Food Control 35 (2014) 192e199

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Comparative simulation of Escherichia coli O157:H7 behaviour in

packaged fresh-cut lettuce distributed in a typical Canadian supply
chain in the summer and winter
Robin C. McKellar a, Denyse I. LeBlanc b, Fernando Pérez Rodríguez c, Pascal Delaquis a, *
a
Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, 4200 Highway 97 South, Summerland, BC, Canada V0H 1Z0
b
Atlantic Food and Horticulture Research Centre, c/o Food Research Centre, Université de Moncton, Moncton, NB, Canada E1A 3E9
c
Department of Food Science and Technology, University of Córdoba, Córdoba 14014, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The ability to predict the risk associated with Escherichia coli O157:H7 in packaged fresh-cut lettuce
Received 25 September 2012 hinges on the availability of realistic times and temperatures encountered in commercial supply chains.
Received in revised form We report here on temperature profiles measured in winter and summer months in a retail supply chain
30 May 2013
and their predicted impact on the fate of the pathogen in fresh-cut lettuce. Time and temperature data
Accepted 4 June 2013
were recorded in individual packages within cases of lettuce in the bottom, middle and top layers of
pallets distributed from a processing facility to retail storage. Measurements indicated there were oc-
Keywords:
casional increases in temperature during either season and that temperatures were
5  C for longer
E. coli O157:H7
Predictive modeling
periods of time during the summer. A stochastic simulation model based on time and temperature
Fresh-cut lettuce distributions was constructed in @RISKÔ to predict the fate of E. coli O157:H7. The output from the model
Distribution demonstrated a range of possible outcomes for the time-temperature profiles measured in the com-
mercial supply chain, ranging from slight growth to die-off. The outputs of the stochastic simulations
were similar to analytical simulations based on actual times and temperatures experienced by product in
the supply chain. The distributions presented in the current model could be incorporated in quantitative
risk assessments to improve predictions about the prevalence and fate of E. coli O157:H7 in fresh-cut
lettuce supply chains.
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction
Several outbreaks of illness caused by the human pathogen
Escherichia coli O157:H7 have been associated with the consump-
tion of packaged fresh-cut lettuce (Lynch, Tauxe, & Hedberg, 2009).
Packaged fresh-cut lettuce is a highly perishable commodity that
should ideally be stored below 4  C to maintain overall organoleptic
quality (Jacxsens, Develieghere, & Debevere, 2002). Laboratory data
indicate that most enteric bacterial pathogens do not grow below
5  C and this temperature has been recommended as a means to
minimize the risk of proliferation during the distribution of leafy
vegetables through commercial supply chains (USFDA, 2010). The
fate of E. coli O157:H7 in packaged fresh-cut lettuce is strongly
affected by temperature (Delaquis, Bach, & Dinu, 2007). A predic-
tive mathematical model derived from aggregate literature data
* Corresponding author. Tel.: þ1 250 494 6367.
E-mail address: pascal.delaquis@agr.gc.ca (P. Delaquis).
suggests that growth can occur above 5  C while death is likely at
lower values (McKellar & Delaquis, 2011). Temperatures in com-
mercial supply chains are known to fluctuate between optimum
values or recommended standards and “abusive” levels where they
exceed the minimum required for growth of bacterial pathogens
(Rediers, Claes, Peeters, & Willems, 2009; Luo, He, & McEvoy, 2010).
The effect of temperature fluctuations on E. coli O157:H7 pop-
ulations in fresh-cut lettuce and their implication for the risk to
public health are not well understood. A recent study performed in
Canada showed that conditions conducive to either growth or
death were encountered at different stages along a supply chain
that included storage at the processing facility, distribution centres
and retail outlets and transportation between each stage (McKellar,
LeBlanc, Lu, & Delaquis, 2012). The temperature profiles used in this
work were collected during winter months. However, temperatures
in supply chains are known to vary with season (LeBlanc, Stark,
MacNeil, Goguen, & Beaulieu, 1996). More accurate predictions of
E. coli O157:H7 behaviour in packaged fresh-cut lettuce and the
ensuing assessment of risks to public health will require the

