Food Research International 75 (2015) 233–243

Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Review

The challenge of challenge testing to monitor Listeria monocytogenes

growth on ready-to-eat foods in Europe by following the European
Commission (2014) Technical Guidance document
Avelino Álvarez-Ordóñez a,⁎, Dara Leong a, Bernadette Hickey b, Annie Beaufort c, Kieran Jordan a
a
Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
b
Department of Agriculture, Food and the Marine, Dairy Science Laboratory, Backweston Campus, Celbridge, Co. Kildare, Ireland
c
AB Consultant, 23 Rue Jean Guy Labarbe, 94130 Nogent-Sur-Marne, France

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

Article history: European Regulation (EC) No. 2073/2005 lays down the microbiological criteria for certain microorganisms in
Received 4 March 2015 foods and the implementing rules to be complied with by food business operators (FBOs) in Europe when
Received in revised form 28 May 2015 implementing general and specific hygiene measures. In relation to Listeria monocytogenes, this regulation covers
Accepted 3 June 2015
primarily ready-to-eat (RTE) food products, and requires different microbiological criteria depending on the
Available online 6 June 2015
ability of the food product to support growth of L. monocytogenes. In addition, this regulation establishes that
Keywords:
food safety is the responsibility of the FBO. The FBO can conduct studies to evaluate the growth of
Listeria monocytogenes L. monocytogenes that may be present in the product during the shelf-life under reasonably foreseeable storage
Challenge tests conditions of distribution, storage and use in order to investigate compliance with the criteria throughout the
Foods shelf-life of the product. The European Union Community Reference Laboratory for L. monocytogenes published
Growth potential a revised technical guidance document in June 2014 for conducting shelf-life studies on L. monocytogenes in
European guidelines RTE foods. This review article describes the recently published European guidance document, with special
Food safety focus on the design of challenge studies to determine the growth potential of L. monocytogenes on foods. Informa-
tion is given particularly on what a challenge test is and when one is advisable. The factors to be considered and
the laboratory methodology to be applied when performing a challenge test to determine the growth potential of
L. monocytogenes in a defined food matrix are also described. Results of recent research articles applying chal-
lenge tests to determine the growth of L. monocytogenes in a range of foodstuffs are summarized and discussed.
Finally, recommendations for obtaining data that can contribute to any further revision of the guidance document
and for addressing the main challenges of challenge testing for FBOs are presented.
© 2015 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
2. Application of challenge tests on L. monocytogenes in the food industry following the EU Technical Guidance document . . . . . . . . . . . . . 234
3. Available literature on application of challenge tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
3.1. Challenge tests in vegetables and salads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
3.2. Challenge tests in meat products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
3.3. Challenge tests in seafood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
3.4. Challenge tests in cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
3.5. Challenge tests in other prepared meals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
4. Concluding remarks and recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

⁎ Corresponding author.
E-mail address: avelino.alvarez-ordonez@teagasc.ie (A. Álvarez-Ordóñez).

