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Supplement Article

Role of the intestinal microbiome in health and disease: from

correlation to causation
Willem M de Vos and Elisabeth AJ de Vos

Recorded observations indicating an association between intestinal microbes

and health are long-standing in terms of specific diseases, but emerging
high-throughput technologies that characterize microbial communities in the
intestinal tract are suggesting new roles for the supposedly normal microbiome. This
review considers the nature of the evidence supporting a relationship between the
microbiota and the predisposition to disease as associative, correlative, or causal.
Altogether, indirect or associative support currently dominates the evidence base,
which now suggests that the intestinal microbiome can be linked to a growing
number of over 25 diseases or syndromes. While only a handful of cause-and-effect
studies have been performed, this form of evidence is increasing. The results of such
studies are expected to be useful in monitoring disease development, in providing a
basis for personalized treatments, and in indicating future therapeutic avenues.
© 2012 International Life Sciences Institute

INTRODUCTION personalized human organ with a metabolic activity

second only to that of the liver.4 It also resulted in a
Virtually every day we are all confronted with the activity change in terminology: what was first known as “micro-
of our intestine, and it is no surprise that at least some of flora” – a term still found in some publications and
us have developed a fascination for our intestinal condi- medical textbooks – has now been renamed “microbiota”
tion and its relation to health and disease. Following his on the basis of the diversity of microorganisms revealed
discovery of microbial life, Antonie van Leeuwenhoek mainly by microbial ecologists, who used molecular sys-
reported in 1681 the first observation relating a disturbed tematics and positioned microbes in ancestral evolution-
microbial composition to the diarrhea he experienced, ary terms far away from plants.5 With the implementation
possibly after drinking polluted Amsterdam canal water.1 of genomics-based approaches, the exploration of the
The account that his watery excrements contained more human intestinal microbiome, defined here as all micro-
and different “little animals,” as he called the bacteria, is biota in the intestinal tract, has begun. The collective
largely correct. It reflects the association between some genomes within the microbiome have been found to
forms of diarrhea and a sudden shedding of intestinal contain more than 3 million unique genes.6
microbes, including those that inhabit the mucosa. As we It is known that the intestinal microbiota shows a
now know, the mucosal microbial communities may specific spatial organization,2 but as the vast majority of
differ in composition and abundance from those present microbes are found in the colon, virtually all present-day
in the colon.2 This hallmark discovery of several centuries studies focus on the microbiome that is recovered from
ago was not followed up by further well-documented fecal samples. An understanding of the human intestinal
work until the end of the last century, when interest in the microbiome is now rapidly developing, and in many cases
intestinal microbes experienced a real renaissance.3 It led the relationship between the microbiome and health and
to the notion that intestinal microbes can be considered a disease is being explored. In most instances, however, this

Affiliations: WM de Vos is with the Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands, and the
Departments of Veterinary Biosciences and Bacteriology and Immunology, Helsinki University, Helsinki, Finland. EAJ de Vos is with the
Medical School, Free University Amsterdam, Amsterdam, The Netherlands.
Correspondence: WM de Vos, Laboratory of Microbiology, Dreijenplein 10, NL-6703 HB Wageningen, The Netherlands. E-mail:
[email protected]. Phone: +31-653735635.
Key words: disease, health, high-throughput approaches, microbiota

doi:10.1111/j.1753-4887.2012.00505.x
Nutrition Reviews® Vol. 70(Suppl. 1):S45–S56 S45
has simply meant an analysis of associations with disease MICROBIOTA COMPOSITION, THE COMMON CORE, AND
or functional disturbances, and only in special cases are A CALL FOR CAUTION
specific correlations described in which specific microbial
groups relate to a healthy or a diseased state in a manner A main driver for the increased understanding of the
that implies a linear relationship. Finally, there are only intestinal microbiome has been the development of
a handful of examples in which the cause-and-effect molecular and high-throughput tools that obviated the
relations satisfying Koch’s postulates apply, but even need for culturing and permitted the analysis of micro-
these relate mainly to studies in animal models, thereby bial function.7 The application of these tools reinforced
providing hypotheses for human disease and human the conclusions of a decade ago that humans are colo-
intervention tests. While one may argue that probiotic nized from birth by a developing intestinal microbiota
interventions can be seen as providing causal evidence for that, in adult life, is highly individual, temporally stable,
their roles, these interventions involve the use of specific and similar in monozygotic twins and other genetically
bacteria that may have a direct effect on the host rather related subjects.8–10 Moreover, metagenomic develop-
than on the intestinal microbiota per se. Probiotics are ments with next-generation technology (NGT) sequenc-
therefore not considered here, particularly as they have ing approaches have now provided a catalog of over 3
been reviewed exhaustively elsewhere. million genes, which, in terms of the average microbiota
Following a short overview of the present knowledge composition, are derived mainly from prokaryotic Bacte-
of the human microbiota and the available high- ria and, to a lesser extent, Archaea, with only a few fungal
throughput analytical approaches along with their genes encountered.6 This and other analyses based on
promise and pitfalls, the most noteworthy studies that quantitative analysis11 showed the human intestinal com-
relate to the human microbiome in health and its predis- munities to be highly complex, predicted to contain more
position to disease are summarized. Where possible, evi- than 1,000 different prokaryotic species belonging to a
dence suggesting simply correlations is differentiated limited set of a dozen taxa and dominated by gram-
from that suggesting actual causal relationships. It is positive anaerobes (Figure 1). Further computational
expected that this information will 1) provide a concep- analysis has led to the notion that the apparently diverse
tual basis for how the human intestinal microbiome microbial communities can be grouped into three
affects disease, 2) contribute to the development of tech- so-called enterotypes, consisting of networks between
niques for monitoring disease development, 3) provide a different microbial groups that are robust and evident in
basis for personalized treatment, and 4) indicate future subjects from different continents.12 It has been suggested
therapeutic avenues. that these enterotypes may well affect the response of

