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Minireview
Human distal gut microbiomeemi_2574 3088..3102
Julian R. Marchesi*School of Biosciences, Museum Avenue,
CardiffUniversity, Cardiff CF10 3AX, UK.
Summary
The distal gut and its associated microbiota is a newfrontier in
the quest to understand human biology andevolution. The renaissance
in this field has beenpartly driven by advances in sequencing
technologyand also by the application of a variety of omic
tech-nologies in a systems biology framework. In the initialstages
of understanding what constitutes the gut,culture-independent
methods, primarily inventoriesof 16S rRNA genes, have provided a
clear view of themain taxonomic groups of Bacteria in the distal
gutand we are now moving towards defining the func-tions that
reside in the distal gut microbiome. Thisreview will explore recent
advances in the area of thedistal gut and the use of a variety of
omic approachesto determine what constitutes this fascinating
collec-tion of microbes.
Introduction
Gut or intestinal microbiology has undergone a mini-renaissance
in the past 10 years. In a comprehensivereview of the role of the
gut microbiota in the health of thehost, Sekirov and colleagues
(Sekirov et al., 2010)charted the number of publications for the
period from1990 to 2009. Their data show that in this period there
hasbeen a near fivefold increase in the yearly publicationrate. In
fact the overall number they show is an underes-timation, if the
ISI Web of Knowledge database is queriedwith similar keywords (Fig.
1), the trend is the same;however, the number of publications,
which now includes2010, is nearly twice the figure and currently
peaks at1366 for 2010. Several reasons are responsible for
thisincreased attention, the recognition that the gut micro-biota
plays a central role in host health, as well as
thecross-pollination of ideas from microbiologists working in
the varied areas of environmental microbiology. As a dis-cipline
environmental microbiology has always been chal-lenged by what has
been referred to as the the great platecount anomaly and which
describes the disparitybetween what we can grow in the laboratory,
on conven-tional microbiology media and what we can directly
count(Staley and Konopka, 1985). This challenge has resultedin a
dramatic (and some may say it is a swing too far awayfrom
culturing) shift away from culturing to
developingculture-independent approaches to investigate ecosys-tem
function and the role that microbes play (Amann andKuhl, 1998).
However, microbiologists working in thehuman body, have been
relatively fortunate because asignificant proportion of the
microbial community in thesesystems are culturable, a fact that
delayed the introduc-tion of culture-independent approaches to
analyse thisecosystem. The suite of methods that have been used
arevariations on the genomic, transcriptomic, proteomic
andmetabolomic methods. The most commonly used are themetagenomic
and 16S/18S rRNA gene-based methods todetermine the functions in
the microbiome and thespecies 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 gutand oral cavity, because
logistically they are the easiest toaccess. In both instances the
proportions of the microbialcommunity that are as yet uncultivated
are between 30%and 50% (Wade, 2002; Eckburg et al., 2005; Duncanet
al., 2007), which provides researchers with a significantculturable
microbial biomass for investigation. When thisfigure is compared
with environmental ecosystems suchas the deep biosphere or soil
where the culturable fractioncan be between < 0.1% and 1%
respectively (Hugenholtzet al., 1998; Fry et al., 2008) it becomes
clear howresearchers in these areas needed to create a suite
oftools to help in developing a more complete picture ofmicrobial
contributions to ecosystem function. The currentburst of interest
in the gut ecosystem and how itsmicrobes influence host
function/physiology has in someway been driven by microbiologists
adopting the tools ofenvironmental microbiologists and implementing
them in
Received 13 May 2011; accepted 20 July 2011. *For
correspondence.E-mail [email protected]; Tel. (+44) 29208
74188; Fax(+44) 29208 74305.
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a new setting. Without these methods we would not haverealized
that the gut microbiota is so diverse betweendifferent individuals,
or resilient to perturbations, or thatkey functions in some
instances are redundant, whileother they are not. These initial
forays into the gut eco-system using culture-independent methods
paved theway for developing hypotheses in which the gut micro-biota
are drivers of health and disease. Hence in light ofthe numerous
reviews on the microbiota of the human gut(524 for 20082010) this
review will concentrate on themost 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), thecolon or distal gut
(subdivided into the ascending, trans-verse and descending colons)
and rectum (Fig. 2). Whilethe physiological role of the gut is to
process and digestthe food we ingest, it also offers a niche for
colonization bya variety of microbes. Each niche harbours a
specificmicrobial community, which to some extent reflects
thedynamics of that compartment. The numbers of microbesin each
niche increases as one moves from the stomachto the rectum
resulting in one of the most densely popu-lated ecosystems being
found in the distal gut or colon,which contains between 10111012
bacteria per gram ofluminal material. Because the distal gut
contains one ofthe densest communities known (Whitman et al.,
1998)and is very easy to access [up to 55% of a stool sample
isbacterial biomass (Cummings and Macfarlane, 1997)] it
has received the majority of the attention. However, thisdoes
not mean it is a robust representation of the wholecolon or small
intestine; moreover, the mucosal surfacecontains a microbiota that
is significantly different to thatfound in a stool sample from the
same subject(Momozawa et al., 2011). Data obtained from analysis
offaecal material must be considered in light of where thissample
comes from and conclusions based on this datamust be tempered
appropriately; however, these data stillprovide a very valuable
insight into functions and speciespresent in the gut.
The current census of the inhabitants of the distalgut: the
early years of the distal gut
Unlike many environmental ecosystems being investi-gated, the
establishment of the climax community in thegut is played out time
and time again with every birth;moreover, it can very easily be
perturbed and involves animmunological dialogue with the system in
which itresides. Many of the major ecosystems that are
studied,marine, terrestrial, deep-biosphere and atmosphere havebeen
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 sludgecontaining cultured isolates of
Mycoplasma hominis,Streptococcus mutans and Aspergillus flavus has
beenobserved (Espinoza et al., 2005; Romero et al., 2008)]and
immediately upon exit from the mother start to be
Fig. 1. The number of publications retrieved from the ISI Web of
Knowledge database (http://apps.isiknowledge.com/), obtained by
using thefollowing keywords and Boolean operators: intestinal
microbiota OR gut microbiota OR intestinal flora OR gut flora OR
intestinalmicroflora OR gut microflora OR gut microbiome OR
intestinal microbiome (the addition of the word gut microbiome and
intestinalmicrobiome, not used by Sekirov and colleagues, added 92
publications compared with the same search without).
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colonized by microbes. There is a significant interest
inunderstanding what drives this colonization process andhow much
nature or nurture plays an influencing role.Specifically, because
we have a poor understanding ofwhether early life events, which may
alter the gut micro-biotas composition, can have ramifications for
later lifehealth. The climax community seems to be
establishedwithin the first 2 years of life and after the first
year, it hasstarted to converge and reflects a generalized adult
distalgut community (Palmer et al., 2007). The factors that
influ-ence this process are the maternal microbiota(Dominguez-Bello
et al., 2010), diet (breast fed vs.formula fed; Favier et al.,
2003), mode of delivery (normalvs. 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 (Zhanget al., 2009) technically this paper
shows the impact ofsurgery on the adult gut)]. This progression has
also beenconfirmed when using metagenomic DNA (mgDNA)instead of the
16S rRNA gene. Koenig and co-workers(Koenig et al., 2011) created
inventories of the 16S rRNAgene (from 60 infants) and used this
information to select12 infants for a sequence based metagenomic
analysison the Roche 454 platform. The data they generated
wereprocessed using MEGAN (Huson et al., 2007) andMG-RAST (Meyer et
al., 2008) to assess the taxonomic
Fig. 2. The anatomy of the gastrointestinal tract, major
bacterial phyla and their abundance in each niche. The information
for this figure wascompiled from (Eckburg et al., 2005; Bik et al.,
2006; OHara and Shanahan, 2006; McConnell et al., 2008; van den
Bogert et al., 2011).
