Internalization pathogens roots

							Internalization of pathogens through roots and translocation to aerial tissues
(last updated 8/15/2012)
"F" after the reference citation signifies it was a field study whereas "G" signifies that the study was conducted in a laboratory or growth chamber.



Bernstein, N., S. Sela, and S. Neder-Lavon. 2007. Assessment of contamination potential of lettuce by Salmonella
enterica serovar Newport added to the plant growing medium. J. Food Prot. 70:1717-1722. G
17-, 20-, and 33-day old plants were exposed to contaminated potting medium (106 CFU/g). Bacterial internalization was
evaluated 2 and 7 days after inoculation of the potting medium. (The plant material was not surface sterilized to prevent
possible penetration of the sterilizing agent to internal cut parts of plant tissues and thus elimination of putative intratissue
bacteria.) Apparent internalization of Salmonella via the root to above-ground parts was identified in 33- but not 17- or 20-
day-old plants and was stimulated by root decapitation. Leaves of lettuce plants with intact and damaged roots harbored
Salmonella at 500 and 5,130 CFU/g of leaf, respectively, at 2 days postinoculation but not 5 days later.
DiCaprio, E., Y. Ma., A. Purgianto, J. Hughes, and J. Li. 2012. Internalization and dissemination of human norovirus
and animal caliciviruses in hydroponically grown Romaine lettuce. Appl. Environ. Microbiol. 78:6143-6152. G
Twenty-day-old romaine lettuce plants were exposed for 14 days to hydroponic feed water containing either human
norovirus (NoV), Tulane virus (TV) or murine norovirus (MNV-1) at initial levels of ~6 log RNA copies or PFU/ml. A
higher level of Tulane virus (4-5 log RNA copies/g) were detected in leaves and shoots at days 1 and 2 postinoculation using
real-time PCR compared to a relatively low level of infectious viral particles determined by plaque assay. Decreased RNA
detection in samples treated with RNase suggested that detection of naked viral RNA, originating from virus particles
damaged within the plant tissues, also occurred. A high level of human NoV RNA (5 log RNA copies/g) was detected in leaf
tissue of lettuce on day 1 post inoculation and these levels remained stable over the 14-day period. RNase treatment led to a
decrease of 0.5 to 1.5 log in human NoV RNA copies in lettuce tissue from days 2 to 14, suggesting that naked human NoV
RNA was also present in the samples. In contrast to human NoV and Tulane virus, internalization of MNV-1 was less
efficient with an average titer of 5.7 PFU/g on day 1. By day 7, the viral titer of MNV-1 increased to ~5 log PFU/g. Surface
disinfection of plant tissues with chlorine confirmed that virus contamination of leaves and shoots was internal.
Erickson, M.C., C.C. Webb, J.C. Diaz-Perez, S.C. Phatak, J.J. Silvoy, L. Davey, A.S. Payton, J. Liao, L. Ma, and M.P.
Doyle. 2010. Infrequent internalization of Escherichia coli O157:H7 into field-grown leafy greens. J. Food Prot.
73:500-506. F
In one set of experimental trials, irrigation water containing E. coli O157:H7 at 2, 4, or 6 log CFU/ml was applied to the base
of field-grown plants at 0, 55, and 69 days after transplantation and then plants analyzed at weekly intervals up to 3 weeks
after application. During the early or late portion of the growing season, internalized E. coli O157:H7 was not detected in
leaves or roots exposed to any of the inoculum concentrations. In mid-season exposed plants, internalized E. coli O157:H7
was detected by enrichment in surface-disinfected roots 7 days after exposure (5 of 30 samples) but was not detected in roots
14 or 22 days after exposure.
In the second set of experimental trials, E. coli O157:H7-contaminated compost (3 or 5 log CFU/g) was applied to field plots
prior to transplantation of spinach, lettuce, or parsley plants and then plants collected and analyzed 2, 7, 13, and 49 days later.
Internalized E. coli O157:H7 was not detected in leaves or roots of plants (surface sterilized with 1% silver nitrate) collected
at any of the sampling times.
Esseili, M.A., Q. Wang, Z. Zhang, and L.J. Saif. 2012. Internalization of sapovirus, a surrogate for norovirus, in
Romaine lettuce and the effect of lettuce latex on virus infectivity. Appl. Environ. Microbiol. 78:6271-6279. G
Lettuce roots of 4-week old plants were submerged in a 2-ml sapovirus (SaV) stock solution (6 log 50% tissue culture
infective dose) for 2 h before replanting in sterile soil. The SaV RNA persisted on the roots and was also detected in the
leaves until postinoculation day (PID) 28; however, the RNA levels were near or below the infectivity detection limit.
Mature plants (8 weeks of age) were also exposed to SaV (5 ml of stock solution diluted in 25 ml sterile water) in sterile soil.
