Prospects & Overviews

Review essays
Intestinal colonization: How key
microbial players become established
in this dynamic process
Microbial metabolic activities and the interplay between the host and microbes

Sahar El Aidy1)
, Pieter Van den Abbeele2), Tom Van de Wiele2), Petra Louis3) and
Michiel Kleerebezem4)

In this review, we provide an overview of the dynamic Introduction: The human gut microbiome
changes within the microbiota and its metabolites that are can be regarded as a metabolically
implicated in establishing and maintaining gastrointestinal active organ
homeostasis during various stages of microbial colonization.
The gradual conversion of the gut microbiota toward a Our knowledge of host-gut microbe interrelationships has
mutualistic microbial community involves replacement of rapidly accelerated by expanding genomics-based molecular
pioneer gut colonizers with bacterial taxa that are charac- techniques such as transcriptomics, metabonomics, and
teristic for the adult gut. An important microbial signature of metagenomics, especially in combination with the use of in
homeostasis in the adult gut is the prevalence and activity of vivo host models such as germfree animals [1–3]. Diverse
research interests have converged on the crucial impact of the
a diverse spectrum of bacterial species that produce interplay between the gut microbiota, diet, host metabolism,
beneficial metabolites through metabolic interactions and immune system on the human health status [4].
between microbial groups. Deciphering these microbial The human gut is colonized by a complex microbiota
signatures and their metabolites that govern short and long- comprising an enormous reservoir of genes, overwhelming the
term equilibrium, as well as imbalances in host-microbial host genome by a factor of 150 [5, 6]. Despite the fact that
only two phyla, the Firmicutes and Bacteroidetes, account for
relationships, may provide novel diagnostic tools and/or
90% of the microbial species of this ecosystem in humans,
therapeutic targets for specific disorders associated with high diversity and significant interpersonal variability results
intestinal dysbiosis and loss of homeostasis. from the hundreds of species and thousands of strains that

.
Keywords:
butyrate; Clostridium cluster IV and XIVa; core
microbiome; cross-feeding; fucosylation; mucus; sulfate
reducing bacteria
are comprised within these phyla [6–8]. The revolution in
analytical tools to investigate the human microbiome has
resulted in a better understanding of the composition and
functioning of this microbiome in health and disease. A recent
study, with an unprecedented large cohort of US (n ¼ 316),

DOI 10.1002/bies.201300073

1)

Laboratory of Microbiology, Wageningen University, Wageningen, Corresponding authors:
The Netherlands Sahar El Aidy
2)
Laboratory of Microbial Ecology and Technology (LabMET), Ghent E-mail: [email protected]
University, Ghent, Belgium Michiel Kleerebezem
3)
Microbiology Group, Rowett Institute of Nutrition and Health, University of E-mail: [email protected]
Aberdeen, Aberdeen, UK
4)
Host-Microbe Interactomics Group, Wageningen University, Wageningen, Abbreviations:
The Netherlands IBD, inflammatory bowel disease; OTU, operational taxonomic unit; SCFA,
short chain fatty acid; SRB, sulfate reducing bacteria.

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 S. El Aidy et al. Prospects & Overviews ....
Malawian (n ¼ 115), and Amerindian (n ¼ 100) subjects, um clusters IV and XIVa, since these bacterial groups appear
elucidated that the fecal microbiome is unique to certain age to be vital in achieving homeostasis in the adult gut, as
groups and geographical locations, likely reflecting differ- illustrated from their close involvement in many of the
Review essays

