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Detection of Listeria spp. in liquid egg products and in the egg breaking plants
environment and tracking of Listeria monocytogenes by PFGE

Article  in  International Journal of Food Microbiology · June 2013

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 International Journal of Food Microbiology 166 (2013) 109–116

Contents lists available at SciVerse ScienceDirect

International Journal of Food Microbiology

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

Detection of Listeria spp. in liquid egg products and in the egg breaking
plants environment and tracking of Listeria monocytogenes by PFGE
Katell Rivoal ⁎, Aurore Fablet, Céline Courtillon, Stéphanie Bougeard, Marianne Chemaly, Jocelyne Protais
Anses, French Agency for Food, Environmental and Occupational Health and Safety, Ploufragan/Plouzané Laboratory, BP 53, 22 440 Ploufragan, France
Université Européenne de Bretagne, France

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

Article history: Human listeriosis, caused by Listeria monocytogenes, is a severe bacterial infection that can lead to meningitis,
Received 21 March 2013 cerebromeningitis, bacteremia or septicemia, with acute lethality and potentially leading to death. A study has
Received in revised form 14 June 2013 shown that 29.5% of the caged laying hens in France are contaminated by L. monocytogenes (Chemaly et al.,
Accepted 15 June 2013
2008). However, very little information regarding egg and egg product contamination is currently available.
Available online 20 June 2013
The objective of this study is to determine the sanitary status of egg products and egg breaking plants in
Keywords:
France regarding Listeria spp. and L. monocytogenes contaminations. The sampling scheme performed in five
Listeria monocytogenes egg breaking plants in Western France during one year have revealed that 8.5% of raw egg products were
Egg products contaminated by L. monocytogenes. No pasteurized egg products have been shown to be contaminated by
Environment L. monocytogenes. However, a high level of contamination by Listeria spp., and particularly by L. innocua, has
PFGE characterization been shown with 26.2% and 1.8% of raw and pasteurized egg products contaminated, respectively. This work
has also revealed the presence of Listeria spp. and L. monocytogenes in the environment of egg breaking plants
with 65.1% and 8.0% of contaminated samples, respectively.
The typing of 253 isolates of L. monocytogenes by PFGE using ApaI and AscI enzymes has revealed a high diversity
with 46 different pulsotypes and has shown that the raw material is a source of contamination of egg breaking
plants. One L. monocytogenes cluster was dominant in the 5 egg-breaking plants during the four seasons studied.
The issue of which strains are better adapted to egg products must be considered and studied in depth by com-
paring them to pulsotypes from strains of other chains. However, the traceability of L. monocytogenes in plants
during the various seasons has also made it possible to highlight the presence of strains that are specific to egg
breaking plants. The study of cleaning and disinfection methods in these plants as well as the recurring bacteria's
resistance to disinfectants could provide answers to the egg product industry.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction Frequently contaminated foods include milk and dairy products,

Listeria monocytogenes is a food-borne pathogen of serious public
health concern (Schlech et al., 1983). This pathogen can cause listeriosis,
an illness that may result in encephalitis or septicemia in adults. In preg-
nant women, the infection can lead to miscarriages, premature births or
neonatal infections. In France, the disease remains rare with less than 4
cases per million, but the mortality rates can raise up to 30% (Vaillant et
al., 2005; EFSA, 2012). In the European Union (EU), a total of 1601 con-
firmed human cases of listeriosis (0.35/100,000) were reported in 2010
(EFSA, 2012). Since 2006, an increasing number of listeriosis cases have
been noted in several EU countries, including France, predominantly in el-
derly people (Goulet et al., 2008; EFSA, 2012).
⁎ Corresponding author at: Hygiene & Quality of Poultry and Pig Products, French Agency
for Food, Environmental and Occupational Health and Safety, BP 53, F-22 440, Ploufragan,
France. Tel.: +33 2 96 01 62 22; fax: +33 2 96 01 85 38.
E-mail address: katell.rivoal@anses.fr (K. Rivoal).
meat and meat products, plant products, fish and fishery products;
however, eggs and egg-products have never been involved in out-
break or sporadic cases of listeriosis.
Data concerning egg product contamination by L. monocytogenes
was obtained in France by a study on the detection of pathogenic
bacteria and alterations in the liquid matrix of eggs (Protais et al.,
2007). L. monocytogenes was found in 17.4% of raw whole egg products
and 2.1% of pasteurized egg products (Rivoal et al., 2010). The contam-
ination of raw egg products could be explained by the use of eggs with
shells which were contaminated by L. monocytogenes in the hen house
environment. In fact, Chemaly et al. (2008) found L. monocytogenes
in the dust samples of 31 laying hen farms out of 200 (15.5%).
L. monocytogenes seems to show a relative resistance to storage condi-
tions and handling of table eggs (Brackett and Beuchat, 1992).
L. monocytogenes is able of surviving shell egg cleaning procedures
and can be present in the water used for cleaning (Laird et al., 1991;
Jones et al., 2006). In fact, this pathogen, which is ubiquitous and fre-
quently encountered in food industry environments, could be found
in egg breaking plants (Gudbjörnsdottir et al., 2004).