http://dx.doi.org/10.1016/j.foodres.2015.06.004
0963-9969/© 2015 Elsevier Ltd. All rights reserved.
234 A. Álvarez-Ordóñez et al. / Food Research International 75 (2015) 233–243
1. Introduction
Listeria monocytogenes is a Gram-positive, facultatively anaerobic,
non-sporeforming rod involved in cases of food-borne listeriosis infec-
tion. L. monocytogenes is widely distributed in the environment and
has been isolated from a variety of sources, including soil, vegetation,
silage, faecal material, sewage and water. It is frequently present in
raw foods of both plant and animal origin, and it can be found in cooked
foods due to post-processing contamination. Thus, it has been isolated
from foods such as raw and unpasteurized milk, cheese, ice cream,
raw vegetables, fermented meats and cooked sausages, raw and cooked
poultry, raw meats, and raw and smoked seafood. In addition, its ubiq-
uitous presence also leads to the potential for contamination of the
food processing environment, where occurrence and persistence of
L. monocytogenes is frequent (Fox, Hunt, O'Brien, & Jordan, 2011; Nakari
et al., 2014; Vongkamjan, Roof, Stasiewicz, & Wiedmann, 2013).
Although listeriosis is a relatively rare disease, it can be life threaten-
ing, and is of particular concern for pregnant women, the elderly and im-
munocompromised individuals, with fatality rates of 20 to 30% being
common among hospitalised patients (Vazquez-Boland et al., 2001). Lis-
teriosis occurs in different forms: neuromeningeal, maternal-neonatal
and febrile gastroenteritis, and in serious cases it can lead to brain in-
fection and even death. According to the latest EU summary report on
zoonoses, zoonotic agents and food-borne outbreaks (European Food
Safety Authority [EFSA], 2014), 1642 confirmed human cases of listerio-
sis were reported in the European Union in 2012, representing a 10.5%
increase compared with 2011. The EU notification rate was 0.41 cases
per 100,000 population, with the highest member state specific notifica-
tion rates observed in Finland, Spain and Denmark. On average, 91.6% of
the cases were hospitalised. This is the highest proportion of hospitalised
cases of all zoonoses under EU surveillance. A total of 198 deaths due to
listeriosis were reported by 18 member states in 2012, which was the
highest number of fatal cases reported since 2006.
European Regulation (EC) No. 2073/2005 (European Commission
(EC), 2005) lays down the microbiological criteria for certain microor-
ganisms in foods and the implementing rules to be complied with by
food business operators (FBOs) when implementing general and specif-
ic hygiene measures. In relation to L. monocytogenes, this regulation
covers primarily RTE food products, and requires the following: (i) in
RTE products intended for infants and for special medical purposes
L. monocytogenes must not be present in 10 × 25 g; and (ii) in RTE prod-
ucts other than those for infants and special medical purposes different
microbiological criteria apply depending on the ability of the food prod-
uct to support growth of L. monocytogenes. Thus, for RTE foods unable to
support the growth of L. monocytogenes, the levels should be b 100 CFU/
g throughout the shelf-life of the product (n = 5; c = 0). On the other
hand, in RTE foods that are able to support the growth of the bacterium,
L. monocytogenes must not be present in 5 × 25 g samples at the time of
leaving the production plant; however, if the producer can demonstrate,
to the satisfaction of the competent authority, that the product will not
exceed the limit of 100 CFU/g throughout its shelf-life, the level should
be b 100 CFU/g throughout the shelf life of the product (n = 5, c = 0). In
addition, this regulation establishes that the safety of the food is the re-
sponsibility of the FBO who can conduct studies to evaluate the growth
of L. monocytogenes that may be present in the product during the shelf-
life under reasonably foreseeable storage conditions of distribution,
storage and use in order to investigate compliance with the criteria
throughout the shelf-life of the product. This triggers the question on
how the FBO decides if the product is able or unable to support the
growth of L. monocytogenes, and how compliance with the 100 CFU/g
limit throughout the shelf-life can be demonstrated. In this regard, the
Directorate-General of Health and Consumers (DG SANCO) of the
European Commission published a document directed at Food Business
Operators who produce ready-to-eat (RTE) foods aimed to help them to
demonstrate to the satisfaction of the competent authority that their
products comply with the Community Regulation, to understand the
range of different approaches available to help establish a safe product
shelf-life in relation to L. monocytogenes, and to classify their products
into RTE foods in which growth of L. monocytogenes can occur or in
RTE foods in which growth of L. monocytogenes will not occur during
their shelf-life (DG SANCO, 2008).
Determining the ability of foods to support the growth of
L. monocytogenes is not simple since many RTE foods are traditionally
produced in local regions using variable formulations which may have
an impact on the fate of L. monocytogenes. The Food Standards Agency
of New Zealand has recently published guidelines for undertaking chal-
lenge studies (Food Standards Agency New Zealand [FSANZ], 2014),
although this document is not specifically related to L. monocytogenes.
On the other hand, Canada also has guidelines which specifically relate
to L. monocytogenes (Health Canada, 2012). In Europe, in order to facil-
itate the task of performing challenge studies, the European Union Com-
munity Reference Laboratory for L. monocytogenes prepared a technical
guidance document in 2008 in collaboration with seven laboratories,
including six National Reference Laboratories for L. monocytogenes
(European Commission [EC], 2008). This guidance document was
aimed at describing the microbiological procedures for determining
growth of L. monocytogenes using challenge tests in the frame of the
application of the Regulation (EC) No. 2073/2005. The content of this
technical guidance document has been reviewed by Beaufort (2011).
However, feedback from food processors and independent laboratories
indicated a need for the revision of the guidance document and to de-
velop a more user-friendly set of guidelines to facilitate such analyses.
In September 2012 the revision of the “EURL Lm Technical Guidance
document for conducting shelf-life studies on L. monocytogenes in
ready-to-eat foods” commenced. The European Union Community Ref-
erence Laboratory for L. monocytogenes established a working group of
representatives of 10 national reference laboratories, 1 associate nation-
al reference laboratory and 1 laboratory on behalf of a national reference
laboratory, and the updated version of the technical guidance document
has been recently published (European Commission (EC), 2014).
This review article describes the above mentioned, recently published
European guidance document, with special focus on the design of chal-
lenge studies to determine the growth potential of L. monocytogenes on
foods. Particularly, information is given on what a challenge test is,
when one is advisable, the factors to be considered and the laboratory
methodology to be applied when performing a challenge test to deter-
