom leaves that were in contact with
the soil, whereas upper leaves could be pathogen free if there
were no disturbances of the soil. If contact with soil or
disturbance of soil is avoided, the risk of contamination of
leaves would likely be reduced.
Surface-sanitized lettuce leaves and roots were macerated and analyzed for E. coli O157:H7 to determine if
internalization of E. coli O157:H7 occurs. All 512 surfacesanitized leaf samples, regardless of the age of plants when
inoculated or analyzed, type of lettuce, strain of E. coli
O157:H7 in the inoculum, or water or cow manure extract
used as an inoculum carrier, were negative for E. coli
O157:H7. Only 2 of 512 surface-disinfected root samples
were positive. The two positive samples likely resulted from
inadequate surface sanitizing or infiltration of E. coli
O157:H7 into wounds. Results indicate that internalization
of E. coli O157:H7 into lettuce tissues after inoculation of
leaves or soil in which plants were grown did not occur.

2034

ZHANG ET AL.

J. Food Prot., Vol. 72, No. 10

TABLE 7. Occurrence of Escherichia coli O157:H7 on lettuce grown in soil inoculated with E. coli O157:H7 at 6 log CFU/g a
Time between inoculation
and analysis (days)

Strainb

17

ATCC 43888

EO122

K3995

K4992

F4546

Control

45

ATCC 43888

EO122

K3995

K4992

F4546

Control

60

ATCC 43888

EO122

K3995

K4992

F4546

Control

Lettuce type

Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg

Soilc

Leaf surfaced

Macerated
leavese

Rhizospheref

2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0
0
0
4
4
3
3
3
3
3
4
3
4
4
4
4
4
4
0
0
0
4
4
4
4
4
4
0
1
0
3
2
4
4
3
4
0
0
0

3
4
4
3
4
4
2
0
4
4
3
4
4
2
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
0
0
0
4
4
3
4
2
3
4
4
4
4
3
3
4
4
4
0
0
0
3
2
1
1
2
2
0
0
1
3
2
3
3
3
2
0
0
0

Macerated
rootsg

0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

Occurrence ~ number of positive samples/total samples analyzed. With the exception of analyzing only two samples of soil 17 days after
inoculation, four samples from all other combinations of test parameters were analyzed.
b
Strain sources: ATCC 43888, human feces; EO122, bovine; K3995, spinach; K4492, lettuce; and F4546, alfalfa sprout outbreak.
c
Soil in which lettuce plants were grown.
d
Surfaces of uninoculated leaves from inoculated and control (uninoculated) plants.
e
Macerated leaves (uninoculated) that were surface sanitized.
f
Unwashed root surfaces, including soil attached to roots.
g
Macerated roots that were surface sanitized.

2035

LACK OF INTERNALIZATION OF E. COLI O157:H7 IN LETTUCE

J. Food Prot., Vol. 72, No. 10

TABLE 8. Occurrence of Escherichia coli O157:H7 on lettuce grown in soil inoculated with E. coli O157:H7 at 3 log CFU/g a
Time between inoculation
and analysis (days)

Strain

Lettuce type

Soil

Leaf surface

Macerated
leaves

Rhizosphere

Macerated
roots

26

ATCC 43888

Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg
Leaf
Romaine
Iceberg

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
0
0
0
2
1
1
0
2
2
2
1
2
1
1
0
1
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

0
1
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

EO122

K3995

K4992

F4546

Control

60

ATCC 43888

EO122

K3995

K4992

F4546

Control

Occurrence ~ number of positive samples/4 samples analyzed. See Table 7 for description of headings.

This is in partial agreement with the observation of Hora et al.