0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijfoodmicro.2013.06.014
110 K. Rivoal et al. / International Journal of Food Microbiology 166 (2013) 109–116
The contamination of pasteurized egg products is more surprising.
Pasteurization should ensure the total inactivation of Listeria spp. in
the whole egg, especially when found in small quantities in the raw
egg (Moore and Madden, 1993). Parameters relative to the resistance
of L. monocytogenes could intervene, such as the physiological state of
L. monocytogenes cells (age, stress…) present in the egg product
before applying any type of sanitary treatment, the ability of the
bacteria to revitalize after non-lethally damaged in normal or abusive
preservation conditions (Augustin, 1996). Finally, post-pasteurization
contamination of the samples cannot be ignored, particularly in view
of the ability of L. monocytogenes to persist in the environment of food
industry plants and to grow at low temperatures, which poses a real dif-
ficulty for the food industry (Tasara and Stephan, 2006; Warriner and
Namvar, 2009).
Discriminative molecular subtyping methods have been used to
characterize L. monocytogenes in order to understand the routes of con-
tamination in plants (Wiedmann, 2002). Phenotypic typing methods
such as sero- and phage-typing have low discriminatory power. Molec-
ular subtyping methods have been extensively applied for tracking
L. monocytogenes (Giovannacci et al., 1999). Although new methods
are being investigated, Pulsed-Field Gel Electrophoresis is still currently
the ‘gold standard’ subtyping method for L. monocytogenes, due to its
high reproducibility, robutstness and high discriminating power
(Graves and Swaminathan, 2001).
The objective of this project is to describe Listeria spp. contamination,
particularly L. monocytogenes contamination, in French egg breaking
plants during different seasons, and to characterize L. monocytogenes by
PFGE in order to describe plant contamination and to trace the origin of
the contamination.
2. Materials and methods
2.1. Sampling
This study was carried out over a one-year period in 5 egg breaking
plants in North Western France. The five plants, distant from each other
by 200 km, belong to five different companies and account for the
production of 75% of French liquid whole egg products. Each plant was
visited and sampled 4 times, one visit per season.
The presence of Listeria spp. was investigated in 4 different types
of samples, represented by:
a) egg shells (one or several egg batches) by rubbing (about 300 eggs
by wet swabs) and after breaking (25 g of about 300 egg shells),
b) environment of the egg breaking plant (wet swabs were done in
reception room including cells, doors, the parts of egg breakers
in contact with the egg product, piling and crushing of the shells),
c) raw egg products (whole egg, egg white and egg yolk; natural,
sweetened and salted) corresponding to the egg batches previously
sampled (see point a), immediately after breaking and shell filtering.
The egg shell filter was also analyzed;
d) pasteurized egg products, matching the batches of eggs and raw egg
products previously sampled (point a and c), although correspon-
dence cannot be entirely exact due to high volumes.
Samplings of salt, sugar and other additives to egg products were
also analyzed.
A total of 1557 samples were collected, consisting of 564 egg
products, 260 egg shell samples and 733 environment samples.
Samples were kept at 2 °C ± 1 during transport and temperature
was checked upon arrival at the laboratory.
2.2. L. monocytogenes isolation and identification
Detection of Listeria spp. and L. monocytogenes were performed in
accordance with the NF EN ISO 11290-1-February 2007 standard
using two plating media (Aloa and Palcam; AES, Combourg, France).
Briefly described, the egg product samples (10 ml) are diluted to
the tenth in half-Fraser broth (AES) and then incubated for 24 h at
30 °C. This first enrichment is streaked onto two selective agars
which are incubated for 48 h at 37 °C. A second enrichment is then
performed using 0.1 ml of the pre-enrichment into 10 ml of Fraser
broth (AES) for incubation at 37 °C for 48 h. Isolations are then
performed on Palcam and Aloa (incubation at 37 °C up to 48 h).
A second dilution is performed to 1/40 in half-Fraser broth on the
egg white products in order to reduce the action of antibacterial com-
ponents contained in the egg whites.
For environmental samples, 150 ml of half-Fraser is added to each
swab. The protocol of isolation previously described is then applied.
Five characteristic colonies were isolated from the selective agar
plates, and genus confirmation was then performed (catalase, Gram
staining, mobility) prior to species identification (hemolysis, use of
sugars, Camp test).
Quantitative assessment of L. monocytogenes was also performed on
all raw egg products at their arrival in the lab. Quantitative analysis was
performed by preparing tenfold dilution from half-Fraser broth enrich-
ments before incubation performed for qualitative analysis. One ml of
undiluted enrichment and 0.1 ml of each dilution were spread on Aloa
plates. After incubation at 37 °C for 48 h, characteristic colonies of
L. monocytogenes were counted. The detection threshold for the method
used was 10 CFU/ml. When a thermally treated sample of egg product
or a sugar and/or salt sample was positive, an enumeration was then
performed on this product stored at 4 °C.
Finally, one to 6 isolates per positive sample were identified, for a
total of 4119 Listeria spp. isolates collected.
2.3. Selection and typing of L. monocytogenes isolates
After speciation of Listeria spp. isolates (n = 4119), 595
L. monocytogenes were identified and serogrouped using the PCR
method perfected by Doumith et al. (2004). A selection of 253
isolates was performed among these 595 isolates collected from 87
samples positive for PFGE typing. For each positive sample, one
isolate was collected per positive enrichment media and per positive
selective media. Moreover, if different serotypes were revealed in
a single sample, one isolate of each serotype was typed, for a total
of 253 isolates. These isolates were subjected to PFGE using the
restriction enzymes ApaI and AscI (BioLabs, Beverly, MA) according
to the protocol described by PulseNet (Graves and Swaminathan,
2001).
The profiles obtained were analyzed using BioNumerics software
version 6.5 (Applied Maths, Gand, Belgium) considering maximum
optimization of 1%, and based on Dice similarity of bands, with max-
imum position tolerance of 1.5%. Unweighted Pair Group Method
using Averages (UPGMA) was used for profile clustering and dendro-
gram construction. Two profiles were considered to be different if
they differed by one band. Profiles with an overall level of similarity
>80%, calculated using BioNumerics software, are grouped into cluster
(Gianfranceschi et al., 2009). The Simpson index (D) was determined
(Hunter, 1990) to assess the genetic diversity of Listeria monocytogenes
populations (Boscher et al., 2012).
2.4. Statistical analysis
Results of egg products contamination were analyzed by performing
two logistic regressions to explain the following variables: Listeria spp.
and L. monocytogenes using 3 main effects: egg breaking plant, season
and matrix (whole egg, egg yolk or egg white).
For environmental samples, several models were tested. Two logis-
tic regressions to explain the different variables (contamination by
Listeria spp. and by L. monocytogenes) using 3 main effects: egg breaking
plant, season and sampling type (environment, machine or shells) were
 K. Rivoal et al. / International Journal of Food Microbiology 166 (2013) 109–116 111

