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Seminars in Immunology 19 (2007) 94–105

Review

What immunologists should know about bacterial communitiesof the human bowel

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

bstract

The human bowel is home to a bacterial community of much complexity. This article summarizes current bacteriological knowledge of theommunity and highlights topics of potential interest to innovative immunologists. The role of the bacterial community in the development andegulation of the immune system of neonates seems likely to be a particularly important area of future research.

2006 Elsevier Ltd. All rights reserved.

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eywords: Bacteria; Bowel; Microbiota; Gut; Microbial ecology

. The company that you keep

Homo sapiens, it seems, first walked the Earth about 130,000ears ago. Our distant ancestors presumably carried within theirowels a large and complex community of microbes just aso their primate relations, modern descendants and other ani-al species represented in nature today. Whether this microbial

ommunity has the same composition in humans of the 21stentury as it did when associated with our ancient progeni-ors, somewhere in Africa, will perhaps never be determined.umans inhabiting developed countries today are, however,uch less likely to be colonized by parasitic worms (helminthes)

nd it is possible that the more hygienic lifestyles of recentecades have resulted in additional alterations to the compositionf the bowel community because exposure to certain bacteriaay be less common [1,2]. Even so, extant bowel communities

f humans are impressive both qualitatively (biodiversity) anduantitatively. Large bowel contents and feces contain about011 bacterial cells per gram (wet weight) and bacterial cellsomprise about 50% of fecal mass [3]. Four bacterial phyla areepresented (Firmicutes [Gram-positive], Bacteroidetes [Gram-egative], Actinobacteria [Gram-positive], and ProteobacteriaGram-negative]) and three groups, still phyogenetically broad,

ach containing many genera and species, are numerically dom-nant in the feces of healthy humans (Clostridium coccoidesroup, Clostridium leptum subgroup, Bacteroides-Prevotella

∗ Tel.: +64 3 479 7713; fax: +64 3 479 8540.E-mail address: gerald.tannock@stonebow.otago.ac.nz.

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044-5323/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.smim.2006.09.001

roup) [4,5]. Bacteria, then, dominate the bowel community.ungi and Archaea may also be resident, but comprise less than.05% and 1% of the total inhabitants, respectively [6,7]. Fur-her information concerning the phylogenetic composition ofhe bowel community is provided in Fig. 1. Much of this infor-

ation has been generated through the application of nucleiccid-based methodologies, most of which target the nucleotidease sequence of small ribosomal subunit RNA (16S rRNA inhe case of bacteria) which provides a cornerstone of micro-ial taxonomy. An earlier estimate of the number of bacterialpecies that might be resident in the human large bowel wasased on bacteriological culture. Four hundred species seemedlikely number by extrapolation from what had already been

ultured [12,13]. Nucleic acid-based methods of detection sug-est that about 50% of the bacterial cells seen microscopicallyn feces cannot yet be cultured in the laboratory, even whenccounting for the fact that some of the bacteria are dead [14,15].his phenomenon, manifest even more dramatically in terrestrialnd aquatic ecosystems was, based on traditional bacteriolog-cal experience, totally unexpected and has been called “thereat plate count anomaly” [16]. “Operational taxonomic units”OTU; molecular species) never encountered in culture-basedacteriology are detectable by the molecular methods, revealingnew world remaining to be investigated by bacteriologists of

he future. Estimates of biodiversity now seem to continuallynflate, probably because many of the 16S rRNA sequences in

atabanks differ by one nucleotide base. These are likely to beequences representing the same OTU but containing sequenc-ng errors. Curiously, therefore, we do not really know what,n any detail, our modern bowel community is composed of let

G.W. Tannock / Seminars in Imm

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ig. 1. Where and what types of bacteria are detected in the human gut [8–11].

lone what that of ancient humans contained. What we do know,nd all that matters really, is that these bowel inhabitants haverofound effects upon us.

. Unseen and unfelt

A chemostat is a culture apparatus that is used to maintain therowth of bacterial cultures continuously and at a constant ratender laboratory conditions. Culture medium is fed into a cultureessel containing the bacterial cells at a rate controlled by a flowegulator. The culture volume is held constant by means of anverflow tube that enables spent medium (effluent) to pass fromhe culture vessel. The human large bowel is the equivalent of ahemostat. It is fed with culture medium derived from the undi-ested components of the diet that pass from the small bowel,s well as substances produced endogenously by the humanost. Hence this culture medium is particularly rich in com-lex carbohydrates derived from plant cell walls and complexlycoproteins from mucus [17,18]. The ileo-cecal valve regu-ates the flow of culture medium into the large bowel; feces arehe effluent. Under these circumstances, a continuous fermen-ation of exogenous and endogenous substrates by consortia ofacteria proceeds, resulting in the formation of short chain fatty

cids (mainly acetic, propionic, and butyric), amines, phenols,ndoles, and gases as the major products [17]. The absorptionf the fatty acids from the bowel lumen once probably providedmportant caloric and carbon sources for humans, just as they do

