Environmental Microbiology (2011) 13(12), 30883102

doi:10.1111/j.1462-2920.2011.02574.x

Minireview
Human distal gut microbiome

Julian R. Marchesi*
School of Biosciences, Museum Avenue, Cardiff
University, Cardiff CF10 3AX, UK.
Summary
The distal gut and its associated microbiota is a new
frontier in the quest to understand human biology and
evolution. The renaissance in this field has been
partly driven by advances in sequencing technology
and also by the application of a variety of omic technologies in a systems biology framework. In the initial
stages of understanding what constitutes the gut,
culture-independent methods, primarily inventories
of 16S rRNA genes, have provided a clear view of the
main taxonomic groups of Bacteria in the distal gut
and we are now moving towards defining the functions that reside in the distal gut microbiome. This
review will explore recent advances in the area of the
distal gut and the use of a variety of omic approaches
to determine what constitutes this fascinating collection of microbes.
Introduction
Gut or intestinal microbiology has undergone a minirenaissance in the past 10 years. In a comprehensive
review of the role of the gut microbiota in the health of the
host, Sekirov and colleagues (Sekirov et al., 2010)
charted the number of publications for the period from
1990 to 2009. Their data show that in this period there has
been a near fivefold increase in the yearly publication
rate. In fact the overall number they show is an underestimation, if the ISI Web of Knowledge database is queried
with similar keywords (Fig. 1), the trend is the same;
however, the number of publications, which now includes
2010, is nearly twice the figure and currently peaks at
1366 for 2010. Several reasons are responsible for this
increased attention, the recognition that the gut microbiota plays a central role in host health, as well as the
cross-pollination of ideas from microbiologists working in
Received 13 May 2011; accepted 20 July 2011. *For correspondence.
E-mail marchesijr@cardiff.ac.uk; Tel. (+44) 29208 74188; Fax
(+44) 29208 74305.

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the varied areas of environmental microbiology. As a discipline environmental microbiology has always been challenged by what has been referred to as the the great plate
count anomaly and which describes the disparity
between what we can grow in the laboratory, on conventional microbiology media and what we can directly count
(Staley and Konopka, 1985). This challenge has resulted
in a dramatic (and some may say it is a swing too far away
from culturing) shift away from culturing to developing
culture-independent approaches to investigate ecosystem function and the role that microbes play (Amann and
Kuhl, 1998). However, microbiologists working in the
human body, have been relatively fortunate because a
significant proportion of the microbial community in these
systems are culturable, a fact that delayed the introduction of culture-independent approaches to analyse this
ecosystem. The suite of methods that have been used are
variations on the genomic, transcriptomic, proteomic and
metabolomic methods. The most commonly used are the
metagenomic and 16S/18S rRNA gene-based methods to
determine the functions in the microbiome and the
species present. While metatranscriptomic {Gosalbes,
2011 #17381; Bomar, 2011 #17475} and metaproteomic
{Verberkmoes, 2009 #15794} {Rooijers, 2011 #17579;
Klaassens, 2007 #6600} methods are been implemented,
but to a much lesser extent. Using the gut as an example,
the two most commonly studied niches are the distal gut
and oral cavity, because logistically they are the easiest to
access. In both instances the proportions of the microbial
community that are as yet uncultivated are between 30%
and 50% (Wade, 2002; Eckburg et al., 2005; Duncan
et al., 2007), which provides researchers with a significant
culturable microbial biomass for investigation. When this
figure is compared with environmental ecosystems such
as the deep biosphere or soil where the culturable fraction
can be between < 0.1% and 1% respectively (Hugenholtz
et al., 1998; Fry et al., 2008) it becomes clear how
researchers in these areas needed to create a suite of
tools to help in developing a more complete picture of
microbial contributions to ecosystem function. The current
burst of interest in the gut ecosystem and how its
microbes influence host function/physiology has in some
way been driven by microbiologists adopting the tools of
environmental microbiologists and implementing them in

Human microbiome 3089

Fig. 1. The number of publications retrieved from the ISI Web of Knowledge database (http://apps.isiknowledge.com/), obtained by using the
following keywords and Boolean operators: intestinal microbiota OR gut microbiota OR intestinal flora OR gut flora OR intestinal
microflora OR gut microflora OR gut microbiome OR intestinal microbiome (the addition of the word gut microbiome and intestinal
microbiome, not used by Sekirov and colleagues, added 92 publications compared with the same search without).

