Attachment pathogens to aerial tissues

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					ATTACHMENT (CONTAMINATION) OF LEAFY GREEN AERIAL TISSUES
(last updated 4/5/2012)

"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.

Alum, A., C. Enriquez, and C.P. Gerba. 2011. Impact of drip irrigation method, soil, and virus type on tomato and
cucumber contamination. Food Environ. Virol. 4:78-85. G
Tomatoes and cucumbers were grown in Pima clay loam or Brazito sandy loam soils and irrigated using either surface drip
irrigation or subsurface drip irrigation with secondary wastewater effluent seeded with bacteriophages MS-2 and P22,
poliovirus type 1 (PV1), enteric adenovirus 40 (Ead 40), and hepatitis A virus. Viral contamination always occurred on
above ground parts with both crops whereas no viruses were detected in any of the above ground plant surfaces with
subsurface drip irrigation. Plant stems had a greater contamination than plant roots when surface drip irrigation was applied
whereas a greater contamination of plant roots than plant stems occurred when subsurface drip irrigation was applied.
Arthurson, V., A. Sessitsch, and L. Jäderlund. 2011. Persistence and spread of Salmonella enterica serovar
Weltebreden in soil and on spinach plants. FEMS Microbiol. Lett. 314:67-74. G
Significantly higher numbers of S. Weltebreden inoculated into manure and applied to soil before planting spinach were
found in soil than in pot culture, where the pathogen had been inoculated directly into soil 14 days postplanting. May be
attributed to a better developed spinach root system in the latter case and leading to more pronounced effects of the
rhizosphere on S. Weltebreden stimulation. Moreover, the pathogen appeared to be mobilized from manure to spinach roots,
as the number of contaminated pot cultures steadily increased throughout the evaluation period. When pathogen cells were
added to the soil in a manure mixture, no Salmonella cells were recovered above the threshold level (4 log cells/g) on leaves
when added to the soil in a manure mixture (used a molecular method for detection). When bacteria were added in saline
solution and added directly to soil 14 days after sowing and fertilization, cells were detected in all replicate pots on days 0
and 8 postinoculation. Moreover, Salmonella persisted on leaves in some of the replicates up to 21 days postinoculation.
Baldotto, L.E.B. and F.L. Olivares. 2008. Phylloepiphytic interaction between bacteria and different plant species in a
tropical agricultural system. Can. J. Microbiol. 54:918-931. G
Leaf blades of 47 plant species distributed in 27 botanical families were collected on a typical Brazilian farm. All plant
species studies were colonized by phyllooepiphytic bacteria, which were observed as solitary cells, microcolonies, and
biofilms. Independent of the family, the plant species differed in the pattern of phyllosphere colonization, as reflected in
bacteria frequency and presence or absence of anatomical features that would favor the association. Phylloepiphytic bacteria
were preferentially established on the following sites: epidermal cell wall junctions, glandular and nonglandular trichomes,
veins, stomata, and epidermal cell wall surface.
Fungi seemed to alter the bacteria colonization pattern, most probably by microenvironmental modifications. The trichome
type and density as well as the presence of epicuticular wax on the leaf blade surface seemed to be the most determinant
anatomical features for the pattern of phyllosphere colonization. The presence of trichomes has a favorable, and epicuticular
wax an unfavorable influence on the plant-bacteria interaction.
Stomata seemed to play a secondary role as an anatomical structure that improves epiphytic fitness. In most cases, no
difference between the upper and lower leaf surfaces could be seem, despite the differential distribution of these structures in
the plants under investigation.
Barak, J.D., A. Liang, and K-E. Narm. 2008. Differential attachment to and subsequent contamination of agricultural
crops by Salmonella enterica. Appl. Environ. Microbiol. 74:5568-5570. G
Surface-sanitized seeds were sown in the contaminated soil (enriched potting soil contaminated with 4 log CFU/ml water).
Pots were irrigated every other day with 25 ml of sterile water, which resulted in no drainage. Preliminary experiments
revealed no differences among rhizoplane S. enterica populations on different plants. Thus, only the phyllosphere was
sampled at the one true leaf stage. Radish, turnip, and broccoli (family Brassicaceae) seedlings had a significantly higher
contamination incidence than those of carrot, lettuce, and tomato. Radicchio and endive had a significantly higher
contamination incidence than lettuce; however, there were no significant differences among lettuce cultivars.
Lettuce had the highest phyllosphere populations of the mixed leafy greens. These data suggest that lettuce may not be
naturally contaminated via soil but once attached, high populations may be attached in the phyllosphere (3.1 log CFU/g).
Berger, C.N., R.K. Shaw, F. Ruiz-Perez, J.P. Nataro, I.R. Henderson, M.J. Pallen, and G. Frankel. 2009. Interaction
of enteroaggregative Escherichia coli with salad leaves. Environ. Microbiol. Rept. 1:234-239. G
Binding of clinical enteroaggregative Escherichia coli (EAEC) isolates to leaves from Eruca vesicaria (commonly known as
rocket or arugula) were divided into high, moderate and low adherent phenotypes. While EAEC O42 was found associated
with the epidermis of lettuce, basil, and spinach, adhesion of the stomata was seen in only 71%, 0%, and 0%, respectively.
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Adhesion of EAEC O42 to epithelial cells is mediated by the AAF pilus. Compared the binding efficiency of wild-type O42
and O42aafA. This revealed that the mutant was highly attenuated in its leaf attachment capability but retained stomatal
adherence.
Dinu, L.-D. and S. Bach. 2011. Induction of viable for nonculturable Escherichia coli O157:H7 in the phyllosphere of
lettuce: a food safety risk factor. Appl. Environ. Microbiol. 77:8295-8302. G
Two E. coli O157:H7 strains maintained culturability when spot inoculated on lettuce leaves held in a growth chamber at
16°C but if the plants were held at 8°C, the bacterial populations evolved toward the viable but nonculturable (VNBC) state.
