TIMI 1461 No.

of Pages 23

Review
Gut Microbiota Dysbiosis in
Postweaning Piglets:
Understanding the Keys to
Health
Raphaële Gresse,1,2 Frédérique Chaucheyras-Durand,2
Mickaël Alain Fleury,3 Tom Van de Wiele,4 Evelyne Forano,1
and Stéphanie Blanquet-Diot1,*
Weaning is a critical event in the pig’s life cycle, frequently associated with
Trends
severe enteric infections and overuse of antibiotics; this raises serious eco-
Weaning is the most critical phase in
nomic and public health concerns. In this review, we explain why gut micro- pig production, generally associated
biota dysbiosis, induced by abrupt changes in the diet and environment of with enteric infections, and it requires
substantial use of antibiotics.
piglets, emerges as a leading cause of post-weaning diarrhea, even if the
exact underlying mechanisms remain unclear. Then, we focus on nonantimi- Overuse of antibiotics raises serious
crobial alternatives, such as zinc oxide, essential oils, and prebiotics or pro- public health concerns due to the
increasing emergence of multidrug-
biotics, which are currently evaluated to restore intestinal balance and allow a resistant bacteria.
better management of the crucial weaning transition. Finally, we discuss how
in vitro models of the piglet gut could be advantageously used as a comple- Gut microbiota dysbiosis has emerged
as a leading cause of postweaning
ment to ex vivo and in vivo studies for the development and testing of new feed diarrhea and associated infections in
additives. piglets.

A current challenge is finding new

effective nonantibiotic alternatives to
restore gut microbial balance in wean-
The Swine Industry and Antibiotic Resistance
ing piglets.
Pork is the world’s most consumed meat from terrestrial animals [http://www.fao.org/ag/
againfo/themes/en/meat/background.html]. In modern swine breeding conditions, weaning is In vitro models of the piglet gastroin-
the main critical period in the course of the pig’s life due to sudden dietary, social, and testinal tract at weaning would accel-
erate the understanding of dysbiosis
environmental changes [1]. Multiple stressors encountered at piglet weaning induce transient
etiology and the development of new
anorexia, intestinal inflammation, and unbalanced gut microbiota [1,2]. The circumstances of feed additives.
weaning transition generally cause gastrointestinal (GI) infections, mainly colibacillosis diarrhea
[3], that have been associated with the death of around 17% of piglets born in Europe [1]. 1
Université Clermont Auvergne, UMR
Despite the ban on antibiotic growth promoters in the European Union since 2006, antimicro- 454 MEDIS UCA-INRA, F-63000
bials are still massively used in the swine industry for therapeutic purposes, but are also used as Clermont-Ferrand, France
2
Lallemand Animal Nutrition, F-31702
prophylactic or metaphylactic treatments to prevent GI infections in farms and their associated
Blagnac Cedex, France
economic losses [4]. The overuse of antibiotics is closely related to the growing number of 3
Institut de l’élevage IDELE, F-14310
antimicrobial-resistant agents and raises important concerns about animal and also human Villers Bocage, France
4
Ghent University, Center for Microbial
health (Box 1). For instance, a new Clostridium difficile resistant strain causing life-threatening
Ecology and Technology, B-9000,
diarrhea is estimated to provoke 14 000 deaths per year in the USA [5]. Additionally, some Gent, Belgium
bacterial families, such as Enterobacteriaceae, are resistant to all, or nearly all, antibiotics,
including last-resort drugs [5]. Regarding this pessimistic context, finding antibiotic alternatives
*Correspondence:
to both maintain piglet health at the critical weaning period and preserve public health becomes
[email protected]
a real emergency. (S. Blanquet-Diot).

Trends in Microbiology, Month Year, Vol. xx, No. yy http://dx.doi.org/10.1016/j.tim.2017.05.004 1

© 2017 Elsevier Ltd. All rights reserved.
 TIMI 1461 No. of Pages 23

Box 1. Antimicrobial Resistance: A Major Public Health Concern

Antibiotics have been recognized as one of the most successful therapies in medicine but are now compromised by the growing number of antibiotic-resistant
bacteria. Antibiotics are administered in both human and veterinary medicine and lead to the development of resistant bacterial strains within human and animal gut
microbiota (Figure I). Bacteria become resistant to antibiotics when a mutation affects (i) the production of enzymes that can inactivate the antibiotic molecule, (ii) the
recognition pathway between antibiotics and bacterial cells, or (iii) the mechanisms of entry and transport of the antibiotic molecule through the cells. When resistant
strains are extracted from their host, they are disseminated into the environment, and increase the spread of resistant genes among bacterial populations [116,117].

Development of resistant bacteria inside

Prevenve or curave
human gut microbiota
anbioc treatments

(A) Conjugaon (C) Transformaon

DNA

Plasmid

Donor cell Recipient cell Lysis of resistant cell

Release of resistance
gene into the
(B) Transducon Phage carrying environment
resistance gene
Development of Lysis
resistant bacteria inside
pig gut microbiot
Infected donor cell Recipient cell Recipient cell

Resistance spread to
general community

Hospitalisaon
Contaminaon of meat

Contaminaon of the
environment Transmission of resistance to human Resistance spread to other paents

Figure I. Mechanisms of Antibiotic Resistance Dissemination. Several mechanisms allow bacteria to acquire antimicrobial resistance such as (A) conjugation
or cell fusion occurring through cell contact between donor and recipient, (B) phage transduction, or (C) transformation from DNA fragments taken up directly from
the environment.

In this review, we first describe the main characteristics of weaning in the swine industry with a
focus on diet and intestinal physiology. Secondly, we discuss the hypothesis surrounding the
disruption of piglet intestinal microbial balance at weaning and its possible involvement in
postweaning infections. Then, we examine nonantibiotic solutions to maintain gut ecosystem
integrity in newly weaned piglets – such as zinc oxide, essential oils, prebiotics or probiotics.
Finally, the usefulness of pig gut models in the development and testing of such nonantibiotic
solutions is developed.

2 Trends in Microbiology, Month Year, Vol. xx, No. yy

 TIMI 1461 No. of Pages 23

Weaning As a Critical Part of Pig Life

In the modern swine industry, weaning is generally practiced at around 3–4 weeks of age,
although natural weaning would occur around 17 weeks after birth [6]. Weaning is a sudden,
stressful, short, and complex event characterized by changes in diet, social, and environmental
life conditions, which profoundly impacts piglet health and leads to decreased performance
and sometimes mortality [7]. In intensive production, piglets can even be weaned at only 1 or 2
weeks of age, which consistently amplifies the physiological and behavioral effects of weaning
[8–11]. Social and environmental stresses are generated by separation from the mother,
handling, transport, different physical environments, and mixing litters [7,9]. These diverse
factors induce vocalizations and even fighting, and are associated with elevated heart rates and
the secretion of stress-linked factors [9,12]. The switch from highly digestible liquid milk to a
less-digestible, more complex solid feed has also critical consequences on piglet behavior and
the physiology of their still-immature GI tract [1]. Dietary change is associated with low and
erratic feed and water intake, resulting in a fasting period of mostly 24 to 48 h – and thus a
transiently reduced growth rate [1,9]. Weaning anorexia has been shown to contribute to local
inflammation in the piglet's small intestine [13]. Numbers of other GI changes have been
associated with the overall weaning transition, such as activation of pathways related to
inflammatory responses, changes in hormonal activity, reduction in gastric motility, induction
of small-intestine atrophy and reduced villous height, reduction in nutrient, fluid and electrolyte
absorption, and increased permeability to antigens and toxins [14–17]. Finally, it is now more
and more accepted that dietary transition and environmental changes at weaning are linked to
modifications in piglet intestinal microbiota which could be involved in the etiology of post-
weaning diarrhea and enteric infections [15].