Figure 1 Phylogenetic tree of the most important microbial taxa in the human intestinal tract, along with their
relative contributions.

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subjects to dietary and pharmaceutical interventions, and factor has been the depth of the analysis that can be seen
hence it is of interest that a biased distribution was in terms of the rRNA sequences that can be reliably quan-
observed in irritable bowel syndrome (IBS) patients13 and tified. Phylogenetic microarrays such as the HITChip may
that the enterotypes were reportedly affected by diet.14 quantify the microbiota in a highly reproducible way,
The intestinal microbial communities are highly representing a depth of 10-4 to 10-5, which is comparable
complex; therefore, the analytical coverage of this com- to over 200,000 reads (approximately 100 Mb of sequence
plexity, its reproducibility, and its accuracy are important information) on an NGT sequencer.18 Since the fraction
factors that determine the quality of the analytical assess- of single microbial groups may vary 100- to 1,000-fold, it
ment. Yet, despite the rapid developments of NGT is critical to have a detection limit as low as possible to
sequencing systems, deep metagenomic sequence analysis obtain good discrimination in the microbiota analysis.
is still time-consuming, costly, and rather challenging This is exemplified in a simple experiment in which
from a bioinformatics and data storage point of view. the microbiotas of 65 healthy subjects of different nation-
Hence, most studies addressing the intestinal Bacteria and alities were analyzed using the HITChip17 (Figure 2). In a
Archaea have focused on the 1.5-kb bacterial 16S rRNA single subject, around 900 phylotype-like species could be
gene sequences that are well-established phylogenetic detected, which is slightly higher than the number found
markers.5 There is already a growing number of over a by ultra-deep metagenomic analysis,6 which emphasizes
million entries in accessible databases. Only a fraction the depth of the HITChip analysis. Similar studies have
derive from the human intestinal microbiota, and these been reported with different-sized groups of subjects, and
have been curated some years ago, leading to the conclu- shared phylotype-like sequences were identified as what
sion that only approximately 400 species have been cul- is known as the “common core.”20,21 It is evident from the
tured from human intestine.15 Since only microbes that displayed plot of sequences that this common core is
have been cultured can be characterized taxonomically, dependent not only on the number of subjects but,
this leads to the sometimes difficult situation that a spe- notably, on the depth of the analysis (Figure 2A). In these
cific 16S rRNA sequence or signature is now found to be healthy subjects, a common core of over 450 species-like
associated with disease or a healthy state, but this rRNA taxa could be defined. Detailed analysis of this common
sequence cannot be assigned to a species. These may then core showed it to consist of a series of well-known species
be termed species-level phylotypes or operational taxo- or genera, including those belonging to Bifidobacterium,
nomic units that are identified only by their complete 16S Clostridium, Colinsella, Dorea, Eubacterium, Para-
rRNA sequence and usually defined as sharing 97% or less bacteroides, Prevotella, Ruminococcus, and Streptococcus
sequence identity with other entries in the ribosomal spp. (Figure 2B). The vast majority of this common core
databases.16 Applying this strict criterion has led to a data- (a total of 387, representing 85%), however, included
base of around 1,200 different phylotypes that provide a microbes that have not yet been cultured and hence are
systematic framework of the microbial diversity in the phylotypes that show similarity to 16S rRNA sequences
human gastrointestinal tract.15 This information is of from bacteria that have not yet been cultured.
great value in defining a healthy microbiota and compar- In spite of the many technological advances in the last
ing it with that of diseased subjects. Moreover, this data- decade, there are still considerable challenges that call for
base has been instrumental in the design of high- caution. Several problems are related to the inappropriate
throughput approaches such as the Human Intestinal use of technologies, the interpretation of results, or other
Tract Chip (HITChip), a phylogenetic DNA microarray systematic errors.22 DNA isolation and PCR amplification
for the comprehensive analysis of gastrointestinal tract seem trivial but have been shown to provide biased views
microbiota at multiple levels of taxonomic resolution.7,17 that may explain retrospectively the early observations
A wide range of high-throughput approaches, mainly that adult samples lack Bacteroides spp. or that samples
based on microarray hybridization, polymerase chain from babies do not contain bifidobacteria.23 Moreover, the
reaction (PCR), and NGT sequence analysis as well as interpretation of 16S rRNA sequence information should
combinations thereof, have been applied successfully to be based on the appropriate knowledge and use of this
monitor the human intestinal microbiota, and these have phylogenetic biomarker.5 A recent careful analysis of
been reviewed extensively.7 While instrumental in pro- mock mixtures of microbial DNA by NGT sequencing
viding deep insight, they all suffer from inherent biases methods revealed a large range of inaccuracies and indi-
that vary from sequence errors, i.e., PCR chimers or cated that many new taxa are incorrectly identified due to
cloning artifacts, to cross-hybridization. However, several chimer formation. This can now be avoided by new algo-
of these approaches have been compared to each other, rithms, but these are still imperfect, and hence low-
and, overall, highly similar results were obtained by abundance taxa should be treated with caution and as
HITChip analysis and NGT sequencing of diagnostic potential artifacts.24 Another issue is that many prokary-
regions of 16S rRNA amplicons.18,19 A discriminating otes have multiple copies of 16S rRNA in their genomes.