0956-7135/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.06.002
 R.C. McKellar et al. / Food Control 35 (2014) 192e199
availability of temperature profiles at different times of the year.
We report here on differences in temperature profiles measured in
winter and summer months in a Canadian supply chain and their
predicted impact on the fate of E. coli O157:H7 in fresh-cut lettuce.
2. Materials and methods
2.1. Time-temperature data
Nine cases containing six 454 g clam shells of ready-to-eat
baby leaf lettuce were instrumented with thermocouple (TC)
probes connected to data-loggers. Instrumented cases were posi-
tioned at the top, in the middle and on the bottom of stacked
pallets, as described previously (McKellar et al., 2012). The data-
loggers recorded temperatures during distribution in a commer-
cial retail supply chain that consisted of five main stages: storage
at the processor, transportation to distribution centre (DC), storage
at the DC, transportation to retail and storage at retail. Four
separate trials were carried out during the summer (July/August
2011) season. A total of 36 cases of lettuce were instrumented over
the four trials, although some data was lost due to recorder mal-
function. Consequently, reliable temperature profiles were ob-
tained for 33 cases of lettuce. Corresponding data collected in the
winter (January/February 2010) was derived from measurements
in 35 cases distributed in the same supply chain (McKellar et al.
2012). The main difference between the two series of trials was
the ambient temperature. In Canada, transport trailers are equip-
ped with refrigeration units to minimize product warming during
the summer months and to prevent product freezing during the
winter.
2.2. Growth/death model
The development of the growth/death model for E. coli O157:H7
was described by McKellar and Delaquis (2011). The equations used
here were modified from McKellar et al. (2012) and consist of a
square root model for growth, and a simple linear die-off coefficient
for death:
Rate ¼ if ðT
5; Growth; DeathÞ (1)
Growth ¼ ðbðT  Tmin ÞÞ2 (1a)
Death ¼ k (1b)
Tmin ¼ 1:34  5:77b (2)
log Nt þ Dt ¼ log Nt þ ðRate  Dt=2:303Þ (3)
where Nt ¼ cell number at time ¼ t; b ¼ temperature coefficient
(distribution); Tmin ¼ notional minimum growth temperature
(  C); T ¼ temperature (  C); k ¼ die-off coefficient (distribution).
Growth and death are both in units of natural log (ln) cfu h1. A
temperature of 5  C was set as the cut-off point at which the
growth and death models change. Parameter values are given in
Table 1, and are the same as those used in a previous study
(McKellar et al., 2012), with the exception that the lognormal
distribution for the die-off parameter (k) was truncated at 0.03.
The lognormal distribution provided the best fit for the k param-
eter values although unreasonably high predictions were noted in
some cases, which would lead to situations where more die-off is
predicted than would actually occur. Consequently, the truncation
was fixed at the highest value found experimentally (McKellar &
Delaquis, 2011).
193
Table 1
Parameter values for growth and death models (McKellar & Delaquis, 2011).
Parameter Value
Lower Mean Upper
Growth
b 0.01 0.023 0.035
Tmin Calculated Calculated Calculated
Death
k (mean)
0.013
SD
0.010
Shift
0.001
Truncation
0.030
Lower and upper parameter values refer to the 95% prediction limits.
2.3. Distribution fitting to time and temperature data
Lettuce temperatures were recorded at 5 min intervals in indi-
vidual cases as they moved through the five stages of the retail supply
chain. All of the product temperature readings for summer or winter
trials were pooled per stage for analysis. The number of temperature
readings per stage for each season varied from 4000 to 22,000.
Probability distributions were fitted to the pooled temperature data
using @RISKÔ 6.1 (Palisade Corp., Ithaca, New York, USA, http://www.
palisade.com/decisiontools_suite/) in MicrosoftÒ Excel, and the most
appropriate distribution was selected based on the Chi Square
Goodness of Fit test. The total time spent by each case of product at
each stage during the summer or winter was also pooled. The number
of time values for each stage varied between 33 and 35 depending on
the season (i.e. one time value per instrumented case). Due to the
limited number of time values, the fitting procedure was not optimal
and empirical cumulative distributions were defined based on
measured time. The empirical probability distributions were used to
simulate the duration of each stage of the supply chain.
2.4. Stochastic simulations
In order to simulate the effect of the supply chain on E. coli
O157:H7 populations in fresh-cut lettuce (expressed in terms of
change in log cell number per unit of lettuce), the fitted tempera-
ture and time distributions at each stage were simulated with 5000
iterations using @RISKÔ. The input for the model was fixed at zero
log cfu per unit of lettuce as initial concentration. A different time-
temperature profile was established for each iteration by sampling
the temperature and time distributions for each of the five stages.