a new setting. Without these methods we would not have

realized that the gut microbiota is so diverse between
different individuals, or resilient to perturbations, or that
key functions in some instances are redundant, while
other they are not. These initial forays into the gut ecosystem using culture-independent methods paved the
way for developing hypotheses in which the gut microbiota are drivers of health and disease. Hence in light of
the numerous reviews on the microbiota of the human gut
(524 for 20082010) this review will concentrate on the
most recent and significant findings in the literature.
Anatomically, the human gut is divided into six sections,
the oral cavity, oesophagus, stomach, small intestine
(subdivided into the duodenum, jejunum and ileum), the
colon or distal gut (subdivided into the ascending, transverse and descending colons) and rectum (Fig. 2). While
the physiological role of the gut is to process and digest
the food we ingest, it also offers a niche for colonization by
a variety of microbes. Each niche harbours a specific
microbial community, which to some extent reflects the
dynamics of that compartment. The numbers of microbes
in each niche increases as one moves from the stomach
to the rectum resulting in one of the most densely populated ecosystems being found in the distal gut or colon,
which contains between 10111012 bacteria per gram of
luminal material. Because the distal gut contains one of
the densest communities known (Whitman et al., 1998)
and is very easy to access [up to 55% of a stool sample is
bacterial biomass (Cummings and Macfarlane, 1997)] it

has received the majority of the attention. However, this

does not mean it is a robust representation of the whole
colon or small intestine; moreover, the mucosal surface
contains a microbiota that is significantly different to that
found in a stool sample from the same subject
(Momozawa et al., 2011). Data obtained from analysis of
faecal material must be considered in light of where this
sample comes from and conclusions based on this data
must be tempered appropriately; however, these data still
provide a very valuable insight into functions and species
present in the gut.
The current census of the inhabitants of the distal
gut: the early years of the distal gut
Unlike many environmental ecosystems being investigated, the establishment of the climax community in the
gut is played out time and time again with every birth;
moreover, it can very easily be perturbed and involves an
immunological dialogue with the system in which it
resides. Many of the major ecosystems that are studied,
marine, terrestrial, deep-biosphere and atmosphere have
been colonized for many millions to billions of years.
However, in the majority of cases humans are born sterile
[cases have been reported in which amniotic fluid sludge
containing cultured isolates of Mycoplasma hominis,
Streptococcus mutans and Aspergillus flavus has been
observed (Espinoza et al., 2005; Romero et al., 2008)]
and immediately upon exit from the mother start to be

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3090 J. R. Marchesi

Fig. 2. The anatomy of the gastrointestinal tract, major bacterial phyla and their abundance in each niche. The information for this figure was
compiled from (Eckburg et al., 2005; Bik et al., 2006; OHara and Shanahan, 2006; McConnell et al., 2008; van den Bogert et al., 2011).

colonized by microbes. There is a significant interest in

understanding what drives this colonization process and
how much nature or nurture plays an influencing role.
Specifically, because we have a poor understanding of
whether early life events, which may alter the gut microbiotas composition, can have ramifications for later life
health. The climax community seems to be established
within the first 2 years of life and after the first year, it has
started to converge and reflects a generalized adult distal
gut community (Palmer et al., 2007). The factors that influence this process are the maternal microbiota
(Dominguez-Bello et al., 2010), diet (breast fed vs.
formula fed; Favier et al., 2003), mode of delivery (normal
vs. caesarean; Biasucci et al., 2010; Dominguez-Bello

et al., 2010), full or preterm gestation (Schwiertz et al.,

2003; Morowitz et al., 2011), environmental exposure
(Palmer et al., 2007) and clinical interventions [antibiotics
(Palmer et al., 2007) or gastrointestinal surgery (Zhang
et al., 2009) technically this paper shows the impact of
surgery on the adult gut)]. This progression has also been
confirmed when using metagenomic DNA (mgDNA)
instead of the 16S rRNA gene. Koenig and co-workers
(Koenig et al., 2011) created inventories of the 16S rRNA
gene (from 60 infants) and used this information to select
12 infants for a sequence based metagenomic analysis
on the Roche 454 platform. The data they generated were
processed using MEGAN (Huson et al., 2007) and
MG-RAST (Meyer et al., 2008) to assess the taxonomic

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Human microbiome 3091

Fig. 3. Taxonomic distribution of
metagenomic sequences isolated from infant
faecal DNA [adapted from Koenig et al. (2011)
with data kindly provided by Prof Ruth Ley].

source and functions contained within the mgDNA respectively. Using mgDNA the same succession was seen as
with 16S rRNA gene data (Fig. 3) and other groups have
also confirmed that random sequence reads can be used
in lieu of taxonomically relevant genes such as the 16S
rRNA gene (Manichanh et al., 2008; Ghosh et al., 2010;
Gori et al., 2011). The consensus of opinion from these
studies seems to be that the trajectory of the colonization
process is towards a similar outcome, i.e. a distal gut
microbiota, which after the age of 2 is stable and colonized predominantly by Firmicutes and Bacteroidetes
(see below). However, we do lack the information of which
factors are driving this process, how the colonization
process in different ethnic groups proceeds and to what
extent the functions in the gut are established. Hence
there is a clear need to continue to determine the key
events that influence the establishment of the climax
community.
The adult and ageing distal gut microbiota
One of the most comprehensive early cultureindependent analyses (using clone libraries of 16S rRNA
genes and Sanger or first-generation sequencing platforms) was carried out on the distal gut by Eckburg and
colleagues (Eckburg et al., 2005). This study revealed
that while there were many bacteria in the gut they were
actually not as diverse as soil or marine ecosystems. In

