Food Microbiology 99 (2021) 103823

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

The changing microbiome of poultry meat; from farm to fridge

M. Marmion a, b, *, M.T. Ferone a, b, P. Whyte c, d, A.G.M. Scannell a, b, d
a
UCD School of Agriculture Food Science and Veterinary Medicine, Belfield, Dublin 4, D04, V1W8, Ireland
b
UCD Institute of Food and Health, Belfield, Dublin 4, D04, V1W8, Ireland
c
UCD School of Veterinary Medicine, Belfield, Dublin 4, D04, V1W8, Ireland
d
UCD Centre for Food Safety, University College Dublin, Belfield, Dublin 4, D04, V1W8, Ireland

A R T I C L E I N F O A B S T R A C T

Keywords: Chickens play host to a diverse community of microorganisms which constitute the microflora of the live bird.
Meat microflora Factors such as diet, genetics and immune system activity affect this complex population within the bird, while
Processing external influences including weather and exposure to other animals alter the development of the microbiome.
Campylobacter
Bacteria from these settings including Campylobacter and Salmonella play an important role in the quality and
Broilers
safety of end-products from these birds. Further steps, including washing and chilling, within the production
cycle aim to control the proliferation of these microbes as well as those which cause product spoilage. These steps
impose specific selective pressures upon the microflora of the meat product. Within the next decade, it is forecast
that poultry meat, particularly chicken will become the most consumed meat globally. However, as poultry meat
is a frequently cited reservoir of zoonotic disease, understanding the development of its microflora is key to
controlling the proliferation of important spoilage and pathogenic bacterial groups present on the bird. Whilst
several excellent reviews exist detailing the microbiome of poultry during primary production, others focus on
fate of important poultry pathogens such as Campylobacter and Salmonella spp. At farm and retail level, and yet
others describe the evolution of spoilage microbes during spoilage. This review seeks to provide the poultry
industry and research scientists unfamiliar with food technology process with a holistic overview of the key
changes to the microflora of broiler chickens at each stage of the production and retail cycle.

1. Introduction
Established prior to hatching, the poultry microbiome is a complex
microbial community found within each individual bird which exerts
influence over its health, growth, intestinal physiology and resistance to
infection for life (Clavijo and Vives Flórez, 2018). This community of
protists, fungi, archaea, viridplantae and bacteria constitutes a dynamic
proto-organ which develops throughout the bird’s life. The composition
of the microbiome is subject to both host genetics and the environment
(Fig. 1) in which the bird exists (Broom and Kogut, 2018). The avian
microbiome contributes to the health of the host, influencing a range of
processes from complex carbohydrate breakdown and fermentation to
vitamin production and immune system stability (Broom and Kogut,
2018). Most microorganisms exist as passive commensals within the
avian host, including human pathogens such as E. coli, Campylobacter
and Salmonella. Fluctuations in the composition of the microbiota can
lead to long-term effects for the host bird, as well as within associated
products. Imbalance in the microbiome, or dysbiosis, can be problematic
for the health of the bird as it is linked to poor GIT barrier function, poor
nutrient uptake and gastric inflammation, leading to symptoms such as
diarrhoea which can spread disease when combined with high stocking
densities (Shang et al., 2018). Dysbiosis can cause blooms in putative
pathogens such as Avian Pathogenic Escherichia coli (APEC), a possible
relative of human Extraintestinal Pathogenic E. coli (ExPEC), and Clos­
tridium perfringens, which may impact upon both the host and the human
consumers (Clavijo and Vives Flórez 2018; Kers et al., 2018; Manges,
2016; Oakley et al., 2014; Shang et al., 2018).
Poultry slaughter and processing inadvertently cause the spread of
GIT associated microorganisms between carcasses and cause contami­
nation of processing surfaces and environment, especially during the
defeathering and evisceration steps (Boubendir et al., 2021; Buess et al.,
2019). These microbes affect the quality of poultry meat, reducing the
shelf-life, and potentially causing outbreaks of foodborne campylo­
bacteriosis and salmonellosis if the meat is not handled correctly by the
consumer (EFSA, 2018). Meat is a nutrient-rich matrix which supports
the growth of bacteria, fungi, and protists. The extended storage times

* Corresponding author. UCD School of Agriculture Food Science and Veterinary Medicine, Belfield, Dublin 4, D04, V1W8, Ireland.
E-mail address: matthew.marmion@ucdconnect.ie (M. Marmion).

https://doi.org/10.1016/j.fm.2021.103823
Received 15 January 2021; Received in revised form 24 April 2021; Accepted 27 April 2021
Available online 1 May 2021
0740-0020/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
M. Marmion et al. Food Microbiology 99 (2021) 103823

that chicken meat undergoes can allow for the proliferation of different that is found throughout the broiler microbiome is Lactobacillus, which is
microbial species on meat surfaces, depending on the given conditions. especially dominant in the crop and gizzard, while other important
Factors such as storage temperature and packaging atmosphere can Firmicute genera include populations of the Clostridium, Flavonifractor,
affect the time it takes for chicken to lose its appeal to customers as it the Faecalibacterium and Ruminococcus genera (Broom and Kogut, 2018;
meat is stored for extended periods. The nature of the packaging ap­ Oakley et al., 2013; Shang et al., 2018). However, considerable variation
proaches also dictate the nature of the spoilage that transpires with in the composition of the broiler microbiome can be observed (Table 1).
respect to the dominant microflora of the end-product (Conte-Junior A range of factors can affect the microflora of live broilers. The
et al., 2020; Rouger et al., 2018). It is therefore clear that controlling the bacterial taxa dominating within the GIT of the host is influenced by age,
composition of the microbiome will benefit poultry producers, meat sex, diet, production system and hormones, as well as extant commensal
processors, retailers, and downstream consumers, potentially improving microbes and exposed host cell ligands, which can contribute to the
the both the quality and safety of the poultry meat. establishment of complex and dynamic bacterial populations in unique
Whilst a number of excellent reviews exist detailing the microbiome niches throughout the chicken gastrointestinal tract (GIT), affecting bird
of poultry during primary production (Clavijo and Vives Flórez, 2018; health, and the quality of any meat products derived from said birds.
Pandit et al., 2018), and the fate of important poultry pathogens such as Some of these factors can fluctuate throughout the bird’s life, contrib­
Campylobacter and Salmonella spp. At farm and retail level have been uting to alterations in the gastrointestinal microbiota (Borda-Molina
reported (Rivera-Pérez et al., 2014; Skarp et al., 2016), and further et al., 2018; Broom, 2019).
studies look at the evolution of spoilage microbes during spoilage (Chen
et al., 2020; James et al., 2006), this review takes a more holistic 2.1. Host breed
approach. We aim to assess the impact of various factors on changes in
the microbiome of live broiler chickens, and how these influences might Individual bird species and genetics have been found to be the largest
affect the microflora of end-product, as well as ascertaining the micro­ determinant of the microbiome composition of an individual. Factors
bial influences that occur throughout the meat manufacturing process such as feed conversion, physiology, immune system activity and
and refrigerated storage. These influences may originate in the live bird behaviour, the host phenotype acts as a strong determinant upon the
or be introduced at various stages during the food chain.
Table 1
2. The live poultry microbiome Important bacterial genera of the broiler microflora.
Phylum Genera Reference
The poultry microbiome undergoes many changes throughout the
life of the host. The dominance of a single phylum, genus or species is Firmicutes Anaerostipes, Blautia, Butyrivibrio, (Borda-Molina et al., 2018;
Clostridium, Ethanoligenes, Broom and Kogut, 2018;
usually transient. Overall, over 117 genera have been found within the
Eubacteria, Flavonifractor, Clavijo and Vives Flórez,
broiler microbiome. However, this community is diverse and dynamic. It Hespellia, Lachnospiraceae, 2018)
has been well-established that, while Proteobacteria dominate the early Lactobacillus, Leuconostoc,
broiler chick microbiome, by the time of slaughter, around 32 days old Megamonas, Pseudoflavonifractor,
for conventionally produced broilers, the microbiome is dominated by Roseburia, Ruminococcus,
Streptococcus, Veillonella
genera from the phylum Firmicutes which make up 70–78% of caecal
Bacteroidetes Bacteroides, Paraprevotella, Clavijo & Vives Flórez
microbes. Genera from the phylum Bacteroidetes forming a large mi­ Prevotella, Riemerella, Tannerella (2018)
nority (~11%) of the remaining bacterial genera. Other important phyla Proteobacteria Campylobacter, Desulfohalobium, (Ae Kim et al., 2017; Bailey
at this stage of life include Proteobacteria, and Actinobacter, while ev­ Escherichia, Gallibacterium, et al., 2018; Clavijo and
Neisseria, Pseudomonas, Vives Flórez, 2018)
idence for another 9 phyla has been found in other metagenomic studies.
Salmonella, Shigella, Vibrio,
By the age of slaughter, the broiler caecum can have as many as 1011 Yersinia
Colony Forming Units (CFU)/g of caecal contents as a mature and Actinobacteria Bifidobacterium, Corynebacterium, (Shang et al., 2018; Teng
diverse microbiome develops (Broom and Kogut, 2018; Clavijo and Streptomyces and Kim, 2018)
Vives Flórez, 2018; Oakley et al., 2013). The most dominant single genus

Fig. 1. Key Factors that influence the poultry microbiome *MAP = Modified Atmosphere Packaging.

2
M. Marmion et al.
favouring of certain bacterial species within a host (Pandit et al., 2018;
Shi et al. 2019). This can extend further to breed characteristics. For
example, the extensively used modern Cobb breed of broilers has a lower
Bacteroidetes:Actinobacter ratio than is found in another widely used
commercial line, the Ross broiler. Meanwhile, some breeds have shown
increased susceptibility to colonisation by antibiotic resistant Escherichia
coli (Kers et al., 2018; Waite and Taylor, 2014).
