Intestinal Microbiota in Functional Bowel Disorders

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Intestinal microbiota in functionalbowel disorders: a Rome foundationreportMagnus Simren,1 Giovanni Barbara,2 Harry J Flint,3 Brennan M R Spiegel,4

Robin C Spiller,5 Stephen Vanner,6 Elena F Verdu,7 Peter J Whorwell,8

Erwin G Zoetendal9

ABSTRACT

It is increasingly perceived that gut hostemicrobialinteractions are important elements in the pathogenesisof functional gastrointestinal disorders (FGID). The mostconvincing evidence to date is the finding that functionaldyspepsia and irritable bowel syndrome (IBS) may

develop in predisposed individuals following a bout of

infectious gastroenteritis. There has been a great deal ofinterest in the potential clinical and therapeutic

implications of small intestinal bacterial overgrowth inIBS. However, this theory has generated much debatebecause the evidence is largely based on breath testswhich have not been validated. The introduction ofculture-independent molecular techniques providesa major advancement in our understanding of themicrobial community in FGID. Results from 16S rRNA-based microbiota profiling approaches demonstrate bothquantitative and qualitative changes of mucosal andfaecal gut microbiota, particularly in IBS. Investigatorsare also starting to measure hostemicrobial interactionsin IBS. The current working hypothesis is that abnormalmicrobiota activate mucosal innate immune responseswhich increase epithelial permeability, activatenociceptive sensory pathways and dysregulate theenteric nervous system. While we await importantinsights in this field, the microbiota is alreadya therapeutic target. Existing controlled trials of dietarymanipulation, prebiotics, probiotics, synbiotics andnon-absorbable antibiotics are promising, although most

are limited by suboptimal design and small sample size.In this article, the authors provide a critical review ofcurrent hypotheses regarding the pathogenetic

involvement of microbiota in FGID and evaluate theresults of microbiota-directed interventions. The authors

also provide clinical guidance on modulation of gutmicrobiota in IBS.

INTRODUCTION

Functional gastrointestinal disorders (FGIDs) aredened by symptom-based diagnostic criteria thatcombine chronic or recurrent symptoms attribut-able to the GI tract in the absence of other patho-logically-based disorders.1 The FGIDs are classiedinto six major categories for adults: oesophageal,gastroduodenal, bowel, functional abdominal pain

syndrome, biliary and anorectal. Of these, the

functional bowel disorders (FBD) constitute one ofthe most common reasons for seeking healthcare,2

and they are associated with poor health-relatedquality of life3e5 and substantial costs to society.6e9

The pathophysiological mechanisms underlyingthese disorders are incompletely known, butabnormal gastrointestinal (GI) motility, visceral

hypersensitivity, altered braine

gut function, low-grade inammation, psychosocial disturbance andintestinal microbes may contribute.10e12

The human body is inhabited by a complexcommunity of microbes, collectively referred to asmicrobiota.13 It is estimated that the humanmicrobiota contains 1014 cells, which outnumberthe human cells in our bodies by a factor of ten.14Avast majority of these are found in the GI tract,with a continuum from 101e103 bacteria per gramof content in the stomach and duodenum to1011e1012 cells per gram in the colon.15 Moreover,the microbial composition differs between thesesites,16 and there are also signicant differencesbetween the microbiota present in the gut lumenand the microbiota attached to and embedded inthe mucus layer of the GI tract.17 The microbiota istaxonomically classied via the traditional biolog-ical nomenclature (phylumdclassdorderdfamilydgenusdspecies) and currently more than50 bacterial phyla have been described, of which 10inhabit the colon and three predominate: theFirmicutes, Bacteroidetes and the Actinobacteria;other sites display a different microbial composi-tion.18 19 A challenge for researchers and cliniciansis that most of the microbial diversity in thehuman GI tract is not currently represented by

available cultured species,20

but during recent years,the use of culture-independent techniques to studythe gut microbiota has increased the understandingof the role of gut microbiota in health and disease. 14

Several lines of evidence indicate that bacteriamay be involved in the pathogenesis and patho-physiology of FBD, through the metabolic capacityof the luminal microbiota, and the potential of themucosa-associated microbiota to inuence the hostvia immuneemicrobial interactions.21 For instance,many subjects with irritable bowel syndrome (IBS)report symptom onset following an enteric infec-tion.22 There are also studies reporting positiveeffects of treatments directed at gut microbiota in

patients with FBD.23 24 Moreover, small intestinal

< An additional material ispublished online only. To viewthis file please visit the journalonline (http://dx.doi.org/10.1136/gutjnl-2012-302167).

For numbered affiliations seeend of article.

Correspondence toProfessor Magnus Simren,Department of Internal Medicine,Institute of Medicine,Sahlgrenska Academy, Universityof Gothenburg, GothenburgS-41345, Sweden; [email protected]

Recent advances in clinical practice

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Published Online First10 July 2012


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bacterial overgrowth (SIBO)25 and altered intestinalmicrobiota26 are implicated in subgroups of FBDpatients. However, the clinical relevance of thesendings remains unclear and, therefore, we soughtto critically review the existing literature on therole of intestinal microbiota in FBD, focusingpredominantly on IBS, and to provide recommen-dations for how to implement the current knowl-

edge into clinical practice and to guide futureresearch.

This manuscript is a synthesis of the endeavourof the Rome Foundation Committee Report. Moreindepth description of the work produced by thisteam is provided as online supplementary material.