Gut Microbiota Dysbiosis and Postweaning Enteric Infections

The gut microbiota of mammals has numerous roles benefiting the host – such as digestion and
fermentation of carbohydrates, production of vitamins, maintenance of normal functions of the
intestinal villi, regulation of the immune responses, and protection from pathogenic bacteria
[18,19]. Among the physiological and GI factors impacted by the weaning transition, gut
microbiota disruption is likely to be recognized as one of the keys leading to postweaning
diarrhea. The pig gut microbiota is a very complex ecosystem showing dynamic composition
and diversity which shifts over time and along the entire GI tract [20]. Colonization is initiated at
birth and is shaped by consumption of the sow’s milk – which provides nutritional advantages
to the population of lactic acid bacteria, building a milk-oriented microbiome [21]. Escherichia
coli and Streptococcus spp. create an anaerobic environment favoring the establishment of
other colonizers such as Bacteroides, Lactobacillus, Bifidobacterium, and Clostridium [22].
During the suckling period, the breed and nursing mother lead to further differentiation of the
fecal microbiota of piglets [23]. A study by Bian et al. [23], conducted in suckling animals,
highlighted that the switch of nursing mother milk and host genetics strongly influenced the
development of the gut bacterial community. Therefore, the suckling period offers a peculiar
window of gut microbiota modifications.

At weaning, piglets are suddenly fed with a diet containing cereals and a relatively high
concentration of crude proteins, although modern diets tend to pay more attention to amino
acid balance. In addition, a prestarted diet is generally proposed for suckling piglets with the
aim of attenuating the abrupt change of food encountered at weaning. Most of the studies
conducted during the weaning transition have reported a decrease in bacteria of the Lactoba-
cillus group and a loss of microbial diversity, whereas Clostridium spp., Prevotella spp. or
facultative anaerobes such as Proteobacteriaceae, including E. coli, were positively impacted
(Table 1). Levels and sources of proteins or fibers shape the diversity and composition of the gut
microbiome of weaning piglets [24,25]. For instance, in weaning pigs, enriched pectin diets as
well as a soybean meal decreased the relative abundance of Lactobacillus and increased that

Trends in Microbiology, Month Year, Vol. xx, No. yy 3

4

TIMI 1461 No. of Pages 23

Table 1. Influence of Weaning Transition on the Characteristics of Piglet Gut Microbiota
Trends in Microbiology, Month Year, Vol. xx, No. yy

Age of piglets Diet and breeding Origin of Method of microbiota Qualitative modifications Quantitative composition of gut microbiota Refs
when conditions samples analysis of gut microbiota
sampling

Before weaning After weaning

Healthy piglets

19 days 32 piglets weaned with a Ileum PCR-DGGE Decrease in Lactobacillus in the ileum No clones of E. coli in the ileum 30% of clones highly similar to [31]
cereal and protein-based Colon qPCR and colon E. coli and Shigella flexneri
diet 16S amplicon 7.1  1.3  107 of total 4.4  0.5  105 of total
sequencing Lactobacillus in ileal samples Lactobacillus in ileal samples
of unweaned piglets 2 days postweaning

7 to 35 days 30 piglets from 3 different Stomach PCR-DGGE Decrease in Lactobacillus spp. relative Streptococcus suis not 107 copies/g of potentially [118]
litters, fed with cereal- Jejunum qPCR abundance in the stomach, jejunum detected harmful Streptococcus suis
based diet and no Ileum 16S amplicon and ileum
antibiotics sequencing

4 to 6 weeks 15 piglets weaned at Feces 16S amplicon Increase in Prevotella relative 54.0% of Firmicutes 35.8% of Firmicutes [119]
28 days and housed in sequencing abundance 38.7% of Bacteroidetes 59.6% of Bacteroidetes
controlled environmental (454- pyrosequencing) Increase in Shannon-Weaver diversity 4.2% of Proteobacteria 1.0% of Proteobacteria
conditions index 0.7% of Spirochates 2.0% of Spirochates
Shift from Firmicutes to Bacteroidetes
New species of Clostridium detected

7 to 32 days Piglets weaned at 25 days, Stomach PCR DGGE Decrease in diversity in the ileum and NDa ND [28]
randomly divided into 4 Ileum Colon qPCR colon
litters No change in the stomach

14 to 70 days 31 piglets weaned at Feces 16S amplicon Increase in Acetivibrio, Dialister, At 14 days: At 36 days: [120]
 
28 days fed with sequencing (454- Oribacerium and Prevotella 9% of Bacteroides 0% of Bacteroides
 
carbohydrate-based pyrosequencing) 2% of Escherichia/Shigella 0% of Escherichia/Shigella
 
concentrate 3% of Lactobacillus 0.5% of Lactobacillus
 
0.5% of Prevotella 28% of Prevotella

21 days to 3 36 crossbred castrated Feces qPCR Increase in Proteobacteria relative ND 40.8–45.8% of Bacteroidetes [121]
weeks male pigs weaned at 16S amplicon abundance at 1 week postweaning and 35.8–45.1% of Firmicutes
21 days and fed with mash sequencing (Illumina decrease at 3 weeks postweaning 0.9–12.9% of Proteobacteria
diet MiSeq) Increase in anaerobic fiber-fermenting 0.7–5.2% of Tenericutes
Firmicutes 1.4–3.1% of Spirochaetes
0–1.3% of Planctomycetes
 
0 to 7 weeks 40 Meishan and Yorkshire Feces 16S amplicon Disappearance of Fusobacterium 45–70% of Firmicutes 55–70% of Firmicutes [23]
 
piglets weaned at 28 days sequencing (454- Lower relative abundance of 20–40% of Bacteroidetes 20–30% of Bacteroidetes
 
and separated in mixed pyrosequencing) Lactobacillus 3–20% of Fusobacterium 0–10% of Fusobacterium
groups at birth Prevotella, Ruminococcaceae,
Spirochaetaceae more abundant

35 days 120 piglets weaned at Jejunum qPCR Decrease in Lactobacillus ND ND [46]

2 days and fed with basal Increase in Enterococcus and
diet for 14 days Escherichia coli
 Table 1. (continued)

TIMI 1461 No. of Pages 23

Age of piglets Diet and breeding Origin of Method of microbiota Qualitative modifications Quantitative composition of gut microbiota Refs
when conditions samples analysis of gut microbiota
sampling

Before weaning After weaning

Healthy piglets treated with antibiotics

3 to 5 weeks 6 piglets weaned at 3 Feces 16S amplicon Dramatic increase in E. coli populations ND ND [32]
weeks, 3 of the 6 sequencing (454- and Proteobacteria
supplemented with 100 g/ pyrosequencing) Increase in Succinivibrio and
ton of chlortetracycline Ruminococcus
+ 100 g/ton of Decrease in Bacteroidetes,
sulfamethazone + 100 g/ Anaerobacter, Barnesiella,
ton of penicillin Sporacetigeniuma and Sarcina