The two strains also exhibited different responses with one strain losing culturability within 7 days but the other strain did
not start to lose culturability until 15 days after inoculation. When a high-cell-density inoculum was applied to the leaves,
the number of cells entering the VNBC state was approximately 55% but approximately 70% of the cells entered the VNBC
state when a low-density inoculum was applied to the leaves.
Elhariry, H.M. 2011. Attachment strength and biofilm forming ability of Bacillus cereus on green-leafy vegetables:
Cabbage and lettuce. Food Microbiology 28:1266-1274. G
A reference strain (DSMZ 345) and five biofilm-producing strains (aquatic strains: TUB8, TUB30, TUB31, TUB32, and
TUB33) were investigated. The attachment strength of both spores and vegetative cells of the studied strains to the lettuce
surface was higher than that of the cabbage surface. The highest biofilm formation on cabbage and lettuce surfaces was
obtained by spores and vegetative cells of all tested strains on the 4 th hour of the incubation period. Scanning electron
microscopy exposed random distribution of cells either on the surface or cut edge, without clear obvious affinity to the
surface structures. Significant enhancement for in vitro biofilm formation by vegetative cells of B. cereus was recorded in
the diluted TSB.
Ells, T.C. and L.T. Hansen. 2006. Strain and growth temperature influence Listeria spp. attachment to intact and cut
cabbage. Int. J. Food Microbiol. 111:34-42. G
Cabbage coupons suspended in Listeria spp. inocula for various periods of time. Generally, samples were pulled from their
respective suspensions after 3 h and rinsed by two successive dips in phosphate buffered saline. The rate at which cells
attached to the cabbage was very rapid with numbers approached 4.3 log/cm2 for cells cultivated at 10°C after only 5 min of
exposure. Numbers of attached Listeria cells to disc-shaped cabbage coupons ranged from 3.84 to 4.78 log CFU/cm2 and
4.82 to 5.90 log CFU/cm2 on intact and cut cabbage, respectively. Strains with the highest numbers of attached cells on
intact cabbage were also the most efficient in attaching to the cut surfaces.The distribution of cells found on the intact
surfaces appeared to be randomly distributed with no apparent affinity for specialized surface structures such as stomata.
In general, the number of attached cells on the cabbage increased with longer exposure times for all cultures regardless of
cultivation temperature. However, there was a significant decline in the actual rate of attachment after the initial 5 min of
exposure. Between 4 and 24 h of exposure, approximately one-half of the noted increases in attachment of cells were
actually due to cells proliferating on the cabbage surface. SEM analysis also revealed the increased presence of large
clusters of cells on leaf surfaces after 4 and 24 h. These cell aggregates appeared to be in the early stages of biofilm
development.
The culture growth temperature was shown to significantly affect the strength of attachment during the first 24 h of exposure
with cells cultivated at 37°C more easily removed from leaf surfaces than those cells cultivated at 10 or 22°C. The lack of
production of flagella at this temperature which has been reported elsewhere was suggested as the possible cause of the
differences in attachment of cells cultivated at different temperatures.
No differences were observed for the numbers of Listeria cells attached to abaxial or adaxial leaf surfaces. However, areas
on the surface with small abrasions or tears frequently housed numerous cells, indicating a preference for exposed or
damaged tissues.
Esseili, M.A., Q. Wang, and L.J. Saif. 2012. Binding of human GII.4 norovirus virus-like particles to carbohydrates of
Romaine lettuce leaf cell wall materials. Appl. Environ. Microbiol. 78:786-794. G
Immunofluorescence analysis showed that the virus-like particles (VLPs) attached to the leaf surface, especially to cut edges,
stomata, and along minor veins. Binding was quantified using enzyme-linked immunosorbent assay (ELISA) performed on
cell wall materials (CWM) from innermost younger leaves and outermost lamina of older leaves. The binding to CWM of
older leaves was significantly higher (1.5- to 2-fold) than that to CWM of younger leaves. Collectively, our results indicate
that norovirus VLPs bind to lettuce CWM by utilizing multiple carbohydrate moietieis.
Fink, R.C., E.P. Black, Z. Hou, M. Sugawara, M.J. Sadowsky, and F. Diez-Gonzalez. 2012. Transcriptional responses
of Escherichia coli K-12 and O157:H7 associated with lettuce leaves. Appl. Environ. Microbiol. 78:1752-1764.
Proposed that when interacting with lettuce leaves, E. coli K-12 and O157:H7 first attach to the leaf surface using curli
fibers, shift down their metabolism to adapt to the nutrient-deprived environment of the leaf, and suppress the formation of a
permanent biofilm by increasing their intracellular indole concentration.
Gutiérrez-Rodriguez, A. Gundersen, A.O. Sbodio, and T.V. Suslow. 2011. Variable agronomic practices, cultivar,
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strain source and initial contamination dose differentially affect survival of Escherichia coli on spinach. J. Appl.
Microbiol. 112:109-118. F
E. coli O157:H7 was spray inoculated to deliver 1.45 or 3.4 log CFU/m2 on spinach plants in the field. After 14 days, E. coli
O157:H7 populations had leveled off at 1.2 log CFU/g. The pathogen was detected more consistently on basal shoot tissue
than on leaves. Decreased persistence in soil was associated with decreased moisture content in the soil. E. coli showed
greater environmental fitness than E. coli O157:H7.
Ibekwe, A.M., P.M. Watt, P.J. Shouse, and C.M. Grieve. 2004. Fate of Escherichia coli O157:H7 in irrigation water on
soils and plants as validated by culture method and real-time PCR. Can. J. Microbiol. 50:1007-1014. G
E. coli O157:H7 (1 L of 7 log CFU/ml) was applied to either clay or sandy soil through drip lines. On the average, about 170
CFU of E. coli O157:H7 was recovered from lettuce phyllosphere grown on clay soil and about 67 CFU from lettuce
phyllosphere grown on sandy soil after the first 12 d. E. coli O157:H7 concentrations were higher in the rhizosphere than in
the non-rhizosphere soils and leaf surfaces, and persisted longer in clay soil.