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source and functions contained within the mgDNA respec-tively.
Using mgDNA the same succession was seen aswith 16S rRNA gene data
(Fig. 3) and other groups havealso confirmed that random sequence
reads can be usedin lieu of taxonomically relevant genes such as
the 16SrRNA gene (Manichanh et al., 2008; Ghosh et al., 2010;Gori
et al., 2011). The consensus of opinion from thesestudies seems to
be that the trajectory of the colonizationprocess is towards a
similar outcome, i.e. a distal gutmicrobiota, which after the age
of 2 is stable and colo-nized predominantly by Firmicutes and
Bacteroidetes(see below). However, we do lack the information of
whichfactors are driving this process, how the colonizationprocess
in different ethnic groups proceeds and to whatextent the functions
in the gut are established. Hencethere is a clear need to continue
to determine the keyevents that influence the establishment of the
climaxcommunity.
The adult and ageing distal gut microbiota
One of the most comprehensive early culture-independent analyses
(using clone libraries of 16S rRNAgenes and Sanger or
first-generation sequencing plat-forms) was carried out on the
distal gut by Eckburg andcolleagues (Eckburg et al., 2005). This
study revealedthat while there were many bacteria in the gut they
wereactually not as diverse as soil or marine ecosystems. In
the majority of mammals the two main phyla present arethe
Bacteriodetes and Firmicutes (Ley et al., 2008) and itseems that
members of these two phyla contributeapproximately 90% of the
species in the distal gut. Thenumber of species estimated to be
present in the distalgut is relatively small [compared with soil in
which millionsof species are estimated to exist in 10 g (Gans et
al.,2005)] and is in the hundreds (Qin et al., 2010), while alarger
degree of diversity exists at the strain level, whichmaybe in the
thousands (Ley et al., 2006). The impor-tance of the strain
diversity may only be significant whenthe functions that the strain
carries are non-redundant.For example, there are two
hydrogenotrophic groups, themethanogens and sulphate reducing
bacteria, which arerepresented by very few species and strains in
the distalgut {Scanlan, 2009 #16220} {Dridi, 2009 #17393}. In
thisscenario, it would be easy to lose the functions theseorganisms
provide, whether health promoting or detrimen-tal remains to be
seen, to the host. A further consequenceof this strain diversity is
that phylogenetic trees of the guttend to have few branches, which
are not deep, but havea large degree of radiance at the ends.
However, thiscensus is based on a very small number of samples
andto put it into perspective a recent search for single
nucle-otide polymorphisms that correlate with adult heightscreened
183 727 individuals to determine statisticallysignificant
correlations (Lango Allen et al., 2010); in con-trast, the majority
of the studies that have been under-
Fig. 3. Taxonomic distribution ofmetagenomic sequences isolated
from infantfaecal DNA [adapted from Koenig et al. (2011)with data
kindly provided by Prof Ruth Ley].
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taken to determine the composition of the gut microbiotause
small cohorts that can be counted in the tens ratherthan thousands.
One recent study that has sampled hun-dreds of individuals has
shown why we need large cohortsof subjects. While we can take
averages of the numbersof species present in the distal gut and
conclude that twophyla predominate, if the sample size is increased
theproportions of these phyla can tell a very different story.
Inthe Eldermet project (http://eldermet.ucc.ie/) being under-taken
in University College Cork, Ireland, the investigatorsprofiled the
distal gut of 386 > 65 year old individualsusing
second-generation 454 pyrosequencing andobtained approximately 40
000 reads per sample andwhich spanned the V4 region of the 16S rRNA
gene. Onceagain the composite picture of the distal gut was one
inwhich gene sequences from the Bacteriodetes and Firmi-cutes
contributed 97% of the overall sequences obtained(57% and 40%
respectively; Claesson et al., 2011).However, when the individual
profiles were plotted andordered an entirely different picture was
obtained (Fig. 4).The distribution of 16S rRNA genes showed that
withinthe cohort there was a continuum, at one endBacteroidetes
made up nearly 90% of the distal gut micro-biota while at the other
Firmicutes made up more than95% of the sequences recovered. This
larger study furtherhighlights the necessity to increase the cohort
size andmove away from small studies of 23 subjects. The factthat
the distal gut microbiota is so variable at the phylumlevel does
make one wonder whether it is this variablefurther down the
taxonomic levels, for example, at the
genus level? To answer this question, several groupshave been
exploring the concept of the core microbiotaand whether there exist
a group of species found in alldistal guts regardless of geography,
ethnicity, age, genderor diet. While it would be safe to say that
there is a coremicrobiota at the phylum level, i.e. all humans
possesmembers of the Bacteroidetes and Firmicutes, when wedrill
down the taxonomic levels it seems that this conceptbecomes more
sketchy and different studies and methodsprovide different answers.
Tap and colleagues undertooka de novo analysis of the composition
of the distal gutmicrobiota using first-generation sequencing and
PCRamplification and cloning of the 16S rRNA gene (Tapet al.,
2009). They generated 10 456 16S rRNA genesequences from 17 human
faecal DNA samples andanalysed them to determine which sequences
wereshared and which were unique. In their conclusions, theystate
that on average each individual contains 259 opera-tional taxonomic
units (OTUs at the 98% level), but therange was large (159383) and
in total 3180 OTUs wereidentified from the total pool of 16S rRNA
genesequences. Approximately, 79% of the OTUs were onlyfound in one
sample and 21% were found at least twice;however, no OTUs was found
in all 17 distal guts. Theyshowed that 66 OTUs were found in 50% of
the samplesand proposed that these may in some way constitute acore
microbiota. These OTUs belonged to 18 genera andthese were
affiliated predominantly with the Firmicutes(57/66). In a study
with a similar goal, to determine themembers of the core microbiota
of the distal gut, Rajilic-
Fig. 4. Proportions of main bacterial phyla in 386 Eldermet
faecal samples, the two main phyla are shown in the figure while
the remainingphyla 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).
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Stojanovic and co-workers used a DNA array approach todetermine
the composition of the distal gut (Rajilic-Stojanovic et al.,
2009). While the aims were comparablethe methods are not, even if
they both target the samegene 16S rRNA. DNA arrays have the
advantage ofbeing more sensitive and are able to detect sequences
ina mixture at much lower levels than a random samplingwithout
replacement strategy, e.g. analysis of a clonelibrary or
second-generation amplicon sequencing (Har-rington et al., 2008;
Paliy et al., 2009; Rigsbee et al.,2011). However, they are only as
good as the databaseused to design the array probes and any novel
sequencesin the samples, which are not represented on the chip,
willnot be detected. Bearing this in mind Rajilic-Stojanovicand
co-workers used their human intestinal tract chip(HITChip) to
profile the distal gut of five young and fiveelderly volunteers.
Their HITchip can measure the abun-dance of 1140 unique microbial
phylotypes and they con-cluded that there was a common core between
all 10individuals, which consisted mainly of probes from threephyla
(Actinobacteria, Bacteroidetes and Firmicutes)found in the distal
gut and confirms that we possess adistal gut microbiota that is
host specific. In addition, theywere also able to show that the
young and elderly gutssamples clustered according to the hosts age
with all theyoung and elderly samples found in their
respectiveclades. In the Eldermet study (Claesson et al.,
2011),there was also a significant difference between the
elderlyand young distal gut. In the elderly distal gut more
thanhalf of the core microbiota (53%) were from theBacteroidetes,
from the genera Bacteroides (29%), Alisti-pes (17%) and
Parabacteroides (7%), while in theyounger distal gut this figure
dropped to between 8% and27%. Furthermore, the core clostridial
species were pre-dominantly in the Clostridium cluster IV for the
elderlywhereas cluster XIVa was more prevalent in the
youngercohort, again highlighting the need for longitudinal
studiesrather than snapshots of the distal gut composition. In
thestudy conducted by Biagi and co-workers (Biagi et al.,2010),
which looked at young (Y), elderly (E) and cente-narians (C)
(groups of 20, 22 and 21 and average ages of31, 72.7 and 100.5
respectively) using the HITChip plat-form and quantitative PCR, the
trend for a variation in themain groups was also seen (Fig. 5).