Nutrition Reviews® Vol. 70(Suppl. 1):S45–S56 S47

 A B

Known Core Genus/Species Number of Species

Akkermansia muciniphila 1
Alistipes finegoldii 1
Anaerotruncus colihominis 1
Bacteroides spp. 11
Bifidobacterium spp. 2
Clostridium spp. 13
Colinsella spp. 2
Dorea spp. 2
Eubacterium spp. 9
Faecalibacterium prausnitzii 1
Lactobacillus lactis 1
Lachnobacterium spp. 1
Lachnospira pectinoschiza 1
Parabacteroides spp. 3
Prevotella spp. 2
Roseburia intestinalis 1
Ruminococcus spp. 13
Streptococcus spp. 18
Subdoligranulum variabile 1
Sutterella wadsworthia 1
Uncultured Phylotypes 387

Figure 2 The common core microbiota in healthy subjects. A. The perspective plot shows the common core size of phylo-
types (vertical) as a function of the prevalence in the number of healthy subjects (all controlled for quality of life) and the depth
of the analysis as a log intensity of the hybridization signals. B. The composition of the common core of over 50 healthy
subjects; the number of species-like phylotypes is indicated. The data were processed as described previously.20

This should be taken into consideration when addressing ing health, however, is much more complex than defining
quantitative effects. Moreover, in some cases, significant disease, as has been recently emphasized.27 The World
sequence heterogeneity exists in the copies of the 16S Health Organization (WHO) has defined health as a state
rRNA genes, such as in Bifidobacterium adolescentis, an of complete physical, mental, and social well-being and
important inhabitant of the adult colon.25 Similarly, not merely as the absence of disease or infirmity.28 It is of
metagenomic studies also suffer from biases, albeit of a interest to note that, in addition to the physical state, the
different kind, such as inaccurate quantifications of bacte- mental and social aspects of well-being are also included.
rial populations, limitations of databases or computing These are of relevance to the intestinal tract, which is
capacity,and incorrect assembly of short reads or repeated composed of a single layer of epithelial cells surrounded
sequences,including rRNA operons.6,26 However,the most by the enteric nervous system, the largest reservoir of
important factors that may explain most of the differences nerve cells in the human body apart from that in the
in the present literature include the limited number of brain. Hence, the intestinal tract is part of the brain-gut
samples analyzed, the great variety of analysis platforms, axis and has also been recognized as the second brain.29 In
and the differences between experimental procedures. recent years, it has become evident that the intestinal
While this emphasizes the need for standardization, it also microbes communicate with the epithelial cells, as shown
implies that caution should be used when comparing by the extension of early observations in monoassociated
various studies unless rigorous tests for robustness and mouse models30 to new studies in healthy human volun-
reproducibility have been performed. teers.31 It has also now emerged that specific intestinal
microbes also interact with the enteric nervous system. So
MICROBIOTA IN HEALTH: COMPOSITION, ACTIVITY, far, this has only been reported for animal models, but the
AND STABILITY impact on animal behavior and anxiety is striking.32–34 It is
therefore appropriate to consider the intestinal micro-
When addressing the intestinal microbiota in disease, it is biota in the context of the WHO definition of health by
essential to know the baseline in healthy subjects. Defin- including mental and social aspects. The recent conclu-

