Intestinal microbiota in functional bowel disorders: a Rome foundation report

 [bib_ref] Health-related quality of life in patients attending a gastroenterology outpatient clinic: functional..., Simren [/bib_ref] [bib_ref] Systematic review: impact of constipation on quality of life in adults and..., Belsey [/bib_ref] [bib_ref] The impact of irritable bowel syndrome on health-related quality of life, Gralnek [/bib_ref] [bib_ref] Health-related quality of life in patients attending a gastroenterology outpatient clinic: functional..., Simren [/bib_ref] [bib_ref] The burden of IBS: looking at metrics, Spiegel [/bib_ref] [bib_ref] Societal costs for irritable bowel syndrome--a population based study, Hillila [/bib_ref] [bib_ref] Health care seeking for abdominal bloating and visible distention, Jiang [/bib_ref] [bib_ref] Use of health care resources and cost of care for adults with..., Singh [/bib_ref] [bib_ref] Functional bowel disorders, Longstreth [/bib_ref] [bib_ref] Peripheral factors in the pathophysiology of irritable bowel syndrome, Gunnarsson [/bib_ref] [bib_ref] New insights into the pathogenesis and pathophysiology of irritable bowel syndrome, Ohman [/bib_ref] [bib_ref] Pathogenesis of IBS: role of inflammation, immunity and neuroimmune interactions, Ohman [/bib_ref] [bib_ref] Overview of the gastrointestinal microbiota, Young [/bib_ref] [bib_ref] Gut microbiota in health and disease, Sekirov [/bib_ref] [bib_ref] Molecularphylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases, O&apos;hara [/bib_ref] [bib_ref] Spatial organization of bacterial flora in normal and inflamed intestine: a fluorescence..., Swidsinski [/bib_ref] [bib_ref] An ecological and evolutionary perspective on human-microbe mutualism and disease, Dethlefsen [/bib_ref] [bib_ref] High-throughput diversity and functionality analysis of the gastrointestinal tract microbiota, Zoetendal [/bib_ref] [bib_ref] An ecological and evolutionary perspective on human-microbe mutualism and disease, Dethlefsen [/bib_ref] [bib_ref] High-throughput diversity and functionality analysis of the gastrointestinal tract microbiota, Zoetendal [/bib_ref] [bib_ref] Host-bacterial mutualism in the human intestine, Backhed [/bib_ref] [bib_ref] Diversity of the human intestinal microbial flora, Eckburg [/bib_ref] [bib_ref] Gut microbiota in health and disease, Sekirov [/bib_ref] [bib_ref] Gastrointestinal microbiota in irritable bowel syndrome: their role in its pathogenesis and..., Parkes [/bib_ref] [bib_ref] Postinfectious irritable bowel syndrome, Spiller [/bib_ref] [bib_ref] Therapies aimed at the gut microbiota and inflammation: antibiotics, prebiotics, probiotics, synbiotics,..., Quigley [/bib_ref] [bib_ref] Review article: the treatment of functional abdominal bloating and distension, Schmulson [/bib_ref] [bib_ref] Small intestinal bacterial overgrowth in irritable bowel syndrome: systematic review and meta-analysis, Ford [/bib_ref] [bib_ref] Gastrointestinal microbiota in irritable bowel syndrome: present state and perspectives, Salonen [/bib_ref] 

# A. introduction
Years of co-evolution have allowed the host and the intestinal microbiota to peacefully coexist under steady state conditions. Evidence, mostly from animal, but also from some human studies, supports the concept of a bidirectional interaction between the host and its intestinal microbiome. A conceptual framework is emerging where host genetic and immune, as well as environmental factors, influence intestinal microbiota composition; while, in turn, the intestinal microbiota contributes to shape host immunity and physiology, within and beyond the gut.

## B. complexity of the human gut microbiota
Humans acquire bacteria randomly from their environment (initially mainly from the mother in a conventional birth) and whether these bacteria are transients or become permanent colonizers depends on genetic makeup, diet, age, infection (reviewed in chapter 6) and lifestyle, including drug intake such as antibiotics. There is a dominant intestinal microbiota community in mammals consisting of thousands of bacterial species that belong to a small number of phyla [bib_ref] An ecological and evolutionary perspective on human-microbe mutualism and disease, Dethlefsen [/bib_ref] [bib_ref] Evolution of mammals and their gut microbes, Ley [/bib_ref] [bib_ref] Ecological and evolutionary forces shaping microbial diversity in the human intestine, Ley [/bib_ref]. These include Firmicutes, Bacteroidetes, and Actinobacteria; with a substantially greater number of Firmicutes compared to the other two in both humans and mice [bib_ref] Obesity alters gut microbial ecology, Ley [/bib_ref]. However, despite conservation at the highest taxonomic ranks, most studies find considerable inter-individual variability as assessed by similarity indices. It is not surprising that several human studies using sequence analysis of cloned small-subunit ribosomal RNA genes [16S ribosomal DNA] have demonstrated a hitherto unimagined complexity of the human gut microbiota of which most identified bacteria have not previously been cultured [bib_ref] Diversity of the human intestinal microbial flora, Eckburg [/bib_ref].
It needs to be emphasized that most studies investigating microbiota composition have been performed using fecal samples. A study in healthy volunteers detected differences in the total number of cultivatable aerobic bacteria in faeces and at the mucosal surface. However, in IBS-D patients the difference found between faecal and mucosal sites was less pronounced [bib_ref] Molecular analysis of the luminal-and mucosal-associated intestinal microbiota in diarrheapredominant irritable bowel..., Carroll [/bib_ref]. Thus, comparisons between luminal and mucosal-associated intestinal microbiota may yield important pathophysiological insights.

## C. effect of age on the intestinal microbiota
The inter-individual differences in intestinal microbiota composition are maximum in the neonatal period and decrease with ageing. Babies are in general considered to be born with sterile intestines but are rapidly colonised. Those born vaginally acquire maternal vaginal and fecal bacteria while those born by caesarean section have reduced Bifidobacterium spp., Lactobacillus spp. [bib_ref] Fecal microflora in healthy infants born by different methods of delivery: permanent..., Gronlund [/bib_ref] and
Bacteroides fragilis with increased risk of Clostridium difficile at 1 month of age [bib_ref] Factors influencing the composition of the intestinal microbiota in early infancy, Penders [/bib_ref]. Early studies using conventional culture techniques showed breast-fed babies had a simpler microbiota during the first few months dominated by Bifidobacterium spp and few enterobacteria while formula-fed babies had more strict anaerobes and a higher diversity of bacteria [bib_ref] The microbial ecology of the large bowel of breast-fed and formula-fed infants..., Stark [/bib_ref]. Introduction of solid food caused a successive appearance of first enterococci, followed by the strict anaerobes such as Bacteroides spp., Clostridium spp. and anaerobic streptococci [bib_ref] The microbial ecology of the large bowel of breast-fed and formula-fed infants..., Stark [/bib_ref]. A more detailed analysis of healthy babies using a microarray followed by sequencing showed a rapid increase in total bacterial counts from 10 4 to 16S ribosomal RNA (rRNA) gene copies per gram faeces [bib_ref] Effects of alternative dietary substrates on competition between human colonic bacteria in..., Duncan [/bib_ref] , rapidly reduced by courses of antibiotics.
The dominant phyla were in all cases Firmicutes, Bacteroidetes and Proteobacteria , but the precise species were unique to each individual and relatively stable. Early colonisers were mainly aerobes (staphylococci, streptococci and enterobacteria while late colonisers were strict anaerobes (Eubacterium spp. and clostridia). As babies grew, "uneven" profiles where one species dominated became less common and their profiles approached the more complex adult pattern. Early sudden changes in individual bacterial numbers were not uncommon even in those not receiving antibiotics, possibly reflecting phage attacks. The same study showed that the efficiency of PCR for bifidobacteria was 8 fold less than for clostridia, leading to systematic underestimation of numbers, a problem that is frequently seen in 16S rRNA gene PCR-based approaches, but often neglected in the biological interpretation.
Denaturing Gradient Gel Electrophoresis (DGGE) of PCR amplified 16S rRNA genes demonstrated that in the first 4-weeks of life preterm infants had rather simple band patterns that increased in similarity (11-57%) with other preterm babies with time. By contrast breast-fed remained more individual with only 11% interindividual similarity [bib_ref] Development of the intestinal bacterial composition in hospitalized preterm infants in comparison..., Schwiertz [/bib_ref]. The KOALA project in the Netherlands showed exclusively formula fed babies had more E. coli, C. difficile and B. fragilis, a difference which is lost if the formula contains oligosaccharides as prebiotics [bib_ref] Factors influencing the composition of the intestinal microbiota in early infancy, Penders [/bib_ref].
There are few studies of the evolution of faecal microbiota from 1 year to adulthood. One study from India, where most were lactovegetarians, showed relatively stable Bifidobacterium spp. up to age 17 followed by a steep decline in adults, while from 2-17 there was a gradual increase in Bacteroides spp. to adult levels [bib_ref] Bacterial succession in the colon during childhood and adolescence: molecular studies in..., Balamurugan [/bib_ref]. Lactobacillus spp. declined markedly after age 5 years while Faecalibacterium prausnitzii increased and then declined. These changes may well reflect changes in diet though this has yet to be proven.
Extreme old age is associated with a decrease in Bacteroides spp. and increases in Enterococcus spp.
and E. coli [bib_ref] The effects of ageing on the colonic bacterial microflora in adults, Enck [/bib_ref].

## D. effect of diet on the intestinal microbiota
Diet is a modifiable factor significantly affecting human health. The interaction between diet and intestinal bacteria begins soon after birth and is a complex process. Human milk contains up to microbes/L including staphylococci, bifidobacteria [bib_ref] Isolation of bifidobacteria from breast milk and assessment of the bifidobacterial population..., Martin [/bib_ref] and lactic acid bacteria (LAB) [bib_ref] Diversity of the Lactobacillus group in breast milk and vagina of healthy..., Martin [/bib_ref]. It also contains oligosaccharides which act as prebiotics, increasing the number of bifidobacteria in the gut [bib_ref] The first prebiotics in humans: human milk oligosaccharides, Coppa [/bib_ref].
Therefore, galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS) are now commonly added to baby formula because of their perceived benefits. On weaning, young babies are traditionally fed fermented dairy products containing LAB such as yogurt and cheese. A microbiological study of a randomized controlled trial (RCT) of the effect of a probiotic L. paracaseii A in 12-24 month old infants showed minimal changes overall in the faecal microbiota with a small decline in Clostridium spp. over 4 weeks and increase in Lactobacillus spp. [bib_ref] Lactobacillus paracasei A survives gastrointestinal passage and affects the fecal microbiota of..., Marzotto [/bib_ref]. African children living in rural areas with a polysaccharide-rich diet, when compared with Italian city children, showed a significant enrichment in Bacteroidetes and depletion in Firmicutes together with an increase in Prevotella spp. and Xylanibacter spp., bacteria known to contain genes for cellulose and xylan hydrolysis [bib_ref] Impact of diet in shaping gut microbiota revealed by a comparative study..., De Filippo [/bib_ref]. See [bib_ref] Brussels sprouts, inulin and fermented milk alter the faecal microbiota of human..., Humblot [/bib_ref].
There have been several RCTS looking at the prebiotic, oligofructose which increases faecal Bifidobacterium spp. and reduces time to resolution of C. difficile-associated diarrhoea during antibiotic treatment from 6 to 3 days 53 .
Lactulose is a synthetic disaccharide, not absorbed by the small intestine but metabolized by colonic bacteria and widely used as a laxative. The ingestion of lactulose acidifies the proximal colon 54 , stimulating propulsive motility [bib_ref] Influence of chronic lactulose ingestion on the colonic metabolism of lactulose in..., Florent [/bib_ref] [bib_ref] Low-dose lactulose produces a tonic contraction in the human colon, Jouet [/bib_ref] and leading to an increase in Bifidobacterium spp. counts in healthy humans [bib_ref] Effects of lactulose and lactitol on colonic microflora and enzymatic activity, Ballongue [/bib_ref] [bib_ref] Lactulose ingestion increases faecal bifidobacterial counts: a randomised double-blind study in healthy..., Bouhnik [/bib_ref]. A prospective, randomized parallel-group trial to evaluate the effects of lactulose and polyethylene glycol-4000 on colonic microbiota in chronic idiopathic constipation showed that lactulose induced a significant increase in faecal Bifidobacterium spp. counts and bgalactosidase activity. The metabolic activity of the faecal microbiota remained stable in the lactulose group, but a significant decrease in total short-chain fatty acids, acetate, butyrate and faecal bacterial mass was observed in the PEG-treated group [bib_ref] Prospective, randomized, parallel-group trial to evaluate the effects of lactulose and polyethylene..., Bouhnik [/bib_ref]. It is unclear whether this matter since PEG is at least as effective as lactulose and generally better tolerated [bib_ref] Comparison of a low dose polyethylene glycol electrolyte solution with lactulose for..., Attar [/bib_ref].
Other prebiotics such as lupin kernel can increase Bifidobacterium spp. in humans 60 as can inulincontaining juices [bib_ref] Prebiotic effect of fruit and vegetable shots containing Jerusalem artichoke inulin: a..., Ramnani [/bib_ref] and 10 gm of arabinoxylan-oligosaccharides [bib_ref] Tolerance of arabinoxylan-oligosaccharides and their prebiotic activity in healthy subjects: a randomised,..., Cloetens [/bib_ref].