3 to 5 weeks 48 piglets weaned at 3 Ileal mucosa 16S amplicon Dramatic decrease in Lactobacillus ND 88.5% of Firmicutes [33]
weeks and fed with high sequencing (454- Increase in Clostridium and 21.9% of Lactobacillus
complexity diet with 2.73 g pyrosequencing) Proteobacteriaceae, including E. coli 49% of Clostridium
of chlortetracycline 9.9% of Proteobacteriaceae
8.4% of Escherichia

0 to 7 weeks 27 piglets housed in Feces 16S amplicon Decrease in Lactobacillus, 0.3% of Prevotellaceae 14.8% of Prevotellaceae [21]
pathogen- free facilities sequencing (Illumina Bacteroidaceae and 15.4% of Bacteroidaceae 1.4% of Bacteroidaceae
and weaned at 21 days MiSeq) Enterobacteriaceae 1.7% of Ruminococcaceae 9.6% of Ruminococcaceae
with oat-based diet Increase in Prevotellaceae,
+ 444 mg/kg Lactobacillaceae, Ruminococcaceae,
chlortetracycline + 39 mg/ and Veillonellaceae
kg tiamulin

Infected weaning piglets

25 days 24 piglets weaned at Ileum 16S amplicon Top genera of infected piglets: ND ND [122]
21 days and fed with sequencing (FLX Clostridium, Staphylococcus,
commercial diet, among pyrosequencing) Helicobacter and Lactobacillus
them 2 experimentally
infected and 2 naturally
infected with S.
typhimurium
Trends in Microbiology, Month Year, Vol. xx, No. yy

Piglets suffering from postweaning diarrhea

7 to 47 days 20 piglets weaned at Feces qPCR High relative abundance of ND ND [29]

21 days housed in poor 16S amplicon Enterobacteriaceae in D pigs after
conditions, among them 13 sequencing (Illumina weaning
suffered from postweaning MiSeq) High relative abundance of
diarrhea (D) and 7 were Bacteroidetes in H pigs after weaning
healthy (H) Lower Simpson diversity index in D
than H pigs
Decrease in evenness index before
weaning in both group, and after
weaning in D pigs
Higher evenness in D pigs before
weaning
5

a
ND, not determined.
 TIMI 1461 No. of Pages 23

of Prevotella in the colon [26], and a fish protein source was linked to a large expansion of the
Escherichia/Shigella group [27]. Such disturbances of the gut microbial ecosystem and loss of
diversity at early times of life [28,29] can dramatically increase the risk of GI diseases [30]. In
particular, as Lactobacillus spp. are major players in disease prevention, their abrupt decrease
during weaning transition can contribute to an increase in the risk of disease [31]. In-feed
antibiotics can also introduce differences in piglet gut microbiota at weaning [21,32,33] due to
their wide spectrum activity and thus their potential ability to kill or prevent the growth of both
pathogenic and beneficial microbes. The diversity of the microbiota may be even more
decreased [32,34]. Prolonged use of subtherapeutic doses of antibiotics, but also therapeutic
treatments, can increase opportunities for pathogenic microorganisms to colonize and trigger
diseases [30,35]. The two major pathogens impacting the swine industry are Salmonella
enterica serovar Typhimurium (S. Typhimurium) and E. coli [36]. Among pathogenic E. coli,
enterotoxigenic E. coli (ETEC) is the main infectious agent of postweaning diarrhea in piglets,
being responsible for 50% of piglet deaths worldwide per year [37]. The genetic background of
the host, which has a key role in driving the settlement of the gut microbiota, also represents a
predisposing factor to infections in piglets. Indeed, F4 ETEC and F18+ pathogenic E. coli-high
susceptibility phenotypes have been recently detected, for instance in Flemish pigs [38]. On the
other hand, a polymorphism within an intron of the Mucin 4 gene on the porcine genome can
invalidate the production of the specific ETEC F4 receptor and subsequently protect these pigs
from ETEC F4 infections [39]. ETEC-susceptible pigs (based on Mucin 4 gene polymorphism)
also display a reduced gut bacterial diversity compared to nonsusceptible pigs [40].

Hence, in piglets, weaning transition has been associated with a disrupted state of the
microbiota that can be referred to as ‘dysbiosis’ [1]. The characteristics of such a state are
not completely clear, although dysbiosis has been defined in mammals as a gut microbial
imbalance identified by a marked decrease in the representation of obligate anaerobic bacteria,
such as members of the classes Clostridia and Bacteroidia, and an increased relative abun-
dance of facultative anaerobic bacteria such as members of Enterobacteriaceae [41]. The
process leading to dysbiosis and GI infections is, at this moment, poorly documented in piglets.
However, there are several hypotheses which can be translated from in vitro, mice, or human
studies (Figure 1). The decrease in gut microbial diversity at weaning makes the glycans which
compose the mucus layer protecting the gut epithelium more available for pathogenic micro-
organisms [42]. The degradation of mucus polysaccharides by commensals can release
sugars, such as fucose, galactose, or mannose, which can promote the growth of pathogenic
species. For instance, an in vitro study has revealed that the commensal species Bacteroides
thetaiotaomicron produces fucose from mucus carbohydrates, used by enterohaemorrhagic E
coli to activate type III secretion system (T3SS) gene expression [43]. T3SS is the system
deployed by some pathogenic E. coli and Salmonella to sense and adhere to host enterocytes
[36]. Besides a loss of gut microbiota diversity, weaning transition is also associated with an
increase in permeability which could favor the crossing of toxins and pathogens through the
epithelium [14]. Also, change of diet and stresses occurring at weaning promote intestinal
inflammation that can be exacerbated by enteric infections [42]. The inflamed gut appears to
provide a favorable environment for expansion of Enterobacteriaceae [44]. Indeed, gut inflam-
matory host-response produces reactive species such as nitric oxide (NO) which shows
antimicrobial properties [42,44]. However, NO released into the intestinal lumen is rapidly
transformed into nitrate [42,44]. The nitrate-rich environment of the inflamed gut confers growth
advantages on strains of E. coli which possess nitrate reductase genes that are absent in
species of Clostridia or Bacteroidia [41,45]. Interestingly, a recent study in piglets supports this
hypothesis by reporting an increased concentration of reactive oxygen species in the intestine
coupled with an expansion of the E. coli population 7 days after weaning [46]. Additionally, the
concentration of oxygen in the inflamed intestine, which is enhanced by the higher blood flow,
may favor the bloom of facultative anaerobes such as members of the Enterobacteriaceae,