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sion from experimental animals that their social behavior ics, such as metatranscriptomics, metaproteomics, and
changes in relation to the microbiota also allows new metabolomics.7 While metatranscriptomics is a high-
experimental approaches to test the nature of microbiota/ throughput method that exploits NGT sequence analysis,
behavioral relationships.35 the recovery of mRNA from the intestinal tract is a great
In operational terms, it is not easy to apply the com- challenge because the half-life of mRNA in prokaryotes is
plete WHO definition of health. In most cases, compara- in the order of minutes. So far, metatranscriptomic
tive studies are performed with control groups of healthy approaches have been successfully applied to intestinal
subjects who have been selected on the basis of the systems with a sufficiently high flux that permits rapid
absence of disease and infirmity. In some cases, however, sampling and processing. These include the intestinal
extensive and validated questionnaires have been used tracts of babies and ileostoma patients, in whom vitamin
that address the quality of life (QoL) and incorporate the production and sugar metabolism by bifidobacteria and
physical, social, and mental aspects in some way. These streptococci, respectively, were found to be among the
are particularly useful in differentiating healthy subjects most abundant functions.41,42 Metaproteomics capitalizes
from those who suffer from IBS, a frequently occurring on the fast and global mass spectrometry analysis of pro-
aberration that involves the brain-gut axis.36 In the data teins that are generally much more stable than transcripts.
presented above (Figure 2), QoL questionnaires were While exploiting the rapidly growing metagenome data-
administered to the subjects, so the common core defined bases, metaproteomics has developed into an established
in this group may represent a healthy microbiota. This tool to assess microbial function in the complex ecosys-
work was recently extended in a similar analysis of over tem of the intestinal tract.43–46 The potential of a new
100 healthy subjects using the same HITChip platform.21 metaproteomics annotation approach has been illus-
When the same approach was applied to the intestinal trated by revealing the in situ activity of mucus-degrading
microbiota of patients with ulcerative colitis (UC), Akkermansia spp., a member of the Verrucomicrobia
however, one of the forms of inflammatory bowel disease (Figure 1), in healthy volunteers.44 Last, but not least,
(IBD; see below), a much smaller general core was found. there is the metabolomics approach, which is a powerful
Moreover, the common core found in UC patients was tool that has been used in a great variety of studies
markedly different from that in the healthy subjects, illus- addressing the impact of the intestinal microbiota on
trating the power of this comparative approach.21 Apart human health.47 Notably, urine and blood metabolomics
from detecting compositional differences in health and provided new insight into microbiota function, leading to
disease, the diversity of the intestinal microbiota is also the recent discovery of the involvement of intestinal
often addressed. Diversity is here defined in the ecological microbes in promoting suitable diets for patients with
terms of species richness and evenness, reflecting the atherosclerosis.48 One of the limitations of the present-
number of phylotypes and their relative abundance. This day metabolomics, however, lies in identifying the
is particularly important because there is a strong corre- observed metabolites and, in some cases, reliably deter-
lation between species diversity and resilience in many mining their concentration.
ecosystems, and there is no reason to assume this is dif- While these functional metagenomic tools will be
ferent for the healthy intestinal microbiota. While the instrumental in analyzing the relation between micro-
microbial diversity increases rapidly in early life, it has biota function and health, they have not yet been applied
been found to stabilize during adulthood and is main- in large-scale studies comparing healthy and diseased
tained stably throughout all later phases, though it may subjects. Hence, in the section below, the main focus is
decrease slightly in subjects over 100 years of age.37 In on global microbiota analysis based on high-throughput
several diseases, there is a marked effect on the microbial approaches. It is expected, however, that in the near
diversity, the most prominent being that observed in future such studies will be paralleled by functional
patients with recurrent Clostridium difficile infection approaches that expand beyond only the composition of
(CDI).38 the microbiota.
Apart from the microbial composition, the activity of Apart from the composition and function of the
the intestinal microbiota is a major contributing factor to microbiota, a third factor needs to be addressed, and that
health and disease. Various ways to define the activity of is time. The temporal variation of the microbiota compo-
the intestinal microbiota have been described.39 By using sition in healthy subjects has been addressed in various
cell sorting combined with specific dyes, it was found that time windows, varying from weeks to months to
fecal samples on average contain approximately 30% dead years.17,20,49 In all cases, a high temporal stability was
and 20% injured cells that show a nonstochastic phyloge- observed that resulted in the maintenance of a recogniz-
netic distribution, probably because some bacterial able individual microbiota composition for periods of
groups are more easily damaged than others.40 More over 10 years. However, the immediate effects of anti-
global approaches capitalize on functional metagenom- biotic use were observed and affected the temporal