## E. effect of transit on the intestinal microbiota
Modulating intestinal transit by accelerating it with senna or slowing it with loperamide has been used to explore how this alters the microbiota. Faster transit, which may impair absorption in the small bowel and increase available nutrients in the colon, increased faecal sulphate, sulphide, bile acids, methionine and the production rates of acetic acid but reduced fecal methanogens and methane production. The reverse effects were seen with loperamide 63 .
Cisapride-reduced transit time was associated with a significant rise in the concentrations of total Activation of TLRs by commensals is important for the maintenance of gut homeostasis via the stimulation of cytokines and epithelial reparative factors [bib_ref] Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis, Rakoff-Nahoum [/bib_ref] , and adaptive immunity cooperates with innate immunity to maintain host-commensal homeostasis [bib_ref] Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism, Slack [/bib_ref]. The absence of normal secretory IgA (IgAs) has been shown to lead to a significant shift in anaerobe populations in the small intestine in mice [bib_ref] Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut, Suzuki [/bib_ref]. In addition, IgAs limit translocation of commensal bacteria to mesenteric lymph nodes [bib_ref] Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria, Macpherson [/bib_ref] , and mice lacking sIgA develop low-grade inflammation in the gut [bib_ref] Absence of epithelial immunoglobulin A transport, with increased mucosal leakiness, in polymeric..., Johansen [/bib_ref].
h. Host-microbiota bidirectional interaction:

## H.i. the intestinal microbiome is a driving force for gut immunity and physiology
Discrimination between commensals and pathogens is in part achieved by a family of receptors that recognize bacterial components. Two major classes of innate receptors have been identified: 1) TLRs involved in the detection of extracellular bacterial components [bib_ref] The role of Toll-like receptors and Nod proteins in bacterial infection, Philpott [/bib_ref] [bib_ref] A family of human receptors structurally related to Drosophila Toll, Rock [/bib_ref] [bib_ref] The expression of toll-like receptor 2 and 4 mRNA in local tissues..., Zhang [/bib_ref] 

## H.ii. the intestinal microbiome and the gut-brain axis
Gastrointestinal function is controlled by the central nervous system (CNS). The gut brain axis is a bidirectional communication system that integrates brain and gastrointestinal (GI) functions.
Evidence for a microbiota-gut-brain axis that influences brain biochemistry and modulates behaviour in adult mice comes from a recent study showing that transient perturbation of the microbiota with antimicrobials increased hippocampal BDNF and exploratory behavior. These changes were reversible upon normalization of the microbiota after antimicrobial treatment.
Interestingly, antimicrobials did not affect behaviour in germ-free mice, but colonization of germ-free mice with a specific pathogen free (SPF) microbiota altered behaviour. Moreover, using two germ free mouse strains as recipients, and a cross-over design, SPF microbiota from mouse strains with opposite behavioural phenotype, determined behaviour and brain BDNF level in the recipient mice [bib_ref] The Intestinal Microbiota Affect Central Levels of Brain-Derived Neurotropic Factor and Behavior..., Bercik [/bib_ref]. This is an exciting new area that raises the hypothesis of a critical contribution of the intestinal microbiota in behaviour and central neurotrophin expression. These results may have important clinical implications for the understanding of IBS and its psycho-social co morbidity (reviewed in Chapter 7). Gut microbiota and the intrinsic and extrinsic factors that can affect its distribution and composition. A number of host mechanisms participate in gut microbiota modulation, including gastric acid secretion, fluid, anti-commensal sIgA and antimicrobial peptide production, and GI motility. Drugs that block acid secretion and affect GI motility, can indirectly alter the microbiota. Antibiotics, depending on spectrum and dosage, will directly affect microbiota composition inducing dysbiosis. Dietary modifications, including probiotic and fibre supplements will also affect microbiota composition. (MMC: migrating motor complex; PPI: proton pump inhibitor; NSAID: non steroidal anti-inflammatory drugs).

## Approaches to the study of microbiota

## Key points
 Breath tests are not validated to accurately detect SIBO.
 Rapid molecular approaches have largely replaced cultural approaches for enumeration of the dominant GI tract microbiota; nevertheless 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 have revolutionized our insight into this complex microbial ecosystem.
 The possibilities of using high throughput approaches and their depth of analysis are increasing explosively, but it is important they are applied with careful reference to welldefined scientific questions.
Interest in the GI tract microbiota in relation to health and disease is increasing as it is evident that the microbiota plays a crucial role. For more than a decade insights into the GI tract microbiota have increased which is in part due to the emerging developments in culture-independent technologies to study different aspects of the microbiota. Other sugar substrates and radio-labelled sugars, including fructose, sucrose, and xylose, as well as bile acids have also been used in a small number of studies. They do not offer any apparent advantages 106 and will not be discussed further. The results of the LBT in IBS studies, and to a lesser degree the GBT, were pivotal in developing the concept that SIBO is associated with the irritable bowel syndrome (IBS) [bib_ref] Irritable bowel syndrome hypothesis: implications for treatment, Vanner [/bib_ref] [bib_ref] Eradication of small intestinal bacterial overgrowth reduces symptoms of irritable bowel syndrome, Pimentel [/bib_ref] [bib_ref] Normalization of lactulose breath testing correlates with symptom improvement in irritable bowel..., Pimentel [/bib_ref] [bib_ref] Small intestinal bacterial overgrowth: a framework for understanding irritable bowel syndrome, Lin [/bib_ref] [bib_ref] Irritable bowel syndrome and small intestinal bacterial overgrowth, Yamini [/bib_ref]. This has led to much greater use of these diagnostic tests in North America and Europe for the assessment of patients with IBS. While the results continue to be promoted as a rationale for using antibiotics to treat these patients [bib_ref] Irritable bowel syndrome and small intestinal bacterial overgrowth, Yamini [/bib_ref] [bib_ref] Proton pump inhibitor therapy does not affect hydrogen production on lactulose breath..., Law [/bib_ref] [bib_ref] An evidence-based treatment algorithm for IBS based on a bacterial/SIBO hypothesis: Part..., Pimentel [/bib_ref] , there is considerable controversy concerning their accuracy for diagnosing SIBO in IBS patients and hence their contribution to understanding the pathogenesis of IBS [bib_ref] The lactulose breath test for diagnosing SIBO in IBS patients: another nail..., Vanner [/bib_ref] [bib_ref] Combined oro-caecal scintigraphy and lactulose hydrogen breath testing demonstrate that breath testing..., Yu [/bib_ref] [bib_ref] Small intestinal bacterial overgrowth in patients with irritable bowel syndrome, Posserud [/bib_ref] [bib_ref] Lactulose breath testing does not discriminate patients with irritable bowel syndrome from..., Bratten [/bib_ref].

## A.ii. rationale underlying the use of the gbt and lbt in ibs patients.
The proposed rationale for using the LBT and GBT tests is based on the fact that H 2 and CH 4 gas are produced almost exclusively in the gastrointestinal tract [bib_ref] Use and abuse of hydrogen breath tests, Simren [/bib_ref] and that bacteria (colonic microbiota) can produce measureable changes in breath H 2 gas levels within a few minutes of contact with small quantities of these sugar substrates [bib_ref] Investigation of small bowel transit time in man utilizing pulmonary hydrogen (H2)..., Bond [/bib_ref] [bib_ref] Interpretation of the breath hydrogen profile obtained after ingesting a solid meal..., Read [/bib_ref]. For example, in healthy control subjects where lactulose was infused directly into the cecum or just a few cm proximally within the distal terminal ileum, and simultaneous measurements of breath H 2 were obtained, infusion of 0.5 g lactulose (i.e. 5% of 10 g test dose) was sufficient to increase H 2 gas levels 5-10 parts per million (PPM) in breath samples within 2 min 118 .
Thus, if abnormal numbers and/or possibly microbial species were present in the small intestine this would theoretically lead to an early rise in H 2 and/or CH 4 production. The use of the LBT versus the GBT also has implications for detecting SIBO in IBS because of the differences in the transit of glucose and lactulose in the small bowel [bib_ref] Irritable bowel syndrome hypothesis: implications for treatment, Vanner [/bib_ref] [bib_ref] Small intestinal bacterial overgrowth: a framework for understanding irritable bowel syndrome, Lin [/bib_ref]. Glucose has been reported to provide a better measure of fermentation in the proximal small intestine because it can be absorbed before reaching the distal small bowel whereas lactulose is a non-absorbable sugar and therefore also provides a measure of fermentation in the distal small bowel or colon. Proponents of the SIBO hypothesis in IBS have suggested the pivotal event is the migration of colonic microbiota into the distal small intestine in these patients and therefore the LBT would be a much more accurate means of detection [bib_ref] Small intestinal bacterial overgrowth: a framework for understanding irritable bowel syndrome, Lin [/bib_ref] [bib_ref] Irritable bowel syndrome and small intestinal bacterial overgrowth, Yamini [/bib_ref] [bib_ref] Use and abuse of hydrogen breath tests, Simren [/bib_ref].
This reasoning however has not been consistently applied in the literature. For example, the same authors have argued that using glucose instead of lactulose can be problematic [bib_ref] A systematic review of diagnostic tests for small intestinal bacterial overgrowth, Khoshini [/bib_ref] [bib_ref] Irritable bowel syndrome and small intestinal bacterial overgrowth, Yamini [/bib_ref] and may explain why others have not found evidence for SIBO in IBS 120 , yet in their recent meta-analysis of breathing testing in IBS, 6 of the 11 studies examined used glucose or a related sugar other than lactulose. The results of this analysis were then used to suggest, despite their conclusion in yet another study that currently there are no tests that can accurately diagnose SIBO [bib_ref] A systematic review of diagnostic tests for small intestinal bacterial overgrowth, Khoshini [/bib_ref] , that breath testing is merited in IBS and that the results support the idea that SIBO is important in IBS [bib_ref] Abnormal breath testing in IBS: a meta-analysis, Shah [/bib_ref] [bib_ref] Irritable bowel syndrome and small intestinal bacterial overgrowth, Yamini [/bib_ref].
The separate measurement of CH 4 gas in LBTs and GBTs may also be important because changes in this gas were strongly correlated with constipation-predominant IBS 121 , methanogens may falsely lower H 2 gas levels, CH 4 gas may play a pathogenic role in constipation, possibly by inhibiting motility [bib_ref] Methane, a gas produced by enteric bacteria, slows intestinal transit and augments..., Pimentel [/bib_ref] , and may provide a useful measurement in patients where changes in H 2 gas is not detectable. However, this association is also found in non-IBS constipated subjects and in high numbers of controls and thus the significance of this finding in IBS remains to be clarified [bib_ref] Irritable bowel syndrome hypothesis: implications for treatment, Vanner [/bib_ref].