the majority of mammals the two main phyla present are

the Bacteriodetes and Firmicutes (Ley et al., 2008) and it
seems that members of these two phyla contribute
approximately 90% of the species in the distal gut. The
number of species estimated to be present in the distal
gut is relatively small [compared with soil in which millions
of species are estimated to exist in 10 g (Gans et al.,
2005)] and is in the hundreds (Qin et al., 2010), while a
larger degree of diversity exists at the strain level, which
maybe in the thousands (Ley et al., 2006). The importance of the strain diversity may only be significant when
the functions that the strain carries are non-redundant.
For example, there are two hydrogenotrophic groups, the
methanogens and sulphate reducing bacteria, which are
represented by very few species and strains in the distal
gut {Scanlan, 2009 #16220} {Dridi, 2009 #17393}. In this
scenario, it would be easy to lose the functions these
organisms provide, whether health promoting or detrimental remains to be seen, to the host. A further consequence
of this strain diversity is that phylogenetic trees of the gut
tend to have few branches, which are not deep, but have
a large degree of radiance at the ends. However, this
census is based on a very small number of samples and
to put it into perspective a recent search for single nucleotide polymorphisms that correlate with adult height
screened 183 727 individuals to determine statistically
significant correlations (Lango Allen et al., 2010); in contrast, the majority of the studies that have been under-

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3092 J. R. Marchesi

Fig. 4. Proportions of main bacterial phyla in 386 Eldermet faecal samples, the two main phyla are shown in the figure while the remaining
phyla were the Proteobacteria, Actinobacteria, Lentisphaerae and Verrucomicrobia. The inset pie-chart shows the mean values for the phyla
(F Firmicutes and B Bacteroidetes) isolated from the distal guts of the elderly individuals, the category others includes the following phyla
Proteobacteria, Actinobacteria, Lentisphaerae and Verrucomicrobia (data to construct this figure were kindly supplied by Dr Paul OToole,
University College Cork, Ireland and Eldermet principle investigator).

taken to determine the composition of the gut microbiota

use small cohorts that can be counted in the tens rather
than thousands. One recent study that has sampled hundreds of individuals has shown why we need large cohorts
of subjects. While we can take averages of the numbers
of species present in the distal gut and conclude that two
phyla predominate, if the sample size is increased the
proportions of these phyla can tell a very different story. In
the Eldermet project (http://eldermet.ucc.ie/) being undertaken in University College Cork, Ireland, the investigators
profiled the distal gut of 386 > 65 year old individuals
using second-generation 454 pyrosequencing and
obtained approximately 40 000 reads per sample and
which spanned the V4 region of the 16S rRNA gene. Once
again the composite picture of the distal gut was one in
which gene sequences from the Bacteriodetes and Firmicutes contributed 97% of the overall sequences obtained
(57% and 40% respectively; Claesson et al., 2011).
However, when the individual profiles were plotted and
ordered an entirely different picture was obtained (Fig. 4).
The distribution of 16S rRNA genes showed that within
the cohort there was a continuum, at one end
Bacteroidetes made up nearly 90% of the distal gut microbiota while at the other Firmicutes made up more than
95% of the sequences recovered. This larger study further
highlights the necessity to increase the cohort size and
move away from small studies of 23 subjects. The fact
that the distal gut microbiota is so variable at the phylum
level does make one wonder whether it is this variable
further down the taxonomic levels, for example, at the

genus level? To answer this question, several groups

have been exploring the concept of the core microbiota
and whether there exist a group of species found in all
distal guts regardless of geography, ethnicity, age, gender
or diet. While it would be safe to say that there is a core
microbiota at the phylum level, i.e. all humans posses
members of the Bacteroidetes and Firmicutes, when we
drill down the taxonomic levels it seems that this concept
becomes more sketchy and different studies and methods
provide different answers. Tap and colleagues undertook
a de novo analysis of the composition of the distal gut
microbiota using first-generation sequencing and PCR
amplification and cloning of the 16S rRNA gene (Tap
et al., 2009). They generated 10 456 16S rRNA gene
sequences from 17 human faecal DNA samples and
analysed them to determine which sequences were
shared and which were unique. In their conclusions, they
state that on average each individual contains 259 operational taxonomic units (OTUs at the 98% level), but the
range was large (159383) and in total 3180 OTUs were
identified from the total pool of 16S rRNA gene
sequences. Approximately, 79% of the OTUs were only
found in one sample and 21% were found at least twice;
however, no OTUs was found in all 17 distal guts. They
showed that 66 OTUs were found in 50% of the samples
and proposed that these may in some way constitute a
core microbiota. These OTUs belonged to 18 genera and
these were affiliated predominantly with the Firmicutes
(57/66). In a study with a similar goal, to determine the
members of the core microbiota of the distal gut, Rajilic-