The sampled temperature was assumed to remain constant for the
duration of the stage. For each iteration of the model, the rates for
either growth or death (growth when temperature
5  C) were
calculated using Eqs. (1) and (2) for each of the five stages of the
cold chain using the model parameters (Table 1). Changes in log cell
numbers were calculated using the rate derived from the corre-
sponding temperature (Eq. (3)). The resulting predicted changes in
log cell numbers were added to the initial concentration of cells
(zero log cfu per unit of lettuce) and expressed as distributions.
Because a higher temperature could be matched with a longer
time period when time and temperature were sampled separately
during a simulation, there was a possibility that the model could
overestimate growth. A series of simulations was therefore carried
out where the times and temperatures selected from the input
distributions were assigned a correlation using the @RISKÔ Risk-
CorrMat function to anticipate such situations.
Spearman rank correlation coefficients were determined using
@RISKÔ to evaluate the extent to which each input variable (e.g.
194 R.C. McKellar et al. / Food Control 35 (2014) 192e199
temperature, time, etc.) influenced the final output (log change in
cell numbers). This coefficient varied between -1 and 1, indicating,
respectively, strong positive and negative relationship between
input and output (Cullen & Frey, 1999).
2.5. Analytical simulations
Estimates of E. coli O157:H7 populations in lettuce distributed
through the supply chain were also carried out using the temper-
atures experienced under each experimental condition over time
(i.e. timeetemperature profiles), with the model parameters
(Table 1 and Eqs. (1)e(3)) for growth and death as described pre-
viously (McKellar et al., 2012). The initial cell numbers for these
simulations was also zero log cfu per unit of lettuce. For each of the
35 timeetemperature profiles recorded in instrumented cases
during winter and 33 profiles recorded during summer, the simu-
lated cumulative growth and die-off was calculated and the output
distribution expressed as log change in cell numbers. Each simu-
lated case gave rise to an output distribution. In order to obtain a
single output distribution for the winter and summer seasons the
individual analytical output distributions were sampled using a
discrete distribution with equal statistical weighting. This enabled
the comparison of the winter and summer output distributions for
both the stochastic and analytical simulations.
2.6. Scenario analysis
A scenario analysis with multiple simulations was carried out
to assess the effect of different initial cell concentrations on the
final percentage of contaminated units of lettuce after exposure to
either summer or winter storage and transportation conditions
using the stochastic simulation approach described above. For the
purpose of this scenario a unit was defined as a 500 g package of
lettuce. Five scenarios (with 5000 iterations each) were simulated
with nominal initial concentrations of 1, 2, 3, 5 and 10 cfu unit1.
The Poisson distribution was used to model cell distribution based
Fig. 1. Temperature profiles of fresh-cut lettuce delivered to three retail outlets. Two trials (a and b) are shown. Dashed lines indicate the location of the instrumented case in the
pallet. Rectangles of different shades indicate duration of each stage (width of rectangle) and mean temperature of the three cases for each stage (height of rectangle).
on the assumption that E. coli O157:H7 cells are distributed
homogenously throughout the lettuce. By applying this distribu-
tion, changes in the number of prevalent units could be simulated
along the supply chain when concentrations were very low
(<1 cfu unit1). The Poisson (l) distribution is defined by l, which
corresponds to the nominal value for cfu unit1 for each scenario.
The change in cell numbers unit1 were calculated after simulated
exposure to either winter or summer conditions, and a Poisson
distribution was used to simulate the number of residual cells per
unit. The percent of iterations giving rise to contaminated units
was then calculated using the @RISKÔ RiskTargetD (cellref, target
value) function where cellref was remaining cells unit1 and target
value was zero.
3. Results
3.1. Temperature profiles
Lettuce temperatures recorded in cases delivered to three retail
outlets on four separate occasions (a, b, c and d) during summer are
shown in Figs. 1 and 2. Temperature profile lines with different
dashes indicate the location of the instrumented cases in the pallet
(bottom, middle and top). Rectangles of different shade are
included in Figs. 1 and 2 to show the duration of each stage (width)
and the mean temperature of the three cases for each stage
(height). Comparison with profiles collected during winter alone
(McKellar et al., 2012) indicated that lettuce was exposed to tem-
peratures
5  C for longer periods of time during the summer
season.
As seen in Figs. 1 and 2, the 33 cases of lettuce experienced quite