mine the growth ability of L. monocytogenes in a defined food matrix.
Moreover, results of recent research articles applying challenge tests to
determine the growth of L. monocytogenes in a range of foodstuffs are
summarized and discussed. Finally, recommendations for obtaining
data that can contribute to any further revision of the guidance document
are presented.
2. Application of challenge tests on L. monocytogenes in the food
industry following the EU Technical Guidance document
The growth ability of L. monocytogenes in food products may be esti-
mated based on specifications of physico-chemical characteristics of the
product, consultation of the available scientific literature, or predictive
mathematical modelling. However, in most cases growth assessment
will involve laboratory-based studies, so-called challenge tests. A chal-
lenge test can be defined as a laboratory-based study that measures
the growth of L. monocytogenes in artificially contaminated food stored
under foreseeable abuse conditions of transportation, storage at retail
and at consumer level. As a primary objective, challenge tests aim to de-
termine whether or not a particular food product has the ability to sup-
port growth of L. monocytogenes. An indication of the growth potential is
obtained from the difference between the log10 CFU/g at the end of the
shelf-life and the log10 CFU/g at the beginning of the test. When this dif-
ference is greater than 0.5 log10 CFU/g the food is classified into RTE
foods able to support the growth of L. monocytogenes. Alternatively,
 A. Álvarez-Ordóñez et al. / Food Research International 75 (2015) 233–243
when the difference is less than 0.5 log10 CFU/g, the food is classified
into RTE foods unable to support the growth of L. monocytogenes.
Performance of challenge tests is not needed for many food prod-
ucts. Consultation of available scientific literature and specifications
of physico-chemical characteristics of the product will help decide
whether a challenge test is required or not, based on the evidence
that L. monocytogenes does not represent a risk or does not have
the ability to grow in the product (Fig. 1). Indeed, challenge tests
for L. monocytogenes would not be needed for the following food
categories:
- Foods which are intended to be cooked or subjected to any other
bacterial inactivation step before human consumption.
- Foods which have received heat treatment or other processing
effective to eliminate L. monocytogenes, when recontamination is
not possible after this treatment (e.g. products treated in their final
package).
- Fresh, uncut and unprocessed vegetables and fruits, excluding
sprouted seeds (these are classified under primary production).
- Bread, biscuits and similar products.
- Bottled or packed waters, soft drinks, beer, cider, wine, spirits and
similar products.
- Sugar, honey and confectionery, including chocolate and cocoa
products.
- Bivalve molluscs.
- Food grade salt.
- Frozen products.
- Foods with pH ≤ 4.4 or aw ≤ 0.92 or pH ≤ 5.0 and aw ≤ 0.94, conditions
which are already known as unable to support the growth of
L. monocytogenes.
Fig. 1. Decision tree showing the schematic steps to follow to determine whether a challenge study is necessary.
235
Also, historical data on prevalence of L. monocytogenes in the specific
RTE food at the end of shelf-life and particularly on results of durability
studies (the number of samples exceeding 100 CFU/g) and outputs of
predictive microbiology modules may be useful in deciding whether a
challenge test is required or not for a particular foodstuff.
The following factors must be considered when performing a labora-
tory challenge test to assess growth potential by following the updated
version of the EURL Lm Technical Guidance document for conducting
shelf-life studies on L. monocytogenes in RTE foods (Table 1):
(i) Number of batches: the number of batches to be included in the
design of the challenge test will depend on the available infor-
mation on probability of growth and inter-batch variability of
pH and water activity (aw). Predictive microbiology tools such
as growth/no growth boundary modules or “inter-batch variabil-
ity” calculators can be used for this purpose. If the growth prob-
ability is low or the inter-batch variability of pH and aw
regarding the growth of L. monocytogenes can be considered neg-
ligible it is possible to limit the study to one single batch. On the
other hand, if the growth probability and inter-batch variability
are high at least three batches need to be tested.
(ii) Bacterial strains: to account for variation in growth and survival
among strains of L. monocytogenes, challenge tests must be per-
formed with a mixture of at least two strains. One of them
must be a strain with known growth characteristics, while the
other strain/s can be freely chosen and will ideally be originally
isolated from the food product being analysed. This second strain
can also be isolated from environments, outbreaks or can be a
collection strain. The European Union reference laboratory for
L. monocytogenes has recently constituted a set of strains from
236 A. Álvarez-Ordóñez et al. / Food Research International 75 (2015) 233–243

various origins (meat, dairy products, fish) and various
genoserotypes (II and IV). These strains were selected for
their growth ability in harsh conditions of temperature, pH
and aw, according to the literature. The growth of these strains
under harsh conditions (8 °C, pH = 5 or aw = 0.95) has been
characterized and their use is recommended when perfor-
ming challenge tests (European Union Reference Laboratory
for Listeria monocytogenes (EURL Lm), 2013).
(iii) Inoculum preparation: bacterial strains must be firstly inocu-
lated in a non-selective medium (e.g. Brain Heart Infusion
[BHI] broth) incubated at an optimal temperature (e.g. 30 or
37 °C) for the required time to reach the early stationary
phase of growth (e.g. overnight), and then they must be
subcultured in non-selective medium and incubated at a tem-
perature close to the actual storage temperature of the prod-
uct to be tested (e.g. 7 °C, or 10 °C when considering
refrigerated RTE foods) for the required time to reach early
stationary phase. This allows for bacterial adaptation to the
environmental temperature conditions prevailing during the
challenge test in the food product. Extra stresses of relevance
may be also added. Finally, individual cultures must be com-
bined in equal quantities and serial dilutions must be pre-
pared to obtain an inoculum at the expected concentration
to be used for inoculation of the food.
(iv) Food inoculation: the method of inoculation of the food prod-
uct with the cocktail of L. monocytogenes strains must be in
such a way that it does not compromise the intrinsic proper-
ties (physico-chemical characteristics) of the food. For this
reason, the inoculum volume must not exceed 1% of the
mass (or volume) of the test unit. In addition, the inoculation
must mimic realistic scenarios of food contamination by
L. monocytogenes. In order to minimize the measurement un-
certainty, the contamination level must be targeted at around
100 CFU/g. Several methods of inoculation can be considered.
Inoculation can be performed at surface to mimic contamina-
tion of a specific part of the food product along the food chain.