## A.iii. summary of test criteria and study results.
In IBS studies, a number of criteria have been used to define an abnormal breath test. In studies employing the LBT, the most commonly used criteria for H 2 gas were: 1) a double peak rise in H 2 (variably defined), 2) a rise in H 2 > 20 PPM by 180 min, or
3) a rise in H 2 >20 PPM by 90 min (which currently appears to be most widely recommended) [bib_ref] Irritable bowel syndrome hypothesis: implications for treatment, Vanner [/bib_ref] [bib_ref] Evaluating a bacterial hypothesis in IBS using a modification of Koch's postulates:..., Pimentel [/bib_ref].
Criteria for changes in CH 4 gas have also been variable between studies with some studies using a rise of 1 PPM or more by 90 min after lactulose ingestion 108 or a rise in CH 4 >20 PPM 121 in the same time period. Similarly, various criteria for H 2 gas have been used to define an abnormal GBT but most commonly it has been defined as a rise in H 2 of 12 PPM above baseline . When these criteria were applied in LBTs in the early clinical trials, a very strong association between SIBO and IBS was reported, with prevalence rate as high as 84% [bib_ref] Evaluating a bacterial hypothesis in IBS using a modification of Koch's postulates:..., Pimentel [/bib_ref]. On the other hand, it appears that the most abundant phylotypes are also the most likely to have been cultured. Walker et al [bib_ref] Dominant and diet-responsive groups of bacteria within the human colonic microbiota, Walker [/bib_ref] found that 66% of phylotypes that individually accounted for >0.5% of sequences corresponded to cultured species, while only 30% of those accounting for <0.5% of total sequences corresponded to cultured species. This suggests that the majority of bacterial cells in normal faecal samples will be related to cultured species, whilst it is the rarer species that are underrepresented by cultures. This also suggests that a lack of well-characterized isolates, rather than any intrinsic resistance to cultivation, may be an additional reason for the low coverage of gut microbial diversity by cultured strains.
With the advent of sequence-based molecular methodologies there is a tendency to dismiss culturebased approaches as 'old technology'. In the past enormous effort went into describing the human faecal microbiota using careful anaerobic cultivation methods (eg. [bib_ref] Intestinal floras of populations that have a high risk of colon cancer, Moore [/bib_ref] [bib_ref] Comparison of fecal microflora of elderly persons in rural and urban areas..., Benno [/bib_ref]. Although it has been widely assumed that cultivation methods must severely bias the bacteria that can be recovered, recent work suggests that the dominant species described in such studies correspond quite closely to the most dominant species detected by molecular surveys [bib_ref] Dominant and diet-responsive groups of bacteria within the human colonic microbiota, Walker [/bib_ref]. Conversely clone libraries of amplified 16S rRNA sequences have tended to underestimate certain groups such as bifidobacteria that are readily recovered by cultivation [bib_ref] Critical evaluation of two primers commonly used for amplification of bacterial 16S..., Frank [/bib_ref].
The sheer laboriousness of cultural approaches makes it unlikely that large scale anaerobic culture work will be undertaken as an approach to describing microbial diversity in the future. On the other hand, viable counts offer a straightforward estimate of total bacterial numbers in gut samples. Total anaerobe counts estimated in faecal samples from healthy adults using 'non-selective' media typically yield >10 11 cfu/g compared with around 10 8 /g for facultative anaerobes 132 although facultative numbers are higher in the elderly [bib_ref] Comparison of compositions and metabolic activities of fecal microbiotas in young adults..., Woodmansey [/bib_ref]. Total bacterial numbers can also be estimated by DAPI (4',6-diamidino-2-phenylindole) staining and by FISH (fluorescence in situ hybridization) microscopy using total bacterial probes, giving numbers that are of the same order 132 or somewhat higher 134 than those obtained by viable counting. Most other molecular methods such as qPCR and direct sequencing either do not give reliable estimates of total numbers, or give only relative
proportions. Selective plating methodologies using anaerobic media include Beerens medium, considered selective for bifidobacteria, and MRS medium considered selective for lactobacilli and lactococci [bib_ref] Comparison of compositions and metabolic activities of fecal microbiotas in young adults..., Woodmansey [/bib_ref] [bib_ref] Bifidobacterial diversity determined by culturing and by 16S rDNA sequence analysis in..., Delgado [/bib_ref] [bib_ref] Age and disease related changes in intestinal bacterial populations assessed by cell..., Hopkins [/bib_ref]. Verifying that counts obtained on such media correspond to a given phylogenetic group, however, requires further molecular analysis of the colonies obtained. Another problem is in verifying that all representatives of a given groups are equally likely to be recovered on the selective medium. For these reasons anaerobic selective plating has been largely discontinued as an approach to the enumeration of particular phylogenetic groups, being replaced by molecular methodologies, although its value in describing functional groups of gut bacteria remains crucial as discussed further below. In principle, improved selective approaches could be developed in the future for enumerating key groups of obligately anaerobic bacteria. This would however require a detailed knowledge of growth characteristics that is currently lacking. It has proved possible to detect single strains of butyrate-producing strict anaerobes when introduced into a background of total human gut bacteria in fermentor simulations, but this relied on introducing antibiotic resistance mutations [bib_ref] Effects of alternative dietary substrates on competition between human colonic bacteria in..., Duncan [/bib_ref].
In contrast, selective culture-based approaches remain of great value for the enumeration of specific groups, especially the many important pathogens that are facultative anaerobes. The predominant obligate anaerobes are eliminated under aerobic conditions, and media can be made highly selective for the relevant target organisms. Furthermore they allow detailed strain typing and characterization, which is particularly important for pathogens. Magnetic beads carrying antibodies that selectively bind a given serotype have also been used to enhance recovery of bacteria present in low numbers eg. for E. coli O157 138 .

## C. isolation and function-based molecular detection
An important benefit from the ability to culture micro-organisms is that functions can be identified and putatively associated with phylogroups defined by 16S rRNA (or other) sequences [bib_ref] Interactions and competition within the microbial community of the human colon: links..., Flint [/bib_ref]. Relevant functions that appear to show limited phylogenetic distribution among organisms that colonise the human large intestine include methanogenesis (confined to certain Archaea) and sulphate reduction (certain genera of Proteobacteria). Functions such as butyrate production or lactate utilization occur within several different phylogenetic groups, but particular subgroups that are responsible for these traits can be identified and independently targeted [bib_ref] Development of a semiquantitative degenerate real-time pcr-based assay for estimation of numbers..., Louis [/bib_ref]. 16S rRNA -based probes and PCR detection methods that target relevant functional/phylogenetic groups have now provided important information on responses to dietary change and disease states in a number of studies .
The use of selective culture media provides an important approach for estimating populations of organisms concerned with particular functions, especially the utilization of specific substrates [bib_ref] Assessment of metabolic diversity within the intestinal microbiota from healthy humans using..., Chassard [/bib_ref] [bib_ref] Characterization of the xylan-degrading microbial community from human faeces, Chassard [/bib_ref] or cross-feeding of fermentation products [bib_ref] Most probable number enumeration of H2-utilizing acetogenic bacteria from the digestive tract..., Dore [/bib_ref]. This has led to the isolation and description of new species with defined functions, e.g. the utilization of lactate [bib_ref] Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major..., Duncan [/bib_ref] , mucin 147 or cellulose [bib_ref] The cellulolytic microflora of the human colon: evidence of microcrystalline cellulose-degrading bacteria..., Robert [/bib_ref]. Isolations from non-selective media have also been used to identify organisms that produce specific short chain fatty acids [bib_ref] Phylogenetic relationships of butyrate-producing bacteria from the human gut, Barcenilla [/bib_ref]. Isolated strains are providing fundamental information on substrate utilization, and on microbe-microbe and microbe-host interactions for key members of the microbial community [bib_ref] Interactions and competition within the microbial community of the human colon: links..., Flint [/bib_ref] [bib_ref] How host-microbial interactions shape the nutrient environment of the mammalian intestine, Hooper [/bib_ref].
Valuable functional information can also be obtained from studies that combine cultivation of the mixed community with molecular analyses. One such approach is stable isotope probing, in which the substrate of interest is labelled, usually with 13 C, and incubated with mixed faecal microorganisms. Organisms that utilize the substrate will incorporate 13 C label into their RNA and DNA, and the labelled nucleic acid can be recovered after density gradient centrifugation.
Amplification and sequencing of 16S rRNA genes can then be used to identify the organisms involved.
This approach has been applied successfully to utilization of starch [bib_ref] Linking phylogenetic identities of bacteria to starch fermentation in an in vitro..., Kovatcheva-Datchary [/bib_ref]. Another approach allows identification of organisms that selectively adhere to insoluble substrates [bib_ref] Selective colonization of insoluble substrates by human faecal bacteria, Leitch [/bib_ref]. Mixed faecal bacteria are incubated with substrate in a fermentor system, and attached bacteria again identified by 16S rRNA sequencing.