ences in diet and lifestyle [8]. This study also showed that processes presented below.
variation in the adult fecal microbiome is driven by a trade-off
between two bacterial groups; Prevotella and Bacteroides
species, rather than being governed by distinct fecal enter-
otypes as identified by Arumugam et al., [7] i.e. the Dynamic establishment of the large
Bacteroides, Prevotella, and Ruminococcus enterotype. intestinal microbial ecosystem
Microbial colonization induces changes in gut epithelial
homeostasis, such as increases in cell proliferation and in The mammalian host-microbial interplay stimulates the
the relative number of secretory cells [1, 9–11], maturation of maturation of an effective intestinal barrier that promotes
the gut epithelium including major shifts in epithelial surface colonization by commensal bacteria and opposes colonization
expressed glycans [1, 10, 12], and an increased number of by pathogens. Additionally, the nutritional value of the diet is
gut-associated immune cells [1, 3, 13]. Among the reported enhanced by microbiota-facilitated digestion, while the diet in
beneficial contributions of the intestinal microbes to human turn also shapes the gut microbial community [24]. During
health, their metabolic function is crucial for host health [14]. the initial stage of colonization, the gut microbiota is highly
It has even been considered as the prominent evolutionary unstable and the temporal pattern in which it evolves is
force for the incorporation of microbes in the gut and for characterized by a remarkable interindividual variation.
acquiring host defense mechanisms [15]. Consequently, the This has been reported both in humans [25] and in mouse
microbiota was proposed to have a “collective metabolic models [26]. Despite the highly unstable early stage of
activity equal to a virtual organ within an organ” inside the microbial colonization, the establishment of the gut ecosys-
human body [16]. The main substrates that are metabolized tem seems to follow a conventional program. In humans, an
by colonic microbes include a variety of polysaccharides, adult-like microbiota is generally established three years after
such as plant cell storage glycans (starch and fructans), plant birth [8]. Yatsunenko et al. [8] observed a lower microbial
cell wall glycans (cellulose, hemicelluloses, and pectin), diversity in US residents when compared to other human
and host-derived salivary and colonic mucin O- and N-linked subjects living in ancient geographic locations. This lower
glycans and glycosaminoglycans. The involved microbes are microbial diversity is caused by several typical Western
phylogenetically distantly related but closely interact with society practices. For instance, cesarean delivery, which
one another to ferment these substrates [17, 18]. Versatile represents almost one third of the births in the US [27],
microbes, such as Bacteroides thetaiotaomicron, are com- deprives the newborn from being exposed to a broad spectrum
monly regarded as primary degraders. The genome of of vaginal microbes, resulting in lower intestinal microbial
B. thetaiotaomicron is highly enriched with genes encoding diversity in the newborn [28]. Replacing breast feeding by
cell envelope-associated multiprotein systems, called starch formula milk is another major contributor in the establish-
utilization systems (Sus) [19]. Recent studies, however, ment of the gut ecosystem. Breast milk is essential for the
indicate that Bacteroides spp. may mainly be involved in early microbial development by providing nutrients for early-
soluble carbohydrate breakdown, while primary degradation life symbionts (e.g. Bifidobacteria) [29] and by providing
of insoluble substrates may mainly be carried out by members live bacteria [30]. Misuse of antibiotic treatments, in particular
of the Firmicutes phylum [20]. Further, the degradation of in the first years of a newborn’s life, which are considered
specific substrates may also depend on specific primary critical in the subsequent development of a balanced gut
degraders, termed keystone species, as demonstrated for microbiota [31], further contribute to the decreased microbial
Ruminococcus bromii in the case of resistant starch degrada- diversity in the human gut microbiome.
tion [21–23]. Finally, complex microbe-microbe interactions Over time, the fitness advantage of microbial groups that
are required to fully metabolize a given substrate, depending typically dominate the adult intestinal microbiota overcomes
on the type of this substrate. the initial advantage of early-colonizing microbes that are less
In view of the significant role of the gut microbiota in adapted to the intestinal environment (Fig. 1). This is due to
establishing and maintaining a homeostatic relationship with intrinsic developmental changes in the intestinal mucosa, as
the host, one could imagine that the microbial colonization of well as changes in the gut microbiota itself and the gut
the initial sterile intestine evolves under tightly regulated environment [32–34]. These factors allow only microbial
molecular responses and strong selective pressure, acting on populations that are capable of establishing a mutualistic
both the intestinal niche and its microbial inhabitants. Here, relation with the host to be maintained in the homeostatic gut
we survey the succession of microbial colonization of the ecosystem [9], creating a habitat that exerts restrictive
intestinal tract and the corresponding changes in the host- selection on its microbial inhabitants, a phenomenon
microbial metabolic interactions. Following an overview of commonly referred to as colonization resistance [34].
how the large intestinal microbiota is established, this review The progression toward a stable adult-like microbiota
will focus on two processes that play a crucial role during the composition in a germfree animal model involves replacement
dynamic microbial colonization, i.e. modulation of mucosal of facultative, aerotolerant Bacilli, Bacteroidetes, and Clos-
binding sites and microbial metabolic interactions. Further, tridium clusters XVI, XVII, and XVIII, which dominate among
we will focus on host immune responses and elaborate on the low diversity, initial colonizers of the host sterile intestine
novel findings regarding microbes belonging to the Clostridi- (Fig. 1) [26]. The abundance of oxygen during the early days of

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Figure 1. Dynamic establishment of the large