Table 1
Main differences between the European Technical Guidance documents of 2008 (EC, 2008) and 2014 (EC, 2014).

2008 European guidance document 2014 European guidance document

Number of batches At least 3. - If growth probability is low or inter-batch variability of

Choice of strains
Inoculum preparation
Food inoculation
Storage conditions
Analysis of inoculated test units
Analysis of non inoculated test units
Calculation of standard deviation of cell The standard deviation of the results obtained for
numbers (log 10 CFU/g) at day 0
Calculation of the growth potential
A mixture of at least 3 strains. One must be a
reference strain. The other strains must be isolated
from the same or a similar food matrix.
First subculture in a non-selective medium at a
temperature favourable to optimal growth of
L. monocytogenes (37 °C).
Second subculture at a temperature close to the temperature of
the product, in order to adapt the strain to the storage conditions.
The inoculum should not exceed 1% of the
volume of the test unit.
The contamination level must be targeted at 50 CFU/g
and should not exceed 100 CFU/g.
Several methods of inoculation can be considered
depending on the product tested.
- When national information available, the use of this
information is preferred to select the storage time and
storage temperature to be used.
- If no data are available: 8 °C (1/3 of shelf life), 12 °C
(1/3 of shelf life), and 12 °C (1/3 of shelf life).
- Enumeration of L. monocytogenes: at least at the beginning of
the challenge test and at the end of the shelf life of the product
(3 test units at each time) by following the
standard method EN ISO 11290-2.
- Associated microflora: at the start and end of the
challenge test following relevant standard methodology.
- Physico-chemical characteristics of the food
(at least pH and aw): at least at the
beginning and end of the challenge test.
Analysis of 3 test units following relevant standard
methodology for:
- Detection of L. monocytogenes (EN ISO 11290-1);
- Microflora;
- Physico-chemical characteristics of the food
(at least pH and aw).
enumeration of L. monocytogenes on the inoculated batches at
day 0 should not exceed 0.3 log10 CFU/g. If this value is
exceeded the challenge study is unacceptable.
The growth potential (log10 CFU/g) is the difference in the
median of the results at the end of the challenge test and the
median of the results at the beginning of the challenge test.
pH and water activity (aw) is negligible: 1 batch.
- If growth probability or inter-batch variability are
high: at least three batches.
A mixture of at least two strains. One of them must be a
strain with known growth characteristics (EURL Lm strain
collection available to this aim). The other strain/s can be
freely chosen from food, environment, outbreak, or collection.
First subculture in a non-selective medium at an optimal
temperature (e.g. 30 or 37 °C).
Second subculture at a temperature close to the actual
storage temperature of the product.
Inoculum volume must not exceed 1% of the mass
(or volume) of the test unit.
The contamination level must be targeted at around 100 CFU/g.
Several methods of inoculation can be considered
depending on the product tested. The inoculation
procedure should mimic natural contamination.
- When FBO has its own data on the first two stages of the
cold chain (from manufacturing to retail, and storage at retail) or
there exists national information available, the use of this
information is preferred to select the
storage temperature to be used.
- If no data are available: 8 °C (1/3 of shelf life), 12 °C
(1/3 of shelf life), and 12 °C (1/3 of shelf life).
- Enumeration of L. monocytogenes: at least at the
beginning of the challenge test and at the end of the
shelf life of the product (3 test units at each time)
by following the standard method EN ISO 11290-2.
- Associated microflora: at the start and end of
the challenge test following relevant
standard methodology.
- Physico-chemical characteristics of the food
(at least pH and aw): at least at the
beginning and end of the challenge test.
Analysis of 1 test unit following relevant standard
methodology for:
- Detection of L. monocytogenes (EN ISO 11290-1);
- Associated microflora;
- Physico-chemical characteristics of the food
(at least pH and aw);
- Gas concentration.
The standard deviation of the results obtained for
enumeration of L. monocytogenes on the inoculated
batches at day 0 should not exceed 0.5 log10 CFU/g.
If this value is exceeded the challenge study is unacceptable
The growth potential (log10 CFU/g) is the difference in
the median of the results at the end of the challenge
test and the median of the results at the beginning of
the challenge test.
 A. Álvarez-Ordóñez et al. / Food Research International 75 (2015) 233–243
However, for foods considered to be homogeneous (e.g.
ground food) or foods prepared by mixing several materials
(e.g. mixed salad) inoculation “in depth” would be the best
option. Other techniques (e.g. dipping) can be used if it can
be demonstrated that the intrinsic properties of the food are
not changed. Packaged foods can be removed from their pack-
aging, inoculated and then repacked under similar gas condi-
tions as an unopened pack (consumer pack), or maintained
in its packaging and contaminated through a septum.
(v) Storage conditions: conditions of storage (temperature, time
and package) of inoculated foods must comply with the condi-
tions to which the product is most likely to be subjected in the
food chain, until its final consumption. Storage time must be
equivalent to the shelf life of the food product. Regarding stor-
age temperature, abuse temperature(s) must be considered in
order to avoid underestimation of L. monocytogenes growth.
When the FBO has its own data on the first two stages of the
cold chain (from manufacturing to retail, and storage at retail)
or national information is available, the use of this information
is preferred to select the storage times/temperatures to be
used. In that case, the 75th percentile of the observed data
should be used. However, if no data are available and the
shelf-life of the product is ≤ 21 days the following default con-
ditions must be used: 8 °C for one third of the total shelf life of
the product (representing from manufacturing to retail), 12 °C
for the second third of the total shelf life of the product
(representing storage at retail), and 12 °C for the last third of
the total shelf life of the product (representing consumer stor-
age). If the shelf life is N 21 days the following default storage
conditions must be used: 8 °C for 7 days (manufacturing to re-
tail), 12 °C for half of the remaining shelf life (storage at retail)
and 12 °C for the other half of the remaining shelf life (storage
at consumer).
(vi) Analysis of inoculated test units: numbers of L. monocytogenes
must be determined at least at the beginning of the challenge
test and at the end of the shelf life of the product by following
the standard method EN ISO 11290-2 for enumeration of
L. monocytogenes. Additionally, further test points can be
included in the experimental design in order to detect poten-
tial peaks in growth/inactivation across the shelf life. The use
of alternative analytical methods is acceptable when the
methods are validated against the reference method and if a
proprietary method, certified by a third party in accordance
with the protocol set out in EN/ISO Standard 16140 or other
internationally accepted similar protocols, is used. Other
methods shall be validated according to internationally ac-
cepted protocols and their use authorised by the Competent
Authority. Associated microflora of the product must also be
enumerated at the start and end of the challenge test follow-
ing relevant standard methodology for the organisms and
food type concerned. Physico-chemical characteristics of the
food (at least pH and aw [alternatively NaCl content or mois-
ture]) must be also determined. In the case of foods packed
under modified atmosphere or vacuum packed it is desirable
to also monitor gas atmosphere at day “0” and day “end” of
the challenge test.
(vii) Analysis of non-inoculated test units: non-inoculated test
units must be checked for the presence of L. monocytogenes
by following the standard method EN ISO 11290-1 for detec-
tion of L. monocytogenes. Only those batches showing absence
of L. monocytogenes must be subjected to artificial contamina-
tion and challenge testing. Some uninoculated samples can be
kept and in case of a positive detection of L. monocytogenes,
durability studies on naturally contaminated food may be un-
dertaken by determining bacterial numbers over time (under
foreseeable storage conditions) by following the EN ISO11290
237
methodology. Associated microflora of the product and
physico-chemical characteristics of the food must be also de-
termined for non-inoculated samples.
(viii) Calculation of growth potential: for each batch, the growth
potential (in log10 CFU/g) is estimated as the difference be-
tween the median of L. monocytogenes numbers at the end
of the challenge test and the median of L. monocytogenes
numbers at the beginning of the challenge test. The highest
value obtained among all tested batches is retained as the
growth potential. When the growth potential calculated is N
0.5 log10 CFU/g it is considered that the food product supports
growth of L. monocytogenes.
3. Available literature on application of challenge tests
Since the publication of the first EURL Lm Technical Guidance docu-
ment for conducting shelf-life studies on L. monocytogenes in RTE foods
in 2008, a number of studies have been conducted applying challenge
tests to determine the ability of a wide range of foodstuffs to support
L. monocytogenes growth (Table 2). However, strict adherence to the
Guidelines was not observed in most cases — one or more of the criteria
were modified. This section of the manuscript summarises the main
findings of recent research articles on challenge studies.
3.1. Challenge tests in vegetables and salads
Several different types of vegetables and salads have been subjected
to challenge testing in recent years, with variable results.
Uyttendaele et al. (2009) analysed 182 mayonnaise-based deli salads
(including egg salads, meat salads, fish salads and vegetable salads) after
inoculation in-depth with a cocktail of three L. monocytogenes strains,
packing under air, or modified atmosphere conditions as indicated by
the FBO, and incubation for the actual shelf life of the product at 4
or 7 °C or a variable temperature schedule (e.g. 1/3 of shelf life at
4 °C and 2/3 of shelf life at 7–8 °C) as defined by the FBO. Growth
of L. monocytogenes was supported by 18 of the 182 salads tested.
The majority of the deli-salads subjected to challenge testing were
formulated to have an acid pH (usually pH 5.0–5.5) in combination
with an aw of 0.96–0.98. Under these conditions, no growth of
L. monocytogenes was observed during prolonged shelf-life (up to
35–42 days) at 4–7 °C. Mayonnaise-based deli salads often contain
organic acids, predominantly acetic acid. This acid has a strong inhib-
itory effect on L. monocytogenes growth even at very low concentra-
tions [0.2% (w/w)] (Vermeulen et al., 2007). Other organic acids,
such as sorbic and benzoic acid, are often added to deli-salads as
chemical preservatives and may prevent L. monocytogenes growth.
However, in salads with pH ≥ 5.5 and aw ≥ 0.97 there was clearly a po-
tential for L. monocytogenes growth.
Skalina and Nikolajeva (2010) performed challenge tests on
shrimp–tomato salad, smoked ham salad and garlic cheese salad artifi-
cially inoculated individually with three strains of L. monocytogenes
(one reference strain and two field isolates) and stored at refrigerated
temperatures (3 °C and 7 °C) for 48 h. They showed that all three salads,
and especially garlic cheese salad and smoked ham salad, were able to
support L. monocytogenes growth, and that the growth potential in-
creased with increasing temperature.
Sant'Ana, Barbosa, Destro, Landgraf, and Franco (2012) used chal-
lenge tests to evaluate the ability of different types of RTE vegetables (es-
carole, collard green, spinach, watercress, arugula, grated carrot, green
salad, and mix for yakisoba) to support growth of L. monocytogenes.
RTE vegetables were inoculated with a cocktail of five strains isolated
from RTE vegetables marketed in Sao Paulo, Brazil, and stored at refriger-
ation (7 °C) and abuse temperature (15 °C). All RTE vegetables, except
carrots, supported L. monocytogenes growth and the highest growth
 238
Table 2
Methodological approach followed in research publications published to date on challenge tests of L. monocytogenes in RTE foods.