Further studies comparing Ross308 and Cobb400 broilers with less
intensively bred native birds, such as Indian Kadaknath and Aseel lines,
raised on the same diet and in the same location indicate that Bacter­
oidetes and Firmicutes dominate the GIT microbiome on a phylum level
across all lines analysed by Pandit et al. (2018). These phyla covered
76.6–90.8% of GIT operational taxonomic units (OTU), a measure of
species diversity, within the broiler lines of interest, but variation at
class and genus level was evident beyond this. Bacteroidia were found to
dominate at a class level in each line, except in the case of Cobb400, in
which Clostridiales species dominated. Higher overall GIT OTUs were
reported in commercial lines, with positive correlations reported for
most bacteria in Ross308 and Cobb400 birds (Pandit et al., 2018).
However, birds from the Kadaknath and Aseel lines were reported to
carry more unique genera than modern broiler breeds, with Caulobacte,
Geobacillus, Cyclobacterium, Caldicellulosiruptor, Desulfovibrio and Cyto­
phaga found frequently in Kadaknath lines, and Slackia, Cronobacter,
Phascolarctobacterium, Oceanimonas, Deferribacter, Tepidibacterium, Ato­
pobium, Tannerella, Zunongwangia, Acetobacterium, unclassified Alphap­
roteobacteria, Candidatus and Phytoplasma found in the Aseel breed
(Pandit et al., 2018). Less unique genera were reported in modern
breeds, with 10 distinct genera observed in Cobb400 and only 2 seen in
Ross308 birds. The heterogeneity of the immune system of breeds such
as Jerusalem and Virginia broiler lines may also contribute to host control
over GIT colonisation, especially when compared to the more homoge­
nous immune system activity of breeds such as the Wageningen line (van
der Most et al., 2011).
The different breeds of bird can require different nutrients, housing,
and handling. While it is rare to eat laying hens due to changes in the
meat flavour profile, some of these birds may still be eaten in ethnic
cuisine. Laying birds are typically exposed to cyclical hormone levels
and allowed to reach greater ages, while over a century of selective
breeding has had genetic effects on factors such as Feed Conversion Rate
(FCR), immunoglobulin expression, and the deposition of muscle (Kers
et al., 2018). The burden of varying factors and their effect on differ­
ences between the microbiota of laying birds and in broilers are difficult
to fully establish, especially in meta-analyses. This is due to the preva­
lence of male-only studies of the broiler chicken microbiome, conducted
due to difficulties in replicability caused by hormone fluctuations in
female broiler chickens (Videnska et al., 2014).
2.2. Influence of host genetics
The influence of host genotype on the microbiome composition ex­
tends from immune system activity to FCR. The ability of a bird to
convert feed to bodyweight is one of the most important factors in
selecting suitable breeds and is strongly associated with the microbiome
composition. Broilers have been selectively bred for over a century to
produce the heaviest birds with the lowest amount of food consumed, i.
e., to have a lower FCR. This contributes to metabolic gene alterations
while lower FCR have a microbiome in broilers when compared to layer
hens. This can have knock-on effects for the gastrointestinal microbiota,
which have become adapted to produce increased quantities of short
chain fatty acids, due to higher numbers of Firmicutes and Bacteroides,
as well as enzymes including α-amylase, α-1,2-mannosidase and endo-
1,4-β-mannosidase produced by the Bacteroides phylum. These bacteria
supplement host metabolic activities on dietary starch and polymers
(Pandit et al., 2018). High FCR birds have been found to have higher
numbers of Acinetobacter, Bacteroides, Clostridium, Lactobacillus and
Streptococcus species, higher in species from the Salmonella, Escherichia
3
Food Microbiology 99 (2021) 103823
and Shigella genera. Microbes in these birds tend to have altered genetic
expression with enriched transport and metabolism genes, including
those involved in the citrate cycle as well as lipid storage and
Peroxisome-Proliferator Activated Receptor (PPAR) expression (Ding
et al., 2016). It has also been reported that low FCR birds carry higher
numbers of Lactobacilli (Borda-Molina et al., 2018; Kers et al., 2018).
Microbes can also impact FCR. For example, male broilers supple­
mented with probiotic Bacillus subtilis CGMCC1.1086 showed an
improved FCR and increased weight gain when compared to control
birds (Maki et al., 2019). Lactobacilli are frequently found in the crop of
broiler chickens where they contribute to the production of lactate via
starch hydrolysis and fermentation. For this reason, they have frequently
been investigated for use as probiotics (Arsi et al., 2015). This glucose
precursor is associated with bird weight gain and underlines the
importance of supporting the microbiome when feeding livestock, as
this can contribute to the overall wellbeing and quality of the animal
product (Borda-Molina et al., 2018). Lactobacilli have been associated
with low FCR broilers regardless of gender across several studies (Du
et al., 2020; Maki et al., 2019; Stanley et al., 2016).
The use of selectively bred high yield/low FCR broilers has become
widespread in modern agricultural practice, leading to larger birds with
a metabolism primed for production of consistently high-value meat.
These birds display altered phenotypes from their ancestors, as well as a
divergent microbiome. A comparison of the microbiome of a historic
breed i.e., Athens Canadian Random Bred (ACR) line, with modern high-
yield and multipurpose birds (HY/MP) indicates that, while both ACR
and HY/MP lines exhibited microbial instability early in life, until day 8,
there were distinct shifts in the microbiome of the bird lines after this
point depending on the bird breed. By the time of slaughter, there was
less than 50% similarity between MP and ACR breed microbiome,
regardless of diet (Lumpkins et al., 2010). By day 21 of life, distinct
microbiomes had developed in each line (ACR, HY and MP) due to the
maturation and stabilisation of the GIT environment. Approximately
90% microbiome similarity was reported between MP and HY birds,
with divergence emerging due to potential differences in characteristics
such as villus height and mucosal crypt depth, local pH, mucin pro­
duction and local enzyme production (Lumpkins et al., 2010). Further
factors linked to host genotype which may cause microbiome differen­
tiation include obesity, the genotype for which can affect the micro­
biome in mice (Ley et al., 2005; Lumpkins et al., 2010).
2.3. Host immune system
The poultry immune system exerts host protection within the GIT of
birds over their lifetime. For example, the immune response at the
mucosal level can prevent microbial adherence. This is facilitated by
structures such as tight junctions between epithelial cells which form a
physical barrier to microbial entry, while Pattern Recognition Receptors
(PRR) on the surface of both antigen detecting cells and epithelial cells
continuously sample luminal contents, acting to induce humoral and
cellular responses should threats be detected. The nutrient-rich niches of
the GIT are coated in a layer of mucins, primarily MUC2, to inhibit
microbe adhesion to mucosal surfaces. Within this mucous, antibodies
such as immunoglobulin A (IgA) further prevent pathogen binding to
epithelial surfaces, as well as providing humoral defences against these
microbes (Willson et al., 2018).
Maternal antibodies, such as IgA and IgY, are frequently found in the
mucosal layer of the gastrointestinal tract of chickens during the first
two to three weeks of life and are an important early determinant of the
chick microbiome which are present in the yolk of egg. These antibodies
provide specific immunity to prevent early gastrointestinal colonisation
by microbes, including several viruses and putative pathogens such as
Campylobacter (Gharaibeh and Mahmoud 2013; Kers et al., 2018; Van­
marsenille et al., 2018), thus influencing the viriome and microbiome of
the chicken, and consequently impacting the health status of the bird.
The production of immunoglobulin A (IgA) by GIT lamina propria
M. Marmion et al.
plasma cells is a crucial element in this control (Shang et al., 2018).
While the development of the broiler adaptive and innate immune
systems in utero is dictated by host genetics, post-hatch immune matu­
ration requires ‘training’ by microfloral antigen signals from the surface
of commensal microbes. This development includes inducing changes in
both mucosal structure, antimicrobial peptide production and microbe-
specific IgA production by lamina propria plasma cells (Broom and
Kogut, 2018; Clavijo and Vives Flórez, 2018). This training is vital for
the development of a specific, mature immune system within a healthy
host.
The presence of microbes within the chicken GIT environment pre­
sents host epithelial and antigen-detecting cells with a variety of
Microbe-Associated Molecular Patterns (MAMP). When microbes bind
to host PRRs, the MAMPs induce production of both cytokines and
chemokines, via macrophage encounters with microbes near the host
mucosa, as well as the expression of antimicrobial proteins within the
lamina propria of the GIT. This can create a low level of inflammation
within the gut producing an environment into which heterophil and
other leukocytes migrate (Oakley and Kogut, 2016). These non-specific
immune cells are recruited by the interleukin (Il)-8 -like chemokine
CXCLi1 to the site of infection, and have roles in the phagocytic control
of invasive microbes, such as Salmonella spp. Through degranulation,
oxidative burst and extracellular traps which kill local microbial threats
(Broom, 2019). The presence of high numbers of heterophils correlates
with less systemic invasion by members of the poultry microbiome such
as Salmonella spp., while heterophil deficient individuals are also
observed to have higher mortality. Taken together, it is clear that these
cells play a vital role in control over the host microbiome (Broom, 2019).
Genera such as Bifidobacteria are associated with improved GIT
health, while families such as Lachnospiraceae and species such as Fae­
calibacterium prausnitzii have been shown to actively influence the pro­
duction of short-chain fatty acids (SCFAs) such as butyrate through
fermentation of complex carbohydrates within the caecum (Clavijo and
Vives Flórez, 2018). SCFAs act as energy sources for host enterocytes
and induce mucin production within the GIT. These nutrients can then
enhance overall GIT health and integrity (Borda-Molina et al., 2018;
Shang et al., 2018; Thibodeau et al., 2015). Important functions such as
improving nutrient availability and vitamin synthesis are also provided
by the GIT microbiota, influencing host traits such as growth rate and fat
deposition. The avian GIT microbiome further influences the conversion
of uric acid to ammonia, which provides the host chicken with material
with which to make amino acids (Shang et al., 2018).
