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10
risks of spoilage and salmonella contamination of table eggs
AbstractConsumers and food business operators
are more and more aware of food safety
issues. Food safety can broadly be divided
into microbiological and chemical food
safety. Microbiological contamination of
eggs has important implications. For table
eggs, internal contamination may occur,
leading to spoilage and in the case of a pa-
thogen to human disease and food safety
risks. Eggs are one of the main sources of
contamination cited in relation to human
salmonellosis, with Salmonella Enteritidis
(SE) being the most frequently isolated
Salmonella serovar. There are two possible
routes of bacterial infection of table eggs:
either vertically or horizontally. In the verti-
cal transmission the egg content is directly
contaminated as a result of bacterial infec-
tion of the reproductive organs, i.e. ovaries
or oviduct tissue (transovarian route). In the
horizontal transmission the micro-orga-
nisms penetrate through the eggshell. SE
seems to have special capabilities to coloni-
ze the hen’s reproductive tract and to survi-
ve in the egg albumen compared to other
Salmonella serovars and other bacteria. Se-
veral studies support also the idea that the
vertical contamination route may be more
important for SE. The causative agent of
strong-evidence human outbreaks caused
by eggs and egg products in the EU in 2012
is SE, with 66.7% of the cases.
Different factors play a role in the risks
for spoilage and Salmonella contaminati-
on of eggs. Some of those factors will be
discussed, starting with different physical
and chemical defence mechanisms that
protect egg contents from microbial inva-
sion and multiplication. Egg factors (eggs-
hell and egg content), egg freshness, egg
cooling, eggshell condensation, amount
of bacterial load on the shell, bacterial
species, housing system, extending the
self-live of eggs, … are important factors.
Finally risk factors for SE contamination of
laying hen farms and the impact of the
Salmonella control programs in the EU,
with or without compulsory vaccination
against SE, will be discussed.
IntroductionThe egg has several physical and chemi-
cal defence mechanisms that protect its
contents from microbial invasion and mul-
tiplication. While the eggshell and shell
membranes physically hinder microbial
penetration into the egg albumen, the
vitelline membrane reduces penetration
Risks of spoilage and Salmonella contamination of table eggs
Koen De reu
to contact the author: Mail at [email protected]
Koen de reu is senior researcher and food Microbiologist at the ‘institute for Agricultural and fis-heries research’ or “iLVo”, in Melle, Belgium. his research group works in the broad field of food mi-crobiology with special attention for poultry microbiology, shiga-toxine producing e. coli, cleaning and disinfection, biocide and antibiotic resistance and biofilms. he is author of more than 50 peer review international scientific papers and has at this moment the supervision on 4 Phd students.
Vol. 50 (2), September 2015 | LOHMANN Information
11
into the most nutritious compartment of
the egg, the egg yolk. The chalazae also
act as physical barriers against bacterial
penetration into the egg yolk maintaining
the yolk in a central position. Finally, the
various antimicrobial properties of egg al-
bumen, its viscosity and alkaline pH inhibit
bacterial proliferation and contribute to
hindering egg yolk invasion. The proper-
ties of the main antimicrobial proteins pre-
sent in the albumen are listed in Table 1.
material and methodsTo study the bacterial eggshell penetration
and egg content contamination and the
risks factors for eggshell penetration and
egg content contamination different lab
scale methods can be used and found in
literature. Methods which, however, are
often applied are following: 1) An agar
method that can be used to study and
visualize the bacterial eggshell penetra-
tion and 2) An intact egg method to stu-
dy the egg content contamination. Both
methods are described by De Reu et al.
(2006b and 2006c). Bacterial inoculation of
the egg shell can be done by immersion of
the egg (agar filled or intact) in solutions
containing a certain concentration (e.g. 105
- 106 CFU/ml) of the bacterial target spe-
cies. More details of this inoculation me-
thod is described by De Reu et al. (2006b
and 2006c). Inoculation can also be done
using a cotton swab. After immersion of
the swab in an overnight bacterial culture,
the contaminated swab can be used to in-
oculate the eggshell surface area. In short,
the agar method (figure 1) consists of re-
placing the egg content by sterile molten
Nutrient Agar, containing streptomycin,
cycloheximide (preventing yeast and mold
growth) and the indicator 2,3,5- triphenyl-
tetrazolium-chloride. The addition of strep-
tomycin to the agar assures that only the
on the shell inoculated streptomycin resis-
tant bacterial species are able to grow on
the agar. Where bacterial eggshell penet-
ration occurs, the target bacterium grows
on the agar and reduces the TTC to the red
colored formazan (figure 1).