6 Trends in Microbiology, Month Year, Vol. xx, No. yy

 TIMI 1461 No. of Pages 23

Resident microbiota
Loss of bacterial diversity

Enterobacteriaceae Lumen
Enterobacteriaceae
Nitrate re
spiraon

Nitrite
Nitrate
Obligate
Differences in anaerobe
Weaning transion

mucus availability Fucose

NO O2
B. thetaiotaomicron

Mucus layer

Virulence
gene Epithelium
expression

Infecon
Permeability Inflammaon
Reduced
feeding
Macrophage
Stress

Solid-based In-feed Anbioc

diet anbiocs treatment
Sow milk

Symbiosis Intesnal dysbiosis Postweaning diarrhea

Birth 3/4 weeks 1 to 10 days post-weaning

Figure 1. Impact of Weaning Transition on Piglet Gut Microbiota and Expansion of Infectious Agents. At weaning, piglets undergo the abrupt change from
sow’s milk to solid feed as well as social and environmental stresses. These modifications result in disruption of gut microbiota composition and intestinal inflammation
that can lead to the expansion of enteric pathogens and postweaning diarrhea. Some hypotheses have been raised to explain underlying mechanisms. During weaning,
the nutritional landscape of the piglet gut is modified, and mucus polysaccharides may be more available for commensal bacteria (such as Bacteroides thetaio-
taomicron). Long-term feed antibiotics and therapeutic doses of antimicrobials may contribute to this vicious circle by decreasing bacterial diversity and increasing
intestinal inflammation.

decrease obligate anaerobes, and consequently induce a loss of bacterial diversity [44]. Thus,
the succession of postweaning events in piglets could drive a real vicious circle, leading to
enteric infections (Figure 1). As antibiotics can promote intestinal inflammation [44,47], and are
associated with decreased microbiota diversity, antimicrobial compounds may fuel and exac-
erbate the vicious circle process during weaning transition.

Trends in Microbiology, Month Year, Vol. xx, No. yy 7

 TIMI 1461 No. of Pages 23

Nonantibiotic Alternatives to Restore Gut Microbial Balance and Prevent

Infections
Given the major role of antibiotics in gut microbiota dysbiosis, and the rise in important public
health concerns about the spread of multiresistant bacteria, there is an urgent need for
developing nonantibiotic alternative strategies to restore microbial balance and control GI
infections associated with weaning transition in piglets. Several kinds of solution exist to fight
against dysbiosis and infections at weaning.

For instance, a live oral vaccine against ETEC F4 is already commercialized and recommended
for healthy piglets from 17 days of age [48]. Weaned piglets have already been successfully
vaccinated using this product. The vaccine, administered in drinking water, resulted in a
significant reduction in the incidence of diarrhea, ileal colonization, and fecal shedding of ETEC
F4 in challenged postweaning piglets [49]. However, protection occurs several days after
weaning, meaning that piglets are not protected during the short period in which ETEC
infections strongly arise [48]. Vaccination should ideally take place during the suckling period,
but vaccines would be susceptible to neutralization by the high level of maternal IgA antibodies
present in the suckling piglet gut [48]. Then, there is a need for clarifications regarding the use of
vaccination, which, in addition, has for now been developed mostly to target one particular
strain or species of pathogen.

Another solution is the use of phages which generally attack a specific bacterium or a narrow
group of bacteria without negatively affecting autochthonous bacteria [50]. The oral admin-
istration of a bacteriophage cocktail in postweaning piglets challenged with ETEC K88 has
been proposed to alleviate the symptoms of infection [51]. In another recent study, a
bacteriophage cocktail increased the relative abundance of Bifidobacterium spp. and Lac-
tobacillus spp. and decreased coliforms and Clostridium spp. in weaning piglets [52].
Nonetheless, the disadvantages of this therapy are (i) the requirement for a large number
of target bacteria, (ii) the need for rapid administration of the phages after infection, (iii)
neutralization of the phages by the host immune system, and (iv) the possible development of
resistance [50].

The nutritional components of the animal diet can also be adjusted using various feed additives
showing specific properties. Up to now, most of the nutritional studies conducted in weaning
piglets have focused on increased feed intake, daily gain weight, improved immune function, or
enhanced digestive functions and metabolism [16]. Indeed, rather few in vivo studies have
investigated the effects of feed additives on piglet intestinal microbiota at weaning, as reviewed
in Table 2 and described below.

Zinc Oxide
Zinc oxide (ZnO) has been proposed as one of the most effective feed additives to replace
antibiotics and is already widely commercialized in several countries. High levels of ZnO have
shown antimicrobial properties and are used to fight against postweaning infections [53].
Nevertheless, the effect of high levels of ZnO on resident microbiota of newly weaned piglets
remains controversial. Starke et al. [53] showed a reduction in E. coli and Enterobacteriaceae in
the stomach and small intestine, while Vahjen et al. [54] and Højberg et al. [55] noticed opposite
trends. In addition, all of these studies reported a reduction in health-associated lactic acid
bacteria such as Lactobacillus spp. [53–56]. Thus, the beneficial effects of ZnO are not
universally acknowledged, especially since it can negatively impact animal health due to the
accumulation of Zn in the liver, pancreas, and kidney [57], as well as human health by increasing
the proportion of multiresistant E. coli in the GI tract of piglets [58]. Moreover, the European
legislation limits the use of ZnO in animal production to a maximum of 150 mg/kg because of
suspected environmental pollution [53].

8 Trends in Microbiology, Month Year, Vol. xx, No. yy

 Table 2. Effects of the Main Nonantibiotic Alternatives on the Gut Microbiota of Healthy or Pathogen-Challenged Piglets at Weaning

TIMI 1461 No. of Pages 23

Age of piglets Diet and rearing conditions Origin of Method of Additives Dose Modification of intestinal microbiota or Other observations Refs
when sampling samples microbiota in the diet effect on pathogen colonization
analysis

Zinc oxide

42 days 32 piglets weaned at 28 days Caecum Culture Zinc oxide 2500 mg/kg Reduction in lactic acid bacteria Reduction of ATP [55]
housed individually Colon Increase in coliforms and enterococci accumulation

42 days 208 piglets weaned at around Ileum Culture Zinc oxide 3100 mg/kg Reduction of anaerobic and lactic acid No effect on [56]
22 days with a basal diet and bacteria performance
housed in pens No effects on Escherichia coli

40 to 42 days 12 piglets weaned at 28 days and Ileum 16S amplicon Zinc oxide 3042 mg/kg Increase in Enterobacteriaceae relative NDa [54]
housed in controlled conditions sequencing abundance and diversity
(454- Reduction of Lactobacillus reuteri relative
pyrosequencing) abundance
Increase in Weisella cibaria, Weisella
confuse, Leuconostoc citreum,
Streptococcus equinus and
Streptococcus lutetiensis relative
abundances

25 to 53 days Piglets weaned at 25 to 26 days Stomach qPCR Zinc oxide 2420 mg/kg Reduction of Enterobacteriaceae, E. coli Reduction of [53]
and housed in pens Jejunum and Lactobacillus spp. especially in the bacterial
Ileum stomach and small intestine 1 week metabolites such as
Colon postweaning SCFAb
Lower lactate
concentrations

Essential oils

Healthy piglets

3 to 5 weeks 6 pens of 6 piglets per treatment, Ileum Culture Herbal extract 0.75% Reduction of coliforms at day 14 No effect on [60]
weaned at 16 to 19 days Colon PCR DGGE containing No difference in the carriage in E. coli K88 intestinal
feces cinnamon, morphology
thyme and
oregano extract
Trends in Microbiology, Month Year, Vol. xx, No. yy

0 to 35 days 96 piglets weaned with a basal Caecum Culture Essential oil 0.01% Reduction of E. coli number in the Lower level of [61]
post-weaning diet and housed in pens with Colon blend with 18% caecum, colon and rectum Interleukin 6
controlled conditions Rectum thymol and Lactobacilli to E. coli ratio increased in Higher level of tumor
cinnamaldehyde the colon necrosis factor-a
Higher villous height
to crept depth ratio
in the jejunum