Table 1
Contamination of raw and pasteurized egg products by L. monocytogenes and Listeria spp. during the various seasons.

Egg products Winter Spring Summer Fall Total

Raw L. monocytogenes 5/76 (6.6%) 9/71 (12.7%) 8/72 (11.1%) 3/74 (4.1 %) 25/293 (8.5%)
Listeria spp. 17/76 (22.4%) 19/71 (26.8%) 23/72 (32%) 18/74 (24.3%) 77/293 (26,2%)
Pasteurized L. monocytogenes 0/67 0/70 0/65 0/69 0/271
Listeria spp. 3/67 (4.5%) 0/70 1/65 (1.5%) 1/69 (1.5%) 5/271 (1.8%)

performed. Interactions were then tested. Only significant interactions
were kept for the final model.
Processing was performed using SAS software (SAS Institute Inc.).
3. Results
Out of 1557 samples analyzed, 646 were positive for Listeria spp.
(41.5%) and 87 for L. monocytogenes (5.6%; Tables 1 and 2). From these
samples, 4119 isolates were collected and identified. Four species were
found: Listeria innocua (3515 isolates), L. monocytogenes (595 isolates),
Listeria seeligeri (8 isolates) and Listeria ivanovii (1 isolate).
Three serogroups were observed among L. monocytogenes isolates:
IIa for 461 isolates (77.5%), IIb for 82 (13.8%) and IVb for 52 (8.7%).
Table 3 shows the distribution of these serogroups according to the
type of samples.
3.1. Egg product contamination by Listeria spp.
Twenty-five samples out of the 293 raw samples (8.5%) were
positive for L. monocytogenes (Table 1). Less than 10 CFU/ml of
L. monocytogenes were found in 60% of these samples. Twenty-five
percent of the samples were contaminated at concentrations varying
between 10 and 100 CFU/ml. Only 5 samples showed contaminations
higher than 100 CFU/ml. L. monocytogenes was never found in pas-
teurized egg product samples.
L. innocua was found in 68 (23.2%) and 5 (1.8%) raw and pasteurized
samples, respectively. Among these 5 pasteurized samples contaminat-
ed by L. innocua, 3 egg products (1 natural whole egg, 2 natural egg
yolks) were sampled in the winter, one (natural egg yolk) in the sum-
mer and the other one (natural egg white) in the fall.
No significant association of egg product contamination by
L. monocytogenes with egg breaking plant (p = 0.7251) or season
(p = 0.2562) was revealed (Table 4).
However, a significant difference in contamination (p = 0.02,
Table 4) between the matrix has been shown: the whole liquid egg
and the egg yolk products have 12 [1.5–97.6]95% and 18 times [2.3–
144.2]95% more risk of being contaminated by L. monocytogenes than
the egg white, respectively (Table 5). There is no significant differ-
ence between the whole egg and the egg yolk (p = 0.097).
A significant difference (p = 0.0008, Table 4) in Listeria spp. con-
tamination was shown between the egg breaking plants. In plant 5,
the contamination is significantly different from that observed in
plants 1 and 4 (p = 0.0001 and p = 0.0014; data not shown). Plant
1 has shown the smallest risk of contamination by Listeria spp.
The effect of the matrix is also significant (p b 0.0001) on the
Listeria spp. contamination risk (Table 4). Liquid whole egg products
show a contamination risk 3 times [1.5–6.1]95% higher than egg yolk
and 9.8 times [4.2–23.2]95% higher than egg white (Table 5). Contam-
ination risk of egg yolk is 3 times [1.3–7.9]95% higher than egg white
(Table 5).
As for L. monocytogenes contaminations, seasons have no signifi-
cant effect (p = 0.1316, Table 4) on egg product contamination by
Listeria spp.
3.2. Environmental contamination of egg breaking plants by Listeria spp.
Sixty-two (42.2%) eggshell samples were positive for Listeria spp.
detection, while only 3 (2%) were positive for L. monocytogenes
(Table 2).
In the plant environment, 477 samples (65.1%) tested positive for
Listeria spp. (Table 2) and 59 samples (8.0%) for L. monocytogenes
(Table 2). The three tested effects (plants, sampling types and sea-
sons) were shown to have a significant impact on contamination by
L. monocytogenes and Listeria spp. (Table 6).
Environmental contamination by L. monocytogenes is significantly
(p = 0.0003) higher in the plant 2 (14.7%) than in the plants 1, 3, 4
and 5, in which 4.8%, 7.1%, 3.1% and 3.2% of the samples collected
from the environment were contaminated, respectively. With regard
to contaminations by Listeria spp., plant 5 indicates a lower contami-
nation than the other 4 plants with a contamination level of 26.5%,
while the plants 1, 2, 3 and 4 show respectively 66.9%, 86.7%, 60%
and 66.9% of contaminated samples.
The statistical analysis also reveals that the environment, whether
of the plant's premises or the egg breakers, is significantly more
contaminated than the egg shells, whether that contamination is
caused by L. monocytogenes (p = 0.0004, Table 6) or by Listeria spp.
(p b 0.0001, Table 6).
A seasonal effect on the environmental contamination by
L monocytogenes and by Listeria spp. has also been highlighted. There-
fore, whether contamination is due to L. monocytogenes or to Listeria
spp., the environmental samples collected during winter showed
a higher level of contamination than those collected during the
3 other seasons. The risk of discovering positive samples for
L. monocytogenes in the environment during winter is 5.2 [2.2–
12.5]95% times higher than in fall, 2.7 [1.3–5.5]95% times higher than
in spring, and 3 [1.5–6.2]95% times higher than in summer. Similar
results were observed for Listeria spp. contaminations.

Table 2
Contamination of eggshells and environment by L. monocytogenes and Listeria spp. in egg breaking plants during the various seasons.

Winter Spring Summer Fall Total

Egg shells L. monocytogenes 0/34 2/41 (4.9%) 1/35 (2.9%) 0/37 3/147 (2.0%)
Listeria spp. 17/34 (50%) 15/41 (36.6%) 11/35 (31.4%) 19/37 (51.4%) 62/147 (42.2%)
Ground shellsa L. monocytogenes / 0/41 0/35 0/37 0/113
Listeria spp. / 8/41 (19.5%) 5/35 (14.3%) 12/37 (14.3%) 25/113 (22.1%)
Environment L. monocytogenes 28/181 (15.5%) 13/191 (6.8%) 11/188 (5.9%) 7/173 (4.0%) 59/733 (8.0%)
Listeria spp. 125/181 (69.1%) 122/191 (63.9%) 115/188 (61.2%) 115/173 (67.1%) 477/733 (65.1%)
a
Following the initial sample collection performed during the winter season, none of the samples collected by shell rubbing were contaminated by the bacteria. In order to in-
crease the L. monocytogenes detection probability, we added analyses on ground shells (previously rubbed) during the following three seasons. However, this sampling mode was
shown to be significantly less effective (p b 0.01) to detect the presence of Listeria spp. on eggshells.
112 K. Rivoal et al. / International Journal of Food Microbiology 166 (2013) 109–116

Table 3 Table 5
Distribution of the number of samples as a function of the serogroup and its origin. Value of odd-ratios according to matrix.