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stability,20 confirming model experiments with a small The majority of the studies relating microbiota and
number of volunteers.50 Moreover, in a weekly follow-up disease concern only a few aberrations that have a promi-
study of QoL-controlled healthy subjects, it was observed nent effect on health; these include IBD, IBS, and CDI
that traveling across time zones may affect the temporal (Table 1). The two main IBD conditions, Crohn’s disease
stability of the microbiota.20 In addition, by linking intes- (CD) and UC, have been associated with genetic predis-
tinal health to the microbiota, it could be established that positions, and several dozens of host genes have been
abdominal pain was inversely correlated with the described, reflecting the complexity of these diseases.51
amounts of bifidobacteria.20 These unexpected observa- The intestinal microbiota associations in IBD have been
tions testify to the power of these global approaches and studied by comparing healthy and compromised subjects.
provide a basis for further prospective studies to establish To correct for the genetic impact, however, monozygotic
cause-and-effect relationships. Moreover, they underline and dizygotic twins discordant for the disease are often
the need to incorporate time as an additional factor to studied. Both CD and UC are associated with a reduced
take account of the temporal stability of the intestinal diversity of the intestinal microbiota. There are, however,
microbiota. marked differences between the two diseases that reflect
their very different nature. In CD, reduced numbers of
STRONG ASSOCIATIONS OF MICROBIOTA AND DISEASE: Faecalibacterium prausnitzii have been repeatedly
CAUSE-AND-EFFECT STUDIES observed, and this anaerobic butyrate producer is
reported to have anti-inflammatory properties in a mouse
In recent years, associations with varying degrees of model.52 Similarly, in a comparative study of IBD-
support have been established between human intestinal discordant twins, an increased level of Faecalibacterium
microbiota and an increasing number of over 25 diseases, prausnitzii was also found in the CD patients, but the
syndromes, or functional aberrations. The support for microbiota of the UC patients were similar to those of
these associations can vary from anecdotal indications, their healthy twin siblings.53 This latter finding may be
such as those described below, to much firmer evidence attributed to the limited depth of the analysis or to other
obtained from large cohorts. Here, the focus is on 10 of technical factors, since other studies showed marked dif-
the strongest associations that are supported by multiple ferences in the microbiota of UC patients.35,55 Moreover,
studies (Table 1). Specific correlations between function in a recent study that specifically addressed the mucosal
or disease and intestinal microbes and, where possible, bacteria in IBD patients, the level of Akkermansia
causation are also described and, in some cases, are sup- muciniphila was reported to be 10-fold reduced in CD
ported by studies in animal models. patients and 100-fold reduced in UC patients, and it was

Table 1 Intestinal microbiota-associated diseases, syndromes, or other aberrations, with summaries of multiple
studies that support an association between the microbiota and the indicated aberration.
Aberration Most relevant observations and References
potential correlation
Crohn’s disease Diversity decrease – reduced F. prausnitzii Kaser et al. 201051; Sokol et al. 200952;
Willing et al. 201053
Ulcerative colitis Diversity decrease – reduced A. muciniphila Png et al. 201054; Kaser et al. 201051 ;
Lepage et al. 201155
Irritable bowel Global signatures – increased Dorea and Salonen et al. 201036; Saulnier et al. 201156;
syndrome Ruminococcus Rajilić-Stojanović et al. 201113
Clostridium difficile Strong diversity decrease – presence of C. difficile Grehan et al. 201057; Khoruts et al. 201058
infection
Colorectal cancer Variation in Bacteroides spp. – increased Sobhani et al. 201159; Wang et al. 201260;
fusobacteria Marchesi et al. 201161
Allergy/atopy Altered diversity – specific signatures Stsepetova et al. 200762; Bisgaard et al.
201163; Storrø et al. 201164
Celiac disease Altered composition, notably in small intestine Nistal et al. 201265; Di Cagno et al. 201166;
Kalliomäki et al. 201267
Type 1 diabetes Signature differences Vaarela 201168; Giongo et al. 201169; Brown
et al. 201170
Type 2 diabetes Signature differences Larssen et al. 201071; Wu et al. 201072;
Kootte et al. 201273
Obesity Specific bacterial ratios (Bacteroidetes/Firmicutes) Ley et al. 200674; Turnbaugh et al. 200910;
Musso et al. 201175