Johannessen, G.S., R.B. Froseth, L. Solemdal, J. Jarp, Y. Wasteson, and L.M. Rorvik. 2004. Influence of bovine
manure as fertilizer on the bacteriological quality of organic Iceberg lettuce. J. Appl. Microbiol. 96:787-794. F
E. coli O157:H7 was isolated from firm manure and slurry, and soils fertilized with the respective fertilizers the second year.
E. coli O157 recovered from manure and from soil samples 1 week after application of manure but was not recovered from
the lettuce.
Keraita, B., F. Konradsen, P. Drechsel, and R.C. Abaidoo. 2007. Reducing microbial contamination on wastewater-
irrigated lettuce by cessation of irrigation before harvesting. Trop. Med. Intl. Hlth. 12:8-14. F
Objective – to assess the effectiveness of cessation of irrigation (2, 4, and 6 days) before harvesting in reducing microbial
contamination of lettuce irrigated with wastewater in urban vegetable farming in Ghana. Samples taken in the dry season
just after rains often recorded higher levels of contamination than those taken during the wet season, especially when it had
not rained few days previous to the sampling day. Discarding outer leaves that are more in contact with contaminated soils
and less exposed to direct sunlight can also help in reducing vegetable contamination. Found an average difference of 1.8
log units of thermotolerant coliforms between the outer and inner leaves of lettuce.
If irrigation occurred on the same day as harvest, helminth concentrations in lettuce ranged from 2.2-2.7 eggs/L in the dry
season and 1.5-1.8 eggs/L in the wet season. When irrigation was stopped 2 days prior to harvest, helminth concentrations in
lettuce ranged from 1.6-2.1 eggs/L in the dry season and 1.1-1.2 eggs/L in the wet season.
When irrigation was stopped 4 days prior to harvest, helminth concentrations in lettuce ranged from 0.8-1.3 eggs/L in the dry
season and 0.5-0.8 eggs/L in the wet season.
When irrigation was stopped 6 days prior to harvest, helminth concentrations in lettuce ranged from 0.2-0.4 eggs/L in the dry
season and 0.2-0.3 eggs/L in the wet season.
Kroupitski, Y., R. Pinto, E. Belausov, and S. Sela. 2011. Distribution of Salmonella typhimuriurm in romaine lettuce
leaves. Food Microbiol. 28:990-997. G
Lettuce discs were submerged in GFP-tagged Salmonella suspension (only one strain) at 8 log CFU/ml for 2 h at 25°C.
Rinsed twice before stomaching for analysis of leaf-associated Salmonella. Attachment of Salmonella to different leaf
regions was highly variable; yet, a higher attachment level was observed on leaf regions localized close to the petiole (7.7 log
CFU/g) compared to surfaces at the far-end region of the leaf blade (6.2 log CFU/g). Attachment to surfaces located at a
central leaf region demonstrated intermediate attachment level (7.0 log CFU/g). Salmonella displayed higher affinity toward
the abaxial side compared to the adaxial side of the same leaf region. Rarely, Salmonella cells were also visualized
underneath stomata within the parenchymal tissue.
Comparison of attachment to leaves of different ages showed that Salmonella displayed higher affinity to older compared to
younger leaves (1.5 log). Scanning electron microscopy revealed a more complex topography on the surface of older leaves,
as well as on the abaxial side of the examined leaf tissue supporting the notion that a higher attachment level might be
correlated with a more composite leaf landscape.
Kroupitski, Y., R. Pinto, M.T. Brandl, E. Belausov, and S. Sela. 2009. Interactions of Salmonella enterica with lettuce
leaves. J. Appl. Microbiol. 106:1876-1885. G
Lettuce pieces were submerged in the Salmonella suspension (7 log CFU/ml) for 2 h at 25°C. The lettuce pieces were then
rinsed twice by gentle immersion for 1 min in 25 ml sterile water to remove unattached bacteria. Strong biofilm producers
were found to attach better to intact Romaine lettuce leaf tissue compared to weak producers. Association of the pathogen
with lettuce leaves facilitates its persistence during storage and enhances its acid tolerance.
Lapidot, A., U. Romling, and S. Yaron. 2006. Biofilm formation and the survival of Salmonella Typhimurium on
parsley. Int. J. Food Microbiol. 109:229-233. G
Parsley was immersed in a suspension of 7 log CFU/ml. Plants were removed and air-dried in a laminar flow hood for 2 h.
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After this, the parsley samples were immersed in 100 ml deionized sterile water or chlorinated water containing sodium
hypochlorite at concentrations of 100, 200, 800 and 1600 mg/L in stomacher bags.
The wild type and the mutants (that either constitutively produce the biofilm matrix components or absent the ability to form
biofilm) bound the parsley in an average of ca. 7 log CFU/g parsley, and rinsing with water removed about 1 log of the
attached cells. Results indicated that the ability to produce cellulose or curli does not provide an advantage during the initial
step of adhesion, and that the wild type as well as a biofilm-deficient mutant were strongly attached to parsley. The highest
chlorine concentration reduced Salmonella levels (including tightly and loosely bound cells) by 3 logs.
After sitting on the tissue for a week, attached cells that were able to produce the biofilm matrix were significantly more
resistant to the disinfection treatments. Storage of the mutant at 25°C improved the adhesion of the cells to the plant (higher
recovery after rinsing with water), but did not afford significant protection to chlorination.
Lopez-Velasco, G., H.A. Tydings, R.R. Boyer, J.O. Falkinham III, and M.A. Ponder. 2012. Characterization of
interactions between Escherichia coli O157:H7 with epiphytic bacteria in vitro and on spinach leaf surfaces. Int. J.
