618

Journal of Food Protection, Vol. 72, No. 3, 2009, Pages 618–623

Copyright 䊚, International Association for Food Protection

Research Note

Transfer of Salmonella enterica Serovar Typhimurium from

Contaminated Irrigation Water to Parsley Is Dependent on Curli
and Cellulose, the Biofilm Matrix Components
ANAT LAPIDOT AND SIMA YARON*

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Faculty of Biotechnology and Food Engineering, Technion, Israel Institute of Technology, Haifa 32000, Israel

MS 08-237: Received 27 May 2008/Accepted 26 September 2008

ABSTRACT
Enteric pathogens can contaminate fresh produce, and this contaminated produce can be a significant potential source of
human illness. The objective of this study was to determine a possible mode of transfer of Salmonella Typhimurium from
contaminated irrigation water to mature parsley plants and to investigate the role of bacterial cellulose and curli. Parsley plants
were drip irrigated with water containing green fluorescent protein–labeled Salmonella Typhimurium. Stems and leaves were
harvested 1 day after the third irrigation and examined for the presence of Salmonella Typhimurium. Three weeks after
harvesting, the presence of Salmonella was again confirmed in the regrown plants. During this period, bacterial numbers on
leaves declined from 4.1 (⫾0.3) to 2.3 (⫾0.1) log CFU g⫺1 (P ⬍ 0.05). Numbers in the soil were constant (5 log CFU g⫺1).
Results demonstrated the ability of Salmonella Typhimurium to transfer from irrigation water to the edible parts of the plants.
Confocal laser scanning microscopic images revealed that Salmonella Typhimurium formed aggregates at a depth of 8 to 32
␮m beneath the leaf surface. Penetration might be achieved through the roots or the phyllosphere. The importance of the
bacterial cellulose and curli was determined by comparing the wild-type strain with its mutants, which lack the ability to
synthesize cellulose and curli. Counts of the double mutant were 2-log higher in the soil but 1-log lower in the leaves (P ⬍
0.05). Deletion of the agfBA gene (for curli) was more effective than deletion of bcsA (for cellulose). Thus, curli and cellulose
play a role in the transfer or survival of Salmonella Typhimurium in the plant, as they do for plant pathogens.

