Download Salmonella must be viable in order to attach to the

Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Salmonella must be viable in order to attach to the surface
of prepared vegetable tissues
E.J. Saggers, C.R. Waspe, M.L. Parker, K.W. Waldron and T.F. Brocklehurst
Institute of Food Research, Norwich Research Park, Norwich, UK
Keywords
ecology, food, food processing, horticulture,
microbial-cell interaction.
Correspondence
Elizabeth J. Saggers, Institute of Food
Research, Norwich Research Park, Colney,
Norwich, NR4 7UA, UK.
E-mail: [email protected]
2007 ⁄ 1425: received 3 September 2007,
revised 4 January 2008 and accepted 24
January 2008
doi:10.1111/j.1365-2672.2008.03795.x
Abstract
Aims: The aims of the current study were to explore the site of bacterial
attachment to vegetable tissues and to investigate the hypothesis that Salmonella must be living in order to attach to this site(s).
Methods and Results: Scanning electron micrographs of intact potato cells
showed that Salm. serotype Typhimurium attached to cell-wall junctions; suggesting a high-level of site selectivity. Inactivation of Salm. Typhimurium using
heat, ethanol, formalin or Kanamycin resulted in cells that could be no longer
attached to these sites. Attachment of a Gfp+ strain of Salm. Typhimurium to
cell-wall material (CWM) was examined via flow cytometric analysis. Only live
Salm. Typhimurium attached to the CWM.
Conclusions: Salmonella serotype Typhimurium must be metabolically active
to ensure attachment to vegetable tissues. Attachment preferentially occurs at
the plant cell-wall junction and the cell-wall components found here, including
pectate, may provide a receptor site for bacterial attachment.
Significance and Impact of the Study: Further studies into individual plant cellwall components may yield the specific bacterial receptor site in vegetable tissues.
This information could in turn lead to the development of more targeted and
effective decontamination protocols that block this site of attachment.
Introduction
Minimally processed fruits and vegetables have recently
undergone an increase in consumer demand because of
their healthy image and convenience. The product range
includes ready-to-eat prepared vegetable tissues, such as
carrot sticks and shredded lettuce and prepared fruits.
Microbiological safety is a key issue for the entire product
range, as they are intended for consumption raw, without
further preparation or cooking. Contamination of these
products is predominantly by Gram-negative bacteria, in
particular, members of the Pseudomonadaceae and
Enterobacteriaceae (Brocklehurst et al. 1987; Brocklehurst
1994). In addition, it is well established that some products can also contain potential pathogens (Nguyen-the
and Carlin 1994; Beuchat 1996; Francis et al. 1999), and
some have been implicated in an increasing number of
outbreaks of food-borne illness (Long et al. 2002).
Between 1st January 1992 and 31st December 2000, 5Æ6%
of outbreaks of food-borne illness in England and Wales
were attributed to salad vegetables and fruit, with Salmonella being the most frequently implicated bacterial pathogen (Long et al. 2002). Lettuce, in particular, has been
connected to outbreaks of Salmonella serotype Typhimurium (Horby et al. 2003), Escherichia coli (Ackers et al.
1998; Hilborn et al. 1999) and Shigella sonnei (Kapperud
et al. 1995).
We regard bacterial colonization of prepared fruit and
vegetable tissues to be the result of three phases: an initial
attachment phase, a consolidation phase, which may
involve the production of extracellular polymer, and subsequent growth to form microcolonies. Most studies of
colonized vegetable tissue surfaces concentrate on the
growth of bacteria on the product rather than on the
initial attachment and consolidation phase. However,
commercial decontamination processes are usually applied
to prepared vegetable tissues within a few minutes of dicing, chopping or shredding, i.e. during the initial attachment and colonization phases. It is recognized that the
use of biocides as decontaminants is not always successful
ª 2008 The Authors
Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1239–1245
1239
Attachment of Salmonella to vegetable tissues
(Adams et al. 1989; Garg et al. 1990; Brocklehurst 1994;
Zhang and Farber 1996). Accordingly this work is part of
a wider ongoing study to understand the mechanisms
used by bacteria for attachment to tissues. In particular,
this paper focuses on the competencies of the organism
in the attachment process with a view to exploiting this
knowledge to improve postharvest technologies and yield
more effective decontamination protocols.