8 weeks 14 piglets weaned at 28 days Caecum Culture Essential oil 0.025% Decrease in E. coli and total anaerobes in Increase in average [59]
receiving a control diet of Colon blend with 4.5% the colon and rectum daily gain
3400 kcal/kg for 28 days Rectum cinnamaldehyde Greater jejunum
and 13.5% villus height
thymol
9
 Table 2. (continued)
10

TIMI 1461 No. of Pages 23

Age of piglets Diet and rearing conditions Origin of Method of Additives Dose Modification of intestinal microbiota or Other observations Refs
Trends in Microbiology, Month Year, Vol. xx, No. yy

when sampling samples microbiota in the diet effect on pathogen colonization

analysis

35 days 120 piglets weaned at 21 days Jejunum qPCR Carvacol-thymol 100 mg/ kg Increase in Lactobacillus Decrease mRNA [46]
with the basal diet + essential oil blend Decrease in Enterococcus spp. and E. levels of TNF-a
for 14 days coli

Piglets challenged with pathogens

32 to 59 days 64 weaned piglets of 24 days Feces Culture Thymol 1% No change in fecal excretion in S. Reduction of feed [62]
challenged with 109 CFU of S. Typhimurium intake
Typhimurium Higher
immunoglobulin
concentrations in
serum before
challenge

Organic acids

Healthy piglets

3 to 5 weeks 6 pens of 6 piglets per treatment, Ileum Culture Acid 1: Acetic, 1.1% for each Lower fecal counts of coliforms at day 4 No effect on [60]
weaned at 16 to 19 days Colon PCR DGGE formic, mixture for both treatment intestinal
Feces propionic, No difference in the carriage of E. coli morphology
phosphoric, and K88
citric acids
Acid 2: 50% of
lactic acid and
50% of acid
mixture

4 weeks Piglets weaned at 16 to 19 days Ileum PCR DGGE Acid blend 1.1 to 2.1% No change in bacterial diversity ND [67]
and housed in standard pens qPCR Increase in Lactobacillus relative
16S amplicon abundance
sequencing
(454-
pyrosequencing)

53 to 55 days 96 male piglets of 25 days old Jejunum qPCR Organic acids OA: 0.416% OA: Increase in Bacteroides, OAc: decrease in [68]
castrated and housed in pens Ileum (OA) fumaric acid and Porphyromonas, Prevotella and intestinal pH
Colon Medium chain 0.328% lactic Clostridium cluster XIVa, I and IV in the
fatty acids acid stomach
(MCFA) MCFA: 0.15% Decrease in Streptococcus in the colon
caprylic and MCFA: Increase in Escherichia, Hafnia,
capric acids Shigella groups in the jejunum

Piglets challenged with pathogens

26 to 37 days 27 piglets weaned at 19 to Stomach Culture Fumaric or citric 1.5% No effect on E. coli carriage or Higher volatile fatty [69]
23 days and challenged with 1010 Jejunum acids commensal microbiota acids in the jejunum
CFU of ETEC K88 Caecum
Colon
Feces
 Table 2. (continued)

TIMI 1461 No. of Pages 23

Age of piglets Diet and rearing conditions Origin of Method of Additives Dose Modification of intestinal microbiota or Other observations Refs
when sampling samples microbiota in the diet effect on pathogen colonization
analysis

Prebiotics

Healthy piglets

3 to 6 weeks 128 piglets weaned at 18 to Jejunum Culture Mannan- 0.2% Decrease in Enterobacteriaceae in the No difference in [73]
22 days and housed in pens qPCR oligosaccharides jejunum ileum weight or crypt
No difference for Lactobacillus spp. depth

24 to 49 days 240 piglets weaned at 24 days Feces Culture Lactose (L) L: 150 or 250 g/ Reduced counts of E. coli with SWE or Higher average daily [74]
and housed in pens Seaweed (SWE) kg high L diet gain with high L diet
containing SWE: 2.8 g/kg Increase in nitrogen
laminarin and digestibility and
fucoidan gross energy with
SWE

3 to 5 weeks 144 piglets weaned at 21 days Jejunum Culture Cello- 1.5, 3.0 and Increase in Lactobacillus Increase in villus [72]
and housed in pens oligosaccharides 4.5 g/kg Decrease in Clostridium height
No effect on Bifidobacterium and E. coli Increase in villus
surface area

Piglets challenged with pathogens

21 to 49 days Piglets weaned at 12 days orally Ileum PFGE Fructooligosac 1% in water or Tendency of reduction of S. Typhimurium ND [75]
challenged with 107 CFU of S. Colon charides (FOS) feed shedding in feces when FOS
Typhimurium administered in water only

27 to 37 days 105 piglets weaned at 25 to Ileum qPCR Sugar beet pulp 15% of DDGS No effect on infection or S. Typhimurium ND [76]
32 days kept in level 2 Caecum (SBP) 6% of SBP shedding
biocontainment facilities and orally Colon Wheat distillers
challenged with 109 CFU of S. dried grains with
Typhimurium solubles (DDGS)

4 to 7 weeks 11 piglets weaned at 28 days Ileum qPCR Βeta glucans ND No prevention of Salmonella colonization No change in SCFA [77]
challenged with 109 CFU of S. Colon hulless barley or persistance concentrations
Typhimurium
Trends in Microbiology, Month Year, Vol. xx, No. yy

4 to 6 weeks 72 weaned pigs of 23 to 27 days Ileum Colon qPCR Lactulose 10 g/kg Increase in total lactobacilli Increase in daily [78]
challenged with 109 CFU of ETEC Feces No effect on ETEC carriage weight gain
K88 Ileal mucosa Increase in the
amount of butyrate
in the colon

Probiotics

Healthy piglets

52 to 54 days 360 piglets weaned at 24 to Feces PCR DGGE Enterococcus 0.5109, 1.0 Increase in Lactobacillus Lower incidence of [81]
26 days and housed in pens Real-time PCR faecalis 109 or 2.5109 diarrhea
CFU/kg of feed Higher average daily
gain
Higher feed
conversion
efficiency
11
 Table 2. (continued)
12

TIMI 1461 No. of Pages 23

Age of piglets Diet and rearing conditions Origin of Method of Additives Dose Modification of intestinal microbiota or Other observations Refs
Trends in Microbiology, Month Year, Vol. xx, No. yy

when sampling samples microbiota in the diet effect on pathogen colonization

analysis

42 days 24 piglets weaned at 21 days fed Feces Culture Lactobacillus 5107 or 108 Increase in Lactobacillus Higher feed intake [82]
with a basal diet and probiotic and johnosii or CFU/g/piglet/ Decrease in E. coli Higher body weight
housed in controlled conditions Lactobacillus day of each gain
mucosae strain

10 to 37 days Piglets receiving from birth Ileum T-RFLP profiling Pediococcus 2109 CFU/kg Decrease in ileal diversity with PA ND [123]
probiotic treatment diluted in 2 mL Colon acidilactici (PA) of feed of each treatment
of peptone. At weaning, piglets Feces or strain Increase in Firmicutes with PA treatment
continued to receive probiotic in a Saccharomyces Increase in Porphyromonadaceae and
basal diet cerevisiae (SC) Ruminococcus in the colon with SC
treatment