Fresh fruits and vegetables are significant sources of
foodborne disease outbreaks, and are associated with 7 to
15% of the identified cases of foodborne diseases in many
countries (1, 37). Salmonella enterica is among the most
common pathogens (6). Salmonella-related outbreaks have
been associated with the consumption of fruits, vegetables,
sprouts, and leafy vegetables such as parsley (6, 7). Con-
tamination of the plants may take place in the field or dur-
ing postharvest processing and storage. Water present at
each of these stages may be the source of contamination at
any given point.
Enteric pathogens colonize both warm- and cold-
blooded animals. They are usually exposed to a new host
via contaminated foods or water and are shed back into the
environment. Thus, plants can play a role as vehicles for
transfer of pathogens from the environment into the gut of
a new host, and enteric pathogens may survive in plants
long enough and in sufficient numbers to ensure the infec-
tion of the new host (22). Intimate interactions between
enteric strains and plants are dependent on both plant and
bacterial factors and have been described in the literature.
Some S. enterica strains can adhere to different plant parts,
survive for long periods, and then grow (8, 9, 13, 16). Spe-
* Author for correspondence. Tel: 972-4-829-2940; Fax: 972-4-829-3399;
E-mail: simay@tx.technion.ac.il.
cific genes of S. enterica such as yihO, bcsA, rpoS, and
agfD are required for attachment to and colonization of
plants (3, 4). S. enterica and other enteric pathogens can
also become endophytic, i.e., they can invade internal plant
parts (13, 19–21, 34, 36). The ability to colonize plants
endophytically is serovar and cultivar dependant (20, 22).
Internalization of S. enterica serovars was extensively ob-
served in cut leaves or during germination of young sprouts
(12, 13, 19). However, reports of internalization of S. en-
terica in roots are contradictory. Internalization was re-
ported for tomato plants grown hydroponically (15) and in
33-day-old but not in 20-day-old soil-grown Romaine let-
tuce plants (5). However, no internalization was apparent
in another experiment with soil-grown iceberg lettuce (11).
Although internalization of S. enterica in plant roots
and its transfer into the edible parts of the plants mainly
has been studied in developing seedlings grown in the lab
or in a greenhouse under controlled conditions of temper-
ature or humidity (5, 12, 19), information regarding con-
tamination of mature plants grown in soil is scarce. The
objective of this study was to determine the possibility of
transfer of S. enterica serovar Typhimurium from contam-
inated irrigation water to the edible parts of mature parsley
plants. Because extracellular components of the biofilm
such as cellulose and aggregating fimbriae are important
for adhesion and internalization of plant pathogens and
J. Food Prot., Vol. 72, No. 3 TRANSFER OF SALMONELLA FROM WATER TO PARSLEY
TABLE 1. S. enterica serovar Typhimurium strains used in this study
Strain Genotype
ATCC 14028 UMR1 ATCC 14028-1s Nalr (wild type)
MAE52 UMR1 PagfD1
MAE97 MAE52 ⌬agfBA
MAE150 MAE52 bcsA101::MudJ
MAE190 MAE52 ⌬agfBA bcsA101::MudJ
symbiotic bacteria (24–26, 30), we investigated whether
these factors also play a role in the interactions of Salmo-
nella Typhimurium with parsley plants.
MATERIALS AND METHODS
Bacterial strains. S. enterica serovar Typhimurium ATCC
14028-1s (UMR1) and its mutants MAE52, MAE97, MAE150,
and MAE190 are described in Table 1. The Salmonella Typhi-
murium strains were transformed with the pGFP plasmid (Clon-
tech, Palo Alto, CA) to obtain green fluorescent protein (GFP)–
labeled cells.
Plasmid stability assay. The stability of the pGFP plasmid
in the bacteria was examined by tracing the GFP expression as
previously described (19) but with a few modifications. The GFP-
labeled bacteria were inoculated into Luria-Bertani (LB) broth
without ampicillin. Samples of the cultures were diluted (1:100)
in fresh medium daily, incubated for 24 h, and transferred again.
Plate counting on LB agar plates with or without 100 ␮g ml⫺1
ampicillin was performed daily to quantify the functional stability
of the plasmid. The plasmid was still present in more than 85%
of the colonies up to day 11.
Parsley growth and production. Parsley (flat Petroselinum
crispum) seeds were disseminated in planters (1-m diameter) in a
level 2 greenhouse in the Technion Ecological Garden. The plant-
ers contained local commercial nonsterile potting soil (Hagarin,
Yavne, Israel) and were watered twice daily until germination (10
to 14 days). Plants were then automatically irrigated with tap wa-
ter (400 ml) once daily. Plants were grown under a controlled
lighting system of strip fluorescent lights 50 cm above the planters
with photoperiods of 12 h per day. Temperature and relative hu-
midity were not controlled during the study, and the plants in the
greenhouse were subject to the weather changes in Haifa between
September and November 2006 to 2007.
Preparation of contaminated water and irrigation pro-
cedure. Overnight Salmonella cultures were diluted (1:100) in
fresh LB broth supplemented with ampicillin and were incubated