Viral RNA was detected in the roots through at least PID 14; however, viral RNA was detected only in the leaves of 3 of 9
plants tested initially and on PID 2. The detection of SaV RNA in xylem sap suggested that SaV was transferred to leaves
from roots via the xylem vessels. Outer leaves (6 to 7 per plant) of mature plants were spot inoculated with 1 ml SaV stock
solution and then both inner and outer leaves sampled up to PID 14. Although viral RNA continued to be detected on outer
leaves, infectious viruses could only be detected initially (2 h postinoculation). The SaV RNA was not detected in any of the
inner leaves of infected plants. Aqueous extracts of lettuce leaves incubated with SaV did not significantly reduce SaV
infectivity or antigenicity but latex sap did reduce SaV infectivity.
Franz, E., A.A. Visser, A.D. Van Diepeningen, M.M. Klerks, A.J. Termorshuizen, and A.H.C. van Bruggen. 2007.
Quantification of contamination of lettuce by GFP-expressing Escherichia coli O157:H7 and Salmonella enterica
serovar Typhimurium. Food Microbiol. 24:106-112. G
Lettuce seedlings were either grown in an inoculated hydroponic system (plants harvested 18 d after intial exposure) or

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Internalization of pathogens through roots and translocation to aerial tissues
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seedlings planted in potting soil and exposed to pathogen through soil that was irrigated with contaminated water 14 and 18
days after seeding (plants harvested 35 days after initial exposure). Leaf samples were sterilized with silver nitrate in order
to detect internalized pathogens. With introduction of pathogens via the hydroponic system, internal colonization of lettuce
only occurred at high densities with S. Typhimurium MAE 199. With introduction of pathogens via the soil system, E. coli
O157:H7, S. Typhimurium 110 and S. Typhimurium 119 were found at considerable densities in sterilized leaf samples
(3.95, 2.57 and 2.37 log cfu/g on average, respectively) with prevalences of 0.29, 0.23 and 0.15, respectively. No statistical
differences were observed between the Salmonella strains; however, the degree of internal contamination was statistically
higher for E. coli O157:H7 than for Salmonella spp.
For each pathogen it was observed that those plants which showed internal contamination weighed less than plants without
internal contamination.
Franz, E., A.D. van Diepeningen, O.J. de Vos, and A.H.C. van Bruggen. 2005. Effects of cattle feeding regimen and
soil management type on the fate of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in
manure, manure-amended soil, and lettuce. Appl. Environ. Microbiol. 71:6165-6174. G
E. coli O157:H7 and Salmonella added to manure and mixed with loamy and sandy soil 56 and 28 days later, respectively.
After another 14 days, iceberg lettuce seedlings were planted and then checked for pathogens after 21 days of growth. To
distinguish between epiphytic and endophytic contamination, one-half of leaf and root samples surface sterilized with silver
nitrate. No pathogens were detected in the edible lettuce parts. Only one root sample showed the presence of E. coli
O157:H7 however, this sample had not been surface sterilized.
Ge, C., C. Lee, and J. Lee. 2012. The impact of extreme weather events on Salmonella internalization in lettuce and
green onion. Food Res. Int. 45:1118-1122. G
Three treatments were applied to lettuce and green onion plants: 1) optimal group (moisture content of 2.2. g/dw or 69% of
wet weight); 2) heavy rain (over-irrigation) group was watered with 1500 ml/pot for 2 days giving 3.73 g/g dw or 79% of wet
weight; and 32) drought group stopped watering of plants for 2 days post-inoculation giving 0.83 g/g dw or 45% of wet
weight. Each batch of plants was contaminated in the rhizosphere with a Salmonella suspension to give 5, 7, 8, or 9 log
CFU/g. After 2 days, entire plants were dug out from the soil, disinfected with ethanol and 1% silver nitrate, cut into leaves
and roots, and then stomached for 2 min in presence of peptone water. No internalization was detected in either plant when
soil was contaminated with 6 log CFU/g soil. In the case of green onions, 8 log CFU/g soil contamination led to green onion
internalization of 4 log CFU/g which was 1-3 log higher than when soil had been contaminated with 7 log CFU/g and root
samples had a significantly higher internalization level than the leafy parts. In contrast, the internalization of Salmonella was
only found in the leafy parts irrespective of the water stress. No effect on the level of internalization in green onions was
observed with water stress whereas water stress increased internalization in lettuce. At a soil contamination level of 8 log
CFU/g, the drought group showed more internalization than the optimal group at a soil contamination level of 9 log CFU/g,
the storm group showed greater internalization than the optimal group.
Habteselassie, M.Y., M. Bischoff, B. Applegate, B. Reuhs, and R.F. Turco. 2010. Understanding the role of
agricultural practices in the potential colonization and contamination by Escherichia coli in the rhizospheres of fresh
produce. J. Food Prot. 73:2001-2009. G
A strain expressing bioluminescence (lux) was used to monitor metabolic activity, survival, and distribution in an agar-based
growth system. Radish and lettuce seeds were inoculated with O157 lux by soaking approximately 3 to 5 g seeds in 20 ml (7
log CFU/ml) for 20 min before placing in an agar growth system. The numbers of O157 lux introduced onto the seeds before
germination were 6.6 and 6.5 CFU/g for radish and lettuce, respectively. High levels of bacterial luminescence associated
with the root demonstrate the potential of the rhizosphere to support metabolism of E. coli O157:H7 during seed germination.