## D. functionally relevant gene targets
One application of functional information from cultured strains has been to identify functional gene targets that can be used for culture-independent molecular enumeration. In this approach, the molecular characterization of the community is not based on species or phylotype identity, but on functional properties. This is extremely useful for functions that can be performed by microorganisms from different phylogenetic groups or functions that are only represented in specific strains from a species, since these functions cannot be directly extrapolated from species or phylotype data. For example, it has been shown that most isolated human colonic bacteria that produce butyrate rely on the butyryl CoA: CoA transferase gene for the final step in butyrate formation [bib_ref] Restricted distribution of the butyrate kinase pathway among butyrate-producing bacteria from the..., Louis [/bib_ref] [bib_ref] A novel class of CoA-transferase involved in short-chain fatty acid metabolism in..., Charrier [/bib_ref]. Degenerate PCR primers have now been developed that allow the amplification of this gene from phylogenetically diverse Gram-positive bacteria [bib_ref] Diversity of human colonic butyrate-producing bacteria revealed by analysis of the butyryl-CoA:acetate..., Louis [/bib_ref]. Other examples of this approach are provided by the sulfatase [bib_ref] Molecular ecological analysis of the succession and diversity of sulfate-reducing bacteria in..., Deplancke [/bib_ref] and Archaeal mcrA genes 157 . While the same information could in principle be obtained by metagenomics, such targeted approaches can be quicker and cheaper and remove the need for extensive bioinformatics.
e. From culturing to 16S rRNA gene characterization of microbial communities.
Although large numbers of bacterial strains are continuously isolated with help of recent innovations in high throughput microbial cultivation [bib_ref] The micro-Petri dish, a million-well growth chip for the culture and high-throughput..., Ingham [/bib_ref] and targeted cultivation of microbial groups with specific functions such as butyrate production 142 or mucin degradation [bib_ref] Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium, Derrien [/bib_ref] , it is evident that laborious cultivation approaches are not suitable for studying the diversity and population dynamics of the gastrointestinal tract microbiota. Therefore, approaches that do not rely on selective cultivation are indispensable to obtain insight into the true diversity. About two decades ago, Carl Woese [bib_ref] Bacterial evolution, Woese [/bib_ref] [bib_ref] Phylogenetic structure of the prokaryotic domain: the primary kingdoms, Woese [/bib_ref] [bib_ref] Comparative cataloging of 16S ribosomal ribonucleic acid: molecular approach to procaryotic systematics, Fox [/bib_ref] discovered ribosomal RNA (rRNA) to be an extremely useful phylogenetic marker for microbial identification and systematics and this has led to the discovery of the Archaea as the third domain of life and the construction of the famous tree of life [bib_ref] Bacterial evolution, Woese [/bib_ref] [bib_ref] Towards a natural system of organisms: proposal for the domains Archaea, Bacteria,..., Woese [/bib_ref]. rRNA is part of the ribosomes, which are also called the "factories" of protein synthesis. Since ribosomes are universal in structure and function in all life forms, it has very low mutation and transformation rates. Therefore, differences in rRNA sequences are inversely correlated to the relatedness between the organisms they derive from and hence, rRNA is ideal for phylogenetic identification of organisms. From the three rRNA molecules the small subunit (SSU) RNA or (16S rRNA in Bacteria and Archaea, 18S rRNA in Eukarya) is the most widely used marker due to its manageable size (approximately 1.5 kb) and its discriminative power. It contains nine hypervariable regions that enable identification and differentiation of specific species.
Since 16S rRNA and its corresponding gene can be directly obtained from any environmental sample without cultivation procedures, it has been recognized as a marker to detect and identify basically all members of the respective ecosystem, including the microbes that have never been obtained in culture. This has led to the new research expertise called molecular microbial ecology and as a result the number of nearly complete 16S rRNA sequences in the databases is approximately 1.5 million, which is far more than what can be found for any other gene [bib_ref] The Ribosomal Database Project: improved alignments and new tools for rRNA analysis, Cole [/bib_ref]. A variety of 16S rRNA based approaches has been developed and include classical cloning and sequencing, fingerprinting, FISH, and qPCR, which have been employed to determine the bacterial diversity of ecosytems or to detect and quantify specific bacterial groups in the human GI tract [bib_ref] Molecular ecological analysis of the gastrointestinal microbiota: a review, Zoetendal [/bib_ref]. It has to be realized that none of the approaches gives an unbiased view of the diversity of the microbiota and abundance of its different members. Therefore, it should be noted that these nowadays called classical approaches are not competitors of each other, but can best be seen as complementing approaches as they differ in terms of sensitivity, selectivity and phylogenetic resolution .
The most commonly used 16S rRNA approach is the PCR amplification of 16S rRNA genes followed by subsequent cloning and sequencing to directly access the phylogenetic diversity of bacteria within the GI tract using universal primers that cover most currently known microbes [bib_ref] Molecular ecological analysis of the gastrointestinal microbiota: a review, Zoetendal [/bib_ref]. This classical approach still provides the highest phylogenetic information as nearly complete 16S rRNA genes are sequenced. Exploring diversity based on sequencing 16S rRNA genes requires a novel different classification system because cultivation is not involved. Therefore, classical taxonomic approaches, which include physiological characterization, cannot be applied to classify uncultured microbes into species. As the 16S rRNA gene is the only marker to detect uncultured microbes, classification is based on comparative sequence analysis that results in species-level phylogenetic types or phylotypes. Phylotypes are defined as groups of 16S rRNA gene sequences with a certain level of similarity. Phylotype definition is based on a cut-off value of 16S rRNA sequence similarity and currently that is not consistently used with variations between 97 and 99%. As a consequence different cut-off values will result in different estimates of diversity. Moreover, it has to be emphasized that 16S rRNA gene sequences and their copy numbers may vary within the same microbial genome and hence it is difficult to extrapolate 16S rRNA gene numbers to actual cell numbers. Cloning and sequencing of 16S rRNA genes has been used to characterize a wide variety of ecosystems, including the GI tract.
Sequence specific fingerprinting of 16S rRNA genes has also been frequently applied in studying the GI tract microbiota composition and population dynamics [bib_ref] Molecular ecological analysis of the gastrointestinal microbiota: a review, Zoetendal [/bib_ref] provide information about the overall community and its dynamics. Moreover, fingerprinting can monitor population dynamics of the total community as well as that of specific groups of microbes, which can be determined by the choice of primers [bib_ref] Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the..., Heilig [/bib_ref] [bib_ref] Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by..., Walter [/bib_ref] [bib_ref] Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient..., Satokari [/bib_ref] [bib_ref] DGGE show diversity and short-term temporal stability in the Clostridium coccoides-Eubacterium rectale..., Maukonen [/bib_ref].
Fingerpinting of 16S rRNA genes or other approaches based on classical PCR do not provide quantitative information about the microbiota. Real-time or quantitative PCR (qPCR) on the other hand is a method that is used to quantify the amount of DNA or a gene-of-interest present in biological samples. During qPCR the amount of DNA produced at the end of each amplification cycle is quantified using fluorescent technology. In contrast to regular PCR, qPCR focuses on the onset of the logarithmic phase (threshold) of PCR product accumulation rather than the end-point abundance of PCR product since this is a more accurate estimate of the amount of PCR product obtained because it is less affected by the amplification efficiency of the reaction or depletion of reagents. The power of 16S rRNA gene-specific qPCR is that it can be used to quantify any population of interest and can target even low abundance microbes [bib_ref] Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of..., Matsuki [/bib_ref] [bib_ref] Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR, Matsuda [/bib_ref]. However, it remains difficult to extrapolate 16S rRNA gene copies to actual numbers due to various numbers of gene copies per species and cell lysis efficiency that can be different in environmental samples compared to pure cultures. Another 16S rRNA-specific quantification approach is Fluorescent in situ hybridization (FISH). This is a technique using a specific fluorescent oligonucleotide probe binding to rRNA in whole cells to detect, identify and enumerate bacteria in complex ecosystems. FISH using an array of 16S rRNA oligonucleotide probes can be combined with automated microscopy-based enumeration or flow cytometric enumeration [bib_ref] Extensive set of 16S rRNA-based probes for detection of bacteria in human..., Harmsen [/bib_ref] [bib_ref] Quantification of uncultured Ruminococcus obeum-like bacteria in human fecal samples by fluorescent..., Zoetendal [/bib_ref] [bib_ref] Fluorescent hybridisation combined with flow cytometry and hybridisation of total RNA to..., Rigottier-Gois [/bib_ref]. FISH is the currently the most accurate quantification method as it targets whole cells rather than extracted DNA. However, cell permeability and ribosome accessibility are crucial for FISH and the efficiency differs between species. Moreover, FISH quantification of bacteria in tissue is difficult.
These classical 16S rRNA (gene)-based approaches have already been frequently applied to characterize the human GI tract microbiota and due to their relatively low throughput the approach of choice depended largely on the research question to be addressed. The combination of these approaches in the different studies resulted in a phylogenetic framework of the GI tract microbiota.
The pioneering study by Suau and colleagues demonstrated the high diversity of the GI tract microbiota of which the fast majority has not been identified by culturing [bib_ref] Direct analysis of genes encoding 16S rRNA from complex communities reveals many..., Suau [/bib_ref]. These findings have been confirmed repeatedly, including a recent extensive study that looked at faecal and colonic mucosa-associated communities [bib_ref] Diversity of the human intestinal microbial flora, Eckburg [/bib_ref]. A striking observation with most of the studies of cloning and sequencing approaches have underestimated the number of bifidobacteria and other bacteria belonging to the phylum Actinobacteria, which is likely due to the mismatches of the most frequently used "universal primers" to target them in combination with the high G+C content in the genomes of Actinobacteria. This became evident when FISH is used to quantify different groups of microbes, as has been demonstrated by FISH using SSU rRNA-targeted oligonucleotide probes [bib_ref] Extensive set of 16S rRNA-based probes for detection of bacteria in human..., Harmsen [/bib_ref] [bib_ref] Colonic microbiota signatures across five northern European countries, Lay [/bib_ref]. The first sequence-specific fingerprinting of GI tract communities demonstrated that the dominant microbes are host-specific and relatively stable in time in healthy adults [bib_ref] Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples..., Zoetendal [/bib_ref] , which has been confirmed frequently. In contrast, GI tract community structure and that the microbiota composition is unstable when individuals are suffering from intestinal disorders, such as inflammatory bowel diseases (IBD) [bib_ref] Alterations of the dominant faecal bacterial groups in patients with Crohn's disease..., Seksik [/bib_ref] [bib_ref] Terminal restriction fragment length polymorphism analysis of the diversity of fecal microbiota..., Andoh [/bib_ref] [bib_ref] Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome..., Maukonen [/bib_ref] [bib_ref] Composition and temporal stability of gastrointestinal microbiota in irritable bowel syndrome--a longitudinal..., Matto [/bib_ref] [bib_ref] Unstable composition of the fecal microbiota in ulcerative colitis during clinical remission, Martinez [/bib_ref].
Faecal samples have the benefit that they contain a dense microbiota and that they can be easily collected without invasive procedures. However, a major disadvantage is that feces only represents the end of the colon as has been demonstrated by comparative analysis with mucosal biopsies from the intestine [bib_ref] Diversity of the human intestinal microbial flora, Eckburg [/bib_ref] [bib_ref] Molecular characterization of the microbial species that colonize human ileal and colonic..., Wang [/bib_ref] [bib_ref] Comparison of bacterial diversity along the human intestinal tract by direct cloning..., Wang [/bib_ref] [bib_ref] Molecular analysis of jejunal, ileal, caecal and recto-sigmoidal human colonic microbiota using..., Hayashi [/bib_ref] [bib_ref] Mucosa-associated bacteria in the human gastrointestinal tract are uniformly distributed along the..., Zoetendal [/bib_ref] [bib_ref] Biodiversity of the mucosa-associated microbiota is stable along the distal digestive tract..., Lepage [/bib_ref]. Moreover, microbiotas of the small intestine are different in composition compared to those from the colon, which is most likely explained by the different conditions microbes encounter in the respective parts of the GI tract [bib_ref] Molecular analysis of jejunal, ileal, caecal and recto-sigmoidal human colonic microbiota using..., Hayashi [/bib_ref] [bib_ref] Human gut microbiome adopts an alternative state following small bowel transplantation, Hartman [/bib_ref] [bib_ref] High temporal and inter-individual variation detected in the human ileal microbiota, Booijink [/bib_ref]. This indicates that care must be taken when conclusions about the microbiota in relation to health, disease and diet are solely based on fecal sample analysis.

## F. high throughput 16s rrna characterization of the microbiota
Since the past years classical 16S rRNA gene based approaches have been complemented by novel high throughput approaches. These novel approaches provide deeper insight into the microbiota composition and enable characterization of multiple samples in a high throughput manner, allowing more and better powered observations. These high throughput approaches include phylogenetic microarraying and barcoded pyrosequencing. Phylogenetic microarray analysis is based on arrays spotted with 16S rRNA targeting oligonucleotides that are developed on 16S rRNA gene sequences that exist in DNA databases. The barcoded pyrosequencing approach is different because it does not rely on current 16S rRNA gene sequences in databases, but is based on the novel sequencing tools, [bib_ref] Prescript-assist probiotic-prebiotic treatment for irritable bowel syndrome: an open-label, partially controlled, 1-year..., Bittner [/bib_ref] pyrosequencing, and hence can be considered as de novo community profiling.
The principle of phylogenetic microarray analysis is similar to those of comparative genomics and transcriptomics. The principle of phylogenetic microarraying is based on a DNA microarray, a glass or membrane surface of a microscopic slide that is spotted with thousands of covalently linked 16S rRNA-gene specific probes, which are subsequently hybridized with DNA or RNA for diversity analysis.
In this way thousands of 16S rRNAs or their respective genes can be specifically identified and quantified in a single experiment. After publication of the first phylogenetic microarray, specific for nitrifying bacteria, these microarrays have been implemented in a variety of studies focusing the microbial diversity different ecosystems, varying from specific microarrays focusing on sulphate reducers as well as general microarrays covering thousands of microbes [bib_ref] Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology, Guschin [/bib_ref] [bib_ref] High-density universal 16S rRNA microarray analysis reveals broader diversity than typical clone..., Desantis [/bib_ref]. Besides general or group specific microarrays, ecosystem-specific microarrays have also been developed including those focusing on the human GI tract. The first microarray was developed to monitor 40 bacterial species in the GI Tract [bib_ref] Design and evaluation of oligonucleotidemicroarray method for the detection of human intestinal..., Wang [/bib_ref]. Afterwards, several phylogenetic microarrays have been developed and applied to study the human GI tract microarray in a high throughput matter [bib_ref] Rapid quantitative profiling of complex microbial populations, Palmer [/bib_ref] [bib_ref] Development and application of the human intestinal tract chip, a phylogenetic microarray:..., Rajilic-Stojanovic [/bib_ref] [bib_ref] High-throughput quantitative analysis of the human intestinal microbiota with a phylogenetic microarray, Paliy [/bib_ref] [bib_ref] Dysbiosis of fecal microbiota in Crohn's disease patients as revealed by a..., Kang [/bib_ref]. These studies have demonstrated the power of phylogenetic microarraying since it combines the power of fingerprinting and phylogenetic analysis with the quantitative power of FISH in a single analysis per sample.
Another major benefit of phylogenetic microarray analysis is that the array data can be stored in a database and this allows multiple sample comparisons as has been demonstrated in a recent metaanalytic study on phylogenetic profiles originating from 1,000 samples [bib_ref] Advanced approaches to characterize the human intestinal microbiota by computational meta-analysis, Nikkila [/bib_ref].
Another high throughput 16S rRNA gene approach that is increasing in popularity is [bib_ref] Prescript-assist probiotic-prebiotic treatment for irritable bowel syndrome: an open-label, partially controlled, 1-year..., Bittner [/bib_ref] [bib_ref] Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community..., Claesson [/bib_ref]. Nevertheless it has to be mentioned that the dynamic range of the HITChip compares to approximately 200,000 pyrosequences per sample and that both approaches provide more distinct profiles for the small intestinal microbiota [bib_ref] Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community..., Claesson [/bib_ref].
Nevertheless, it is evident that both, phylogenetic microarraying and high throughput sequencing of 16S rRNA genes are promising approaches which will help researchers to link specific members of the microbiota to host characteristics in health and disease. the discovery of multiple genes or operons, potentially linked to phylogenetic markers that supplement specific functional pathways to the cloning host, which is in most cases Escherichia coli [bib_ref] Cloning the metagenome: Culture-independent access to the diversity and functions of the..., Handelsman [/bib_ref]. The benefit of direct random sequencing is that DNA can be directly assessed without any cloning procedure as indicated previously. In general, metagenomics-based studies can be divided into two main strategies: sequence-based screening and function-based screening [bib_ref] Metagenomics: application of genomics to uncultured microorganisms, Handelsman [/bib_ref] [bib_ref] Microbial communities in the human small intestine: coupling diversity to metagenomics, Booijink [/bib_ref]. Sequence-based screening employs large scale sequence determination and mining to unravel gene functions in order to describe and understand the microbial community [bib_ref] Molecular eco-systems biology: towards an understanding of community function, Raes [/bib_ref] , while function-based screening mainly focuses on the identification of gene sequences of clones that were screened for a particular function or activity in a heterologous expression host.