Serogroups OR IC− IC+

IIa IIb IVb L. monocytogenes Whole vs white 12.119 1.505 97.559

Egg shells 1 1
Production line 15 3 3
Egg products 25 1 1
Environment 25 9 11
Totala 66 14
a
Some samples are contaminated by 2 or 3 serogroups of L. monocytogenes.
3.3. Genetic diversity of Listeria monocytogenes
Macrorestriction of L. monocytogenes isolates with ApaI and AscI
generated 46 and 34 different profiles, respectively. The observed
Simpson index was 0.9652 for ApaI and 0.9572 for AscI.
Forty-six combined pulsotypes (named P1 to P46), grouped at
54.9% similarity level, were observed (Fig. 1). The combined ApaI–
AscI Simpson Index was 0.9652. Isolates were grouped by serogroups
(Fig. 1). Twelve clusters were identified, grouping all pulsotypes ex-
cept three (P13, P24 and P25). Isolates belonging to serogroup IIa
were divided into 8 clusters. Cluster 3, grouping 6 pulsotypes (P7 to
P12), includes 26.8% of all analyzed isolates (n = 68) collected from
23 different samples. This cluster is also the only one to group
pulsotypes that were observed in all five studied plants during all
the seasons (Table 7). Cluster 8 groups 12.2% of isolates (n = 31), be-
longing to 6 pulsotypes (P26 to P31), observed in 3 different plants.
Clusters 4, 5 and 6 gather 7.5% (n = 19), 7.1% (n = 18), and 6.7%
(n = 17) of typed isolates, respectively. Clusters 4 and 6 included iso-
lates that were found in different plants, while cluster 5 is the only
cluster that groups pulsotypes found in a single plant (plant 3). Clus-
ters 1, 2 and 7 group the remaining isolates of serogroups IIa. Isolates
belonging to serogroup IIb were divided into 2 clusters. Clusters 11
and 12 group 11.5% (n = 29) and 1.6% (n = 4) of typed isolates
collected from 12 and 2 samples, respectively. Isolates belonging to
serogroup IVb were divided into 2 clusters. Clusters 9 and 10 group
10.7% (n = 27) and 2.4% (n = 6) of typed isolates collected from
14 and 4 samples, respectively.
Yolk vs white 18.320 2.328 144.199
1
Listeria spp. Whole vs yolk 3.057 1.521 6.144
Whole vs white 9.896 4.227 23.165
Yolk vs white 3.237 1.326 7.901
16
in winter and summer and strains of cluster 5 (P16, P17 and P18) in
spring, summer and fall. In plant 4, all positive samples (egg products
and environmental samples) were contaminated by strains of cluster
3 (P7 and P10) and in one egg product pulsotype P20 was also isolated.
In plant 5, strains of cluster 3 (P9 and P11) were found in winter, spring
and fall, in egg product and environmental samples. Strains of cluster 8
(P28 and P31) were collected in winter, spring and summer. Pulsotype
P36 (cluster 10) was also isolated in spring and fall.
Moreover, some identical strains or clusters were collected in differ-
ent plants. Thus, strains of cluster 3 were isolated from egg products and
environmental samples in the 5 plants during all the year. Clusters 9 and
11 were found in all plants (except plant 4) and cluster 8 was isolated in
plants 1, 2 and 5. All the others clusters were found at least in two dif-
ferent plants except cluster 5 and pulsotype P24 that were only isolated
from plant 3.
Our results showed that eggs in shell are one of the sources of con-
tamination of the plant's environment and of the egg product. Thus, in
plant 3, pulsotype P17 (cluster 5) was isolated from shell at the arrival
in the plant during summer and in the egg product during fall. Similarly,
in plant 2, pulsotype P40 (cluster 11) was found on shell during spring
and in the plant's environment during the same season. In plant 5,
pulsotype P36 (cluster 10) was found on the shell during spring and
in the plant's environment in fall.
Moreover, we observed cross-contaminations between the environ-
ment and the egg products. For example, in plant 1, during summer,
pulsotype P14 (cluster 4) was isolated from the environment, the egg
breakers and the egg products. In the same way, in plant 3, pulsotype
P24 was collected in several environment samples and in egg products
during winter.