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suggested that this bacterium could be a health biomar- considered as indicating that the altered intestinal micro-
ker.54 Akkermansia muciniphila is a mucus-degrading biota in IBD and IBS are a cause rather than an effect,
and propionate-producing bacterium belonging to the larger sets of fecal transplantation patients, criteria that
Verrucomicrobia (Figure 1) that notably stimulates the define an optimal donor microbiota, and better descrip-
immune system and the barrier function in a mouse tions of the medical conditions, the efficacy, and the
model.76,77 Very recently, it was shown in a mouse model changes in the microbiota are needed to support that
of IBD with human-relevant disease-susceptibility muta- conclusion.
tions that Koch’s postulates were fulfilled by common Another important disease for which a series of
commensal Bacteroides spp. but not by members of the recent studies support an association with the microbiota
Enterobacteriaceae.78 Remarkably, the latter were > 100- is colorectal cancer (CRC), a life-threatening disease that
fold enriched in the IBD model but were not associated in some cases is linked to colitis, the so-called colitis-
with disease, so this elegant experiment stresses the need associated cancer (Table 1). It is assumed that dietary
for cause-and-effect rather than association studies. components, such as nitrate, which is a precursor for
While IBD affects only a fraction of the population, carcinogenic nitrosamines, can be converted into (pro)
IBS is a highly prevalent aberration that may affect over carcinogens by enzymes of the intestinal microbiota and
5% of the population and can be differentiated into hence can promote the onset of CRC, as has been recently
several different types that relate to bowel habits.35 Two reviewed.82 Testifying to the interest in this area is the very
recent studies in adults and children have produced a recent insight gained in a series of studies in which
series of global signatures that differentiate IBS subjects the fecal and, in some cases, the mucosal microbiota of
from healthy individuals.56 In spite of the differences in CRC patients were compared with those of healthy
analysis platforms (microarrays, quantitative PCR, and subjects.59–61 In the fecal microbiota of over several hun-
NGT sequencing), a consistent reduction in anaerobic dreds of CRC patients and healthy controls, an increased
gram-positive bacteria belonging to Dorea and Rumino- level of bacteria belonging to the Bacteroides/Prevotella
coccus spp. was found to be associated with IBS. Remark- was observed in France using a quantitative PCR
ably, the healthy controls of the adult study were found to approach, while in a global study in China, a more
contain an increased number of Bifidobacterium spp. and complex pattern was observed that was characterized by a
Faecalibacterium prausnitzii that had previously been reduction of potentially pathogenic gram-negative bacte-
associated with an absence of abdominal pain and inflam- ria in CRC patients, an unequal distribution of some
mation (see above). Bacteroides spp., and a reduced number of butyrate-
While there is no direct support for the involvement producing bacteria.59,60 More detailed differences were
of the intestinal microbiota in IBD and IBS in humans, observed by comparing the microbiota on tumor biopsies
there is such evidence in the case of CDI. Patients with with those of the neighboring healthy tissues, and this
recurrent CDI are usually on antibiotic therapy, and the revealed a set of global differences.61 Similar approaches
intestinal microbiota of antibiotic-treated patients show with fewer patients but more powerful genomic
highly reduced diversity.38 In many cases, however, the approaches revealed a variety of differences that system-
use of antibiotics is not effective, and patients can only be atically included an overrepresentation of Fusobacterium
rescued by a rigorous but highly effective (approximately spp. in the tumor sites.83,84 A strain of Fusobacterium
90% effective) treatment known as fecal transplantation nucleatum was isolated, genomically characterized, and
or bacteriotherapy, in which the patient’s microbiota is found to be invasive in a human cell line.84 Remarkably,
replaced by that from a healthy donor.79–81 In two studies, close inspection of the earlier reported study with biop-
single cases of transplantation events that led to CDI sies of CRC patients also showed an increase in fusobac-
eradication have been investigated, and colonization by teria in the tumor samples.61 Whether the fusobacteria
the donor organisms for up to 24 weeks was described.57,58 really are involved in the onset of CRC, however, remains
In a more systematic study, a dozen patients were moni- to be established, and it is well possible that these gram-
tored for up to 2 months, and a consistent pattern of positive bacteria, which are rather common intestinal
increased diversity indistinguishable from the donor colonizers, just prefer affected tissue. Inflamed appendi-
microbiota was observed in the cured patients (van Nood ces removed after appendicitis were also found to contain
et al., unpublished data, 2012). These cases all represent much more fusobacteria, and microscopic evidence of
clear examples of a cause-and-effect relationship in which invasion of fusobacteria into the enterocytes was pro-
a diverse donor microbiota is stably established in low- vided85; moreover, the level of bacteria related to the
diversity recipients with CDI who were thereby cured of mucus-degrading Akkermansia muciniphila was greatly
the disease. There are some single case reports of success- reduced, 85 as in IBD (see above).54 Similarly, it was found
ful fecal transplantation in patients with UC and other that there was a relation between the level and invasive-
IBD, even IBS.81 While these observations can also be ness of Fusobacterium nucleatum and the severity of