## G.i. sequence-based metagenomics
Sequence-based metagenomics, or random sequencing of DNA fragments from environmental samples, can be performed in several ways. One way is to construct a metagomic library and screen for sequences of interest via PCR or microarray strategies [bib_ref] Metagenomics: application of genomics to uncultured microorganisms, Handelsman [/bib_ref]. When 16S rRNA gene is used as a screening target, such an approach will unravel some of the genetic potential of specific (uncultured) microbes as genes flanking the 16S rRNA genes will be sequenced. However, this way of screening can also be used to screen for the diversity of specific genes, such as those encoding butyryl-CoA:acetate-CoA transferase in the fecal microbiota [bib_ref] Development of a semiquantitative degenerate real-time pcr-based assay for estimation of numbers..., Louis [/bib_ref].
Most sequence-based sequencing strategies are based on random shotgun sequencing or random pyrosequencing or solexa sequencing. The latter ones have been primed by the explosive developments in sequencing technologies as has been discussed earlier. Random sequencing strategies generate vast amounts of sequence information which require advanced computational approaches for assembly and gene-function assignment [bib_ref] Molecular eco-systems biology: towards an understanding of community function, Raes [/bib_ref] and a large variety of reference genome sequences from GI tract bacterial species. The first shotgun sequencing of the human GI tract microbiota revealed the discovery of genes that complement our human genome with respect to GI tract functionality [bib_ref] Metagenomic analysis of the human distal gut microbiome, Gill [/bib_ref] [bib_ref] Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes, Kurokawa [/bib_ref]. The latter study also demonstrated that microbiotas of different individuals are rather diverse in functional categories in infants, while those in adults are more similar 212 , especially between family relatives [bib_ref] A core gut microbiome in obese and lean twins, Turnbaugh [/bib_ref]. Recently, deep paired end solexa sequencing of the fecal microbiota of more than 100 individuals enabled the definition of an intestinal microbiota coremetagenome, and allowed the detection of gene sets specifically enriched within this microbial consortium.
Besides random sequencing of DNA, more specified sequencing of ecosystem members has also been performed. Transposon-aided capture (TRACA) of plasmids from the human GI tract microbiota has been demonstrated to get insight into the mobile metagenome of the human GI tract microbiota [bib_ref] Transposon-aided capture (TRACA) of plasmids resident in the human gut mobile metagenome, Jones [/bib_ref] and recently the enormous diversity of our virome has also been revealed by metagenomic sequencing of virus-like particles isolated from human faeces [bib_ref] Viruses in the faecal microbiota of monozygotic twins and their mothers, Reyes [/bib_ref].
It is evident that sequence-based metagenomics of the human GI tract microbiota will expand drastically the coming years. There are worldwide many initiatives in sequencing this microbiota all around the world, including the EU FP7 project MetaHIT (http://www.metahit.eu) and the Human Microbiome Initiative from the USA (http://nihroadmap.nih.gov/hmp/). All these efforts will result in a large catalogue of microbial genes that are associated with our GI tract.

## G.ii. function-based metagenomics
Function-based metagenomics is based on construction of small or large-insert libraries in a cloning host. In contrast to the random sequencing approach in sequence-based metagenomics, this approach focuses on screening metagenomes for functional properties that are encoded on cloned DNA fragments. In order to enable detection of functions, heterologous expression of genes located in the insert as well as suitable screening assays are needed [bib_ref] Metagenomics: application of genomics to uncultured microorganisms, Handelsman [/bib_ref]. This might be complicated for functions that involve multi-gene pathways or that need to be transported across the membrane(s).
Despite these restrictions, function-based metagomics has been successfully applied to screen for microbial functions in the human GI tract. Screening the human GI tract metagenome library for bile salt hydrolase (BSH) activity has revealed that it is a conserved activity of the microbiota of individual human hosts that can be found in Bacteria and Archaea [bib_ref] Functional and comparative metagenomic analysis of bile salt hydrolase activity in the..., Jones [/bib_ref]. In another study, two cloned inserts encoding mucin degradation capacity were identified in a fosmid library derived from a human ileum biopsy specimen of which one of the inserts encoded a putative novel pathway for mucin degradation with highest similarity to genes from Entercoccus 216 .
One of the major problems with functional screening is that large numbers of clones have to be screened in order to identify one or a few positive clones and therefore, high throughput screening technologies are crucial. Substrate-Induced Gene Expression (SIGEX), which employs a cloning vector for operon-trapping and fusion to green fluorescent protein (GFP), is one way of high throughput screening as it can be coupled to Fluorescence Activated Cell Sorting (FACS) of positive cells [bib_ref] Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes, Uchiyama [/bib_ref]. In a similar way, a high throughput "intracellular" screening called "metabolite-regulated expression"
METREX was developed, in which metagenomic DNA is cloned into a host cell containing a biosensor for compounds that induce bacterial quorum sensing [bib_ref] Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing..., Williamson [/bib_ref]. Neither of these screening systems has been applied to the GI tract ecosystem yet, but could be attractive to set-up a screening assay for induction of microbial gene expression by host-derived substrates such as mucus or specific bacterial metabolites such as short chain fatty acids.
A novel way to unravel microbial functions in the GI tract is by cell-line based host-response phenotype screening [bib_ref] Development of high-throughput phenotyping of metagenomic clones from the human gut microbiome..., Gloux [/bib_ref]. In this approach fosmid clones were screened for their capacity to induce or inhibit epithelial-cell proliferation. Specific genes in the fosmid clones were identified by transposonmutagenesis and included ABC systems, a glutamate synthase subunit, a RecD homologue, and a Vtype ATPase subunit. In a similar way, several fosmid clones were detected that has an impact on the NF-B signaling pathway [bib_ref] Functional metagenomics: a high throughput screening method to decipher microbiotadriven NF-kappaB modulation..., Lakhdari [/bib_ref]. This revealed the identification of genes encoding an ABC transport system and lipoprotein with highest similarity to Bacteroides. These examples indicate the power of these screening approaches to couple microbial genes to specific functions in the GI tract. Since metagenomics uses DNA as a target, it only provides a catalogue of the genetic potential within an ecosystem. It has to be realized that the presence of a gene or function does not indicate that it will be expressed in situ. It is even possible that the genetic information originates from dead cells. To gain insight into the activity of microbes, markers of activity, such as messenger RNA (mRNA), proteins or metabolites need to serve as targets.

## H. metatranscriptomics
Metatranscriptomics covers the overall or gene-subset-specific transcriptome analysis of microbes within an ecosystem [bib_ref] A perspective: metatranscriptomics as a tool for the discovery of novel biocatalysts, Warnecke [/bib_ref]. With metatranscriptomics the overall gene-expression within a community is determined which reflects its overall activity. Since bacterial RNA is rather instable, proper storage of samples, and a direct and efficient RNA isolation protocol are crucial to detect genes that are expressed in an ecosystem, such as the GI tract, accurately [bib_ref] Isolation of RNA from bacterial samples of the human gastrointestinal tract, Zoetendal [/bib_ref]. Moreover, enrichment of mRNA, which reflects the actual transcript activity, is required since more than 95% of total bacterial RNA consists of rRNA. This can be done by selective capturing of rRNA or selective exonuclease reaction to digest specifically rRNA [bib_ref] A perspective: metatranscriptomics as a tool for the discovery of novel biocatalysts, Warnecke [/bib_ref] [bib_ref] Metatranscriptome analysis of the human fecal microbiota reveals subject-specific expression profiles, with..., Booijink [/bib_ref].
Classical transcriptomics is basically done with DNA microarrays or gene-specific Reverse transcription qPCR (RT-qPCR) which are both well established. However, designing microrrays or gene-specific qPCR assays for metatranscriptomics of diverse communities is very challenging, because these require prior knowledge of the genes that are present in the community.
One way to reduce this complexity is designing microarrays that target a specific subgroup of the community and determine its gene expression in situ. Such an approach has been recently applied by determining the gene-expression pattern of bifidobacteria in the infant gut by using a mixed species

## I. metaproteomics
Metaproteomics encompasses the study of the proteome produced by the community in an ecosystem and is a very powerful strategy for understanding overall microbial-ecosystem functioning [bib_ref] Microbial community proteomics: elucidating the catalysts and metabolic mechanisms that drive the..., Wilmes [/bib_ref]. Similar to metatranscriptomics, metaproteomics provides insights into the microbiota overall activity as reflected by its protein profiles. Classical proteome consists of two-dimensional (2D) gel electrophoresis, often combined matrix assisted laser desorption ionization-time of flight mass spectometry (MALDI-TOF MS) and the first gut metaproteomics study of the infant microbiota revealed its metaproteome dynamics and the identification of a bifidobacterial transaldolase 232 .
However, a major impetus in metaproteomics of the human GI tract microbiota came from a novel high-throughput, non-targeted mass spectrometry (MS) based on the shotgun metaproteomic approach for detection and identification of all proteins without gel-based separation [bib_ref] Shotgun metaproteomics of the human distal gut microbiota, Verberkmoes [/bib_ref]. A high abundance of proteins involved in translation, energy production and carbohydrate metabolism were among the dominantly produced proteins by the GI tract microbiota. Recently, it has been demonstrated that metaproteomics data analysis can even be enhanced by using an approach based on a synthetic metagenome that combines the power of sequence diversity contained in metagenomic diversity and the high reliability of predicted proteins [bib_ref] An iterative workflow for mining the human intestinal metaproteome, Rooijers [/bib_ref]. Although metaproteomics is still in its infancy, the technological developments are increasing fast and hence, it is expected that its application in studying the human GI tract microbiota will increase in the near future.

## J. metabonomics/metabolomics
Metabolite profiling can be divided into metabolomics and metabonomics. With metabolomics small metabolite molecules in complex biological samples, are characterized and quantified, while with metabonomics the global and dynamic metabolic response to environmental factors or genetic manipulations of ecosystems or other multicellular biological systems are measured [bib_ref] Systems biology: Metabonomics, Nicholson [/bib_ref]. In contrast to the other meta-omics approaches, metabonomics and metabolomics focus on the produced metabolites and therefore, cannot be linked to any genetic information. Hence, they do not provide details about the microbes that are responsible for the produced metabolites. Although the field of metabolite profiling in ecology is recent, it has already been applied to associate variation in human metabolic phenotypes to several factors, such as host genotype, age, sex, lifestyle, nutrition, health status and commensal microbial [bib_ref] Metabolomics, a novel tool for studies of nutrition, metabolism and lipid dysfunction, Oresic [/bib_ref]. Metabolic profiling of faecal samples from IBD patients and healthy controls indicated marked metabolic differences between these groups [bib_ref] Rapid and noninvasive metabonomic characterization of inflammatory bowel disease, Marchesi [/bib_ref] shifts have been detected in faecal metabolites in obese subjects on high protein, low carbohydrate weight loss diets [bib_ref] High-protein, reduced-carbohydrate weight-loss diets promote metabolite profiles likely to be detrimental to..., Russell [/bib_ref] [bib_ref] Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease, Wang [/bib_ref]. It is evident that these metabonomics studies, combined with other metaomics' approaches will improve our understanding of host-microbe interactions and how that affects our global metabolism as a superorganism of human and microbial cells.

## Differences in the microbiome in functional bowel disorders

## Key points
 Very little is known about small bowel commensals.
 Using a conventional cut-off of >10 5 cfu/ml in duodenal aspirate the incidence of small intestinal bacterial overgrowth is <5% in IBS, no different from healthy controls  The lactulose breath test is not validated for diagnosing SIBO and many of the abnormalities may reflect rapid oro-caecal transit.
 A number of confounding factors, including acid inhibition by concomitant proton pump inhibitors, may also account for abnormal lactulose breath tests and/or differences in numbers of bacteria in the small intestine.
 Colonic commensals form complex interrelated communities.
 Heterogeneity of IBS and variation in methods used to study the fecal microbiota has resulted in conflicting reports of differences from healthy controls  There are no data on changes in postinfectious IBS but studies of acute gastroenteritis show a "dysbiosis" with loss of diversity, depletion of anaerobes often associated with overgrowth of enterobacteria.
 The microbiome may contribute to IBS symptoms by altering gut neuromotor-sensory function, barrier function, and/or the brain-gut axis.