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unology 19 (2007) 94–105 95

or other animal species even today [19]. Well-fed New Zealan-ers and North Americans, as examples, can doubtless manageithout this nutritional contribution much better than their pre-istoric forebears whose diet was impoverished by comparison.evertheless, nutritionists invoke butyric acid as an important

uel for colonocytes [20]. One would imagine that the bacterialulture responsible for the fermentation would be the same fromuman to human since bowel biochemistry follows the same pat-ern from one person to the next [5]. Yet, again on the basis ofolecular methods of analysis, it appears that bowel communi-

ies are about 30% dissimilar between humans (Fig. 2). Thoughsurprising fact when first encountered, the uniqueness of bowelommunities is likely to be due to extensive functional redun-ancy in the bacterial World: more than one bacterial speciesan carry out a particular catabolic process. Thus, just as there isnormal range of blood chemistry values in the human popula-

ion, there is also a range of inhabitants that can fill the ecologicaliches responsible for the overall bowel fermentation.

. It’s a wise man that knows his own father

According to Sophocles, Oedipus did not know hisntecedents and, because of this, in all innocence, murdered hisather and married his mother. A colossal tragedy resulting fromenealogical ignorance. We do not know where the phylogeneticrigins of the members of bacterial communities in the bowel lie,ut an educated guess would indicate that their ancestors werelant-associated, and regularly ingested by mammals. Cellulose,ectins, arabino-xylans, starch and beta-glucans, all substancesf plant origin, figure prominently as substrates for the predom-nant bowel bacteria [22–24]. Genomic sequences of residentsf the human bowel (Bacteroides and Bifidobacterium species)how that these bacteria devote much of their genomes to genesncoding hydrolytic enzymes, often contained in self-regulatedperons, that catalyze the degradation of oligosaccharides andlycoproteins [22,23]. There are also many genes associatedith signal transduction by which the bacteria can detect and

eact to changes in the nutrient milieu. Responses to qualitativehanges in substrate availability, and well-regulated metabolicathways that conserve cellular energy, help to explain the eco-ogical fitness of these bacteria and the phylogenetic stabilityf bowel communities [25]. The numerically predominant bac-eria of bowel communities are obligate anaerobes. This sug-ests a long-distant temporal origin when Earth’s atmosphereas anoxic, but their biochemical evolution must have con-

inued after the appearance of plants with complex cell wallsnd vascular systems that provide their nutritional substratesoday. Perhaps they were decomposers in sediments and natu-al composts, or similar anaerobic environments rich in complexarbohydrates? Some of these environmental bacteria must havedapted to the bowel environment so that residence, still on theutside (bowel lumen), but sequestered inside, of the humanody was established. The human bowel is daily contaminated

ith bacterial cells present in food and water. Some of these bac-

eria survive and transit the digestive tract and are detectable inhe feces [26]. The same applies to “probiotic” bacteria that arententionally consumed in self-care health products [27]. These

96 G.W. Tannock / Seminars in Immunology 19 (2007) 94–105

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Fig. 2. The composition of the fecal bacteri

allochthonous” bacteria do not become part of the bowel com-unity because they are no longer detected in feces once the

ource of their ingestion is removed. “Autochthonous” bacteria,n the other hand, have a habitat in the bowel. They have long-erm associations with the human host, form stable populationsf characteristic size, and have demonstrable means of earningliving (an ecological function or niche) in the distal bowel

28]. Researchers interested in the bowel community need to beware of the Oedipus tragedy; would not it be embarrassing to

laim that the digestive tract was colonized by a vast array ofacteria when, in fact, only the DNA of allochthonous microbesad been detected? Unraveling the secrets of life in the bowelould be impossible if an allochthonous fecal isolate was chosen

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munity varies from human to human [21].

s the model organism for study! Snapshots of the composi-ion of bowel communities are only the beginning of ecologicalnvestigations and DNA-based methods reveal only “who mightave been there”. Temporal (quantitative sequential measure-ents) and function-based (transcriptomics, proteomics) obser-

ations are necessary to define bacterial communities. We needo know “what the bowel residents are doing and how they dot”. It would also be very interesting to know how the transi-ion from allochthonous to autochthonous status in relation to

he bowel was achieved. The highly competitive nature of theowel community may have represented, and perhaps still does,hotbed of evolution where novel attributes developed and wereelected. The molecular (genetic) processes by which bacteria

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G.W. Tannock / Seminars in

dapted from a free-living lifestyle outside of the body to aost-dependent life in the bowel would provide the basis of aascinating story.

. The child is father of the man

The gut of newborn human infants resembles that of theermfree animal because it does not yet harbor a bacterial orther microbial community. This germfree state is short-livedecause within minutes of birth the baby is exposed to bac-eria in the feces that have been involuntarily expelled by the

other during labor, as well as to environmental microbes.uckling, kissing and caressing the infant after birth providedditional assurance that maternal microbes are transmitted fromne human generation to another. Regulatory mechanisms gen-rated within the ecosystem (autogenic factors) and by external

orces (allogenic factors) permit the episodic persistence of someacterial populations, but the elimination of others in a classicaliological succession [29]. It takes several years to produce a cli-ax community resembling that of adults (Figs. 3 and 4; [30]).

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ig. 3. The composition of the bowel bacterial community takes years to stabilize. Cecal samples collected at intervals during the first 6 years of life. The communityomplex. Stability in composition is not apparent until about 4 years of age (Munro,

unology 19 (2007) 94–105 97

embers of the bacterial genus Bifidobacterium are numeri-ally predominant in the gut of infants during the first monthsf life. Nucleic acid-based methods of analysis show that bifi-obacteria form between 60% and 91% of the total bacterialommunity in the feces of breast-fed babies and 28–75% (aver-ge 50%) in formula-fed infants whereas they comprise only aew percent of the fecal community of adult humans [31]. Thenfant during early life is therefore almost a monoassociatednotobiote, and bifidobacterial antigens may be important insti-ators of immunological development. By analogy, Rene Dubosnd colleagues at Rockefeller University carried out pioneeringtudies of the impact of the bacterial community of the bowelf infant mice. As recalled by Dwayne Savage, the NCS (Newolony Swiss) mouse colony derived at The Rockefeller Uni-ersity was the first murine colony in the World in which thenimals, while harboring a bowel community, were yet free of

ertain mouse pathogens and could be bred in sufficient quantityor major experiments [32]. Dubos and colleagues soon realizedhat NCS mice differed in several characteristics from SS miceStandard Swiss from which the NCS colony had been derived).

omparison of PCR/DGGE profiles of the fecal community of a human infant.is relatively simple until after weaning and then becomes progressively moreBateup and Tannock, unpublished).

98 G.W. Tannock / Seminars in Imm

Fig. 4. Babies are almost monoassociated gnotobiotes. Quantification of bifi-dobacteria relative to total community composition in the feces of the childwhose bacterial community profiles are shown in Fig. 3. Bifidobacteria com-prised almost 100% of the total community early in life, gradually reducing tolevels characteristic of adults (Munro, Bateup, Tannock, Harmsen and Welling,ua

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a[otpib“AarincAtpoStcw2apsccrtfdwloZlipiaedbtcocaesarean sections, special-care baby unit admissions, smaller

npublished). Quantification by fluorescence in situ hybridization/computer-ssisted microscopy.

CS mothers bore on average more infants per litter, and theirffspring grew faster and were larger than SS mice (even whened diets low in lysine and threonine). It was suggested that theifferences between NCS and SS mice were the result of muta-ions (in other words, that a new mouse strain had been selected)ut, astoundingly, NCS mice housed with SS mice reverted to theharacteristics of SS mice in all properties tested. The fecal bac-eria of the two colonies of mice were different when analyzedy bacteriological culture. Unlike the SS mice, NCS animalsid not harbor facultatively anaerobic Gram-negative bacteriauch as Escherichia coli. Remarkably, NCS mice were toleranto parenteral doses of endotoxin that were lethal within a feways of administration to SS mice. Exposure of NCS animals toS mice during early life, or prior injections with small dosesf heat-killed cells of Gram-negative bacteria that thus exposedhe animals to sub-lethal amounts of endotoxin, increased theusceptibility of the NCS mice such that it now matched thatf SS animals. Dubos et al. concluded that the enterobacteria,resent in relatively high numbers in the gut of infant SS mice,ensitized the animals so that, as adults, they were highly sus-eptible to endotoxin [33]. Drawing on these results, Dubos andolleagues concluded that “From all points of view, the child

s truly the father of the man, and for this reason we need toevelop an experimental science that might be called biologicalreudianism. Socially and individually the response of human

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unology 19 (2007) 94–105

eings to the conditions of the present is always conditioned byhe biological remembrance of things past”.

. Immunological Freudianism

In recent decades, many affluent countries have experiencedn increase in the prevalence of atopic diseases, including asthma2]. Several aspects of lifestyle have changed in these countriesver the same period and theories have been advanced to explainhe altered prevalence of allergies. The “Hygiene Hypothesis”roposes that atopic diseases could be prevented by infectionsn early childhood because the neonatal immune system woulde driven towards a T helper 1 (Th1) response, but a specificinfectious protective factor” has never been identified [34,35].ttention has turned to the bacterial community of the bowel

nd the possibility that colonization of the gut by specific bacte-ial species might be more important than the impact of sporadicnfections. For example, the composition of the bowel commu-ity has been reported to differ in the numbers of lactobacilli andlostridia in the feces of Estonian and Swedish children [36].topic diseases were less prevalent in the Estonian compared to

he Swedish population. Ouwehand et al. have reported that therevalence of Bifidobacterium adolescentis differed in the fecesf healthy and allergic Finnish children aged 2–7 months [37].ix out of seven allergic children harbored B. adolescentis in

he faeces whereas this species was not detected in six healthyhildren. Young and colleagues reported the results of a study inhich the fecal populations of bifidobacteria from children aged5–35 days in Ghana (low prevalence of atopy), New Zealandnd the United Kingdom (high prevalence countries) were com-ared. Natal origin influenced the detection of bifidobacterialpecies because fecal samples from Ghana almost exclusivelyontained Bifidobacterium infantis whereas those of the otherhildren did not. Choosing species on the basis of the bacte-iological results, bifidobacterial preparations were tested forheir effect on cell surface markers of dendritic cells harvestedrom cord blood. Bifidobacterial species-specific effects onendritic cell activation were observed in that CD83 expressionas increased by Bifidobacterium bifidum, Bifidobacterium

ongum and Bifidobacterium pseudocatenulatum. One or moref these species were detected in the faeces of 40 out of 46 Newealand and United Kingdom children, but only in a few (B.

ongum: 2/32) of the samples from infants living in Ghana. B.nfantis, common in the feces of babies from Ghana, failed toroduce this effect [38]. Further investigations of the molecularnterplay between bifidobacteria, human dendritic cells, T cellsnd allergens are clearly required and may provide the first clearvidence of “Immunological Freudianism” in relation to humaniseases. The environmental conditions under which babies areorn and nurtured may affect which microbes they are exposedo, and subsequently influence the composition of their bowelommunity. Differences in neonatal bowel communities mightccur due to the common occurrence of hospital deliveries,

amily size, widespread use of antibiotics, good hygiene, andature of the maternal diet in affluent countries [39]. Either theack of exposure of babies to particular bifidobacterial species,

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G.W. Tannock / Seminars in

nd/or elimination of bifidobacterial species from the bowelhrough the use of antibiotics, might reduce the exposure ofhildren in early life to important bacterial antigens at a criticalime in the maturation of the immune system, for examplen removing (immune deviation) the T helper 2 (Th2) skewpparently characteristic of the newborn [40,41].

. Retained but contained

The antigenic burden associated with approximately 2 × 1013

acterial cells (exceeding the human population of Earth about0,000-fold) present at any moment in the large bowel is prob-bly incalculable. Dendritic cells associated with the bowelpithelium may sample this antigenic landscape regularly byxtending dendrites between enterocytes to reach the mucosalurface [42]. M cells of Peyer’s Patches are also sites of antigenptake [43]. Relative to germfree animals, the lamina proprian the gut of animals harboring bacterial communities is mildlynflamed in terms of histopathology [44]. Antibodies reactiveith bowel bacterial antigens are present in human sera [45].hus bacterial antigens are detected by the innate and adaptive

mmune systems, but inflammation is somehow kept at mini-al levels. How “tolerance” (hypo-responsiveness) to the bowel

ommunity is achieved mechanistically should be an interestingopic of immunological research. It can be postulated, though,hat sequestration of the bacterial community away from themmune system in a “virtual tube” within the bowel may belarge part of the answer. The mucosal surface of the bowel is

overed with a blanket of mucus that flows away from the epithe-ium and is continuously replaced by fresh mucus from the gobletells [46]. This moving, sticky blanket cleanses the mucosal sur-ace keeping bacterial cells at bay. The tight junctions formedetween enterocytes render the epithelium a formidable bar-ier to bacterial penetration [47]. Moreover, bacterial cells inhe bowel are coated with sIgA, possibly counteracting effec-ive use of bacterial adhesins [48]. Defensins liberated in thentestinal crypts by Paneth cells complete the “wall” of the tube49]. Everything is designed to stop bacteria associating withhe mucosal surface. Contained within the virtual tube in theowel lumen, the bacterial community can be retained safely.evertheless, it does not seem to be a totally efficacious sys-

em since pathogens can reach the mucosal surface and attachy means of adhesins to specific receptors prior to prolifera-ion or invasion. Even in health, small temporary cracks in theirtual walls of the tube must occur which allow bacterial anti-ens to seep from the tube and make contact with enterocytes,endrites of dendritic cells, and M cells. Although enterocytesre not professional immune cells, contact with bacterial anti-ens may cause a transient induction of “immune responses”ithin them. For example, Ruiz et al. have described inductionf phosphorylation/activation of the NF-�B transcriptionallyctive subunit RelA and the mitogen-activated protein kinase38 in enterocytes by an innocuous bifidobacterial species,

hich resulted in interleukin-6 gene expression [50]. The cor-erstone of innate signaling between bacteria and enterocytesests on pattern recognition receptors (Toll-like receptors [TLR],ucleotide-binding oligomerization domain protein receptors

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unology 19 (2007) 94–105 99

NOD]) that recognize molecular patterns associated with bac-erial cells regardless of whether they originate in the bowelommunity or are pathogens [51]. However, pathogens inducen acute inflammatory response, harmless bacteria do not. Thisifferential effect may be due to the relative location of differ-nt TLR (apical membrane, intracellular, basal membrane ofnterocytes), the rapid turnover of enterocytes at villous tips,r to particular properties of pathogens (virulence factors) thatther bacteria lack. Negative regulators exerted by the entero-ytes themselves might also mediate the differential responseo signals from bowel inhabitants and pathogens [52]. Defininghese differential/regulatory systems is important immunolog-cal research because it impinges on the etiology of chronicmmune inflammatory bowel diseases.

. Surrogate pathogens

Failure to demonstrate convincingly an association betweenspecific pathogen and chronic immune inflammatory condi-

ions of the bowel (Crohn’s disease and ulcerative colitis) hasesulted in the concept that the inflammation is fueled by theacterial bowel community, or at least some members of it [53].his is demonstrably true in experimental animal models of col-

tis in which gene deletion or transgenic manipulation producesdysfunctional immune system that reacts aggressively with

acterial antigens [53]. Under germfree conditions, these ani-als have minimal disease but develop colitis when exposed tospecific-pathogen-free collection of bowel bacteria, or selectedtrains of bowel bacteria. Human patients suffering from inflam-atory bowel diseases (IBD) have a broken tolerance to their

wn bowel bacteria as has been clearly demonstrated by thelassical studies of Duchmann et al. [54]. Familial clusteringf cases and association with specific chromosomal aberrationsat least in a proportion of patients) and with particular humaneukocyte antigen genotypes indicate a genetic predisposition toBD [55,56]. The bowel community may indeed be the antigenicuel for chronic inflammation (in this sense acting as a surro-ate pathogen), but how and what initiates the process? It coulde postulated, perhaps, that the enterocytes and, concurrently orubsequently, immune cells are exposed to excessive amounts ofacterial antigens because the virtual, multi-component defen-ive wall of the bowel of humans predisposed genetically toBD is defective in at least one factor. For example, the alteredhemical composition of the mucus of ulcerative colitis patientsay be linked to the larger number of bacteria associated with

iopsies collected from them (Fig. 5). Genetically based causesf breaches in the wall, possibly occurring very early in lifediagnosis of IBD is characteristically made in young adults),ogether with broken immunological tolerance to bacterial anti-ens, could result in a sensitization to bacterial antigens andhe progression of poorly controlled inflammatory reactions.lthough the initiating factors may well occur early in life, stud-

es of the composition of the distal bowel community represented

n feces have mostly been conducted with adult IBD patients inhe hope that they would reveal specific groups of bacteria that

ight be associated with inflammation. These studies have pro-uced extremely variable results (Table 1). In retrospect, this

100 G.W. Tannock / Seminars in Immunology 19 (2007) 94–105

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s not terribly surprising concerning the approaches that haveeen followed in defiance of the complexity of the ecosystemnder investigation (Fig. 6). Broad-based phylogenetic tools, aolluted 16S rRNA gene database, subjects that are medicated,ack of standardization of biopsy-associated measurements, andarying fecal water content (patients may have loose stools, con-rols formed stools) are among the confounding factors. A much

etter approach may be to detect the substances against whichhe dysfunctional immune system reacts. This will require “cul-ivating the uncultured” members of the bowel community or,ailing that, functional screens (immunoassays) of gene libraries

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accurate picture of the bowel ecosystem?

epresenting the collective genomes of the bacterial communitymetagenomics) [82].

. Give me the children until they are seven and anyoneay have them afterwards

Francis Xavier believed that a Jesuit education until the age of

prepared a child to live a useful Christian life in no matter whatircumstances they later ended up in. Modern educationists, too,nderstand that there are optimal periods during childhood inhich the ability to process certain information (visual, sound,

G.W. Tannock / Seminars in Immunology 19 (2007) 94–105 101

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

Investigation Patients medicated forIBD or administeredantimicrobial drugs?

Observations Reference

Biopsies were collected from five colonic sites as well asthe terminal ileum of nine healthy subjects. Fluorescencein situ hybridization/microscopy was used to target fecalbacterial groups

None Bacterial cells were seen associated with the luminalsurface of mucus and seldom in the mucus itself

[57]

Colonic biopsies from 21 ulcerative colitis patients, 14Crohn’s disease patients, and 24 controls were collectedand treated with dithiothreitol to remove mucus, thenmucosa-associated and intramucosal bacteria (released byhypotonic lysis and gentamicin treatment) were cultured

Yes, IBD treatment Bacteria were cultured more commonly fromsamples from Crohn’s disease patients compared tothose from ulcerative colitis or controls. AdherentEscherichia coli were more commonly detected insamples from Crohn’s disease patients compared tocontrols

[58]

DNA extracted from paraffin-embedded tissue samplescollected from 16 Crohn’s disease, 11 ulcerative colitisand 18 colon cancer patients was used to enumerateMycobacterium species, Bacteroides vulgatus, and E. coliusing real time quantitative PCR

IBD treatment notstated

Mycobacteria were not detected but B. vulgatus andE. coli were detected more frequently and in greaternumbers in samples obtained from Crohn’s diseaseand ulcerative colitis patients compared to coloncancer subjects

[59]

Colonic biopsies were washed and used for culture ofbacteria, and detection of bacteria by electronmicroscopy, fluorescence in situ hybridization, and PCR.The results from 28 patients with self-limiting colitis, 104with indeterminate colitis, 119 with ulcerative colitis, 54with Crohn’s disease, and 40 asymptomatic subjects werecompared

IBD treatment notstated

There were more bacterial cells associated withbiopsies collected from patients than fromasymptomatic controls. The numbers of bacteriaincreased in association with increasedinflammation (colitis)

[60]

Rectal biopsies and feces were collected from 12 Crohn’sdisease patients during periods with or withoutantimicrobial (metronidazole, cotrimoxazole) treatment.Culture-based studies were conducted

Yes, antimicrobialdrugs during part ofthe study

Eight of the 12 patients showed clinicalimprovement when administered antimicrobialdrugs but this could not be correlated withbacteriological results

[61]

Biopsies collected from four sites (ileum, right and leftcolon, rectum) were collected from 20 Crohn’s disease,11 ulcerative colitis, and 4 control subjects. Temporaltemperature gradient gel electrophetic fingerprints ofbiopsy-associated bacteria were prepared

IBD treatment notstated

Similar profiles were obtained from sampling sitesintra-individual, but varied inter-individually.Profiles did not discriminate between subject groups

[62]

Rectal biopsies collected from 9 ulcerative colitis patientsand 10 control subjects were examined by culture andfluorescence in situ hybridization/microscopy

IBD treatment notstated

The bacterial types that were detected variedbetween subjects making it impossible to assign apathogenic role to specific bacteria

[63]

Rectal biopsies collected from 33 ulcerative colitis patients,6 Crohn’s disease, and 14 control subjects were examinedby fluoresence in situ hybridization/microscopy

Yes, IBD treatment Bifidobacterial numbers were lower in ulcerativecolitis patients; E. coli numbers were higher inulcerative colitis and Crohn’s disease patients

[64]

Tissue sections were prepared from resected intestine(terminal ileum, colon) and examined by fluorescence insitu hybridization/microscopy. Samples were collectedfrom 12 ulcerative colitis, 12 Crohn’s disease, and 14control subjects

IBD treatment notstated. Antimicrobialdrugs givenintravenously justprior to surgery

There were higher numbers of bacteria associatedwith tissue collected from ulcerative colitis andCrohn’s disease subjects and there was moreevidence of bacterial invasion of tissue. Samplesfrom Crohn’s disease patients had mostlyproteobacteria and Bacteroides-Prevotellaassociated with the tissue, whereas a more diversecollection of bacteria was present in ulcerativecolitis samples

[65]

Biopsies collected endoscopically or surgically from theterminal ileum of 11 Crohn’s disease and 11 controlsubjects were examined by PCR

IBD treatment notstated. Antimicrobialsprior to surgery

16S rRNA gene sequences similar to those ofHelicobacter species, Mycobacterium aviumsubspecies paratuberculosus, Listeriamonocytogenes and E. coli were detected in three offive biopsies collected surgically from Crohn’sdisease patients. Biospies collected from all subjectsby endoscopy had associated bacteria, but biopsiescollected at surgery from controls were devoid ofbacteria

[66]

Colonic biopsies collected from 13 ulcerative colitispatients and 61 controls were examined by PCRcombined with enrichment culture to detect the presenceof sulphate-reducing bacteria

IBD treatment notstated

All of the biopsies contained sulphate-reducingbacteria

[67]

102 G.W. Tannock / Seminars in Immunology 19 (2007) 94–105

Table 1 (Continued )

Investigation Patients medicated forIBD or administeredantimicrobial drugs?

Observations Reference

Colonic biopsies from 26 Crohn’s disease, 31 ulcerativecolitis, 15 controls with inflammation, and 31non-inflamed control subjects were examined by 16SrRNA gene profiling, the preparation of 16S rRNA geneclone libraries, and real time quantitative PCR

Yes, IBD treatment The diversity of bacterial types was less in samplesfrom Crohn’s disease and ulcerative colitis patientscompared to controls. This was due to a reduction indetection of Bacteroides species, Eubacteriumspecies, and Lactobacillus species

[68]

Rectal biopsies were collected from 19 IBD patients (notdifferentiated) and 14 controls. The biopsies wereexamined by RNA in situ hybridization/microscopy

Yes, IBD treatment Bacterial cells were not observed in the case of 10control and 6 IBD patient biopsies. In the remainingsamples, there were more bacteria associated withIBD biopsies compared to those of controls. Thebacteria were seen within the mucus but not incontact with enterocytes, nor within the laminapropria

[69]

16S rRNA gene clone libraries were prepared from 16biopsies collected from Crohn’s disease patients(aphthous ulcers), 15 surgical samples from Crohn’sdisease patients, and biopsies from controls

Yes, IBD treatment Specific phylogenetic groups of bacteria could notbe associated with Crohn’s disease lesions

[70]

Ileal and colonic tissues from Crohn’s disease patients andcontrols were studied to determine the prevalence ofadherent-invasive strains of E. coli

IBD treatment notstated

Adherent-invasive strains of E. coli were morecommonly detected in association with tissues,particularly from the ileum, of Crohn’s diseasepatients compared to those of controls

[71]

Tissue samples collected at surgery from 20 Crohn’s diseasepatients and 6 controls, and examined by nested PCR andfluorescence in situ hybridization/microscopy for thepresence of Mycobacterium avium subspeciesparatuberculosis

Yes, IBD treament M. avium subsp. paratuberculosis DNA wasdetected more commonly in samples from Crohn’sdisease patients than from those of controls.

[72]

Archival tissue obtained at surgery from 15 Crohn’s diseasepatients and 10 non-Crohn’s granulomatous boweldisease patients were examined. Granulomas weremicrodissected from the tissue and DNA was extracted.Nested PCR was used to detect sequences characteristicof E. coli.

Treatments not stated. E. coli DNA was detected more frequently insamples from Crohn’s disease patients than fromnon-Crohn’s disease patients. The authors concludedthat, since a similar result with respect to M. aviumsubsp. paratuberculosis had been obtained withthese same samples in a previous study, the findingsmay reflect non-specific association of bacteria withgranulomas, rather than cause and effect

[73,74]

Colonic biopsies were collected from 4 ulcerative colitispatients and 2 controls. 16S rRNA gene clone librarieswere prepared and selected libraries were probed with alabeled oligonucleotide probe that detectedBacteroides-Prevotella

Yes, IBD treatment There were more clones representingBacteroides-Prevotella bacteria in libraries fromulcerative colitis patients than from controls

[75]

Pediatric patients with Crohn’s disease (12 subjects),ulcerative colitis [7], other intestinal abnormalities [16],and 7 controls were studied. Biopsies were collectedfrom ileum, cecum and rectum and examined bybacteriological culture and qualitative and quantitativePCR

None Higher numbers of facultatively anaerobic bacteriawere associated with samples from Crohn’s diseaseand ulcerative colitis patients compared to controls.The numbers of Bacteroides vulgatus were lower inthe case of Crohn’s disease and ulcerative colitispatients relative to controls

[76]

16S rRNA gene clone libraries were prepared from biopsiescollected from 20 Crohn’s disease, 15 ulcerative colitispatients, and 14 controls. Patient biopsies were collectedfrom both inflamed and non-inflamed mucosa of eachsubject. Denaturing gradient gel electrophoretic (DGGE)profiles were prepared, and total bacterial numbersassociated with biopsies were enumerated by real timequantitative PCR

None. Patients werenewly diagnosed anduntreated.

DGGE profiles were similar for all sites sampledintra-individually but differed inter-individually.LIBSHUFF statistical comparison showed that 16SrRNA gene clone libraries prepared from Crohn’sdisease, ulcerative colitis and control subjects haddifferent compositions. Collections of bacteriaassociated with inflamed tissue did not differ fromthose of non-inflamed tissue. There were morebacterial cells associated with biopsies collectedfrom ulcerative colitis patients compared to those ofCrohn’s disease and control subjects. Unclassifiedmembers of the Class Bacteroidetes were moreprevalent in the samples from Crohn’s diseasepatients compared to the other groups.

[77]

The fecal bacterial communities of Crohn’s disease (fivepatients) and healthy controls (five) were compared bybacteriological culture

None The number of bacteria belonging to the Bacteroidesfragilis group was higher, and these bacteria weredetected more frequently, in patients than controls

[78]

G.W. Tannock / Seminars in Immunology 19 (2007) 94–105 103

Table 1 (Continued )

Investigation Patients medicated forIBD or administeredantimicrobial drugs?

Observations Reference

Two metagenomic libraries were prepared from fecalsamples obtained from 6 Crohn’s disease patients and 6controls, respectively. 16S rRNA genes detected in thelibraries were sequenced

Yes, IBD treatment Forty three OTU belonging to the phylumFirmicutes were detected in the control library butonly 13 in the Crohn’s disease library. This reduceddiversity in Crohn’s disease subjects was confirmedby fluorescence in situ hybridization/flow cytometryexamination of feces in which a reduction in theClostridum leptum subgroup as a proportion of thetotal bacterial community was observed

[79]

Biopsies were collected from the intestines of 15 Crohn’sdisease patients during flare-ups of disease. Inflamed andnon-inflamed mucosal samples were obtained andexamined by PCR/TTGE

IBD treatment notstated

The bacterial profiles did not differ betweeninflamed and non-inflamed tissues

[80]

Radioactive probes were used to quantify bacterial groupsin the feces of 17 Crohn’s disease patients and 16controls. Community profiles were prepared by temporaltemperature gradient gel electrophoresis (TTGE)

Yes, IBD treatment Enterobacteria were more numerous in the feces ofCrohn’s disease patients, but Bacteroides andClostridium coccoides group numbers weredecreased compared to those of controls. There was

[81]

ntitspsefhichibr

FucwnaFr

(dlactadbt4cw

umbers, language) can be acquired. Comparisons of the charac-eristics of germfree, monoassociated or conventionalized micen short-term experiments show that the presence of bowel bac-eria influences gene expression in the bowel mucosa. Mostpectacular are the impacts on the transcription of genes whoseroducts influence epithelial integrity, angiogenesis, and depo-ition of fat in adipocytes [83–85]. The results of gnotobioticxperiments are certainly dramatic and point to the potentialor cross-talk between bowel bacteria and the tissues of theirost. These studies do not tell us if the same interactions occurn humans but, if they do, they are most likely to occur inhildhood. Gnotobiotic animals mimic the bowel conditions ofuman neonates that are initially bacteria-free but then are, to all

ntents and purposes, monoassociated with bifidobacteria, laterecoming associated with a progressively more complex bacte-ial collection that seems to climax at about 4 years after birth

ig. 6. Comparison of numbers of bacteria associated with biopsies. Mean val-es and standard errors are shown. Bacterial numbers were the same for samplesollected from Crohn’s disease (CD) and healthy subjects, whereas numbersere about doubled (P <0.01) for ulcerative colitis patients (UC). This wasot due to inflammation (I) of the tissue since noninflamed (NI) mucosa wasssociated with similar numbers of bacteria (77; reproduced with permission).ourteen, 11 and 33 biopsies were examined for the CD, UC and healthy groups,espectively.

lotoatita(tfino

R

temporal variation in TTGE profiles of patientscompared to stability in those of controls

Figs. 3 and 4). The neonatal bowel is physiologically immatureuring early life and programming of the immune system seemsikely to be a continuous process during the first years of lifes appropriate windows of opportunity with regards to dietaryomposition and bacterial exposure are opened [86]. If the vir-ual walls of the tube that sequester bacteria and their antigensnd inhibit them from making mucosal contact are not yet fullyeveloped in the first few years of life, one could postulate thatacteria-host conversations, such as occurs in experimental gno-obiotes, might have long-lasting, biological repercussions. By

years of age, the education of the bowel tissue and its asso-iated immune phenomena might all be over [87]. All of theindows may be closed and shuttered. We are unlikely to ever

earn much about the composition of the bacterial communityf antediluvian humans. We do, however, have the opportunityo investigate the developing bowel community and maturationf the immune system of modern children. In Freudianism, thedult conscious is unknowingly influenced by the subconscioushat has developed in early life due to the influences of variousnteractive circumstances (social and environmental). Exposureo a succession of bacterial populations in early life may result inphysiological version of the Freudian iceberg in which the tip

the discernible result) is but a small portion of a gigantic struc-ure, most of which is unseen. Here lies a potentially productiveeld of research for bacteriologists, physiologists and immu-ologists who are interested in neonatal development, allergy,r inflammatory bowel diseases.

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