The spread of light from the inoculated seed to the different parts of the root system suggests increased bacterial numbers on
the seedlings and clearly confirms the observation that the bacteria are able to grow on the nutrients provided during
germination.
In a separate contamination experiment, plants were exposed to E. coli either via soil mixed with 7 log CFU/g contaminated
manure or to contaminated irrigation water (20 ml, 6 log CFU/ml) added to plants in uncontaminated soil on days 17 and 32.
Postharvest (day 41), the numbers of E. coli in the rhizosphere samples in soil receiving the bacteria via manure or via
manure and irrigation water combined were 5.2 and 5.1 log CFU/g, respectively, while the numbers were 5.2 and 5.7 log for
the bulk soil samples (initial inoculum size was 7.2 log).
E. coli was detected on the phyllosphere 10 days after application (day 27) at 1.4 log CFU/g (25 CFU/g), indicating that E.
coli moved to the leaves after irrigation of the soil. E. coli was not, however, detected on the leaves at harvest (day 41) even
though it was present in the rhizosphere and bulk soil at 3.3 and 3.2 log CFU/g, respectively.
Compiled by Marilyn Erickson, Center for Food Safety, University of Georgia                                                                             Page 2
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Internalization of pathogens through roots and translocation to aerial tissues
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Hora, R., K. Warriner, B.J. Shelp, and M.W. Griffiths. 2005. Internalization of Escherichia coli O157:H7 following
biological and mechanical disruption of growing spinach plants. J. Food Prot. 68:2506-2509. G
5-week old spinach plants in commercial grade soil irrigated with 20 ml of E. coli O157:H7 (6 log CFU/ml) then analyzed 7
days after exposure. Samples surface disinfected with chlorine. Mechanical disruption of roots (removal of seminal root and
root hairs) does not result in internalization nor did simultaneous exposure to northern root-knot nematode.
Jablasone, J., K. Warriner, and M. Griffiths. 2005. Interactions of Escherichia coli O157:H7, Salmonella typhimurium
and Listeria monocytogenes plants cultivated in a gnotobiotic system. Int. J. Food Microbiol. 99:7-18. G
Scanning electron micrographs of root seedlings suggested that O157 preferentially colonized the root junctions of seedlings.
Seeds were soaked for 20 min in the pathogen cell suspension (2 log CFU/ml) and air dried. Bacterial loads on seeds ranged
between 3.3 and 4.7 log CFU/g irrespective of the seed or bacterial type. Germinated in one of two systems: 1) on dampened
sterile filter paper disks that were kept moistened; 2) on beds of solidified hydroponic nutrient solution. Tissues surface
disinfected with chlorine.
During seed germination, the numbers of all pathogens significantly increased. Radish and cress supported the growth of
O157 to a greater extent than carrot, lettuce, mustard spinach or tomato. Cress and radish supported greater Salmonella
growth than carrot, lettuce, mustard, spinach, or tomato. L. monocytogenes numbers on germinating seeds were typically
greater than those of E. coli O157:H7 and Salmonella.
In 9-day seedlings, O157 could be recovered from the external and internal plant tissue of all types tested. The internalized
O157 populations of plants decreased to below the level of detection (<1 log CFU/g) by day 49 although the pathogen still
persisted on the exterior.
On day 9, only radish and lettuce seedlings were found to harbor internalized populations of Salmonella. Cultivation of
plants resulted in a significant decrease in Salmonella numbers recovered from the surface and internal tissue of plants.
L. monocytogenes could only be recovered from the surface of seedlings with populations being similar for all types of
seedlings.
To evaluate antagonistic bacteria isolated from rhizosphere, co-inoculated with suspension of pathogen and potential
antagonistic bacteria, both at 2 log CFU/ml. Co-inoculation of isolates recovered from the rhizosphere of plants did not
significantly affect the numbers or persistence of human pathogens. Only with lettuce inoculated with En. cloacae was there
a significant decrease in E. coli O157:H7 or L. monocytogenes numbers relative to controls.
Klerks, M.M., E. Franz, M. van Gent-Pelzer, C. Zijlstra, and A.H.C. van Bruggen. 2007. Differential interaction of
Salmonella enterica serovars with lettuce cultivars and plant-microbe factors influencing the colonization efficiency.
ISME J. 1:620-631. G
Seeds sterilized then planted in S. enterica-contaminated manure-amended soil (7 log CFU/g) and analyzed for endophytic
bacteria by surface disinfecting with 70% ethanol. After 6 weeks, each plant was harvested by cutting the plant at the stem
level. For S. enterica serovar Dublin, 3 out of 28 plants were also positive for endophytic colonization after surface
disinfection. (164 CFU/g for positive samples). Species richness of the natural endophytic microbial community of lettuce
cultivars was negatively correlated to the number of endophytic S. enterica CFUs/g shoot tissue. No endophytic colonization
was observed for surface-disinfected plants grown on soil contaminated with S. enterica serovar Enteritidis or S. enterica
serovar Typhimurium.
To test potential differential colonization, 3 lettuce cultivars were tested with 5 S. enterica serovars in hydroponic system (70
µl of 7 log CFU/ml pipetted near roots in Hoagland’s agar). The number of endophytically colonized plants was
significantly affected by the serovar but not by the lettuce cultivar (Tumburo, Cancan, Nelly). Prevalence of lettuce plants
endophytically colonized were 59%, 85%, 93%, 85%, and 89% for serovars Dublin, Enteritidis, Montevideo, Newport, and
Typhimurium. Chemotaxis experiments revealed that S. enterica serovars actively move toward root exudates of lettuce
cultivar Tamburo. Subsequent micro-array analysis identified genes of S. enterica serovar Typhimurium that were activated
by the root exudates of cultivar Tamburo.
Differentially expressed genes (due to chemotaxis-inducing root exudates) appeared either associated with pathogenicity or
pointed toward a relationship with a sugar-like carbon source. For example, SsaH and SsaM are regulators of the type III
secretion system and SpaO is involved in the surface presentation of antigens that is secretory proteins. The observed
induction of DsrA implies the activation of processes that allow attachment, since it is involved in the synthesis of capsular
polysaccharides, an important factor in the attachment to host cell surfaces. Typically, amino acids could also play an
important role during chemotaxis or survival close to roots, as the MetE gene, induced in the presence of mono-cysteine, was
also upregulated.
Compiled by Marilyn Erickson, Center for Food Safety, University of Georgia                                                                             Page 3
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Internalization of pathogens through roots and translocation to aerial tissues
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Klerks, M.M., M. van Gent-Pelzer, E. Franz, C. Zijlstra, and A.H.C. van Bruggen. 2007. Physiological and molecular
responses of Lactuca sativa to colonization by Salmonella enterica serovar Dublin. Appl. Environ. Microbiol. 73:4905-
4914. G
Lettuce plants were colonized both endophytically (3 of 56 plants) and epiphytically (15 of 56 plants) when lettuce seeds
were germinated and sprouted on S. enterica serovar Dublin-inoculated manure-amended soil (7 log CFU/g). Salmonella
was found to be associated with lettuce plants, mainly from the root-stem transition point up to the leaves, but not in the
leaves. Analyses of the cross sections revealed strong growth of Salmonella on the root surface and near emerging lateral
roots. Moreover, internalization was observed via the intercellular spaces between epidermal cells. With both approaches
(soil and sterile culure), Salmonella was mainly present on the plant surface but was also found endophytically at a ratio of
13:1. Lettuce grown under sterile conditions (Hoagland’s agar) was even more susceptible to colonization by S. enterica
serovar Dublin via the roots (prevalence of 43%) than was lettuce grown in soil (7 log cfu/g). This study also demonstrated
that lettuce plants respond to the presence of S. enterica serovar Dublin at physiological (leaf yellowing, stunting, reduction
in growth) and molecular levels (concurrent expression of pathogenicity-related genes).
Koseki, S., Y. Mizuno, and K. Yamamoto. 2011. Comparison of two possible routes of pathogen contamination of
spinach leaves in a hydroponic cultivation system. J. Food Prot. In Press. G
Three major bacterial pathogens, E. coli O157:H7, Salmonella spp. and Listeria monocytogenes, were inoculated into the
hydroponic solution, in which the spinach was grown to give concentrations of 6 and 3 log CFU/ml. In parallel, the
pathogens were inoculated onto the growing leafy surface by pipetting, to give concentrations of 6 and 3 log CFU/leaf.
Although contamination was observed at a high rate through the root system by the higher inoculum (6 log CFU) for all the
pathogens tested, the contamination was rare when the lower inoculum (3 log) was applied. In contrast, contamination
through the leaf occurred at a very low rate, even when the inoculum level was high. The risk of contamination by L.
monocytogenes was about 0.3 times that of Salmonella enterica subsp. enterica serovars Typhimurium and Enteritidis and E.
coli O157:H7.
Kroupitski, Y., D. Golberg, E. Belausov, R. Pinto,D. Swartzberg, D. Granot, and S. Sela. 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. G
Incubation of gfp-tagged Salmonella enterica with iceberg lettuce leaves (3 x 3 cm leaf piece of leaf submerged in 10 ml of 8
log cfu/ml) in the light resulted in aggregation of bacteria near open stomata and invasion into the inner leaf tissue. In
contrast, incubation in the dark resulted in a scattered attachment pattern and very poor stomatal internalization. Forcing
stomatal opening in the dark by fusicoccin had no significant effect on Salmonella internalization. Results suggested that the
pathogen is attracted to nutrients produced de novo by photosynthetically active cells.
Salmonella internalization assays were conducted in the presence of exogenous sucrose and its monosaccharide metabolites
glucose and fructose. Indeed, all three sugars each at a concentration of 100 mM significantly inhibitied Salmonella
internalization, with fructose having the maximal inhibition effect. Furthermore, leaf extract derived from light-exposed
lettuce also inhibited Salmonella penetration.