for 2.5 h at 37⬚C. Cells were harvested by centrifugation (4,000
⫻ g for 20 min at 4⬚C) and resuspended in 500 ml of tap water
to yield a final concentration of about 7.6 log CFU ml⫺1.
When plants reached an average height of 20 to 40 cm, they
were drip irrigated with the contaminated water. Each planter was
manually drip irrigated three times during a 9-day period (3-day
intervals) with 500 ml of freshly prepared contaminated water by
direct application of the pipettes to the soil at a depth of about 1
cm. In this manner, the water was not allowed to come into contact
with the plants’ aerial parts. Each planter had holes at the bottom,
and water leaving the planter was collected. Control planters were
placed in parallel in the same greenhouse and were treated in the
same way but with Salmonella-free water. After the contamination
process, plants were irrigated once daily with 400 ml of tap water.
619
Production of
cellulose at Production of
28⬚C curli at 28⬚C Reference
⫹ ⫹ 32
⫹ ⫹ 32
⫹ ⫺ 31
⫺ ⫹ 40
⫺ ⫺ 40
Samples collection and analysis. Samples of soil, leaves,
and stems were aseptically collected from each planter 1 day after
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the first and third irrigation steps. The bare plants grew again, and
new parsley was harvested from the same plants at day 30 (i.e.,
21 days after the last irrigation with contaminated water). Soil
from randomly selected plants was aseptically collected from 2
cm below the surface with a sterile spoon and placed into a sterile
plastic bag. Each soil sample was mixed and weighed. A 10-g
soil sample was added to 90 ml of saline solution in a stomacher
bag and vigorously agitated by hand for 30 s. The top stems or
leaves (at least 10 cm above the soil) were aseptically harvested
and weighed. Care was taken to collect only the highest parts of
the plants that had not come into any direct contact with the soil.
A 20-g sample of these plant parts was added to 100 ml of saline
solution in a sterile stomacher bag and pummeled in a stomacher
for 3 min. Serial dilutions (1:10 in saline) were prepared for plate
counts. Colonies of Salmonella Typhimurium, which were fluo-
rescent under UV light, were counted as described (23).
To determine whether the bacteria had penetrated the stems
and leaves, we externally sterilized the upper parts of the stems
with swabs of 70% ethanol for 2 min, cut the stems, and used a
sterile pipette tip to collect the internal liquids that accumulated
on top of the cut stems. The experiment was performed on four
treated and untreated plants. The liquid drops (5 to 10 ␮l) were
plated on LB agar with ampicillin. Ethanol-sterilized whole leaves
and leaves with tiny incisions (15 incisions about 2 to 3 mm long
made with a sterile knife) were placed for 1 h on LB agar plates
so that the upper leaf epidermis layer faced the agar. Fluorescent
colonies were counted after incubation at 37⬚C.
Examination of leaf samples with confocal scanning mi-
croscopy. Leaf samples were washed twice in a saline solution,
placed on glass microscope slides, and examined under a 40⫻/
1.3 oil immersion objective in a confocal laser scanning micro-
scope (Carl Zeiss, Thornwood, NY). The green fluorescence of
pGFP-labeled Salmonella cells was detected using an excitation
wavelength of 488 nm and a BP500/50 filter. Images were ob-
tained with Zeiss LSM image browser software (version 4).
Statistical analysis. Four duplicate experiments were per-
formed, and data were analyzed with a one-way analysis of var-
iance followed by the Tukey-Kramer test when needed. P values
of ⱕ0.05 were considered significant.
RESULTS
Transfer of Salmonella from contaminated water to
the soil and phyllosphere. Initially, we investigated wheth-
er irrigation with contaminated water could lead to bacterial
contamination of the soil or the edible parts of the parsley
plant. Parsley plants were irrigated three times with water
containing wild-type (WT) Salmonella Typhimurium while
preventing direct contact between the dripped water and the
620 LAPIDOT AND YARON
TABLE 2. Presence of wild-type Salmonella Typhimurium in soil
and plant parts after three irrigation steps with contaminated wa-
tera
Mean (SD) Salmonella Typhimurium concn
(log CFU g⫺1 sample) at:
Sample type 1 dayb 21 daysb
Soil 5.5 (0.1) A 5.3 (0.2) A
Stem 3.0 (0.4) E NDc F
Leaves 4.1 (0.3) B 2.3 (0.1) C
a Values are means of four experiments, with duplicate determi-
nations per experiment. Within each row, means not followed
by the same letter are significantly different (P ⬍ 0.05).
b Days after the third irrigation step.
c ND, not detected (limit of detection was 1.7 log CFU g⫺1).
phyllosphere. Samples were collected 1 and 10 days after
the first irrigation for quantification of the Salmonella Ty-
phimurium population associated with the soil, leaves, and
stems. No GFP-expressing colonies were observed in the
control plants irrigated with tap water. Very few colonies
of GFP-expressing Salmonella Typhimurium (about 1.9 to
2.0 log CFU/g of parsley) were seen in samples of leaves
collected 1 day after the first irrigation, just above the de-
tection limit (1.7 log CFU g⫺1). Results of viable counts