different time-temperature profiles as the duration of each stage
varied significantly from trial to trial (a, b, c and d in Figs. 1 and 2)
and the duration of the retail storage stage also varied significantly
for each store within a trial. The cases of lettuce followed in the
same supply chain during winter months experienced as much
variability in their time-temperature profiles (McKellar et al., 2012).
 R.C. McKellar et al. / Food Control 35 (2014) 192e199
Fig. 2. Temperature profiles of fresh-cut lettuce delivered to three retail outlets. Two trials (c and d) are shown. Dashed lines indicate the location of the instrumented case in the
pallet. Rectangles of different shades indicate duration of each stage (width of rectangle) and mean temperature of the three cases for each stage (height of rectangle).
The times and temperatures measured during each of the five
stages of the supply chain were expressed as distributions (Tables 2
and 3, respectively). Because of the limited number of measured
times (i.e. one time per stage for each instrumented case in a trial),
empirical probability distributions were used for time (Table 2). The
fraction of time spent at temperatures
5  C for each of the five
stages is shown in Table 2. During winter, there were few instances
of temperatures
5  C; the longest time spent in this temperature
range was 7.4% at retail storage. During summer, there was
increased time spent above 5  C, with a maximum of 73% of the
time during processor storage (Table 2). Transportation to retail
was also a problem area for elevated temperature, with 53% of the
time spent at
5  C in this stage. Although the proportion of time
above 5  C was longer during summer than during winter, the
amount of time spent at higher temperatures was relatively short
as demonstrated with the various peaks shown in Figs. 1 and 2.
Because there were large numbers of temperature readings
available, the data were fitted to various distributions using
@RISKÔ. Normal distributions were deemed most appropriate for
the winter profiles, with the exception of temperatures during
processor storage where a lognormal distribution was selected by
chi-square goodness of fit test (Table 3). Lognormal distributions
were generally more suitable for the summer temperature profiles,
with the exception of processor storage (Table 3). This may be the
consequence of higher ambient temperatures during the summer,
resulting in a right hand tail on the distribution curve that was
better described by the lognormal distribution (i.e. a skewed dis-
tribution). All of the lognormal distributions were truncated at the
highest temperature found experimentally to prevent the selection
of unreasonably high temperatures when sampled for simulations
(Table 3).
3.2. Stochastic simulations
Stochastic simulations were performed to compare the output
(log change in cell numbers per unit of lettuce) from the winter and
195
summer time and temperature distributions. The output distribu-
tions are shown in Fig. 3. The winter distribution (dark grey, in the
background) defines the location of the 5th and 95th
percentiles: 1.92 and 0.22, respectively. This distribution had an
extended left-hand tail, reflecting the greater proportion of com-
binations of low temperatures and longer times in the winter
supply chain, resulting in greater die-off. The summer output dis-
tribution represented by the overlay graph (light grey) was more
symmetric, and was shifted to the right, relative to the winter
distribution. The summer distribution showed higher overall
growth potential, with a shift of 72.6% of the iterations to above the
95th percentile (0.22) of the winter simulation, likely due to the
preponderance of temperatures
5  C. These observations were
also reflected in the relationship between the means and standard
deviations (SD) for the two output distributions (Table 4). The mean
for the summer distribution was 0.05, indicating a very slight
overall die-off compared to the initial value of zero. The mean of the
winter distribution was 0.84 (Table 4). The greater SD for the
winter distribution compared to the summer (0.55 and 0.47,
respectively) reflects the longer left-hand tail in the winter output
distribution. The 95th percentile for the summer distribution cor-
responded to 0.62 suggesting that E. coli O157:H7 populations could
significantly increase during summer in some few instances.
Indeed, the maximum log-increase obtained in summer was 5.2 in
contrast to the value simulated in winter, which was 0.76.
3.3. Analytical simulations
Simulated output for individual cases of lettuce under winter
conditions using recorded timeetemperature profiles (referred to
as analytical simulations) was described previously (McKellar et al.,
2012). Here we expand this work by generating similar analytical
simulations for summer storage and transportation conditions, and
comparing these to the output of the stochastic simulations. The
comparison of the two simulation approaches is shown in Fig. 4.
The overlay winter analytical simulation (light grey) represents the
196 R.C. McKellar et al. / Food Control 35 (2014) 192e199