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Human microbiome 3093

Stojanovic and co-workers used a DNA array approach to
determine the composition of the distal gut (RajilicStojanovic et al., 2009). While the aims were comparable
the methods are not, even if they both target the same
gene 16S rRNA. DNA arrays have the advantage of
being more sensitive and are able to detect sequences in
a mixture at much lower levels than a random sampling
without replacement strategy, e.g. analysis of a clone
library or second-generation amplicon sequencing (Harrington et al., 2008; Paliy et al., 2009; Rigsbee et al.,
2011). However, they are only as good as the database
used to design the array probes and any novel sequences
in the samples, which are not represented on the chip, will
not be detected. Bearing this in mind Rajilic-Stojanovic
and co-workers used their human intestinal tract chip
(HITChip) to profile the distal gut of five young and five
elderly volunteers. Their HITchip can measure the abundance of 1140 unique microbial phylotypes and they concluded that there was a common core between all 10
individuals, which consisted mainly of probes from three
phyla (Actinobacteria, Bacteroidetes and Firmicutes)
found in the distal gut and confirms that we possess a
distal gut microbiota that is host specific. In addition, they
were also able to show that the young and elderly guts
samples clustered according to the hosts age with all the
young and elderly samples found in their respective
clades. In the Eldermet study (Claesson et al., 2011),
there was also a significant difference between the elderly
and young distal gut. In the elderly distal gut more than
half of the core microbiota (53%) were from the
Bacteroidetes, from the genera Bacteroides (29%), Alistipes (17%) and Parabacteroides (7%), while in the
younger distal gut this figure dropped to between 8% and
27%. Furthermore, the core clostridial species were predominantly in the Clostridium cluster IV for the elderly
whereas cluster XIVa was more prevalent in the younger
cohort, again highlighting the need for longitudinal studies
rather than snapshots of the distal gut composition. In the
study conducted by Biagi and co-workers (Biagi et al.,
2010), which looked at young (Y), elderly (E) and centenarians (C) (groups of 20, 22 and 21 and average ages of
31, 72.7 and 100.5 respectively) using the HITChip platform and quantitative PCR, the trend for a variation in the
main groups was also seen (Fig. 5). However, the
changes in the subgroups within the clostridial group were
not the same as found in the Eldermet project, with an
increase in Clostridium cluster XIVa going from Y to E,
which decreased in the centenarians. The Clostridium
cluster IV remained the same between all three groups,
while between the C and E groups only the Faecalibacterium prausnitzii was significantly different, between C
and Y groups Bifidobacterium spp. differed and between
E and Y members of the genus Akkermansia differed. In a
recent development to describe the core microbiota of the

Fig. 5. Relative abundance of phylum/order phylotypes from

centenarians (C), elderly (E) and young (Y) (adapted from Biagi
et al., 2010).

distal gut Sekelja and colleagues undertook a post hoc

analysis of previously published datasets from pyrosequencing projects targeting the 16S rRNA genes (Sekelja
et al., 2011). They also changed the approach used, by
moving away from defining taxonomic groups and in their
words search for a human core microbiota independent
of both predefined phylogroup depths and phylogenetic
trees. Using an alignment-independent approach, they
analysed 16S rRNA gene sequences (from eight previous
studies and comprising 1 186 272 partial 16S rRNA
sequences from 210 samples) and clustered them using
principal component analysis based on their sequence
similarity [calculated by establishing 5 mer nucleotide frequencies in each sequence (Rudi et al., 2006; 2007)].
From their analysis they report that there were two microbiota cores, which were consistently found in all samples.
Both cores were affiliated to the Firmicutes and were
members of the clostridial family Lachnospiraceae.
Interestingly they concluded that each core appeared at
defined moments in evolution with core 2 co-evolving with
the radiation of vertebrates and core 1 co-evolved with the
mammals. These studies enforce the stochastic nature of
sampling the distal gut and the need for more large-scale
studies to minimize confounding factors such as diet,
environment and genetic/immunological variability of the
host.