Selective breeding of modern broilers has had downstream conse­
quences as these birds exhibit stunted immune development, with hu­
moral immunity frequently underdeveloped. For example, a lower
diversity of antibodies against common avian viral threats can be pro­
duced in these broilers, with effective antibody production only seen
against Newcastle Disease virus. This reduced immune efficacy may be
linked to the energy-dense needs of growth since pro-inflammatory cy­
tokines, produced to control microbial proliferation within a host can
direct energy away from growth, something which is detrimental to
rapid growth rates in broiler birds (Broom, 2019; van der Most et al.,
2011). Alternatively, it could be a result of an unconscious selection
away from phenotypes with an active pro-inflammatory response. The
microbiome of these low FCR birds is subject to lower levels of host
control than their ancestors, reducing the effect of the host immune
system on the GIT microbiome in commercial poultry breeds.
2.4. Bird maturation
While age is not a direct effector on the microbiome composition of a
broiler chicken, the life of the broiler is dictated by age-related processes
that occur within and to the host. Processes such as feed changes,
exposure to external microbes and immune system development are
time-dependent processes which will affect the microbiome. The most
dominant phyla within the avian gastrointestinal microbiome are the
4
Food Microbiology 99 (2021) 103823
Firmicutes, Proteobacteria and Bacteroidetes, with the component
makeup of these phyla being highly dependent upon temporal factors
among others (Shang et al., 2018). The development of broilers with
time has a variety of external influences as the bird matures which can
affect the unique composition of the microbiome. These different in­
fluences over microbiome expand the variation between individuals and
microbial niches with time. Although broiler eggs are frequently cleaned
and fumigated to prevent entry of environmental microbes via shell
pores, upon hatching, domestic chicks are exposed to microbes which
have survived the ultra-hygienic hatchery environment (Meijerink et al.,
2020). Initial microbial exposure is further influenced by diet and con­
tact with human handlers during the early days of a chick’s life. (Kers
et al., 2018; Rothrock et al., 2019). Transport crates, utilised during
transport from the hatchery as well as later towards the abattoir, are a
source of external microbes which can contribute to the establishment of
the bird’s microbiome (Kers et al., 2018; Stanley et al., 2013a, 2013b).
Maternal antibodies, particularly IgA, from the egg yolk protect the
chick against colonisation by external bacteria for the first 14 days of
life. After this point, the microbiome undergoes substantial changes
(Kers et al., 2018). However, the microbes to which the young broiler is
exposed early in life, even transiently, have been found to have a pro­
found effect on the composition of and diversity of the mature broiler’s
microbiome (Meijerink et al., 2020).
Zoonotic pathogenic bacteria, e.g. Salmonella spp., can struggle to
become established in the broiler microbiota once the mature micro­
biota of the broiler GIT has developed due to competitive exclusion from
a preferred niche. Early colonisation of broilers by these human path­
ogens is important for these genera to become established in the broiler
population (Antunes et al., 2016; Cosby et al., 2015).
Comparing the microbes in birds at Day 3 and at Day 49 (Table 2)
demonstrates the microbiome shifts that occur in broilers with
advancing age (Lu et al., 2003; Shang et al., 2018). By day 21 of life, the
microflora reaches a point of maturity, though from this point on the
variation within the microbial communities begins to fall (Feye et al.,
2020a). Studies have established that the early laying hen microbiome is
dominated by Proteobacteria, particularly members of the family
Enterobacteriaceae and genus Escherichia, which can comprise up to 50%
of species in the microbiome of layer chicks during the first week of life.
This period of the chick’s life is also marked by the most variability in
the microbiota. Firmicutes, particularly families Lachnospiraceae (genera
Roseburia and Blautia) and Ruminococcaceae, become dominant after this
point, as levels of Proteobacteria decline in the layer GIT (Broom and
Kogut, 2018; Rothrock et al., 2019; Videnska et al., 2014). This trend,
however, is not seen in broiler individuals, where Firmicutes dominate
following hatch (Clavijo and Vives Flórez, 2018). Flavonifractor, Pseu­
doflavonifractor and Lachospiraceae were found to be the species in
highest abundance until day 7 post-hatch in broiler chickens, with
Faecalibacterium then becoming the most dominant species. The
numbers of other species can continue to fluctuate, with increases in
genus Roseburia, as well as a Lachnospiraceae sequence type by day 42,
however there continues to be considerable variation in the avian
microbiome, even among adjacently reared birds, at this stage (Broom
and Kogut, 2018; Clavijo and Vives Flórez, 2018; Kers et al., 2018; Shang
Table 2
Maturity-dependent changes in poultry microbiomea.
Time Dominant Families, Genera and Species
Point
Day 0–3 Lactobacillus delbruecki, Clostridium perfringens, Campylobacter coli,
Escherichia spp., Enterobacteriaceae, Flavonifractor, Pseudoflavonifractor,
Lachnospiraceae
Day 7–21 Lactobacillus acidophilus, Enterococcus, Streptococcus, Faecilbacterium
Day Lactobacillus spp. Including Lb. Crispatus, Faecilbacterium, Roseburia,
28–49 Lachnospiraceae
a
(Adapted from Lu et al., 2003; Broom and Kogut, 2018; Clavijo and Vives
Flórez, 2018).
M. Marmion et al.
et al., 2018). Production factors in-line with bird age including dietary
changes like the beginning of the finisher diet from day 28 of life
introduce further selective changes within the broiler GIT microbiome
(Feye et al., 2020a). By day 42 of life, the broiler microbiome has un­
dergone diversification on a large scale, as the 50 genera that colonise it
on day 0 of life broaden to over 200 genera (Borda-Molina et al., 2018).
2.5. The external environment of the bird
The microbiome of broilers does not exist in isolation from the
environment in which it is raised. It is influenced by climatic, social, and
condition-related factors which can induce changes in the internal
microflora. Factors such as housing, exposure to the outdoors, pasture-
rearing, rearing alongside adjacent animals, exposure to wild vectors,
litter, farming practices, heat stress, presence of faeces, bird density, and
other influences affect the health, wellbeing, and productivity of the
bird. The microbiome is subject to these influences too, as these factors
affect bird behaviour, for example faecal pecking, water drinking and
movement, and individual exposure to exogenous microbes (Borda-­
Molina et al., 2018; Kers et al., 2018; Shi et al., 2019). The control of
these factors through biosecurity and appropriate bird management
provides a multi-hurdle challenge for microbes to overcome. However,
there remain opportunities and sources for microbes to enter the
food-chain.
Location can have a strong influence over the microbiome of broilers,
due in part to local factors such as weather and local wildlife (Shi et al.,
2019). Commonly utilised broiler lines such as Cobb400 and Ross308
have distinct differences in the carriage of genera across diverse loca­
tions, indicating that these local factors have a strong influence over the
microbiome. For example, while Bacteroides dominated the microbiome
of Ross birds across two Indian regions, Cobb birds saw higher numbers
of Firmicutes at one location, and Bacteroidetes in the other (Pandit
et al., 2018).
Season can have a strong impact on the composition of the micro­
biome of the bird. High summer temperatures have been linked to the
over-proliferation of dominant species, while higher bacterial CFUs are
isolatable from meat in the summer than in the winter (Kim et al., 2019).
The seasonal influence on certain bacteria such as Campylobacter has
also been previously reported with peaks observed in the summer
(Borda-Molina et al., 2018; Hald et al., 2007; Kers et al., 2018). The
summer period is also associated with higher incidences of fly vectors,
which can spread microbes within and between broiler houses, while
high temperatures induce stress responses in chickens that are associ­
ated with higher levels of both Salmonella and Campylobacter species.
The cleanliness of the hatchery, as well as biosecurity approaches
utilised on commercial farms can create opportunities for the creation of
a dysbiotic microbiota. Due to the lack of exposure to parental and adult
microbes in modern flocks, the largest source of microbes becomes both
the environment in which chickens are raised, and the birds with which
an individual is surrounded by. For example, early contamination by
more resilient taxa such as spore-forming Clostridium spp. Can lead to
early colonisation of chicks by these bacteria. This genus includes the
avian pathogen C. perfringens, which may cause necrotic enteritis within
susceptible immune hosts (Ludvigsen et al., 2016). Clostridia, of the
phylum Proteobacteria, can form a major part of the early chick
microbiome (Kers et al., 2018; Ludvigsen et al., 2016). This demon­
strates the susceptibility of the early microbiome to colonisation by
zoonotic pathogens, and putative pathogens.
2.6. Gastrointestinal tract conditions
The highest abundance and diversity of organisms within the
microbiome is found within the gastrointestinal tract (GIT), especially in
the large intestine and caecum as both microbial abundance and di­
versity increase distally along the GIT (Broom and Kogut, 2018). The
preceding organs within the GIT have a distinct influence upon the
5
Food Microbiology 99 (2021) 103823
microbiome of successive regions through affecting characteristics such
as pH, bile salt concentration, oxygenation, available nutrients, and
antimicrobial species within the GIT contents. The different conditions
can also affect the metabolism of microbes within adjacent niches
(Fig. 2). The lower pH and higher levels of digestive enzymes of the
gizzard contribute to reduced starch fermentation by Clostridium and
Lactobacillus species when compared to the saccharolytic activity and
lactate fermentation by the same genera and species in the adjacent crop
(Clavijo and Vives Flórez, 2018). These condition-dependent functions
may go on to contribute to the health of the host through a series of
complex interconnections of the microbiota with host well-being.
The gastrointestinal lumen and mucosa provide disparate environ­
mental challenges, such as high salt, low oxygen, and stable nutrient
contents within the mucosa, or short nutrient retention time and more
pH variation in the lumen. However, as nutrient stability and long
retention time benefit microbial growth within the GIT, it has been
found that a greater diversity can be seen in the mucosa (Borda-Molina
et al., 2018). Up to Day 14 of a chick’s life, the microbial composition of
the ileum has a direct influence upon the composition of the caecal
microbiome, which can considered a subset of that of the ileum (Lu
et al., 2003). However, after Day 14, a large disparity between the two
regions emerges. The long retention time within the caecum, as well as
high concentrations of bioavailable nutrients not utilised by the host and
very low oxygen content contributes to favourable growth conditions for
many microbes within this niche, allowing this region to become
colonised by a more diverse group of organisms when compared to
preceding regions in the GIT. With time, this allows divergence of the
caecal microbiome from that of the ileum. The caecal microbiome is
dominated by the phyla Firmicutes and Bacteroidetes, which includes
Clostridiaceae, Bacteroidaceae Lactobacillaceae and butyrate-producing
Lachnospiraceae family bacteria (Borda-Molina et al., 2018; Shang
et al., 2018).