Table 2
Distributions for time (h).

Season/stage Distribution Proportion of time above 5  C

Winter
Storage processor RiskCumul (17.5,23.7,{18,19, 20,23},{0.257,0.429,0.514,0.743}) 0.001
Transportation DC RiskCumul (12.6,64.6,{13,42,61},{0.257,0.486,0.743}) 0.043
Storage DC RiskCumul (35.9,78.7,{36,40,60},{0.091,0.273,0.727}) 0.006
Transportation retail RiskCumul (10.3,16,{11,12,13,14,15},{0.091,0.182,0.364,0.636,0.818}) 0.002
Storage retail RiskCumul (0.2,146.1,{10,50,100},{0.286,0.571,0.857}) 0.074
Summer
Storage processor RiskCumul (9.4,55.5,{10,20,30,40,50},{0.24,0.24,0.76,0.76,0.76}) 0.73
Transportation DC RiskCumul (7.25,12,{8,9,10,11},{0.48,0.48,0.48,0.73}) 0.40
Storage DC RiskCumul (10.58,60.5,{20,40,60},{0.76,0.76,0.91}) 0.03
Transportation retail RiskCumul (9.7,13.67,{10,11,12,13},{0.083,0.333,0.583,0.667}) 0.53
Storage retail RiskCumul (0.17,175,{10,50,100,150},{0.5,0.81,0.91,0.97}) 0.36

combined distribution of individual simulations. As evidenced by
Fig. 4a, the stochastic (dark grey, in the background) and analytical
simulations for winter are quite similar, with only slightly fewer
(3.6%) of the analytical iterations falling above the stochastic 95th
percentile. The winter analytical simulations (overlay light grey in
Fig. 4a) resulted in 5th and 95th percentiles of 1.95 and 0.24,
respectively, which matched results for stochastic simulations
described above and Table 4. The means and SD values for the two
simulation approaches are also similar (Table 4). The greater dif-
ferences between both approaches were found in the extremes of
the distribution, with the stochastic simulation giving higher
maximum and lower minimum (Table 4).
The summer stochastic and analytical simulations were also
compared (Fig. 4b), and the results were in agreement with those
found for the winter simulations. The proportion of the iterations
from the analytical simulation which fell above the stochastic
simulation 95th percentile of 0.62 was only 4.4%, whilst the pro-
portion below the 5th percentile (0.79) was 3.5%. Means and SD
values for the summer analytical simulation were close to those
found with the stochastic approach (Table 4). Once again, the sto-
chastic simulation gave higher maximum and lower minimum
values (Table 4), reflecting the occasional matching of longer times
with lower or higher temperatures.
3.4. Sensitivity analysis
Sensitivity analysis was performed to assess the relative
contribution of the various input distributions to the output from
the stochastic simulation. A negative correlation means that a
higher value of a distribution gives lower final cell numbers,
resulting from either greater die-off or limited growth. A positive
correlation means that the parameter is related to increased growth
or reduced die-off, overall. Correlations below or equal to 0.1 were
considered weak hence only inputs with correlation values >0.1
were reported.
As expected with the winter simulations, mainly negative cor-
relations were important, since there were few instances of tem-
perature
5  C. The largest negative correlation (0.86) was found
with the parameter k (Table 5), as it is directly related to die-off.
Times for retail storage (0.30), transportation to (0.18) and
storage at DC (0.11) were all negatively correlated with the
output, indicating that the length of time spent in these stages of
the supply chain was sufficiently long in some iterations to influ-
ence the extent of die-off. The difference in negative correlation for
these three stages relates to the variation in the duration of each
stage. During winter, the retail storage stage showed the largest
range of times, followed by the transportation to and storage at DC
(Table 2). The only positive correlation was found with the tem-
perature at retail storage (0.10). This reflects the increased inci-
dence of temperatures
5  C at this stage (McKellar et al., 2012).
The sensitivity analysis for the summer time-temperature
simulation (Table 5) shows a greater influence of temperature
than was found during the winter, which was expected since
temperatures
5  C were more common in summer. Generally,
temperature inputs were positively correlated with output, while
times were negative. As expected, the k parameter again had the
highest negative correlation (0.39). The positive correlations for
temperature during storage at retail (0.34) and at processor (0.31),
and during transportation to retail (0.25) and DC (0.14), indicated
conditions conducive to growth. This is supported by the temper-
ature profiles of Figs. 1 and 2 where temperatures above 5  C were
frequently recorded during these four stages. The variation in
temperatures for the different trials and the different stores were
more pronounced for the retail storage stage which may explain
why the temperature of the product during retail storage had the

Table 3
Distributions for temperature ( C).