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3094 J. R. Marchesi
A bacterial-centric view that needs to
encompass all the microbiota
To date we still have a limited understanding of what
constitutes the core microbiota of the distal gut and as
such cannot define its limits. We still need to expand the
numbers of distal guts sampled, the ethnic groups from
which we obtain the samples and also the compositional
stability of the core. While the previous studies have all
indicated that the concept of a core microbiota is not
dead, we have not reached any consensus as to what
species should be considered as members of this important group of bacteria, but we do agree that main phyla
are the Bacteroidetes and Firmicutes. Furthermore, the
concept of a core microbiota has not been fully inclusive
and maybe should be renamed the core bacteriota as it
has not considered the micro-eukaryotic and viral components. Both of these groups of organisms have been
studied in relation to the distal gut, but in a more limited
fashion. Only a few studies have been undertaken looking
at the human micro-eukaryotic diversity using cultureindependent approaches (Ott et al., 2008; Scanlan and
Marchesi, 2008) and viral diversity in faecal samples
(Breitbart et al., 2003; 2008; Zhang et al., 2005; Reyes
et al., 2010). The micro-eukaryotic diversity and numbers
is several orders of magnitude lower than the Bacteria
and is skewed towards Candida and Saccharomyces spp.
when cultured, but culture-independent approaches using
18S rRNA genes shows that Blastocystis spp. are very
common in the distal gut and yeasts are rarely obtained.
In fact, it may be concluded that micro-eukaryotes are
only really significant when there is a dysbiosis in the gut
(Goldman and Huffnagle, 2009). For the viral component
the story is very different with their numbers being at least
an order of magnitude higher than the bacterial numbers
in the distal gut. Thus we might need to start to consider
the viral component as drivers of community dynamics as
some marine microbiologists do (Suttle, 2007). In fact,
Lepage and colleagues (2008) have hypothesized a role
for distal gut bacteriophage as drivers of dysbiosis in the
distal gut and inflammatory bowel disease. While studies
looking to define the core microbiota have focused on
describing the Bacteria within the distal gut, there is also
a significant number of Archaea in this niche. The most
common species and 16S rRNA gene sequence isolated
from the distal gut come from the Euryarchaeota and in
particular the Methanobacteriaceae family (Scanlan et al.,
2008a; Dridi et al., 2009) with Methanobrevibacter smithii
and Methanosphaera stadtmanae the two predominant
Archaea found. However, other rarer archaeal sequences
have been reported that cluster in the Methanosarcinales
[a methyl coenzyme reductase subunit A (mcrA)
sequence (Scanlan et al., 2008a)], Halobacteriaceae
(Oxley et al., 2010) and a putative sixth archaeal order

(Mihajlovski et al., 2008; 2010). However, in all studies to

date M. smithii and M. stadtmanae are the two main
Archaea (Dridi et al., 2011) and one would question to
what extent the much rarer species are autochthonous
and are actually contaminants from our diet/environment.

The luminal microbiota versus the

mucosal microbiota
One of the major criticisms of many of the studies on the
distal gut is the reliance on stool or faecal material as the
source of microbial biomass or genomic DNA. While it is
quite simple to collect it is clear that faeces do not afford
a robust proxy for the gut microbiota as a whole. In
Eckburg and co-workers 2005 culture-independent
analysis of the distal gut (Eckburg et al., 2005) they
clearly showed that while the microbiota attached to the
mucosa was similar throughout an individuals large intestine it was significantly different to the stool sample from
the same individual, but whether there is any biological
significance in this difference remains to be shown, as the
number of luminal bacteria are between 46 orders of
magnitude less than the mucosally associated bacteria
[MAB; Zoetendal (Zoetendal et al., 2002; Ahmed et al.,
2007; Walker et al., 2011)]. Using a DNA microarray (AusHIT Chip) Aguirre de Carcer and colleagues (de Carcer
et al., 2011) have shown not only a gender difference
between the MAB, but also a qualitative change in the
MAB composition moving from the caecum to the rectum,
via the transverse and sigmoid colon. However, we still
need larger studies to determine what is considered to be
the prevalent species colonizing the different regions of
the colon and at what scale the community starts to
diverge.

Is there a core microbiome?

Qin and co-workers (Qin et al., 2010) and other
sequence-based metagenomic studies have addressed
the issue of whether there is a core microbiome (the
collection of microbial genes) and if so what does it look
like? To date the study of Qin and colleagues is by far the
deepest and largest metagenomic1 sequencing project to
be undertaken; however, two smaller metagenomic
studies do precede it (Kurokawa et al., 2007; Turnbaugh
et al., 2009). In the most recent study the authors used an
Illumina second-generation sequencing platform to generate 0.58 terabases of sequence from 124 volunteers
1

The term metagenomics is routinely confused with creating

inventories of 16S rRNA genes to describe bacterial diversity.
Metagenomics is the analysis of random genomic fragments
either by sequencing or functional analysis.

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Table 1. Comparison of the pros and cons of the two metagenomics methods used to study the functions in an ecosystem.

Screen large amounts of DNA

Provide novelty
Genomic context
Toxic genes
Expression issues
Storage issues

Computational issues

Function-based screen

Sequence-based screen

Yes with the aid of colony picking and

arraying robots
Yes
Yes

Yes with the use of second-generation

sequencing platforms
No
Limited and relies on assembly of reads and
assumptions on pan-genomic nature of gut bacteria.
Yes
No
No

No
Yes
Yes physical storage, one fosmid library
can easily take 650, 384 well plates
and 1950 if stored in triplicate
No