However, thechanges in the subgroups within the clostridial group
werenot the same as found in the Eldermet project, with anincrease
in Clostridium cluster XIVa going from Y to E,which decreased in
the centenarians. The Clostridiumcluster IV remained the same
between all three groups,while between the C and E groups only the
Faecalibac-terium prausnitzii was significantly different, between
Cand Y groups Bifidobacterium spp. differed and betweenE and Y
members of the genus Akkermansia differed. In arecent development
to describe the core microbiota of the
distal gut Sekelja and colleagues undertook a post hocanalysis
of previously published datasets from pyrose-quencing projects
targeting the 16S rRNA genes (Sekeljaet al., 2011). They also
changed the approach used, bymoving away from defining taxonomic
groups and in theirwords search for a human core microbiota
independentof both predefined phylogroup depths and
phylogenetictrees. Using an alignment-independent approach,
theyanalysed 16S rRNA gene sequences (from eight previousstudies
and comprising 1 186 272 partial 16S rRNAsequences from 210
samples) and clustered them usingprincipal component analysis based
on their sequencesimilarity [calculated by establishing 5 mer
nucleotide fre-quencies in each sequence (Rudi et al., 2006;
2007)].From their analysis they report that there were two
micro-biota cores, which were consistently found in all
samples.Both cores were affiliated to the Firmicutes and
weremembers of the clostridial family Lachnospiraceae.Interestingly
they concluded that each core appeared atdefined moments in
evolution with core 2 co-evolving withthe radiation of vertebrates
and core 1 co-evolved with themammals. These studies enforce the
stochastic nature ofsampling the distal gut and the need for more
large-scalestudies to minimize confounding factors such as
diet,environment and genetic/immunological variability of
thehost.
Fig. 5. Relative abundance of phylum/order phylotypes
fromcentenarians (C), elderly (E) and young (Y) (adapted from
Biagiet al., 2010).
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A bacterial-centric view that needs toencompass all the
microbiota
To date we still have a limited understanding of whatconstitutes
the core microbiota of the distal gut and assuch cannot define its
limits. We still need to expand thenumbers of distal guts sampled,
the ethnic groups fromwhich we obtain the samples and also the
compositionalstability of the core. While the previous studies have
allindicated that the concept of a core microbiota is notdead, we
have not reached any consensus as to whatspecies should be
considered as members of this impor-tant group of bacteria, but we
do agree that main phylaare the Bacteroidetes and Firmicutes.
Furthermore, theconcept of a core microbiota has not been fully
inclusiveand maybe should be renamed the core bacteriota as ithas
not considered the micro-eukaryotic and viral compo-nents. Both of
these groups of organisms have beenstudied in relation to the
distal gut, but in a more limitedfashion. Only a few studies have
been undertaken lookingat the human micro-eukaryotic diversity
using culture-independent approaches (Ott et al., 2008; Scanlan
andMarchesi, 2008) and viral diversity in faecal samples(Breitbart
et al., 2003; 2008; Zhang et al., 2005; Reyeset al., 2010). The
micro-eukaryotic diversity and numbersis several orders of
magnitude lower than the Bacteriaand is skewed towards Candida and
Saccharomyces spp.when cultured, but culture-independent approaches
using18S rRNA genes shows that Blastocystis spp. are verycommon in
the distal gut and yeasts are rarely obtained.In fact, it may be
concluded that micro-eukaryotes areonly really significant when
there is a dysbiosis in the gut(Goldman and Huffnagle, 2009). For
the viral componentthe story is very different with their numbers
being at leastan order of magnitude higher than the bacterial
numbersin the distal gut. Thus we might need to start to
considerthe viral component as drivers of community dynamics assome
marine microbiologists do (Suttle, 2007). In fact,Lepage and
colleagues (2008) have hypothesized a rolefor distal gut
bacteriophage as drivers of dysbiosis in thedistal gut and
inflammatory bowel disease. While studieslooking to define the core
microbiota have focused ondescribing the Bacteria within the distal
gut, there is alsoa significant number of Archaea in this niche.
The mostcommon species and 16S rRNA gene sequence isolatedfrom the
distal gut come from the Euryarchaeota and inparticular the
Methanobacteriaceae family (Scanlan et al.,2008a; Dridi et al.,
2009) with Methanobrevibacter smithiiand Methanosphaera stadtmanae
the two predominantArchaea found. However, other rarer archaeal
sequenceshave 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 todate
M. smithii and M. stadtmanae are the two mainArchaea (Dridi et al.,
2011) and one would question towhat extent the much rarer species
are autochthonousand are actually contaminants from our
diet/environment.
The luminal microbiota versus themucosal microbiota
One of the major criticisms of many of the studies on thedistal
gut is the reliance on stool or faecal material as thesource of
microbial biomass or genomic DNA. While it isquite simple to
collect it is clear that faeces do not afforda robust proxy for the
gut microbiota as a whole. InEckburg and co-workers 2005
culture-independentanalysis of the distal gut (Eckburg et al.,
2005) theyclearly showed that while the microbiota attached to
themucosa was similar throughout an individuals large intes-tine it
was significantly different to the stool sample fromthe same
individual, but whether there is any biologicalsignificance in this
difference remains to be shown, as thenumber of luminal bacteria
are between 46 orders ofmagnitude less than the mucosally
associated bacteria[MAB; Zoetendal (Zoetendal et al., 2002; Ahmed
et al.,2007; Walker et al., 2011)]. Using a DNA microarray (Aus-HIT
Chip) Aguirre de Carcer and colleagues (de Carceret al., 2011) have
shown not only a gender differencebetween the MAB, but also a
qualitative change in theMAB composition moving from the caecum to
the rectum,via the transverse and sigmoid colon. However, we
stillneed larger studies to determine what is considered to bethe
prevalent species colonizing the different regions ofthe colon and
at what scale the community starts todiverge.
Is there a core microbiome?
Qin and co-workers (Qin et al., 2010) and othersequence-based
metagenomic studies have addressedthe issue of whether there is a
core microbiome (thecollection of microbial genes) and if so what
does it looklike? To date the study of Qin and colleagues is by far
thedeepest and largest metagenomic1 sequencing project tobe
undertaken; however, two smaller metagenomicstudies do precede it
(Kurokawa et al., 2007; Turnbaughet al., 2009). In the most recent
study the authors used anIllumina second-generation sequencing
platform to gen-erate 0.58 terabases of sequence from 124
volunteers
1The term metagenomics is routinely confused with
creatinginventories of 16S rRNA genes to describe bacterial
diversity.Metagenomics is the analysis of random genomic
fragmentseither by sequencing or functional analysis.
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(approximately 4.5 gigabases per individual with anaverage
read-length of 75 bp) and determined that therewere 3.3 million
non-redundant genes in the distal gutmetagenome. This figure is in
agreement with the previ-ous figure of 9 million genes (Yang et
al., 2009) and thatbetween the different gut samples there were 204
056common genes that comprised 38% of an individuals gutmicrobiome.
In this project these genes were grouped into6313 clusters of
orthologous groups and could be dividedinto house-keeping genes and
gut-specific genes. Whenstudying the gut it would be the genes only
found in thegut that are of key interest as these may play a role
inshaping the relationship between the host and its gutmicrobiota.
While the house-keeping genes were part ofthe main metabolic
pathways commonly associated withbacteria, for example, amino acid
synthesis, nucleic acidprocessing and general secretory processes,
the gut-specific genes were identified as being involved in
adhe-sion to host proteins or catabolizing globoseriesglycolipids.