CURRENT KNOWLEDGE OF THE MICROBIOTAA relationship, often termed symbiosis, has devel-oped between the host and the intestinal micro-biota over millions of years. Host genetic andimmune as well as environmental factors inuenceintestinal microbiota composition which in turnshape host immunity and physiology within andbeyond the gut (gure 1). Recent human studiesdemonstrate a hitherto unimagined complexity ofthe human gut microbiota with hundreds ofphylotypes, of which 80% remain uncultured.19 Ofthe 10 bacterial phyla detected in the gut theFirmicutes, Bacteroidetes and Actinobacteriapredominate, of which the Firmicutes is the mostdominant and diverse phylum in the GI tract.

Facultative anaerobes account for


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changed both our diet and microbiota as evidencedby comparing the faecal microbiota of African ruralchildren with a polysaccharide-rich diet with Italiancity children on a high fat, high protein diet. Africanchildren have a signicant enrichment in Bacter-oidetes, especiallyPrevotellaand Xylanibacter generaknown to contain genes for xylan hydrolysis35

(gure 2). Whole grain cereals,36 resistant starch37 38

and low residue diets profoundly alter the micro-biota.39 Although there is evidence indicating thatobese individuals have an increase in Firmicutes anda decrease in Bacteroidetes (a difference likely relatedin part to different diets40), other studies failed tosupport these observations.41 42 Many dietaryprebiotics including oligofructose,43 lactulose,4 4 4 5

lupin kernel,46 inulin-containing juices47 and arabi-noxylan-oligosaccharides48 signicantly alterhuman faecal microbiota. The concept of poorlyabsorbed but fermentable oligo-, di- and mono-saccharides and polyols (FODMAPs) includes manysubstances which are substrates for bacterialmetabolism and may therefore alter the microbiotabut this has as yet not been studied.

Most high bre diets alter the microbiota andaccelerate transit. Accelerating transit using sennaincreased the production of short chain fatty acids(SCFAs) but reduced faecal methanogens, theopposite to the effect of loperamide.49 Acceleratingtransit with cisapride also increases production ofSCFAs, particularly propionic and butyric acids.50

Acetate, which predominates in the coloniccontents, is largely inhibitory. In contrast, propio-nate and butyrate stimulate motility, activatepropulsive ileal motor patterns in humans51 andensure that bacteria are propelled from the ileum to

the colon. The normal microbiota also stronglyinuence the mucosal immune system52 53 which isunderdeveloped in germ-free animals, who havereduced T cells, immunoglobulin A producing Bcells and intraepithelial Tcells.52 54e56 Twin studiessuggest that the host genotype inuences the gut

microbiota, although results remain conictingbecause of the inability to control for shared envi-ronmental factors.40 57 One of the most importantgenetic effects is mediated via the innate immuneresponse. Thus, mice lacking the bacterial sensingreceptor nucleotide-binding oligomerisationdomain-containing protein-2 showed signicantlymore Bacteroidetes as well as Firmicutes compared

with wild-type mice.58

Modulation of the microbiota induces visceralhypersensitivity in mice, which is reduced by

Lactobacillus paracasei NCC2461 secreted prod-ucts.59 Lactobacillus acidophilus NCFM andLactoba-

cillus paracasei NCC2461 also modulate visceralpain perception in rodents.6 0 6 1 Transient pertur-bation of the microbiota with antimicrobials altersbrain-derived neurotrophic factor expression,exploratory behaviour and colonisation of germ-free mice suggesting that intestinal microbiotaimpact is not limited to the gut and the immunesystem, but may involve the central nervoussystem.62 (Note: this last sentence appears run-onbut I cant quite decipher how to x it.)

APPROACHES TO THE STUDY OF MICROBIOTAApproaches to the study of microbiota and relativeadvantages/pitfalls are reported in box 1. Culture-based studies reveal that the gut microbiota isa highly complex community (box 1).63 Althoughculturing remains valuable for identifying func-tional groups and for selective enumeration (eg, ofpathogens), new culture-independent approachesprovide more powerful and convenient methodolo-gies for monitoring changes in the GI tract

community (table 1). Information on the diversityof microbes that colonise the gut has expandedrapidly over the past 15 years, based largely on theanalysis of the small subunit ribosomal RNA (16SrRNA for Bacteria and Archaea, 18S rRNA forEukaryotes) gene sequences that can be obtained bydirect amplication from nucleic acids extractedfrom gut or stool samples.64 This informationprovides the basis for a range of complimentarytechniques for enumerating gut bacteria, includingngerprinting methods such as denaturing gradientgel electrophoresis65 and targeted methods such asuorescent in situ hybridisation and quantitativePCR. The arrival of new high-throughput

sequencing approaches and 16S rRNA-basedmicroarraying has further accelerated the supply ofdata by allowing amplied 16S rRNA sequences tobe analysed indepth without the need for classicalcloning and sequencing methods.66 67 Althoughculturing may bias against bacteria that are hard togrow in the laboratory, PCR amplication biasesagainst certain groups of gut bacteria. For example,bidobacterial 16S rRNA sequences are oftenunder-represented among amplied products,although more reliably enumerated by 16S rRNA-targeted uorescent in situ hybridisation detectionor quantitative PCR.37 While most molecular

enumeration methods target 16S rRNA, some arebased on more functionally relevant genes, for

Figure 2 Gut microbiota composition in African children living in rural areas with

a polysaccharide-rich diet when compared with Italian city children.35

(Reprinted withpermission from Proc Natl Acad Sci USA).


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example, involved in methanogenesis or butyratesynthesis.