(approximately 4.5 gigabases per individual with an

average read-length of 75 bp) and determined that there
were 3.3 million non-redundant genes in the distal gut
metagenome. This figure is in agreement with the previous figure of 9 million genes (Yang et al., 2009) and that
between the different gut samples there were 204 056
common genes that comprised 38% of an individuals gut
microbiome. In this project these genes were grouped into
6313 clusters of orthologous groups and could be divided
into house-keeping genes and gut-specific genes. When
studying the gut it would be the genes only found in the
gut that are of key interest as these may play a role in
shaping the relationship between the host and its gut
microbiota. While the house-keeping genes were part of
the main metabolic pathways commonly associated with
bacteria, for example, amino acid synthesis, nucleic acid
processing and general secretory processes, the gutspecific genes were identified as being involved in adhesion to host proteins or catabolizing globoseries
glycolipids. However, the majority of the clusters of
orthologous groups (74.3%) were not defined and this fact
highlights a key problem with sequence-based metagenomic projects, they lack the ability to provide novel functions (Table 1). Many of the sequences, when compared
with the current databases will either return hits to annotated functions, hypothetical ORFs or unknowns. In the
case at hand when the supplementary data (tables 10 and
11 from Qin et al., 2010) are searched there are no
reported hits to genes involved in butyrate synthesis
(Louis et al., 2010), bile catabolism (Jones et al., 2008),
glucuronidases (Gloux et al., 2011) and functions, which
are not easily classified, but maybe important to the host,
for example indole-3-propionic acid synthesis (Wikoff
et al., 2009), choline catabolism (Wang et al., 2011) and
NF-kB modulators (Lakhdari et al., 2010). However, this is
not a criticism of the study, but rather an observation of the
difficulty of the task and deciding what should be classified as a core function of the microbiome (genes involved
in bile catabolism and butyrate synthesis are present in
the METAHIT datasets, but are not abundant). Trying to

Yes BLAST searches and data analysis are

becoming bottlenecks in the analysis

determine which genes are important to the host, when

they may be at low levels in the microbiome, cannot be
achieved by simply sequencing. This fact is further
enforced by the recent study of Arumugam and colleagues (Arumugam et al., 2011), which has taken 17
metagenomic datasets from previous studies (Gill et al.,
2006; Kurokawa et al., 2007; Turnbaugh et al., 2009) as
well as 22 they generated using first-generation Sanger
sequencing and statistically and phylogenetically analysed the information. The major outcome of this analysis
was their conclusion that the distal gut is stratified into
three enterotypes, which are predominantly driven by
species composition. Enterotype 1 is dominated by the
genus Bacteroides, enterotype 2 is dominated by the
genera Prevotella while in enterotype 3 the genus Ruminococcus is the discriminatory genus (Fig. 6; see Supporting information for Arumugam et al., 2011 for further
information on genus abundance). Another interesting
finding was that several abundant functions found in the
different enterotypes are not associated with abundant
genera, for example, bacterial pilus assembly were associated with the low-abundance genus Escherichia, while
the hydrogenotrophic functions, which include acetogenesis, sulphate reduction and methanogenesis were not
detected using the functional marker approach. The mcrA
functional gene was only detected in 3 out of the 22
European samples, although the methanogens that
harbour this gene is found in > 95% of individuals (Dridi
et al., 2009). Dridi and colleagues claim that the low incidence of methanogens in previous studies was due to an
inappropriate DNA extraction method and PCR target,
using their modified approach they improved detection
from 19% to 95.7% in the 700 samples studied (Dridi
et al., 2009). Which raises the question of how much bias
is introduced into these studies by such factors as the
method used to extract the DNA? The method used in the
METAHIT study was one developed to obtain high
molecular weight genomic DNA for creating a metagenomic library fosmids and uses a gentle extraction protocol that does not involve any mechanical shearing

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3096 J. R. Marchesi
a

Fig. 6. A. Abundance of the main phylogenetic groups contributing to defining the three enterotypes of the distal gut.
B. Network analysis showing the interrelationships between the main genera in each enterotype (taken from Arumugam et al., 2011).

(Courtois et al., 2003), hence this may explain why some

functions/groups are absent. Furthermore, if genes
involved in hydrogenotrophic processes, which are known
to be important to gut and host function (McNeil, 1984;
Waniewski and Martin, 1998; Attene-Ramos et al., 2006;
Sahakian et al., 2010), cannot be robustly detected is the
data suspect? The authors concede [functional genes]
from these less abundant microbes could barely be identified. However, such studies do provide the wider scientific community with an invaluable resource from which we
can derive hypotheses as to what constitute a core microbiome and these can be tested in either large human
cohorts or animal models of the human gut. However, we
may need to invest in even deeper sequencing projects to
establish the limits of how deep we need to probe in order
to find functions that are of importance to the host and
define the gut ecosystem.
One way to avoid missing functions is to adopt a topdown or reverse genetics strategy to determine the core
functions in the gut (Nicholson et al., 2005; Martin et al.,
2007). The most robust strategy would be to use a metabonomic approach either in a targeted fashion or nontargeted, using either mass spectrometry (hyphenated

with chromatographic separation, e.g. UPLC-MS) or

nuclear magnetic resonance to identify key metabolites
that occur in the gut and can only be derived from microbial processes (see example in Fig. 7). From these
metabolites it would be possible to develop a database of
the core metabonome and work back to microbial genes
that are responsible for synthesizing them. Metabonomic
studies have started to provide an insight into the key
metabolites that are seen constantly in the gut at varying
levels, for example, the short chain fatty acids (Martin
et al., 2009), amines (Wang et al., 2011), amino acids
(Wikoff et al., 2009) and bile salts (Martin et al., 2007).
From these metabolite signals, we can start to develop
strategies to investigate the diversity and expression of
the microbials genes that are responsible for their synthesis. Louis and colleagues investigated the diversity of
butyrl-CoA : acetate CoA-transfereses (Louis et al., 2010)
using a degenerate PCR method and showed that this
gene and its associated function are found in all the
samples studied and shows a large degree of variation.
Thus this function would be considered to be a core
function of the microbiome, because it not only plays a
role in the bacterium, but is a significant factor responsible