However, the majority of the clusters oforthologous groups (74.3%)
were not defined and this facthighlights a key problem with
sequence-based metage-nomic projects, they lack the ability to
provide novel func-tions (Table 1). Many of the sequences, when
comparedwith the current databases will either return hits to
anno-tated functions, hypothetical ORFs or unknowns. In thecase at
hand when the supplementary data (tables 10 and11 from Qin et al.,
2010) are searched there are noreported hits to genes involved in
butyrate synthesis(Louis et al., 2010), bile catabolism (Jones et
al., 2008),glucuronidases (Gloux et al., 2011) and functions,
whichare not easily classified, but maybe important to the host,for
example indole-3-propionic acid synthesis (Wikoffet al., 2009),
choline catabolism (Wang et al., 2011) andNF-kB modulators
(Lakhdari et al., 2010). However, this isnot a criticism of the
study, but rather an observation of thedifficulty of the task and
deciding what should be classi-fied as a core function of the
microbiome (genes involvedin bile catabolism and butyrate synthesis
are present inthe METAHIT datasets, but are not abundant). Trying
to
determine which genes are important to the host, whenthey may be
at low levels in the microbiome, cannot beachieved by simply
sequencing. This fact is furtherenforced by the recent study of
Arumugam and col-leagues (Arumugam et al., 2011), which has taken
17metagenomic datasets from previous studies (Gill et al.,2006;
Kurokawa et al., 2007; Turnbaugh et al., 2009) aswell as 22 they
generated using first-generation Sangersequencing and statistically
and phylogenetically analy-sed the information. The major outcome
of this analysiswas their conclusion that the distal gut is
stratified intothree enterotypes, which are predominantly driven
byspecies composition. Enterotype 1 is dominated by thegenus
Bacteroides, enterotype 2 is dominated by thegenera Prevotella
while in enterotype 3 the genus Rumi-nococcus is the discriminatory
genus (Fig. 6; see Sup-porting information for Arumugam et al.,
2011 for furtherinformation on genus abundance). Another
interestingfinding was that several abundant functions found in
thedifferent enterotypes are not associated with abundantgenera,
for example, bacterial pilus assembly were asso-ciated with the
low-abundance genus Escherichia, whilethe hydrogenotrophic
functions, which include acetogen-esis, sulphate reduction and
methanogenesis were notdetected using the functional marker
approach. The mcrAfunctional gene was only detected in 3 out of the
22European samples, although the methanogens thatharbour this gene
is found in > 95% of individuals (Dridiet al., 2009). Dridi and
colleagues claim that the low inci-dence of methanogens in previous
studies was due to aninappropriate DNA extraction method and PCR
target,using their modified approach they improved detectionfrom
19% to 95.7% in the 700 samples studied (Dridiet al., 2009). Which
raises the question of how much biasis introduced into these
studies by such factors as themethod used to extract the DNA? The
method used in theMETAHIT study was one developed to obtain
highmolecular weight genomic DNA for creating a metage-nomic
library fosmids and uses a gentle extraction pro-tocol that does
not involve any mechanical shearing
Table 1. Comparison of the pros and cons of the two metagenomics
methods used to study the functions in an ecosystem.
Function-based screen Sequence-based screen
Screen large amounts of DNA Yes with the aid of colony picking
andarraying robots
Yes with the use of second-generationsequencing platforms
Provide novelty Yes NoGenomic context Yes Limited and relies on
assembly of reads and
assumptions on pan-genomic nature of gut bacteria.Toxic genes No
YesExpression issues Yes NoStorage issues Yes physical storage, one
fosmid library
can easily take 650, 384 well platesand 1950 if stored in
triplicate
No
Computational issues No Yes BLAST searches and data analysis
arebecoming bottlenecks in the analysis
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(Courtois et al., 2003), hence this may explain why
somefunctions/groups are absent. Furthermore, if genesinvolved in
hydrogenotrophic processes, which are knownto 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 thedata suspect? The authors concede
[functional genes]from these less abundant microbes could barely be
iden-tified. However, such studies do provide the wider scien-tific
community with an invaluable resource from which wecan derive
hypotheses as to what constitute a core micro-biome and these can
be tested in either large humancohorts or animal models of the
human gut. However, wemay need to invest in even deeper sequencing
projects toestablish the limits of how deep we need to probe in
orderto find functions that are of importance to the host anddefine
the gut ecosystem.
One way to avoid missing functions is to adopt a top-down or
reverse genetics strategy to determine the corefunctions in the gut
(Nicholson et al., 2005; Martin et al.,2007). The most robust
strategy would be to use a meta-bonomic approach either in a
targeted fashion or non-targeted, using either mass spectrometry
(hyphenated
with chromatographic separation, e.g. UPLC-MS) ornuclear
magnetic resonance to identify key metabolitesthat occur in the gut
and can only be derived from micro-bial processes (see example in
Fig. 7). From thesemetabolites it would be possible to develop a
database ofthe core metabonome and work back to microbial genesthat
are responsible for synthesizing them. Metabonomicstudies have
started to provide an insight into the keymetabolites that are seen
constantly in the gut at varyinglevels, for example, the short
chain fatty acids (Martinet 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
developstrategies to investigate the diversity and expression ofthe
microbials genes that are responsible for their synthe-sis. Louis
and colleagues investigated the diversity ofbutyrl-CoA : acetate
CoA-transfereses (Louis et al., 2010)using a degenerate PCR method
and showed that thisgene and its associated function are found in
all thesamples studied and shows a large degree of variation.Thus
this function would be considered to be a corefunction of the
microbiome, because it not only plays arole in the bacterium, but
is a significant factor responsible
a
b
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).
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for a key interaction with the host itself. Hence the
defini-tion of a core function may need to be revised so we getaway
from defining the core microbiome as the functions/genes found in a
gut, which include genes found in allbacteria, to one that includes
the need to interact with thehost and is undergoing positive
selection by the host,either directly or indirectly. Using this
definition many func-tions would not be included in the core
microbiome andonly those playing a role in both biological
compartmentswould be considered. Another such example of a
corefunction of the microbiome would be the bile salt hydro-lases
(Jones et al., 2008). In the absence of these geneswe can see that
rodents have reduced bile acid deconju-gation, produce more bile
acids and absorb more choles-terol (Wostmann, 1973; Wilks, 2007).
Furthermore, themicrobial re-colonization of a gnotobiotic animal
providesevidence of the gut microbiomes ability to modulate
bileacid metabolites, which themselves are regulators of
lipidabsorption (Claus et al., 2011). These types of integrativeor
systems biology studies are bringing together the dif-ferent
biological compartments and help to develop abetter understanding
of the what aspects of the coremicrobiome are really important in a
superorganism.
The mobile microbiome or mobilome
In nearly all the functional and sequence driven
humanmetagenomic studies to date, very little regard is paid
togenetic elements involved in gene transfer. However, weknow that
bacteria are frequently transferring DNA viaphage, plasmids,
transposons and other mobile geneticelements (MGEs) (Ochman et al.,
2000). One of the most
commonly isolated functions that are found on these ele-ments
are genes involved in antibiotic resistance (Wright,2007); however,
the methods used to pull out theseMGEs are themselves highly
biased. They tend to isolatebacteria showing a chosen function
[positive screening orendogenous isolation (Smalla and Sobecky,
2002)], thisapproach limits the range of functions that can
bescreened and microbes that can be cultured. Alternativelythe
methods only isolate MGEs that can transfer into asuitable host
[exogenous isolation (Bale et al., 1988)],which tend to be
Gram-negative. Hence the ability toisolate and describe the
functions on MGEs is limited bythe current methods available and
the fact that manyfunctions are not easily maintained or screened
for in asurrogate host (when using functional metagenomics)
orreassembled into a whole plasmid in sequence-basedapproaches.
Moreover, cryptic ORFs on MGEs may notbe recognized as such if the
complete element is notreassembled from the raw data. To this end
otherapproaches have been developed to specifically look atunknown
function on plasmids and these have beenapplied to the distal gut.