MICROBIAL COLONIZATION
intestinal microbial ecosystem. A schematic
model illustrating the succession of the micro-
biota and its metabolites in the colon, and the

Review essays
corresponding alterations in the host gene and
Bacteroidetes
MICROBIOTA

metabolic profiles. Approximate trends of

changes in abundance of the different microbial
taxonomical levels to the overall microbiota in
Lactobacillales the ex-germfree mouse colon, during the pro-
Mollicutes cess of colonization is represented by different
SRB geometrical shapes (adapted from [26]). Similar
Proteobacteria color codes represent correlations computed
LUMEN

between the microbiota and the host mucosal

and urine genes and metabolites as recently
Clostridium clusters IV, XIVa reported by El Aidy et al. [26]. SRB, sulfate
reducing bacteria; Gpr43, G protein coupled
receptor 43; Hmgcs2, hydroxyl-3-methylglutaryl-
Clostridium clusters XVI,XVII,XVIII CoA synthase; Fut2, fucosyltransferase 2.
METABOLITES

Lactate, Succinate

Acetate

Propionate, Butyrate
Gpr43
MUCOSA

X
METABOLITES GENES

Hmgcs2
Fut2, Glycan Biosynthesis

Inositol
Glutamine,Glycine, Alanine
METABOLITES
URINE

Fumarate,Oxaloglutarate,Malate

Methylamines
Tryptophan

gut colonization is a stimulus for the primary colonization by succinate and lactate, from early colonisers provides sub-
aerotolerant bacteria and prevents immediate expansion of strates for some strictly anaerobic and Gram-positive succes-
obligate anaerobes [34]. El Aidy et al. [26] reported that the sive colonizers [26]. A potential driving force for these adult-
accumulation of fermentation metabolites such as lactate like community members to establish themselves stably and
and succinate were characteristic to the primary stage of irreversibly once they colonize a host may include cooperation
colonization. Analogously, the correlation analysis shown in in microbial food networks, where fermentation end products
that study depicted a link between some members of from one organism can act as a substrate for another, a
the primary colonizers belonging to Bacteroidetes and the phenomenon known as “cross-feeding” [35, 36].
accumulation of urine metabolites that are involved in the
TCA cycle (Fig. 1). These results suggest a state of microbial
dysbiosis during the primary stage of colonization. Tran- Establishment of mucosal binding sites aids
scriptome and metabonome studies demonstrated that persistent accommodation of the microbiota
colonization of the pioneer microbes and their metabolites
were not reflected in the mucosal metabolic pattern or other The host-microbe fundamental relationship relies on chemical
molecular responses of the host. However, this primary signaling and nutrient availability [37]. Mammals are born
colonization was paralleled by changes in the mucosal with an intestinal mucosa that is relatively high in sialic
adhesion sites, in particular, increased transcription of the acid-containing cell-surface glycans and relatively low in
fucosyltransferase 2 (Fut2) gene. This reflects expression of fucosylated glycans [38]. Fucosylation is the addition of
fucosylated epithelial surface glycans (Fig. 1) [26], which oligosaccharides (containing fucose sugars) to glycoproteins
facilitate the persistence of specific microbes in the intestine or glycolipids via the activity of fucosyltransferase enzymes
[9]. Likewise, the acculumation of metabolites, such as [39]. Primary microbial colonization induces Fut2 expression

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 S. El Aidy et al. Prospects & Overviews ....
on the colonic epithelium [26, 39, 40]. Glycolipids allow the pioneer colonic colonizer belonging to the Bacteroidetes,
establishment of microbial groups that use terminal fucose as Prevotella [26], also has glycosulfatase activity, which causes
an energy source [41] and are implicated in the activation of desulfation of sulfated-mucus glycoprotein [59, 60]. Increased
Review essays