Season/stage Distribution Mean SD Shift Truncation

Winter
Storage processor Lognormal 3.01 0.823 0.809 5.33
Transportation DC Normal 2.28 1.11
Storage DC Normal 1.96 0.596
Transportation retail Normal 2.91 0.923
Storage retail Normal 4.09 0.670
Summer
Storage processor Normal 5.59 0.905
Transportation DC Lognormal 6.29 1.62 1.46 11.4
Storage DC Lognormal 1.28 1.19 1.70 9.89
Transportation retail Lognormal 5.85 2.62 0.040 15.8 Fig. 3. Comparison of winter (dark grey) and summer (light grey) stochastic simula-
Storage retail Lognormal 3.52 1.59 1.18 11.8 tions. Cross hatching defines the ranges <5th and >95th percentiles. Initial cell con-
centration was zero log cfu per unit of lettuce.
 R.C. McKellar et al. / Food Control 35 (2014) 192e199 197

Table 4 more growth than would be found experimentally. This is sup-

Characteristics of output distributions (log change in cell numbers) for stochastic ported by the earlier observation that maximum values for changes
and analytical simulations.
in log cell numbers were greater in stochastic as compared to
Winter Summer analytical simulations. Applying a negative correlation between
Stochastic Analytical Stochastic Analytical time and temperature in stochastic simulations using @RISKÔ
might result in reduced prediction of excessive growth.
Minimum 3.83 3.62 2.44 2.12
Maximum 0.76 0.11 5.20 1.60 The results (data not shown) indicate that negative correlations
Mean 0.84 0.86 0.05 0.03 up to 1.0 applied to winter and summer stochastic simulations
SD 0.55 0.54 0.47 0.40 shifted the output distributions by only a few percentage points.
5th Percentile 1.92 1.95 0.79 0.70
Correlation also matched low temperatures with longer time,
95th Percentile 0.22 0.24 0.62 0.59
resulting in a general shift of the stochastic distributions to slightly
increased die-off. The values of mean, SD, 5th and 95th percentiles
were similar to those obtained in the stochastic and analytical
most effect on the fate of E. coli O157:H7. The temperature simulations, while the maximum values were reduced with respect
parameter b also had a positive correlation with the output (0.25). to the stochastic simulations. These results and the observation that
This parameter relates the effect of temperature on the growth rate stochastic and analytical simulations do not differ in any significant
(McKellar & Delaquis, 2011). Negative correlations of 0.18 way, suggests that changes of E. coli O157:H7 populations in fresh-
and 0.12 were found for time of storage at DC and retail, respec- cut lettuce in the supply chain could be simulated regardless of
tively. This result suggests that combinations of longer times and correlation between time and temperature, yielding similar results
lower temperatures were frequent in those stages, resulting in to those obtained from the analytical simulation.
negative correlation. The longer the duration of retail and DC
storage, the lower product temperature reached and the more die- 3.6. Scenario analysis applied to initial concentration
off was likely to occur. In keeping with this, Figs. 1 and 2 show that
product temperatures decreased when the retail and DC storage The influence of initial contamination level on prevalence of
stages were sufficiently long. The only positive correlation with contaminated packages was also examined using stochastic
time was for storage at the processor (0.12). Because product simulations. Here we were interested in determining the per-
temperature does not vary much during processor storage and was centage of units which become uncontaminated after exposure
generally
5  C (Figs. 1 and 2), longer storage time at this stage to the winter and summer conditions in the supply chain. The
would result in some growth of E. coli O157:H7. results of these simulations are shown in Table 6. At an initial
concentration of 1 cfu package1, only 17.7%of iterations resulted
3.5. Correlation of time and temperature in stochastic simulations in a contaminated package in the winter simulation, likely
because of cell die-off. Under summer conditions a total of 42.5%
In the stochastic simulation, the time and temperature were of the iterations were positive (Table 6), indicating an increased
sampled independently. It is possible that a higher temperature risk. When initial cell concentration was increased to 2cfupack-
could be matched in the simulation with a longer time, resulting in age-1 the total number of contaminated packages for winter and
summer simulations were 30.2 and 65.1%, respectively. With
each successive increase in initial cell numbers, a proportional
increase in simulated survivors was observed. A tan initial cell
concentration of 10 cfupackage-1 during the summer, there were
still a few iterations (3.5%) which led to some uncontaminated
packages. The predicted number of contaminated lettuce pack-
ages was always less in the winter due to die-off during longer
storage times at temperatures below 5  C, and shorter periods of
time when temperatures exceeded 5  C.