The TRACA method (Jones andMarchesi, 2007) uses an in vitro
transposition eventcoupled with a plasmid-safe DNAse to tag
circular DNA(plasmids and DNA phage) with a selectable marker andan
E. coli plasmid origin of replication. This strategy canbe used to
capture small plasmids (< 15 kb) from the gutmetagenome and
stability maintain them in E. coli withoutthe need for any
selection, apart from that which wasintroduced (in this case
kanamycin), or transfer to a suit-able recipient. Using this
approach, several plasmidshave been isolated from the large
intestine of an individual
Fig. 7. Changes in urinary metabolites due to colonization of
the gut by microbiota as shown by pattern recognition analysis
[principalcomponents (PC) analysis] of partial nuclear magnetic
resonance spectroscopic data from gnotobiotic sequential rat urine
samples. Sampleswere collected for up to 3 weeks during the gut
microbiotal conventionalization process, the mapping position of
five different temporal subsetsare shown (T1T5). One animal
(triangle marked by an asterisk next to d 21 cluster) completed
conventionalization by day 17 (adapted fromNicholson et al.,
2005).
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and sequenced. Coupling these sequence data with bio-informatic
methods, it was possible to use them as a DNAhook to pull out
similar sequences from the metagenomicdatasets deposited in the
public databases (Jones, 2010;Jones et al., 2010). These studies
have started to showthat as with certain gut functions, such as
butyrate pro-duction and bile salt deconjugation, there is also a
coremobilome in the gut. Two of the plasmids isolated,pTRACA10 and
pTRACA22, were found to be enriched inthe metagenomes of the 15
human distal guts (from USA,Europe and Japan), while four others
did not showany significant homology to these datasets (BLASTn,>
100 bp fragments, > 80% identity and E-value of 1e-5).However,
when the same six plasmids were screenedagainst the METAHIT
dataset, all were shown to be rep-resented in these datasets, with
pTRACA22, showing asignificant enrichment compared with the other
five plas-mids. pTRACA22 is a small 5.9 kb mobilizable plasmidthat
most probably originates from Blautia hydro-genotrophica as all
nine ORFs show > 98% identity togenes from this draft genome
(Jones et al., 2010). Themost notable feature of this plasmid is
its RelBE or type IIaddiction module (Van Melderen, 2010) and
thesemodules have been implicated in range of
host-specificfunctions, for example, modulation of gene
expression,formation of persister cells and biofilm
dispersal.However, whether this enrichment of these modules
isbiologically significant and of relevance to the gut or hoststill
needs to be determined, but it does show that even inthe mobilome
the gut does show interesting enrichmentsof some genes and more
thorough investigation of thisgenetic compartment needs to be
undertaken in order toestablish its role in the ecology of this
ecosystem.
The distal gut microbiome as a driver of healthand disease
The whole concept of integrating the core microbiome intohost
biology and physiology is further extended and chal-lenged by
considering it as a driver of disease as well. Ifwe have a core
microbiota, evolved to the hosts needs,and if two individuals share
common features of this corewill 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
dis-eases? Such concepts have been explored in the contextof the
gut microbiota as an environmental factor in func-tional
gastrointestinal diseases, for example, inflamma-tory bowel disease
(Scanlan et al., 2006; Frank et al.,2007), colorectal cancer
(Scanlan et al., 2008b; Sobhaniet al., 2011), irritable bowel
syndrome (Kassinen et al.,2007; OMahony et al., 2009) and
Clostridium difficile-associated diarrhoea (Khoruts et al., 2010)
and morerecently 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 obser-vation {Duncan, 2008 #15503}
{Fleissner, 2010 #17580}{De La Serre, 2010 #16857} {Zhang, 2009
#15740}{Schwiertz, 2009 #16428}) and psychiatric diseases
(Des-bonnet et al., 2008; Rook and Lowry, 2008) {Bercik,
2011#17583}. However, there are several issues that discom-bobulate
the idea of the gut microbiota as an environmen-tal factor in these
and other diseases. First, many of thestudies look at the gut
microbiota after diagnosis of thedisease, hence we are unsure as to
whether we areobserving cause or effect. In order to circumvent
thisissue, large prospective studies need to be undertaken,which
are statistically empowered, in which frequentsamples are taken and
appropriately stored for retrospec-tive analysis. Second, we are
currently developing corre-lations between a disease state and a
snapshot ofmicrobial diversity in the gut or a potential
metabolite, weneed to develop stronger causal links and
mechanisticmodels that are predictive and can be tested in
suitableanimal models. Even with these issues researchers
aredeveloping the view that certain functions and the asso-ciated
microbes are beneficial to the health of the host.Some of the most
commonly seen bacterial metabolites inthe human gut are the SCFA,
butyrate, acetate, lactateand propionate {Saric, 2008 #15051}. The
two bacterialgroups that are mainly responsible for producing
butyrateare the F. prauznitzii and Eubacterium
rectale/Roseburiagroups {Louis, 2010 #17389; Louis, 2009 #15805}.
Thismetabolite has been implicated in large array of effects inthe
intestine that include controlling apoptosis, cytokineproduction,
energy for colonocytes and mucus synthesis{Guilloteau, 2010
#17009}. Hence any changes in thesegroups would potentially have an
impact on this functionand host physiology. Beyond this ubiquitous
function itdoes become an exercise in speculation as to what
bac-terial groups are important to host health. In one
respectmoving away from trying to define a core microbiome to acore
metabonome may aid in defining what we need tostudy and understand
in order to maintain a healthy gutand thus a healthy host.
Concluding remarks
The paradigm of the human distal gut microbiome hasshifted in
recent years, from one that looked upon it as asource of
opportunistic pathogens to one that embraces itas a virtual organ
with the ability to influence the healthstatus of the host.
Taxonomically, we have establishedthat this system is mainly
composed of members of theBacteroidetes and Firmicutes, but we are
still struggling todetermine the key functions that are important
to themicrobes and the host. The ability to catalogue the genes
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present in the distal gut does not equate to defining thecore
microbiome and using a top down approach will helpto determine this
feature in more detail. The question ofcohort sizes needs to be
addressed and to this end weneed to increase the sample sizes used,
in order todevelop a much more complete picture of the functions
inthe gut and start to combine sequence and function-based
metagenomic studies in order to determine thecore microbiome. In
addition, the integration of metabo-nomic data into this model will
help to determine the coremicrobiome and establish how it varies
both inter- andintra-individually. Once we have created this
foundationwe can commence to develop hypothesizes that addresssuch
questions as how does variation in the distal gutmicrobiome
influence host function or can we modulatethe gut microbiome in
order to promote health and shouldwe even try.
Acknowledgements
I wish to acknowledge the help of my colleagues in theEldermet
project in University College Cork for sharing theirdata and
Professor Ruth Ley for kindly providing me with heredata for Fig.
3.
References
Ahmed, S., Macfarlane, G.T., Fite, A., McBain, A.J., Gilbert,P.,
and Macfarlane, S. (2007) Mucosa-associated bacterialdiversity in
relation to human terminal ileum and colonicbiopsy samples. Appl
Environ Microbiol 73: 74357442.
Amann, R., and Kuhl, M. (1998) In situ methods for assess-ment
of microorganisms and their activities. Curr OpinMicrobiol 1:
352358.
Arumugam, M., Raes, J., Pelletier, E., Le Paslier, D.,Yamada,
T., Mende, D.R., et al. (2011) Enterotypes of thehuman gut
microbiome. Nature 473: 174178.
Attene-Ramos, M.S., Wagner, E.D., Plewa, M.J., andGaskins, H.R.
(2006) Evidence that hydrogen sulfide is agenotoxic agent. Mol
Cancer Res 4: 914.
Bale, M.J., Day, M.J., and Fry, J.C. (1988) Novel method
forstudying plasmid transfer in undisturbed river epilithon.Appl
Environ Microbiol 54: 27562758.