Food Microbiol. 153:351-357.
A total of 1512 phylloepiphytic bacterial isolates were screened for their ability to inhibit or to enhance the growth of E. coli
O157:H7 in vitro and on spinach leaves. Reduced numbers of E. coli O157:H7 were recovered from detached spinach leaves
that were co-inoculated with epiphytic isolates belonging to five genera, whereas bacteria belonging to eight different genera
increased numbers of E. coli O157:H7. Inhibitory effects were ascribed to nutrient competition or acid production.
Macarisin, D., J. Patel, G. Bauchon, J.A. Giron, and V.K. Sharma. 2012. Role of curli and cellulose expression in
adherence of Escherichia coli O157:H7 to spinach leaves. Foodborne Path. Dis. 9:160-167. G
Curli-expressing E. coli O157:H7 strains developed stronger association with leaf surface than curli-deficient mutants. The
relative attachment strength of E. coli O157:H7 to spinach increased with incubation time for the curli-expressing strains.
Production of cellulose was not required to develop strong attachment to spinach leaf.
Maloney, P.E., A.H.C. van Bruggen, and S. Hu. 1997. Bacterial community structure in relation to the carbon
environments in lettuce and tomato rhizospheres and in bulk soil. Microbial Ecol. 34:109-117. G
Copiotrophic organisms grow in carbon rich environments. Survival of oligotrophic organisms is dependent on their ability
to multiply and maintain activity in habitats of low C flux. Consistent patterns in the ratio of copiotrophic to oligotrophic
(C:O) bacteria along the roots of lettuce and tomato were obtained and clearly showed that the C:O ratio was different for
these two crop species. With lettuce, C:O ratios were high at the root tip (1.22 to 1.61) and upper mid-root locations (0.90 to
1.30), intermediate at the lower mid-root locations (0.73 to 0.95) and low at the root base (0.56 to 0.76). With tomato, C:O
ratios were low at root tip locations (0.50 to 0.68) and high at mid and base locations along the root (1.20 to 1.28). These
differences may reflect qualitative and quantitative differences in root exudates between these crop species. Nitrogen
concentrations and lateral branch sites, providing C sources, were important factors influencing bacterial populations in the
rhizosphere of lettuce and tomato.
Melloul, A.A., L. Hassani, and L. Rafouk. 2001. Salmonella contamination of vegetables irrigated with untreated
wastewater. World J. Microbiol. Biotechnol. 17:207-209. F
The crops whose edible product develops on the ground surface, such as lettuce and parsley, were more contaminated than
those which grown, above the soil surface, like tomatoes and pimento. Except on lettuce, Salmonella on crops did not persist
beyond 3 days after irrigation.
Minhas, P.S., N. Sharma, R.K. Yadav, and P.K. Joshi. 2006. Prevalence and control of pathogenic contamination in
some sewage irrigated vegetable, forage and cereal grain crops. Bioresource Technol. 97:1174-1178. F
Different crops were exposed for a long-term to sewage irrigation. The coliform counts in vegetables were within
permissible limis by two washings with water, exposing these to sunlight for about 4 h, and removing the two outermost
leaves of cabbage. Also, cutting above some height from ground level in sorghum reduced the load in fodder crops.
Moyne, A.-L., M.R. Sudarshana, T. Blessington, S.T. Koike, and M.D. Cahn. 2011. Fate of Escherichia coli O157:H7
in field-inoculated lettuce. Food Microbiol. 28:1417-1425. F
Attenuated E. coli O157:H7 was inoculated onto the soil beds after seeding with a backpack sprayer or onto 2- or 4-week-old
lettuce plant foliage with a spray bottle at a level of 7 log CFU/ml. When E. coli O157:H7 was inoculated onto 2-week-old
plants, the organism was recovered by enrichment in 1 of 120 or 0 of 240 plants at 21 or 28 days post-inoculation,
respectively. For the four trials where inoculum was applied to 4-week-old plants, the population size of E. coli O157:H7
declined rapidly and by day 7, counts were near or below the limit of detection (10 cells per plant) for 82% or more of the
samples. However, in 3 out 4 field trials E. coli O157:H7 was still detected in lettuce plants by enrichment 4-weeks
postinoculation. Neither drip nor overhead sprinkler irrigation consistently influenced the survival of E. coli O157:H7 on
lettuce.
Natvig, E.E., S.C. Ingham, B.H. Ingham, L.R. Cooperband, and T.R. Roper. 2002. Salmonella enterica serovar

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Typhimurium and Escherichia coli contamination of root and leaf vegetables grown in soils with incorporated bovine
manure. Appl. Environ. Microbiol. 68:2737-2744. G
Salmonella (3 strains) inoculated into manure-fertilized soil (silty clay loam and loamy sand) to give 4 to 5 log CFU/g.
(Contaminated manure was approx. 7 log CFU/g). Simulated environmental conditions for Wisconsin growing season of
March to August and June to Sept. Vegetables were radishes, arugula, and carrots. If contaminated manure was applied in
March, vegetables harvested in August were not contaminated; however, the pathogen could still be detected in the soils. If
contaminated manure was applied in June, radishes and arugulo were contaminated at harvest and were more likely to be
contaminated when harvested from silty clay loam soils than from loamy sand soils. Vegetables had been planted
approximately 9 and 11 weeks after manure applications in March and June, respectively.
Salmonella declined in both soils from initial levels of ca. 4.8 to 1.7 log CFU/g between March application and planting of
vegetables 9 weeks later. It was detected in both soils by enrichment when vegetables were harvested 21 weeks later.
Repeated freeze-thaw cycles were detrimental to the survival of Salmonella and E. coli in manure-fertilized soil.