results and discussion
risks for spoilage and Sal-monella contamination of table eggs
Contamination routes
There are two possible routes of bacteri-
al infection of shell eggs: either vertically
or horizontally. In the transovarian route
(vertical transmission, figure 2), the yolk
(very infrequently the yolk itself ), the al-
bumen and/or the membranes are direct-
ly contaminated as a result of bacterial
infection of the reproductive organs, i.e.
ovaries or oviduct tissue, before the egg is
covered by the shell. Vertical transmission
can originate from infection of the ovari-
es of a laying hen via systemic infection,
or from an ascending infection from the
contaminated cloaca to the vagina and
lower regions of the oviduct. Different
researchers are convinced that the verti-
cal route is the most important route for
Salmonella Enteritidis (SE) contamination
of eggs. This is due to the ability of SE to
table 1: properties of the main antimicrobial proteins present in the albumen (De reu 2006a)
protein Characteristics
ovotransferrin chelating metal ions (particularly fe3+, but also cu3+, Mn2+, co2+, cd2+, Zn2+ and ni2+)
ovomucoid inhibition of trypsin
Lysozyme a) hydrolysis of β(1-4)glycosidic bonds in bacterial cell wall peptidoglycan – ac-ting specifically on the polymer n-acetyl glucosamine n-acetyl muramic acid, splitting the link between them
b) flocculation of bacterial cellsc) formation of oligosaccharides from bacterial cell wall tetrasaccharides by
transglycosylation
ovoinhibitor inhibition of several proteases
ovoflavoprotein chelating riboflavin (or vit. g or vit. B2); rending it unavailable to bacteria that require it
Avidin chelating biotin; rending it unavailable to bacteria that require it
Figure 1: Agar method - Filling up drained eggs with supplemented Nutrient Agar (left); inoculation of eggshell by immersion in a bacterial suspension (middle); visualisation of penetration by candling (right). De reu et al. (2006b and 2006c)
12
colonize the ovary and oviduct of laying
hens long-term. (De Reu et al. 2008)
type of bacterial flora
The microflora of the eggshell is domina-
ted by Gram-positive bacteria, whereas
Gram-negative bacteria are best equipped
to overcome the antimicrobial defences of
the egg content. Because of their toleran-
ce for dry conditions Gram-positive bac-
teria which may originate from dust, soil
or faeces can survive on the shell. Rotten
eggs normally contain a mixed infection
of Gram-negative and a few Gram-positive
organisms. Some of the most common
spoilage types are members of the gene-
ra Alcaligenes, Pseudomonas, Escherichia,
Proteus and Aeromonas (Mayes and Take-
balli, 1983; Board and Tranter, 1995). This
indicates that Gram-negative bacteria are
well equipped to overcome the antimi-
crobial defences of the egg. According to
Board and Tranter (1995), the internal pro-
perties of eggs favour survival and growth
of contaminating organisms which are
Gram-negative, have a relatively simple
nutritional requirement and have the abili-
ty to develop at low temperatures.
De Reu et al. (2006d; 2006e; 2007) have
found that the natural eggshell contami-
nation of table eggs was dominated by
Gram-positive Staphylococcus spp.. Sta-
phyloccoccus also seems to be the most
dominating microflora in the air of the
poultry houses (De Reu et al., 2007). As ma-
jor egg content contaminants Gram-nega-
tive bacteria as Escherichia coli, Salmonel-
la and Alcaligenes sp. were found. Beside
also Gram-positive bacteria like Staphy-
lococcus lentus, Staphylococcus xylosus
and Bacillus sp. were isolated from the egg
content (De Reu et al., 2006e, 2007, 2008).
Influence of bacterial load on eggshell
penetration and egg content conta-
mination
De Reu et al. (2006c) found that the pro-
bability of eggshell penetration is corre-
lated with the eggshell contamination;
but less obviously with the egg content
contamination (figure 3). External eggshell
contamination seems to be important for
the shelf life and the food safety of con-
sumption eggs and egg products. It is hy-
pothesised that an important part of the
bacterial contamination of the egg con-
tent results from the penetration of the
shell by bacteria deposited on the surface
of the egg after it has been laid (horizontal
infection route).