Disparate conditions within the GIT are further influenced by the
mucosal layer that coats much of the surface of the GIT. Mucous pro­
vides stable environment, ideal for the of microbes, leading to higher
levels of diversity (Borda-Molina et al., 2018). The mucous is also ideal
for the proliferation of mucinolytic bacteria and their dependent species.
A micro-aerobic environment is created in the mucosal lining of the GIT
due to oxygen diffusion from the dense capillary network of the GIT into
the mucous. This aids the survival of microaerobic species, such as
Campylobacter spp., within the thick mucous lining, (Zhang et al., 2018).
The mucosa of the GIT is dominated by bacterial species such as Rumi­
nococcus, Turicibacter, and Clostridium XIV a/b, in contrast with the more
turbulent lumen, in which nutrient availability and quality fluctuate
much more, while shedding is also more frequent. This niche tends to be
dominated by Anaeroplasma, Oscillobacter, Pallibacter, Peptococcus and
Subdoligranulum. The lumen environment favours the survival of obli­
gate anaerobic bacterial species due to the low prevalence of oxygen
found in this niche, Lactobacillus spp. Are found in up to three times
higher numbers in this region of the GIT (Borda-Molina et al., 2018;
Zhang et al., 2018). The survival of Lactobacterium spp. Within the du­
odenum and jejunum is further improved through production of bile salt
hydrolase which prevents bile-induced cell damage (Zhang et al., 2018).
The pH changes of GIT contents follow a trend of rapid acidification,
followed by slow neutralisation with time and passage along the GIT
(Gauthier, 2002). The pH of the GIT undergoes distinct shifts due to the
role of acid in the macromolecular breakdown of feed within the gizzard
of the bird. This acidification reduces the survival capacity of many
microbes, while also preventing microbial fermentative metabolism
(Clavijo and Vives Flórez, 2018). Few microbes are adapted to the
extreme acidity of the gizzard, with gastric Helicobacter spp. Being a rare
acid-tolerant species that can proliferate within and colonise this harsh
environment (Hamada et al., 2018; Marcus et al., 2018). Therefore,
while the high acidity in the gizzard limits the proliferation of
pH-sensitive microbes in the GIT prior to the duodenum, there is a
corresponding an increase in microbiome diversity seen from this point
M. Marmion et al.
Fig. 2. Changes in conditions along the avian gastrointestinal tract (Adapted from Duke, 1986; Gauthier, 2002; Clavijo and Vives Flórez, 2018).
as the lumen pH becomes more favourable to a wider variety of species.
At acidic pH, Firmicutes can dominate the microbiome over Bacter­
oidetes. For example, many lactic acid bacteria grow best at a pH of
5.5–6.5, a pH which can be found within the crop [pH 5.5] and after the
duodenum [pH 5–6], but which is not seen in the gizzard [pH 2.5–3.5]
(Gauthier 2002; Pothakos et al., 2015; Zhang et al., 2018). The neutral
pH of the jejunum, ileum and caeca favours the colonisation by luminal
microbes, with members of the Bacteroidetes phylum frequently
outgrowing Firmicutes species as the pH becomes more neutral (Zhang
et al., 2018).
Nutrient availability changes within the host GIT as the host utilises
many constituent carbohydrates prior to access by the microbial popu­
lation. Therefore, the concentrations of easily accessible nutrients such
as simple monosaccharides decline by the time food reaches the lower
GIT. At this stage, most remaining carbohydrates consist primarily of
complex polysaccharides that are inaccessible to the host (Zhang et al.,
2018). Whilst some microbes can utilise these polysaccharides, they
frequently undergo fermentation to release accessible nutrients such as
short chain fatty acids and arabinoxylooligosaccharides (AXOS) (Eeck­
haut et al., 2008). Fermentation occurs both in the crop (primarily by
Lactobacillus and Bifidobacterium spp.) and caecum, locations charac­
terised by a long food retention and a less acidic pH (Borda-Molina et al.
2018; Waite and Taylor 2014).
The contents of the lumen, and the bacteria associated with them, are
important determinants of the microbes that may contaminate meat
end-products. The process of evisceration can perforate the lining of the
intestine, leading to the spread of microbes to both the exterior of the
chicken and the equipment itself, as well as surfaces, workers, and the
air itself within the processor. This can lead to the further contamination
and spread of zoonotic pathogens, e.g. Campylobacter, Salmonella or
E. coli, to adjacently processed carcasses, especially if cleaning protocols
are not adhered to rigorously (Sahin et al., 2015a, 2015b; Seliwiorstow
et al., 2016).
2.7. Diet and feed effects
Broiler chicken rearing requires a diverse diet to allow for fast
growth and adequate weight deposition by the time of slaughter. Mod­
ern feeds and feeding programmes have been developed to favour this
from hatch. Initially, broilers are raised on a high protein starter feed
until circa 21 days of age, when the chicken will switch to a finisher feed,
6
Food Microbiology 99 (2021) 103823
which is lower in protein but higher in carbohydrates, to allow for
weight deposition in the broiler (Anon, 2016). The nature of the feed
will also affect nutrient specificity and availability for the gastrointes­
tinal microbiome. Diet and feed conversion affect nutrient availability
within the host and can lead to blooms and declines in species that are
adept at/deficient in processing the available biomolecules. In mam­
mals, this manifests in major life events such as the switch from maternal
milk to solid food, causing changes in the commensal microbes of the
infant GIT which can lead to health effects (Stanley et al., 2013a, 2013b;
Thompson et al., 2015). The diets of both broiler and layer chickens can
be seen similarly, as dietary contents are crucial determinants of the
microbiome composition. The diet of chickens is typically grain-based
and poorly digestible. This favours microbes which can utilise/absorb
available nutrients within the avian GIT, which may not be readily
accessible. For example, dietary cereals have different levels of arabi­
noxylans, the principle non-starch polysaccharide in cereals found
within plant cell walls. These complex sugars are inaccessible to the
avian host, but act as prebiotic compounds that encourage the growth of
microbial species such as Lactobacilli, non-pathogenic Clostridia, and
Bifidobacteria (Borda-Molina et al., 2018; Duke, 1986; Mendis and
Simsek, 2014). While arabinoxylan undergoes fermentation in the
ileum, AXOS promotes the growth of beneficial microbes such as Bifi­
dobacteria. The increased Bifidobacterium population also acts upon
adjacent microbes through the production of butyric acid which both
encourages strict anaerobe growth, as well as preventing Salmonella
colonisation and invasion (Eeckhaut et al., 2008; Mendis and Simsek,
2014).
Due to their role in microbiota selection, complex polysaccharides
have been proposed as prebiotics, functioning to improve the host
microbiota in three ways: (1). They favour the growth of beneficial
microbial species throughout the host GIT; (2). They are also a principal
source of dietary fibre, acting to increase gastrointestinal content
retention time due to low digestibility. Short retention time has been
linked to increases in the proliferation of fast-growing species such as
Clostridium perfringens, which is frequently associated with avian
necrotic enteritis; and (3) there is evidence that pathogens such as E. coli
and Salmonella can selectively bind to complex carbohydrate prebiotics
with Type-1 fimbriae, allowing their simultaneous egestion from the
host (Borda-Molina et al., 2018; Teng and Kim, 2018). The provision of
low animal-product diets is another nutritional tactic to remove the
burden of potential pathogens, as high animal protein and fats within
M. Marmion et al.
chicken diets have been linked to C. perfringens proliferation within
some individual birds (Borda-Molina et al., 2018; Clavijo and Vives
Flórez, 2018).
Micronutrients are important determinants of microbe survival, with
the presence of phosphates, contributing to the incidence of different
families in the avian GIT. Phosphates are frequently supplemented in the
broiler diet, usually in the form of phytates, to encourage broiler growth
and productivity. The release of phosphates requires endogenous phy­
tases, which are also incorporated as a dietary supplement due to the
poor activity of endogenous broiler phytases. The increased bioavail­
ability of phosphates increases the incidence of Ruminococcaceae and
Bacteroidaceae families, with a concurrent reduction of Eubacterium
(Tilocca et al., 2016).
2.8. Other members of the microbiome
Population dynamics within the microbiome allows microbial com­
munity influences to become important in dictating its own composi­
tion. Production of compounds such as short chain fatty acids (SCFAs; e.
g. butyrate, acetate) act to induce growth and colonisation by members
of the microbiota, while the hydrolytic activities of many genera liberate
nutrients for the growth of other microbes (Luethy et al., 2017; Thibo­
deau et al., 2015). The mucosal environment can also be considered. For
example, hydrolysis of the MUC2 mucin and its related glycans by
genera such as Pseudomonas liberates nutrients such as carbohydrates, or
micronutrients such as sulphur which can be utilised by these microbial
cells. An example of this microbe-dependent growth is the proliferation
of Desulfovibrio spp. In adult chickens due to hydrogen
sulphide-dependent respiration. This requires sulphur released by the
sulfatase activity of Bacteroides thetaiotamicron within the mucous
coating of the GIT contributing to the proliferation of Desulfovibrio at a
specific point of the chicken’s life (Benjdia et al., 2011; Borda-Molina
et al. 2018; Videnska et al., 2014). However, mucinolytic bacteria exert
indirect competitive pressure on species unable to bind the mucosal
surface directly, as they can contribute to mucosal shedding. This con­
tributes to the shedding of some competing microbial species (Clavijo
and Vives Flórez, 2018; Oakley et al., 2014).