Nutrition Reviews® Vol. 70(Suppl. 1):S45–S56 S51

inflammation in mucosal samples of IBD patients.86 This have been researched extensively following the first
supports the hypothesis that invasion precedes inflam- description in human obesity that linked this condition
mation and, in some cases, like colitis-associated cancer, with microbial ecology74 (Table 1). Because of its poten-
may lead to the development of cancer. tially major significance and, possibly, because some of
In several of the diseases described so far, notably the initial observations could not be reproduced by other
IBD and CDI, the recurrent use of antibiotics and a high or the same authors,10 the relation between microbiota,
level of hygiene also have been implicated in their preva- diabetes, and obesity is one of the most extensively
lence. This so-called “hygiene hypothesis”87 also applies to reviewed research domains.75 While both type 1 diabetes
the development of allergy, which is often already devel- (T1D) and type 2 diabetes (T2D) are characterized by
oping in early life and manifested in various forms of high levels of blood glucose, their mechanistic basis
atopy. In a series of comparisons of babies and young differs: T1D patients have a defect in the production of
children with atopy compared with age-matched healthy insulin, while T2D patients are generally insulin resistant.
controls, correlations were found between specific micro- T1D is not a lifestyle disease but is considered to result
biota differences and the manifestation of allergy. In some from autoimmune destruction of the insulin-producing
cases, specific groups of bacteria, including Bifidobacte- beta cells of the pancreas; currently, there are few reports
rium spp., have been implicated, but the development of on an association with the microbiome, and these deal
the diversity also appears to be involved.62–64 A compli- with only a handful of patients. Hence, there is a need for
cating factor is that, in early life, both the infant and the much more robust analysis. Animal experiments,
microbiota are still developing and are subject to large however, have provided clear indications for an impor-
variations, so their proper study will require substantial tant role of the microbiota that relates to its impact on
numbers in the cohorts. In a recent study, over 1,000 immune signaling in T1D.94
babies were monitored over time. It was reported that Similarly, elegant transplantation studies in mice
colonization by Clostridium difficile at the age of 1 month showed a clear causal link between the microbiota and
was associated with wheeze, eczema, and asthma at a later T2D, implying the involvement of Toll-like receptor 5
age.88 Although these cohort study results are promising, signaling.95 The relation between T2D and the microbiota
the analysis of the microbiota was performed by quanti- has been further explored in human subjects; a series of
tative PCR. Analysis by the new all-encompassing tech- microbiota signatures have been associated with T2D,
niques has not yet been performed, which thereby limits but they differ considerably from study to study. A
the significance of the study. Similar aspects of power common theme is a low-grade inflammation that, in
apply to the analysis of celiac disease, a chronic inflam- animal experiments, has been associated with high levels
mation of the mucosa in the small intestine that develops of lipopolysaccharide and an aberrant microbiota.96 T2D
in genetically susceptible persons in response to dietary is linked to obesity, which is usually defined as a body
gluten, present in barley, wheat, and rye. A set of over mass index (BMI) of over 30 and can be easily assessed
1,000 genes have been involved in celiac disease, testifying without complex phenotyping.
to its complexity.89 Another major factor, however, The first hallmark study addressing the association
appears to be the intestinal microbiota, and it has been between the human microbiota and obesity suggested an
proposed that aberrations in early-life colonization result inverse relationship between obesity and the Bacteroides/
in inappropriate host immune responses that lead to Firmicutes ratio and showed that this ratio increased fol-
enteric inflammation.90 There is, however, no clear lowing weight loss.74 This study was accompanied by a
picture yet of the link between the microbiota and celiac seminal experiment showing an increased energy-
disease. A variety of associations between various differ- harvesting capacity of the microbiota of obese mice with
ent microbial groups have been made, especially in small a low Bacteroides/Firmicutes ratio, a property that can be
intestinal biopsies.65–67 Furthermore, connections to the transplanted to lean, germ-free mice, providing a causal
expression of ileal genes, notably those involved in relationship between microbiota and obesity in experi-
immune regulation, have been made.67 A complicating mental animals.97 As both the Bacteroides/Firmicutes ratio
factor is that the current knowledge of early-life coloni- and BMI can be easily measured, many studies with
zation is still limited and not supported by large cohort human subjects have subsequently attempted to address
studies. Moreover, with the involvement of host factors, the relationship between these parameters. Often,
the microbiota, and diet, celiac disease is particularly however, a relationship could not be established, resulting
complex, as noted in several recent reviews.91–93 in inconsistent results (see Diamant et al.98 for a recent
Lifestyle diseases are a last group of highly prevalent extensive review). This may be due to the use of different
aberrations that increasingly impact human life. These analysis platforms, the reproducibility issues of NGT
include diabetes, obesity, and metabolic syndrome. The analyses in particular, and other biases that call for
associations between these diseases and the microbiota caution (see above). Another explanation, however, could

S52 Nutrition Reviews® Vol. 70(Suppl. 1):S45–S56

lie in the fact that the original study monitored severe lyzed for associations with the intestinal microbiota like-
weight loss but did not specify the initial BMI of the obese wise increases. In addition, many models of mice and
subjects, instead reporting only their weight reduction.74 other experimental animals are used in disease models to
It is possible that very obese subjects were selected to study the associations with the intestinal microbiota.
maximize the effect. In a recent study using the HITChip Needless to say, these are very useful in providing mecha-
phylogenetic microarray, an ultra-deep analysis of the nistic insight and do stimulate follow-up studies, but the
intestinal microbiota of morbidly obese subjects with an results cannot be extrapolated to humans. It should be
average BMI of > 45 was performed. The Bacteroides/ noted that humans and mice have considerably different
Firmicutes ratio was found to be significantly lower com- microbiota, as their intestinal architectures, physical-
pared with that in lean subjects (BMI of 25) (Verdam chemical environments, and biological environments are
et al., unpublished data, 2012). This study also indicated very dissimilar. A summary of some of the presently
that a high BMI correlated significantly with a high level anticipated associations that are less strong than those
of proteobacteria, known to produce lipopolysaccharide discussed above is provided in Table 2. Some of the
that was associated with low-grade inflammation in support derives from analyses in human trials and obser-
animal experiments.96 This opens avenues for revisiting vational studies of single patients, and some from a
the association between the microbiota and obesity, BMI, careful analysis of the literature. Other support relates
and other clinical parameters such as nonalcoholic fatty exclusively to trials in animals, often wild-type, mutant, or
liver disease.99–101 This requires deep analysis of the germ-free or antibiotic-treated mice. In many cases, the
microbiota as well as advanced phenotyping and geno- evidence for the associations is rather premature and
typing of the human subjects. Moreover, causal relations relies on single case reports. Several diseases, however, are
need to be established; for example, further mechanistic so important that they are worth including, e.g., the data
insight can be gained from ongoing studies with on nonalcoholic fatty liver disease, which affects over 30%
fecal transplantations of lean donors, which have of the US population.101 Other diseases, such as Alzhe-
shown that the insulin resistance of T2D patients can be imer’s disease, multiple sclerosis, and Parkinson’s disease,
corrected.102 are almost untreatable diseases. Hence, sufficient care
should be taken not to overinterpret the indications in the
IMPLICATIONS OF THE MICROBIOTA IN OTHER summary presented in Table 2. In some cases, other cir-
DISEASES AND ABERRATIONS cumstantial evidence supports the relation to intestinal
microbiota. This is the case with Alzheimer’s disease, mul-
As the number of studies on the intestinal microbiota tiple sclerosis, and Parkinson’s disease, for which non-
increases, the number of diseases and aberrations ana- peer-reviewed but promising results have been reported