Influence of the housing system on the
bacterial contamination of table eggs
With the introduction of alternative
housing systems for laying hens in the EU,
from 2006 until 2012 research focused on
the bacterial contamination of table eggs,
e.g. eggshell and egg content contamina-
tion. Contamination of eggshells with ae-
robic bacteria is generally comparable for
nest eggs derived from non-cage systems
compared to furnished cage nest eggs
or conventional cages. Studies indicate a
higher bacterial load for outside nest eggs
as well from furnished cages as from non-
cage systems. The major differences found
by De Reu et al. (2005) in experimental
studies between furnished cage and non-
cage systems are less pronounced under
commercial conditions (figure 4) (De Reu
et al. 2009). The effect of housing system
on eggshell contamination with specific
groups of bacteria is variable (De Reu et
secondary Plant compounds are more than essential oils
Figure 2: overview of egg contamination by Salmonella (Gantois et al., 2009): a) Salmonella contamination of the reproductive organs of a hen via systemic spread after gut colonization or via an ascending infection; b) horizontal transmission route; c) vertical transmission route; d) survival and growth of Salmonella in the egg contents.
Figure 3: total count of inoculated 7 species on the eggshell of contaminated whole eggs (Y) and non-contaminated whole eggs (N) considering all strains (De reu et al. 2006c).
Vol. 50 (2), September 2015 | LOHMANN Information
13
al. 2009). Limited information is available
on the influence of housing system on
egg content contamination. Research of
De Reu et al. (2006e, 2007, 2008) does not
indicate large differences in egg content
contamination between eggs from cage-
and non-cage systems (ignoring outside
nest and floor eggs).
Studies also show that it is highly unlikely
that a move from conventional cages to
alternative cage systems and non-cage
housing systems will result in an increa-
se in Salmonella infection and shedding,
rather the opposite is expected (Van Hoo-
rebeke et al. 2010).
eggshell factors influencing eggshell
penetration and whole egg contami-
nation by different bacteria, including
Salmonella enteritidis
Trans-shell infection routes and whole
egg contamination of 7 phylogenetically
divers bacterial strains showed interesting
results. Eggshell penetration was correla-
ted with various eggshell characteristics
and the identity of strains. Contrary to the
cuticle deposition (figure 5), the shell sur-
face area, shell thickness and number of
pores did not influence the eggshell pe-
netration (De Reu et al. 2006c).
The results indicate that Gram-negative,
motile and non-clustering bacteria pene-
trated the eggshell most frequently (figure
6a); Pseudomonas sp. and Alcaligenes sp.
(58%) were primary invaders followed by
SE. In comparison with the non-Salmo-
nella strains, SE was a primary invader of
whole eggs suggesting not only the eggs-
hell penetration capability of SE as motile
bacterium but also the capability to sur-
vive the anti-microbial properties of the
albumen (figure 6b). Egg related SE strains
had no special capacity to contaminate
whole eggs compared to other Salmonella
serovars. Penetrated eggshells and conta-
minated whole eggs showed a significant
higher bacterial eggshell contamination
(De Reu et al. 2006c).
Those findings are not always confirmed
by other researchers. The defence of the
cuticular layer has for example questioned
by Nascimento et al. (1992) and Messens
et al. (2005a) using agar-filled eggs. Other
researchers found that SE had special ca-
pabilities to survive in the albumen com-
pared to other Salmonella serovars. On
the other hand attempts made by several
workers to correlate eggshell porosity with
bacterial penetration gave varying results.
The most important contribution of the
shell is to provide a mechanical protection
(Board and Tranter 1995). The shell also
forms a physical barrier against external
microbial contamination. De Reu et al.
(2006c) found no relationship between
shell thickness and the likelihood of bacte-
ria including SE to penetrate the eggshell.
The shell membranes are very effective
barriers to bacterial penetration and at the
same time have antibacterial properties.
Influence of sweating (condensate) and
hen age
During storage and transport until con-
sumption excessive temperature fluctu-
ations should be avoided because this
provokes water condensation and sub-
sequently microbial growth and possible
eggshell penetration. De Reu et al. (2006b)
found a short sweating period of the egg
influences the eggshell penetration (agar
method) but had no impact on the egg
content contamination (intact egg me-
thod). In the study condensation on the
Figure 4: eggshell contamination with total count of aerobic bacteria in furnished cages (7 flocks) compared to non-cage systems (6 flocks) (De reu et al. 2009)
Figure 5: Influence of cuticle deposition on eggshell penetration of 7 bacterial species (lower red color = more cuticle deposition) (De reu et al. 2006c)
Figure 6a: percentage of eggshell penetrati-on (agar method) for each individual bacte-rial strains
Figure 6b: percentage whole egg contami-nation (intact egg method) for each indivi-dual bacterial strains (De reu et al. 2006c)
14
eggshell encouraged the bacterial egg-
shell penetration with SE but it had a smal-
ler and not significant impact on the who-
le egg contamination. The higher survivals
of the pathogen on the eggshells of agar-
filled eggs with condensate might explain
the higher penetration of those eggshells.
