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 Koen.dereu@ilvo.vlaanderen.be

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:

Koen.dereu@ilvo.vlaanderen.be