3.4. Listeria monocytogenes traceability in the various plants 4. Discussion

The distribution of L. monocytogenes pulsotypes according to the
egg breaking plant, the type of samples and the season is described
in Table 7.
Our results showed that some strains or clusters are persistent
within each plant. Thus, in plant 1, pulsotype P9 was isolated from
egg product during winter and summer. Strains of cluster 9 (P32 and
P35) were found in environmental samples during winter and spring.
Similarly, in plant 2, pulsotype P3 was found in the environment during
winter and fall. Strains of cluster 11 (P39, P40 and P41) were collected
during winter and spring, strains of cluster 9 (P33, P34, P35) and of
cluster 3 (P8, P9, P11 and P12) during winter, summer and fall and
strains of cluster 8 (P26, P27, P29 and P30) during winter, spring and
fall. In plant 3, pulsotype P9 (cluster 3) and strains of cluster 11 (P42
and P43) were isolated in winter and spring. Pulsotype P14 was found
Table 4
Influence of different factors on the contamination of raw egg products.
In our study, none of the pasteurized egg products were contami-
nated by L. monocytogenes, nevertheless 8.5% of the raw egg products
were contaminated. Thus, our findings indicate an improvement over
what was observed previously in France (17.4% and 2.1% of contami-
nated raw and pasteurized egg products, respectively; Rivoal et al.,
2010). The observed contamination levels are higher compared to
what was observed by Ohkochi et al. (2009). In Japan, 0.5% of 803
raw egg products analyzed were contaminated by L. monocytogenes.
Similarly, Leasor and Foegeding (1989) indicate a contamination
level by L. monocytogenes of about 5% of egg products analyzed in
the United States. Concerning Listeria spp., both studies reported rel-
atively high levels of contamination in raw egg products with 30% and
Table 6
Influence of various factors on the contamination of the environment.
Variation factor DF Fvalue Pr > Fvalue

L. monocytogenes Egg breaking plant 4 21.39 0.0003

Variation factor DF Fvalue Pr > Fvalue
Typea 2 15.42 0.0004
L. monocytogenes Egg breaking plant 4 2.05 0.7251 Season 3 19.56 0.0002
Matrix 2 7.78 0.0205 Listeria spp. Egg breaking plant 4 144.27 b.0001
Season 3 4.05 0.2562 Type 2 72.14 b.0001
Listeria spp. Egg breaking plant 4 18.83 0.0008 Season 3 9.70 0.0213
Matrix 2 29.80 b.0001 a
The different environment sample collections were differentiated into 3 types: egg
Season 3 5.62 0.1316
shells, egg breaker and more global environment (wall, boots,…).
 K. Rivoal et al. / International Journal of Food Microbiology 166 (2013) 109–116 113

No.of samples
No.of isolates
AscI-ApaI

100
55

60

65

70

75

80

85

90

95
94.1
P1 1 1
88.9 P2 2 1
80.1 P3 4 3
Cluster 1
74.1 P4 2 1
P5 1 1
92.9 Cluster 2
P6 4 1

98.2
P7 16 6
95.3 P8 2 1
71.4
92.3 P9 20 6
Cluster 3
89.5 P10 16 3
80.9
P11 12 6
77.3
P12 2 1
69.3
75.3 P13 2 1
P14 14 6
98.4
Cluster 4
P15 5 1

91.9
P16 4 2 Serogroup IIa
86.2 P17 13 4 . Cluster 5
P18 1 1

88.3
P19 6 1
68.0
83.5 P20 6 1 Cluster 6
78.9 P21 5 1
P22 2 1
78.4
98.4
Cluster 7
P23 2 1
71.9
P24 12 7
P25 2 1

97.1
P26 6 2
71.0
96.2
P27 1 1

98.4
P28 4 1
Cluster 8
89.8
P29 1 1
54.9 86.1
P30 3 2
P31 16 4

90.5
P32 5 2
P33 10 5
80.0
Cluster 9
96.9
P34 7 4
74.9 P35 5 3
Serogroup IVb
98.6
P36 3 2 .
97.8 P37 1 1 Cluster 10
P38 2 1

96.9
P39 3 2
74.2

88.6
P40 6 3
P41 4 2
85.9
98.5
Cluster 11
P42 6 1
82.4
P43 8 3 Serogroup IIb
77.9 P44 2 1

96.9 P45 2 1
P46 2 1 Cluster 12

Fig. 1. Dendrogram of the ApaI–AscI profiles of L. monocytogenes. The four major clusters are indicated by colors that are also used in Table 7. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)

26.6%, respectively. In our study, Listeria spp., particularly L. innocua, In fact, several studies have shown Listeria spp. to be relatively
accounted for 26.2% and 1.8% of raw and pasteurized egg products, re- thermo-resistant (Alvarez-Ordonez et al., 2009; Bartlett and Hawke,
spectively. The presence of L. innocua in pasteurized egg products can 1995; Murphy et al., 2002; Palumbo et al., 1995). Additionally, post-
be explained by its ability to resist to different pasteurization treatments. pasteurization contamination can occur within the plant, particularly
114 K. Rivoal et al. / International Journal of Food Microbiology 166 (2013) 109–116

Table 7
Distribution of L. monocytogenes pulsotypes according to plant, season and types of samples. The colors are the same used in the dendrogram and indicate the major clusters.