Elevated internalization and attachment rates were evident at the the higher termperatures (25 and 37°C) supporting the
involvement of active metabolism in the two processes.
The penetration of fliGHI and cheY mutants defective in motility and chemotaxis was significantly inhibited suggesting that
the two processes are important for Salmonella internalization. In addition, the cheY mutant, also exhibited reduced
attachment, about 50% that of the wild type, implying that flagella are also required for efficient attachment.
Lapidot, A. and S. Yaron. 2009. Transfer of Salmonella enterica serovar Typhimurium from contaminated irrigation
water to parsley is dependent on curli and cellulose, the biofilm matrix components. J. Food Prot. 72:618-623.
G
To parsley plants (avg. hgt of 20 to 40 cm), drip irrigated 3 times during a 9-day period (3-day intervals) with 500 ml of
freshly prepared contaminated water (7.6 log CFU/ml). Added to 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. Care was taken to collect only the highest parts of the
plants that had not come into any direct contact with the soil. Very few colonies of Salmonella were seen in samples of
leaves collected 1 day after the first irrigation (2.0 log CFU/g), just above the detection limit (1.7 log CFU/g). For samples
collected on day 10, Salmonella were detected in all of the experimental treated samples with a trend toward higher
concentration in the leaves (4.1 log CFU/g) than in the stems. Examination of leaves by confocal laser scanning microscopy
revealed colonization of the leaves with both single cells and small aggregates of Salmonella at a depth of 8 to 32 µm

Compiled by Marilyn Erickson, Center for Food Safety, University of Georgia                                                                             Page 4
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Internalization of pathogens through roots and translocation to aerial tissues
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beneath the upper surface of the leaf.
Externally sterilized the upper parts of the stems with swabs of 70% ethanol for 2 min, then cut the stems and used a sterile
pipette tip to collect the internal liquids that accumulated on top of the cut stems. Positive fluorescent colonies were
apparent on the plates of liquids taken from the plants irrigated with Salmonella.
Although the bacterial numbers in the soil were not different 3 weeks after contamination, the bacterial numbers associated
with the regrown plants declined. This reduction may result from death or aggregation of the bacteria or from changes in the
physiology of the harvested plants. 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 contaminate the plant. This hypothesis is supported by previous findings that bacteria in
close contact with soil particles adsorb to the soil particles and can move only short distances, even under saturated
conditions.
The importance of the bacterial cellulose 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.
Mitra, R., E. Cuesta-Alonso, A. Wayadande, J. Talley, S. Gilliland, and J. Fletcher. 2009. Effect of route of
introduction and host cultivar on the colonization, internalization, and movement of the human pathogen Escherichia
coli O157:H7 in spinach. J. Food Prot. 72:1521-1530. G
O157 dropped onto spinach leaf surfaces (10 µl of 6 log inoculum) survived on the phylloplane for at least 14 days, with
increasing titers and colonization over time. After 2 weeks, cv Bordeaux hosted very few bacteria, cv Space had more
bacteria, while cv Tyee had the highest numbers suggesting that cv Tyee may provide protected niches or more nutrients or
may promote stronger bacterial adherence. Stem puncture inoculations, simulating natural wounding rarely resulted in
colonization or multiplication. Bacteria forced into the leaf interior (stab inoculation or pressure inoculation, 10 µl of 6 log
inoculum) survived for at least 14 days in intercellular spaces but did not translocate or multiply. Whole plant samples
taken and surface sterilized with ethanol and 10% sodium chlorite. In soil drench, 10 ml of 6 log inoculums inserted into soil
and dispensed directly in root zone. Internal colonization was observed in 10 of 60 plant samples (primary root and leaves).
Mootian, G., W-H. Wu, and K.R. Matthews. 2009. Transfer of Escherichia coli O157:H7 from soil, water, and manure
contaminated with low numbers of the pathogen to lettuce plants. J. Food Prot. 72:2308-2312. G
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 1, 2, 3, or 4 log CFU O157/g or ml. The inoculum was applied carefully to prevent splashing of the
inoculum onto the edible portion of the lettuce plant. The plants were cut from the root systems approx. 1 cm above the soil
surface to minimize surface contamination of the edible portion of the plant. No samples were positive for O157 after direct
plating of serial dilutions.
Enrichment of all samples from young plants demonstrated that approx. 21% (113 of 552) of plants were positive for O157.
Approx. 30% (36 of 120) of the mature plants initially irrigated with or grown in contaminated soil, were positive for O157.
O157 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 after exposure to contaminated soil. Nearly 63% of plants (non-surface sterilized)
that had been cultivated for 15 days after irrigation with contaminated water were positive for O157 whereas only 13% of
surface-sterilized plants were positive for the target pathogen.
Following enrichment, 9% (26 of 288) of surface-sterilized plants exposed at young age were positive for E. coli O157:H7.
7.5% (9 of 120) of surface-sterilized plants exposed at mature age were positive for E. coli O157:H7.