28 to 180 days 36 piglets weaned at 28 days Feces Culture Lactobacillus 109 CFU/g of Increase in fecal counts in LAB and Higher average daily [83]
acidophilus or fermented feed bifidobacteria weight gain
Pediococcus Decreased in E. coli and clostridia Higher average dry
acidilactici matter intake
Higher gain to feed
ratio
Higher lactic acid
concentration in
feces
Decrease in
incidence of
diarrhea
Increase in villus
height
Decrease in crypt
depth

50 days 8 piglets weaned at 28 days but a Caecum RT-PCR Probiotic mixture 0.5 g of probiotic No effect on the relative amount in Increase in acetic [84]
cereal-based diet with probiotic Colon of 109 CFU of mixture/kg of selected bacterial population acid concentrations
mixture was offered from 10 days Lactococcus feed and total SCFA in
lactis, the caecum
Carnobacterium
divergens,
Lactobacillus
casei and
Lactobacillus
plantarum

40 days 30 piglets weaned at 30 days Jejunum 16S amplicon L. reuteri (LAB) or 2109 CFU/mL Increase in the species richness with LAB ND [85]
supplemented either with a Caecum sequencing chlortetracycline (LAB) compared to ATB where several taxa
probiotic or an antibiotic Colon (Illumina MiSeq) (ATB) 100 mg/kg (ATB) were eliminated
Decrease relative abundance in
Firmicutes and Prevotella in the colon
and caecum with LAB compared to ATB
 Table 2. (continued)

TIMI 1461 No. of Pages 23

Age of piglets Diet and rearing conditions Origin of Method of Additives Dose Modification of intestinal microbiota or Other observations Refs
when sampling samples microbiota in the diet effect on pathogen colonization
analysis

Piglets challenged with pathogens

3 to 4 weeks 16 piglets weaned at 21 days Feces qPCR Lactobacillus 1010 CFU/piglet/ Significant reduction in ETEC levels in the Improvement in daily [86]
challenged with 51010 CFU sobrius day ileum and colon weight gain
ETEC F4

26 to 36 days 18 piglets weaned at 18 days Stomach Culture Lactobacillus 1011 CFU/piglet/ Decrease in fecal coliforms Attenuation of [87]
challenged with 109 CFU of ETEC Ileum rhamnosus day Increase in lactobacilli and bifidobacteria serum IL-6 induced
K88 Caecum by E. coli
Colon Higher
concentrations of
TNF-a
Decrease in diarrhea
incidence

28 to 35 days 36 piglets weaned at 21 days Colon Culture Lactobacillus 1010 CFU/piglet/ Tendency to increase ETEC in the feces No effect on villus to [91]
challenged with 1.5108 CFU of Feces rhamnosus day No effect on lactic acid bacteria, crypt ratio
ETEC F4 enterobacteria, yeasts in the colon Tendency to
decrease villus
height
Reduction of total
IgA in the blood
serum

4 to 6 weeks 72 weaned pigs of 23 to 27 days Ileum Colon qPCR Lactulose (L) 10 g/kg of feed Increase in Lactobacillus plantarum in the Reduction of [78]
challenged with 1010 CFU of Feces with (L) ileum and colon and in total lactobacilli in diarrhea incidence
ETEC K88 Ileal mucosa Lactobacillus 21010 CFU/ the colon
plantarum (LP) piglet/day (LP) No effect on ETEC levels

36 days Piglets weaned at 21 days Feces qPCR Bacillus subtilis 3.9108 or Reduction of E. coli abundance in feces Upregulation of the [88]
challenged with 1010 CFU of and B. 7.8108 CFU/ expression of TLR4,
ETEC F4 lichenformis piglet/day NOD2, iNOS, IL-8
mixture and IL-22
Trends in Microbiology, Month Year, Vol. xx, No. yy

4 to 7 weeks 50 piglets weaned at 24 days Feces Culture Saccharomyces 51010 CFU/kg Reduction of ETEC excretion No effect on other [89]
challenged with 1.5108 CFU of cerevisiae of feed parameters
ETEC F4

a
ND, not determined.
b
SCFA, short-chain fatty acid.
c
OA, organic acid.
13
 TIMI 1461 No. of Pages 23

Essential Oils
Essential oils are a complex mixture of volatile organic compounds obtained from many diverse
plants which have antimicrobial, antioxidant, or antiviral properties [59]. In all available in vivo
studies, the supplementation of piglet diet with essential oils has been associated with an
increase in the Lactobacillus group and a decrease in E. coli or total coliforms [46,59–61]. Li
et al. [61] have even reported a decrease in E. coli carriage in piglets fed with thymol and
cinnamaldehyde at a similar level to that obtained with in-feed antibiotics, suggesting that
essential oils could be a candidate to replace traditional antibiotics. However, in piglets, herbal
extracts and thymol have shown no effect on the colonization of pathogenic strains of E. coli
and S. Typhimurium, respectively [60,62]. The characterization of active principles as well as
the mode of action of essential oils remain relatively unclear, and there is no real agreement
about their effects against Gram-negative or Gram-positive bacteria [63]. Therefore, more
investigations are needed to develop essential oils with adapted antimicrobial properties.
Because of their lipophilic characteristics, essential oil compounds may raise concerns about
their potential toxicity and possible negative impact on animal and human health [64]. At least,
individual constituents of carvacol, citral, and limonene oxide at subinhibitory concentrations
were shown to favor the emergence of a hyper-resistant E. coli strain [65].

Organic Acids
Short- and medium-chain organic acids (OAs), such as citric, propionic, lactic, or fumaric acids,
are already used in high dose in piglet diet for feed preservation because of their bacteriostatic
and bactericidal effects and very low cost [66]. The use of OAs in newly weaned piglets has been
mostly related to a decrease in coliforms [60] and an increase in Lactobacillus [67], although they
seem to impact the microbiota composition from the stomach to the colon in a broader manner
[68]. Despite their reviewed antimicrobial properties, citric and fumaric acids demonstrated a lack
of efficiency toward an ETEC K88 challenge [69], and mixtures of OAs had no influence on the
natural carriage of this pathogenic bacterium [60]. Conversely, they have a demonstrated
antimicrobial effect against Gram-positive bacteria [66]. Besides, it has been reported that
the pathogenic E. coli strain O157:H7 may display a mechanism of resistance against an acetic
acid treatment [70]. Further investigations are thus needed to endorse the interest in OAs, fully
understand their mode of action, and avoid inhibition of the resident microbiota.

Prebiotics
Prebiotics are fibers defined as selectively fermented dietary ingredients that allow specific
changes both in the composition and/or activity of the GI microbiota that confer a beneficial
physiological effect on the host [71]. Most prebiotics belong to nonstarch oligosaccharides
such as fructooligosaccharides (FOS) or galactooligosaccharides (GOS) [71]. They can stimu-
late short-chain fatty acid (SCFA)-producing bacteria, such as butyrate-producing bacteria,
which provide substrates and promote normal proliferation and differentiation of intestinal cells
[30]. Recent investigations reported interesting effects of prebiotics on the intestinal microbiota
of weaning piglets, such as an increased proportion of Lactobacillus [72] and a decreased
amount of potentially harmful groups such as Clostridium [72] and Enterobacteriaceae [73,74].
Unfortunately, no study has yet reported a significant effect of prebiotics on the carriage of
ETEC K88 or S. Typhimurium in orally challenged weaning piglets [75–78]. Future research
should confirm the potential interest of prebiotics in the management of the weaning phase and
outline the specificity of the different available fibers.