The low impact of condensation on the
whole egg contamination can be exp-
lained by the equal survival of SE on the
eggshell of whole eggs with and without
condensation, and by the antimicrobial
defences of the albumen. The higher who-
le egg contamination found at the end of
lay compared to the previous hen ages
showed older eggs might be more su-
sceptible for egg content contamination.
There are many possible reasons for the
higher susceptibility for bacterial infection
of eggs from older hens: cuticle depositi-
on decreases, decrease in shell quality, less
antimicrobial properties of egg albumen,
… Although the various research results
sometimes contradict each other.
Salmonella contamination and Salmo-
nella control programs
SE is considered the only pathogen cur-
rently posing a major risk of egg-borne
diseases in the European Union. Namely,
eggs and egg products are most incrimi-
nated in outbreaks caused by SE but in
few occasions they are also susceptible
to contamination with other pathogens,
such as B. cereus, Staphylococcus aureus,
Listeria monocytogenes and Campylobac-
ter (EFSA 2014). Much of the research on
eggshell and egg content contamination
focuses on Salmonella, and especially SE.
Observed Salmonella prevalence on the
eggshell and in the egg content vary,
depending on the fact whether investi-
gations were based on randomly samp-
led table eggs or on eggs from naturally
infected hens. Dewaele et al. (2012) found
on naturally infected SE farms 0.18 - 1.8%
contaminated eggshells and 0.04% – 0.4%
egg contents. In 2003, the European Com-
mission issued Regulation No. 2160/2003,
requiring member states to take effective
measures to detect and control Salmonella
and other zoonotic agents, not only at the
level of primary production of animals, but
also at other stages in the food production
chain. This legislation specified general re-
quirements to establish a national control
program (NCP) for Salmonella. A subse-
quent Directive (2003/99/EC) aimed at en-
suring that zoonoses, zoonotic agents and
their antimicrobial resistance are properly
monitored, and that food-borne outbreaks
are properly investigated. Later, minimal
demands for NCPs in laying hen holdings
were stated in Regulations No. 1168/2006
and 1177/2006 as amended by Regulation
No. 517/2011. The application of Salmo-
nella control programmes at EU level has
led to a clear decrease in the incidence of
SE infection in laying hen flocks, and of hu-
man infections due to this serovar.
Influence of storage temperature and
timeDe Reu et al. (2008) found the level of eggs-
hell contamination by total aerobic flora
and Gram-negative bacteria was shown to
decrease over a storage period of nine days
or more in both non-refrigerated and ref-
rigerated eggs. The survival of Salmonella
on egg shells is possible and is enhanced
by low temperature. Survival is inversely re-
lated to both storage temperature and re-
lative humidity (Humphrey, 1994; Messens
et al. 2006). In egg albumen SE grows slow-
ly and only to a limited extent because the
albumen contains multiple antimicrobial
components, including bacterial cell wall
and DNA damage (Gantois et al., 2009). At
less than 8°C Salmonella is unable to grow
in albumen (EFSA 2014) and a bacterici-
dal effect is observed at 4°C in a few days.
These findings show that cooling of eggs
is appropriate to reduce bacterial infection
and outgrowth with Salmonella and other
spoiling organisms.
ConclusionsIt is clear that several factors play a role in
spoilage and Salmonella contamination
of table eggs. But under healthy breeding
conditions an egg’s content is generally ste-
rile just after laying and good handling and
preservation further in the agri-food chain
will certainly reduce the risk for microbiolo-
gical contamination to an exception.
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Author / Co-authorsDe Reu, K.1*, Herman, L.1, Heyndrickx, M.1 and De-
waele, I.2
1 ILVO – Institute for Agricultural and Fisheries Re-
search, Brusselsesteenweg 370, 9090 Melle, Belgium
2 Formerly ILVO, now Poulpharm, Prins Albertlaan
111, 8870 Izegem, Belgium
* Corresponding author: