Seminars in Immunology 19 (2007) 94105

Review

What immunologists should know about bacterial communities

of the human bowel
Gerald W. Tannock
Department of Microbiology and Immunology, University of Otago, P.O. Box 56, 720 Cumberland Street, Dunedin, New Zealand

Abstract
The human bowel is home to a bacterial community of much complexity. This article summarizes current bacteriological knowledge of the
community and highlights topics of potential interest to innovative immunologists. The role of the bacterial community in the development and
regulation of the immune system of neonates seems likely to be a particularly important area of future research.
2006 Elsevier Ltd. All rights reserved.
Keywords: Bacteria; Bowel; Microbiota; Gut; Microbial ecology

1. The company that you keep

Homo sapiens, it seems, first walked the Earth about 130,000
years ago. Our distant ancestors presumably carried within their
bowels a large and complex community of microbes just as
do their primate relations, modern descendants and other animal species represented in nature today. Whether this microbial
community has the same composition in humans of the 21st
century as it did when associated with our ancient progenitors, somewhere in Africa, will perhaps never be determined.
Humans inhabiting developed countries today are, however,
much less likely to be colonized by parasitic worms (helminthes)
and it is possible that the more hygienic lifestyles of recent
decades have resulted in additional alterations to the composition
of the bowel community because exposure to certain bacteria
may be less common [1,2]. Even so, extant bowel communities
of humans are impressive both qualitatively (biodiversity) and
quantitatively. Large bowel contents and feces contain about
1011 bacterial cells per gram (wet weight) and bacterial cells
comprise about 50% of fecal mass [3]. Four bacterial phyla are
represented (Firmicutes [Gram-positive], Bacteroidetes [Gramnegative], Actinobacteria [Gram-positive], and Proteobacteria
[Gram-negative]) and three groups, still phyogenetically broad,
each containing many genera and species, are numerically dominant in the feces of healthy humans (Clostridium coccoides
group, Clostridium leptum subgroup, Bacteroides-Prevotella

Tel.: +64 3 479 7713; fax: +64 3 479 8540.

E-mail address: gerald.tannock@stonebow.otago.ac.nz.

1044-5323/$ see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.smim.2006.09.001

group) [4,5]. Bacteria, then, dominate the bowel community.

Fungi and Archaea may also be resident, but comprise less than
0.05% and 1% of the total inhabitants, respectively [6,7]. Further information concerning the phylogenetic composition of
the bowel community is provided in Fig. 1. Much of this information has been generated through the application of nucleic
acid-based methodologies, most of which target the nucleotide
base sequence of small ribosomal subunit RNA (16S rRNA in
the case of bacteria) which provides a cornerstone of microbial taxonomy. An earlier estimate of the number of bacterial
species that might be resident in the human large bowel was
based on bacteriological culture. Four hundred species seemed
a likely number by extrapolation from what had already been
cultured [12,13]. Nucleic acid-based methods of detection suggest that about 50% of the bacterial cells seen microscopically
in feces cannot yet be cultured in the laboratory, even when
accounting for the fact that some of the bacteria are dead [14,15].
This phenomenon, manifest even more dramatically in terrestrial
and aquatic ecosystems was, based on traditional bacteriological experience, totally unexpected and has been called the
great plate count anomaly [16]. Operational taxonomic units
(OTU; molecular species) never encountered in culture-based
bacteriology are detectable by the molecular methods, revealing
a new world remaining to be investigated by bacteriologists of
the future. Estimates of biodiversity now seem to continually
inflate, probably because many of the 16S rRNA sequences in
databanks differ by one nucleotide base. These are likely to be
sequences representing the same OTU but containing sequencing errors. Curiously, therefore, we do not really know what,
in any detail, our modern bowel community is composed of let

G.W. Tannock / Seminars in Immunology 19 (2007) 94105

95

for other animal species even today [19]. Well-fed New Zealanders and North Americans, as examples, can doubtless manage
without this nutritional contribution much better than their prehistoric forebears whose diet was impoverished by comparison.
Nevertheless, nutritionists invoke butyric acid as an important
fuel for colonocytes [20]. One would imagine that the bacterial
culture responsible for the fermentation would be the same from
human to human since bowel biochemistry follows the same pattern from one person to the next [5]. Yet, again on the basis of
molecular methods of analysis, it appears that bowel communities are about 30% dissimilar between humans (Fig. 2). Though
a surprising fact when first encountered, the uniqueness of bowel
communities is likely to be due to extensive functional redundancy in the bacterial World: more than one bacterial species
can carry out a particular catabolic process. Thus, just as there is
a normal range of blood chemistry values in the human population, there is also a range of inhabitants that can fill the ecological
niches responsible for the overall bowel fermentation.
3. Its a wise man that knows his own father

Fig. 1. Where and what types of bacteria are detected in the human gut [811].

alone what that of ancient humans contained. What we do know,

and all that matters really, is that these bowel inhabitants have
profound effects upon us.
2. Unseen and unfelt
A chemostat is a culture apparatus that is used to maintain the
growth of bacterial cultures continuously and at a constant rate
under laboratory conditions. Culture medium is fed into a culture
vessel containing the bacterial cells at a rate controlled by a flow
regulator. The culture volume is held constant by means of an
overflow tube that enables spent medium (effluent) to pass from
the culture vessel. The human large bowel is the equivalent of a
chemostat. It is fed with culture medium derived from the undigested components of the diet that pass from the small bowel,
as well as substances produced endogenously by the human
host. Hence this culture medium is particularly rich in complex carbohydrates derived from plant cell walls and complex
glycoproteins from mucus [17,18]. The ileo-cecal valve regulates the flow of culture medium into the large bowel; feces are
the effluent. Under these circumstances, a continuous fermentation of exogenous and endogenous substrates by consortia of
bacteria proceeds, resulting in the formation of short chain fatty
acids (mainly acetic, propionic, and butyric), amines, phenols,
indoles, and gases as the major products [17]. The absorption
of the fatty acids from the bowel lumen once probably provided
important caloric and carbon sources for humans, just as they do

According to Sophocles, Oedipus did not know his

antecedents and, because of this, in all innocence, murdered his
father and married his mother. A colossal tragedy resulting from
genealogical ignorance. We do not know where the phylogenetic
origins of the members of bacterial communities in the bowel lie,
but an educated guess would indicate that their ancestors were
plant-associated, and regularly ingested by mammals. Cellulose,
pectins, arabino-xylans, starch and beta-glucans, all substances
of plant origin, figure prominently as substrates for the predominant bowel bacteria [2224]. Genomic sequences of residents
of the human bowel (Bacteroides and Bidobacterium species)
show that these bacteria devote much of their genomes to genes
encoding hydrolytic enzymes, often contained in self-regulated
operons, that catalyze the degradation of oligosaccharides and
glycoproteins [22,23]. There are also many genes associated
with signal transduction by which the bacteria can detect and
react to changes in the nutrient milieu. Responses to qualitative
changes in substrate availability, and well-regulated metabolic
pathways that conserve cellular energy, help to explain the ecological fitness of these bacteria and the phylogenetic stability
of bowel communities [25]. The numerically predominant bacteria of bowel communities are obligate anaerobes. This suggests a long-distant temporal origin when Earths atmosphere
was anoxic, but their biochemical evolution must have continued after the appearance of plants with complex cell walls
and vascular systems that provide their nutritional substrates
today. Perhaps they were decomposers in sediments and natural composts, or similar anaerobic environments rich in complex
carbohydrates? Some of these environmental bacteria must have
adapted to the bowel environment so that residence, still on the
outside (bowel lumen), but sequestered inside, of the human
body was established. The human bowel is daily contaminated
with bacterial cells present in food and water. Some of these bacteria survive and transit the digestive tract and are detectable in
the feces [26]. The same applies to probiotic bacteria that are
intentionally consumed in self-care health products [27]. These

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G.W. Tannock / Seminars in Immunology 19 (2007) 94105

Fig. 2. The composition of the fecal bacterial community varies from human to human [21].

allochthonous bacteria do not become part of the bowel community because they are no longer detected in feces once the
source of their ingestion is removed. Autochthonous bacteria,
on the other hand, have a habitat in the bowel. They have longterm associations with the human host, form stable populations
of characteristic size, and have demonstrable means of earning
a living (an ecological function or niche) in the distal bowel
[28]. Researchers interested in the bowel community need to be
aware of the Oedipus tragedy; would not it be embarrassing to
claim that the digestive tract was colonized by a vast array of
bacteria when, in fact, only the DNA of allochthonous microbes
had been detected? Unraveling the secrets of life in the bowel
would be impossible if an allochthonous fecal isolate was chosen

as the model organism for study! Snapshots of the composition of bowel communities are only the beginning of ecological
investigations and DNA-based methods reveal only who might
have been there. Temporal (quantitative sequential measurements) and function-based (transcriptomics, proteomics) observations are necessary to define bacterial communities. We need
to know what the bowel residents are doing and how they do
it. It would also be very interesting to know how the transition from allochthonous to autochthonous status in relation to
the bowel was achieved. The highly competitive nature of the
bowel community may have represented, and perhaps still does,
a hotbed of evolution where novel attributes developed and were
selected. The molecular (genetic) processes by which bacteria

G.W. Tannock / Seminars in Immunology 19 (2007) 94105

adapted from a free-living lifestyle outside of the body to a

host-dependent life in the bowel would provide the basis of a
fascinating story.
4. The child is father of the man
The gut of newborn human infants resembles that of the
germfree animal because it does not yet harbor a bacterial or
other microbial community. This germfree state is short-lived
because within minutes of birth the baby is exposed to bacteria in the feces that have been involuntarily expelled by the
mother during labor, as well as to environmental microbes.
Suckling, kissing and caressing the infant after birth provide
additional assurance that maternal microbes are transmitted from
one human generation to another. Regulatory mechanisms generated within the ecosystem (autogenic factors) and by external
forces (allogenic factors) permit the episodic persistence of some
bacterial populations, but the elimination of others in a classical
biological succession [29]. It takes several years to produce a climax community resembling that of adults (Figs. 3 and 4; [30]).

97

Members of the bacterial genus Bidobacterium are numerically predominant in the gut of infants during the first months
of life. Nucleic acid-based methods of analysis show that bifidobacteria form between 60% and 91% of the total bacterial
community in the feces of breast-fed babies and 2875% (average 50%) in formula-fed infants whereas they comprise only a
few percent of the fecal community of adult humans [31]. The
infant during early life is therefore almost a monoassociated
gnotobiote, and bifidobacterial antigens may be important instigators of immunological development. By analogy, Rene Dubos
and colleagues at Rockefeller University carried out pioneering
studies of the impact of the bacterial community of the bowel
of infant mice. As recalled by Dwayne Savage, the NCS (New
Colony Swiss) mouse colony derived at The Rockefeller University was the first murine colony in the World in which the
animals, while harboring a bowel community, were yet free of
certain mouse pathogens and could be bred in sufficient quantity
for major experiments [32]. Dubos and colleagues soon realized
that NCS mice differed in several characteristics from SS mice
(Standard Swiss from which the NCS colony had been derived).

Fig. 3. The composition of the bowel bacterial community takes years to stabilize. Comparison of PCR/DGGE profiles of the fecal community of a human infant.
Fecal samples collected at intervals during the first 6 years of life. The community is relatively simple until after weaning and then becomes progressively more
complex. Stability in composition is not apparent until about 4 years of age (Munro, Bateup and Tannock, unpublished).

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G.W. Tannock / Seminars in Immunology 19 (2007) 94105

beings to the conditions of the present is always conditioned by

the biological remembrance of things past.
5. Immunological Freudianism

Fig. 4. Babies are almost monoassociated gnotobiotes. Quantification of bifidobacteria relative to total community composition in the feces of the child
whose bacterial community profiles are shown in Fig. 3. Bifidobacteria comprised almost 100% of the total community early in life, gradually reducing to
levels characteristic of adults (Munro, Bateup, Tannock, Harmsen and Welling,
unpublished). Quantification by fluorescence in situ hybridization/computerassisted microscopy.

NCS mothers bore on average more infants per litter, and their
offspring grew faster and were larger than SS mice (even when
fed diets low in lysine and threonine). It was suggested that the
differences between NCS and SS mice were the result of mutations (in other words, that a new mouse strain had been selected)
but, astoundingly, NCS mice housed with SS mice reverted to the
characteristics of SS mice in all properties tested. The fecal bacteria of the two colonies of mice were different when analyzed
by bacteriological culture. Unlike the SS mice, NCS animals
did not harbor facultatively anaerobic Gram-negative bacteria
such as Escherichia coli. Remarkably, NCS mice were tolerant
to parenteral doses of endotoxin that were lethal within a few
days of administration to SS mice. Exposure of NCS animals to
SS mice during early life, or prior injections with small doses
of heat-killed cells of Gram-negative bacteria that thus exposed
the animals to sub-lethal amounts of endotoxin, increased the
susceptibility of the NCS mice such that it now matched that
of SS animals. Dubos et al. concluded that the enterobacteria,
present in relatively high numbers in the gut of infant SS mice,
sensitized the animals so that, as adults, they were highly susceptible to endotoxin [33]. Drawing on these results, Dubos and
colleagues concluded that From all points of view, the child
is truly the father of the man, and for this reason we need to
develop an experimental science that might be called biological
Freudianism. Socially and individually the response of human

In recent decades, many affluent countries have experienced

an increase in the prevalence of atopic diseases, including asthma
[2]. Several aspects of lifestyle have changed in these countries
over the same period and theories have been advanced to explain
the altered prevalence of allergies. The Hygiene Hypothesis
proposes that atopic diseases could be prevented by infections
in early childhood because the neonatal immune system would
be driven towards a T helper 1 (Th1) response, but a specific
infectious protective factor has never been identified [34,35].
Attention has turned to the bacterial community of the bowel
and the possibility that colonization of the gut by specific bacterial species might be more important than the impact of sporadic
infections. For example, the composition of the bowel community has been reported to differ in the numbers of lactobacilli and
clostridia in the feces of Estonian and Swedish children [36].
Atopic diseases were less prevalent in the Estonian compared to
the Swedish population. Ouwehand et al. have reported that the
prevalence of Bidobacterium adolescentis differed in the feces
of healthy and allergic Finnish children aged 27 months [37].
Six out of seven allergic children harbored B. adolescentis in
the faeces whereas this species was not detected in six healthy
children. Young and colleagues reported the results of a study in
which the fecal populations of bifidobacteria from children aged
2535 days in Ghana (low prevalence of atopy), New Zealand
and the United Kingdom (high prevalence countries) were compared. Natal origin influenced the detection of bifidobacterial
species because fecal samples from Ghana almost exclusively
contained Bidobacterium infantis whereas those of the other
children did not. Choosing species on the basis of the bacteriological results, bifidobacterial preparations were tested for
their effect on cell surface markers of dendritic cells harvested
from cord blood. Bifidobacterial species-specific effects on
dendritic cell activation were observed in that CD83 expression
was increased by Bidobacterium bidum, Bidobacterium
longum and Bidobacterium pseudocatenulatum. One or more
of these species were detected in the faeces of 40 out of 46 New
Zealand and United Kingdom children, but only in a few (B.
longum: 2/32) of the samples from infants living in Ghana. B.
infantis, common in the feces of babies from Ghana, failed to
produce this effect [38]. Further investigations of the molecular
interplay between bifidobacteria, human dendritic cells, T cells
and allergens are clearly required and may provide the first clear
evidence of Immunological Freudianism in relation to human
diseases. The environmental conditions under which babies are
born and nurtured may affect which microbes they are exposed
to, and subsequently influence the composition of their bowel
community. Differences in neonatal bowel communities might
occur due to the common occurrence of hospital deliveries,
caesarean sections, special-care baby unit admissions, smaller
family size, widespread use of antibiotics, good hygiene, and
nature of the maternal diet in affluent countries [39]. Either the
lack of exposure of babies to particular bifidobacterial species,

G.W. Tannock / Seminars in Immunology 19 (2007) 94105

and/or elimination of bifidobacterial species from the bowel

through the use of antibiotics, might reduce the exposure of
children in early life to important bacterial antigens at a critical
time in the maturation of the immune system, for example
in removing (immune deviation) the T helper 2 (Th2) skew
apparently characteristic of the newborn [40,41].
6. Retained but contained
The antigenic burden associated with approximately 2 1013
bacterial cells (exceeding the human population of Earth about
10,000-fold) present at any moment in the large bowel is probably incalculable. Dendritic cells associated with the bowel
epithelium may sample this antigenic landscape regularly by
extending dendrites between enterocytes to reach the mucosal
surface [42]. M cells of Peyers Patches are also sites of antigen
uptake [43]. Relative to germfree animals, the lamina propria
in the gut of animals harboring bacterial communities is mildly
inflamed in terms of histopathology [44]. Antibodies reactive
with bowel bacterial antigens are present in human sera [45].
Thus bacterial antigens are detected by the innate and adaptive
immune systems, but inflammation is somehow kept at minimal levels. How tolerance (hypo-responsiveness) to the bowel
community is achieved mechanistically should be an interesting
topic of immunological research. It can be postulated, though,
that sequestration of the bacterial community away from the
immune system in a virtual tube within the bowel may be
a large part of the answer. The mucosal surface of the bowel is
covered with a blanket of mucus that flows away from the epithelium and is continuously replaced by fresh mucus from the goblet
cells [46]. This moving, sticky blanket cleanses the mucosal surface keeping bacterial cells at bay. The tight junctions formed
between enterocytes render the epithelium a formidable barrier to bacterial penetration [47]. Moreover, bacterial cells in
the bowel are coated with sIgA, possibly counteracting effective use of bacterial adhesins [48]. Defensins liberated in the
intestinal crypts by Paneth cells complete the wall of the tube
[49]. Everything is designed to stop bacteria associating with
the mucosal surface. Contained within the virtual tube in the
bowel lumen, the bacterial community can be retained safely.
Nevertheless, it does not seem to be a totally efficacious system since pathogens can reach the mucosal surface and attach
by means of adhesins to specific receptors prior to proliferation or invasion. Even in health, small temporary cracks in the
virtual walls of the tube must occur which allow bacterial antigens to seep from the tube and make contact with enterocytes,
dendrites of dendritic cells, and M cells. Although enterocytes
are not professional immune cells, contact with bacterial antigens may cause a transient induction of immune responses
within them. For example, Ruiz et al. have described induction
of phosphorylation/activation of the NF-B transcriptionally
active subunit RelA and the mitogen-activated protein kinase
p38 in enterocytes by an innocuous bifidobacterial species,
which resulted in interleukin-6 gene expression [50]. The cornerstone of innate signaling between bacteria and enterocytes
rests on pattern recognition receptors (Toll-like receptors [TLR],
nucleotide-binding oligomerization domain protein receptors

99

[NOD]) that recognize molecular patterns associated with bacterial cells regardless of whether they originate in the bowel
community or are pathogens [51]. However, pathogens induce
an acute inflammatory response, harmless bacteria do not. This
differential effect may be due to the relative location of different TLR (apical membrane, intracellular, basal membrane of
enterocytes), the rapid turnover of enterocytes at villous tips,
or to particular properties of pathogens (virulence factors) that
other bacteria lack. Negative regulators exerted by the enterocytes themselves might also mediate the differential response
to signals from bowel inhabitants and pathogens [52]. Defining
these differential/regulatory systems is important immunological research because it impinges on the etiology of chronic
immune inflammatory bowel diseases.
7. Surrogate pathogens
Failure to demonstrate convincingly an association between
a specific pathogen and chronic immune inflammatory conditions of the bowel (Crohns disease and ulcerative colitis) has
resulted in the concept that the inflammation is fueled by the
bacterial bowel community, or at least some members of it [53].
This is demonstrably true in experimental animal models of colitis in which gene deletion or transgenic manipulation produces
a dysfunctional immune system that reacts aggressively with
bacterial antigens [53]. Under germfree conditions, these animals have minimal disease but develop colitis when exposed to
a specific-pathogen-free collection of bowel bacteria, or selected
strains of bowel bacteria. Human patients suffering from inflammatory bowel diseases (IBD) have a broken tolerance to their
own bowel bacteria as has been clearly demonstrated by the
classical studies of Duchmann et al. [54]. Familial clustering
of cases and association with specific chromosomal aberrations
(at least in a proportion of patients) and with particular human
leukocyte antigen genotypes indicate a genetic predisposition to
IBD [55,56]. The bowel community may indeed be the antigenic
fuel for chronic inflammation (in this sense acting as a surrogate pathogen), but how and what initiates the process? It could
be postulated, perhaps, that the enterocytes and, concurrently or
subsequently, immune cells are exposed to excessive amounts of
bacterial antigens because the virtual, multi-component defensive wall of the bowel of humans predisposed genetically to
IBD is defective in at least one factor. For example, the altered
chemical composition of the mucus of ulcerative colitis patients
may be linked to the larger number of bacteria associated with
biopsies collected from them (Fig. 5). Genetically based causes
of breaches in the wall, possibly occurring very early in life
(diagnosis of IBD is characteristically made in young adults),
together with broken immunological tolerance to bacterial antigens, could result in a sensitization to bacterial antigens and
the progression of poorly controlled inflammatory reactions.
Although the initiating factors may well occur early in life, studies of the composition of the distal bowel community represented
in feces have mostly been conducted with adult IBD patients in
the hope that they would reveal specific groups of bacteria that
might be associated with inflammation. These studies have produced extremely variable results (Table 1). In retrospect, this

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G.W. Tannock / Seminars in Immunology 19 (2007) 94105

Fig. 5. Do current sampling procedures reveal an accurate picture of the bowel ecosystem?

is not terribly surprising concerning the approaches that have

been followed in defiance of the complexity of the ecosystem
under investigation (Fig. 6). Broad-based phylogenetic tools, a
polluted 16S rRNA gene database, subjects that are medicated,
lack of standardization of biopsy-associated measurements, and
varying fecal water content (patients may have loose stools, controls formed stools) are among the confounding factors. A much
better approach may be to detect the substances against which
the dysfunctional immune system reacts. This will require cultivating the uncultured members of the bowel community or,
failing that, functional screens (immunoassays) of gene libraries

representing the collective genomes of the bacterial community

(metagenomics) [82].
8. Give me the children until they are seven and anyone
may have them afterwards
Francis Xavier believed that a Jesuit education until the age of
7 prepared a child to live a useful Christian life in no matter what
circumstances they later ended up in. Modern educationists, too,
understand that there are optimal periods during childhood in
which the ability to process certain information (visual, sound,

G.W. Tannock / Seminars in Immunology 19 (2007) 94105

101

Table 1
Summary of investigations of the bowel bacterial community of humans in relation to inflammatory bowel diseases
Investigation

Patients medicated for

IBD or administered
antimicrobial drugs?

Observations

Reference

Biopsies were collected from five colonic sites as well as

the terminal ileum of nine healthy subjects. Fluorescence
in situ hybridization/microscopy was used to target fecal
bacterial groups
Colonic biopsies from 21 ulcerative colitis patients, 14
Crohns disease patients, and 24 controls were collected
and treated with dithiothreitol to remove mucus, then
mucosa-associated and intramucosal bacteria (released by
hypotonic lysis and gentamicin treatment) were cultured

None

Bacterial cells were seen associated with the luminal

surface of mucus and seldom in the mucus itself

[57]

Yes, IBD treatment

[58]

DNA extracted from paraffin-embedded tissue samples

collected from 16 Crohns disease, 11 ulcerative colitis
and 18 colon cancer patients was used to enumerate
Mycobacterium species, Bacteroides vulgatus, and E. coli
using real time quantitative PCR
Colonic biopsies were washed and used for culture of
bacteria, and detection of bacteria by electron
microscopy, fluorescence in situ hybridization, and PCR.
The results from 28 patients with self-limiting colitis, 104
with indeterminate colitis, 119 with ulcerative colitis, 54
with Crohns disease, and 40 asymptomatic subjects were
compared
Rectal biopsies and feces were collected from 12 Crohns
disease patients during periods with or without
antimicrobial (metronidazole, cotrimoxazole) treatment.
Culture-based studies were conducted
Biopsies collected from four sites (ileum, right and left
colon, rectum) were collected from 20 Crohns disease,
11 ulcerative colitis, and 4 control subjects. Temporal
temperature gradient gel electrophetic fingerprints of
biopsy-associated bacteria were prepared
Rectal biopsies collected from 9 ulcerative colitis patients
and 10 control subjects were examined by culture and
fluorescence in situ hybridization/microscopy
Rectal biopsies collected from 33 ulcerative colitis patients,
6 Crohns disease, and 14 control subjects were examined
by fluoresence in situ hybridization/microscopy
Tissue sections were prepared from resected intestine
(terminal ileum, colon) and examined by fluorescence in
situ hybridization/microscopy. Samples were collected
from 12 ulcerative colitis, 12 Crohns disease, and 14
control subjects

IBD treatment not

stated

Bacteria were cultured more commonly from

samples from Crohns disease patients compared to
those from ulcerative colitis or controls. Adherent
Escherichia coli were more commonly detected in
samples from Crohns disease patients compared to
controls
Mycobacteria were not detected but B. vulgatus and
E. coli were detected more frequently and in greater
numbers in samples obtained from Crohns disease
and ulcerative colitis patients compared to colon
cancer subjects
There were more bacterial cells associated with
biopsies collected from patients than from
asymptomatic controls. The numbers of bacteria
increased in association with increased
inflammation (colitis)

Eight of the 12 patients showed clinical

improvement when administered antimicrobial
drugs but this could not be correlated with
bacteriological results
Similar profiles were obtained from sampling sites
intra-individual, but varied inter-individually.
Profiles did not discriminate between subject groups

[61]

[63]

Biopsies collected endoscopically or surgically from the

terminal ileum of 11 Crohns disease and 11 control
subjects were examined by PCR

IBD treatment not

stated. Antimicrobials
prior to surgery

Colonic biopsies collected from 13 ulcerative colitis

patients and 61 controls were examined by PCR
combined with enrichment culture to detect the presence
of sulphate-reducing bacteria

IBD treatment not

stated

The bacterial types that were detected varied

between subjects making it impossible to assign a
pathogenic role to specific bacteria
Bifidobacterial numbers were lower in ulcerative
colitis patients; E. coli numbers were higher in
ulcerative colitis and Crohns disease patients
There were higher numbers of bacteria associated
with tissue collected from ulcerative colitis and
Crohns disease subjects and there was more
evidence of bacterial invasion of tissue. Samples
from Crohns disease patients had mostly
proteobacteria and Bacteroides-Prevotella
associated with the tissue, whereas a more diverse
collection of bacteria was present in ulcerative
colitis samples
16S rRNA gene sequences similar to those of
Helicobacter species, Mycobacterium avium
subspecies paratuberculosus, Listeria
monocytogenes and E. coli were detected in three of
five biopsies collected surgically from Crohns
disease patients. Biospies collected from all subjects
by endoscopy had associated bacteria, but biopsies
collected at surgery from controls were devoid of
bacteria
All of the biopsies contained sulphate-reducing
bacteria

IBD treatment not

stated

Yes, antimicrobial
drugs during part of
the study
IBD treatment not
stated

IBD treatment not

stated
Yes, IBD treatment

IBD treatment not

stated. Antimicrobial
drugs given
intravenously just
prior to surgery

[59]

[60]

[62]

[64]

[65]

[66]

[67]

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G.W. Tannock / Seminars in Immunology 19 (2007) 94105

Table 1 (Continued )
Investigation

Patients medicated for

IBD or administered
antimicrobial drugs?

Observations

Reference

Colonic biopsies from 26 Crohns disease, 31 ulcerative

colitis, 15 controls with inflammation, and 31
non-inflamed control subjects were examined by 16S
rRNA gene profiling, the preparation of 16S rRNA gene
clone libraries, and real time quantitative PCR
Rectal biopsies were collected from 19 IBD patients (not
differentiated) and 14 controls. The biopsies were
examined by RNA in situ hybridization/microscopy

Yes, IBD treatment

The diversity of bacterial types was less in samples

from Crohns disease and ulcerative colitis patients
compared to controls. This was due to a reduction in
detection of Bacteroides species, Eubacterium
species, and Lactobacillus species
Bacterial cells were not observed in the case of 10
control and 6 IBD patient biopsies. In the remaining
samples, there were more bacteria associated with
IBD biopsies compared to those of controls. The
bacteria were seen within the mucus but not in
contact with enterocytes, nor within the lamina
propria
Specific phylogenetic groups of bacteria could not
be associated with Crohns disease lesions

[68]

Adherent-invasive strains of E. coli were more

commonly detected in association with tissues,
particularly from the ileum, of Crohns disease
patients compared to those of controls
M. avium subsp. paratuberculosis DNA was
detected more commonly in samples from Crohns
disease patients than from those of controls.

[71]

E. coli DNA was detected more frequently in

samples from Crohns disease patients than from
non-Crohns disease patients. The authors concluded
that, since a similar result with respect to M. avium
subsp. paratuberculosis had been obtained with
these same samples in a previous study, the findings
may reflect non-specific association of bacteria with
granulomas, rather than cause and effect
There were more clones representing
Bacteroides-Prevotella bacteria in libraries from
ulcerative colitis patients than from controls

[73,74]

Higher numbers of facultatively anaerobic bacteria

were associated with samples from Crohns disease
and ulcerative colitis patients compared to controls.
The numbers of Bacteroides vulgatus were lower in
the case of Crohns disease and ulcerative colitis
patients relative to controls
DGGE profiles were similar for all sites sampled
intra-individually but differed inter-individually.
LIBSHUFF statistical comparison showed that 16S
rRNA gene clone libraries prepared from Crohns
disease, ulcerative colitis and control subjects had
different compositions. Collections of bacteria
associated with inflamed tissue did not differ from
those of non-inflamed tissue. There were more
bacterial cells associated with biopsies collected
from ulcerative colitis patients compared to those of
Crohns disease and control subjects. Unclassified
members of the Class Bacteroidetes were more
prevalent in the samples from Crohns disease
patients compared to the other groups.
The number of bacteria belonging to the Bacteroides
fragilis group was higher, and these bacteria were
detected more frequently, in patients than controls

[76]

Yes, IBD treatment

16S rRNA gene clone libraries were prepared from 16

biopsies collected from Crohns disease patients
(aphthous ulcers), 15 surgical samples from Crohns
disease patients, and biopsies from controls
Ileal and colonic tissues from Crohns disease patients and
controls were studied to determine the prevalence of
adherent-invasive strains of E. coli

Yes, IBD treatment

Tissue samples collected at surgery from 20 Crohns disease

patients and 6 controls, and examined by nested PCR and
fluorescence in situ hybridization/microscopy for the
presence of Mycobacterium avium subspecies
paratuberculosis
Archival tissue obtained at surgery from 15 Crohns disease
patients and 10 non-Crohns granulomatous bowel
disease patients were examined. Granulomas were
microdissected from the tissue and DNA was extracted.
Nested PCR was used to detect sequences characteristic
of E. coli.

Yes, IBD treament

Colonic biopsies were collected from 4 ulcerative colitis

patients and 2 controls. 16S rRNA gene clone libraries
were prepared and selected libraries were probed with a
labeled oligonucleotide probe that detected
Bacteroides-Prevotella
Pediatric patients with Crohns disease (12 subjects),
ulcerative colitis [7], other intestinal abnormalities [16],
and 7 controls were studied. Biopsies were collected
from ileum, cecum and rectum and examined by
bacteriological culture and qualitative and quantitative
PCR
16S rRNA gene clone libraries were prepared from biopsies
collected from 20 Crohns disease, 15 ulcerative colitis
patients, and 14 controls. Patient biopsies were collected
from both inflamed and non-inflamed mucosa of each
subject. Denaturing gradient gel electrophoretic (DGGE)
profiles were prepared, and total bacterial numbers
associated with biopsies were enumerated by real time
quantitative PCR

Yes, IBD treatment

The fecal bacterial communities of Crohns disease (five

patients) and healthy controls (five) were compared by
bacteriological culture

IBD treatment not

stated

Treatments not stated.

None

None. Patients were

newly diagnosed and
untreated.

None

[69]

[70]

[72]

[75]

[77]

[78]

G.W. Tannock / Seminars in Immunology 19 (2007) 94105

103

Table 1 (Continued )
Investigation

Patients medicated for

IBD or administered
antimicrobial drugs?

Observations

Reference

Two metagenomic libraries were prepared from fecal

samples obtained from 6 Crohns disease patients and 6
controls, respectively. 16S rRNA genes detected in the
libraries were sequenced

Yes, IBD treatment

[79]

Biopsies were collected from the intestines of 15 Crohns

disease patients during flare-ups of disease. Inflamed and
non-inflamed mucosal samples were obtained and
examined by PCR/TTGE
Radioactive probes were used to quantify bacterial groups
in the feces of 17 Crohns disease patients and 16
controls. Community profiles were prepared by temporal
temperature gradient gel electrophoresis (TTGE)

IBD treatment not

stated

Forty three OTU belonging to the phylum

Firmicutes were detected in the control library but
only 13 in the Crohns disease library. This reduced
diversity in Crohns disease subjects was confirmed
by fluorescence in situ hybridization/flow cytometry
examination of feces in which a reduction in the
Clostridum leptum subgroup as a proportion of the
total bacterial community was observed
The bacterial profiles did not differ between
inflamed and non-inflamed tissues

Enterobacteria were more numerous in the feces of

Crohns disease patients, but Bacteroides and
Clostridium coccoides group numbers were
decreased compared to those of controls. There was
temporal variation in TTGE profiles of patients
compared to stability in those of controls

[81]

Yes, IBD treatment

numbers, language) can be acquired. Comparisons of the characteristics of germfree, monoassociated or conventionalized mice
in short-term experiments show that the presence of bowel bacteria influences gene expression in the bowel mucosa. Most
spectacular are the impacts on the transcription of genes whose
products influence epithelial integrity, angiogenesis, and deposition of fat in adipocytes [8385]. The results of gnotobiotic
experiments are certainly dramatic and point to the potential
for cross-talk between bowel bacteria and the tissues of their
host. These studies do not tell us if the same interactions occur
in humans but, if they do, they are most likely to occur in
childhood. Gnotobiotic animals mimic the bowel conditions of
human neonates that are initially bacteria-free but then are, to all
intents and purposes, monoassociated with bifidobacteria, later
becoming associated with a progressively more complex bacterial collection that seems to climax at about 4 years after birth

[80]

(Figs. 3 and 4). The neonatal bowel is physiologically immature

during early life and programming of the immune system seems
likely to be a continuous process during the first years of life
as appropriate windows of opportunity with regards to dietary
composition and bacterial exposure are opened [86]. If the virtual walls of the tube that sequester bacteria and their antigens
and inhibit them from making mucosal contact are not yet fully
developed in the first few years of life, one could postulate that
bacteria-host conversations, such as occurs in experimental gnotobiotes, might have long-lasting, biological repercussions. By
4 years of age, the education of the bowel tissue and its associated immune phenomena might all be over [87]. All of the
windows may be closed and shuttered. We are unlikely to ever
learn much about the composition of the bacterial community
of antediluvian humans. We do, however, have the opportunity
to investigate the developing bowel community and maturation
of the immune system of modern children. In Freudianism, the
adult conscious is unknowingly influenced by the subconscious
that has developed in early life due to the influences of various
interactive circumstances (social and environmental). Exposure
to a succession of bacterial populations in early life may result in
a physiological version of the Freudian iceberg in which the tip
(the discernible result) is but a small portion of a gigantic structure, most of which is unseen. Here lies a potentially productive
field of research for bacteriologists, physiologists and immunologists who are interested in neonatal development, allergy,
or inflammatory bowel diseases.
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Fig. 6. Comparison of numbers of bacteria associated with biopsies. Mean values and standard errors are shown. Bacterial numbers were the same for samples
collected from Crohns disease (CD) and healthy subjects, whereas numbers
were about doubled (P <0.01) for ulcerative colitis patients (UC). This was
not due to inflammation (I) of the tissue since noninflamed (NI) mucosa was
associated with similar numbers of bacteria (77; reproduced with permission).
Fourteen, 11 and 33 biopsies were examined for the CD, UC and healthy groups,
respectively.

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