Scanning electron microscopy (SEM) of prepared leaf
tissues has shown that bacteria can be found on the
abaxial and adaxial surfaces (Carmichael et al. 1999).
However, the natural flora of processed lettuce was found
to be concentrated in the intercellular junctions of the leaf
(Carmichael et al. 1999). Further colonization studies
showed that E. coli preferentially attached to the cut
surface of lettuce whereas Pseudomonas fluorescens preferentially attached to the intact surface (Takeuchi et al.
2000). Salmonella serotype Typhimurium attached to both
cut and intact surfaces (Takeuchi et al. 2000). The cut surface, along with other leaf structures such as stomata, may
allow bacteria to become internalized within tissues and,
as a result, become protected from chlorine disinfection
(Seo and Frank 1999; Takeuchi et al. 2000; Takeuchi and
Frank 2001). The above studies indicate the preferential
areas of attachment, but do not investigate the mechanisms of the attachment process. Recent studies by this
laboratory have examined the attachment of the spoilage
bacteria Ps. fluorescens and Pantoea agglomerans (Garrood
et al. 2004) and the pathogenic bacteria Salm. Typhimurium (E.J. Saggers et al., unpublished) to vegetable tissue.
Further studies attempted to prevent attachment of Salmonella to vegetable tissues by masking potential attachment
sites using dead Salmonella. However, attachment of subsequently applied live Salmonella was unaffected. Following the apparent failure of dead cells to mask potential
attachment sites, we hypothesized that attachment may be
an active process requiring the bacteria to be viable.
In the study reported here, we explore the hypothesis
that Salmonella must be living in order to attach to vegetable tissues and present evidence in support of this
hypothesis. We also present evidence that implicates components of the plant cell wall as potential bacterial receptor sites important in the initial attachment of Salmonella
to vegetable tissues.
Materials and methods
Bacteria
Salmonella serotype Typhimurium strain LT-2 (NCIMB
10248) was obtained from the National Collection of
Industrial and Marine Bacteria, Aberdeen, UK. A Gfp+
strain of Salm. Typhimurium SL1344, (strain JH3016)
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E.J. Saggers et al.
was provided by Dr Isabelle Hautefort, Molecular Microbiology Group, Institute of Food Research, UK. This
strain contains a single copy rpsM::gfp+ fusion inserted
chromosomally (Hautefort et al. 2003). This fusion is
expressed in all conditions tested and was used to fluorescently label the Salmonella cells.
Culture media
Stock cultures of Salm. Typhimurium LT-2 were stored
on Heart Infusion agar (Oxoid) slopes at 1C. At monthly
intervals, it was subcultured to fresh Heart Infusion agar
slopes which were incubated at 25C for 24 h and subsequently stored at 1C.
A stock culture of Salm. Typhimurium SL1344 strain
JH3016 was stored at )80C in Tryptone Soya Broth
(Oxoid) plus glycerol (30% w ⁄ v).
Stock cultures were plated onto Trypticase Soy Agar
(Oxoid, Basingstoke, UK) plates which were incubated at
25C for 24 h and subsequently stored at 5C for no
more than 2 weeks.
Inocula were prepared in Trypticase Soy Broth
(Oxoid), and all viable counts were made on Plate Count
Agar (PCA; Oxoid CM325).
The potato variety used throughout the study was
Maris Piper obtained from a local supermarket.
Preparation of live and inactivated inocula
Bacteria were grown successively at 25C for 24 h, and
then at 20C for 24 h. The resultant population of
approx. 109 viable cells ml)1 was diluted in peptone salt
dilution fluid (PSDF) (I.C.M.S.F. 1978) to give a suspension that contained the desired number of viable bacteria.
To provide a heat-inactivated inoculum, the 20C
culture was diluted to 108 CFU ml)1 in preheated (50C)
PSDF and then heated at 50C for 14 min. This resulted
in inactivation of all cells within the inoculum (data not
shown).
To provide an ethanol-inactivated inoculum, the 20C
culture was filtered using a 0Æ22-lm-pore-sized membrane
filter (Millipore, Billerica, MA, USA) to remove cells from
suspension. Cells were then re-suspended in 10 ml 70%
(v ⁄ v) ethanol and incubated at 20C for 5 min, the time
taken (found experimentally) to kill the population (data
not shown). Cells were then filtered again through a 0Æ22lm filter and re-suspended in 10 ml PSDF to remove the
ethanol.
Kanamycin inactivation of the inoculum was achieved
by dilution of the 24 h culture to the desired concentration. Kanamycin (200 lg ml)1) was added and the
culture incubated for 30 min at 20C after which time all
cells were unable to form colonies on PCA (data not
ª 2008 The Authors
Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1239–1245
E.J. Saggers et al.
shown). The cells were then removed from the suspension
by centrifugation and re-suspended in 10 ml phosphatebuffered saline (PBS) to provide the Kanamycin-inactivated inoculum.
Formalin-inactivation of the inoculum was achieved by
fixation in 4% (v ⁄ v) formalin for 2 min. The cells were
removed from the suspension by centrifugation and
re-suspended in 10 ml PBS to provide the formalininactivated inoculum.
Preparation of inoculated potato tissue samples for
scanning electron microscopy
The surface of potatoes was sterilized by spraying with
70% (v ⁄ v) ethanol. A sterile knife was used to remove two
opposing surfaces of the potato revealing an area of sterile
inner potato tissue. A sterile cork borer (8 mm diameter)
was then used to produce cores of sterile potato tissue
4 cm long. The potato cores were blanched by immersion
in boiling water for 30 s. Blanching allowed the potato
cells to separate from each other when snapped but still
remain as intact cells (Parker et al. 2001). Blanching for
30 s resulted in a gradient of cell separation across the
potato tissue core with raw (un-separated) cells in the centre of the core and separated cells at the outer surface.
Post-blanching, cores were rinsed in 100 ml cold sterile
glass distilled water for 5 min to remove excess starch.
After rinsing, each core was aseptically snapped in half
and a 3-mm section aseptically cut from each snapped
end and placed, snapped face uppermost, in a beaker
containing the inoculum to be investigated.
The potato tissue was exposed to the inoculum for
10 min at 20C to allow attachment to the tissue. Tissue
samples were then rinsed twice to remove unattached
cells. Rinsing was by placing tissue samples in a beaker of
100 ml sterile PSDF which was stirred for 1 min at
150 rev min)1 using a magnetic stirrer.
After rinsing, the tissue samples were prepared for SEM
according to a previously described method (Parker and
Waldron 1995). In summary, the potato tissue was fixed
in 3% (w ⁄ v) glutaraldehyde in 0Æ05 mol l)1 cacodylate
buffer (pH 7Æ2) for 2 h, dehydrated in an ethanol series
and transferred to acetone. They were then dried by critical point method using liquid CO2 as the transition fluid
and mounted, snapped surface uppermost, onto aluminium stubs using silver conducting paint. All samples were
then sputter coated with a layer of gold and imaged in a
Leica Cambridge Stereoscan 360 SEM.
Preparation of potato cell-wall material
Cell-wall material (CWM) was prepared based on the
method of Parker and Waldron (1995). In summary, the
Attachment of Salmonella to vegetable tissues
surface of the potato was sterilized as described earlier
and the potato sliced transversely (6 mm thick). Parts
within the vascular ring were cut out from slices from the
middle third of the axis. The tissue was frozen in liquid
nitrogen and stored. Batches of potato tissue (500 g) were
blended for 3 min in 1Æ5% sodium dodecyl sulfate
(1000 ml) + 5 mmol l)1 Na2S2O5 + 5 ml octanol in a
Waring Blender (Christison Particle Technologies Ltd,
Gateshead, UK). The mixture was filtered on a 2-mm
mesh to remove unblended tissue and the filtrate was
homogenized with an Ystral homogenizer (Ystral GmbH,
Dottingen, Germany) at 16 000 rev min)1 for 1 min, then
filtered using a 200-lm mesh nylon cloth (BioDesign Inc.
of New York, Carmel, NY, USA). The material retained
was washed with water and then suspended in 0Æ5%
(w ⁄ v) sodium dodecyl sulfate (500 ml) + 5 mmol l)1
Na2S2O5 + 2Æ5 ml octanol and then ball-milled for 4 h at
60 rev min)1 at 4C in a 2Æ5-l pot (Capco Test Equipment Ltd, Wickham Market, Suffolk, UK). The liquor
was poured out and each ball was washed with water.