## A. small intestine microbiome in healthy controls and ibs patients
The hypothesis that overgrowth of bacteria in the small intestine is an important pathogenic mechanism underlying IBS [bib_ref] Evaluating a bacterial hypothesis in IBS using a modification of Koch's postulates:..., Pimentel [/bib_ref] 

## A.i. small intestinal microbiome in healthy controls
Culture. Despite the interest in the small intestine microbiota there is relatively little known, largely because of the limited access to this region of the GI tract [bib_ref] Metatranscriptome analysis of the human fecal microbiota reveals subject-specific expression profiles, with..., Booijink [/bib_ref]. Most of what we know has been obtained using culture techniques on effluent and mucosal samples obtained using endoscopic and radiologic tube/capsule placement in healthy volunteers, samples obtained from trauma patients and at autopsy, and those from ileostomies. Despite the well-recognized limitations of culture techniques [bib_ref] A systematic review of diagnostic tests for small intestinal bacterial overgrowth, Khoshini [/bib_ref] [bib_ref] Microbial communities in the human small intestine: coupling diversity to metagenomics, Booijink [/bib_ref] Culture-independent analysis. The application of molecular techniques to the study of human small intestine microbiome is in its relative infancy but emerging studies highlight the potential complexity of these microbial communities and suggest that previous consideration of this microbiota may be overly simplistic [bib_ref] Microbial communities in the human small intestine: coupling diversity to metagenomics, Booijink [/bib_ref] 

## A.ii. small intestinal microbiome in ibs
Culture. In addition to the general limitations of this technique, two specific issues related to IBS have presented challenges for diagnosing SIBO in this disorder. Firstly, while many experts suggest that SIBO should be defined as 10 5 cfu/ml of colonic bacteria, this definition has not been uniformly accepted [bib_ref] A systematic review of diagnostic tests for small intestinal bacterial overgrowth, Khoshini [/bib_ref] [bib_ref] Critical evaluation of diagnosing bacterial overgrowth in the proximal small intestine, Kerckhoffs [/bib_ref]. Proponents of the SIBO-IBS hypothesis, suggest that these criteria are derived from studies of major diseases predisposing to SIBO e.g. stagnant loop in Bilroth II gastrectomy with associated steatorrhea and may not be relevant to IBS [bib_ref] A systematic review of diagnostic tests for small intestinal bacterial overgrowth, Khoshini [/bib_ref] [bib_ref] An evidence-based treatment algorithm for IBS based on a bacterial/SIBO hypothesis: Part..., Pimentel [/bib_ref]. Secondly, if the SIBO-IBS hypothesis depends upon migration of colonic bacteria into the ileum and distal jejunum as initially proposed, sampling the more proximal bowel would not detect these changes [bib_ref] Bacterial concepts in irritable bowel syndrome, Lin [/bib_ref]. There is only one large prospective study of well characterized IBS patients [bib_ref] Small intestinal bacterial overgrowth in patients with irritable bowel syndrome, Posserud [/bib_ref]. Together, these studies show the duodenum and proximal jejunum of most IBS patients contains relatively small numbers of bacteria and no obvious differences in content compared to controls. The largest study did show a significantly greater proportion of patients had  5 X 10 3 cfu/ml compared to healthy controls (42% vs. 12%) in a post-hoc analysis but no difference between groups using a  5 X 10 5 cfu/ml cut-off. A recent preliminary report found similar findings using the lower cut-off, but identified the IBS patients using Rome III criteria retrospectively from a group of patients admitted with upper gastrointestinal bleeding [bib_ref] Small Bowel Culture Confirms the Presence of Small Intestinal Bacterial Overgrowth in..., Pyleris [/bib_ref]. Further studies are needed to determine the significance of these finding, particularly as increased numbers have been documented in other diseases [bib_ref] A systematic review of diagnostic tests for small intestinal bacterial overgrowth, Khoshini [/bib_ref] Culture-independent analysis. To date there are very few studies which have applied these techniques to the study of the microbiome in IBS patients but these studies should be important in resolving the debate concerning the role of SIBO in IBS. One study examined jejunal aspirates in small numbers of IBS patients (n=10) and healthy controls using qPCR combined with culture techniques and breath testing [bib_ref] A systematic review of diagnostic tests for small intestinal bacterial overgrowth, Khoshini [/bib_ref]. The molecular analysis did not reveal any differences in total bacterial counts or the composition of the microbiota including the numbers of colonic-type bacteria (see however limitations of qPCR above).

## B.i. confounding factors between sibo and ibs
The lack of consistency in the data linking SIBO to IBS 28 raises the possibility of confounding by known factors such as proton pump inhibitors (PPIs), impaired clearance of fasting secretions by disordered motility or other unknown factors.

## B.ii. could ibs be linked to sibo through ppis?
The use of proton pump inhibitors (PPIs) which is commoner in IBS than controls could be a major confounder [bib_ref] Bacterial overgrowth and irritable bowel syndrome: unifying hypothesis or a spurious consequence..., Spiegel [/bib_ref] since PPI therapy may promote both gastric bacterial overgrowth [bib_ref] Effect of omeprazole on intragastric bacterial counts, nitrates, nitrites, and N-nitroso compounds, Verdu [/bib_ref] [bib_ref] Combined oro-caecal scintigraphy and lactulose hydrogen breath testing demonstrate that breath testing..., Yu [/bib_ref]. A recent survey of duodenal aspirate and culture in 675 patients in a tertiary care setting did find about 2% of IBS had SIBO. They also found a positive relationship between PPIs exposure and indeterminate bacterial growth but not with >10 5 orgs/ml 252 .

## B.iii. could ibs be linked to sibo through underlying dysmotility?
The above studies suggest that around 2-4% of IBS have SIBO. It is likely that IBS symptoms are related, in part, to abnormal intestinal motility which can cause SIBO. Perhaps the most powerful example is scleroderma, where hypomotility and stagnation lead to SIBO, with attendant defecatory symptoms and bloating [bib_ref] Development of a preliminary scleroderma gastrointestinal tract 1.0 quality of life instrument, Khanna [/bib_ref] , some of which can be improved by antibiotic therapy [bib_ref] Small bowel bacterial overgrowth in systemic sclerosis: detection using direct and indirect..., Kaye [/bib_ref]. Recent data suggests that IBS-D is associated with fast small bowel transit and that this accounts for the early rise in breath hydrogen after lactulose as it enters the colon [bib_ref] Combined oro-caecal scintigraphy and lactulose hydrogen breath testing demonstrate that breath testing..., Yu [/bib_ref] [bib_ref] The relationship between symptoms, body mass index, gastrointestinal transit and stool frequency..., Sadik [/bib_ref]. The observation that antibiotics help some IBS patients [bib_ref] Rifaximin therapy for patients with irritable bowel syndrome without constipation, Pimentel [/bib_ref] is not necessarily evidence that SIBO causes IBS, since it is more likely that the antibiotics alter colonic fermentation and that this accounts for the improvement in bloating and flatulence. As shown in . In contrast, the other definitions of SIBO did not correlate IBS symptoms at all, as also found by Grover and colleagues in a separate study 125 .

## B.iv. other potential confounders
There are a range of other confounders that might undermine the causal link between alterations in small bowel microbiota and IBS. These include variations in antibiotic use, probiotic, prebiotic, or other dietary ingestions (e.g. fermentable oligo-, di-and mono-saccharides, and polyols [FODMAP] [bib_ref] Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics, Gibson [/bib_ref] [bib_ref] The FODMAP diet for irritable bowel syndrome: food fad or roadmap to..., Rangnekar [/bib_ref] in IBS patients vs. controls. There are several studies indicating that antibiotic consumption is commoner in IBS than controls [bib_ref] Antibiotic use, childhood affluence and irritable bowel syndrome (IBS), Mendall [/bib_ref] and that their use is a risk factor for developing IBS [bib_ref] Antibiotics increase functional abdominal symptoms, Maxwell [/bib_ref]. Furthermore their use can be followed by persistent abnormalities in the faecal microbiota even after 60 days 271 .
Other factors could involve the effects of low grade inflammation in the small intestine or even altered immune function [bib_ref] Differential expression of toll-like receptors in patients with irritable bowel syndrome, Brint [/bib_ref] [bib_ref] Is irritable bowel syndrome an inflammatory disorder?, De Giorgio [/bib_ref].

## C. large bowel microbiome in healthy controls and ibs patients
The human GI tract microbiota is very complex in composition, can be influenced many factors , is individual specific and consists of numerous uncultured microbes, as discussed in Chapter 4. Ten bacterial phyla have been detected in fecal samples from which the Firmicutes, Bacteroidetes and Actinobacteria dominate [bib_ref] High-throughput diversity and functionality analysis of the gastrointestinal tract microbiota, Zoetendal [/bib_ref]. Despite the individual variation, a recent paper suggested that the human GI tract microbiota can be divided in three robust clusters called enterotypes that are indicated to be driven by groups of species that together contribute to their respective preferred community composition [bib_ref] Enterotypes of the human gut microbiome, Arumugam [/bib_ref]. Remarkably, these enterotypes do not appear to be dependent on features, such as nation, gender, age or body mass index, although these findings are based on relatively small numbers of subjects.
The GI tract microbiota may have an important role in the onset and maintenance of IBS, particularly postinfectious IBS. Several studies have already described the microbiota composition in IBS patientsand although differences from controls have been described these are inconsistent and sometimes contradictory with respect to identification of species associated with IBS.
One of the problems with a meta-analysis of the different IBS studies is the fact that a limited number of subjects is used. In addition, samples are in general single snapshots from the individuals and since IBS symptoms are characteristically erratic, analysis of a single sample may be misleading when it is not linked to symptoms at the time when it is taken.
Another major concern that hampers a detailed analysis is the different methods that have been used to identify the microbiota as they include selective cultivation, specific targeted qPCR and FISH, DGGE, G+C -and microarray profiling. In addition, most studies focused on composition analysis of the microbiota whereas what may be more important is the microbiota function and metabolic and immunomodulatory activity.
Besides microbiota, detailed description of the patients, such as IBS typing beyond the Rome III criteria, IBS symptom scores and diet recordings are often not complete and comparable between different studies. In addition, the number of studies itself is very limited considering that more than half of the studies originate from a single country in which the same cohorts have been used in multiple studies. Temporal fluctuations in microbiota compositions have been observed in IBS [bib_ref] Analysis of the fecal microbiota of irritable bowel syndrome patients and healthy..., Malinen [/bib_ref] and IBD [bib_ref] Unstable composition of the fecal microbiota in ulcerative colitis during clinical remission, Martinez [/bib_ref] , which contrasts to the rather stable community composition in the GI tract of healthy adults .
However, different enterotypes of the gut microbiota exists [bib_ref] Enterotypes of the human gut microbiome, Arumugam [/bib_ref] It should be noted that elevated BMI is associated with accelerated colonic transit in IBS [bib_ref] The relationship between symptoms, body mass index, gastrointestinal transit and stool frequency..., Sadik [/bib_ref] [bib_ref] Lower functional gastrointestinal disorders: evidence of abnormal colonic transit in a 287..., Manabe [/bib_ref] and this may account for the differences in microbiota noted in IBS patients with BMI >25 kg/m .

## D. post infectious ibs and the effect of infections on the gut microbiome

## D.i postinfectious ibs
A healthy commensal microbiota resists pathogen colonisation by occupying the different niches in the GI tract and producing a range of antibacterial products including both antibiotics, as well as metabolites such as short chain fatty acids. Animal studies clearly show that depleting the microbiota by giving antibiotics can break this colonisation resistance [bib_ref] Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis..., Barthel [/bib_ref]. If the normal resistance is overcome, the resulting infective gastroenteritis produces a profound depletion of the commensal microbiota [bib_ref] Hostmediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae, Lupp [/bib_ref] , though for how long this disruption lasts and how completely recovery occurs, is unclear. The incidence of infective gastroenteritis in the UK is 19/100 person years [bib_ref] Study of infectious intestinal disease in England: rates in the community, presenting..., Wheeler [/bib_ref] 

## D.ii effects of gi infections on the microbiome
It is likely that in addition to these effects on gut physiology there will also be changes in the

## E.ii intestinal barrier dysfunction
In addition to being a selective physical barrier, extrinsic factors in the intestinal mucosa such as mucin production [bib_ref] The intestinal epithelium: sensors to effectors in nematode infection, Artis [/bib_ref] and secretory immunogloubulin A (sIgA) [bib_ref] Evolution, development, mechanism and function of IgA in the gut, Fagarasan [/bib_ref] This highlights that bidirectional pathways exist between the host epithelium and its microbiota (Chapter 4). In a study that included patients with IBD, IBS and healthy controls and investigated mucosal and faecal bacteria using FISH, adherent bacterial biofilms were detected in a majority of IBD patients compared to IBS and healthy controls [bib_ref] Spatial organization of bacterial flora in normal and inflamed intestine: a fluorescence..., Swidsinski [/bib_ref]. Mucosal biofilms were also observed in healthy volunteers and IBS patients, but bacterial concentrations in both groups were lower than those in patients with overt inflammation and IBD. No clear bacterial infiltration of the mucosa was observed in any of the patients, except for areas with mucosal breaks [bib_ref] Spatial organization of bacterial flora in normal and inflamed intestine: a fluorescence..., Swidsinski [/bib_ref]. In another study in patients with idiopathic diarrhoea, faecal analysis showed increased mucus strands and reduction in concentrations of Eu. rectale, Bacteroides, and F. prausnitzii groups [bib_ref] Biostructure of fecal microbiota in healthy subjects and patients with chronic idiopathic..., Swidsinski [/bib_ref]. Fecal levels of Human beta-defensin-2 (HBD-2) were increased in IBS compared with healthy controls and were similar to those in the patients with ulcerative colitis [bib_ref] Elevated human beta-defensin-2 levels indicate an activation of the innate immune system..., Langhorst [/bib_ref]. Moreover, increased beta-defensin 2 peptide expression was detected by immunohistochemistry technique in colonic epithelial enterocytes of IBS patients [bib_ref] Elevated human beta-defensin-2 levels indicate an activation of the innate immune system..., Langhorst [/bib_ref].
Overall the results suggest that bacterial-host interactions may be initiated by components of the microbiota that can cross the mucus and adhere to epithelial cells, inducing activation of the mucosal innate defense system even in the absence of mucosal destruction.