Patel, J. and M. Sharma. 2010. Differences in attachment of Salmonella enterica serovars to cabbage and lettuce
leaves. Int. J. Food Microbiol. 139:41-47. G
Discs or strips or lettuce or cabbage (upper and lower epidermis removed) were aseptically submerged into Salmonella
suspensions and incubated at 10°C for 24 h. At specific time intervals (0, 1, 4, and 24 h), pieces were removed and rinsed.
For loosely attached cells, discs or strips were vortexed in presence of PBS and 0.1% Tween 20. For strongly attached cells,
the vortexed pieces were exposed to buffered peptone water and sonicated for 30 s. Attachment strength represents the
percentage of the total population of bacteria associated with produce surface which were strongly attached to produce
surface.
Salmonella Tennessee and S. Thompson produced stronger biofilms on microtiter plates compared to S. Newport, S. Negev,
and S. Braenderup. Biofilm formation was also stronger when Salmonella was grown in diluted TSB (1:10). All Salmonella
serovars attached rapidly on intact and cut produce surfaces. Salmonella spp. attached to Romaine lettuce at significantly
higher numbers than those attached to Iceberg lettuce or cabbage. The overall attachment strength of Salmonella was
significantly lower on cabbage (0.12) followed by Iceberg (0.23) and Romaine lettuce (0.34). Cabbage, intact or cut, did not
support attachment of Salmonella as well as Romaine lettuce.
Patel, J., M. Sharma, and S. Ravishakar. 2011. Effect of curli expression and hydrophobicity of Escherichia coli
O157:H7 on attachment to fresh produce surfaces. J. Appl. Microbiol. 110:737-745. G
Tissue coupons were suspended in 6 log CFU/ml for periods up to 24 h at 10°C. Loosely attached cells were defined by
removal with PBS and 0.1% Tween -20 using vortexing. Vortexed coupons were transferred to buffered peptone water and
sonicated to recover populations of strongly attached O157.
Hydrophobicity was determined using bacterial adherence to hydrocarbons. The Congo red binding assay was used to
determine curli expression. (Curli are very thin, coiled, extracellular structures on cell surface of most E. coli, and their
production can be influenced by low temperature. Most pathogenic E. coli strains do not produce curli when grown at 37°C.)
Biofilm formation was stronger when E. coli O157:H7 were grown in diluted tryptic soy broth (1:10). Salt may interfere with
multi-cellular behavior of E. coli in expression of adhesive extracellular matrix components which may have resulted in
weak biofilm formation when O157 grown in full-strength media.
In general, strong curli-expressing E. coli O157:H7 strains 4406 and 4407 were more hydrophobic and attached to cabbage
and iceberg lettuce surfaces at significantly higher numbers than other weak curli expressing strains. Overall, E. coli
O157:H7 populations attached to cabbage and lettuce (iceberg and Romaine) surfaces were similar (P > 0.05), indicating
produce surfaces did not affect (P < 0.05) bacterial attachment. The difference between O157 populations attached to intact
and cut surfaces was not significant in most cases.
Sadovski, A.Y., B. Fattal, D. Goldberg, E. Katzenelson, and H.I. Shuval. 1978. High levels of microbial contamination
of vegetables irrigated with wastewater by the drip method. Appl. Environ. Microbiol. 36:824-830. F
Cucumbers were grown in field plots and irrigated with one of three drip irrigation systems: 1) the drip lines were exposed
on the soil surface; 2) the soil and drip lines were covered with transparent or dark polyethylene sheets; and 3) drip lines
were buried at a depth of 10 cm in the soil. The densities of microorganisms in the inoculated effluent entering the irrigation
system were 1.0 x 107 CFU of E. coli and 9.0 x 106 PFU of enteroviruses/100 ml. Cucumbers were contaminated with E. coli
on the day of the irrigation event at levels of 4 log CFU/100 g but declined to 65 cfu/100 g on day 8. Enterovirus
contamination on cucumbers was at 3 log PFU/100 g on the initial day of irrigation and declined to 30 PFU/100 g on day 8.
Only a few positive cucumber samples were collected shortly after the irrigation event when soil had been covered with
plastic sheets. Burying the drip lines in the soil beneath the polyethylene cover had no noticeable effect on contamination.
Saldaña, Z., E. Sánchez, J. Xicohtencatl-Cortes, J.L. Puente, and J.A. Girón. 2011. Surface structures involved in
plant stomata and leaf colonization by Shiga-toxigenic Escherichia coli O157:H7. Frontiers Microbiol. 2. Article 119.
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STEC O157:H7 colonizes baby spinach leaves through the coordinated production of curli, the E. coli common pilus,
hemorrhagic coli type 4 pilus, flagella, and T3SS. Electron microscopy analysis of tainted leaves revealed STEC bacteria in
the internal cavity of the stomata, in intercellular spaces, and within vascular tissue (xylem and phloem), where the bacteria
were protected from the bactericidal effect of gentamicin, sodium hypochlorite or ozonated water treatments. We confirmed
that the T3S escN mutant showed a reduced number of bacteria within the stomata suggesting that T3S is required for the
successful colonization of leaves. In agreement, non-pathogenic E. coli K-12 strain DH5α transformed with a plasmid
carrying the locus of enterocyte effacement (LEE) pathogenicity island, harboring the T3SS and effector genes, internalized
into stomata more efficiently than without the LEE.
Shaw, R.K., C.N. Berger, B. Feys, S. Knutton, M.J. Pallen, and G. Frankel. 2008. Enterohemorrhagic Escherichia coli
exploits EspA filaments for attachment to salad leaves. Appl. Environ. Microbiol. 74:2908-2914. G
Freshly excised leaves with a width of 10 mm were immersed in 3 ml of bacterial cultures at 20 or 37°C for 1 h. In order to
quantify adhesion levels, counted adherent bacteria on five independent immunostained leaf sections. Revealed an adhesion
level at 37°C of 2 x 105 bacteria/cm2. Reduced adhesion when leaves incubated at 20°C. No adherent bacteria were
observed following inoculation of leaves with a primed Type III secretion system (T3SS)-defective mutant. These results
suggest that adhesion of EHEC O157 to the phyllosphere is T3SS dependent .