obtained 10 days after the first irrigation are shown in Table
2. At this point, GFP-expressing Salmonella cells were de-
tected in all of the experimental samples (not in the con-
trol), with a trend toward higher concentrations in the
leaves than in the stems. Salmonella was detected in leaves
at a mean (⫾standard deviation [SD]) concentration of 4.1
(⫾0.3) log CFU g⫺1. There was no visible damage to the
contaminated leaves.
Several experiments were conducted to determine
whether the bacteria had penetrated the stems and leaves.
Initially, we cut upper parts of the surface-sterilized stems
and collected the internal liquids that accumulated on top
of the cut stems. Positive fluorescent colonies were appar-
ent on the plates of liquids taken from the plants irrigated
with water containing GFP-expressing Salmonella but not
for the liquids from the control plants. Fluorescent Salmo-
nella colonies also were seen on LB plates around the in-
cisions of the cut leaves that had been irrigated with con-
taminated water. No fluorescent colonies were seen around
the whole leaves of these plants or around cut leaves of the
control plants. Examination of leaves by confocal laser
scanning microscopy revealed colonization of the leaves
with both single cells and small aggregates of Salmonella
Typhimurium at a depth of 8 to 32 ␮m beneath the upper
surface of the leaf (Fig. 1).
Persistence of Salmonella Typhimurium in growing
parsley and in the soil. After harvesting the upper parts
of the plants, we continued watering the planters daily with
tap water (Salmonella free) until the plants regrew. The
contaminated plants grew well and looked healthy. The re-
grown leaves and stems were aseptically harvested after 21
days. During this period, the bacterial numbers associated
with the soil were constant (Table 2). Salmonella Typhi-
J. Food Prot., Vol. 72, No. 3
murium was not evident in the stems of the regrown plants
but was present in the leaves of these plants, although the
numbers were significantly lower than those in leaves col-
lected 1 day after contamination (Table 2).
Transfer of the biofilm-deficient mutant from water
to parsley. When plants were irrigated with water contain-
ing the Salmonella Typhimurium MAE52 mutant (which
forms biofilm constitutively), its presence in the soil and in
the leaves or stems was similar to that of the WT (Table
3). However, the biofilm-deficient mutant MAE190 tended
to accumulate in the soil at concentrations 2 log units higher
than that of the WT (P ⬍ 0.01), and its transfer to the plant
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was approximately 1 log lower than that of the WT (P ⬍
0.05) (Table 3). Counts of mutants MAE97 and MAE150
associated with the plants were higher than the MAE190
counts but lower than the WT counts. The mutant with
deletion of the agfBA genes was more effective in reducing
the bacterial numbers than was the cellulose-deficient mu-
tant (Table 3).
DISCUSSION
To investigate the ability of Salmonella Typhimurium
to transfer from water to soil-grown parsley, mature plants
were drip irrigated with water containing Salmonella Ty-
phimurium and then assayed for Salmonella. Under the
field conditions of our experiments, Salmonella Typhimu-
rium was found in significant numbers in the edible por-
tions of the plants. Some bacteria even invaded the leaf;
however, the mechanism of transfer, i.e., through the roots
or through the surface of the stems and/or leaves, has not
been determined. Colonization by Salmonella serovars of
different plants such as tomatoes, parsley, and lettuce has
been described (15, 17, 20, 21). However, these experi-
ments were usually conducted with seedlings or with ma-
ture plants grown axenically or hydroponically. These con-
ditions may strongly affect the physiology of both the plant
and the bacteria and the absence or presence of rhizosphere
bacteria. Warriner et al. (39) reported root internalization in
hydroponically grown Escherichia coli but not in soil-
grown spinach seedlings. In the present study, the indige-
nous soil bacteria, which had colonized the roots or leaves
of the mature plants before contamination, did not obviate
the accessibility of Salmonella.
Transfer of Salmonella to mature plants occurred in
less than 24 h, but the concentration was higher after two
more irrigation steps. Although the bacterial numbers in the
soil were not different 3 weeks after contamination, the
bacterial numbers associated with the regrown plants de-
clined. This reduction may result from death or aggregation
of the bacteria or from changes in the physiology of the
harvested plants. Whatever the source of the bacteria, either
soil or the remaining plant tissue at the crown, these results
indicated that the transfer from the soil to the plant’s upper
parts at this stage is much lower than the transfer during
irrigation with contaminated water. Thus, bacteria that are
already tightly attached to the soil are less likely to contam-
inate the plant. This hypothesis is supported by previous
findings that bacteria in close contact with soil particles
J. Food Prot., Vol. 72, No. 3 TRANSFER OF SALMONELLA FROM WATER TO PARSLEY 621

