Transcript Effects of mechanical Injuries on leafy greens in

Effects of mechanical injuries on leafy greens in relation to proliferation of Escherichia coli O157:H7
Danyelle Osorio1 , Daniel Labuz1 , Sofia Windstam1 , Beatrix Alsanius2
1 Department of Biological Sciences, SUNY Oswego, Oswego, NY, USA
2 Department of Horticulture, Swedish University of Agricultural Sciences, Alnarp, Sweden
Introduction:
Recently, there has been a rising concern over the ability of enteric bacteria, such as Escherichia coli
O157:H7 (E.coli), to survive on the surface of produce because of the increased global concern with food safety
(Aruscavage et al, 2006).
Enteric pathogens only appear on the phyllosphere (plant leaf surface) after plants have been contaminated by
their environment (Aruscavage et al, 2006, Franz and van Bruggen, 2008) and their persistence is still under evaluation.
In that time, Aruscavage et al. (2008) and Brandl (2008), both found an increase in growth of E.coli when plant leaves
are mechanically damaged.
The purpose of this experiment was to assess under which sugar conditions promoted the most growth of
E.coli, intact vs. mechanically damaged, on the leaves of spinach, arugula, and red chard through the use of cell counts.
Methods:
Harvested baby leaves (Red Chard (Beta vulgaris var.
vulgaris) and Romaine (Lactuca sativa var. longifolia cv. Ovired)
were submerged in solutions of 105 cfu × ml-1 E.coli or 0.085%
NaCl and left intact or mechanically injured using tweezers.
Leaves were incubated at 28˚C for 2,4, and 24 hrs at which point
leaves were transferred to 40 ml of 0.1 M Tris in 50 ml tubes.
Leaves were sonicated at 20˚C for 7 minutes, then vortexed for 20
s before serially diluting suspensions and plating dilutions in
duplicate onto Luria Bertani agar amended with 100 µg × ml-1
ampicillin and 0.2% arabinose. Plates were incubated at 37˚C
overnight before enumerating colonies.
Inoculation
Controla
E. coli
Time (hpi)
0
2
4
24
0
2
4
24
E. coli O157:H7 (log cfu × g-1 leaf)
Intact
Bruised
Ndb
Nd
Nd
Nd
Nd
Nd
4.14 (0.23)
4.78 (0.29)
5.34 (0.05)a
5.39(0.13)a
5.46 (0.07)ab
5.24(0.09)a
5.17 (0.11)ab
5.19(0.19)a
7.00 (0.32)b
7.11(0.14)b
Table 2. E. coli O157:H7 on bruised baby romaine
leaves after incubation at 28˚C for up to 24 hrs
Inoculation
post inoculation (hpi). Each data point is the
Controla
mean of three replicates and the number in
parenthesis is the standard error (S.E.). Different
letters following the mean and S.E. denote that
treatments were significantly different at the level
of p=0.05.
E. coli
aControl
leaves were submerged in 0.085% NaCl
bNd = Not detected
Table 1. E. coli O157:H7 on bruised baby red
chard leaves after incubation at 28˚C for up to 24
hrs post inoculation (hpi). Each data point is the
mean of three replicates and the number in
parenthesis is the standard error (S.E.). Different
letters following the mean and S.E. denote that
treatments were significantly different at the level
of p=0.05.
aControl
leaves were submerged in 0.085% NaCl
bNd = Not detected
Time (hpi)
0
2
4
24
0
2
4
24
E. coli O157:H7 (log cfu × g-1 leaf)
Image 1. Daniel Labuz harvesting the first leaves of
Red Chard plants.
Results:
Time (hpi)
Intact
Bruised
Cut
Shredded
0
4.93 (0.03)a
4.65 (0.05)a
5.47 (0.24)ab
5.09 (0.06)a
2
4.89 (0.03)a
4.76 (0.09)ab
4.92 (0.04)a
4.97 (0.05)a
4
4.95 (0.09)ab
5.00 (0.01)b
5.40 (0.16)a
5.48 (0.08)b
24
5.54 (0.08)b
5.53 (0.21)a
5.71 (0.12)b
6.37 (0.12)c
E. coli O157:H7 (log cfu × g-1 leaf)
Intact
Bruised
Ndb
Nd
Nd
Nd
Nd
Nd
Nd
Nd
5.58 (0.05)ab
5.42 (0.05)a
5.41 (0.04)a
5.00 (0.09)a
5.60 (0.09)ab
5.31 (0.17)ab
6.40 (0.21)b
6.40 (0.18)b
Table 3. E. coli O157:H7 on minimally
processed mature romaine leaves after
incubation at 28˚C for up to 24 hrs post
inoculation (hpi). Each data point is the mean
of three replicates and the number in
parenthesis is the standard error (S.E.).
Different letters following the mean and S.E.
denote that treatments were significantly
different at the level of p=0.05.
Conclusions:
Image 2. 0hr time point plates of
E.coli collected from bruised spinach
leaves. The sample was plated through
the use of a spiral plater.
Romaine: In the end, there were two replicate assays that both had a significant increase of the growth of E.coli on
both intact and bruised leaves over a 24 hour period. The second replicate assay appeared to have a greater growth of bacteria,
but this may have been due to the size of the leaves being greater in the second assay. The mature romaine leaves also had
significant increases over 24 hours over all treatments. Red Chard: There were three replicated assays done, where in the
second assay, in both treatments, there was a significant increase over 24 hours and in the third there was no significant increase
of growth on the intact leaves, but there was on the bruised leaves. There was less growth on the third assay compared to the
second, which also may have been because of the size of the leaves that were used.
References:
Aruscavage, D., Lee, K., Miller, S., and Lejeune, J.T. (2006) Interaction affecting the proliferation and control of human pathogens on
edible plants. Journal of Food Science. 71, 89-99.
Aruscavage, D., Miller, S. A., Lewis Ivey, M. L., Lee, K., and LeJeune, J.. (2008) Survival and dissemination of
Escherichia coli
O157:H7 on physically and biologically damaged lettuce plants. Journal of Food Protection. 71, 2384-2388.
Brandl, M.T. (2008) Plant lesions promote the rapid multiplication of Escherichia coliO157:H7 on postharvest lettuce. Applied and
Environmental Microbiology. 74, 5285-5289.
Franz, E., and van Bruggen, A. H.C.(2008) Ecology of E.coliO157:H7 and Salmonella enterica in the primary vegetable
production chain. Critical Reviews in Microbiology. 34, 143-161.
Images taken by Osorio, D.