Reference Product tested Number of batches Choice of strains Inoculum preparation Food inoculation Storage conditions Microbiological
analysis of inoculated
test units

Mejholm et al. (2008, Shrimps in brine Not stated. Four L. monocytogenes strains Two subcultures at 25 °C for 24 Shrimps in brine: 0.1% Shrimp in brine, and brined Enumeration of L. monocytogenes
2012) and brined and isolated from seafood. h and 10 °C for 2 to 3 days. (vol/wt) of the cocktail (105 and drained MAP shrimp according to ISO 11290-2.
drained shrimps. CFU ml−1). Brined and drained were stored at 7–8 °C or
shrimps: 1% (vol/wt) of the 15 °C.
cocktail (104 CFU ml−1).
Uyttendaele et al. Mayonnaise-based Not stated. Three L. monocytogenes Subculture for 24 h at 30 °C. Inoculation (0.3–1.0 ml) on Packed samples (air, Enumeration of L. monocytogenes

A. Álvarez-Ordóñez et al. / Food Research International 75 (2015) 233–243

(2009) deli-salads, cooked strains. the surface (meat and fish vacuum or modified according to ISO 11290-2 using a
meat products and product) or as in depth atmosphere) kept for their reduced detection limit.
smoked fish. (deli-salad) inoculation in ca. shelf-life at 4 or 7 °C or a
100 g of food sample to obtain variable temperature
a level of ca. 50–100 CFU/g. schedule (1/3 of shelf-life
at 4 °C and 2/3 of shelf-life
at 7–8 °C as defined by
FBO).
Angelidis et al. (2010) Processed cheese. Two batches. Three L. monocytogenes strains Two subcultures at 30 °C for 24 Spreading of 40 ml of the Modified atmosphere Enumeration of L. monocytogenes
(one type strain, one clinical h and 30 °C for 20 h. cocktail over 25 g cheese packed samples were according to ISO 11290-2.
isolate, one processed cheese samples to achieve three levels stored at 4, 12 or 22 °C.
5
isolate). of inoculation: high (6 × 10
CFU/g); medium (6 × 103
CFU/g); low (102 CFU/g).
Garrido et al. (2010) Sliced ready-to-eat One batch. One L. monocytogenes strain Subculture at 30 °C for 18 h. Inoculation (1 ml) on 25 g of Packaged samples stored at Enumeration of L. monocytogenes
ham. isolated from sliced-cooked ham to give a concentration 5 °C and 9 °C for 15 days. according to ISO 11290-2. Total
ham at retail. between 5 and 10 CFU/g. microorganisms, Lactic acid bacteria.
Skalina and Shrimp–tomato Not stated. Three L. monocytogenes strains Two subcultures at Inoculation (0.1 ml) of 10 g of Storage at refrigerator Enumeration of L. monocytogenes
Nikolajeva (2010) salad, smoked ham (a type strains, one isolated 37 °C for 12 h. salad to obtain a level of 20 to temperatures (3 °C and 7 according to ISO 11290-2. TVC,
salad and garlic from frozen Pollock loins, and 40 CFU/g. °C) for 48 h. staphylococci and Enterobacteriaceae.
cheese salad. one isolated from sausages).
Augustin et al. (2011) Pork pie, smoked Pork pie: 1; One L. monocytogenes strain. Exponential cells: 37 °C — 16 Inoculation at the surfaces or Stored at 8 °C for the Enumeration of L. monocytogenes
herring, sliced herring: 4; ham: 7; h, 37 °C — 8 h, 9 °C — 6 days homogeneous contamination shelf life according to ISO 11290-2. Aerobic
cooked ham, chicken: 2; surimi (BHI). Starved cells: 30 °C — depending on the type of microorganisms and mesophilic lactic
cooked chicken, salad: 3 batches. 24 h, 25 °C — 20 h (TSBYE), 30 product. acid bacteria.
and surimi salad. °C — 24 h (0.85% NaCl).
Vermeulen et al. Smoked salmon. Three batches. Three L. monocytogenes strains Two subcultures at 37 °C for 24 Inoculation (200 μl) of 200 g Storage for 8 days at 2 °C, Enumeration of L. monocytogenes
(2011) (one from cheese, one from h and at 7 °C until the early salmon samples with the followed by 10 days at 4 °C according to ISO 11290-2. Total
pate, one from stationary-phase. cocktail to obtain a and 13 days at 8 °C as psychrotrophic count, LAB and
tuna-deli-salad). concentration of ca. 50 CFU/g. agreed upon with the FBO. Enterobacteriaceae.
Kang et al. (2012) Cold-smoked Not stated. Four L. monocytogenes strains 37 °C — 18 h in BHI, 16 °C — Spreading of the bacterial Vacuum-packed samples L. monocytogenes: spiral plating onto
salmon. (two from RTE salmon, one 24 h in a defined minimal suspension to achieve a final were stored at 7 °C for 30 Oxford agar. Lactic acid bacteria.
from RTE meat and one from a medium, and 16 °C — 24 h in concentration of 104 CFU/g. days.
human skin lesion). the defined medium.
Sant'Ana et al. (2012) RTE vegetables — Not stated. Five L. monocytogenes strains Two subcultures at Spot inoculation (0.5 ml) of Packages (modified L. monocytogenes: homogenizing 25 g
escarole, collard isolated from RTE vegetables. 37 °C for 24 h. portions of 25 g of each RTE atmosphere) stored at with 225 ml of 0.1% peptone water,
green, spinach, vegetable. Final concentration: three different conditions: I following decimal dilutions and
watercress, arugula, 103 CFU/g. (100% of shelf-life [6 days] inoculation on Oxford selective agar.
grated carrot, green at 7 °C), II (30% at 7 °C and
salad, and mix for 70% at 15 °C) and III (100%
yakisoba. at 15 °C).
Bernini et al. (2013) Blue-veined Not stated. Five L. monocytogenes strains. Two subcultures at Spread inoculation of the rinds Storage at either 4 °C or 8 Enumeration of L. monocytogenes
cheeses. 37 °C for 18 h. of cheese slices to a final level °C for 55 days. according to ISO 11290-2. Mesophilic
of between 1 log CFU g−1 and bacteria, yeasts and moulds, LAB,
2 log CFU g−1. Enterobacteriaceae, coliforms and
E. coli.
Daelman et al. (2013) Paella. Three batches. Three L. monocytogenes strains Two subcultures at 37 °C for 24 200 μl of the cocktail dispersed Modified atmosphere Enumeration of L. monocytogenes
(one clinical isolate and two h and at 7 °C until the early across the surface of the paella packaged paella stored at 4 according to ISO 11290-2. Total
cheese isolates). stationary-phase. until an inoculum level of ca. °C until the end of shelf life psychrotrophic aerobic count, LAB,
50 CFU/g. 6 days after purchase. yeasts and moulds, and B. cereus.
Everis and Betts Sliced cooked ham. Industry approach: Three L. monocytogenes strains Industry approach: 30 °C for Inoculation (0.1 ml) of 90 g Industry approach: 8 °C for Enumeration of L. monocytogenes
(2013) one batch. AFFSA (one type strain, one chicken 24 h. AFFSA approach: 37 °C through a double sided foam 21 days. AFFSA approach: 8 according to ISO 11290-2.
approach: three isolate, one meat factory for 24 h then 5 °C for 7 days. pad on the outside of the pack. °C for 7 days, then 12 °C for
batches. isolate). Industry approach: level of 103 14 days.
CFU/g. AFFSA approach: level
of 50–100 CFU/g.
Grassi et al. (2013) Cheese and One batch. Three L. monocytogenes strains Not stated. Inoculation of the sauce with 1 Storage for 31 days at two Enumeration of L. monocytogenes
mushroom sauces (one from a soft cheese, one ml of the cocktail in order to different temperatures, 4 according to ISO 11290-2. Lactic acid
for pasta. from Gorgonzola cheese, one reach a final concentration of °C and 8 °C. bacteria.

A. Álvarez-Ordóñez et al. / Food Research International 75 (2015) 233–243

from a meat product). 103 CFU g−1.
Leong et al. (2013) Whole and sliced Three batches. Three L. monocytogenes strains Subculture at 37 °C for 24 h. Mushrooms: 500 ml of the Mushrooms: 10 days at 8 Enumeration of L. monocytogenes
mushrooms, (one clinical isolate, one cocktail poured into 200 g and and 15 °C. Mushroom according to ISO 11290-2.
mushroom casing persistent strain, one isolate mixed for 15 min. Mushroom casing and substrate: 20
and substrate. from a mushroom production casing and substrate: 10 ml of days at 20 °C.
facility). cocktail added to 200 g and
blended for 5 min.
Manios et al. (2013) Romaine lettuce Two batches. One L. monocytogenes strain Two subcultures at Spot-inoculation of 10 g at low Storage at 8 °C. L. monocytogenes: enumeration on
and white cabbage. (type strain). 30 °C for 24 h. (−1 to −0.4 log CFU/g) or ALOA agar. TVC, Pseudomonas spp.,
high (2 log CFU/g) level. Enterobacteriaceae.
Samapundo et al. Cooked ham and Not stated. One L. monocytogenes strain Two subcultures at 30 °C for 24 Ham: spreading (50 ml) of 50 Modified atmosphere Enumeration of L. monocytogenes
(2013) white sauce isolated from cooked ham. h and 30 °C for 16 h followed g to a level of ~102 CFU/g. packaged ham samples and according to ISO 11290-2. Total
products with by incubation at 7 °C for 6–8 h. White sauce: inoculation of white sauce samples were aerobic and anaerobic plate count,
reduced NaCl 200 g samples to a level of stored at 7 °C. yeasts and moulds, LAB.
levels. ~103 CFU/g.
Sant'Ana et al. (2013) Fresh Lettuce Two batches. Three L. monocytogenes strains Two subcultures at Vegetables were soaked in the Packages (modified L. monocytogenes: homogenizing 25 g
(different varieties) isolated from RTE vegetables. 37 °C for 24 h. cocktail for 15 min and then atmosphere) stored at four with 225 ml of 0.1% peptone water,
and collard greens. were spun in a sanitized salad different conditions: I following decimal dilutions and
spinner. Final concentration: (100% of shelf life [6 days] inoculation on Oxford selective agar.
101–102 CFU/g. at 7 °C), II (70% at 7 °C and
30% at 15 °C), III (30% at 7
°C and 70% at 15 °C), IV
(100% at 15 °C).
Wemmenhove et al. Gouda cheese. One batch per Three L. monocytogenes strains Not stated. L. monocytogenes strains were Storage at 12 °C for up to L. monocytogenes: dilutions in
(2013) strain. (one from cheese, one from a added to separate batches of 52 weeks. peptone physiological NaCl and
cheese factory, and one type milk before cheese making to a plating on PALCAM-selective agar.
strain). final level of approximately
107 CFU ml−1.

239
240 A. Álvarez-Ordóñez et al. / Food Research International 75 (2015) 233–243
potential was observed for collard greens and arugula stored at 15 °C for
6 days. That research group also determined the effects of different stor-
age scenarios [100% of the shelf life (6 days) at 7 °C; 70% of the shelf life at
7 °C and 30% at 15 °C; 30% of the shelf life at 7 °C and 70% at 15 °C; 100% of
the shelf life at 15 °C] on the growth ability of L. monocytogenes in RTE
mixes of iceberg and crisp lettuces and collard greens artificially contam-
inated to achieve 101 and 102 CFU/g with a cocktail of three strains isolat-
ed from RTE vegetables in Brazil. The samples were packed under
modified atmosphere and in perforated film (Sant'Ana, Landgraf,
Destro, & Franco, 2013). They observed that both lettuce and collard
greens supported the growth of L. monocytogenes although the growth
observed was strongly dependent on the storage temperature. Thus,
even contamination as low as 101 CFU/g led to high populations when
temperature abuse during storage occurred (15 °C).
Manios, Konstantinidis, Gounadaki, and Skandamis (2013) studied
the growth variability of low or high populations of L. monocytogenes in
fresh-cut lettuce and cabbage. Salads were inoculated with a few (1–4)
or 1000 cells/sample of one single L. monocytogenes strain and stored at
8 °C. With an inoculum of 1000 cells/sample, cell numbers increased
with limited variation (SD b 0.5 log CFU/g) on vegetable salads, as op-
posed to the great variability (b0.7–3.4 log CFU/g) in the growth observed
from an inoculum of 1 to 4 cells/sample. The total logarithmic increase of
L. monocytogenes on the salads ranged from 1.8 to 2.1 log CFU/g for high
population samples and 2.7 to 3.4 log CFU/g for low population samples.
The authors concluded that fail-dangerous implications may derive from
challenge tests with unrealistically high inocula.
Leong, Alvarez-Ordóñez, Guillas, and Jordan (2013) evaluated
growth of L. monocytogenes on mushrooms. These authors immersed
three independent batches of whole mushrooms and sliced mushrooms
into a three-strain mixture of L. monocytogenes to achieve a concentra-
tion of about 100–1000 CFU/g, and incubated the artificially contami-
nated batches at 15 °C. They demonstrated that both sliced and whole
mushrooms supported growth of L. monocytogenes. In a subsequent ex-
periment, Leong, Alvarez-Ordóñez and Jordan (2015) have shown that,
using a different inoculation procedure (surface spread rather than dip-
ping), whole mushrooms did not support growth of L. monocytogenes.
3.2. Challenge tests in meat products
Various cooked meat products have been subjected to challenge
tests for determination of L. monocytogenes growth, and, in most cases,
growth of L. monocytogenes was observed.
Uyttendaele et al. (2009) performed challenge tests with three
L. monocytogenes strains inoculated on the surface of 92 cooked
meat products including pâté, cooked ham, cooked pork tongue,
cooked pork meat and luncheon meat, and detected growth of
L. monocytogenes on 61 of the 92 meat products tested. In general,
two factors influenced growth potential in cooked meat products,
i.e. the a w and the presence of CO2 in the packaging atmosphere.
There was significant growth of L. monocytogenes in the investigated
cooked meat products at aw values higher than 0.96 unless the
cooked meat samples were packed under modified atmosphere. For
example, if packed under modified atmosphere, L. monocytogenes did
not grow on pâté (aw 0.961) during 42 days. These authors also concluded
that cooked meat products tend to support the growth of
L. monocytogenes mainly because of their intrinsically higher pH (6.0–6.5).
Garrido, García-Jalón, and Vitas (2010) monitored the growth of a
single L. monocytogenes isolate in sliced-cooked ham using low inoc-
ulum (b10 CFU/g) and different storage temperatures (5 °C and
9 °C), representative of domestic refrigerator temperatures. The crit-
ical concentration of 100 CFU/g L. monocytogenes was reached after
the second and the third day of storage at 9 °C and 5 °C, respectively.
When storage time was extended to 5 days, the pathogen achieved
values of N 103 CFU/g at both storage temperatures.
Augustin et al. (2011) carried out challenge testing in vacuum-
packed pork pies, sliced cooked ham packed under modified
atmosphere, and cooked chicken artificially surface inoculated at
~102 CFU/g with a single L. monocytogenes strain. Afterwards, contami-
nated food samples were packed and stored at 8 °C. Growth of
L. monocytogenes was supported by all meat samples tested.
Everis and Betts (2013) used sliced cooked ham to compare two ap-
proaches for performing challenge testing. In the first approach the pro-
tocol of the 2008 European Technical Guidance document on shelf life
studies for L. monocytogenes in RTE foods was followed. This protocol re-
quired testing of three batches of product, an inoculum level of no more
than 100 CFU/g, cultures pre-adapted to chill conditions and a storage
regime of 7 days at 8 °C followed by 14 days at 12 °C. The second ap-
proach was a more standard industry protocol using a single batch of
product, an inoculum level of between 100 and 1000 CFU/g, cultures
grown overnight at optimal temperature and a storage regime of
21 days at 8 °C. In both cases, a cocktail of three L. monocytogenes strains
(one reference strain, an isolate from chicken, and an isolate from a
meat factory) was used to inoculate the sliced cooked ham. The growth
potential, calculated as the median log CFU/g at end of shelf-life minus
the median log CFU/g at day 0, was N6.0 log CFU/g for the first approach
and N 4.0 log CFU/g for the industry approach between days 0 and 21,
while between days 0 and 3 the growth potential was N0.5 log CFU/g
for both approaches. The results of the trial showed that the standard in-
dustry approach gave similar results to that of the more complex
European guidance approach with regards to growth potential, lag
time and time for a 2 log increase.
Samapundo et al. (2013) performed challenge test studies in cooked
ham products made with reduced NaCl levels (28% less NaCl than com-
monly produced cooked ham). Cooked ham samples were surface-
spread inoculated with a single L. monocytogenes strain, originally isolated
from cooked ham, at a level of ~102 CFU/g. After inoculation, the samples
were immediately placed in high oxygen barrier bags and packaged with
an atmosphere of 30% CO2 and 70% N2. After packaging, the samples were
incubated at 7 °C. The results showed that both the reference cooked ham
and the cooked ham with reduced NaCl levels supported growth of
L. monocytogenes and that there were no differences in the growth pro-
files of L. monocytogenes observed in both cooked ham products.
3.3. Challenge tests in seafood
Most of the studies on challenge tests of L. monocytogenes carried out
in seafood have been made in smoked fish, although other seafood
products, such as brined products, have been also analysed.
Challenge testing of smoked fish by Uyttendaele et al. (2009)
showed growth of L. monocytogenes in 12 of 25 samples inoculated
with three different strains and stored for 3–4 weeks at 4 °C. Most of
the smoked fish samples analysed by these authors had a rather neutral
pH (6.0–6.5) combined with overall quite high aW values (0.96–0.98)
(except for one with an aW of ca. 0.94), which represent favourable
physico-chemical conditions for the growth of L. monocytogenes. On
the other hand, when smoked fish presented pH values of 5.5–6.0 com-
bined with lower aW values of 0.93–0.94 a growth limitation of
L. monocytogenes was found. Uyttendaele et al. also subjected 45 non-
inoculated smoked fish samples (13 of which were naturally contami-
nated by L. monocytogenes) to durability studies, and observed that
L. monocytogenes numbers exceeded 100 CFU/g in only one sample
after storage until the end of shelf-life.
Mejholm et al. (2008) performed challenge tests to examine the
growth of L. monocytogenes in brined shrimp. These authors used
shrimp in brine as well as brined and drained shrimp in modified atmo-
sphere packaging (MAP) produced using different brine recipes to study
the effect of preserving parameters such as organic acids (benzoic, citric
and sorbic acids, or acetic, citric and lactic acids), pH and salt. They inoc-
ulated samples with a mixture of four L. monocytogenes isolates pre-
viously obtained from seafood. After inoculation they packed brined
and drained shrimp samples in a modified atmosphere initially con-
taining 40% CO2 and 60% N2, and stored the samples at 7–8 °C for the
 A. Álvarez-Ordóñez et al. / Food Research International 75 (2015) 233–243
shelf-life of the product. They reported that shrimp in brine with benzoic,
citric and sorbic acids prevented growth of L. monocytogenes during more
than 40 days at 7 °C when the preserving parameters resembled those of