The proliferation of Campylobacter is aided in the GIT by microbiome
changes that suit the growth of this pathogen. It is noted that the pres­
ence of Firmicutes genera such as Megamonas is associated with
Campylobacter exclusion from niches (Oakley et al., 2013). Some mem­
bers of the microbiome produce metabolites such as lactic acids which
can inhibit Campylobacter proliferation. Mucin-fermenting species such
as Ruminococci and Clostridia further hamper the ability of Campylo­
bacter to colonise the GIT by degrading the thin mucous cover of the
caecum, exposing the bacterium to antimicrobial peptides and immu­
noglobulins. As such, reductions in these species favour Campylobacter
colonisation (Taha-Abdelaziz et al., 2018). Campylobacter has advan­
tages over other microbes as it can utilise SCFAs and L-fucose as colo­
nisation location indicators, preferentially colonising low lactate, high
butyrate/acetate regions, and energy sources to aid colonisation, allow
it to successfully outcompete the native microbiota (Awad et al., 2016;
Luethy et al., 2017). The established microbiome can aid in the exclu­
sion of other species, such as Salmonella. As mentioned previously Sal­
monella spp. Can readily colonise newly hatched chicks, but the
existence of a resident established microbiome prevents the colonisation
of broilers with Salmonella through competition for nutrients and niches
(Bailey et al., 1991; Cosby et al., 2015). Competitive exclusion has been
explored as a method of preventing the colonisation of broilers with this
pathogen to increase the end-product safety (Bailey et al., 1991;
Braukmann et al., 2016).
3. The microbiome of poultry processing
Consumer demand for chicken meat has led to the development of
large-scale processing plants to produce the quantities of chicken
7
Food Microbiology 99 (2021) 103823
demanded by the public. Hygienic practices during processing are
essential to control the quality and safety of chicken products. The
processing plant is kept free of dust, dirt, and chicken-borne contami­
nants through frequent and vigorous cleaning. It is particularly essential
to control the spread of faeces and caecal content within the processing
environment, as these are the primary reservoirs for enteric pathogens
such as Salmonella, E. coli and Campylobacter (Demirok et al., 2013;
Reich et al., 2018). However, modern production practices prioritise
line-speed to meet demand, and this can lead to issues with controlling
contamination (Feye et al., 2020b). In excess of 21 phyla and 280 genera
have been found to colonise broiler meat, which may impact its safety,
quality or consumer appeal (Ae Kim et al., 2017). Bacterial contami­
nation can build up on equipment and be spread across the facility by
on-surface cross-contamination, workers, as well as in the air. Therefore,
it is vital to understand how the microflora of the end-product is affected
by different steps of the process, and where it is most prudent to
implement antimicrobial interventions within the production sequence
(Fig. 3). From the point of slaughter, a large part of the initial microbial
burden is established. Equipment and surfaces are frequently found to be
difficult to adequately clean. Therefore, equipment acts as a consistent
reservoir for the introduction of microbes to the broiler carcass from
early in the meat production process (Casaburi et al., 2014; De Filippis
et al., 2013; Samapundo et al., 2019).
3.1. Selection and transport
Approximately 24 h prior to slaughter, feed is withdrawn, and the
chicken will enter a state of fasting. This acts to prevent defecation
during transport and the associated microbial spread between chickens
in the transport crates en-route to slaughter, as well as to reduce quan­
tities of faecal material and associated contamination during processing
(Hanning et al., 2012). This precaution reduces the potential for
equipment contamination should the lining of the gastrointestinal tract
be perforated. However, excess feed-withdrawal time has also been
linked to the weakening of the lining of the GIT, leading to increased risk
of perforation, content spillage and contamination of the processor
environment (García-Sánchez et al., 2017; Seliwiorstow et al., 2016).
Transport can be a stressful time for broilers, increasing the pro­
duction of corticosterone and stress-related hormones. These hormones
induce behavioural changes such as increased pecking and defecation,
while also reducing gastrointestinal function. The presence of faeces,
which may contain zoonotic pathogens, in this environment can in­
crease surface contaminants and can adversely affect poultry product
safety. Heat stress and packing stress play major roles at this point,
inducing two separate but related physiological responses within indi­
vidual birds. Heat stress is worsened in the tightly packed confines of
transport crates. Factors such as feather covers and a low ability to sweat
exacerbates heat stress in individual birds. To counter this, blood-flow is
diverted from the GIT to the skin to increase heat dissipation. However,
a loss of blood-flow from the GIT increases its permeability as tight
junctions between epithelial cells loosen (Goo et al., 2019). Corticoste­
rone can also increase GIT permeability, which is related to increased
systemic inflammation due to dissipation of endotoxins such as lipo­
polysaccharide into the bloodstream, and infiltration by bacteria
(Musavian et al., 2014; Scanes, 2016). Stress-related reduction in the
GIT integrity is linked to the increased systemic invasion by Salmonella
enterica ser. Enteriditis in broilers (Quinteiro-Filho et al., 2012). These
bacteria can disseminate through the host organism, and potentially
enter the food chain.
3.2. Slaughter
On arrival at the processing facility, broilers undergo several steps
that prepare them for slaughter. In pre-stunning steps, crates containing
broiler chickens are stored in a dark quiet warehouse room to allow
them to settle. This step is followed by stunning, which can take several
M. Marmion et al.
Fig. 3. A Summary of the industrial poultry processing line. Shading indicates zones of increasing and decreasing contamination. * Indicates steps at which microbial
growth is controlled.
forms. In modern facilities, the crates are passed through chambers
which reduce the level of available oxygen causing the birds to become
drowsy and slip into a coma. Other facilities utilise electrical or me­
chanical stunning. The birds are then hung by the legs on a conveyer belt
and transported to the slaughter point. At this point, broilers are
slaughtered, and the carcasses allowed to bleed out (Nielsen et al.,
2019).
Slaughter and bleeding are also documented points at which around
60% of carcasses may carry the highest burden of Salmonella cells, circa
6.1 log CFU/g, found during the processing chain (Boubendir et al.,
2021; Rivera-Pérez et al., 2014). The bloodborne nature of Salmonella in
some broilers can permit its spread on the exterior of the broiler carcass,
and pose a contamination risk to subsequently processed carcasses
(Boubendir et al., 2021; Goo et al., 2019). However, it is also notable
that at this stage of processing, the carcass remains highly contaminated
by faeces, dirt, feathers, and other external contaminants, while other
factors such as equipment adulteration can facilitate the spread of this
higher abundance of microbes across broiler carcasses. Control steps
taken hereafter effectively act to significantly reduce the carriage of this
pathogen into the homes of consumers (Boubendir et al., 2021; River­
a-Pérez et al., 2014).
High pressure water jets used to clean the carcass at this stage leads
to the production and spread of aerosolised and airborne microbial
contaminants, as well as agitating and spreading floor-borne microbes
which have adapted to the chilled slaughter room. This can contribute to
the spread of aerosolised particles which may contain spoilage microbes
such as lactic acid bacteria (LAB). The resulting humidity of this part of
the slaughterhouse has been documented as conducive for the survival
of bacteria (Casaburi et al., 2014; Chen et al., 2020; Samapundo et al.,
2019). The levels of airborne LAB within this area increase exponen­
tially in the course of a processing day in one reported incidence
increasing from 69 CFU/m3 to 1510 CFU/m3 in 4 h on a given day
(Samapundo et al., 2019). A lack of airflow control in the early poultry
production chain can contribute further to the end-microbiome of
poultry meat, as microbes from this point can circulate in the factory
environment. This includes groups such as psychrophilic LAB which
play an important role in the anaerobic spoilage of poultry meat prod­
ucts (Chen et al., 2020; Samapundo et al., 2019).
3.3. Scalding and defeathering
Following slaughter, carcasses are immersed in a scald tank con­
taining hot water to facilitate defeathering. The feathers and skin of the
broiler become highly contaminated with faeces and dirt in the course of
8
Food Microbiology 99 (2021) 103823
the broiler lifespan, especially during the stressful events of picking and
transport, which spreads to the scald waters and defeatherer (Mulder
et al., 1978; Scanes, 2016). The immediate post-slaughter composition
of the carcass microbiome is dominated by Firmicutes bacteria, with
over 76.54% of microbes being part of this phylum. At this point, aerobic
bacterial plate counts (APC) on the carcass surface can be found at levels
of around 10.16 log CFU/carcass (Ae Kim et al., 2017). Proteobacteria,
which include important spoilage and pathogenic bacterial genera, were
present at levels similar to the those of the live bird i.e. less than 3.5%.
(Ae Kim et al., 2017; Oakley et al., 2014). However, intense selection
processes during processing begin to exert influence upon the meat
microbiome composition at this stage.
Carcass scalding occurs at temperatures of around 52.5 C, reducing
◦
the microbial carriage by up to 2.7 log CFU/g on the surface of carcasses,
as well as softening skin to aid the removal of feathers (Althaus et al.,
2017). However, temperature may be insufficient to prevent the spread
of microbes to carcasses. Approximately, 20 phyla are associated with
scalding tank survival and subsequent product contamination, including
Firmicutes, Proteobacteria and Actinobacteria which combined consti­
tute between 60 and 70% of microbial species in the tank (Rothrock
et al., 2016). Scald tank water contamination increases initially at the
start of a production day before equilibrating after several hours during
which phylum-specific changes occur i.e., Proteobacteria numbers
remain stable, Bacteroidetes numbers fall, and Firmicutes species
increase.
Contamination with Proteobacteria during the latter stages of
chicken meat processing is especially influential on product quality, as
spoilage microbes from genera such as Pseudomonas and Acinetobacter
increase in scald tank waters, while members of the Enterobacteriaceae
family, which may include pathogens, also increase in abundance during
the day (Chen et al., 2020). Simultaneously, Firmicutes such as Anox­
ybacillus and Erysipelotrichaceae experience growth in the same period,
potentially expanding into niches previously occupied by Streptococcus
and Staphylococcus which undergo concurrent declines in the scalder
environment (Rothrock et al., 2016). Pathogens such as E. coli, Salmo­
nella and Campylobacter are also recoverable from this source, and it is
thought to be potentially beneficial to employ a secondary scalding
process to increase its efficacy (Althaus et al., 2017). The microbial
burden of carcasses at this stage when a single scald is used can increase
slightly from 10.16 to 10.37 log CFU/carcass (Ae Kim et al., 2017).