Shaw, R.K., C.N. Berger, M.J. Pallen, Å. Sjöling, and G. Frankel. 2011. Flagella mediate attachment of
enterotoxigenic Escherichia coli to fresh salad leaves. Environ. Microbiol. Rept. 3:112-117. G
Inoculated pieces of cut E. vesicaria leaves with clinical ETEC isolates grown in Luria broth at 20°C, conditions that are not
permissive for expression of the plasmid-encoded colonization factors and hence mimic the actual transmission pathways of
ETEC through intake of contaminated food.
Examination of representative strains by scanning electron microscopy revealed that they adhere to the E. vesicaria (rocket
lettuce) as well as basil and spinach leaf surface in a diffuse pattern by extended filaments resembling flagella. Binding was
dependent on flagella as a fliC mutant attached to leaves at a much lower efficiency. EtpA, which forms a flagellar tip
structure, and colonization factor I are dispensable for leaf attachment.
Of the 23 clinical strains examined, could be divided into high, medium and low adherence.
Shuval, H., Y. Lampert, and B. Fattal. 1997. Development of a risk assessment approach for evaluating wastewater
reuse standards for agriculture. Wat. Sci. Technol. 35(11-12):15-20. G
It was determined that 10.8 ml of irrigation water is expected to attach to each 100 g of long-leaf lettuce during irrigation. It
was assumed then that any microorganism contained in the residual wastewater remaining on the irrigated crop would cling
to the leaf even after the wastewater itself evaporated.
Solomon, E.B., C.J. Potenski, and K.R. Matthews. 2002. Effect of irrigation method on transmission to and
persistence of Escherichia coli O157:H7 on lettuce. J. Food Prot. 65:673-676. G
Ten plants each exposed to either 200 ml (surface irrigation) or 100 ml (spray irrigation) of a 7 log CFU/ml inoculum of E.
coli O157:H7 on 20, 30 and 40 days, and then analyzed at 40 days. The number of plants testing positive following a single
exposure to E. coli O157:H7 through spray irrigation (29 of 32 plants) was larger than the number testing positive following
surface irrigation (6 of 32 plants). E. coli O157:H7 persisted on 9 of 11 plants for 20 days following spray irrigation with
contaminated water. Immersion of harvested lettuce heads for 1 min in a 200 ppm chlorine solution did not eliminate all E.
coli O157:H7 cells.
Solomon, E.B., H-J. Pang, and K.R. Matthews. 2003. Persistence of Escherichia coli O157:H7 on lettuce plants
following spray irrigation with contaminated water. J. Food Prot. 66:2198-2202. G
30-day old plants sprayed in laboratory with ca. 100 ml of contaminated water with a handheld sprayer. Plants placed under
two 400-W halide growth lamps that supplied light for 14/day. Temperature maintained at 23 to 26°C. Leaves stomached.
Only plants exposed to 102 CFU/ml on day 1 did not harbor the pathogen at the end of the sampling period (30 days). All
other treatments resulted in contaminated plants at harvest. Plants irrigated with 10 4 CFU/ml contained up to 5 log CFU/g at
harvest (60 days). Repeated exposure increases the pathogen level on the plant.
Song, I., S.W. Stine, C.Y. Choi, and C.P. Gerba. 2006. Comparison of crop contamination by microorganisms during
subsurface drip and furrow irrigation. J. Environ. Engr. 132:1243-1248. F
Approx. 2.58 x 1013 PFU and 5.93 x 108 cfu of PRD-1, E. coli , and C. perfringens, respectively were applied to each
irrigation plot. Subsurface soil samples were taken at 10 cm depth. Outer 2 to 3 leaves of lettuce heads were removed to
reflect standard industry harvesting techniques.
Significantly greater PRD-1 contamination of lettuce was observed in furrow-irrigated plots than in subsurface drip-irrigated
plots, while the difference in E. coli contamination was not significant. The relatively smaller size of PRD-1 as compared to
E. coli seemed to facilitate its transport through the soil matrix via irrigation water movement, thus resulting in greater crop
contamination. Direct contact of lettuce leaves with irrigation water was observed in furrow-irrigated plots.

                                                              Page 6
Coliphage PRD-1 showed greater persistence than E. coli in soil, while C. perfringens experiences little inactivation during
the experiment periods.
Stine, S.W., I. Song, C.Y. Choi, and C.P. Gerba. 2011. Application of pesticide sprays to fresh produce: A risk
assessment for hepatitis A and Salmonella. Food Environ. Virol. 3:86-91. F
Transfer of pathogens to iceberg lettuce, cantaloupe, and bell peppers from water used to dilute pesticide spray during spray
application averaged 2.1, 0.2, and 0.4% for E. coli (surrogate for Salmonella), respectively and 8.1 x 10-3, 2.1 x 10-4, and
0.5% for PRD1 coliphage (model for hepatitis A), respectively. Based on the transfer rates and reported concentrations of
enteric viruses in surface and ground waters in the U.S., a 1:10,000 annual risk of infection could easily be exceeded with
some groundwater sources. To produce the same risk of infection but harvesting 14 days after the pesticide application
would require that Salmonella populations in water be greater by up to 5 orders of magnitude and HAV numbers be
increased by up to two orders of magnitude.
Stine, S.W., I. Song, C.Y. Choi, and C.P. Gerba. 2005. Application of microbial risk assessment to the development of
standards for enteric pathogens in water used to irrigate fresh produce. J. Food Prot. 68:913-918. F
Irrigation method and type of produce influenced the transfer of organisms from water to produce surfaces via irrigation.