High-throughput DNA sequencing providescompletely new possibilities for -omics-basedanalyses of the gut microbiota.19 Draft genomes ofcultured gut bacteria can now be produced rapidlyand at little cost.68 In addition, these methods canbe applied to DNA recovered from gut or stoolsamples, and the analysis of the resulting complexmixture of sequences is referred to as meta-genomics.69 70 The ability to analyse multiple genesequences from large numbers of samples, comple-

mented with functional screening and character-isation of randomly cloned DNA fragments fromthe GI tract, is currently being exploited to uncoverchanges in disease states including in inammatorybowel disease (IBD). A related technology, meta-transcriptomics, uses high-throughput sequencingor microarray analysis to examine RNA expressedin GI tract samples, thus focusing on bacteria thatare transcriptionally active. Another potentiallypowerful tool, metaproteomics, employs proteinseparation and sequencing techniques to describethe major proteins present in gut or stoolsamples.71 72 These meta-omicsapproaches rely inprimary sequencing and annotation data.73 74 Thus,

they rely heavily on the availability of genomesequences and functional information fromcultured reference bacteria, which means there areconsiderable benets from combining differentapproaches to gut microbiota analysis. A nalomics approach, metabonomics, is not linkeddirectly to genetic information of the microbes, butexamines the metabolite proles resulting fromtotal microbial activity in the gut. Since many ofthese metabolites exert biological effects (somepositive, some negative) on the host, such analysiscan provide a direct measure of the consequences ofmicrobial activity in the gut, although excluding

cell-mediated effects and direct identi

cation toa target microbial species.

Breath testing has been used to detect SIBO inIBS patients by non-invasively detecting hydrogenproducing bacteria or methane producing archaeawithin the gut lumen. The breath test is based onthe concept that hydrogen gases are produced bycolonic bacterial fermentation in response toingestion of a test sugar. They rapidly diffuse intothe blood, are excreted by breath, and can be

collected and quantied.75

If SIBO exists, thetiming of this fermentation would be alteredbut the criteria for abnormal tests lack validity(gure 3).

DIFFERENCES IN THE MICROBIOTA IN FBD ANDTHE LINK TO PATHOPHYSIOLOGYThere is little known about the small intestinalmicrobiota as the small intestine is relatively inac-cessible (summarised in table 2; box 2).75e85

Culture studies show considerably fewer bacteriacompared with the colon with a marked gradientfrom duodenum to distal ileum. The bacteria aretypically Gram-positive aerobes proximally andGram-negative and Gram-positive anaerobes andfacultative anaerobes in the terminal ileum.Culture-independent studies of the small intestinalmicrobiota are in their infancy but suggestcomplexity not appreciated by standard culturetechniques, including marked individual differ-ences,uctuations over time (even within the sameday), age-related differences and several phylotypesnot previously identied.86e88 Moreover, a recentpaper indicated that the small intestinal microbiotaare driven by a rapid uptake and conversion ofavailable simple carbohydrates in whichStreptococcusspp. play an important role.89

The role of SIBO in the pathogenesis of IBS isvery controversial because the breath testsemployed to establish this role have not been vali-dated.90 91 Even the validity of the gold standard,jejunal cultures >105 cfu/ml with colonic-typebacteria, has been challenged, largely because thiscut-off was established from samples followingsurgical diversion.91 Studies in IBS patients showedrelatively few bacteria in the duodenum and prox-imal jejunum and no obvious differences fromcontrols (table 2). Preliminary studies suggest thatmore IBS patients have SIBO when a lower cut-offof>103 cfu/ml is used but well-designed studies areneeded.82 85 Available molecular studies are not

adequately designed to establish whether SIBO isinvolved in IBS but have signicant potential.

Several confounding factors, including acidsuppression by proton pump inhibitors (PPIs) andaltered motility, have been implicated in the studiesof SIBO and IBS.92e94 Some studies suggest thatPPI use might lead to symptomatic SIBO or at leastincreased numbers of bacteria and that followingantibiotics they accelerate recurrence, but thisdepends on the tests employed and criteriaapplied.95Although the link between SIBO and IBSis largely based on breath testing, most positivelactulose breath tests reect rapid transit to the

caecum rather than true SIBO94

(

gure 4). Otherfactors such as antibiotics, probiotics and

Box 1 Approaches to the study of intestinal microbiota

< Breath tests are not validated to accurately detect small intestinal bacterialovergrowth.

< Rapid molecular approaches have largely replaced cultural approaches forenumeration of the dominant gastrointestinal (GI) tract microbiota.

< Cultural microbiology remains crucial for investigating microbial diversity and

for the selective isolation of representatives of key functional groups,including pathogens.

< Culture-independent approaches to study the GI tract microbiota can answerthe questions:

Which microbes are present in the GI tract? (16S rRNA gene-basedapproaches)

What microbial genes are present in the GI tract? (metagenomics) What are GI tract microbes doing? (metatranscriptomics, metaproteo-

mics, metabonomics/metabolomics).< The possibilities of using high-throughput approaches and their depth of

analysis are increasing rapidly, but it is important they are applied withcareful reference to well-defined scientific questions.


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prebiotics, and other dietary items such asFODMAPs could also inuence microbiota in IBSpatients and result in a potentially spuriousassociation.

Earlier culture-based assessment of faecal micro-biota obtained from patients with IBS demon-strated decreased faecal lactobacilli andbidobacteria, and increased facultative bacteria

dominated by streptococci and Escherichia coli aswell as higher counts of anaerobic organisms suchas Clostridium.9 6 9 7 Studies using molecular-basedtechniques reveal changes in faecal microbiotacomposition in IBS versus controls (table 3). Inter-estingly, a recent study demonstrated that faecalmicrobiota of IBS patients could be grouped ina cluster which was completely different from thatof healthy controls.114 Nonetheless, results to dateare inconsistent and sometimes contradictory(table 3). This may reect differences in moleculartechniques employed, the use of single samples thatare not linked to uctuating symptoms (especiallyas studies suggest IBS faecal microbiomes are lessstable), and probably other factors such as diet andphenotypic characterisation of patients. In addi-tion, it should be realised that faecal samples do notnecessarily reect other parts of the GI tract.

The nding that IBS can develop followinginfective gastroenteritis prompted studies evaluatingthe role of inammation in IBS, but there are fewerstudies that focus on the associated changes in gutmicrobiota, which might be just as signicant.Infective gastroenteritis produces a profound deple-tion of the commensal microbiota,118 whoseproduction of metabolites such as SCFAs and anti-biotics normally inhibits pathogen colonisation, as

can be seen from the loss of colonisation resistanceafter antibiotics.119 It is unclear just how completelyand over what time span recovery occurs.

Infective gastroenteritis is common, with anincidence of 19/100 person years in the UK.120 Athird of episodes are viral (Norovirus/Rotavirusbeing the commonest). The commonest bacterialinfections, Campylobacter and Salmonella, accountfor 10% and 3%, respectively. Onset of new IBSsymptoms after a bout of infective gastroenteritis isrelatively common, reported by 6%e17% of IBSpatients,121 while a recent internet survey reported18%,122 with around 40% beginning while travel-ling. The clinical features of post-infectious-IBS

are predominantly those of IBS-diarrhoea(IBS-D).123 124 A recent meta-analysis pooling 18studies indicated a relative increased risk of devel-oping IBS 1 year after bacterial gastroenteritis(mostly Shigella, Campylobacter and Salmonella),RR6.5 CI (2.6e15.4), an effect still apparent at36 months, RR3.9 (3.0e5.0).125 Viral gastroenter-itis, in keeping with the lesser tissue injury, showsa reduced incidence of post-infectious-IBScompared with bacterial infections126 127 in whichthe strongest risk factors are bacterial toxicity,128

prolonged duration of diarrhoea,124 rectalbleeding129 and fever.125Acute enteritis is associated

with a prolonged increase in mucosal cytotoxic Tlymphocytes and increase in enteroendocrineTable1

Mainfeaturesofculture-independentdetectionmethodsofgutmicrobiota

Question

Target

Approach

Datagenera

ted

Canmicrobesbe

identifieddirectly?

Main

benefit

Mainlimitation

Whichmicrobesare

presentintheGItract?

Isolates

Cultivation

Phenotypicc

haracterisation

Yes

Accuratespeciesidentification

Notrepres

entative

16SrRNAgene

Cloningandsangersequencing

Phylogenetic

identification

Yes

Complete16SrRNAgenesequencedata

Cloningbias

16SrRNAgene

High-throughputsequencing

Phylogenetic

identification

Yes

High-throughputdatageneration

Shortreads

16SrRNAgene

Fingerpinting

Communityp

rofile

No

Fast

comparisonbetweencommunities

Nodirectlinkwithphylogeny

16SrRNA

FISH

Cellnumbers

Yes

Accurateenumeration

Dependent

on16SrRNAdatabases

16SrRNAgene

qPCR

16SrRNAgeneabundances

Yes

Wide

dynamicrange

Dependent

on16SrRNAdatabases

16SrRNAgene

Phylogeneticmicroarray

Phylogenetic

identification

Yes

High-throughputphylogeneticprofiling

Dependent

on16SrRNAdatabases

Wha

tmicrobialgenes

arepresentintheGItract?

CommunityDNA

Sequence-basedmetagenomics

Genesequen

ces

Notalways

High-throughputdatageneration

Functionm

ainlybasedonpredictions

CommunityDNA

Function-basedmetagenomics

Functionalproperties

encodedonDNAfragment

Notalways

FunctionalpropertieslinkedtoDNA

sequences

Suitablecloninghost/systemand

screeningassaysneeded

Wha

tareGItract

microbesdoing?

mRNA

Metatranscriptomics

Communityg

eneexpression

Notalways

Direc

tinformationaboutmicrobialactivity

CommunityRNAextractionchallenging

Proteins

Metaproteomics

Communityp

roteinproduction

Notalways

Direc

tinformationaboutmicrobialactivity

Nouniform

protocolforallcellfractions

Metabonomics

Metabonomics/metabolomics

Communitym

etabolityprofiles

No

Microbiotaactivityrepresentation

Nolinkwithmicrobesoritsfunction

Lactulosehydrogen

breathtest

MeasuringGItractgasproduction

Hydrogenandmethanebreath

content

No

Uncle

ar,simpletestbutnotvalidatedfor

diagn

osingSIBO

Maysimplymeasuresmallintestinal

transittimetocaecum

Glucosehydrogen

breathtest

MeasuringGItractgasproduction

Hydrogenbreathcontent

No

Sameasabove

Poorsensitivity;missesdistalSIBO

FISH,

fluorescentinsituhybridisation;GI,gastrointestinal;

qPCR,quantitativePCR;SIBO,smallintestinalbacteria

lovergrowth.


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cells.123 Other studies have shown the importance

of increased 5HT containing cells in IBS-D130

andincreased sensitivity in IBS-D with increased EC cellcounts,131 accelerated gut transit and visceral

hypersensitivity.132 These effects on gut physiologywill impact on the gut microbiota environment. Anearly study of children with acute gastroenteritisdemonstrated alkalinisation of stool pH, likely dueto the decrease in bacterial metabolites (SCFAs) anda fall in numbers of Bacteroides, Bidobacterium,

Lactobacillus and Eubacterium.133 Conventionalenumeration of faecal bacteria showed a 10-fold fall

in anaerobes (Bacteroidaceae and Eubacterium), littlechange in aerobes, but 109 cfu/g of pathogens.

Another study using conventional culture methodsshowed a reversal of the normal anaerobe/aerobedominance during acute infection.134 More recenthuman studies using modern culture-independentmethods tended to conrm these ndings.135 136

PCR-denaturing gradient gel electrophoresisproling of 16S rRNA genes showed a reduceddiversity, often associated with a dominant bandsuggesting overgrowth of one subtype, which maynot always be the original pathogen. A recent clin-ical trial of an oral rehydration solution containinga prebiotic, amylase resistant starch in acute diar-rhoea in India, including children aged 3 months to5 years, used PCR primers directed at selectedbacteria, for example, Eubacterium spp. and Faecali-bacterium prausnitzii, key bacteria involved in starchfermentation. These studies showed a decline insome anaerobes (Bacteroides spp., Eubacterium spp.and Faecalibacterium prausnitzii) while other generaincluding Bidobacterium spp. were unchanged.135

This depletion of anaerobes could be due to accel-eration of transit, which could lead to a loss of theanaerobic niche. Since these are the key bacteriainvolved in colonic salvage of unabsorbed carbohy-drate,137 this may also contribute to the diarrhoea

phenotype by preventing fermentation to SCFAs,which are known to stimulate colonic salt andwater absorption, both directly and by inducing

Figure 3 The lactulose hydrogen breath test (LHBT) predominantly measures smallintestinal transit rather than small intestinal bacterial overgrowth (SIBO) in irritable bowelsyndrome (IBS) patients. Upper schematic shows ingestion of test meal with subsequentserial measurement of both H2gas, resulting from fermentation of the lactulose by intestinalbacteria, and Tc99 scanning in the caecum. This latter measurement detects when the testmeal has reached the caecum. The stylised drawing below shows a representative resultfrom an IBS patient with serial measurements over time. The Tc99 had already reached thecaecum in large quantities before the H2 PPM level has reached the threshold for anabnormal test. This demonstrates that the increased H2production results from fermentationby colonic bacteria, not by abnormal bacteria small intestine (ie, SIBO).94

Table 2 Summary of studies culturing small bowel microbiome

Study Number of patients Sample type Microbiology results Comments

Drasar and Shiner76 13 Diarrhoea, all investigationsnegative

Jejunal cap sule No di fference f rom contr ols;no increased numbers of pathogensor non-pathogens

Possible IBS but not defined as IBS

Rumessen et al77 60 Patients suspected of SIBO Proximal jejunal aspirate 15 With no predisposing causehad no evidence of SIBO; of 23with SIBO, 4 had no predisposingcause

Groups poorly defined, 8 IBS identifiedand all negative for SIBO; 22 casesconsidered inconclusive

Corazza et al78 31 Chronic diarrhoea, nopredisposing cause

Proximal jejunal aspirate 10 Had SIBO ($106 cfu/ml orcolonic bacteria), 2 IBS, 8 other

multiple other diagnoses

IBS not defined, and total IBS not clear

Bardhan et al79 10 Controls; 4 irritable colon;22 other

Endoscopic aspirates fromproximal jejunum

No positive cultures in irritablecolon

Positive cultures in 11 cases, manypostsurgical

Lewis et al80 23 With functional boweldisorders

Duodenal endoscopic aspirate Mean control count 3.23102

cfu/ml, no anaerobes, no sterilesamples

No specific IBS, defined as functionalbowel disorders

Sullivan et al81 7 IBS; 20 contro ls Proximal jejunal b io psy usingWatson capsule

No differences, flora similar tonormal oropharyngeal flora

Colonic pathogen in 2 healthy subjects

Posserud et al82 1 62 IBS; 42 c ontrols Prox im al j ejuna l as pirate 4%$105 cfu, same as controls.Subanalysis using $53103,43% IBS vs 12% controls

No correlation with motor pattern inIBS group

Kerckhoffs et al83 8 IBS; 9 contr ols Proximal jejunal aspi rate No di fferent number diagnosedwith SIBO using multiple definitions

No differences also using molecular-basedcounts

Choung et al84 148 IBS; 542 otherindications to test for SIBO

Duodenal endoscopic aspirate 2% IBS >105 cfu/ml 10% in otherindications

Retrospective study 18% IBS>0103 cfu/ml 15.11% non-IBS All investigated because of UGI bleed

IBS, irritable bowel syndrome; SIBO, small intestinal bacterial overgrowth; UGI, upper gastrointestinal.


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increased expression of transporters.138e140 Previousearlier studies in IBS-D suggest impaired SCFAconcentrations and production rates in ex vivoincubation, which may also reect reduced anaer-obes.141

Another cause of depletion of anaerobes is broad-spectrum antibiotics. There are no RCTs, but

epidemiological studies show an associationbetween antibiotic use and an increased risk of PI-IBS.142 A study of children showed that 3 monthsafter Salmonella infection, vomiting, abdominalpain and diarrhoea were reported by 9.5% of thosetreated with antibiotics but only 2.9% of thosewho received no antibiotics.142

Changes in the interaction between intestinalmicrobiota and host factors (eg, age, diet, transit,host genetic factors, antibiotics) could be importantfor IBS pathophysiology. These factors, in turn, couldbe related to changes in homeostatic pathwaysincluding barrier function, neuromotor sensoryfunction and the brainegut axis.143 144 For example,

bidirectional signalling between the microbiota andthe epithelium regulates epithelial secretion of mucusas well as other defence factors involved in regulatingthe microbiota. Changes in these factors (eg, changesin mucus layer and increased b-defensin-2 peptide)have been detected in patients with IBS and func-tional diarrhoea and suggest a microbiotaehostimmune system engagement.145 146 In line with thisconcept, there is also recent demonstration that IBSpatients have increased colonic mucosal expression of

receptors recognising specic microbiota-relatedsubstances (such as Toll-like receptor-4 which recog-nises bacterial lipopolysaccharides)147 or increasedtitres of circulating antibodies against components ofthe indigenous microbiota (ie, antiagellin anti-bodies).148 Several studies demonstrated low-gradeactivation of innate and adaptive mucosal immuneresponse in large subgroups of patients with

IBS.12 149

Increased activated mast cells, CD3+ve,CD4+ve and CD8+ve T cells have been detected inboth postinfectious IBS and non-specic IBS.12 149

The relative importance of mast cells in this setting isdemonstrated by the abundance of this immune celltype over other immunocytes and by increasedrelease from mucosal biopsies of histamine, tryptaseand prostaglandins.150 151 Mast cells were located incloser vicinity to mucosal innervation and correlatedwith the severity and frequency of abdominal pain inpatients with IBS.152 There are potential implicationsof mucosal immune activation for sensorimotordysfunction of patients with IBS. Histamine andtryptase released from mucosal biopsies of patientswith IBS evoked increased mesenteric sensoryafferent activation and induced visceral hyspersensi-tivity via histamine-1 receptors and proteinaseactivated-2 receptors when applied to recipientrats.150 151 Intestinal microbiota may well be anactive participant in this scenario through stimula-tion of the immune system,153 likely in the subgroupof subjects showing increased epithelial permeabilitywhich could154 expose the immune system to anabnormal microbial antigenic load. Overall, theresults suggest that bacterialehost interactions maybe initiated by components of the microbiota thatcan cross the mucus and adhere to epithelial cells,

inducing activation of the mucosal innate defencesystem even in the absence of mucosal destruction.

The use of probiotics, particularly in animalmodels, also demonstrates that their secretedproducts or metabolites can modulate contractilityof intestinal smooth muscle and visceralsensitivity.59e61 Moreover, application of probioticscan recover neuromotor-sensory dysfunction inIBS-like models.

Modulation of the brainegut axis is particularlyrelevant in IBS because psychological comorbidityis common. Some forms of psychological stress inanimal studies can induce shifts in the bacterialcomposition of the gut that is accompanied by

systemic cytokine response and increased intestinalpermeability.155 The interplay may be bidirectionalas suggested by animal studies showing that themicrobiota can affect brain chemistry and behav-iour.156 Nonetheless, for the time, the potentialrelevance of brainemicrobiota interactions have yetto be shown in humans in general and in FBD inparticular.

GI DISORDERS MIMICKING AND OVERLAPPINGWITH FBDS

Although celiac disease, IBD or diverticulitis can

coexist with IBS, an

IBSdiagnosis in the presenceof an organic disease may be challenging.

Box 2 Relevance of studies showing changes in microbiota in irritable

bowel syndrome

< The relevance of small intestinal bacterial overgrowth in irritable bowelsyndrome (IBS) remains unclear due to methodological problems, influence of

confounding factors and large differences between studies.< Heterogeneity of IBS and variation in methods used to study the faecal

microbiota have resulted in conflicting reports of differences from healthycontrols.

< The microbiome may contribute to IBS symptoms by altering gut neuromotor-sensory function, barrier function and/or the brainegut axis.

Figure 4 Plot chart of currently available strategies formodifying gut microbiota aiming to demonstrate therelationship between the effectiveness and invasiveness/safety of the proposed approach. FODMAP, fermentable

oligo-, di- and mono-saccharides and polyols; PPI, protonpump inhibitor.


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Gluten causes coeliac disease in geneticallysusceptible people and causes gut dysfunction inmice and can generate IBS symptoms in theabsence of coeliac disease.157 Some patients withIBS lack tissue transglutaminase antibodies orhistological markers of coeliac disease yet stillrespond symptomatically to a gluten-free diet. Thisentity is termed non-coeliac gluten sensitivity orgluten sensitive IBS.

157e159

The underlyingmechanisms in humans remain unclear. Mousemodels indicate that gluten can induce activationof innate immunity, increased small intestinalpermeability,160 neuro-muscular dysfunction159 anddysbiosis161 in the absence of autoimmunity.

IBS-like symptoms are common in IBD patientsin long-standing remission, or are frequentlyreported in patients before the diagnosis ofIBD.162 163 It is possible that IBS and IBD coexistwith a higher than expected frequency, or may existon a continuum, with IBS and IBD at different endsof the inammatory spectrum. A study investi-gating IBS symptoms in IBD patients who werethought to be in clinical remission demonstratedhigh levels of calprotectin levels; this suggests thatin most cases IBS symptoms are the result ofundetected ongoing inammation.164 Underlyingmechanistic links are lacking but it is tempting toraise the hypothesis that the intestinal microbiotamay be a common factor in both diseases.165 Infact, as with IBS (tables 2 and 3), faecal166e171 andmucosal-associated dysbiosis1 67 1 72e178 has beendescribed IBD.

A high proportion of patients hospitalised withacute diverticulitis continue to have persistentsymptoms that mimic IBS179 despite the absence of

complications.180

Some uncontrolled studies claimbenet from antibiotics and/or mesalazinesuggesting a role for the microbiota in thissyndrome.181

TREATMENT IMPLICATIONS: ANTIBIOTICS,PROBIOTICS, PREBIOTICS AND SYNBIOTICS

As the microbiota may be disturbed in functionalGI disorders, a potential treatment approach is totry to correct dysbiosis either by the administrationof an antibiotic or a preparation of benecialbacteria (box 3).

Antibiotics

Despite evidence that previous antibiotic use maybe related to the development of IBS,182 183 and thefact that antibiotic treatment may increase thedevelopment of long-term digestive symptomsafter bacterial gastroenteritis,142 poorly absorbableantibiotics might still have therapeutic potential inthis condition.184 Neomycin was the originalchoice184 185 although interest is now focused ona non-absorbed derivative of rifampicin calledrifaximin.186

There are three fully-published, double blind,placebo controlled trials of rifaximin in FBD187e189

and the data suggest an improvement in symp-

toms, especially bloating and

atulence forapproximately 10 weeks following treatment187 189Table3

continued

Stud

y

Subject

Sample

Method

Patientgroup

Mainfinding

Countryofstudy

Carrolletal115

IBS-D(n16)Ctrls(n21)

FaecesColonicmucosa

T-RFLPfingerprintingof16SrRNA-PCR

IBS-D

Diminishedmicrobialbiodiversityinfaecalsamp

les

USA

Parkesetal116

IBS-D(n27)IBS-C(n26)

Ctrls(n26)

Colonicmucosa

FISH

Confocalm

icroscopy

IBS

Expansionofmucosa-associatedmicrobiota;ma

inly

bacteroidesandclostridia;associationwithIBS

subgroups

andsymptoms

UK

Jeffe

ryetal117

IBS(n37)Ctrls(n20)

Faeces

Pyroseque

ncing16SrRNA

ClusteringofIBSpatientsdnormal-likeversusa

bnormal

microbiotacomposition(increasedratioofFirmicutesto

Bacteroidetes);associationwithsymptomprofile

Sweden

n,numberofrandomisedsubjects.

B,Bifidobacterium;C,constipation;C,

Clostridium;Cl,Clostridium;ctrls,controls;D,

diarrhoea;DGGE,

denaturing

gradientgelelectrophoresis;FISH,

fluorescentinsituhybridisation;IBS,

irritablebowelsyndrome;L,Lactoba

cillus;qPCR,quantitativePCR;

R,Ruminococcus;S,

Staphylococcus;T-RFLP,

terminalrestrictionfragmentlengthpolymorphism.


Gut2013;62:159176. doi:10.1136/gutjnl-2012-302167 167


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with a therapeutic advantage over placebo around10%. The doses used in these and other studies varybetween 600 and 2400 mg daily for 7e14days190e195 but there remain concerns about anti-biotic resistance and possible Clostridium difcileinfection although so far these issues have notappeared to be a problem.196e200

Thus, a short course of gut-specic antibioticsmay have utility in some patients with IBS but we

need to know more about predictors of treatmentresponsiveness, antibiotic resistance, the efcacyand safety of re-treatment schedules as well as theoptimal dosing regimen.201 202

ProbioticsProbiotics are live microorganisms which whenadministered in adequate amounts confer a health

benet on the host203

with the most commonlyused being the lactobacilli and bidobacteria.Probiotics can be packaged in many formulationscontaining just one organism or a mixture and havea wide range of activities with evidence supportingan effect on at least some of the putative patho-physiological mechanisms implicated in IBS, suchas visceral hypersensitivity,59 60 204 205 GIdysmotility,206e210 intestinal permeability,204 211 212

the intestinal microbiota213 214 and immune func-tion215 although these effects can differ consider-ably between one organism and another. Thus, justbecause one organism is benecial, this does notmean that related organisms will behave similarly.For use in gastroenterology, it is important thata preparation contains sufcient quantities of

Box 3 Modulation of intestinal microbiota in functional bowel disorders

< A short course of a non-absorbable antibiotic such as rifaximin has beenshown to moderately improve the symptoms of irritable bowel syndrome(IBS), particularly bloating and flatulence. Improvement persists after thecessation of treatment but the exact duration of this effect remains uncertain.

< The majority of trials of probiotics in IBS show some degree of efficacy

although some of the early studies were of very poor quality.< Prebiotics and synbiotics should theoretically have the potential in treating

functional gastrointestinal disorders but there are as yet no reliable data tosupport this view.

Table 4 Placebo controlled clinical trials of single or mixed probiotic preparations in IBS