the intestinal immune system [42]. B. thetaiotaomicron, for sulfatase activity has been detected in patients with
example, was shown to produce multiple fucosidases that inflammatory bowel disease (IBD) [61, 62].
cleave fucose from host glycans, resulting in high fucose Desulfovibrio piger, one of the most abundant SRB found
availability in the gut lumen, which is partially used by B. in the human colon and a potentially important contributor to
thetaiotaomicron itself for the production of propionate [43, sulfide formation in the gut [63], was shown to compete very
44]. Microbial profiling during the process of microbial effectively with other lactate utilizers [48]. This situation
colonization in germfree animals showed that the abrupt is similar to that generally found in the healthy gut where
increase in the ratio of fucosylated to sialylated glycans during lactate remains at low concentrations as a result of efficient
the initial stage of colonization correlates with the establish- utilization. D. piger oxidizes lactate to acetate and carbon
ment of Bacteroidetes members [26, 45]. dioxide. The end product of sulfate reduction during
The induction of fucosylated epithelial surface glycans anaerobic respiration by SRB is hydrogen sulfide (H2S) [64,
helps the succession of microbial colonization through 65]. Low sulfide concentrations were reported to have positive
binding of fucose-adherent bacteria (such as B. thetaiotao- effects on the mitochondria of mammalian intestinal cells [66]
micron and B. fragilis) that may act as the pioneer species for and to affect intestinal motility [67]. Although it plays a role in
succession of gut microbiota. This process results in a the healthy gut, H2S is highly toxic to the colonic mucosa and
balanced intestinal ecosystem [39]. Such binding allows the it has been implicated in conditions such as ankylosing
microbiota to prevent the potential invasion of the gut mucosa spondylitis and chronic intestinal disorders as evoked by
by pathogens, a phenomenon known as “the barrier effect” or data indicating increased numbers of intestinal SRB and
“competitive exclusion” [46, 47]. rates of sulfidogenesis in IBD patients compared to healthy
humans [68–72]. Moreover, H2S may create cellular energy
deficiency. One aspect of H2S that remains underdeveloped
is that it stimulates degradation of disulfide bonds in the
Metabolic cross-feeding leads to mucus layer, which may facilitate microbial penetration into
maturation of key gut microbial players the mucus layer and degradation of the barrier. Moreover,
H2S compromises the epithelial cell barrier by inhibiting
Deciphering the dynamic process of establishing host-microbe the b-oxidation of butyrate. In humans, diminished butyrate
homeostasis in germfree animals suggested that development metabolism contributes to the development of colorectal
of the stable adult-like microbiota is dependent on primary disease [73–75]. Similarly, in germfree animals, the initial
intestinal colonizers and their metabolites [1, 26]. Lactate, for bloom of SRB, which has not yet been balanced by the
instance, can be utilized by several members of the gut colonization of an intact stable microbial community, was
microbial community including sulfate reducing bacteria associated with a transient impairment in b-oxidation. This
(SRB) [48, 49], as well as bacteria that convert lactate to was evidenced by transcriptome studies that illustrated the
butyrate or propionate [35, 50–54]. Given its low pKa value (¼ repression of the mitochondrial hydroxyl-3-methylglutaryl-
3.86), lactate is easily deprotonated at physiological pH in the CoA synthase (Hmgcs2) (Fig. 1) [26]. This enzyme mediates the
human body. Therefore lactate utilization is crucial to avoid first and rate-limiting step of ketone body and acetyl-CoA
metabolic acidosis. Effective lactate utilization by diverse synthesis via the b-oxidation pathway.
bacterial groups explains why lactate concentrations typically
remain low in healthy subjects. In contrast, lactate accumu-
lation is associated with its restrained utilization due to Maturation of the cross-feeding gut microbiome
dysbiosis, as observed during the initial stage of microbial
colonization in germfree animals [26] as well as in fecal The above-mentioned impairment of the b-oxidation pathway
samples of patients with severe ulcerative colitis [55]. is restored with the full maturation of the gut microbial
ecosystem and the accumulation of the bacterial fermentation
product, n-butyrate, the primary fuel for colonocytes in
Initial bloom of sulfate reducing bacteria b-oxidation [76]. Microbial profiling of the ex-germfree mouse
large intestine during the establishment of microbiota-
Studies on germfree animals suggested that the simultaneous accommodating homeostasis indicated that the stable, highly
establishment of lactate-producing (e.g. Bifidobacterium diverse, late colonizers, were dominated by the Gram-
adolescentis) and sulfate-liberating (e.g. Bacteroides spp.) positive Firmicutes, in particular, Clostridium clusters IV
bacteria during the initial stage of microbial colonization, as (also known as Clostridium leptum group, often belonging to
well as the availability of endogenous sulfate sources secreted the Ruminococcaceae family) and XIVa (or C. coccoides/
by the host goblet cells, promotes a bloom of SRB in the Eubacterium rectale group, often members of the Lachnospir-
colonic milieu [1, 56]. One of the pioneer colonic colonizers, aceae family), many of which are butyrate-producing
Bacteriodes fragilis, for example, breaks down sulfomucin bacteria. In addition to butyrate kinase, which is responsible
generated from the mucin-producing goblet cells. This for butyrate production in a minority of butyrate producers,
releases short chain fatty acids (SCFAs) and sulfate into the butyryl-CoA:acetate CoA-transferase performs the final step in
lumen, thus permitting growth of SRB [57, 58]. Another butyrate synthesis in the majority of human gut bacteria [77].