Most of the starch was removed at this stage by filtering
on a 100-lm mesh nylon cloth. The material was washed
with 10 l of water, re-suspended in water and homogenized (16 000 rev min)1 for 1 min), and then filtered and
washed again on a 100-lm mesh nylon cloth. Absence of
starch was assessed by staining with a solution of iodine
in potassium iodide. The CWM was re-suspended in
water and frozen at )20C.
Measurement of the attachment of Salmonella serotype
Typhimurium to potato CWM by flow cytometry
Flow cytometry was used to enumerate the number of
live or dead Salm. Typhimurium JH3016 attached to
potato CWM. Frozen stock CWM was defrosted and
washed with PBS through a 30-lm nylon mesh filter
using a Nalgene filter unit. All PBS used in this part of
the study was filtered through a 0Æ22-lm-pore-sized filter
(Millipore) to reduce the background noise during flow
cytometry analysis. The CWM was divided equally into
aliquots, and each was re-hydrated for 2 h in 10 ml PBS
at 20C rotating at 120 rev min)1 on an orbital shaker.
Hundred microlitres of viable, formalin- or Kanamycin-inactivated Salm. Typhimurium JH3016 were added
to an aliquot (2Æ5 g wet weight) of the re-hydrated CWM
and incubated at 20C, 120 rev min)1 for 30 min to allow
attachment. After this time, samples were washed using
100 ml PBS through a 30-lm filter to retain bacteria
attached to the CWM. The CWM was then collected into
a stomacher bag and stomached (Seward 80 Biomaster;
Fisher Scientific, Loughborough, UK) for 60 s to remove
the attached bacteria. The sample was washed through a
30-lm filter and the filtrate washed through a 5-lm filter
ª 2008 The Authors
Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1239–1245
1241
Attachment of Salmonella to vegetable tissues
to remove any residual CWM. The number of bacteria in
the filtrate, and therefore previously attached to the
CWM, was then enumerated by flow cytometry. For this,
the sample containing viable cells was fixed in 4% (v ⁄ v)
formalin for 2 min. After fixation of the ‘viable’ sample,
and in the case of all samples of inactivated cells, the filtrate was centrifuged at 15 000 g for 5 min and washed
twice by centrifugation with PBS. Samples were then
immediately analysed by flow cytometry. This used a
FACScalibur flow cytometer (Becton Dickinson, Franklin
Lakes, NJ, USA) equipped with a 15-mW air-cooled
argon ion laser as the excitation light source (488 nm). In
order to perform absolute counts, samples were mixed
with a known number of FluoSpheres carboxylate-modified crimson fluorescent microspheres (Molecular probes
F-8816; Invitrogen, Paisley, UK). The beads were detected
on a separate channel from the constitutively GFP+expressing Salmonella strain JH3016. All parameters were
collected by using amplification gains set on LOG mode.
Acquisition was stopped when 10 000 beads were
counted, allowing the volume of sample used to be determined. The number of GFP+-expressing Salmonella cells
detected in that same volume was subsequently calculated
after analysis with CellQuest 3Æ3 software (Becton Dickinson) and converted into number of Salmonella cells per
millilitre. Control samples of filtered PBS and uninoculated CWM were also analysed to establish background
noise within the samples.
To validate the absolute counts obtained from calculations of bead and GFP+ fluorescence, a viable count was
performed on each sample before fixation for flow
cytometry. Fifty microlitres of the sample were inocu-
(a)
(c)
1242
E.J. Saggers et al.
lated onto the surface of duplicate plates of PCA using
a Spiral Plate Maker (Don Whitley Scientific, Shipley,
UK). PCA plates were incubated at 30C for 24 h before
enumeration.
Results
Salmonella serotype Typhimurium must be viable in
order to attach to potato tissue and cell-wall material
The mechanism of Salmonella attachment to vegetable
tissue is unknown. Previous unpublished work by this
laboratory led to the hypothesis that cells must be viable
in order to attach to vegetable tissue. To explore this
hypothesis, viable and inactivated cells of Salm. Typhimurium were incubated with potato tissue to allow attachment to occur and the tissues were visualized using SEM.
Figure 1 shows that only viable cells of Salm. Typhimurium attached to the potato tissue. The method of inactivation appeared to be irrelevant; both heat-inactivated
(Fig. 1b,d) and ethanol-inactivated (data not shown)
organisms failed to attach. Figure 1(a) also identified the
plant cell wall as the preferred site of attachment for
Salm. Typhimurium. In particular, they appeared to
attach to the pectin layer at the cell-wall junction.