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Human microbiome 3097

Fig. 7. Changes in urinary metabolites due to colonization of the gut by microbiota as shown by pattern recognition analysis [principal
components (PC) analysis] of partial nuclear magnetic resonance spectroscopic data from gnotobiotic sequential rat urine samples. Samples
were collected for up to 3 weeks during the gut microbiotal conventionalization process, the mapping position of five different temporal subsets
are shown (T1T5). One animal (triangle marked by an asterisk next to d 21 cluster) completed conventionalization by day 17 (adapted from
Nicholson et al., 2005).

for a key interaction with the host itself. Hence the definition of a core function may need to be revised so we get
away from defining the core microbiome as the functions/
genes found in a gut, which include genes found in all
bacteria, to one that includes the need to interact with the
host and is undergoing positive selection by the host,
either directly or indirectly. Using this definition many functions would not be included in the core microbiome and
only those playing a role in both biological compartments
would be considered. Another such example of a core
function of the microbiome would be the bile salt hydrolases (Jones et al., 2008). In the absence of these genes
we can see that rodents have reduced bile acid deconjugation, produce more bile acids and absorb more cholesterol (Wostmann, 1973; Wilks, 2007). Furthermore, the
microbial re-colonization of a gnotobiotic animal provides
evidence of the gut microbiomes ability to modulate bile
acid metabolites, which themselves are regulators of lipid
absorption (Claus et al., 2011). These types of integrative
or systems biology studies are bringing together the different biological compartments and help to develop a
better understanding of the what aspects of the core
microbiome are really important in a superorganism.
The mobile microbiome or mobilome
In nearly all the functional and sequence driven human
metagenomic studies to date, very little regard is paid to
genetic elements involved in gene transfer. However, we
know that bacteria are frequently transferring DNA via
phage, plasmids, transposons and other mobile genetic
elements (MGEs) (Ochman et al., 2000). One of the most

commonly isolated functions that are found on these elements are genes involved in antibiotic resistance (Wright,
2007); however, the methods used to pull out these
MGEs are themselves highly biased. They tend to isolate
bacteria showing a chosen function [positive screening or
endogenous isolation (Smalla and Sobecky, 2002)], this
approach limits the range of functions that can be
screened and microbes that can be cultured. Alternatively
the methods only isolate MGEs that can transfer into a
suitable host [exogenous isolation (Bale et al., 1988)],
which tend to be Gram-negative. Hence the ability to
isolate and describe the functions on MGEs is limited by
the current methods available and the fact that many
functions are not easily maintained or screened for in a
surrogate host (when using functional metagenomics) or
reassembled into a whole plasmid in sequence-based
approaches. Moreover, cryptic ORFs on MGEs may not
be recognized as such if the complete element is not
reassembled from the raw data. To this end other
approaches have been developed to specifically look at
unknown function on plasmids and these have been
applied to the distal gut. The TRACA method (Jones and
Marchesi, 2007) uses an in vitro transposition event
coupled with a plasmid-safe DNAse to tag circular DNA
(plasmids and DNA phage) with a selectable marker and
an E. coli plasmid origin of replication. This strategy can
be used to capture small plasmids (< 15 kb) from the gut
metagenome and stability maintain them in E. coli without
the need for any selection, apart from that which was
introduced (in this case kanamycin), or transfer to a suitable recipient. Using this approach, several plasmids
have been isolated from the large intestine of an individual