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FIGURE 1. Representative confocal laser scanning micrographs of thin sections of parsley leaf contaminated with GFP-labeled Sal-
monella Typhimurium. The arrows indicate foci of invading Salmonella Typhimurium cells. The bacteria were observed on the leaves
as aggregates or as single cells.

adsorb to the soil particles and can move only short dis-
tances, even under saturated conditions (33).
Binding of plant pathogens or symbiotic bacteria such
as Agrobacterium tumefaciens or Rhizobium leguminosa-
rum to plants occurs in a two-step process: reversible ad-
sorption and primary adhesion (27). The reversible adsorp-
tion is probably affected by van der Waal interactions and
hydrogen binding. Structures on the bacterial surface such
as flagella, pili, and fimbriae may influence the first step of
bacterial attachment (30). The second step, which is also
affected by electrostatic forces, depends on the synthesis of
cellulose fibrils by the bacteria. This step results in a tight
binding of the bacterial cell, which can no longer be re-
moved from the plant cell by shear force (24, 26, 37). A

TABLE 3. Presence of Salmonella Typhimurium in upper stems and leaves of parsley and in the soil 1 day after the third contamination
stepa
Mean (SD) Salmonella Typhimurium concn (log CFU g⫺1 sample)

MAE52 MAE190 MAE97 MAE150

Sample type WT (biofilm⫹) (curli⫺, cellulose⫺) (curli⫺) (cellulose⫺)

Soil 5.5 (0.1) A 5.6 (0.3) A 7.4 (0.5) B NAb NA

Stems 3.0 (0.4) C 3.1 (0.1) C 1.9 (0.3) D 2.0 (0.2) D 2.1 (0.2) D
Leaves 4.1 (0.3) E 4.2 (0.3) E 3.2 (0.3) F 3.5 (0.1) F 3.8 (0.1) E

a Values are means of four experiments, with duplicate determinations per experiment. Within each row, means not followed by the
same letter are significantly different (P ⬍ 0.05).
b NA, not analyzed.
622 LAPIDOT AND YARON
linear correlation between bacterial cell surface charge and
hydrophobicity and the strength of bacterial attachment was
demonstrated in cantaloupes (38). Solomon et al. (35)
found that high numbers of produce-related Salmonella iso-
lates produce curli and cellulose, both of which are main
components of the Salmonella biofilms and usually are reg-
ulated similarly (14, 31, 40).
To determine whether Salmonella has a similar adhe-
sion process and to investigate whether curli and cellulose
are important for transfer of Salmonella into the plant, we
used mutants that do not produce curli and/or cellulose.
Bacterial numbers in the edible portions of parsley were
significantly higher (P ⬍ 0.05) when plants were irrigated
with water containing the WT strain or MAE52 (biofilm⫹)
than with water containing MAE190 (curli⫺, cellulose⫺).
Therefore, we believe that, as with plant pathogens, cellu-
lose and curli are important for strengthening the adhesion
of Salmonella to parsley. The importance of curli is sup-
ported by results of previous studies in which the attach-
ment of S. enterica and E. coli O157:H7 to alfalfa sprouts
was examined (3, 8, 12). The absence of cellulose had a
lesser effect on the colonization on plants, as also found in
other studies (3, 4, 25). However, the role of both compo-
nents in colonization of Salmonella on soil-grown plants
was more significant in the double mutant. Another hy-
pothesis is that the difference in colonization capability
among strains is due to differences in their ability to survive
in the plant. Bacteria that produce cellulose and fimbriae
may be better protected against stress conditions in the
plant than are biofilm-deficient mutants, which no longer
have the ability to form biofilms (2, 28, 29, 31).
In general, the Salmonella numbers applied to soil in
these experiments were high and may not be realistic in
terms of contamination levels that possibly occur in the
environment; thus, they represent a worse-case scenario.
This high number of cells enabled us to identify and com-
pare the different mutants. However, water or soil contam-
ination caused by contact with fecal material containing a
large population of Salmonella could realistically occur (10,
18). Currently, more than 7 million dry tonnes of sewage
sludge are produced annually in the United States, and 54%
of this reaches the soil (33). Sporadic releases of human
sewage may contaminate irrigation water, and runoff from
animal manure can contaminate crops. The results of this
study indicate that water contaminated with human patho-
gens can be a source for inoculation of food crops and
highlight the importance of disinfection when waste water
is used for irrigation in agricultural settings. The transfer
of Salmonella from water to plants depends on the produc-
tion of the extracellular structures, curli and cellulose, sim-
ilar to the situation for plant-associated bacteria. In our
study, we confirmed that Salmonella was able to invade the
plant. Although the mechanism is not clear, pathogen in-
vasion into growing vegetables that may be eaten raw is of
primary concern, because these bacteria are not removed
when the plant is washed. Future work should focus on the
regulation of the biofilm-encoding genes in bacteria in soil
and plants.
J. Food Prot., Vol. 72, No. 3
ACKNOWLEDGMENTS
This research was supported by Bracha Funds and the Israeli Min-
istry of Security. We thank D. Shachar and the Technion Ecological Gar-
den team for their technical assistance and U. Romling for the Salmonella
mutants.
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