4. Discussion and conclusions

The present study has confirmed and extended previous work

Fig. 4. Comparison of winter (a) and summer (b) stochastic (dark grey) and analytical
(light grey) simulations. Cross hatching defines the <5th and >95th percentiles. Initial
cell concentration was zero log cfu per unit of lettuce.
which described the influence of winter time and temperature
conditions on behaviour of E. coli O157:H7 in fresh-cut lettuce
distributed in a Canadian supply chain (McKellar et al., 2012). In
that study, the growth or death of the pathogen was simulated with
times and temperatures corresponding to those experienced by
packaged lettuce during different storage and transportation con-
ditions. The most significant finding was that the extent of die-off
was positively correlated with the total time spent in the supply
chain. In the present work we show that temperatures are often
higher during the summer months. A novel approach was taken to
combine times and temperatures from each stage of the supply
chain into distributions which, when incorporated into an @RISKÔ
simulation, were able to predict the influence of initial cell numbers
on the prevalence of contaminated packages. In addition, it was
possible to use sensitivity analysis to show which of the stages of
the supply chain had the greatest influence on E. coli survival, and
where problems might arise.
198 R.C. McKellar et al. / Food Control 35 (2014) 192e199

Table 5
Spearman rank correlation coefficients assessing the impact of input distributions on the output distribution.

Winter Summer

Stage Factor Coefficient Stage Factor Coefficient

Negative Positive Negative Positive

k 0.86 k 0.39
Storage retail Time 0.30 Storage retail Temperature 0.34
Transportation DC Time 0.18 Storage processor Temperature 0.31
Storage DC Time 0.11 b 0.28
Storage retail Temperature 0.10 Transportation retail Temperature 0.25
Storage DC Time 0.18
Transportation DC Temperature 0.14
Storage retail Time 0.12
Storage processor Time 0.12

DC, Distribution Centre.