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3098 J. R. Marchesi
and sequenced. Coupling these sequence data with bioinformatic methods, it was possible to use them as a DNA
hook to pull out similar sequences from the metagenomic
datasets deposited in the public databases (Jones, 2010;
Jones et al., 2010). These studies have started to show
that as with certain gut functions, such as butyrate production and bile salt deconjugation, there is also a core
mobilome in the gut. Two of the plasmids isolated,
pTRACA10 and pTRACA22, were found to be enriched in
the metagenomes of the 15 human distal guts (from USA,
Europe and Japan), while four others did not show
any significant homology to these datasets (BLASTn,
> 100 bp fragments, > 80% identity and E-value of 1e-5).
However, when the same six plasmids were screened
against the METAHIT dataset, all were shown to be represented in these datasets, with pTRACA22, showing a
significant enrichment compared with the other five plasmids. pTRACA22 is a small 5.9 kb mobilizable plasmid
that most probably originates from Blautia hydrogenotrophica as all nine ORFs show > 98% identity to
genes from this draft genome (Jones et al., 2010). The
most notable feature of this plasmid is its RelBE or type II
addiction module (Van Melderen, 2010) and these
modules have been implicated in range of host-specific
functions, for example, modulation of gene expression,
formation of persister cells and biofilm dispersal.
However, whether this enrichment of these modules is
biologically significant and of relevance to the gut or host
still needs to be determined, but it does show that even in
the mobilome the gut does show interesting enrichments
of some genes and more thorough investigation of this
genetic compartment needs to be undertaken in order to
establish its role in the ecology of this ecosystem.
The distal gut microbiome as a driver of health
and disease
The whole concept of integrating the core microbiome into
host biology and physiology is further extended and challenged by considering it as a driver of disease as well. If
we have a core microbiota, evolved to the hosts needs,
and if two individuals share common features of this core
will they also share common emergent properties too?
Furthermore, if there is a dysbiosis in the gut microbiota,
does this lead to the development of gastrointestinal diseases? Such concepts have been explored in the context
of the gut microbiota as an environmental factor in functional gastrointestinal diseases, for example, inflammatory bowel disease (Scanlan et al., 2006; Frank et al.,
2007), colorectal cancer (Scanlan et al., 2008b; Sobhani
et al., 2011), irritable bowel syndrome (Kassinen et al.,
2007; OMahony et al., 2009) and Clostridium difficileassociated diarrhoea (Khoruts et al., 2010) and more
recently in ex-intestinal diseases such as cardiovascular

disease (Wang et al., 2011), obesity {Turnbaugh, 2008

#15541; Ley, 2005 #7445; Backhed, 2004 #501}
(however, others have been unable to confirm this observation {Duncan, 2008 #15503} {Fleissner, 2010 #17580}
{De La Serre, 2010 #16857} {Zhang, 2009 #15740}
{Schwiertz, 2009 #16428}) and psychiatric diseases (Desbonnet et al., 2008; Rook and Lowry, 2008) {Bercik, 2011
#17583}. However, there are several issues that discombobulate the idea of the gut microbiota as an environmental factor in these and other diseases. First, many of the
studies look at the gut microbiota after diagnosis of the
disease, hence we are unsure as to whether we are
observing cause or effect. In order to circumvent this
issue, large prospective studies need to be undertaken,
which are statistically empowered, in which frequent
samples are taken and appropriately stored for retrospective analysis. Second, we are currently developing correlations between a disease state and a snapshot of
microbial diversity in the gut or a potential metabolite, we
need to develop stronger causal links and mechanistic
models that are predictive and can be tested in suitable
animal models. Even with these issues researchers are
developing the view that certain functions and the associated microbes are beneficial to the health of the host.
Some of the most commonly seen bacterial metabolites in
the human gut are the SCFA, butyrate, acetate, lactate
and propionate {Saric, 2008 #15051}. The two bacterial
groups that are mainly responsible for producing butyrate
are the F. prauznitzii and Eubacterium rectale/Roseburia
groups {Louis, 2010 #17389; Louis, 2009 #15805}. This
metabolite has been implicated in large array of effects in
the intestine that include controlling apoptosis, cytokine
production, energy for colonocytes and mucus synthesis
{Guilloteau, 2010 #17009}. Hence any changes in these
groups would potentially have an impact on this function
and host physiology. Beyond this ubiquitous function it
does become an exercise in speculation as to what bacterial groups are important to host health. In one respect
moving away from trying to define a core microbiome to a
core metabonome may aid in defining what we need to
study and understand in order to maintain a healthy gut
and thus a healthy host.
Concluding remarks
The paradigm of the human distal gut microbiome has
shifted in recent years, from one that looked upon it as a
source of opportunistic pathogens to one that embraces it
as a virtual organ with the ability to influence the health
status of the host. Taxonomically, we have established
that this system is mainly composed of members of the
Bacteroidetes and Firmicutes, but we are still struggling to
determine the key functions that are important to the
microbes and the host. The ability to catalogue the genes

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Human microbiome 3099

present in the distal gut does not equate to defining the
core microbiome and using a top down approach will help
to determine this feature in more detail. The question of
cohort sizes needs to be addressed and to this end we
need to increase the sample sizes used, in order to
develop a much more complete picture of the functions in
the gut and start to combine sequence and functionbased metagenomic studies in order to determine the
core microbiome. In addition, the integration of metabonomic data into this model will help to determine the core
microbiome and establish how it varies both inter- and
intra-individually. Once we have created this foundation
we can commence to develop hypothesizes that address
such questions as how does variation in the distal gut
microbiome influence host function or can we modulate
the gut microbiome in order to promote health and should
we even try.
Acknowledgements
I wish to acknowledge the help of my colleagues in the
Eldermet project in University College Cork for sharing their
data and Professor Ruth Ley for kindly providing me with here
data for Fig. 3.

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