Biagi, E., Nylund, L., Candela, M., Ostan, R., Bucci, L.,
Pini,E., et al. (2010) Through ageing, and beyond: gut micro-biota
and inflammatory status in seniors and centenarians.PLoS ONE 5:
e10667.
Biasucci, G., Rubini, M., Riboni, S., Retetangos, C.,
Morelli,L., and Bessi, E. (2010) Mode of delivery affects the
bac-terial community in the newborn gut. Early Hum Dev 86(Suppl.
1): 1315.
Bik, E.M., Eckburg, P.B., Gill, S.R., Nelson, K.E., Purdom,E.A.,
Francois, F., et al. (2006) Molecular analysis of thebacterial
microbiota in the human stomach. Proc Natl AcadSci USA 103:
732737.
van den Bogert, B., de Vos, W.M., Zoetendal, E.G.,
andKleerebezem, M. (2011) Microarray analysis and
barcodedpyrosequencing provide consistent microbial profiles
depending on the source of human intestinal samples. ApplEnviron
Microbiol 77: 20712080.
Breitbart, M., Hewson, I., Felts, B., Mahaffy, J.M., Nulton,
J.,Salamon, P., and Rohwer, F. (2003) Metagenomic analy-ses of an
uncultured viral community from human feces.J Bacteriol 185:
62206223.
Breitbart, M., Haynes, M., Kelley, S., Angly, F., Edwards,R.A.,
Felts, B., et al. (2008) Viral diversity and dynamics inan infant
gut. Res Microbiol 159: 367373.
de Carcer, D.A., Cuiv, P.O., Wang, T., Kang, S., Worthley,
D.,Whitehall, V., et al. (2011) Numerical ecology validates
abiogeographical distribution and gender-based effect
onmucosa-associated bacteria along the human colon. ISMEJ 5:
801809.
Claesson, M.J., Cusack, S., OSullivan, O., Greene-Diniz, R.,de
Weerd, H., Flannery, E., et al. (2011) Composition, vari-ability,
and temporal stability of the intestinal microbiota ofthe elderly.
Proc Natl Acad Sci USA 108: 45864591.
Claus, S.P., Ellero, S.L., Berger, B., Krause, L., Bruttin,
A.,Molina, J., et al. (2011) Colonization-induced host-gutmicrobial
metabolic interaction. mBio 2: e0027110.
Courtois, S., Cappellano, C.M., Ball, M., Francou, F.X.,Normand,
P., Helynck, G., et al. (2003) Recombinant envi-ronmental libraries
provide access to microbial diversity fordrug discovery from
natural products. Appl Environ Micro-biol 69: 4955.
Cummings, J.H., and Macfarlane, G.T. (1997) Colonic micro-flora:
nutrition and health. Nutrition 13: 476478.
Desbonnet, L., Garrett, L., Clarke, G., Bienenstock, J.,
andDinan, T.G. (2008) The probiotic Bifidobacteria infantis:
anassessment of potential antidepressant properties in therat. J
Psychiatr Res 43: 164174.
Dominguez-Bello, M.G., Costello, E.K., Contreras, M.,Magris, M.,
Hidalgo, G., Fierer, N., and Knight, R. (2010)Delivery mode shapes
the acquisition and structure of theinitial microbiota across
multiple body habitats in new-borns. Proc Natl Acad Sci USA 107:
1197111975.
Dridi, B., Henry, M., El Khechine, A., Raoult, D., and
Dran-court, M. (2009) High prevalence of Methanobrevibactersmithii
and Methanosphaera stadtmanae detected in thehuman gut using an
improved DNA detection protocol.PLoS ONE 4: e7063.
Dridi, B., Raoult, D., and Drancourt, M. (2011) Archaea
asemerging organisms in complex human microbiomes.Anaerobe 17:
5663.
Duncan, S.H., Louis, P., and Flint, H.J. (2007)
Cultivablebacterial diversity from the human colon. Lett Appl
Micro-biol 44: 343350.
Eckburg, P.B., Bik, E.M., Bernstein, C.N., Purdom, E.,
Deth-lefsen, L., Sargent, M., et al. (2005) Diversity of the
humanintestinal microbial flora. Science 308: 16351638.
Espinoza, J., Goncalves, L.F., Romero, R., Nien, J.K.,
Stites,S., Kim, Y.M., et al. (2005) The prevalence and
clinicalsignificance of amniotic fluid sludge in patients
withpreterm labor and intact membranes. Ultrasound ObstetGynecol
25: 346352.
Favier, C.F., de Vos, W.M., and Akkermans, A.D.L.
(2003)Development of bacterial and bifidobacterial communitiesin
feces of newborn babies. Anaerobe 9: 219229.
Frank, D.N., St Amand, A.L., Feldman, R.A., Boedeker,
E.C.,Harpaz, N., and Pace, N.R. (2007) Molecular-phylogenetic
Human microbiome 3099
2011 Society for Applied Microbiology and Blackwell Publishing
Ltd, Environmental Microbiology, 13, 30883102
-
characterization of microbial community imbalances inhuman
inflammatory bowel diseases. Proc Natl Acad SciUSA 104:
1378013785.
Fry, J.C., Parkes, R.J., Cragg, B.A., Weightman, A.J.,
andWebster, G. (2008) Prokaryotic biodiversity and activity inthe
deep subseafloor biosphere. FEMS Microbiol Ecol 66:181196.
Gans, J., Wolinsky, M., and Dunbar, J. (2005)
Computationalimprovements reveal great bacterial diversity and
highmetal toxicity in soil. Science 309: 13871390.
Ghosh, T.S., Monzoorul Haque, M., and Mande, S.S.
(2010)DiScRIBinATE: a rapid method for accurate
taxonomicclassification of metagenomic sequences. BMC
Bioinfor-matics 11 (Suppl. 7): S14.
Gill, S.R., Pop, M., Deboy, R.T., Eckburg, P.B., Turnbaugh,P.J.,
Samuel, B.S., et al. (2006) Metagenomic analysis ofthe human distal
gut microbiome. Science 312: 13551359.
Gloux, K., Berteau, O., El Oumami, H., Beguet, F., Leclerc,M.,
and Dore, J. (2011) A metagenomic beta-glucuronidaseuncovers a core
adaptive function of the human intestinalmicrobiome. Proc Natl Acad
Sci USA 108 (Suppl. 1): 45394546.
Goldman, D.L., and Huffnagle, G.B. (2009) Potential
contri-bution of fungal infection and colonization to the
develop-ment of allergy. Med Mycol 47: 445456.
Gori, F., Folino, G., Jetten, M.S., and Marchiori, E. (2011)MTR:
taxonomic annotation of short metagenomic readsusing clustering at
multiple taxonomic ranks. Bioinformat-ics 27: 196203.
Harrington, C.R., Lucchini, S., Ridgway, K.P., Wegmann,
U.,Eaton, T.J., Hinton, J.C.D., et al. (2008) A
short-oligonucleotide microarray that allows improved detectionof
gastrointestinal tract microbial communities. BMC Micro-biol 8:
195.
Hugenholtz, P., Goebel, B.M., and Pace, N.R. (1998) Impactof
culture-independent studies on the emerging phyloge-netic view of
bacterial diversity. J Bacteriol 180: 47654774.
Huson, D.H., Auch, A.F., Qi, J., and Schuster, S.C. (2007)MEGAN
analysis of metagenomic data. Genome Res 17:377386.
Jones, B.V. (2010) The human gut mobile metagenome, ametazoan
perspective. Gut Microbes 1: 415431.
Jones, B.V., and Marchesi, J.R. (2007) Transposon-aidedcapture
(TRACA) of plasmids resident in the human gutmobile metagenome. Nat
Methods 4: 5561.
Jones, B.V., Begley, M., Hill, C., Gahan, C.G.M., andMarchesi,
J.R. (2008) Functional and comparative metage-nomic analysis of
bile salt hydrolase activity in the humangut microbiome. Proc Natl
Acad Sci USA 105: 1358013585.