Contamination of lettuce by PRD1 did not occur in subsurface drip irrigation plots, whereas E. coli contamination was two to
three orders of magnitude lower in subsurface drip plots than those that were furrow irrigated. The degree of cantaloupe
contamination was one to two orders of magnitude higher for furrow irrigation compared with subsurface drip irrigation.
Contamination of bell peppers contaminated by irrigation water was below the limit of detection. The most notable
difference between viral and bacterial contamination was observed on lettuce irrigated with furrow irrigation. The
contamination of fresh produce by contact with irrigation water is dependent on the physical properties of the edible portion
of the plant, such as surface texture. As with bell peppers, location of the edible portion of the plant in reference to the
irrigation water also plays a significant role in microbial contamination.
Talley, J.L., A.C. Wayadande, L.P. Wasala, A.C. Gerry, J. Fletcher, U. DeSilva, and S.E. Gilliland. 2009. Association
of Escherichia coli O157:H7 with filth flies captured in leafy greens fields and experimental transmission of E. coli
O157:H7 to spinach leaves by house flies. J. Food Prot. 72:1547-1552. G
Under laboratory experimental conditions, flies were capable of contaminating leafy greens in that GFP-tagged bacteria were
detected on surfaces of 50 to 100% of leaves examined by fluorescence microscopy and in 100% of samples tested by PCR.
Flies allowed access to lawns of GFP-tagged E. coli on LB plates left large deposites of bacteria on the spinach.
Aggregations of fluorescent bacteria were fewer and less concentrated on spinach exposed to the GFP-manure (5 ml 106
CFU/ml added to 50 g manure) mixture compared with those exposed to GFP-tagged E. coli O157:H7 plants. The number of
bacteria deposited by flies exposed to plates containing E. coli was artificially high. A more natural situation is illustrated by
the scattered bacterial colonies and aggregations observed on plants exposed to flies that acquired bacteria from manure.
Agromyzid flies, anthomyiid flies, and leafhoppers were caught consistently in both rangeland and leafy greens production
fields at all sites. An unexpected number of flies (n = 34) in the Muscidae and Calliphoridae families (known as filth flies)
were caught in one leafy greens field. A subset of these filth flies were positive (11 of 18 flies) for E. coli O157:H7 by PCR
amplification (eae primer). Hypothesized that the filth flies collected from the leafy greens fields originated from nearby
rangelands that contained fresh cattle manure. The site had an active composting operation near the site of collection.
Several factors could be involved in the presence of filth flies in leafy greens fields. The flies could have been attracted by
“honeydew” secreted by aphids infesting the heads of lettuce.
Tierney, J.T., R. Sullivan, and E.P. Larkin. 1977. Persistence of poliovirus 1 in soil and on vegetables grown in soil
previously flooded with inoculated sewage sludge or effluent. Appl. Environ. Microbiol. 33:109-113. F
Lettuce and radishes were planted in sludge- or effluent-flooded soil either 1 day prior to the flooding event or 3 days after
the plots were flooded. Poliovirus survived on lettuce for 23 days after flooding with wastewater occurred on the outdoor
plots.
Vega, E., J. Garland, S.D. Pillai, and D.J. Suresh. 2008. Electrostatic forces control nonspecific virus attachment to
lettuce. J. Food Prot. 71:522-529. G
Three different conditions, namely 1) 1% Tween 80, (2) 1 M NaCl, and 3) 1% Tween 80 with 1 M NaCl were investigated to
determine the role of hydrophobic, electrostatic, and combined hydrophobic and electrostatic forces, respectively. Overall, 1
M NaCl was the most effective treatment in desorbing viruses (feline calicivirus and echovirus 11) from the surface of lettuce
at pH 7 and 8 implying that electrostatic forces play a major role. The bacteriophage phi X174 was not significantly
affected by any treatment.
Vega, E., J. Smith, J. Garland, A. Matos, and S.D. Pillai. 2005. Variability of virus attachment patterns to butterhead
lettuce. J. Food Prot. 68:2112-2117. G
Echovirus had the highest affinity to lettuce surface. Echovirus 11 appeared to exhibit reversible attachment above its pI
whereas below its pI strong adsorption was observed. Adsorption of FCV was at its maximum above its pI. Bacteriophage
                                                              Page 7
X174 attachment occurred only at the pH extremes (pH 3.0 and 8.0).
Wachtel, M.R., L.C. Whitehand, and R.E. Mandrell. 2002. Association of Escherichia coli O157:H7 with preharvest
leaf lettuce upon exposure to contaminated irrigation water. J. Food Prot. 65:18-25. G
In hydroponic system, seedlings exposed to 6 log CFU/ml and incubated overnight with gentle agitation. Seedlings for
visualization by microscopy were inoculated at a concentration of 8 log CFU/ml.
In soil systems, used seeds as subject. Soil systems were watered with bacterial suspensions (2, 4, 6, and 8 log CFU/ml).
Seeds then sown in soil. The 8 log CFU/ml dose appeared to saturate bacterial binding sites with the highest bacterial
numbers reaching 2 x 104 CFU in roots. The aggregate seen on roots suggests that the bacteria bound as an aggregate, or that
the bacteria grew after initial binding, potentially around a favorable nutrient source.
Bacteria adhered preferentially to plant roots in both model systems and to seed coats in the hydroponic system. Two of five
nonpathogenic E. coli strains showed decreased adherence to seedling roots in the hydroponic system. Adherence of the
nonpathogenic strains to roots was ~15% that of the pathogenic strains.
Investigated whether EHEC adherence to seeds and seedlings was dependent on prior colonization by natural epiphytes;
however, found that lettuce seeds and seedlings typically contained very few bacteria (on average <10 CFU/seed or seedling.
Adherence levels to seed coats sterilized with 0.2% calcium hypochlorite were similar to those on unsterilized seeds for all
strains tested. EHEC attached within the deep grooves and tips of seed coats. Emergence of the radicle during germination
split the seed coat, allowing association of EHEC with the broken edges. In addition, the mature zone of seedling roots was
demarcated by extensive association of EHEC along the root hairs. Though EHEC adherence was greatest to the roots,
significant bacterial numbers were found associated with the edible portion of the plant, both singly and in aggregates.
Wachtel, M.R., L.C. Whitehand, and R.E. Mandrell. 2002. Prevalence of Escherichia coli associated with a cabbage
crop inadvertently irrigated with partially treated sewage wastewater. J. Food Prot. 65:471-475. F
Cabbage samples collected 7 days after the plants came into contact with partially treated sewage wastewater. E. coli was
isolated from the roots of these plants but not from the edible portion of the cabbage. The E. coli field strains displayed
variable patterns of adherence to lettuce seedlings.
In hydroponic attachment studies, seedling roots exposed to 6 log CFU/ml and incubated overnight. Strains evaluated
included one strain representing each of the 6 nonpathogenic E. coli serotypes isolated from contaminated water, an E. coli
O157:H7 lettuce outbreak strain F6460 and a nonpathogenic E. coli strain KSU1, isolated from a cattle feedlot.
The nonpathogenic E. coli strains displayed significantly higher levels of adherence to lettuce seedlings than did the O157
strain.
Ward, B.K. and L.G. Irving. 1987. Virus survival on vegetables spray-irrigated with wastewater. Wat. Res. 21:57-63.
F
Spray irrigated celery, spinach, lettuce, and tomato crops with 3-5 log infectious units/L poliovirus or adenovirus seeded
wastewater. Poliovirus was rapidly inactivated within 48 h in the field environment, with low levels still detected on spinach
after 13 days, on celery and tomato for 4 and 6 days, respectively, but undetectable levels on lettuce after 48 h. Adenovirus
was inactivated quickly and became undetectable as early as 24 h on lettuce after irrigation.
Warriner, K., F. Ibrahim, M. Dickinson, C. Wright, and W.M. Waites. 2005. Seed decontamination as an intervention
step for eliminating Escherichia coli on salad vegetables and herbs. J. Sci. Food Agric. 85:2307-2313. G
Seeds (celery, coriander, lettuce, spinach, and watercress) were inoculated at a level of 3-5 log CFU/g with either E. coli or L.
monocytogenes and decontaminated with ozone gas, acidified sodium chlorite or quaternary ammonium salt preparation.
None of the treatments applied were initially effective at inactivating E. coli on lettuce or spinach seeds as the bacterium
could be recovered on subsequent seedlings. E. coli persisted on the surface of lettuce and spinach leaves from inoculated
non-treated seeds throughout a 42-day cultivation period.
No L. monocytogenes was recovered from any of the seedlings and was conjectured to be due to the growth-suppressing
effect of endogenous microflora.
Wießner, B. Thiel, J. Krämer, and U. Köpke. 2009. Hygienic quality of head lettuce: Effects of organic and mineral
fertilizers. Food Control 20:881-886. F
Different fertilizer treatments used were fresh farmyard manure, composted farmyard manure, stinging nettle extract and
calcium ammonium nitrate. No Salmonella was detected in any soil, fertilizer, or lettuce sample, while E. coli was randomly
detected in low concentrations independent from fertilizer treatment.
Xicohtencatl-Cortes, J., E.S. Chacon, Z. Saldana, E. Freer, and J.A. Giron. 2009. Interaction of Escherichica coli
O157:H7 with leafy green produce. J. Food Prot. 72:1531-1537. G
Adherence assays carried out between 0 and 24 h at 4 to 42°C in the presence of 7 log bacteria. No differences in the results
were found between the three methods of infection: intact leaf in petri dishes, 1-cm leaf section in 24-well plates, or spot
infection in humid chamber. Significant increase in bacterial counts between 6 and 9 h of incubation.
                                                             Page 8
In general, higher bacterial counts were obtained from lettuce than from spinach leaves. We compared bacterial counts from
spinach and lettuce leaves in DMEM versus those in filter-sterilized tap water. The bacteria were able to colonize these leafy
greens in medium, although as expected higher bacterial counts were obtained when they were performed with tap water.
Findings indicated that the bacteria would be able to replicate and survive on the leaves in the presence of municipal water.
Independently of the production of Shiga toxin, EHEC O157:H7 colonizes the leaf surface via flagella and the type 3
secretion system. The latter is a specific plant colonization factor not present in nonpathogenic E. coli strains. Mutation of
the fliC flagellin gene in EHEC rendered the bacteria significantly less adherent suggesting the involvement of flagella in the
bacteria-leaf interaction. Mutation in the escN (ATPase) gene associated with the function of the TS33 but not in the eae
(intimin adhesion) gene required for adherence to host intestinal cells had significantly reduced adherence compared with
that of the parental strain.
Yamazaki, A., J. Li, W.C. Hutchins, L. Wang, J. Ma, A.M. Ibekwe, and C.-H. Yang. 2011. Commensal effect of
pectate lyases secreted from Dickeya dadantii on proliferation of Escherichia coli O157:H7 EDL933 on lettuce leaves.
Appl. Environ. Microbiol. 77:156-162.    G
A type II secretion system (T2SS)-deficient mutant of D. dadantii 3937, A1919 (outC), lost the capability to promote the
multiplication of Escherichia coli O157:H7 EDL933, whereas Ech159 (rpoS), a stress-responsive-factor RpoS-deficient
mutant, increased EDL933 proliferation on lettuce leaves. A spectrophotometric enzyme activity assay revealed that A1919
(outC) was completely deficient in the secretion of pectate lyases (Pels), which play a major role in plant tissue maceration.
These results suggest that the pectinolytic activity of D. dadantii 3937 is the dominant determinant of enhanced EDL933
proliferation on the lettuce leaves.




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