## E.iii alterations in gut-brain axis
Psychological co-morbidity is common in gut functional disorders [bib_ref] Psychosocial factors are linked to functional gastrointestinal disorders: a population based nested..., Locke [/bib_ref] [bib_ref] Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression..., Bravo [/bib_ref]. The results support the concept that components of the intestinal microbiota can affect the brain biochemistry and behaviour in adult mice and raise the hypothesis that modulation of the microbiota is a potential therapeutic approach in psychiatric co-morbidity in IBS. To date, no direct evidence of brain to microbiome interplay has been reported in humans.
There is, however, indirect evidence that the gut microbiome can affect CNS function in humans.
Oral antibiotics and laxatives, that affect the microbiota, have long been used in clinical practice to treat hepatic encephalopathy [bib_ref] The treatment of hepatic encephalopathy, Morgan [/bib_ref]. The mechanism underlying this dramatic improvement in brain function in patients is unclear, but animal studies have shown that bacteria can produce a ligand for the benzodiazepine receptor [bib_ref] Gut bacteria provide precursors of benzodiazepine receptor ligands in a rat model..., Yurdaydin [/bib_ref]. On the other hand, patients with depression have altered profiles of breath hydrogen excretion after ingestion of fructose, and one uncontrolled trial showed that bowel symptoms and depression scores improved after an elimination diet but whether this is secondary to improved bowel symptoms or a direct effect is unclear [bib_ref] Fructose-and sorbitol-reduced diet improves mood and gastrointestinal disturbances in fructose malabsorbers, Ledochowski [/bib_ref]. Moreover, fructose malabsorption, which modifies gut physiology and the composition of the microbiome 341 , has been associated with decreased plasma tryptophan levels 342 .

## The relationship between ibs and other chronic gastrointetsinal disorders.

## Key points:
 Although diverticulitis, IBD or coeliac disease can co-exist with IBS, an "IBS" diagnosis in the presence of an organic disease may be challenging.
 Symptomatic uncomplicated diverticular disease, celiac disease, and IBD can mimic IBS symptoms, which should encourage active case finding in people with IBS. Once the diagnosis of an organic disease is made, "IBS" is generally ruled out, even though coexistence of IBS and organic diseases seems to be relatively common.
 Chronic GI disorders share common pathogenic factors, and the insight gained from coeliac disease and IBD pathogenesis will help us unravel novel mechanisms involved in symptom generation in IBS. For instance, gluten, the storage protein in wheat that causes celiac disease in genetically susceptible people, has recently been shown to cause gut dysfunction in mice and IBS symptoms in the absence of coeliac disease.

# A. introduction.
The most common chronic disorders of the gastrointestinal (GI) tract, such as IBS, IBD and coeliac disease share common pathophysiological factors: genetic susceptibility that predisposes to abnormal or dysregulated host responses to a number of environmental triggers [fig_ref] Fig 4. 1: Gut microbiota composition in African children living in rural areas with a... [/fig_ref]. For most disorders, such as IBS and IBD, it has been difficult to link precisely how these factors interact to increase disease susceptibility. In the case of coeliac disease we have gained tremendous understanding on how the triggering agent (gluten in wheat) interacts with genetic factors (HLA-DQ2/8) to induce a T-cell mediated immune response that causes villous atrophy.
Co-existence of more than one chronic GI disorder or development of a second illness after diagnosis of a first one can occur. Moreover, in some cases IBD and celiac disease may initially mimic IBS symptoms and it may be challenging to distinguish clinically between these disorders. Not surprisingly, proposed underlying mechanisms such as abnormal immune, barrier or neuro-motor responses to microbial or dietary antigens are shared between these disorders. [fig_ref] Fig 4. 1: Gut microbiota composition in African children living in rural areas with a... [/fig_ref] b. IBS and co-morbidities: Symptom mimicking, overlap or both?

## B.i. ibs, coeliac disease and gluten sensitivity
There is discussion as to whether (a) IBS symptoms and full-blown coeliac disease coexist as two separate entities, (b) coeliac disease mimics IBS symptoms ruling out "IBS" as a functional entity when coeliac disease is diagnosed or (c) milder forms of gluten intolerance that may belong to the spectrum of coeliac disease ("potential celiac disease") or not (innate or allergic immune responses to gluten that will never develop into full blown celiac disease), can cause IBS symptoms [fig_ref] Fig 4. 2: Fig 4 [/fig_ref].
Coeliac disease is a chronic inflammatory disorder of the small intestine in genetically susceptible individuals, and the only one in which the exact trigger, gluten, has been identified. Its prevalence has increased substantially in recent years, and this includes both European and North American populations [bib_ref] Increasing prevalence of coeliac disease over time, Lohi [/bib_ref] [bib_ref] Increased prevalence and mortality in undiagnosed celiac disease, Rubio-Tapia [/bib_ref]. The increase in prevalence may not only be explained by improved diagnostic accuracy, and, consequently, environmental factors, some of which may involve alterations in the intestinal microbiota, have been proposed [bib_ref] Increased prevalence and mortality in undiagnosed celiac disease, Rubio-Tapia [/bib_ref] [bib_ref] Effects of a gluten-free diet on gut microbiota and immune function in..., Palma [/bib_ref]. Clinically, coeliac disease can present at any age, with a variety of gastrointestinal (GI) and non-GI manifestations [bib_ref] Small-intestinal histopathology and mortality risk in celiac disease, Ludvigsson [/bib_ref] [bib_ref] The spectrum of celiac disease: epidemiology, clinical aspects and treatment, Tack [/bib_ref]. Some patients have symptoms of IBS that respond well to a gluten-free diet but they do not have tissue translgutaminase autoantibodies or histological markers (mucosal atrophy) of celiac disease. This entity termed "noncoeliac gluten intolerance" or "gluten sensitive IBS" is believed to be very common [bib_ref] Divergence of gut permeability and mucosal immune gene expression in two gluten-associated..., Sapone [/bib_ref] [bib_ref] Celiac disease-like abnormalities in a subgroup of patients with irritable bowel syndrome, Wahnschaffe [/bib_ref] [bib_ref] Editorial: Can gluten contribute to irritable bowel syndrome?, Verdu [/bib_ref] [bib_ref] Between celiac disease and irritable bowel syndrome: the "no man's land" of..., Verdu [/bib_ref] 

## B.ii. ibs and inflammatory bowel disease
Recently several lines of evidence support some commonalities in the pathogenesis of IBS and IBD.
The role of the intestinal microbiota in IBD has long been recognized. It is generally accepted that IBD results from a dysregulated immune response to intestinal microbiota, in genetically susceptible hosts. Consequently, several studies have investigated bacterial communities in IBD. The intestinal microbiota is altered in patients with IBD compared to healthy controls. Faecal samples from CD patients display greater temporal instability 11 and decreased number of commensal bacteria with reduction in the Firmicutes phylum, particularly Clostridium leptum group [bib_ref] Culture-independent analyses of temporal variation of the dominant fecal microbiota and targeted..., Scanlan [/bib_ref]. A significant decrease of bifidobacteria and lactobacilli has also been reported [bib_ref] Gut-associated bacterial microbiota in paediatric patients with inflammatory bowel disease, Conte [/bib_ref] [bib_ref] The assessment of faecal flora in patients with inflammatory bowel disease by..., Giaffer [/bib_ref]. On the contrary, patients with active CD had larger Escherichia coli faecal populations than did patients with quiescent disease or normal controls [bib_ref] The assessment of faecal flora in patients with inflammatory bowel disease by..., Giaffer [/bib_ref] , with specific increase in enteroadherent E. coli [bib_ref] Virulence properties of Escherichia coli strains isolated from patients with inflammatory bowel..., Giaffer [/bib_ref]. Studies investigating mucosaassociated microbiota showed larger numbers of bacteria in biopsies from CD patients compared to controls 357, 360-365 but their biodiversity was reduced with less prevalent members of Firmicutes [bib_ref] Differences between tissue-associated intestinal microfloras of patients with Crohn's disease and ulcerative..., Gophna [/bib_ref] [bib_ref] Faecalibacterium prausnitzii is an antiinflammatory commensal bacterium identified by gut microbiota analysis..., Sokol [/bib_ref].
In particular, decreases in members of Firmicutes with anti-inflammatory capacity, such as Faecalibacterium prausnitzii [bib_ref] Faecalibacterium prausnitzii is an antiinflammatory commensal bacterium identified by gut microbiota analysis..., Sokol [/bib_ref] It has been suggested that IBS-like symptoms are common in IBD patients in long-standing remission [bib_ref] Quality of life in inflammatory bowel disease in remission: the impact of..., Simren [/bib_ref] [bib_ref] Symptoms of irritable bowel syndrome in ulcerative colitis in remission, Isgar [/bib_ref]. There is also clinical overlap between IBS and IBD, with IBS-like symptoms frequently reported in patients before the diagnosis of IBD [bib_ref] Identification of a prodromal period in Crohn's disease but not ulcerative colitis, Pimentel [/bib_ref]. It is possible that IBS and IBD coexist with a higher than expected frequency, or may exist on a continuum, with IBS and IBD at different ends of the inflammatory spectrum. In fact, a study investigating IBS symptoms in IBD patients who were thought to be in clinical remission, demonstrated high levels of calprotectin levels, suggesting that in most cases, IBS symptoms are the result of undetected ongoing inflammation [bib_ref] Irritable bowel syndrome-type symptoms in patients with inflammatory bowel disease: a real..., Keohane [/bib_ref]. Underlying mechanistic links are lacking but the intestinal microbiota may be an important factor in both diseases [bib_ref] Commensal bacteria: the link between IBS and IBD?, Quigley [/bib_ref].

## B.iii. ibs-like symptoms following diverticulitis
While for many patients diverticulosis is an asymptomatic condition, around 1 in 5 have complications including acute diverticulitis, perforation, abscess/ fistula formation and stricturing [bib_ref] Colonic diverticular disease, Humes [/bib_ref] [bib_ref] A populationbased study of perforated diverticular disease incidence and associated mortality, Humes [/bib_ref]. Complications are thought to originate from microabscess formation resulting from ingress of faecal bacteria into the lamina propria via breaches in the epithelial lining of the diverticulum, possibly due to mechanical factors like impaction of faecal pellets. Risk factors for developing diverticulitis include lack of exercise 379 , obesity [bib_ref] Obesity increases the risks of diverticulitis and diverticular bleeding, Strate [/bib_ref] and low fibre/ fruit and vegetable intake [bib_ref] A prospective study of diet and the risk of symptomatic diverticular disease..., Aldoori [/bib_ref]. The dietary risk factors, as well as obesity, would certainly alter the faecal microbiota as well as the consistency of stool, though which is more important in pathogenesis is unknown. Most attention so far has been on evidence that fibre softens stool with no studies describing the changes in faecal microbiota in diverticular disease.
A high proportion of those hospitalised with acute diverticulitis continue to have persistent symptoms [bib_ref] Patterns of pain in diverticular disease and the influence of acute diverticulitis, Simpson [/bib_ref]. The precise mechanism is unclear since many do not have any of the above complications yet have recurrent short lived pain which in some features mimics IBS [bib_ref] Psychological and colonic factors in painful diverticulosis, Humes [/bib_ref]. There is evidence that inflammation induces prolonged changes in neurochemical coding in experimental colitis [bib_ref] Prolonged elevation of galanin and tachykinin expression in mucosal and myenteric enteric..., Simpson [/bib_ref] and diverticular disease 385 with upregulation of tachykinins which in animals can be linked to visceral hypersensitivity. An alternative explanation involving changes in the microbiota has been suggested based on some flawed uncontrolled studies claiming benefit from antibiotics and/or mesalazine but better studies are needed before any definitive conclusion can be drawn 377 .

## Treatment implications -antibiotics, probiotics, prebiotics and symbiotics

## Key points
 A short course of a non-absorbable antibiotic such as rifaximin has been shown to improve the symptoms of IBS, particularly bloating and flatulence. Improvement persists after the cessation 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 it has to be acknowledged that some of the early studies were of very poor quality. It should also be noted that different symptoms respond to different probiotics and some appear to be much more effective than others.
 Prebiotics and synbiotics should theoretically have potential in treating functional gastrointestinal disorders but there is as yet no reliable data to support this view.
As a consequence of the emerging evidence that has already been presented, one potential treatment approach is to alter the microbiota towards normal, where possible. This goal might be achieved either by administering an antibiotic or some form of "beneficial" bacteria, usually referred to as a probiotic. In addition, prebiotics and synbiotics are attracting attention as treatments for patients with functional GI disorders.