The post-mortem removal of feathers may provide an opportunity for
the spread of contamination between adjacently processed flocks. The
stiff rubber fingers of defeathering equipment can act to harbour phys­
ical contaminants such as faeces, dust and dirt, disseminating any
M. Marmion et al.
associated microbes among products (Hutchison et al., 2017; Pachole­
wicz et al., 2015b). Microbes which have a particular affinity for the skin
surface, such as Campylobacter can remain adhered to the skin surface at
this point, aiding the spread and proliferation of these pathogens on
meat surfaces (Pacholewicz et al., 2015b; Hutchison et al., 2017). This
potential for the spread of microbes means that defeathering can be
considered the first of two major points of contamination where bacteria
can spread between carcasses, the second being evisceration. These
points inform meat producers of the best possible practices to implement
downstream control points in order to prevent the entry of adulterated
products into the food-chain (Ae Kim et al., 2017). Due to the enclosed
nature of the defeathering equipment, there are few opportunities to
adequately remove contaminants within the defeatherer. Small alter­
ations like alternating brush spin direction and slowing rotation speeds
can diminish the spread of bacteria in this process, while a subsequent
wash step may be employed to remove most physical contaminants from
the carcass surface. However, the most prescient step for the control of
bacterial proliferation during defeathering is the effective and rigorous
cleaning of machinery and equipment through chemical compounds and
washing (Cason et al., 2004).
3.4. Evisceration
The removal of internal viscera is a delicate process in poultry meat
production due to the relatively small size of the bird. Manual and
mechanical evisceration may lead to perforation of the caecal lining,
spreading undesirable caecal contents as well as their microbial load
throughout the eviscerator environment. As mentioned previously, pre-
slaughter factors such as feed withdrawal are important to retain the
integrity of the caecal lining thereby reducing the risk of puncture
during evisceration. The high burden (circa 11 log CFU/g) of bacteria
per gram of caecal content provides a strong contamination potential
should the processor environment become exposed (Waite and Taylor,
2014). Evisceration is a frequently reported point of contamination
within the plant environment, as pathogen numbers for both Campylo­
bacter and Salmonella increase substantially at this point (Gruntar et al.,
2015; Rivera-Pérez et al., 2014). Equipment and surfaces are particu­
larly vulnerable to carcass cross-contamination in this part of the
factory.
Airborne contaminants within this section can also be considered an
important source of exogenous microbes on poultry carcasses (Sama­
pundo et al., 2019), meat bacterial levels reaching their highest levels at
this point of the process, often exceeding 10.55 log CFU/carcass (Ae Kim
et al., 2017). This is characterised by an increase in Firmicutes burden
and a corresponding decline in the abundance of Proteobacteria (Ae Kim
et al., 2017). Steps such as inside-out washing are employed immedi­
ately after evisceration to remove carcass surface contaminants,
including caecal contents, acquired and spread during evisceration.
3.5. Washing
The washing of broiler carcasses allows the removal of external soil
and loosely associated microbes from the surface of the carcasses which
may have adhered to the skin or feathers of the carcass. Washing can
take place at many stages through the production cycle, but the most
important washes come after defeathering and carcass evisceration. This
can take two main forms, i.e., rinse washing and inside/outside washing.
These may, depending on the jurisdiction, utilise chemical antimicro­
bials including chlorine, trisodium phosphate, organic acids, peroxides,
hypochlorites, cetylprydium chloride and monosodium phosphate
(Buess et al., 2019; Loretz et al., 2010). However, European law forbids
the use of many of these additives at levels higher than those permitted
in potable water, currently 5 ppm for chlorine (Burfoot et al., 2015). The
inclusion of chemicals is also disputed as an improvement to carcass
washing in clean, cold water, as no significant difference was seen across
several studies comparing the efficacy of the two approaches (Buess
9
Food Microbiology 99 (2021) 103823
et al., 2019). Wash steps can effectively reduce the carriage of pathogens
such as E. coli, Campylobacter and members of the Enterobacteriaceae
family by between 0.8 and 1.43 log CFU/g (Buess et al., 2019; Hutchison
et al., 2017).
3.6. Chilling
Chilling is an effective intervention that can be broadly applied to
control microbial growth within both the processor environment and
domestic/retail settings. From this point in the meat production chain,
broiler meat is kept at a low temperature by maintaining a monitored
chill chain through portioning, packaging, transport, and retail storage.
Retail chicken will also include chill storage instructions for consumers/
end users to ensure that the shelf-life of the product is maximised.
Effective chilling can reduce bacterial levels that may have become
elevated during carcass processing, particularly during the defeathering
and evisceration steps, to levels similar to those seen prior to plucking
(Althaus et al., 2017). This is particularly important in the case of
mesophilic poultry-borne microbes such as Salmonella, E. coli and
Campylobacter, as these pathogens often display high thermosensitivity
at chill temperatures that do not reflect their preferred niche in the
broiler GIT (Pacholewicz et al. 2015a, 2015b). However, its effect is
dependent upon factors such as initial microbial load, chemical addi­
tions, chilling system, and cooling capacity (Demirok et al., 2013).
Air chilling (AC) and immersion chilling (IC) are chilling approaches
employed in industry, with AC the preferred chilling approach in
Europe, Canada and Brazil. AC has a lower potential for cross-
contamination and improves product tenderness, but it can have
reduced direct antimicrobial effect against Salmonella and Campylo­
bacter when compared to IC (Demirok et al., 2013; Vanantwerpen et al.,
2016). Campylobacter numbers are more affected by the characteristic
desiccation effect of AC (Pacholewicz et al., 2015b). This drying effect
can be ameliorated through the use of water sprays and chemical ad­
ditives like cetylprydinium chloride, where permitted (Boubendir et al.,
2021). AC does not wholly prevent the spread of aerobic psychrotrophs
such as Paeniglutamicibacter, Chryseobacterium Pseudarthtobacter and the
prevalent spoilage genus Pseudomonas, which is seen on up to 83.51% of
post-AC carcasses (Chen et al., 2020). This is of particular concern when
spray chilling is employed. However, the use of air-chilling can reduce
the carriage of Salmonella on carcasses from around 38% of carcasses
prior to chilling to 0–2.5% (Boubendir et al., 2021). The AC process also
causes the development of wall condensation and floor dripping, within
which psychrophilic LAB, Pseudomonas and pathogenic Listeria spp. Can
proliferate, may become aerosolised during cleaning. Which may affect
the shelf-life of the poultry products (Chen et al., 2020; Keeratipibul and
Techaruwichit 2012; Samapundo et al., 2019).
IC is employed more frequently in the U.S.A, and it has been docu­
mented to be more effective that AC in the reduction of bacterial levels
as it is frequently used in conjunction with antimicrobials, such as
chlorine and peracetic acid. Because of EU restrictions of chemical
addition in poultry processing, the use of IC in the European Union is not
widespread. The antimicrobial efficacy of IC can be improved somewhat
through the implementation of a contraflow current (Boubendir et al.,
2021), however, there is a potential for the spread of loosely-associated
contaminants between carcasses during the immersion process (Bou­
bendir et al., 2021; Demirok et al., 2013; Misra and Jo, 2017). As with
the scald tank, there tends to be an increase in the microbial levels in the
immersion tank as IC processing progresses with up to 5-fold more LAB
species, such as Streptobacillus and Lactobacillus, isolated from chiller
tanks by the end of a processing day, even with chlorination (Rothrock
et al., 2016). IC may also be associated with the spread of Salmonella if
precautions such as chemical usage or a contraflow system are not uti­
lised (Boubendir et al., 2021). Chlorination will reduce this significantly,
along with levels of Pseudomonas and Acinetobacter-related spoilage or­
ganisms that are frequently isolated from this chiller environment
(Rothrock et al., 2016).
M. Marmion et al.
Irrespective of which method is used, analysis of meat bacterial
carriage after chilling indicate that spoilage-associated levels of mi­
crobes are reached at the same point, circa Day 10 of the product shelf-
life (Demirok et al., 2013). Psychrophilic or thermotolerant species such
as Pseudomonas and LAB, and some pathogens such as L. monocytogenes,
can multiply, albeit more slowly, at temperatures as low as 0 ◦ C (Keer­
atipibul and Techaruwichit, 2012). Overall, it was found that carcass
operational taxonomic units (OTU) fell almost ten-fold from 222 OTUs
prior to chilling to 23 OTUs (Handley et al., 2018).
Crust-freezing is a relatively recent novel decontamination approach
in processing broiler carcasses, which is as yet has not been broadly
implemented in the global chicken industry (Chaves et al., 2011;
Haughton et al., 2012a, 2012b). This approach uses of freezing tem­
peratures on the surface of the carcass to control microbe growth on the
skin, causing high levels of bacterial inactivation through cellular water
loss (Hutchison et al., 2017). Campylobacter levels, for example, can be
reduced by up to 2 log CFU/g, through crust freezing (Cox and Pavic,
2010; Hansson et al., 2018; Loretz et al., 2010), without affecting meat
appearance (Haughton et al., 2012a, 2012b). However, the freezing
process can have damaging effects on the structures of the skin (Chaves
et al., 2011).
3.7. Portioning
The portioning step is crucial, as it can be regarded as the final point
at which external contamination can occur prior to packaging and dis­
tribution. Poultry carcasses are processed depending on the desired
products, e.g., whole chickens and chicken portions. Particularly in the
case of the smaller cuts, there is frequent handling and manipulation of
the carcass, making it a key point in the introduction of spoilage mi­
crobes and LAB into poultry products. Both workers and equipment act
as cross-contamination vectors for these spoilage microbes. As
mentioned in section 2 above, cutting equipment in particular poses a
risk, due to its propensity for having a constant low level of bacteria that
can spread between bacteria that in situ cleaning practices do not always
remove (Casaburi et al., 2014; Sahin et al., 2015a, 2015b; Samapundo
et al., 2019). The cutting of meat also has the potential to introduce
bacteria to previously sterile parts of the flesh (Samapundo et al., 2019).
Once portioned and prepared as necessary for chicken products are
packaged and stored at chill temperatures in preparation for sale. In­
terventions such as modified atmosphere packaging (MAP) and chilling
are used to maintain microbial stability through to retail, however,
characteristic changes in the microbial profile will occur, as a direct
consequence of the packaging interventions chosen, right through the
shelf life of the product.