Nthenge, A.K., J.S. Weese, M. Carter, C.-I. Wei, and T.-S. Huang. 2007. Efficacy of gamma radiation and aqueous
chlorine on Escherichia coli O157:H7 in hydroponically grown lettuce plants. J. Food Prot. 70:748-752. G
Lettuce seedlings were planted in contaminated Hoagland’s nutrient solution (5 or 7 log CFU/ml) and thereafter subjected to
gamma radiation at 0.25, 0.5 and 0.75 kGy and 200 ppm chlorine. Leaves disinfected with 80% ethanol and 0.1% mercuric
chloride.
In absence of irradiation, 5 of 24 and 5 of 23 leaves were positive for internalized E. coli O157:H7 when plants were exposed
to a solution containing the pathogen at 5 or 7 log CFU/ml, respectively.
Gamma radiation at 0.25 and 0.5 kGy, and aqueous chlorine at 200 ppm failed to eliminate E. coli O157:H7 in lettuce tissue
completely; however, the bacteria were not detected in 0.75 kGy-treated plants. The presence of E. coli O157:H7 in lettuce
leaves is an indication that the pathogen migrated from the contaminated hydroponic system through the roots to the internal
locations of lettuce tissue.

Compiled by Marilyn Erickson, Center for Food Safety, University of Georgia                                                                             Page 5
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Internalization of pathogens through roots and translocation to aerial tissues
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Ongeng, D., G.A. Vasquez, C. Muyanja, J. Ryckeboer, A.H. Geeraerd, and D. Springael. 2011. Transfer and
internalization of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in cabbage cultivated on
contaminated manure-amended soil under tropical field conditions in Sub-Saharan Africa. Int. J. Food Microbiol.
145:301-310. F
E. coli O157:H7 or S. Typhimurium-contaminated manure was incorporated into soil at point of transplantation, 56 or 105
days post-transplantation, to give 4 or 7 log CFU/g and then cabbage leaves analyzed at harvest (120 days post-
transplantation). No internalization in cabbage leaf tissues (surface sterilized with 1% silver nitrate) when soil was
contaminated at 4 log CFU/g initially. When soil was contaminated at the point of transplantation to give 7 log CFU/g,
internalized E. coli O157:H7 and S. Typhimurium was observed in leaves at harvest (18 of 18).
Pu, S., J.C. Beaulieu, W. Prinyawiwatkul, and B. Ge. 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. G
Two levels (3 and 7 log CFU/ml) of an O157 gfp-expressing strain were introduced into the plant growth media weekly for a
total of 5 times. Among 120 spinach plant samples examined for internal leaf contamination (collected only aerial tissue;
sanitized with 2% calcium hypochlorite), only one yielded a positive result (from 7 log inoculum) and was collected on day 3
from 3rd week inoculation. Surface leaf contamination occurred occasionally and clustered between 3 and 5 weeks of age, but
not among leaves younger than 3 weeks of age (11 of 60 [21.7%] plants inoculated at the 3 log level were contaminated
compared with 13 of 60 [21.7%] plants at the 7 log level. Most of the contamination indices were detected after enrichment,
indicating levels lower than the detection limit (10 CFU/ml) for direct plating. The five spinach leaf samples with countable
numbers by direct plating were the total leaf samples for all three plants that were inoculated at the 7 log level in the 3 rd week
(day 23) and harvested in the same week (day 25).
When inoculated at the 7 log level, the O157 strain survived the entire cultivation period and could be enumerated by direct
plating from soil with one exception. As the gap between inoculation and harvest increased, a gradually reduced level of
contamination was observed.
Quilliam, R.S., A.P. Williams, and D.L. Jones. 2012. Lettuce cultivar mediates both phyllosphere and rhizosphere
activity of Escherichia coli O157:H7. PLoS One 7:e33842.
Just prior to sowing seeds from 12 different lettuce cultivars (Vaila-winter gem, Lobjoits green, Marshall, Little gem, Dazzle,
Unrivalled, Rosetta, Lakeland, Regina del ghiacci, Webbs wonderful, Set (iceberg), and Lollo rossa), a 1 ml suspension of 8
log CFU of a lux-marked strain of E. coli O157:H7 was pipetted evenly over the surface of the agar in each tray and allowed
to infiltrate for 20 min. Twenty days after sowing seeds and growing gnotobiotically, the root and shoot sections were
individually assayed for colonization by E. coli O157:H7 and exhibited differential cultivar-specific response. Two of the
cultivars that had consistently high (Vaila-winter gem) and low (Dazzle) bioluminescence when grown gnotobiotically were
also grown in potting compost with the bioluminescne of the endophytic/tightly bound cells colonizing the cultivar Vaila
significantly higher than those colonizing Dazzle. In contrast, the influence of cultivar in the rhizosphere was the opposite to
that in the phyllosphere with the higher number and activity of E. coli O157:H7 cells occurring in the rhizosphere of Dazzle
and possibly as a consequence of the inability of the cells to gain entry to the plant as effectively as Vaila.
Sharma, M., D.T. Ingram, J.R. Patel, P.D. Millner, X. Wang, A.E. Hull, and M.S. Donnenberg. 2009. A novel
approach to investigate the uptake and internalization of Escherichia coli O157:H7 in spinach cultivated in soil and
hydroponic medium. J. Food Prot. 72:1513-1520. G
Three E. coli O157:H7 isolates were compared for their ability to be internalized within spinach plants when cultivated in
soil or hydroponic systems. All 3 isolates were adapted for growth in dairy manure.
E. coli O157:H7 was applied at 3 log or 7 log/g to pasteurized soils in which baby spinach seedlings were planted. No
pathogen was recovered by enumeration from surface-sanitized internal tissues of spinach plant (used mercury chloride for
disinfection). At a low inoculum, O157 recovered on day 7 by enrichment of shoot tissue from one of 3 plants for rep 1
(pathogenic isolates from leafy green outbreaks). In another replicate (pathogenic isolate from ground beef outbreak), on day
28, one of three shoot tissue samples and 5 of 6 root tissue samples were positive by enrichment. At the high inoculums, on
day 0, one of 3 enriched shoot tissue samples grown in soil for one of the 3 reps was positive by enrichment for E. coli. On
day 28, one of 6 enriched root tissue samples was positive.
Fluorescent cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils.
No internalized cells were observed in shoot tissue of plants grown in inoculated soil.
The outbreak strain of O157 survived at significantly higher populations in the soil than did a nonpathogenic strain of E. coli.

Compiled by Marilyn Erickson, Center for Food Safety, University of Georgia                                                                             Page 6
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Internalization of pathogens through roots and translocation to aerial tissues
(last updated 8/15/2012)
"F" after the reference citation signifies it was a field study whereas "G" signifies that the study was conducted in a laboratory or growth chamber.



In hydroponic systems, seedlings had been germinated from disinfected seeds prior to transferring to sterile hydroponic
growth media containing low (3.5-4.0 log CFU/ml) or high (6.7-6.9 log CFU/ml) E. coli. E. coli O157:H7 was internalized
sporadically into tissues of spinach plants grown in hydroponic medium inoculated with low bacterial populations. E. coli
O157:H7 applied at ca. 7 log CFU/ml to hydroponic medium was consistently recovered from the shoot tissues of spinach
plants after 14 days (3.7 log CFU/shoot) and 21 days (4.4 log CFU/shoot).
Solomon, E.B. and K.R. Matthews. 2005. Use of fluorescent microspheres as a tool to investigate bacterial interactions
with growing plants. J. Food Prot. 68:870-873. G
45-day old lettuce plants in soil exposed to either 30 ml of 8 log CFU E. coli O157:H7/ml or 8 log FluoSpheres/ml. Laser
scanning confocal microscopy revealed that FluoSpheres were present within the root tissue and leaf stem tissue. The
numbers of FluoSpheres in plant tissue were similar to the numbers of E. coli O157:H7.
Solomon, E.B., S. Yaron, and K.R. Matthews. 2002. Transmission of Escherichia coli O157:H7 from contaminated
manure and irrigation water to lettuce plant tissue and its subsequent internalization. Appl. Environ. Microbiol.
68:397-400. G
In one set of treatments, seeds were placed in soil inoculated with 4, 6, or 8 log CFU E. coli O157:H7/g and then seedlings
sampled 3, 6, and 9 days postplanting. In another set of treatments, 200 ml of a 7 log CFU E. coli O157:H7/ml inoculum
added to soil of mature plants (avoided splash) and then plants harvested on days 1, 3, and 5 days postinoculation. Seedlings
were surfaced disinfected with 80% ethanol followed by 0.1% mercury chloride whereas mature plants were not surface
disinfected.
Using confocal microscopy, the target pathogen was visualized at depths up to 45 µm below the tissue surface of seedlings
but cells were restricted to the intercellular space. In seedlings, viable cells were recovered from 0 of 22, 4 of 22, and 6 of 22
seedlings that had been exposed to soil containing 4, 6, and 8 log CFU/g, respectively. With mature plants exposed to
contaminated irrigation water, 40 to 80% of the samples were positive for E. coli O157:H7.
Urbanucci, A., M. Myrmel, I. Berg, C-H. von Bonsdorff, and L. Maunula. 2009. Potential internalisation of
caliciviruses in lettuce. Int. J. Food Microbiol. 135:175-178. G
The roots of lettuce growing either in hydroponic culture or in soil were exposed to canine calicivirus and a human
genogroup 2 norovirus. When the lettuce plants were exposed to very high concentrations (6 to 10 log PCR-U) of canine
caliciviruses (CaCV), the virus was detected in lettuce leaves, indicating contamination via the roots, but the frequency of
positive results was low. 5 of 12 leaf samples were positive for canine calicivirus when plants exposed in hydroponic
system, whereas 1 of 12 leaf samples positive for virus when plants exposed in soil system.
Internalization occurred in both seedlings and grown plants, in both hydroponic and soil cultures and occurred whether the
roots were intact or damaged. Root damage apparently did not increase internalization of viruses, as positive samples were
found only occasionally in the vascular liquid and the leaves of plants with damaged roots. In these studies, no difference
beween soil and hydroponic cultures was observed regarding virus internalization.
Consistent with other results, viruses were not found to be circulating in high amounts in the vascular tissue of the plants in
our study.
Internalization of HuNoV was not detected in any of the experimental setups, although the concentrations to which the plants
were exposed were relatively high (5 log PCR-U).
Warriner, K., F. Ibrahim, M. Dickinson, C. Wright, and W.M. Waites. 2003. Interaction of Escherichia coli with
growing spinach plants. J. Food Prot. 66:1790-1797. G
Three treatments were examined.
In the first treatment, spinach seeds were submerged in 7 log CFU E. coli/ml for 30 min prior to sowing and growing in soil
systems. After a 35-day growing period, plants were analyzed. Internalized E. coli could not be recovered from leaves but
could be recovered from roots (1.9 CFU/g).
In the second treatment, 13-day-old seedlings were exposed to a E. coli-contaminated hydroponic solution (2 or 3 log
CFU/ml) for 16 days prior to plant tissue analysis. E. coli was recovered from surface-sterilized root tissue (1.4-1.6 log
CFU/g) but not from within leaves.
In the third treatment, 20-day-old seedlings were transplanted into E. coli-contaminated soil (2 log CFU/g) and then plants
harvested periodically up to day 42. On day 42, E. coli was recovered from surface-sterilized root tissues (3.8 log CFU/g)
but not from within leaves.
Wei, J., Y. Jin, T. Sims, and K.E. Kniel. 2011. Internalization of murine norovirus 1 by Lactuca sativa during
Compiled by Marilyn Erickson, Center for Food Safety, University of Georgia                                                                             Page 7
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Internalization of pathogens through roots and translocation to aerial tissues
(last updated 8/15/2012)
"F" after the reference citation signifies it was a field study whereas "G" signifies that the study was conducted in a laboratory or growth chamber.



irrigation. Appl. Environ. Microbiol. 77:2508-2512. G
Lettuce was challenged with murine norovirus 1 (MNV) either delivering 8 log reverse transcriptase quantitative PCR (RT-
qPCR) U/ml in nutrient buffer at the roots in one dose followed by 4 days of exposure to virus-free buffer, or delivering 5 log
RT-qPCR U/ml of MNV to the roots on five successive days. With hydroponically grown lettuce challenged with the one-
high dose, ~3.4 log RT-qPCR U of viral DNA/50 mg of plant tissue was detected in leaf samples, and this level was
significantly higher than the level found in leaves (~2.6 log) whose plants had been challenged repetitively with the lower
dose. With lettuce grown in soil, only 2 log RT-qPCR U of viral RNA/50 mg of plant tissue was detected in leaves with no
significant differences being found with challenge dose possibly as a result of the tendency of virus particles to adsorb to soil
particles and not remain suspended in nutrient buffer. Infectious MNV was found in lettuce samples challenged with high
virus inoculums grown either hydroponically or in soil but not in lettuce grown with low virus inoculums. Transpiration rate
was suggested as a possible contributor to internalization of virus into lettuce as more lettuce samples were found positive for
MNV when plants were grown in environments of 70% relative humidity compared to plants grown at 99% relative
humidity.
Zhang, G., L. Ma, L.R. Beuchat, M.C. Erickson, V.H. Phelan, and M.P. Doyle. 2009. Lack of internalization of
Escherichia coli O157:H7 in lettuce (Lactuca sativa L.) after leaf surface and soil inoculation. J. Food Prot. 72:2028-
2037. G
Iceberg, romaine, and leaf lettuce seedlings were transplanted into pots and then an E. coli O157:H7 suspension added to soil
to give 3 or 6 log CFU/g with root and leaf samples taken up to 60 days after seedlings were transplanted. All 270 leaf
samples (surface sanitized with 80% ethanol followed by 0.1% mercury chloride) were negative for internalized E. coli
O157:H7. All surface-sanitized root samples from 3 log CFU/g exposure and 148 of 150 surface-sanitized root samples from
6 log CFU/g exposure were negative for E. coli O157:H7.
Zhang, G., L. Ma., L.R. Beuchat, M.C. Erickson, V.H. Phelan, and M.P. Doyle. 2009. Heat and drought stress during
growth of lettuce (Lactuca sativa L.) does not promote internalization of Escherichia coli O157:H7. J. Food Prot.
72:2471-2475. G
Thirty days after transplanting romaine and iceberg lettuce seedlings, an E. coli O157:H7 suspension was added to the soil to
give 4 or 6 log CFU/g and then plants were exposed during the day (12 h) to either 23°C (3 days), 32°C (3 days) or 36°C (2
days) followed by analysis of root and leaf tissue. All leaf samples (surface sanitized with 80% ethanol followed by 0.1%
mercury chloride) were negative for internalized E. coli O157:H7. 143 of 144 surface-sanitized root samples were negative
for E. coli O157:H7.




Compiled by Marilyn Erickson, Center for Food Safety, University of Georgia                                                                             Page 8
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