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Similarly in mice, amplification of the butyryl-CoA:acetate which may underlie microbiota composition changes [83, 84].
CoA-transferase gene in colonic samples of conventionalized Moreover, SCFAs were shown to regulate AMP production
mice underpinned the abundance of butyrate producing in the colon [85] and to attenuate inflammation in several

Review essays
bacteria [26]. Clostridial cluster IV and XIVa E. rectale, mouse models [86]. In particular, butyrate has been the focus
Roseburia intestinalis, and Faecalibacterium prausnitzii of many studies aiming to unravel its suggested role in cancer
were also detected by phylogenetic chip analysis, all of prevention [87, 88] and overall impact on human physiology.
which are considered abundant butyrate-producing groups Butyrate plays a vital role in epithelial cell proliferation
in the human large intestine [17]. and differentiation, and is known to inhibit the growth
In conclusion, lactate and succinate utilizers appear to be of colorectal cancer cells [89]. SCFAs signal via the G-protein
important for depleting high energy metabolites that initially coupled receptors 41 and 43 (Gpr43). Gpr43 plays an
accumulated during the early unstable stage of microbial important role in immune modulation [86] and has a pivotal
colonization [26]. The efficient depletion of these metabolites role in the regulation of energy balance [90]. Recent findings
was followed by the production of propionate and butyrate, illustrated that the production of SCFAs during the late
which are likely produced via two routes: (1) cross-feeding stage of microbial colonization of the germfree mouse colon
of early metabolites; (2) from carbohydrate breakdown by activates Gpr43, which may be linked to global changes in the
propionate and butyrate producers. host metabolic profile including carbohydrate (glycolysis),
amino acid, nucleotide, and choline metabolism in the
colonic mucosa (Fig. 1) [26].
Besides the saccharolytic microbial activity that promotes
Gut colonization by microbial species SCFA production, the proteolytic activity of other microbial
and metabolites drives the regulation of groups leads to accumulation of methylamine metabolites.
host immune and metabolic responses Elevated levels of methylamine metabolites characterize the
metabolic profiles of colon mucosa and urine of convention-
Recently, it has been shown that the succession of the Gram- alized animals (Fig. 1) [26]. Methylamine production by gut
positive bacteria, in particular, Clostridial clusters IV and microbiota has recently been reported to promote atheroscle-
XIVa, appears to coincide with temporal changes reported in rosis and cardiovascular diseases, providing an example of
the local and systemic host transcriptional and metabolic the detrimental effects the gut microbiota can cause to its
profiles during the colonization period [1, 26]. Transcriptome host [91].
studies indicated that during this process of microbial
colonization, inflammatory tissue responses were counter-
acted by the induction of the regulatory arm of T cells,
including (among others) forkhead box P3 (Foxp3) and
Novel insights into the ecology of
interleukin10 (Il10) markers for tolerance-promoting regula- Clostridium cluster IV and XIVa species,
tory T cells (Tregs) that were induced at the late stage of key players in the establishment of a
colonization in the ex-germfree mouse colon [1]. Intriguingly, homeostatic host-microbe relationship
the expression of these tolerance markers paralleled the
colonization of the gut by Clostridium cluster IV and XIVa Clostridium cluster IV/XIVa species are
species, which were previously reported to stimulate the prominent members of the core microbiome
expansion the of Tregs repertoire in the mouse colon [78].
Atarashi et al. showed that the induction of colonic but not The importance of the butyrate-producing gut inhabitants for
small intestinal Tregs is dependent on the gut microbiota, in human health also follows from their prevalence in the
particular Clostridial species that belong to clusters IV human fecal microbiota (Table 1). Flint et al. [18] demon-
and XIVa, which are underrepresented in the microbial strated that 8 of the 25 most abundant bacterial species are
community from IBD patients [79]. Some of these species, for potent butyrate producers. This was in agreement with a
example E. rectale and R. intestinalis, possess flagella [17] and study by Tap et al. [92] in which it was shown that 20 of the
may be capable of reaching the gut barrier because they can 49 operational taxonomic units (OTUs) that constituted a
strongly colonize the mucus which overlies the colonic putative core microbiota were butyrate producers belonging
epithelium [80]. Therefore, a possible mechanism by which to the Clostridium clusters IV or XIVa. Besides these 16S
these bacteria can induce colonic Treg cell maturation is rRNA-based studies, Qin et al. [6] demonstrated that also
via the flagellin-induced toll-like receptor 5 (TLR5)-mediated many microbial genes belonging to butyrate producers are
signaling [81]. shared among human subjects and are thus vital for the
However, there is a large gap in our knowledge about the “human core microbiome”. Indeed, from the 49 most
exact microbiota-derived metabolites that can induce host abundant microbial genomes, 14 belong to butyrate-produc-
immune responses. SCFAs, the end products of the fermenta- ing Clostridium cluster IV and XIVa members. The impor-
tion of non-digestible carbohydrates and the main source of tance of the butyrate producers that were implicated in the
energy to the colonocytes, represent a potential target. SCFAs above-mentioned studies was confirmed by Louis et al. [93]
provide an excellent example of how host diet and nutrient in which the butyryl-CoA:acetate-CoA transferase gene
fermentation by the microbiota can shape immune responses sequences in 10 human individuals were targeted. These
in the mucosa [24]. SCFAs function as a fuel for the large results indicate an important interaction between humans
intestinal epithelium [82], and cause lowering of the local pH, and butyrate-producing gut microbial species.