Organism n Outcome Reference

Studies in adult patients

S faecium 54 Y Global score Gade et al216

Lactobacillus acidophilus 18 Y Global score Halpern et al217

Lactobacillus plantarum 299V 60 Y Flatulence Nobaek et al218

L plantarum299V 20 Y Pain, all IBS symptoms Niedzielin et al219

L plantarum299V 12 Negative Sen et al220

L plantarumMF1298 16 Deterioration of symptoms Ligaarden et al221

L ramnosusGG 25 Negative OSullivan et al240

L reuteriiATCC 55730 54 Negative Niv et al223

L salivariusUCC4331 75 Negative OMahony et al215

Bifidobacterium infantis 35624 75 Y Pain and composite score OMahony et al215

B infantis 35624 362 Y Pain and composite score Whorwell et al224

Bifidobacterium lactis DN-173-010 274 Y Digestive discomfort Guyonnet et al225

B lactis DN-173-010 34 Y Maximum distensi on & pain Agrawal et al208

Bifidobacterium bifidum MIMBb75 122 Y Global score Guglielmetti et al226

Bacillus coagulansGBI-30, 6086 52 Y Bowel movements Dolin222

Escherichia coliNissle 1917 120 [ Treatment satisfaction Kruis et al227

VSL#3 (x8)* 25 Y Bloating Kim et al209

VSL#3 (x8)* 48 Y Flatulence Kim et al210

Medilac DS(x2)* 40 Y Pain Kim et al228

Mixture (x4)* 103 Y Global score Kajander et al229

Mixture (x4)* 86 Y Global score Kajander et al214

LAB4 (x4)* 52 Y Global score Williams et al230

Mixture (x4)* 106 Negative Drouault-Holowacz et al231

Mixture (x2)* 40 Y Pain Sinn et al232

ProSymbioFlor (x2)* 297 Y Global score Enck et al233

Cultura (x3)* 74 Negative Simren et al234

Cultura (x3)* 52 Negative Sondergaard et al235

Mixture (x4)* 70 Y Pain Hong et al236

Studies in paediatric patients

L ramnosusGG 50 Y Abdominal distens ion Ba usserman a nd Micha il237

L ramnosusGG 104 Y Pain Gawronska et al238

L ramnosusGG 141 Y Pain Francavilla et al212

VSL#3 (x8)* 59 Y Global score Guandalini et al239

*Number of organisms in a mixture.n, number of randomised subjects.

IBS, irritable bowel syndrome; L ramnosus, Lactobacillus ramnosus; L reuterii, Lactobacillus reuterii; L salivarius, Lactobacillus salivarius; S faecium,Streptococcus faecium.


168 Gut2013;62:159176. doi:10.1136/gutjnl-2012-302167


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microbes which need to be acid and enzyme resis-tant with good mucosal adherence also being anadvantage.

Table 4 lists the results of the fully published placebo controlled probiotic trials todate.208e210 212 214e240 Unfortunately, their designsvary considerably;241e244 some of the older studiesare of poor quality, and few attempt to dene the

mechanism of action or assess whether symptom-atic improvement is accompanied by a change inthe microbiota. A recent systematic review reportedthat studies of poorer quality tended to show largereffects and published data indicate a publicationbias, with non-reporting of negative effects in smalltrials.244 Around three-quarters of these studieswere positive, of which four were in children,although different symptoms improved and thetherapeutic gain over placebo was generallymodest. Furthermore, it remains unclear whichorganisms are most effective as, for instance, somemainly reduce bloating and atulence,2 09 2 10 2 18

whereas others improve bowel frequency,222 andsome have a positive effect on global symptomscores.214 215 224 226 229 230 233 In some of the betterquality trials bidobacteria, such as Bidobacteriuminfantis 35624,215 224 241 Bidobacterium lactis DN1730102 08 2 25 and Bidobacterium bidum MIM-Bb75,226 seem to be advantageous and in othersprobiotic mixtures appear to be useful.214 229 233

In only one study was there symptom deteriora-tion221 although some large, high quality trialshave been negative.221 231 234 235

Diet, fibre, prebiotics and synbioticsThere are few proper randomised, placebo controlled

trials of diet modication because of the difculty incontrolling for the placebo effect. One randomisedcontrolled trial showed bran aggravated symp-toms;245 excluding bran should help, and manypatients believe this is true.246A prebiotic is a product

that, on ingestion, stimulates the growth of benecialbacteria already present in the host, which promotesthe health of the individual.247 248 A variety ofoligosaccharides serve this function and a synbioticis a combined prebiotic and probiotic. One of theearliest prebiotics was lactulose, an unabsorbabledisaccharide laxative that increases the faecalconcentrations ofBidobacterium spp.45 249 as does

inulin which, like lactulose, increases atulence47

and thus makes it unlikely it will help IBS patients.To date, there has only been one double blind,

placebo controlled trial of a prebiotic in IBS whichused a trans-galactooligosaccharide mixture.250

Compared with placebo this prebiotic reducedsymptoms and stimulated the growth of bido-bacteria but clearly more research is required ondosing and the relative merits of other compounds.

With regard to synbiotics, there are some studiesbut their design is not sufciently robust to drawany rm conclusions251e255 although the conceptof combining a prebiotic and probiotic is theoreti-cally attractive. Thus, attempting to modify themicrobiota in patients with functional GI disordersshows some promise. However, we need to knowhow symptomatic improvement is achieved: is itmirrored by a change in gut microbiota or is someother mechanism involved?

CLINICAL GUIDANCE REGARDING MODULATIONOF INTESTINAL MICROBIOTA IN IBS

While the science regarding the role of microbiotain FGIDs remains in its infancy, patients areexposed to conicting claims concerning thesymptomatic benet from modulating gut micro-biota. This section aims to help clinicians give the

best advice, despite limited evidence (box 4).Diet profoundly alters the microbiota. Reducing

intake of bre256 or FODMAPs257 is one of thesimplest and safest ways of altering gut microbiota,which can lead to improvement in bloating anddiarrhoea, an effect which may last for years.258

However, so far the evidence to support widespreaduse of FODMAP reduction in patients with IBS islimited and comes mainly from one research group.Systematic exclusion diets may also help258 but arelaborious; targeted exclusion of regularly consumedsuspects, such as dairy, wheat, fruit and vegetables,may be more practical.

The safety of probiotics in IBS is acceptable but

some aggravate symptoms221 and so patientsshould be warned of this possibility. At present, thestrongest evidence is for Bidobacterium infantis35624 at a dose of 13108 cfu/day taken for at least4 weeks.224 It remains unclear who benets fromwhich variety of probiotic since there are manyincompletely answered questions surrounding thistherapeutic approach, including:< Are single organisms better than mixtures or

vice versa?< Do some mixtures of organisms contain strains

that are competitive or antagonistic withoutadditive effects?

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Intestinal Microbiota in Functional Bowel Disorders

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