Plant 1 Plant 2 Plant 3 Plant 4 Plant 5

Number of
Number of positive samples 13 24 25 10 15 Pulsotypes
Number of typed isolates 38 52 74 38 51

Shell / / / / /

Environment P1 (1-1)a ; P32 (1-3) P3 (1-1) ; P11 (1-1) ; P4 (1-2) ; P24 (4-8) ; P7 (2-7) P11 (1-2)
P27 (1-1) ; P30 (2-3) ; P43 (2-4)
P33 (1-2) ; P34 (3-5) ;
Winter P37 (1-1) ; P39 (1-1) ;
P40 (1-2)
Egg breaking P32 (1-2) ; P45 (1-2) / P2 (1-2) ; P14 (1-2) ; P7 (3-6) /
22
machine P24 (2-3) ; P43 (1-4)

Egg product P9 (1-3) / P5 (1-1) ; P9 (1-4) P7 (1-3) P31 (1-2)

P24 (1-1) ; P33 (1-2)

Shell / P40 (1-2) / / P36 (1-2)

Environment / P12 (1-2) ; P38 (1-2) ; P9 (1-2) ; P18 (1-1) / P11 (1-2) ; P31 (1-4)
P39 (1-2) ; P40 (1-2) ; P34 (1-2) ; P35 (1-2) ;
P41 (1-1)
Spring
Egg breaking P35 (1-2) ; P41 (1-3) / P16 (1-2) / P31 (1-4)
machine 19

Egg product P6 (1-4) P26 (1-4) P26 (1-2) P16 (1-2) ; P19 (1-6) P10 (1-4) ; P20 (1-6) P11 (2-5) ; P28 (1-4)
P42 (1-6) ;
Shell / / P17 (1-4) / /

Environment P14 (1-2) P22 (1-2) ; P33 (1-2) P14 (1-2) / /

P35 (1-1)
Summer Egg breaking P14 (2-6) P8 (1-2) ; P33 (1-2) P17 (2-4) / /
12
machine

Egg product P9 (1-4) ; P14 (1-2) P11 (1-2) P21 (1-5) P10 (2-12) P15 (1-5) ; P31 (1-6)

Shell / / / / /

Environment / P3 (2-3) ; P9 (1-3) / / P36 (1-1) ; P44 (1-2) ;

P25 (1-2) ; P29 (1-1) P46 (1-2)
Fall P33 (1-2)
Egg breaking 11
/ / P23 (1-2) / /
machine

Egg product / / P17 (1-5) / P9 (1-4) ; P13 (1-2)

Number of
9 19 15 3 11
Pulsotypes
a
The first number indicates the number of samples contaminated by the pulsotype and the second the number of isolates typed.