4. Post-processing microflora: retail to fridge
Cold storage is employed from the chill step of the poultry produc­
tion process up until the consumption of the product, which, as well as
the packaging gas composition, serves as the primary selective pressure.
The evolution of the microflora on fresh poultry meat products is a
continuous process, until, over time, bacterial populations proliferate
and grow to levels that cause product spoilage, even under chilled
conditions (Rouger et al., 2017b). Spoilage of broiler meat is
multi-faceted, causing noticeable detrimental changes in the quality of
meat, affecting its colour, flavour, structure, and odour. Poultry meat is
considered to be spoiled when bacterial numbers exceed 107 CFU/g,
while noticeable odour changes occur at 108 CFU/g (Naveena et al.,
2017; Rouger et al., 2018; Wang et al., 2016). At these levels, microbes
produce noticeable compounds such as organic acids, for example lactic
acid and thiobarbituric acid, and biogenic amines, such as putrescine
and cadaverine, which alter the taste and aroma of the food irreversibly
(Balamatsia et al., 2006; Casaburi et al., 2014; Lee et al., 2017; Pothakos
et al., 2015; Wang et al., 2016).
Microbial spoilage occurs at different rates depending on the species
10
Food Microbiology 99 (2021) 103823
involved, but it can be influenced by temperature, pH, oxygenation and
bacterial biodiversity on meat surfaces. These changes may be attributed
to either intrinsic factors, such as enzymes or chemical action, or
extrinsic ones, such as microbial spoilage and enzyme production, which
results in the degradation of meat structure, and production of metab­
olites such as aldehydes, sulphur-containing compounds and organic
acids as putrefaction progresses (Farahnaz et al., 2016; Rouger, Tresse,
& Zagorec, 2017a, 2017b).
4.1. Spoilage microflora
The initial microbiome of meat during packaging is composed pri­
marily of Pseudomonas spp., Enterobacteriaceae, Lactobacillus spp., Aci­
netobacter spp. And B. thermosphacta. It is at this point that the packaging
atmosphere becomes important in determining the final product
microbiome. Psychotropic Pseudomonas species effectively grow to
dominate the microbiome of poultry meat when it is stored aerobically,
while Shewanella spp. Plays a minor role in aerobic spoilage (Li et al.,
2020). However, modified atmosphere and low oxygen packaging in­
hibits this proliferation. While Pseudomonas spp. May be present initially
upon the meat surface, it will be unable to proliferate effectively if
enclosed in an oxygen-deficient packaged environment, even if
cold-adapted. Furthermore, carbon dioxide actively inhibits its growth
(Carrizosa et al., 2017; Casaburi et al., 2014; Demirhan and Candoğan
2016; Pothakos et al., 2015). Instead, a more diverse variety of
cold-adapted genera, including Aeromonas, Buttiauxella, Carnobacterium,
Enterobacter, Hafnia, Lactobacillus, Lactococcus, Leuconostoc, Pseudo­
monas, Serratia, Shewanella, and Yersinia, proliferate on meat and
contribute to the production of spoilage compounds such as lactic acid
and ethanol (Casaburi et al., 2014; Li et al., 2020; Pothakos et al., 2015).
The dominant spoilage microflora becomes established within 4 days of
production, and typically causes noticeable spoilage when bacterial
levels reach 107 cells/cm2. Some species have even lower thresholds to
produce noticeable spoilage effects, with B. thermosphacta producing
noticeable product odour changes at levels as low as 105 cells/cm2
(Betts, 2006; Lauritsen et al., 2019). These changes take time to occur at
the low temperatures used in chill storage, but irredeemable meat pu­
trefaction is found to occur around day 10, at which point the product
microbiome has completely reduced the appeal of the product (Sama­
pundo et al., 2019). Furthermore, poor refrigeration hygiene and prac­
tices can significantly accelerate the proliferation of bacterial groups
including Total Viable Counts, E. coli and S. aureus on chicken meat
stored at low temperatures (Masoumbeigi et al., 2017).
4.2. Poultry-linked pathogens
In terms of retail chicken, the key microbial health concern is the
presence of zoonotic human pathogens which form part of the chicken
microbiome. While some, such as Salmonella, are less frequently detec­
ted, other pathogens such as Campylobacter and some variants of
Escherichia coli can occur at high prevalence, with 100% incidence being
reported in some flocks (Bolton, 2015; Kim et al., 2019; Manges, 2016).
These microbes, particularly Campylobacter and Salmonella cause much
of the zoonotic disease each year in developed countries.
Campylobacter infection is principally linked to campylobacteriosis, a
mild self-limiting gastroenteritis associated with the consumption of
undercooked and improperly prepared food (EFSA, 2018; Ijaz et al.,
2018). Campylobacter is a genus of microaerobic bacterium that can
proliferate within the microaerobic environment of the caecum, with
cell densities of 108 CFU/g (Bolton 2015; Taha-Abdelaziz et al., 2018). It
is a common commensal in commercial broilers, with 60–80% of flocks
testing positive for Campylobacter prior to slaughter. This can spread the
pathogen to the end product through faeces and surface contamination
of meat (Carron et al., 2018; Jansen et al. 2014; Reich et al., 2018).
Campylobacter spp., particularly C. jejuni, was implicated in around 70%
of bacterial zoonotic disease in Europe in 2018, causing over 246,000
M. Marmion et al.
cases of clinically-reported illness (EFSA, 2019). There are potentially
up to 9 million cases of Campylobacter infection per year in the E.U.
alone, though this is hard to quantify due to the sporadic and
self-limiting nature of the disease. It is estimated that only 2.1% of cases
are reported each year (Bolton, 2015; Economou et al., 2015; OECD-­
FAO, 2018; Kagambèga et al., 2018). Of this, 40% of cases can be traced
to the direct consumption of chicken meat, and 60–80% of global
campylobacteriosis cases can be traced to the poultry reservoir (Her­
mans et al., 2011; Skarp et al., 2016).
Non-typhoidal Salmonella enterica (NTS) serovars play a large role in
the public consciousness of food-poisoning. Salmonella spp. Is a part of
the broiler gastrointestinal microbiome, and spreads to adjacent birds
through close contact and faeces (Abdi et al., 2017; Álvarez-Fernández
et al., 2012). It does not tend to cause disease in broiler chickens but
plays an important role in human NTS infection. Within Europe, over 91,
000 clinical cases of Salmonellosis were reported in 2017, while 1.03
million cases are estimated to occur per year in the U.S.A (EFSA, 2018;
Foley et al., 2013). Worldwide, 1.6 billion cases of non-typhoidal Sal­
monella infection are estimated to occur annually, with around 3 million
deaths linked to this pathogen. Frequently, salmonellosis is associated
with the consumption of pork and poultry products (Trimoulinard et al.,
2017). In the developed world, between 4 and 20% of chicken meat is
contaminated with NTS, while the incidence of NTS in the developing
world can be as high as 62.5% (Khan et al., 2018). Prevalent poultry
borne NTS serovars include S. enterica serovar Enteriditis, (monophasic)
S. enterica ser. Typhimurium, S. enterica ser. Infantis, S. enterica ser.
Newport, S. enterica ser. Agona and S. enterica ser. Derby. 24.2% of
Salmonella cases can be linked to the poultry reservoir; howeverIKJM,
only 2.2% directly relate to broiler product consumption (EFSA, 2018).
Listeria monocytogenes is a high-risk Gram positive bacterial pathogen
that can cause conditions ranging from influenza-like symptoms to
septicaemia and septic abortions (Ristori et al., 2014). L. monocytogenes
is found throughout the environment with both animals and soil acting
as vectors. L. monocytogenes can then enter the food chain through
contamination of chicken meat products and production surfaces (Ris­
tori et al., 2014). Other pathogens linked to the poultry microbiome
include Clostridium perfringens, Hafnia spp. And Citrobacter spp., which
pose a particular threat to immunocompromised hosts (Clavijo and
Vives Flórez, 2018; Kim et al., 2019).
The growing prevalence of antimicrobial resistance in poultry-
associated bacteria, with extended-spectrum beta-lactamase (ESBL)-
producing Enterobacteriaceae and Salmonella-associated drug resistance
growing in prevalence in some regions, while there is also increased
prevalence of fluoroquinolone and macrolide-resistant Campylobacter
seen in human infections, linked to the use of these drugs in a veteri­
narian context (McDermott et al., 2018; Murphy et al., 2018; Pachole­
wicz et al., 2015a, 2015b). The use of antimicrobial compounds in the
rearing of broilers and sanitation of production settings has led to the
growth in prevalence of potentially resistant serovars in the food-chain,
which can spread potentially dangerous pathogens to consumers (Cosby
Fig. 4. Different packaging types employed for commercially produced chicken products including saran wrap (A), modified atmosphere packaging (B) and vacuum-
packaging (C).
11
Food Microbiology 99 (2021) 103823
et al., 2015; Gantzhorn et al., 2014; West et al., 2018).
4.3. Impact of packaging on poultry microflora
The type of packaging is key to the subsequent microflora develop­
ment during chilled retail storage. Retail chicken products are either
wrapped in PET (Saran) film (Fig. 4A) or packed in Modified Atmo­
sphere Packaging (Fig. 4B). Vacuum packaging or shrink-wrap tends
only to be used for luxury/high-end products, e.g., Duck breasts
(Fig. 4C).
Chicken products exposed to air or saran wrapped are susceptible to
spoilage by aerobic microbes. The most prevalent bacterial genus on
chicken meat stored under aerobic conditions is proteolytic Pseudomonas
spp., accounting for 58.5% of bacteria (Lee et al., 2017). Pseudomonas is
a broad genus of aerobic Gram-negative bacteria that frequently forms
an important and resilient part of the chicken meat microbiome, with
particularly prevalent species including P. aeruginosa, P. fragi,
P. lundensis and P. flourescens. Over 111 Pseudomonas species have been
identified on poultry meat (Estepa et al., 2015; Lee et al., 2017; Wang
et al., 2017). This common spoilage microbe is often sourced from the
environment, especially the meat processing plant. The growth of
Pseudomonas spp. Is most evident in high-oxygen environments and
packaging systems, while they also show tolerance to a range of tem­
peratures. Pseudomonas are most active at neutral pH, degrading amino
acids to produce off-flavours through secretion of a zinc metal­
loproteinase (Lee et al., 2017; Rouger et al., 2017a, 2017b; Wang et al.,
2017). They remain able to grow, albeit more slowly, in oxygen deficient
packaging systems including MAP and vacuum packing, and may still
contribute to the spoilage when present (Li et al., 2020; Rossaint et al.,
2015). It has similarly been found that B. thermosphacta can proliferate
and to meat spoilage under aerobic conditions, and may even outcom­
pete Pseudomonads for available substrates on the meat surface (Rossaint
et al., 2015).