Jones, B.V., Sun, F., and Marchesi, J.R. (2010)
Comparativemetagenomic analysis of plasmid encoded functions in
thehuman gut microbiome. BMC Genomics 11: 46.
Kassinen, A., Krogius-Kurikka, L., Makivuokko, H., Rinttila,T.,
Paulin, L., Corander, J., et al. (2007) The fecal micro-biota of
irritable bowel syndrome patients differs signifi-cantly from that
of healthy subjects. Gastroenterology 133:2433.
Khoruts, A., Dicksved, J., Jansson, J.K., and Sadowsky, M.J.
(2010) Changes in the composition of the human fecalmicrobiome
after bacteriotherapy for recurrent clostridiumdifficile-associated
diarrhea. J Clin Gastroenterol 44: 354360.
Koenig, J.E., Spor, A., Scalfone, N., Fricker, A.D., Stom-baugh,
J., Knight, R., et al. (2011) Succession of microbialconsortia in
the developing infant gut microbiome. ProcNatl Acad Sci USA 108:
45784585.
Kurokawa, K., Itoh, T., Kuwahara, T., Oshima, K., Toh,
H.,Toyoda, A., et al. (2007) Comparative metagenomicsrevealed
commonly enriched gene sets in human gutmicrobiomes. DNA Res 14:
169181.
Lakhdari, O., Cultrone, A., Tap, J., Gloux, K., Bernard,
F.,Ehrlich, S.D., et al. (2010) Functional metagenomics: ahigh
throughput screening method to decipher microbiota-driven NF-kappaB
modulation in the human gut. PLoSONE 5: e13092.
Lango Allen, H., Estrada, K., Lettre, G., Berndt, S.I.,
Weedon,M.N., Rivadeneira, F., et al. (2010) Hundreds of
variantsclustered in genomic loci and biological pathways
affecthuman height. Nature 467: 832838.
Lepage, P., Colombet, J., Marteau, P., Sime-Ngando, T.,Dor, J.,
and Leclerc, M. (2008) Dysbiosis in inflammatorybowel disease: a
role for bacteriophages? Gut 57: 424425.
Ley, R.E., Peterson, D.A., and Gordon, J.I. (2006) Ecologicaland
evolutionary forces shaping microbial diversity in thehuman
intestine. Cell 124: 837848.
Ley, R.E., Hamady, M., Lozupone, C., Turnbaugh, P.J.,Ramey,
R.R., Bircher, J.S., et al. (2008) Evolution ofmammals and their
gut microbes. Science 320: 16471651.
Louis, P., Young, P., Holtrop, G., and Flint, H.J. (2010)
Diver-sity of human colonic butyrate-producing bacteria revealedby
analysis of the butyryl-CoA:acetate CoA-transferasegene. Environ
Microbiol 12: 304314.
McConnell, E.L., Fadda, H.M., and Basit, A.W. (2008)
Gutinstincts: explorations in intestinal physiology and
drugdelivery. Int J Pharm 364: 213226.
McNeil, N.I. (1984) The contribution of the large intestine
toenergy supplies in man. Am J Clin Nutr 39: 338342.
Manichanh, C., Chapple, C.E., Frangeul, L., Gloux, K.,Guigo, R.,
and Dore, J. (2008) A comparison of randomsequence reads versus 16S
rDNA sequences for estimat-ing the biodiversity of a metagenomic
library. Nucleic AcidRes 36: 51805188.
Martin, F.P., Dumas, M.E., Wang, Y., Legido-Quigley, C.,
Yap,I.K., Tang, H., et al. (2007) A top-down systems biologyview of
microbiome-mammalian metabolic interactions in amouse model. Mol
Syst Biol 3: 112.
Martin, F.P.J., Sprenger, N., Yap, I.K.S., Wang, Y., Bibiloni,
R.,Rochat, F., et al. (2009) Panorganismal gut microbiome-host
metabolic crosstalk. J Proteome Res 8: 20902105.
Meyer, F., Paarmann, D., DSouza, M., Olson, R., Glass,E.M.,
Kubal, M., et al. (2008) The metagenomics RASTserver a public
resource for the automatic phylogeneticand functional analysis of
metagenomes. BMC Bioinfor-matics 9: 386.
Mihajlovski, A., Alric, M., and Brugre, J.-F. (2008) A
putativenew order of methanogenic Archaea inhabiting the human
3100 J. R. Marchesi
2011 Society for Applied Microbiology and Blackwell Publishing
Ltd, Environmental Microbiology, 13, 30883102
-
gut, as revealed by molecular analyses of the mcrA gene.Res
Microbiol 159: 516521.
Mihajlovski, A., Dor, J., Levenez, F., Alric, M., and
Brugre,J.F. (2010) Molecular evaluation of the human gut
metha-nogenic archaeal microbiota reveals an age-associatedincrease
of the diversity. Environ Microbiol Rep 2: 272280.
Momozawa, Y., Deffontaine, V., Louis, E., and Medrano,J.F.
(2011) Characterization of bacteria in biopsies of colonand stools
by high throughput sequencing of the V2 regionof bacterial 16S rRNA
gene in human. PLoS ONE 6:e16952.
Morowitz, M.J., Denef, V.J., Costello, E.K., Thomas,
B.C.,Poroyko, V., Relman, D.A., and Banfield, J.F. (2011)
Strain-resolved community genomic analysis of gut
microbialcolonization in a premature infant. Proc Natl Acad Sci
USA108: 11281133.
Nicholson, J.K., Holmes, E., and Wilson, I.D. (2005)
Gutmicroorganisms, mammalian metabolism and personal-ized health
care. Nat Rev Microbiol 3: 431438.
Ochman, H., Lawrence, J.G., and Groisman, E.A. (2000)Lateral
gene transfer and the nature of bacterial innovation.Nature 405:
299304.
OHara, A.M., and Shanahan, F. (2006) The gut flora as aforgotten
organ. EMBO Rep 7: 688693.
OMahony, S.M., Marchesi, J.R., Scully, P., Codling, C.,Ceolho,
A.M., Quigley, E.M., et al. (2009) Early life stressalters
behavior, immunity, and microbiota in rats: implica-tions for
irritable bowel syndrome and psychiatric illnesses.Biol Psychiatry
65: 263267.
Ott, S.J., Khbacher, T., Musfeldt, M., Rosenstiel, P.,
Hellmig,S., Rehman, A., et al. (2008) Fungi and inflammatorybowel
diseases: alterations of composition and diversity.Scand J
Gastroenterol 43: 831841.
Oxley, A.P., Lanfranconi, M.P., Wurdemann, D., Ott,
S.,Schreiber, S., McGenity, T.J., et al. (2010) Halophilicarchaea
in the human intestinal mucosa. Environ Microbiol12: 23982410.
Paliy, O., Kenche, H., Abernathy, F., and Michail, S.
(2009)High-throughput quantitative analysis of the human
intes-tinal microbiota with a phylogenetic microarray. ApplEnviron
Microbiol 75: 35723579.
Palmer, C., Bik, E.M., DiGiulio, D.B., Relman, D.A., andBrown,
P.O. (2007) Development of the human infant intes-tinal microbiota.
PLoS Biol 5: e177.
Qin, J., Li, R., Raes, J., Arumugam, M., Burgdorf, K.S.,
Man-ichanh, C., et al. (2010) A human gut microbial gene cata-logue
established by metagenomic sequencing. Nature464: 5965.
Rajilic-Stojanovic, M., Heilig, H.G.H., Molenaar, D.,
Kajander,K., Surakka, A., Smidt, H., and De Vos, W.M. (2009)
Devel-opment and application of the human intestinal tract chip,
aphylogenetic microarray: analysis of universally
conservedphylotypes in the abundant microbiota of young and
elderlyadults. Environ Microbiol 11: 17361751.