Probiotics
Probiotics are defined as living microorganisms that, when administered in adequate amounts,
confer a health benefit to the host [79]. The effects of probiotics on weaning piglets are widely
documented. Lactic acid bacteria such as Lactobacillus, Bifidobacterium, Enterococcus, or
Streptococcus, and yeasts from the genus Saccharomyces, are the most frequently used

14 Trends in Microbiology, Month Year, Vol. xx, No. yy

 TIMI 1461 No. of Pages 23

microorganisms [80]. Several recent studies with lactic acid bacteria, run in newly weaned
piglets, noted an increased abundance of Lactobacillus or Bifidobacterium spp., a decrease in
E. coli, or a higher production of SCFA [81–85]. However, a consensus cannot be established yet
as there are noticeable differences between bacteria and yeasts regarding their effects, which, in
addition, remain strain-dependent. The ability of probiotics (lactic acid bacteria and Saccharo-
myces cerevisiae) to inhibit colonization by ETEC K88/F4 has been also demonstrated in a
number of studies [78,86–89], as recently reviewed by Roussel et al. [90]. Again, the effect of
probiotics on ETEC seems to be strain-specific as Trevisi et al. [91] found that Lactobacillus
rhamnosus GG (L. rhamnosus GG) impairs the health of ETEC K4-challenged piglets. A study
from Li et al. [92] also reported that a high dose of L. rhamnosus in piglets negated the preventive
effect against ETEC F4 compared to the administration of a 100-times lower dose.

Among the available alternatives, probiotics seem to have the highest potential as they
constitute the only feed additive that is efficient towards pathogenic strains in piglets. Then,
probiotics could represent a safe opportunity to fight against postweaning dysbiosis and
enteric infections in the swine industry. Although the underlying mechanisms are not fully
understood, protection of piglets from postweaning infections by probiotics could notably
occur through inhibition of pathogen growth and adhesion to intestinal mucosa, stimulation of
the piglet immune system, or modulation of the composition and activity of the resident
microbiota (Figure 2). Regarding the last mechanism, the composition of a healthy and
disturbed microbiota needs to be more accurately defined to select relevant probiotics able
to restore gut microbial balance at weaning. The composition of the healthy piglet gut micro-
biota is not well established yet. In a broader manner, the early definition of a healthy gut
microbiota generally focused on sets of taxa that might be expected to be found in a healthy
individual [93]. Investigations in the last decade tended to modify this definition towards a set of
functional core which should include housekeeping functions necessary for individual microbial
life and processes that are not carried out by host cells, thus building the symbiotic host–
microbiota relationship [93]. Yet, an important part of gut microbial gene families remains
functionally uncharacterized, which represents a huge knowledge gap [93]. Taken together,
these findings suggest that further research is needed to investigate the actual effectiveness
and dose for probiotic treatments in weaning piglets.

In Vitro Modelisation of the Pig Gut Ecosystem: A Key Towards Better

Understanding
The extreme complexity of the pig’s gut favors the use of animal experiments – which remain
the best strategy for evaluating phages, feed additives, or probiotic effectiveness. Ex vivo
experiments, consisting in the isolation of a living organ from the animal to carry out analyses,
are an alternative to in vivo studies. Such practices would take the immaturity of the piglet GI
tract and immune system into account, being representative of the weaning phase conditions.
For instance, ex vivo models of the small intestine or colon were considered as particularly
suitable for investigations on the immune response of piglets toward some feed additives [94] or
ETEC infections [95]. Nevertheless, ex vivo techniques do not maintain the integrity of the gut
microbiota, and they are still invasive because they require the sacrifice of the animals. For
technical, cost, and ethical reasons, especially when pathogenic strains are involved, in vitro
models can be advantageously used as an alternative to ex vivo and in vivo assays [96,97].
Such in vitro techniques can save labor and time, offer flexibility, and allow a good reproduc-
ibility between experiments due to standardized conditions, and getting rid of inter- or intra-
individual variability. In addition, in vitro alternatives are fully in line with the 2010/63/EU directive
which aims to replace, reduce, and refine the use of animals in research.

Hitherto, a restrictive number of in vitro models of the pig’s gut have been developed to mimic
the upper or lower GI tract of pigs (Table 3). Currently, the most relevant model of the upper GI

Trends in Microbiology, Month Year, Vol. xx, No. yy 15

 TIMI 1461 No. of Pages 23

Direct inhibion of pathogens Smulaon of resident microbiota

Inhibion of pathogen growth Producon

of SCFA

Secreon of
anbacterial
molecules Lacc acid
producon

Producon pH
of anmicrobial
pepdes
Modulaon
Mucin of microbiota
degradaon

Inhibion of
pathogen
virulence genes Lumen
Smulaon of piglet
immune system
Compeon of Improvement of
sites of adherence
intesnal barrier integrity
Producon
Tight of anbodies
juncons
Mucus layer

Epithelium

Legend

Probiocs
An-inflammatory Smulaoin
response of lymphocytes
Producon of Detecon by
Commensals cytokins
immune cells

Pathogens Acon via host physiology

Figure 2. Possible Mechanisms of Probiotic Strains to Fight against Postweaning Infections in Piglets. Several in vivo studies have established the
beneficial effects of probiotics in healthy and pathogen-challenged piglets. Probiotics may act through three different mechanisms: (i) direct inhibition of pathogen
growth and virulence by secretion of antimicrobial substances, (ii) modulation of resident microbiota composition and activity, and (iii) stimulation of the host immune
system and improvement in intestinal barrier function. SCFA, short-chain fatty acid.

16 Trends in Microbiology, Month Year, Vol. xx, No. yy

 a,b,c

TIMI 1461 No. of Pages 23

Table 3. Main In Vitro Models of the Pig Digestive Tract and Their CharacteristicsIn Vitro
Types of model Simulated parameters Refs

Body Gastric pH Intestinal Gastric Intestinal Digestive Chyme Intestinal Intestinal Anaerobic Simulated
T pH emptying transit secretions mixing microbiota absorption conditions age

In vitro models of the pig upper GIT

Batch systems

GJ : gastric juice, PJ : pancreatic juice 39  C pH 2 pH 6.8 – – Porcine pepsin Stirring – – – ND [100,124]

Porcine
pancreatin

Figure adapted from [64]

Multicompartmental dynamic systems

TIM (TNO Gastro-Intestinal Model) 39  C Kinetic of Intestinal Half-time Half-time of Saliva mixed Water – Use of – ND [98,99]
gastric pH fall pH of stomach ileal with solid food pressure hollow
(addition of controlled emptying: emptying: Gastric juice fibers
hydrochloric with sodium 150 min 650 min Panceatic
acid) bicarbonate juice
pH/time (min) duodenum: Bile
6.0/5 pH 5 Electrolytes
3.5/30 jejunum:
3.0/120 pH 6.5
2.5/180 ileum:
2.0/240 pH 6.5

Figure from [64]