via the environment or contaminated material. This latter hypothesis is
supported by the fact that a large part (65.1%) of environmental samples
was positive for L. innocua.
Moreover, the high level of contamination of the raw egg products
by L. innocua must be considered because several authors show that
the significant presence of the L. innocua species can conceal the
presence of L. monocytogenes (Curiale and Lewus, 1994; Petran and
Swanson, 1993). Thus, Carvalheira et al. (2010) showed that the growth
of L. monocytogenes can be inhibited by high concentrations of L. innocua
in pasteurized milk. These results confirm those of Cornu et al. (2002)
who showed that the significant growth of L. innocua during the enrich-
ment process could be due to a double phenomenon: a better growth
capacity of L. innocua in the enrichment media used but also the growth
inhibiting production for L. monocytogenes.
Contrary to what was previously observed (Rivoal et al., 2010), nei-
ther the egg breaking plant nor the season had any significant impact on
the contamination of egg products by L. monocytogenes. Previous find-
ings indicated a significantly higher risk of contamination of liquid
whole egg products in the summer and winter, compared to fall
(Rivoal et al., 2010). In the current study, we observed the same trend
with decreasing levels of contamination in the spring, in the summer
and in the winter with, respectively, 12.7%, 11% and 6% of the egg prod-
ucts contaminated, the fall remaining the season with the lowest level
of contamination (4%). Nevertheless, the environmental contamination
of the plants is significantly more important in winter, and the risk de-
creases significantly from spring to fall.
The statistical analysis of the results has also revealed the contami-
nation risk by L. monocytogenes to be more important in whole egg
and egg yolk products than in egg whites. With regard to contamina-
tions by Listeria spp., the whole egg product shows 3 to 10 times more
chances of being contaminated than the egg yolk or egg white respec-
tively and the yolk egg product has 3 times more chances of being con-
taminated than the egg white. These results confirm the fact that the
egg yolk and the whole egg are favorable media for the development
of microorganisms while the egg white has bacteriostatic or bactericidal
effects (Notermans et al., 1991, Schuman and Sheldon, 2003; Wang and
Shelef, 1991).
Concerning typing of L. monocytogenes, 77.5% of isolates belonged
to the IIa serogroup. Our results are in line with what was previously
reported by Rivoal et al. (2010) and Chemaly et al. (2008), who found
 K. Rivoal et al. / International Journal of Food Microbiology 166 (2013) 109–116
serotype 1/2a in 94% and 74.3% of analyzed egg products and environ-
mental samples of laying hens, respectively. This is of particular
concern as human cases are linked to L. monocytogenes of serotypes
1/2a, 1/2b and 4b with an increase of cases associated to serotype
1/2a (Allerberger and Wagner, 2010; Parihar et al., 2008).
A large genetic diversity was observed within the isolates of
L. monocytogenes typed with 46 pulsotypes for 87 positive samples,
which may indicate multiple contamination sources. This diversity
is as important in the egg products with 20 different pulsotypes out
of 25 positive samples as in the environment with 35 pulsotypes
among the 59 positive samples for L. monocytogenes. Furthermore,
several samples were contaminated by strains of L. monocytogenes
with different serogroups and pulsotypes. These results confirm the
important diversity that was highlighted during our previous study in
which we had identified 21 different pulsotypes for L. monocytogenes
from 31 egg product samples (Rivoal et al., 2010).
Our study showed that one of the sources of contamination of egg
products was mainly due to raw material, i.e. the eggs and the shells.
Actually, thanks to molecular typing, pulsotypes of L. monocytogenes
identified on eggshell were systematically found later in the plant
environment or in the raw egg products. The shell eggs are certainly
one of the paths of entry for the bacteria in the plants, but it is important
to note that the environment can also be a source of contamination of
the raw egg products. Our study highlighted cross-contamination path-
ways between the plant environment and the raw egg products. Other
authors have reported the same contamination routes (Strydom et al.,