Reyes, A., Haynes, M., Hanson, N., Angly, F.E., Heath,
A.C.,Rohwer, F., and Gordon, J.I. (2010) Viruses in the
faecalmicrobiota of monozygotic twins and their mothers. Nature466:
334338.
Rigsbee, L., Agans, R., Foy, B.D., and Paliy, O.
(2011)Optimizing the analysis of human intestinal microbiota
with phylogenetic microarray. FEMS Microbiol Ecol 75:332342.
Romero, R., Schaudinn, C., Kusanovic, J.P., Gorur, A.,Gotsch,
F., Webster, P., et al. (2008) Detection of a micro-bial biofilm in
intraamniotic infection. Am J Obstet Gynecol198:
135.e131135.e135.
Rook, G.A., and Lowry, C.A. (2008) The hygiene hypothesisand
psychiatric disorders. Trends Immunol 29: 150158.
Rudi, K., Zimonja, M., and Naes, T. (2006) Alignment-independent
bilinear multivariate modelling (AIBIMM) forglobal analyses of 16S
rRNA gene phylogeny. Int J SystEvol Microbiol 56: 15651575.
Rudi, K., Zimonja, M., Kvenshagen, B., Rugtveit, J.,
Midtvedt,T., and Eggesbo, M. (2007) Alignment-Independent
com-parisons of human gastrointestinal tract microbial commu-nities
in a multidimensional 16S rRNA gene evolutionaryspace. Appl Environ
Microbiol 73: 27272734.
Sahakian, A.B., Jee, S.R., and Pimentel, M. (2010) Methaneand
the gastrointestinal tract. Dig Dis Sci 55: 21352143.
Scanlan, P.D., and Marchesi, J.R. (2008)
Micro-eukaryoticdiversity of the human distal gut microbiota:
qualitativeassessment using culture-dependent and
-independentanalysis of faeces. ISME J 2: 11831193.
Scanlan, P.D., Shanahan, F., OMahony, C., and Marchesi,J.R.
(2006) Culture-independent analyses of the temporalvariation of the
dominant faecal microbiota and targetedbacterial sub-groups in
Crohns disease. J Clin Microbiol44: 39803988.
Scanlan, P.D., Shanahan, F., and Marchesi, J.R. (2008a)Human
methanogen diversity and incidence in healthy anddiseased colonic
groups using mcrA gene analysis. BMCMicrobiol 8: 79.
Scanlan, P.D., Shanahan, F., Clune, Y., Collins, J.K.,OSullivan,
G.C., ORiordan, M., et al. (2008b) Culture-independent analysis of
the gut microbiota in colorectalcancer and polyposis. Environ
Microbiol 10: 789798.
Schwiertz, A., Gruhl, B., Lobnitz, M., Michel, P., Radke, M.,and
Blaut, M. (2003) Development of the intestinal bacte-rial
composition in hospitalized preterm infants in compari-son with
breast-fed, full-term infants. Pediatr Res 54: 393399.
Sekelja, M., Berget, I., Ns, T., and Rudi, K. (2011) Unveilingan
abundant core microbiota in the human adult colon by
aphylogroup-independent searching approach. ISME J 5:519531.
Sekirov, I., Russell, S.L., Antunes, L.C.M., and Finlay,
B.B.(2010) Gut microbiota in health and disease. Physiol Rev90:
859904.
Smalla, K., and Sobecky, P.A. (2002) The prevalence anddiversity
of mobile genetic elements in bacterial communi-ties of different
environmental habitats: insights gainedfrom different
methodological approaches. FEMS MicrobiolEcol 42: 165175.
Sobhani, I., Tap, J., Roudot-Thoraval, F., Roperch,
J.P.,Letulle, S., Langella, P., et al. (2011) Microbial dysbiosis
incolorectal cancer (CRC) patients. PLoS ONE 6: e16393.
Staley, J.T., and Konopka, A. (1985) Measurement of in
situactivities of nonphotosynthetic microorganisms in aquaticand
terrestrial habitats. Annu Rev Microbiol 39: 321346.
Human microbiome 3101
2011 Society for Applied Microbiology and Blackwell Publishing
Ltd, Environmental Microbiology, 13, 30883102
-
Suttle, C.A. (2007) Marine viruses major players in theglobal
ecosystem. Nat Rev Microbiol 5: 801812.
Tap, J., Mondot, S., Levenez, F., Pelletier, E., Caron,
C.,Furet, J.P., et al. (2009) Towards the human
intestinalmicrobiota phylogenetic core. Environ Microbiol 11:
25742584.
Turnbaugh, P.J., Hamady, M., Yatsunenko, T., Cantarel,
B.L.,Duncan, A., Ley, R.E., et al. (2009) A core gut microbiomein
obese and lean twins. Nature 457: 480484.
Van Melderen, L. (2010) Toxin-antitoxin systems: why somany,
what for? Curr Opin Microbiol 13: 781785.
Wade, W. (2002) Unculturable bacteria the
uncharacterizedorganisms that cause oral infections. J R Soc Med
95:8183.
Walker, A.W., Sanderson, J.D., Churcher, C., Parkes,
G.C.,Hudspith, B.N., Rayment, N., et al. (2011)
High-throughputclone library analysis of the mucosa-associated
microbiotareveals dysbiosis and differences between inflamed
andnon-inflamed regions of the intestine in inflammatory
boweldisease. BMC Microbiol 11: 7.
Wang, Z., Klipfell, E., Bennett, B.J., Koeth, R., Levison,
B.S.,DuGar, B., et al. (2011) Gut flora metabolism of
phosphati-dylcholine promotes cardiovascular disease. Nature
472:5763.
Waniewski, R.A., and Martin, D.L. (1998) Preferential
utiliza-tion of acetate by astrocytes is attributable to
transport.J Neurosci 18: 52255233.
Whitman, W.B., Coleman, D.C., and Wiebe, W.J. (1998)Prokaryotes:
the unseen majority. Proc Natl Acad Sci USA95: 65786583.
Wikoff, W.R., Anfora, A.T., Liu, J., Schultz, P.G., Lesley,
S.A.,Peters, E.C., and Siuzdak, G. (2009) Metabolomics analy-sis
reveals large effects of gut microflora on mammalianblood
metabolites. Proc Natl Acad Sci USA 106: 36983703.
Wilks, M. (2007) Bacteria and early human development.Early Hum
Dev 83: 165170.
Wostmann, B.S. (1973) Intestinal bile acids and
cholesterolabsorption in the germfree. J Nutr 103: 982990.
Wright, G.D. (2007) The antibiotic resistome: the nexus
ofchemical and genetic diversity. Nat Rev Microbiol 5: 175186.
Yang, X., Xie, L., Li, Y., and Wei, C. (2009) More than9,000,000
unique genes in human gut bacterial commu-nity: estimating gene
numbers inside a human body. PLoSONE 4: e6074.
Zhang, T., Breitbart, M., Lee, W.H., Run, J.-Q., Wei, C.L.,Soh,
S.W.L., et al. (2005) RNA Viral community in humanfeces: prevalence
of plant pathogenic viruses. PLoS Biol 4:e3.
Zhang, H., Dibaise, J.K., Zuccolo, A., Kudrna, D., Braidotti,M.,
Yu, Y., et al. (2009) Human gut microbiota in obesityand after
gastric bypass. Proc Natl Acad Sci USA 106:23652370.
Zoetendal, E.G., von Wright, A., Vilpponen-Salmela, T., BenAmor,
K., Akkermans, A.D., and de Vos, W.M. (2002)Mucosa-associated
bacteria in the human gastrointestinaltract are uniformly
distributed along the colon and differfrom the community recovered
from feces. Appl EnvironMicrobiol 68: 34013407.
3102 J. R. Marchesi
2011 Society for Applied Microbiology and Blackwell Publishing
Ltd, Environmental Microbiology, 13, 30883102
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