Table 2 Indications for associations between the microbiota and health aberrations, provided as an alphabetical
listing of the aberrations suggested to be associated with the intestinal microbiota, along with support for such
an association.
Disease or aberration Type of support Reference*
Alzheimer’s disease Microbiota in a mouse model of Alzheimer’s disease Karri et al. 2010103
Atherosclerosis Analysis of plaques in humans Koren et al. 2011104
Autistic spectrum disorders Analysis of mucosa in children with autism spectrum Williams et al. 2011105
disorders
Chronic fatigue syndrome Cultured microbiota in patients with chronic fatigue Sheedy et al. 2009106
syndrome
Colic babies Longitudinal analysis of colic babies cohort de Weerth et al. 2012
unpublished data
Cardiovascular disease Cardiovascular-diseased mice and microbial metabolism Wang et al. 201148
Depression and anxiety Probiotic intervention in stressed mice Bravo et al. 201134
Frailty Analysis of elderly and high frailty scores van Tongeren et al. 2005107
Graft-vs-host disease Review of human data on graft-vs-host disease Murphy et al. 2011108
Multiple sclerosis Involvement of microbiota in mice with multiple sclerosis Berer et al. 2011109
Nonalcoholic fatty liver disease Effect of choline depletion in humans Spencer et al. 2011101
Parkinson’s disease Role of enteric nervous system and review of Parkinson’s Braak et al. 2003110
disease development
Rheumatoid arthritis Microbiota as predisposing factor in rheumatoid arthritis Scher and Abramson 2011111
Retrovirus infection Mouse retrovirus infection relies on microbiota Kane et al. 2011112
Poliovirus infection Mouse microbiota promotes poliovirus infection Kuss et al. 2011113
* The most recent single reference is given.

Nutrition Reviews® Vol. 70(Suppl. 1):S45–S56 S53

in single cases by using fecal transplantations.114,115 In specified recently, continuing support from gastroenter-
addition, it has been reported that the levels of Bifidobac- ologists is needed.118
terium spp. and Akkermansia muciniphila, previously Once an association with the microbiota has been
identified as a health-related bacteria (see above), were established, there are various obvious next steps to be
decreased in children with autism spectrum disorders taken that include biomarker development, early diag-
compared with healthy controls.116 nostics, and monitoring of disease development. More-
A set of other studies provide completely new over, specific segmentation of the patients may be
mechanistic insight that can be applied to the two recent possible using biomarkers, such as those based on the
studies involving the role of intestinal microbiota in virus enterotypes, which may lead to personalized treatments.
replication in mouse models.112,113 These breakthrough Finally, especially when causal effects have been estab-
results provide a radically new vision of the development lished, specific therapeutic avenues can be developed on
and progression of viral infections, the involvement of the basis of health-promoting microbes or their biomol-
the immune system, and the contribution of the intestinal ecules. Given the importance of the diseases described
microbiota. It will of great interest to see the impact of the here, it is imperative to 1) build on the established asso-
intestinal microbiota on viral infections in compromised ciations between intestinal microbiota and the predispo-
and healthy humans. While these studies may provide sition to disease, 2) elucidate new associations, and 3)
support for the efficacy of antibiotic treatments in viral establish causality.
infections, it should be remembered that repeated antibi-
otic treatment disturbs the intestinal microbiota.50
Acknowledgments
CONCLUSION
The authors are grateful to Dr. Mirjana Rajilić-Stojanović
and Jonna Jalanka-Tuovinen for assistance with Figures 1
A total of over 25 diseases, syndromes, or other aberra-
and 2, respectively.
tions have now been associated with the intestinal micro-
biota. The most robust ones discussed here are supported
by multiple and robust studies in humans (Table 1). In Funding. This work was partially supported by project
addition, a series of other less convincing or first reports 40274/06 of TEKES, Finland, project PSG 93609004 and
are listed as well (Table 2). In several cases, specific the unrestricted Spinoza Award of the Netherlands Orga-
correlations have been made with specific potentially nization of Scientific Research, grant 25.0.1.7 from the
health-associated bacteria, such as some Bifidobacterium European Research Council (MicrobesInside) and
spp., Faecalibacterium prausnitzii, and Akkermansia research grants 118165, 137389 and 141130 from the
muciniphila. As expected, these all belong to the common Academy of Finland.
core of QoL-controlled healthy subjects (Figure 2).
Cause-and-effect relationships, however, are scarce, but it Declaration of interest. The authors have no relevant
is of interest to see a Koch’s postulate applied to Bacteroi- interests to declare.
des strains in a mouse IBD model.78 Moreover, there is a
large body of literature on the use of single or multiple
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