2013; Borucki et al., 2005).
Moreover, we demonstrated that certain strains or clusters seem to
be established in the plants. Thus, some pulsotypes or clusters persist in
the same egg breaking plant. We showed previously, that some strains
of L. monocytogenes were able to persist in the same egg breaking plant
for up to several weeks (Rivoal et al., 2010). The presence of persistent
strains of L. monocytogenes in the plant environment enhances the
possibility of contamination of egg products from the processing envi-
ronment. This fact has been described by other authors, who reported
that the contamination of the final product usually occurs during pro-
cessing and is a result of the presence of persistent strains in the envi-
ronment (Autio et al., 2002; Heir et al., 2004; Thévenot et al., 2006).
The persistence of L. monocytogenes in food processing environments
occurs through the interaction of two main factors. Firstly due to ineffi-
cient measures of cleaning and disinfection, since for the cleaning to be
effective must reach the site of contamination in sufficient quantities
and duration (Mendonça et al., 2012). Several authors have shown a
high resistance of L.monocytogenes to different cleaning and disinfection
procedures in the food industry (Aase et al., 2000, To et al. 2002,
Moretro and Langsrud 2004, Kastbjerg et al., 2010). In addition, another
factor that may contribute to persistence is the nature of the strains, in
view of the fact that some strains are better adapted to industrial envi-
ronments. Thus, in our study, some strains or clusters have been found
in several plants during the different season. For example, cluster 3
(serotype IIa) which groups 5 different pulsotypes, has been found in
23 different samples in the 5 egg breaking plants and in all four seasons.
It has been isolated as much in the environment of the egg-breaking
plants as on the egg breakers and several times in the raw egg products.
This cluster seems to be very common in the egg product chain.
5. Conclusions
The objective of this study is to determine the sanitary status of
egg products and environment of egg breaking plants with regard
to Listeria spp. and L. monocytogenes contamination. Raw egg product
contamination by L. monocytogenes is weak with 8.5% of the samples
contaminated. Pasteurized egg-products did not indicate any sign of
contamination. However, a high level of contamination by Listeria
spp. and particularly by L. innocua has been shown with 26.2% and
1.8% of raw and pasteurized egg products contaminated respectively.
115
This study has made it possible to reveal that the contamination risk
by L. monocytogenes varies with the nature of the egg product. Thus
egg white products have relatively less risks of being contaminated by
L. monocytogenes than whole egg products or yolk products.
This work has also highlighted the presence of Listeria spp. in the en-
vironment of egg breaking plants. It is important to note that the shell
eggs are shown to be mildly contaminated by L monocytogenes (2%)
despite a prevalence of 29.5% in the caged laying hen farms (Chemaly
et al., 2008). However, 42.2% of the shell eggs were contaminated by
Listeria spp. Raw material is undoubtedly a source of entry for the bac-
teria in the plants for egg product productions. The molecular typing
has also shown an important diversity within L. monocytogenes present
in the environment of the egg breaking plants and in the egg products.
The presence of L. monocytogenes and of Listeria spp. in the environment
of the plants is a contamination source of raw egg products and certain-
ly that of pasteurized egg products, notably by cross-contamination.
Strains of L. monocytogenes are found in all the plants, whatever the
season. On the other hand, the presence of strains that are specific to
certain egg breaking plants has also been shown. The study of cleaning
and disinfection methods applied in these plants and the resistance to
disinfectants of the reoccurring bacteria would make it possible to
better control the risk of L. monocytogenes contamination in the egg
product chain.
Acknowledgments
This study was funded by the Pôle Agronomique de l'Ouest
(including Région Bretagne and Région Pays de la Loire). The authors
would like to thank all those who have made this work possible, partic-
ularly the five manufacturers who prepared and submitted the samples.
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