commercial products. However, they showed that small changes in the
preserving parameters and, particularly, reduced concentrations of
benzoic acid led to growth of L. monocytogenes in brined shrimp. Similar-
ly, this research group also performed challenge tests using mixtures of
Lactobacillus sakei (4 isolates), L. monocytogenes (4 isolates), Salmonella
Enteritidis, Salmonella Weltevreden and Staphylococcus aureus (2 iso-
lates) in brined shrimps prepared by using two different types of brine
(the first containing sodium acetate, sodium lactate and sodium chloride;
the second containing sodium benzoate, citric acid, potassium sorbate
and sodium chloride) (Mejholm, Devitt, & Dalgaard, 2012). Following
marination, they packed brined and drained shrimps in a modified atmo-
sphere initially containing 40% CO2 and 60% N2, and stored samples at 7
and 15 °C. These experiments showed that L. monocytogenes was not
able to grow at 7 °C in brined and drained shrimp that resembled com-
mercial products with either acetic and lactic acids or benzoic, citric
and sorbic acids. However, reducing the concentration of acetic and lactic
acids by 50% or 75% resulted in relatively fast growth.
Augustin et al. (2011) monitored the growth of L. monocytogenes in
vacuum-packed smoked herring and surimi salad after inoculation at
~ 102 CFU/g with a single L. monocytogenes strain and incubation at
8 °C, and reported growth of the pathogen in both products tested.
Vermeulen, Devlieghere, De Loy-Hendrickx, and Uyttendaele (2011)
used a case study for cold smoked salmon in order to evaluate the
2008 EU-Technical Guidance document on shelf-life studies for
L. monocytogenes on RTE foods. They inoculated three batches of
cold smoked salmon with a cocktail of three L. monocytogenes strains.
Samples were then repacked under vacuum and incubated under a
reasonably foreseen temperature profile, as agreed upon with the
FBO, i.e. 8 days at 2 °C (internal storage at the production factory),
10 days at 4 °C (storage at retail) and 13 days at 8 °C (storage in a do-
mestic refrigerator). The results showed that cold smoked salmon
had the ability to support growth of L. monocytogenes, with a 1.3 to
2.8 log increase in L. monocytogenes at the end of shelf-life. They
also concluded that interbatch variability was significantly higher
than intrabatch variability and that the best approach to use (simple
challenge tests determining numbers at start and end of shelf-life vs
modelling and predictive microbiology) will depend on the particu-
lar context. Thus, they recognized simple challenge tests as cost-
effective tests for small and medium enterprises that can be set up
rather rapidly, but that provide a single outcome and has to be re-
established if changes occur in the composition or the production
process. On the other hand, they described the modelling approaches
as more expensive, since they require larger set ups and more com-
plicated data analysis, but more advantageous, since they provide
more information and allow prediction of growth/no growth under
different circumstances.
Kang et al. (2012) investigated the effect of curing method and
freeze-thawing of cold-smoked salmon on subsequent growth of genet-
ically diverse strains of L. monocytogenes (inoculated after freeze-
thawing). Wet-cured and dry-cured cold-smoked salmon fillets were
cut into slices of ~25 g and surface-spread inoculated with individual
L. monocytogenes strains at a final concentration of 104 CFU/g. Inoculat-
ed salmon slices were air dried, vacuum packed and incubated at 7 °C.
Cold smoked salmon supported growth of L. monocytogenes and
freeze-thawing the salmon fillets prior to inoculation led to pronounced
growth of L. monocytogenes at 7 °C. The authors also observed variation
in growth among L. monocytogenes strains, which indicates the signifi-
cance of assessing multiple strains.
3.4. Challenge tests in cheese
Some authors have monitored the growth ability of L. monocytogenes
in RTE cheese after either inoculation of milk, prior to manufacture, or
241
surface inoculation of the cheese with L. monocytogenes. Other studies
have followed growth/survival of naturally occurring L. monocytogenes.
In most cases, growth of L. monocytogenes was not observed in RTE
cheese.
Angelidis, Boutsiouki, and Papageorgiou (2010) inoculated batches of
processed cheese independently with three different L. monocytogenes
strains (one reference strain, one clinical isolate associated with a
soft-cheese listeriosis outbreak and one strain isolated from proc-
essed cheese). The inoculum was spread drop-wise as uniformly as
possible over the cheese mass to yield three levels of inoculation
(high [6 × 105 CFU/g]; medium [6 × 103 CFU/g]; low [102 CFU/g]),
and afterwards the cheeses were packed under a controlled atmosphere
environment (30% CO2/70% N2) in order to mimic the product's commer-
cial atmospheric packaging conditions. Cheeses were stored at 4, 12 or
22 °C. Growth of L. monocytogenes was not observed by Angelidis et al.
in any of the experimental trials (experiments involving different combi-
nations of strains, inoculum level and storage temperature) throughout
the storage period. On the contrary, L. monocytogenes populations de-
creased over time at a rate that was strain- and storage temperature-
dependent. Kinetics of L. monocytogenes cell reduction in processed
cheese were subsequently characterized through the use of modelling
tools (Angelidis, Papageorgiou, Tyrovouzis, & Stoforos, 2013).
Wemmenhove, Stampelou, van Hooijdonk, Zwietering, and Wells-
Bennik (2013) inoculated three different L. monocytogenes isolates (a
cheese isolate, an environmental isolate from a cheese factory and a
reference strain) into cheese milk, and following starter-induced curd
formation, ripened the resulting Gouda cheeses for a period of up to
12 months. L. monocytogenes viable counts were determined at the dif-
ferent stages of cheese making and ripening. They observed that during
curd formation, viable numbers of L. monocytogenes increased by
0.5 log CFU/g, resulting from entrapment in the curd. However, no
growth was observed during the first 8 weeks of ripening and a signifi-
cant decline in the viable numbers of L. monocytogenes was observed in
Gouda cheese after ripening for more than 8 weeks.
Bernini et al. (2013) performed challenge testing in blue-veined
cheese rinds artificially contaminated with a cocktail of five
L. monocytogenes strains originally isolated from the same cheese
type. Inoculation was carried out at two contamination levels (101
and 102 CFU/g) by spreading an appropriate dilution of the cocktail
of strains on the rinds of 25 slices of cheese and allowing them to
dry before incubation at either 4 °C or 8 °C for 55 days. Increases in
L. monocytogenes numbers of 1.80 and 1.70 log CFU/g after 10 days
and of 2.48 and 2.93 log CFU/g after 30 days at 4 °C and 8 °C, respec-
tively, were observed.
Dalmasso and Jordan (2014) monitored the growth of L.
monocytogenes in two independent batches of naturally contaminated
Cheddar cheese throughout a five month ripening period. For the first
batch, L. monocytogenes was detected by enrichment during the first
three months of ripening, but bacterial numbers were always below
the detection limit (10 CFU/g). For the second batch, numbers of
L. monocytogenes never exceeded 20 CFU/g for the first two months of
ripening, while at three months of ripening numbers dropped to levels
below the quantification limit, although L. monocytogenes was still de-
tected by enrichment. After five months of ripening L. monocytogenes
could not be detected in either cheese batches by direct count or by en-
richment, which demonstrated that growth of L. monocytogenes is not
supported by that farmhouse Cheddar cheese.
3.5. Challenge tests in other prepared meals
A study by Daelman, Jacxsens, Devlieghere, and Uyttendaele
(2013) evaluating the microbial safety and quality of various types
of cooked chilled foods reported challenge tests for three batches of
a ready-to-heat type paella composed of meat, rice, chicken and veg-
etables. Paella batches were surface inoculated with a cocktail of
three L. monocytogenes isolates, packed under modified atmosphere
242 A. Álvarez-Ordóñez et al. / Food Research International 75 (2015) 233–243
consisting of a 50:50 mixture of N2 and CO2 and stored at 4 °C (as rec-
ommended on the label) until the end of shelf life (6 days). Numbers
of L. monocytogenes increased by 0.63 log CFU/g at the end of the in-
cubation period, which means that the product supported the
growth of L. monocytogenes even at temperatures of 4 °C.
Grassi, Nucera, Lomonaco, and Civera (2013) determined the
growth potential of L. monocytogenes in two types of fresh unpasteur-
ized sauces for pasta (cheese and in mushroom sauces). They inoculated
both types of sauce with a mixed bacterial suspension made up of three