In vitro models of the pig lower GIT

Batch systems

39  C NA – NA – – Stirring Microbiota – Flushing with ND [100,101,

from pig CO2 or N2 111]
Trends in Microbiology, Month Year, Vol. xx, No. yy

feces
17
18

Table 3. (continued)

TIMI 1461 No. of Pages 23

Trends in Microbiology, Month Year, Vol. xx, No. yy

Types of model Simulated parameters Refs

Body Gastric pH Intestinal Gastric Intestinal Digestive Chyme Intestinal Intestinal Anaerobic Simulated
T pH emptying transit secretions mixing microbiota absorption conditions age

Continuous fermentation models

PolyFermS 38  C NA Controlled NA Retention – Stirring at Immobilized – Flushing with 5 months [102,109]

pH at 6.0 time of 9 h 120 rpm microbiota CO2
with sodium (swine from sow
hydroxyde proximal feces
colon)

Figure from [90]

PigutIVM (Piglet Gut in Vitro Model) 39  C NA Controlled NA Retention – Stirring at Microbiota – Anaerobiosis 8 weeks [103]
pH at 6.2 time of 24 h 400 rpm from piglet self-
with sodium (entire feces maintained by
hydroxide colon) the activity of
resident
microbiota

Figure from [92]

a
NA, not applicable.
b
ND, not determined.
c
–, not reproduced in the model.
 TIMI 1461 No. of Pages 23

tract is the TNO Gastrointestinal Model (TIM) that has been adapted by Meunier et al. [98,99] to
simulate the stomach and small intestine of growing pigs. The major feature of this dynamic
porcine digestion model is its ability to simulate the main digestive parameters of the stomach,
duodenum, jejunum, and ileum, namely body temperature, kinetics of pH, peristaltic mixing and
transit, salivary, gastric, biliary, and pancreatic secretions, as well as absorption of small
molecules and water. It should be emphasised that, even though the TIM is a very pertinent
model for studying physicochemical parameters of the GI tract, it does not include resident
microbiota. In favor of their low cost and simple use, batch systems have been largely used to
reproduce the pig colonic environment and related microbial fermentations [36,100,101].
However, batch cultures are restricted by substrate depletion and the continuous change
in pH and redox potential which limit the experimental duration to only several hours [96].
Continuous in vitro fermentation models can more closely mimic pig colonic physiology due to
integration of essential digestive parameters, such as realistic transit time and the supply of nutrient
medium simulating ileal/caecal effluents while maintaining a functional microbiota [102–105].
Such kinds of model are designed for long-term experimental periods – up to 54 days for the
PolyFermS model developed by Tanner et al. [102] – which allows increased microbiota stability
through immobilization on gel beads. None of these models reproduces the specific colonic
conditions of the piglet, except for the PigutIVM (Piglet Gut In vitro Model) recently set up by Fleury
et al. [103]. The PigutIVM has been validated compared to in vivo data in 8-week-old piglets under
untreated conditions, and when colistin, an antibiotic widely used to treat colibacillosis diarrhea,
was administered. The distinctive feature of this model is the reproduction of anaerobiosis by the
sole activity of resident microbiota, and not by flushing with CO2 or N2 as is usually done.

In vitro models of the pig gut have been used in a large number of nutritional applications aiming
to study carbohydrate and protein digestibility [99,106] or follow microbial fermentation of
starch and fibers (including prebiotics) [101,104,107]. Regarding microbiological applications,
pig digestion models have been used to assess the effect of probiotic yeast strains on pig [108]
or piglet [103] microbiota, as well as to study pathogen inhibition by medium-chain fatty acids
[105] or probiotics [109]. For instance, in the PigutIVM, the addition of Saccharomyces boulardii
led to a significant decrease in E. coli in the resident in vitro microbiota, as observed during
colistin treatment [103]. In the PolyFermS, Bifidobacterium thermophilum, combined with FOS
and GOS, inhibited colonization of S. typhimurium in the swine proximal colon [109].

To keep the peculiarity of the lumen and mucosal environment and facilitate host–microbiota
interactions under representative conditions of the GI–microbe interface, piglet in vitro models
could be optimized to integrate a mucosal microenvironment [110,111]. A study by Tran et al.
[111] revealed that adding mucins to a pig in vitro batch fermentation model increased the
proportion of some genera, such as members of the Proteobacteriaceae, which rely on the
mucus layer to increase their activity. In addition, to include the interactions between the host,
the immune system, and the gut microbiota, future in vitro digestion models should be coupled
with intestinal porcine cells in culture such as the IPEC-J2 cell line, isolated from neonatal piglet
jejunum [112] and porcine immune cell lines [113]. It would be of great interest to adapt in vitro
models of the pig GI tract to the particular conditions encountered during the critical weaning
stage. Such development would allow a better understanding of dysbiosis etiology by testing, in
an independent manner, the different parameters that are supposed to be involved (e.g.,
anorexia, dietary changes, and antibiotics). Such in vitro models of the weaning phase could be
also advantageously used by researchers, feed producers, or veterinarians for an efficient and
cost-effective development and testing of nonantibiotic alternative strategies such as phages,
feed ingredients, prebiotics, or probiotics. They could provide additional information on the
cellular and molecular mechanisms of action of new in-feed compounds and their appropriate
conditions of use (dose and mode of administration), which are questions that need to be
addressed before commercialization.

Trends in Microbiology, Month Year, Vol. xx, No. yy 19

 TIMI 1461 No. of Pages 23

Besides, due to the high similarity between the porcine and human gut, the pig can be used as a Outstanding Questions
disease model for humans, especially for infants [114]. For instance, a piglet model of intestinal What is the relative importance of gut
failure, a state of inadequate intestinal surface area following the removal of necrotic intestine, microbiota dysbiosis in the etiology of
postweaning diarrhea?
has already been used for studying the impact of prebiotics on ileal mucosa [115]. Thus, it is
conceivable that a piglet model of dysbiosis could also bring translatable knowledge to the field Would restoration of microbial balance
of pediatric nutritional research. in the piglet gut be sufficient to counter
gastrointestinal disorders at weaning?
Concluding Remarks
Would new nonantibiotic solutions be
Weaning is a critical event in the swine industry which frequently leads to severe intestinal
efficient enough to replace antimicro-
disorders and the overuse of antibiotics, raising serious economic and public health concerns. bials in the management of weaning
Gut microbiota dysbiosis induced by changes in diet and environment of piglets is prone to be transition?
one of the main causes of postweaning diarrhea and enteric infections. However, further
research should be undertaken to better understand the succession of events leading to Can in vitro models accurately repro-
duce the specific gastrointestinal con-
dysbiosis, as well as the relative involvement of influencing factors (see Outstanding Questions).
ditions encountered at weaning?
For a better management of the weaning transition, it is of the utmost importance to find
nonantibiotic strategies that are able to restore a balanced gut microbiota – such as prebiotics
and probiotics. Even though in vivo experiments are the gold standard for evaluating such
compounds, in vitro digestion models of the piglet gut constitute first-choice alternatives for
ethical, technical, cost, and regulatory reasons. The forthcoming development of a ‘dysbiosis’
model – integrating the key parameters encountered in the field at weaning and host dimension
– would be a great help for a better understanding of the etiology of postweaning disorders and
evaluation of new in-feed additives.

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