Within anaerobic environments, the largest contributors to meat
putrefaction are lactic acid and ethanol. A range of facultative and
obligate anaerobic species contribute to the spoilage of poultry meat
products in the absence of/under low abundance of oxygens. Anaerobic
conditions can be achieved in two ways. MAP packaging can utilise non-
oxygen inert gases such as nitrogen and carbon dioxide to reduce the
aerobic spoilage of meat, while vacuum packaged meat produces an
anaerobic environments environment through evacuating all the gas
from the packaging. This can be done using gas impermeable bags or
shrink wrap and tray. Prevalent spoilage species under these conditions
include Brochothrix thermosphacta, Lactobacillus spp., and Carnobacte­
rium spp., while bacteria from genera such as Weissella, Shewanella,
Acinetobacter and Klebsiella further contribute to the spoilage of the
chicken meat product (EFSA, 2016; Rouger et al., 2018). These spe­
cies/genera may be predominantly sourced from the microbiome of the
host animal, while others are found within the meat processing envi­
ronment (Table 3).
M. Marmion et al.
Table 3
Principle sources of important chicken meat-borne microbes derived endoge­
nously from the broiler microbiome (A) and the processing environment (B).
Pathogenic Bacteria Spoilage Bacteria
A Campylobacter spp., Clostridium Lactobacillus spp., Lactococcus spp.,
perfringens, Escherichia coli, Hafnia Leuconostocspp., Pseudomonas spp.
spp., Listeria monocytogenes,
Salmonella spp., Serratia spp.
B Listeria monocytogenes, Salmonella Brochothrix thermosphacta,
spp. Carnobacterium spp., Enterococcus spp.,
Janthinobacterium spp., Lactobacillus
spp., Lactococcus spp, Leuconostocspp,
Pseudomonas spp.
Adapted from (Cox and Pavic, 2010; Lauritsen et al., 2019; Samapundo et al.,
2019; Voidarou et al., 2011).
Modified Atmosphere Packaging is a process by which the ambient
gas atmosphere is used to control microbial growth and extend product
shelf-life. The three key gases used commercially are: 1.) nitrogen, used
as a filler to maintain package integrity, 2.) oxygen, which is added at
high levels in red meats to provide an oxyhaemoglobin bloom-although
this is less important in white chicken meat (Kim et al., 2019; Sama­
pundo et al., 2019)- and 3.) carbon dioxide, an antimicrobial gas which
acts as a bacterial growth inhibitor, especially against aerobic microbes
such as Pseudomonas (Höll et al., 2016). To maintain product quality, it
is important to ensure that levels of oxygen or carbon dioxide do not
negatively affect meat appearance (Carrizosa et al., 2017; Höll et al.
2016). The concentration of gasses influences the nature of in-package
microbial growth. For raw retail chicken, there are two main MAP ap­
proaches, i.e., low-oxygen MAP [30% CO2, 70% N2], or high-oxygen
MAP [20% CO2, 80% O2], while bulk commercial products are packed
using 100% C02. Off-skin portions of chicken tend to receive
high-oxygen MAP treatment for both retail and commercial bulk sam­
ples, i.e. 70% O2, 30% CO2 (Dansensor, 2014; Demirhan and Candoğan,
2016; Kim et al., 2010).
The gas atmosphere provided has a large effect upon the microbes
that will proliferate upon the meat. The provision of oxygen at levels
similar to atmospheric levels, circa 20%, will allow the growth of aerobic
and aerotolerant bacteria, which includes spoilage microbes such as
Pseudomonas spp., which are especially associated with the production
of foul-smelling metabolites, as well as facultative anaerobes such as
Carnobacterium and B. thermosphacta, while stricter anaerobes are
inhibited, including some LAB (Höll et al., 2016; Pothakos et al., 2015).
Excessive levels of oxygen can also have some antimicrobial effect
against aerobic spoilage microorganisms including Pseudomonas spp.
(Conte-Junior et al., 2020; Jaberi et al., 2019) and yeast (Jacxsens et al.,
2001). In the absence of oxygen, there is a definite shift in the dominant
microbiota of the meat as it becomes more diverse at a genus level,
which includes those bacteria responsible for product spoilage including
Aeromonas, Buttiauxella, Enterobacter, Hafnia, Pseudomonas, Serratia,
Shewanella, Yersinia, and LAB, which includes genera such as Lactoba­
cillus, Lactococcus, Enterococcus, Leuconostoc, Weissella and, in particular,
Carnobacterium (Höll et al., 2016; Li et al., 2020; Pothakos et al., 2015).
Carnobacterium is a genus of major spoilage bacteria in modified atmo­
sphere packaging containing high concentrations of carbon dioxide,
while other facultative anaerobes such as B. thermosphacta and LAB also
play a major role in the production of lactic acid and ethanol in the
anaerobic spoilage of meat (McLeod et al., 2018; Patange et al., 2017;
Pothakos et al., 2015).
5. Concluding remarks
The broiler microbiome is established from the day of hatch but re­
mains dynamic to the day of slaughter and through to final product
packaging, distribution until cooked and eaten by consumers. The living
broiler microbiome is dominated by bacteria from the Firmicutes and
12
Food Microbiology 99 (2021) 103823
Bacteroides phyla come the time of slaughter. However, conditions
within the processor cause rapid selective alterations in the carcass
microbiota.
Understanding the influences of hygiene, biosecurity and antimi­
crobial interventions is a crucial tool in ensuring poultry quality and
safety for the consumer. However, it is only with a full grasp over the
contributory factors associated with the chicken meat microbiome and
its impacts upon the end-product that effective interventions and stra­
tegies for its management can be implemented. The poultry production
chain (Fig. 5) has been optimised at every point to reduce the oppor­
tunity for zoonotic organisms to enter the food-chain. However, despite
the employment of on-farm interventions such as biosecurity ap­
proaches, targeted in-feed interventions like antibiotics, probiotics and
pre-biotics, vermin and insect exclusion, and different broiler husbandry
approaches, human pathogens including Salmonella and Campylobacter
can become established within the microbiome successive broiler flocks
(Doyle and Erickson, 2006; Lu et al., 2021). Once present within flocks,
these bacteria can spread among adjacently reared animals and enter the
food-chain as these bacteria become tough to eradicate in live birds.
The key steps for dictating the end-product microflora thus occur
during the final steps of the meat-production plant. Carcass bleed-out,
defeathering and evisceration lead to the spread of zoonotic bacteria
among adjacently and subsequently processed carcasses (Pacholewicz
et al., 2015b; Rivera-Pérez et al., 2014; Rothrock et al., 2016). These
contamination-prone points require rigorous follow-up, through carcass
washing and chilling, to control the proliferation of disease-related
microbes which tend to be more sensitive to these steps. The
processing-line is maintained at low temperatures from the this point on
to minimise the proliferation of zoonotic pathogens like Campylobacter,
which display low cold-tolerance (Gonçalves-Tenório et al., 2018; Lu
et al., 2019). However, the extensive use of cold selects for a
cold-tolerant product microbiome, derived from the processing plant
microbiome, which tend to predominate on the end-product and which
can cause product spoilage and reduced product shelf-life when pur­
chased by the consumer (Chen et al., 2020; Handley et al., 2018).
Steps such as cold-chain use and the employment of various pack­
aging strategies have been optimised to extend the shelf-life of the
product. The packaging of meat represents the final opportunity for cells
to contaminate the end-product under ideal circumstances as physical
barriers can prevent the further entry of exogenous cells onto the
product. However, the extant cells can proliferate and cause spoilage
over the given shelf-life (Rouger et al., 2018). While maintaining the
cold chain is a key element in ensuring that poultry products remain safe
for the duration of their shelf-life, other steps include controlling the
packaging atmosphere through exclusion of oxygen or incorporation of
high concentrations of carbon dioxide to prevent the growth of some
bacterial species. Cold-tolerant Pseudomonas spp. Tend to predominate
in packaging in which oxygen is present at or near atmospheric levels,
while a more diverse microbiota including many LAB and anaerobic
species is established in low oxygen packaging (Chen et al., 2020;
Conte-Junior et al., 2020; Rossaint et al., 2015).
Current research is focussed on further reducing spoilage microor­
ganisms and eliminating pathogens from packaged chicken products
using a range of innovative in-line strategies, including cold-plasma
(Soro et al., 2020; Wang et al., 2018), ultraviolet light (Haughton
et al., 2012a, 2012b), active packaging (Demirhan and Candoğan, 2016;
Hakeem et al., 2020), sonication (Piñon et al., 2020), and high-pressure
processing (Bechstein et al., 2019).
However, barriers such as regional legislation, implementation cost
and unfavourable consumer perceptions have complicated the imple­
mentation of these in-package approaches (Lu et al., 2019). Nonetheless,
it remains imperative that these strategies are investigated and where
possible and practical, implemented to control in-package proliferation
of spoilage and pathogenic microorganisms during chilled refrigerated
storage in retail and consumer refrigerators. By actively controlling the
poultry microbiome at all stages from farm to fridge, it is possible and
M. Marmion et al.
Fig. 5. A Farm-to-Fridge overview of the poultry-meat production chain, indicating the impact of key processing steps on the fate of bacteria in the
poultry microbiome.
further improve product quality and safety, and maintain consumer
confidence in this high protein food source.
Funding statement
This project was funded by the Department of Food Agriculture and
the Marine (Ireland) (Grant no. 17/F/275), under the Food Institutional
Research Measure (FIRM).
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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