(10). They found that mechanical damage of roots of plants
grown in inoculated soil did not result in internalization of E.
coli O157:H7 into the aerial tissue of spinach, although the
bacterium was recovered from surface-sterilized root tissues.
Jablasone et al. (12) reported that E. coli O157:H7 did not
internalize in mature lettuce plants grown in solidified,
hydroponic nutrient solution. Franz et al. (6) did not detect E.
coli O157:H7 or Salmonella in the edible parts of lettuce
grown in manure-amended soil (6). However, there are
reports that E. coli O157:H7 and Salmonella colonized
interior tissues of lettuce plants (7, 15, 16). Different
pathogen strains, lettuce cultivars and types, surfacedisinfection methods, and plant cultivation methods were
used in these experiments. Without the uniform application of
experimental methods, it is difficult to directly compare the
results from different studies. The present study used a
stringent surface-disinfection method (28), three types of
lettuce, five E. coli O157:H7 isolates, leaf and soil

inoculation, inoculation of abaxial and adaxial sides of

leaves, and inoculation of different ages of plants to address
in one experiment a wide array of potential factors that may
contribute to internalization of E. coli O157:H7. With all of
the factors studied, E. coli O157:H7 internalization of lettuce
did not occur. Since a plant pathogen colonizes only a
specific group of plants, it is reasonable that a foodborne
pathogen such as E. coli O157:H7, under typical circumstances, does not internalize or colonize lettuce.
Genetically different E. coli O157:H7 isolates may, in
theory, differ in their ability to internalize in lettuce. All five
isolates used in our study failed to internalize in lettuce. Our
results indicated there was no difference among the E. coli
O157:H7 isolates in their ability to be internalized by
lettuce. However, studies with tomatoes revealed that there
were variations among Salmonella serotypes in their ability
to be internalized within tomato fruits (9, 18).
During the growth and development of plants, the
physiology of plants change, and their leaf surfaces also

2036

ZHANG ET AL.

change. Such changes may affect the internalization of E.

coli O157:H7 in lettuce. Lettuce leaves were inoculated 3,
30, and 60 days after transplanting into pots. Regardless of
the age of lettuce plants at inoculation, E. coli O157:H7 did
not internalize into lettuce tissues.
There are substantial phenotypic and genotypic differences among iceberg, romaine, and leaf lettuces, although
they belong to the same species. Hence, they may respond
differently to foodborne pathogens. Their susceptibility to
internalization by E. coli O157:H7 was compared in our soil
inoculation study; however, no differences in susceptibility
were observed among the three types of lettuce.
If soil is contaminated with E. coli O157:H7 by
irrigation water or manure, the longer the bacteria survive,
the higher the risk of contamination of lettuce. In both lowand high-inoculation trials, E. coli O157:H7 was detected in
soil 60 days postinoculation. This indicates that lettuce
grown in soil containing E. coli O157:H7 may become
contaminated throughout the growing season. A previous
study revealed that E. coli O157:H7 survived 25 to 41 days
in fallow soils; longer than 500 days in frozen soil; and 47 to
96 days on rye (Secale cereale L.), alfalfa (Medicago
sativa), hairy vetch (Vicia sativa L.), and crimson clover
(Trifolium incarnatum L.) (8). In an E. coli O157:H7
survival study of 36 Dutch soils, the calculated detection
time by the Weibull model was 54 to 105 days. E. coli
O157:H7 populations declined more rapidly under more
oligotrophic soil conditions, which can be achieved by using
manure with a relatively high carbon-to-nitrogen ratio and
consequently, a relatively low rate of nutrient release (5).
The pH and fiber contents of manure used in soils also
affected the survival of E. coli O157:H7 in soil (6). Soil
composition, temperature, and moisture content can influence pathogen survival, with nutritionally rich soil in
combination with moisture significantly increasing the
persistence of E. coli O157:H7 in soil (24). Considering
these factors that can contribute to E. coli O157:H7
contamination and persistence, measures should be taken
to prevent contamination of soil with E. coli O157:H7 from
the time of preplanting to harvesting to reduce the risk of
contamination of lettuce at the time of harvest.
ACKNOWLEDGMENTS
Financial support for this study was provided by Fresh Express, Inc.,
Salinas, CA. We also thank Kellie McKoon and Kora Anderson for their
technical assistance.

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