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Table 1. Recent studies using various detection methods to determine the putative human core microbiome revealed that
butyrate-producing species are omnipresent in the human fecal gut microbiome
Review essays

Reference [18]a [92] [6] [93]

Type of analysis 16S rRNA clone 16S rRNA clone Metagenomic Clone library of butyryl-CoA:
library library sequencing acetate CoA-transferase gene
(Illumina)
Number of human subjects 6 (obese) 17 (healthy: omnivorous, 124 (healthy, obese, 10 (healthy)
vegetarian) IBD)
Outcome Core with 25 most Core with 49 OTUs Core with 57 most 32 most abundant BCoA:A CoAT
dominant bacterial abundant bacterial OTUsb
species genomes
Butyrate producing OTUs/ 8/25 OTUs 20/49 OTUs 14/57 OTUs 32/32 OTUs
total OTUs
Butyrate-producing OTU OTU rank of the species/strain in order of decreasing abundance
Faecalibacterium prausnitzii 1 1; 8; 12; 20; 21; 31 13 5
Eubacterium rectale 2 19; 23; 24; 45 26 1
Anaerostipes hadrusc 6 – 7 2
Eubacterium hallii 8 15; 42 9 4
Roseburia faecis 11 – – 3
Subdoligranulum variabile 13 7; 10; 43 34 –
Coprococcus eutactusd 19 41 40 –
Roseburia inulinivorans 24 – – 8
Anaeristipes caccae – 2 – 9
Roseburia intestinalis – 13; 26 18 6
Eubacterium ramulus – 30 – –
Unknown species SS3/4 – – 11 –
Coprococcus comes – – 28 –
Eubacterium ventriosum – – 31 –
Clostridium sp. M62/1 – – 41 9
Butyrivibrio crossotus – – 43 –
Clostridium sp. L2-50 – – 46 –
Anaerotruncus colihominis – – 49 –
Roseburia hominis – – – 7

a
based on Walker et al. [120]; bThe 32 reported OTUs, reported by Louis et al. [93] were combined per bacterial species. Further, we omitted the unknown OTU
sequences reported by Louis et al., which may belong to (1) unidentified species but (2) also to known species of which the butyryl-CoA: acetate-CoA transferase
gene is yet to be sequenced; cincludes SS2/1 and SSC/2; dincludes A2-166.