Cooking (via blanching) separates cells by partially breaking down this pectin layer resulting in a larger exposure
of pectin-rich material. The image of cooked tissue
(Fig. 1c) presented here shows less pectin present at the
cell-wall junction and also less Salmonella attachment. It
is, therefore, possible that pectin is one component of the
cell wall that is a bacterial attachment site.
(b)
(d)
Figure 1 Scanning electron micrographs of
Salmonella serotype Typhimurium LT-2
attached to raw and blanched (cooked)
potato tissue. (a) Live Salm. Typhimurium LT-2
incubated with raw potato tissue showing
attachment to cell-wall junction. (b) Heatinactivated Salm. Typhimurium LT-2 incubated
with raw potato tissue; no attachment
occurred. (c) Live Salm. Typhimurium LT-2
incubated with blanched potato tissue;
blanched potato tissue showing less pectin
present and less attachment. (d) Heat-inactivated Salm. Typhimurium LT-2 incubated with
blanched potato tissue; no attachment
occurred. Scale bar is 10 lm.
ª 2008 The Authors
Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1239–1245
E.J. Saggers et al.
Attachment of Salmonella to vegetable tissues
Plant cell-wall components are a site of bacterial
attachment to potato tissue
The SEM images of intact potato tissue indicated the
plant cell-wall junction was the preferred site of attachment for live Salm. Typhimurium (Fig. 1a,c). Purified
CWM contains only cell-wall components and no other
cellular material (Parker and Waldron 1995) and so can
be used to confirm that (i) a component of the cell wall
was the site of Salmonella attachment to potato tissue and
(ii) that this attachment required Salmonella to be viable.
Attachment of Salmonella to potato CWM was determined via flow cytometric analysis using a GFP+ strain of
Salm. Typhimurium, strain JH3016. Samples derived from
CWM incubated with live JH3016 fluoresced in the GFP+
range indicating organisms were present in this sample
and therefore had attached to the potato CWM (Fig. 2a).
The count of fluorescent particles was used to enumerate
the beads and GFP+-expressing Salmonella. The number
of viable cells derived from this count was 4Æ1 ·
106 CFU g)1. This count was validated by the number
derived from the conventional viable counts carried out
simultaneously (3Æ3 · 106 CFU g)1).
In contrast, in the samples derived from CWM incubated with formalin-inactivated JH3016, only small
counts of fluorescent particles were seen (Fig. 2b), equating to 2Æ4 · 104 CFU g)1. Parallel traditional viable
counts did not recover any viable Salmonella; therefore,
these counts may have been other particles that fluoresced
in the GFP+ range. The same was observed in samples
incubated with Kanamycin-inactivated JH3016 (data not
10 000
shown) and in the control samples; CWM only and PBS
(Fig. 2c,d). These results confirm that the plant cell wall
contains a receptor site for bacterial attachment to potato
tissue and that for this attachment to occur the bacteria
must be viable.
Discussion
Bacterial contamination of ready-to-eat vegetable tissues
is an emerging issue with regards to the safety of these
products. Many studies have concentrated on the growth
of bacteria in the products rather than on the initial
attachment phase and the subsequent consolidation event.
With decontamination protocols occurring within minutes of processing, the understanding of the initial phases
of attachment and consolidation are key to the development of new, more effective decontamination protocols.
Previously, Salmonella Typhimurium has been shown
to attach equally to both cut and intact surfaces of lettuce
tissue (Takeuchi et al. 2000). This study demonstrates
Salm. Typhimurium preferentially binds to material at the
cell-wall junctions of intact potato tissue. It was also
shown to attach to isolated CWM. These results indicate
that a plant cell-wall component is one potential bacterial
receptor site for Salmonella. Studies to identify the particular component of the plant cell wall are ongoing.
Although a potential site of attachment has been identified, the mechanism by which Salmonella attach to vegetable tissues is unknown. Attempts to mask receptor sites on
potato tissue with dead Salmonella lead to the hypothesis
that attachment requires cells to be metabolically active.