different L. monocytogenes strains originally isolated from foods (one
from a soft cheese, one from Gorgonzola cheese, and one from a meat
product) at ~ 103 CFU/g, and incubated the contaminated samples at
two different temperatures, 4 °C and 8 °C. At 4 °C, the increase in
L. monocytogenes viable counts was b0.5 log CFU/g for both sauces,
while at 8 °C the cheese sauce supported growth of L. monocytogenes,
with increases in bacterial population by N0.5 log CFU/g.
Samapundo et al. (2013) assessed the effect of NaCl reduction or
replacement on the growth of L. monocytogenes in white sauce. White
sauce, composed of water, milk powder, wheat flour, modified starch,
margarine, NaCl and potassium sorbate, was inoculated with a single
strain of L. monocytogenes to achieve an initial level of 103 CFU/g. Ino-
culated sauces were incubated at 7 °C and growth was monitored over
time. The results showed that white sauce supported growth of
L. monocytogenes and that the NaCl reduction or replacement did not
significantly influence bacterial growth.
4. Concluding remarks and recommendations
Determination of the ability of RTE foods to support the growth of
L. monocytogenes under reasonably foreseeable storage conditions is
very important for Food Business Operators in order to demonstrate
compliance with the criteria laid down under European Regulation
(EC) No. 2073/2005. However, few of the challenge studies detailed
above have strictly followed all aspects of the EURL Guidelines available
at the moment of their publication, i.e. the EURL Guidelines of 2008.
While these may be valid research studies, the failure to follow the
Guidelines means that they are of limited value to the competent au-
thorities, who have the ultimate decision on which category a food fits
into, and to FBOs, who cannot use the published results to infer
L. monocytogenes growth potential and as a consequence are forced
into carrying out expensive challenge studies. Challenge tests described
in the literature so far in a range of meat, seafood, dairy, vegetable and
prepared meal products have been conducted following significantly
different methodologies. While cocktails of three to five strains
have been normally used, a couple of studies have used a single
L. monocytogenes isolate or have inoculated various strains individually
into separate batches. Temperature and time of incubation for the inoc-
ulum preparation varied widely among studies. While some authors
grew the bacterial strains at an optimum temperature, others per-
formed an adaptation step at low temperatures of ≤ 10 °C. Surface in-
oculation with a low volume of inoculum was the preferred method of
inoculation, but other methods such as deep inoculation, dipping or im-
mersion into the inoculum suspension, have been also employed. Stor-
age time and temperature following inoculation also differed among
studies and were generally agreed with the FBO. Therefore, although
European regulation permits the use of scientific literature to estimate
L. monocytogenes growth ability on particular foodstuffs, the lack of
available studies carried out following a harmonized approach and
conforming to the EU guidance documents impedes its utilization
with this aim. This lack of harmonization, in addition to the fact that
data obtained from challenge studies carried out on behalf of FBOs be-
long to the FBOs themselves and are not generally published, leads to
an information and knowledge void.
Although the recently published European guidelines are undefined
to some extent when describing the methodology to be followed for
some of the processes (e.g. inoculation of the food), their application
will guarantee a more harmonized approach and will make the compar-
ison of results among laboratories easier. Future investigations
analysing the growth of L. monocytogenes in particular foodstuffs should
therefore be carried out following these guidelines if they are to be valid
from a regulatory viewpoint. Nevertheless, studies focused on compar-
ing the proposed methodology with any alternative and simpler meth-
odology are also very valuable (for example, Everis & Betts, 2013) and
could contribute to any further revision of the guidance document in
the future. For that, both the standard methodology and the alternative
methodology must be followed in parallel, and the performance of both
approaches must be compared. Results obtained following an alterna-
tive methodology will not be considered valid otherwise.
The recently published (2014) EURL Lm Technical Guidance docu-
ment for conducting shelf-life studies on L. monocytogenes in RTE
foods is an improvement on the 2008 version, and is a valuable docu-
ment which will give FBOs the opportunity to have challenge studies
undertaken in a harmonized manner. However, FBOs willing to deter-
mine growth potential of L. monocytogenes on their products face sever-
al major challenges which are not yet resolved. These are mainly
regarding expertise and economic constraints. The industry stake-
holders (in some cases artisanal food producers and operators of small
and medium enterprises) do not usually have the technical knowledge,
expertise and resources required to effectively undertake challenge
tests. Since they do not usually have adequate laboratory facilities and
equipment and will have difficulties with understanding and strictly ad-
hering to the European Technical Guidance document (i.e. they may not
have access to scientific literature on L. monocytogenes growth or will
have difficulties in interpreting the results; they may not be able to
use predictive microbiology software or will not have the sufficient
knowledge or skills in “food microbiology” to design and execute a chal-
lenge test), they need to outsource their studies to independent labora-
tories. However, in some countries there are very few laboratories
currently offering this service (with optimised and accredited proto-
cols) and the associated expenses are often too high. In addition, the ex-
penses and efforts may intensify by the need to carry out challenge tests
for all different sorts of RTE foods produced and in all the instances
when a change in product formulation has occurred. Some countries
have a harmonized procedure for the implementation of challenge
tests. For instance, France has a network of laboratories accredited
for L. monocytogenes challenge testing. Such laboratories are
accredited by a working group composed of agents from the compe-
tent authority and agents of the national reference laboratory (NRL)
for L. monocytogenes after the laboratory passes an audit (conducted
by the NRL) which assesses the ability of the laboratory to take into
account the data from the producer and the technical competence
of the laboratory, and after the laboratory gets satisfactory results
to an interlaboratory assay of aptitude organized by the NRL for
L. monocytogenes. An additional major challenge occurring in coun-
tries where a harmonized procedure is not in place is lack of coordi-
nation between regulatory authorities, FBOs and laboratories
carrying out challenge studies. In cases where a flexible interpreta-
tion of the European Technical Guidance document occurs, the re-
sults of a challenge study may not be considered acceptable by
regulatory authorities, which have the final say as to whether the
foodstuff is categorized as a RTE food unable or able to support
L. monocytogenes growth.
To sum up, some FBOs interested in categorizing their RTE foods into
RTE foods that do not support the growth of L. monocytogenes will not be
able to carry out a proper challenge test due to lack of expertise and/or
resources, or will perform a challenge test that will not be considered
valid by the competent regulatory authorities. There is a clear need for
training of FBOs and of independent laboratory employees on the objec-
tives, design, execution and interpretation of results of challenge tests to
determine L. monocytogenes growth potential on food. The coordinated
implementation of National training networks and of networks of
accredited laboratories would help to set up the basis for an improved
 A. Álvarez-Ordóñez et al. / Food Research International 75 (2015) 233–243
application of the European Guidelines. In addition, the establish-
ment of a dialogue with regulatory authorities prior to the execution
of challenge tests is recommended in order to avoid the possibility of
the results being rejected due to a misleading design of the study.
Acknowledgements
This work was supported by the Irish Department of Agriculture
and Food and the Marine under the Food Institutional Research Mea-
sure (FIRM) project number 11F008. A. Alvarez-Ordóñez is a Starting
Investigator Research Fellow funded by Science Foundation Ireland
(SFI) under Grant Number 13/SIRG/2157.
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