Specific Clostridium cluster IV/XIVa species drive layer (with decreasing oxygen levels from blood vessels
mucosal butyrate production toward the intestinal lumen) [104], may further select for
specific butyrate-producing species as demonstrated by
Much of the current knowledge concerning the human Khan et al. [105], who showed that F. prausnitzii employs
intestinal microbiota is restricted to fecal samples, which an extracellular electron shuttle of flavins and thiols to
may not be an appropriate reflection of the interacting transfer electrons to oxygen. This exclusive ability might
microbiota at the mucosal interface. Indeed, specific microbes enable F. prausnitzii to more effectively colonize the mucus
reside in the mucus layer which covers the intestinal layer (which is characterized by an oxygen gradient) over
epithelium, and which consists of a dense, inner and a less other bacteria.
dense, outer layer [94]. Given their proximity to the intestinal While butyrate-producing bacteria are enriched in mucus,
epithelium, mucosa-associated microbes have a great poten- Van den Abbeele et al. [80] showed that this statement is not
tial to closely interact with the host, as compared to their valid for all butyrate producers. Most of the specific mucus
luminal counterparts [95]. Recent in vivo studies revealed an colonizers included R. intestinalis and E. rectale, while
overall enrichment of Firmicutes (especially Clostridium Anaerostipes caccae predominantly colonized the lumen
cluster XIVa) over Bacteroidetes members in biopsies (Table 1). Notably, contrary to the non-motile A. caccae,
compared to luminal or fecal samples, both in rodents [96], R. intestinalis, and E. rectale possess flagella that may allow
and humans [79, 97–100], which appeared to be reflected them to actively penetrate into the mucus layer [17]. Besides
in a sophisticated in vitro model [80]. These microbes allowing a more intimate interaction with the host immune
adhere specifically to mucins [101] or insoluble substrates system as discussed above, colonization of the mucus layer
in general [102]. Mucins may also serve as a growth substrate may lead to exposure of the mucosa to higher butyrate levels
for butyrate-producing Clostridium cluster XIVa species, (Fig. 2), even if luminal butyrate levels remain unaffected.
possibly via cross-feeding with mucin-degrading microbes Such preferential higher mucosal exposure to elevated
that deliver partial breakdown products, succinate, lactate, butyrate concentrations would increase the bio-availability
and/or acetate [103]. Other factors that govern the mucosal of this beneficial microbial metabolite for the host. Stimulat-
environment, such as the oxygen gradient along the mucus ing mucosal butyrate producers for the delivery of butyrate at

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(young adults vs. elderly) is often accompanied by reduced

abundance of Clostridium cluster XIVa species [106, 111].
Remarkably, there is a need for such studies to better

Review essays
characterize the living conditions of the elderly in relation
to their health status (living independently vs. nursing
homes). Interestingly, the microbiota of centenarians [112]
contains fewer butyrate producers that are typically among
the mucosa-associated butyrate producers (R. intestinalis,
E. rectale, F. prausnitzii, Papillibacter cinnamovorans, and
E. ventriosum) [80] (Table 2). In contrast, butyrate producers
that increase during ageing are not enriched in mucus
(E. limosum) or even found to specifically colonize the lumen
(Anaerotruncus colihominis) [80]. The decrease of (mucosal)
butyrate producers during ageing may thus be related to an
earlier proposed hypothesis that mucus production may
itself decrease during ageing [113], negatively affecting the
habitat and fitness of mucosal butyrate producers. Likewise,
IBD and cystic fibrosis are also confronted by disturbed mucus
layers, further supporting the hypothesis that a damaged
mucus layer disturbs the habitat of intestinal butyrate
producers. Similarly, loss of the integrity of the mucus
layer reduces the normal protection afforded by the
barrier [114].

Other environmental factors that determine

butyrate production
Figure 2. Hypothesis regarding mucosal butyrate producers. A: In
case that there are only butyrate producers in the intestinal lumen, It has been demonstrated that recent changes in human
butyrate will diffuse through the mucus layer before reaching the ecology drastically altered the microbes that populate our
host epithelium where it is consumed, resulting in a concentration
bodies. These changes include, among others, the typical
gradient. B: Butyrate-producing microbes that are capable of
colonizing the mucus layer may elevate the mucosal butyrate levels. Western diet and increased use of antibiotics, two factors
Diffusion still takes place but these specific mucosal butyrate which negatively affect butyrate-producing gut microbes.
producers increase the bioavailable fraction for the host. First, De Filippo et al. [115] elegantly investigated the impact of
our Western diet versus a fiber-rich diet (from Burkina Faso)
by comparing the fecal microbiota of European children with
that of children from a rural area of Burkina Faso. Next to a
the mucosal surface may thus be an interesting target for decrease in acetate levels (50% lower in European children),
future interventions that aim to restore or sustain intestinal the Western diet importantly corresponded to an even more
homeostasis. drastic, disproportional decrease in propionate and especially
butyrate levels (80% lower in European children). The
importance of dietary carbohydrate for colonic butyrate
Clostridial cluster IV and XIVa populations are production has also been demonstrated in a fully controlled
altered in ageing and disease human dietary trial in which a fourfold decrease in fecal
butyrate levels was observed in response to a high protein/low
Changes in the composition of the microbial community, carbohydrate diet [116]. This decrease correlated with a
including decreased populations of Clostridial clusters IV and decrease in butyrate-producing bacteria related to the
XIVa, have been observed in the elderly [106], as well as in Clostridium cluster XIVa group. A range of carbohydrates,
patients with IBD [79] and cystic fibrosis [107]. This altered including starch, xylan, and inulin, can be degraded by
microbial composition is likely to have a negative impact on different butyrate producers [17], and the lower gut pH
the supply of colonocytes with their major source of energy – expected to result from carbohydrate fermentation is also
butyrate. likely to provide a competitive advantage to many butyrate
A potential explanation of the observed low abundance in producers over other dominant bacterial groups such as the
IBD is the altered immune response and the presence of Bacteroidetes [117].
antibodies that target flagellar antigens of bacteria related to In addition to the Western diet, antibiotics can also
E. rectale and Roseburia spp. [108]. In addition to targeting the drastically decrease the butyrate production. Willing
flagellar bacteria, reduced numbers of F. prausnitzii, which et al. [118] provided a comprehensive overview of the potential
produces an anti-inflammatory factor [109], have been impact of antibiotics on the gut microbiota, showing that
reported in fecal samples and on the gut mucosa of patients butyrate production is strongly decreased during antibiotic
with Crohn’s disease [110]. It has been observed that ageing treatments and may even be responsible for the inflammatory