The significantly different times and temperatures that cases of
fresh-cut lettuce experienced during separate shipments in the
same supply chain (Figs. 1 and 2) complicated the choice of time-
temperature profiles for use in risk assessment simulations.
Because the duration of each stage varied from one shipment to the
next and from one retail store to the other, it was not possible to use
the Franz, Tromp, Rijgersberg, and van der Fels-Klerx (2010)
method for combining time-temperature profiles. Instead, a
method similar to that proposed by Tromp, Rijgersberg, and Franz
(2010) for stages of variable duration was used to obtain temper-
ature profiles. In our stochastic simulation approach, a product was
assigned a particular temperature for each stage (sampled from the
distribution of temperatures for each stage e Table 3) and each
product remained at this temperature for the duration of the stage
(which was sampled from the appropriate distribution of times for
the stage e Table 2). The temperature profiles obtained with this
technique are likely representative of those encountered in other
retail supply chains in Canada. In addition, because the temperature
of fresh-cut lettuce was followed in the same supply chain during
two seasons, one warm (Figs. 1 and 2) and one cold (McKellar et al.,
2012), seasonal variation can be considered for the risk assessment
scenarios.
The use of time and temperature distributions derived from
individual TC readings has allowed us to develop the means to
stochastically simulate changes in cell numbers based on condi-
tions found during winter and summer months in the supply chain.
Comparison of the stochastic and the analytical simulations
revealed that, for both summer and winter conditions, the method
of randomly assigning times and temperatures in the various stages
(stochastic simulation) accurately reflects what the individual units
of lettuce would be experiencing (analytical simulation). For both
simulation approaches, output distributions revealed higher
simulated growth during the summer, which could lead to signif-
icant log-increases of E. coli O157:H7; the 95th percentile values
obtained in the stochastic and analytical simulations corresponded
to 0.62 and 0.59 log-increase in cell numbers, respectively. This
significant growth was due to the increased incidence of temper-
atures above the growth-permissive minimum of5  C in the supply
chain. In contrast, for winter, stochastic and analytical simulations
evidenced a significant reduction of populations of E. coli O157:H7
in the supply chain, as shown by the 95th percentiles, which was
around 0.24 log decrease in cell numbers. Thus, the stochastic
simulation approach is appropriate for inclusion into quantitative
risk assessments.
In practice we would expect that exposure of produce to
elevated temperatures would be limited, since control systems are
in place in the supply chain. Spoilage would also occur quickly
under temperature abuse conditions, acting as an indicator of
increased risk. However, if a stochastic simulation was being used
to assess the level of risk, it is possible that matching higher tem-
peratures with unreasonably long time periods could lead to a
prediction of excessive growth, if the times and temperatures are
not correlated. The introduction of negative correlation had little
effect on the output distributions in stochastic simulations and,
therefore it was not considered in our analysis. Moreover, the
analytical simulations, based on actual times and temperatures
experienced by experimental packages gave very similar output
distributions to the stochastic simulations. These results suggest
that conditions in which a high temperature would be matched
with a longer time occur infrequently, and would not be expected
to significantly increase the risk. The maximum and minimum
values for the stochastic simulations are greater than the corre-
sponding values for the analytical simulations; however, these are
due to a few iterations in which more extreme combinations of
time, temperature, and growth or die-off coefficients are selected.
Nonetheless, these values defining the extreme of the distributions
are not expected to affect the interpretation of results considering,
as criteria, more representative statistical parameters, such as 5th
and 95th percentile, mean or SD.
We have also used rank correlations to determine the impact of
the various inputs on the stochastic simulation output. Overall, the
die-off coefficient had the greatest negative influence on cell
numbers for both winter and summer, which is expected as it
directly relates to the extent of die-off. This result indicates that the
uncertainty derived from predictive models had the greatest
impact on the output, while underlining the importance of
applying accurate predictive models to obtain more precise esti-
mates. In winter simulations, times for storage at DC and retail, and
transportation to DC were negatively correlated with change in cell
numbers, reflecting the earlier observation that lower cell numbers
were highly correlated to time in the cold chain (McKellar et al.,
2012). During summer, temperatures in several stages of the sup-
ply chain were positively correlated with change in cell numbers,
due to the prediction of growth occurring in the summer in more of
the iterations. In fact, the summer stochastic simulations predicted
Table 6
Simulated percent of iterations giving contaminated bags.
Initial cfu bag1 Percent of iterations
Winter Summer
1 17.7 42.5
2 30.2 65.1
3 40.5 77.8
5 53.7 88.9
10 71.1 96.5
 R.C. McKellar et al. / Food Control 35 (2014) 192e199
some growth in roughly half of the iterations, indicating the strong
possibility of increased risk during summer transportation and
storage.
Our stochastic simulation approach has allowed us to make
predictions regarding the influence of initial cell numbers on the
probability of packages becoming uncontaminated due to inacti-
vation at low temperatures. It is important to note that under
winter conditions where the most die-off is expected, the simula-
tion predicted that a significant number of packages would remain
contaminated given an initial level of 1 cell unit1. This effect was
exacerbated under summer conditions, with as many as 42.5% of
packages being contaminated. This suggests that under typical
storage and distribution conditions found in the Canadian supply
chain, there is still the probability of finding some contaminated
packages of fresh-cut lettuce even at the lowest theoretical level of
contamination with E. coli O157:H7.
In conclusion, the output from the model demonstrates a range
of possible outcomes, based on experimental times and tempera-
tures, in a representative fresh-cut lettuce supply chain. The tem-
perature profiles indicated that there was occasional temperature
abuse during either season, resulting in slight increases in the
predicted pathogen concentration in the units. Nonetheless, tem-
peratures during storage at processor and retail during the summer
season enhanced the probability of growth suggesting that atten-
tion should be focused on these stages in order to mitigate the risk
posed by E. coli O157:H7. The distributions present in the current
model can be incorporated into quantitative risk assessment
models to improve predictions of levels of the pathogen and
probability of illness in fresh-cut lettuce supply chains.
Acknowledgements
The authors would like to thank Defence Research and Devel-
opment Canada for funding this project via the Chemical, Biolog-
ical, Radiological-Nuclear and Explosives Research and Technology
Initiative (CRTI). The authors extend their gratitude to Sabrina
Bergeron-Quirion for conducting the temperature monitoring trials
and Mary Taylor and Bernadette Goguen for data handling and
199
analysis. Sincere thanks are also extended to the processor, distri-
bution centre and retail stores that participated in this project.
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