(a)
Live JH3016
(b)
Formalin-killed
JH3016
(c)
CWM only
(d)
PBS only
1000
Figure 2 Flow cytometric analysis of attachment of Salmonella serotype Typhimurium
JH3016 to potato cell-wall material (CWM).
(a) Fluorescence readings of samples of cellwall material incubated with live JH3016. (b)
Fluorescence readings of samples of CWM
incubated with formalin-inactivated JH3016.
(c) Fluorescence readings of samples of CWM
in phosphate buffered saline (PBS). (d) Fluorescence readings of samples derived from
PBS only. Readings are means of duplicate
experiments.
Beads fluorescence intensity
100
10
0
10 000
1000
100
10
0
0
10
100
1000 10 000 0
10
100
GFP fluorescence intensity
ª 2008 The Authors
Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1239–1245
1000 10 000
1243
Attachment of Salmonella to vegetable tissues
Using four different methods of inactivation, we showed
that Salm. Typhimurium must be viable in order to attach
to vegetable tissues. This was in contrast to studies with
E. coli where both live and inactivated bacteria attached to
lettuce tissue (Soloman and Matthews 2006). In their
study, Soloman and Matthews (2006) used glutaraldehyde
to inactivate the cells. Glutaraldehyde is known to alter
the adhesive properties of the bacterial membrane rendering the membrane more ‘sticky’. This alteration may
account for why inactivated E. coli cells were able to attach
to the tissue. In our study, the inability to attach was
found to be a direct result of viability rather than modification of proteins on the membrane surface. Attachment
by bacteria to plant tissues is believed to occur via surface
structures on the bacterial membrane such as fimbrae, pili
and lipopolysaccharide interacting with proteins on the
plant cell wall (Mandrell et al. 2006). Formalin, ethanol
and heat inactivation alters the protein structures on the
bacterial membrane and so may prevent this recognition.
Therefore, lack of attachment in these inactivated cells
may be as a result of altered membrane structures rather
than lack of metabolic activity. Kanamycin inactivates
bacteria by inhibiting protein synthesis and does not affect
the structure of proteins already present on the bacterial
membrane. The inability of Kanamycin-inactivated Salm.
Typhimurium to attach indicates that the attachment process requires protein synthesis and thus bacteria need to
be viable. The plant pathogen Agrobacterium initially
attaches loosely to the plant cell wall and then synthesizes
cellulose fibrils that bind the organism tightly to the cell
surface (Matthysse 1986). Salmonella serotype Typhimurium has recently been shown to possess the ability to
synthesize cellulose (Zogaj et al. 2001). Therefore, it is
possible that Salmonella uses a similar mechanism to
attach to plant cell walls, i.e. an initial weak attachment by
bacterial surface proteins interacting with the cell wall followed by the synthesis of cellulose to ensure strong attachment. This would explain the need for protein synthesis in
the attachment process. The genes and mechanism
involved in cellulose production in Salmonella are being
elucidated (Zogaj et al. 2001; Barak et al. 2005).
It is recognized that the use of biocides as decontaminants is not always successful (Adams et al. 1989; Garg
et al. 1990; Brocklehurst 1994; Zhang and Farber 1996);
accordingly this work forms part of a wider ongoing study
to understand the mechanisms used by bacteria for attachment to tissues with a view to developing new and more
effective decontamination techniques. In particular, this
paper focuses on the competencies of the organism and
particularly the discovery that organisms must be viable
for attachment to occur. This has implications for novel
decontamination interventions, such as the potential use
of a vaccine-like approach where dead bacteria could be
1244
E.J. Saggers et al.
used to prevent attachment of living bacteria. It is quite
clear that if cells cannot attach when they are not viable,
then the use of such a vaccine approach would be irrelevant. We intend to study further the initial attachment
phase of Salm. Typhimurium to vegetable tissue using the
genomic and proteomic techniques available to us with a
view to elucidating further mechanisms that may be
exploited to create novel and effective decontamination
techniques that can be employed by the food industry.
In summary, this study indicates the pectin-rich area of
the plant cell wall may act as a receptor site for bacterial
attachment to vegetable tissue. Results also illustrate that
in order for attachment to occur bacteria must be metabolically active and capable of protein synthesis.
Acknowledgements
The authors would like to acknowledge the financial support of the Biotechnology and Biological Sciences
Research Council for this work. We also thank Dr Isabelle
Hautefort for the flow cytometry analyses.
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