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 S. El Aidy et al. Prospects & Overviews ....
Table 2. The microbiota of centenarians, as studied by Biagi et al. [112], contains less butyrate producers that are typically
among the mucosa-associated microbiota, as determined by Van den Abbeele et al. [80]
Review essays

Reference [112] [80]

Butyrate producers Preference of butyrate producers for
Study outcome during ageing intestinal lumen versus mucus (in vitro)
Young
Butyrate-producing group adults Centenarians Mucus Lumen
Eubacterium limosum 0.02 0.35
0.07 0.10
Anaerotruncus colihominis 0.66 0.99
0.86 1.94

Faecalibacterium prausnitzii 4.24
2.01 2.20
0.56
Papillibacter cinnamovorans 1.80
1.30 4.31
0.27
Eubacterium hallii 5.76
3.16 0.57
0.24
Eubacterium rectale 3.02
1.68 8.55
0.22
Eubacterium ventriosum 2.62
1.21 1.68
0.11
Roseburia intestinalis 3.21
1.57 6.74
0.08

This decrease of (mucosal) butyrate producers during ageing may reflect that mucus production itself decreases during ageing, negatively
affecting the habitat of mucosal butyrate producers. Both studies employed the HITChip for detection of microbial groups, expressed as
proportion of the entire microbiota (%), allowing a straightforward comparison. A significant increase is indicated by
(determined by the
Wilcoxon test to look for overall differences and then the Mann-Whitney test to check for differences between specific groups).

states that can follow antibiotic treatment. A final factor that the application of holistic tissue-based biological systems
may affect butyrate production is iron (Fe) deficiency. Dostal rather than specific intestinal cell lineages.
et al. [119] recently demonstrated that such deficiencies
may negatively affect butyrate production by the intestinal
microbiota. Acknowledgments
S. El Aidy would like to acknowledge the support of the
European Union through the Interplay project (Grant
Conclusions agreement no. 227549), as well as Cargill’s R&D Center
Europe (Vilvoorde, Belgium). Cargill had no role in decision to
The intestinal microbiota plays a crucial role in metabolic, publish, or preparation of the manuscript. P. Van den Abbeele
nutritional, physiological, and immunological processes in is a Postdoctoral Fellow from FWO-Vlaanderen (Research
the human body. Because the human body contains around Foundation of Flanders, Belgium). His work is partially
3.3 million microbial genes [7], it seems important to study the supported by a GOA (BOF12/GOA/008) and an SBO project
function of these genes as well as the higher-level phenome- (100016) from the Agency for Innovation by Science and
non of microbial diversity and community effects. The Technology (IWT). P. Louis receives support from the Scottish
identification of novel gut microbial isolates, together with Government Rural and Environment Science and Analytical
the characterization of their metabolites and the mechanisms Services Division.
underlying their role in the modulation of the host immune
response and physiology, will help to design potential The authors declare no conflict of interest.
therapeutic targets to prevent and treat human intestinal
disorders including IBD. Deciphering the function of the gut
microbiota requires ecosystem metabolic modeling to unravel
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