## A. antibiotics and functional bowel disorders
Despite evidence that previous antibiotic use may be related to the development of IBS [bib_ref] Antibiotic use, childhood affluence and irritable bowel syndrome (IBS), Mendall [/bib_ref] [bib_ref] Antibiotics increase functional abdominal symptoms, Maxwell [/bib_ref] , as well as the fact that antibiotic treatment does not reduce digestive symptoms after a bacterial gastroenteritis 320 , some nonetheless support use of antibiotics in IBS. The antibiotic approach is based on the contention that a high proportion of patients might exhibit small intestinal bacterial overgrowth, as measured by lactulose hydrogen breath testing [bib_ref] Eradication of small intestinal bacterial overgrowth reduces symptoms of irritable bowel syndrome, Pimentel [/bib_ref]. In an attempt to avoid absorption and side effects, neomycin was the original choice and the clinical response was favourable.
Neomycin led to a 35% improvement in a composite GI symptom score compared to 11% with placebo; the effect was most pronounced in those who normalized their lactulose hydrogen breath test, according to the definition used by the investigators [bib_ref] Eradication of small intestinal bacterial overgrowth reduces symptoms of irritable bowel syndrome, Pimentel [/bib_ref] [bib_ref] Normalization of lactulose breath testing correlates with symptom improvement in irritable bowel..., Pimentel [/bib_ref]. There have also been a number of mainly small and uncontrolled studies using metronidazole to treat functional gut symptoms, with variable success [bib_ref] Giardia lamblia infections become clinically evident by eliciting symptoms of irritable bowel..., D&apos;anchino [/bib_ref] [bib_ref] Do interventions which reduce colonic bacterial fermentation improve symptoms of irritable bowel..., Dear [/bib_ref] [bib_ref] Antibiotic or bacterial therapy in post-giardiasis irritable bowel syndrome, Morken [/bib_ref] [bib_ref] Metronidazole relieves symptoms in irritable bowel syndrome: the confusion with so-called 'chronic..., Nayak [/bib_ref]. However, more recently interest has focused on a non-absorbable derivative of rifampicin, called rifaximin [bib_ref] Rifaximin: a unique gastrointestinal-selective antibiotic for enteric diseases, Koo [/bib_ref].
Rifaximin, which is approved in several counties for treatment of travellers' diarrhoea caused by noninvasive strains of Escherichia coli, and to reduce the risk of recurrent hepatic encephalopathy, has an excellent safety profile and minimal drug interactions [bib_ref] Rifaximin: a unique gastrointestinal-selective antibiotic for enteric diseases, Koo [/bib_ref] [bib_ref] Rifaximin therapy for patients with irritable bowel syndrome without constipation, Pimentel [/bib_ref] , whereas all IBS subgroups were included in the previous study, which was mainly performed at one site [bib_ref] The effect of a nonabsorbed oral antibiotic (rifaximin) on the symptoms of..., Pimentel [/bib_ref] [bib_ref] Treatment for bacterial overgrowth in the irritable bowel syndrome, Drossman [/bib_ref]. The data suggests an improvement in symptoms, especially bloating and flatus over the 10 weeks following cessation of treatment [bib_ref] Rifaximin therapy for patients with irritable bowel syndrome without constipation, Pimentel [/bib_ref] [bib_ref] The effect of a nonabsorbed oral antibiotic (rifaximin) on the symptoms of..., Pimentel [/bib_ref]. In the most recent large study reported in 2011, the therapeutic gain for the primary (i.e. adequate relief of global IBS symptoms) and key secondary endpoint (i.e. adequate relief of bloating) relative to placebo was 9-11%, which is at the lower spectrum of what is usually considered to be clinically relevant [bib_ref] Antibiotic therapy for the irritable bowel syndrome, Tack [/bib_ref]. However, there was diminished efficacy over the ten-week observation period [bib_ref] Rifaximin therapy for patients with irritable bowel syndrome without constipation, Pimentel [/bib_ref] , and at this stage the efficacy and safety of re-treatment is unclear, even though a recent single-centre chart review suggested that re-treatment with rifaximin in IBS was successful (as defined using subjective assessment of retrospective chart review) up to five times without any decrease in duration or effect [bib_ref] Effects of Rifaximin Treatment and Retreatment in Nonconstipated IBS Subjects, Pimentel [/bib_ref] ; these limited data suggest that clinicians will not experience diminishing returns with recurrent cycles of therapy, but require confirmation in larger, prospective, multi-centre studies. There has also been a controlled trial comparing the effect of rifaximin to that of activated charcoal in patients with functional bowel disorders [bib_ref] Non-absorbable antibiotics for managing intestinal gas production and gas-related symptoms, Stefano [/bib_ref] To conclude, at this stage there are promising results suggesting that a subgroup of patients with functional bowel disorders may respond favourably to a short course of gut-specific antibiotics.
Among the symptoms of which patients with functional bowel disorders, bloating and flatulence appear to be especially responsive to non-absorbable antibiotics. However, in order to safely direct these treatment options to the appropriate patients, we need to know more about predictors of treatment responsiveness, the risk of development of antibiotic resistance, the efficacy and safety of re-treatment schedules and the optimal dosing regimen 393, 394 .

## B.probiotics and functional bowel disorders
Probiotics are live microorganisms, which when administered in adequate amounts confer a health benefit on the host [bib_ref] Probiotics and Health: Scientific evidence". Nutricion hospitalaria : organo oficial de la..., Guarner [/bib_ref] Probiotic bacteria can have a wide range of activities, and from animal studies there is evidence supporting a direct effect on at least some of the putative pathophysiological mechanisms implicated in IBS, such as visceral hypersensitivity [bib_ref] Specific probiotic therapy attenuates antibiotic induced visceral hypersensitivity in mice, Verdu [/bib_ref] [bib_ref] Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors, Rousseaux [/bib_ref] [bib_ref] Lactobacillus farciminis treatment suppresses stress induced visceral hypersensitivity: a possible action through..., Ait-Belgnaoui [/bib_ref] [bib_ref] Inhibitory effects of Lactobacillus reuteri on visceral pain induced by colorectal distension..., Kamiya [/bib_ref] , GI dysmotility [bib_ref] Lactobacillus paracasei normalizes muscle hypercontractility in a murine model of postinfective gut..., Verdu [/bib_ref] [bib_ref] Luminal administration ex vivo of a live Lactobacillus species moderates mouse jejunal..., Wang [/bib_ref] , and altered intestinal permeability [bib_ref] Lactobacillus farciminis treatment suppresses stress induced visceral hypersensitivity: a possible action through..., Ait-Belgnaoui [/bib_ref] [bib_ref] Probiotics prevent bacterial translocation and improve intestinal barrier function in rats following..., Zareie [/bib_ref]. There are also a small number of human studies, exploring the potential mechanisms underlying a positive clinical effect, such as effects on gut motility intestinal permeability 414 , composition as well as the stability of intestinal microbiota [bib_ref] Effects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome, Kajander [/bib_ref] [bib_ref] Clinical trial: multispecies probiotic supplementation alleviates the symptoms of irritable bowel syndrome..., Kajander [/bib_ref] and immune modulation [bib_ref] Lactobacillus and bifidobacterium in irritable bowel syndrome: symptom responses and relationship to..., O&apos;mahony [/bib_ref]. However these activities can differ considerably between one organism and another even if, for example, they both belong to the same species. Thus, just because one organism has a beneficial effect in a condition it does not mean that a similar organism will have similar activity. Obviously, for use in the field of gastrointestinal disease a particular bacterium needs to be acid-and enzyme resistant and should survive transit through the gastrointestinal tract maintaining its beneficial activity until the desired anatomical location is reached. For instance, if activity in the small bowel is desirable, then the propensity for good adherence to the mucosa seems to be an advantage and should be maintained. Furthermore, it is also critically important that products contain a sufficient quantity of microorganisms needed to provide benefit to the host, which for some products is questionable [bib_ref] In vitro screening of probiotic characteristics of some italian products, Vecchi [/bib_ref] [bib_ref] Microbiological evaluation of commercial probiotic products available in Italy, Drago [/bib_ref] [bib_ref] Microbiological evaluation of commercial probiotic products available in the USA in 2009, Drago [/bib_ref].
In the light of our understanding of the pathophysiology of IBS, many of the activities attributable to probiotics listed in.1, suggest potential in the treatment in IBS; this has resulted in thirty fullypublished placebo-controlled clinical trials to date, which are listed in.2 although it should be noted that in one study two different probiotics were compared with placebo [bib_ref] Lactobacillus and bifidobacterium in irritable bowel syndrome: symptom responses and relationship to..., O&apos;mahony [/bib_ref]. Unfortunately the design of these trials has varied considerably and some of the older studies do not fully meet the standards that would be demanded today. Furthermore, few of these studies attempted to define the mechanism of action of probiotics or assessed whether any symptomatic change was accompanied by an effect on the microbiota of the gut itself. However, it is possible to draw some conclusions. Of the thirty-one studies reported, twenty-three revealed some degree of symptom improvement, although the individual symptoms varied from study to study, and the therapeutic gain over placebo was generally modest, at best. In line with this reasoning, the general conclusion drawn from systematic reviews is that probiotics appear to be efficacious in IBS, but the magnitude of the benefit remains uncertain and it also remains unclear which strains and species are most effective. Furthermore, there are important limitations in the existing studies, including methodological shortcoming and concerns regarding validity of the endpoints employed in these studies [bib_ref] The utility of probiotics in the treatment of irritable bowel syndrome: a..., Brenner [/bib_ref] [bib_ref] A systematic review and meta-analysis: probiotics in the treatment of irritable bowel..., Hoveyda [/bib_ref] [bib_ref] Meta-analysis of probiotics for the treatment of irritable bowel syndrome, Mcfarland [/bib_ref] [bib_ref] The efficacy of probiotics in the treatment of irritable bowel syndrome: a..., Moayyedi [/bib_ref]. Moreover, there is heterogeneity in the available data and possible evidence of funnel plot asymmetry, suggesting there may be publication bias, with an over-representation of small positive studies in the published literature, and the higher quality studies reported a more modest treatment effect compared with lower quality trials [bib_ref] The efficacy of probiotics in the treatment of irritable bowel syndrome: a..., Moayyedi [/bib_ref]. Thus probiotic bacteria do appear to have a tangible effect in IBS, although it would seem that the choice of organism may depend on which symptom is being targeted. For instance, some products mainly affect bloating and flatulence [bib_ref] A randomized controlled trial of a probiotic, VSL#3, on gut transit and..., Kim [/bib_ref] [bib_ref] A randomized controlled trial of a probiotic combination VSL# 3 and placebo..., Kim [/bib_ref] [bib_ref] Alteration of intestinal microflora is associated with reduction in abdominal bloating and..., Nobaek [/bib_ref] , whereas others improve bowel frequency [bib_ref] Effects of a proprietary Bacillus coagulans preparation on symptoms of diarrheapredominant irritable..., Dolin [/bib_ref] , and some have a positive effect on a global symptom score [bib_ref] Clinical trial: multispecies probiotic supplementation alleviates the symptoms of irritable bowel syndrome..., Kajander [/bib_ref] [bib_ref] Lactobacillus and bifidobacterium in irritable bowel syndrome: symptom responses and relationship to..., O&apos;mahony [/bib_ref] [bib_ref] A mixture of Escherichia coli (DSM 17252) and Enterococcus faecalis (DSM 16440)..., Enck [/bib_ref] [bib_ref] Randomised clinical trial: Bifidobacterium bifidum MIMBb75 significantly alleviates irritable bowel syndrome and..., Guglielmetti [/bib_ref] [bib_ref] A probiotic mixture alleviates symptoms in irritable bowel syndrome patients: a controlled..., Kajander [/bib_ref] [bib_ref] Efficacy of an encapsulated probiotic Bifidobacterium infantis 35624 in women with irritable..., Whorwell [/bib_ref] [bib_ref] Clinical trial: a multistrain probiotic preparation significantly reduces symptoms of irritable bowel..., Williams [/bib_ref]. However there are many questions surrounding this therapeutic approach that still need answering:
 Are single organisms better than mixtures or vice versa?
 Are there any problems associated with taking multiple probiotic preparations?
 What are the best delivery systems in terms of liquids or capsules?
 How can viability and bioavailability be ensured?
 How should optimal doses be determined?
 How long do these preparations have to be given for?
 How often does colonisation of the host occur?
 Given that probiotics are not going to be as potent as pharmacological agents, at what patient group should they best be targeted?
 Are there any potential drawbacks to this treatment approach -it should be noted that one study reported an apparent deterioration of symptoms with active treatment compared to placebo [bib_ref] A candidate probiotic with unfavourable effects in subjects with irritable bowel syndrome:..., Ligaarden [/bib_ref].
 Should different probiotics be given to specific subgroups of IBS patients?
 Which symptoms of IBS should be the main target for therapy with probiotics?
 How should doctors and patients currently be guided regarding advice about their administration?
To conclude, probiotics in general seem to have a positive, albeit modest, effect in both children and adults with functional bowel disorders, especially IBS. However, head-to-head comparisons between different probiotic products would be useful and future trials need to be large scale, high-quality and use valid endpoints. It would also be useful if such trials could include an exploration of the possible mechanisms behind symptom improvement.

## C. pre-and synbiotics in functional bowel disorders
Prebiotics were defined in 1995 as "...nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon, and thus attempt to improve host health..." [bib_ref] Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics, Gibson [/bib_ref]. Recently, this definition was revisited, and modified to "a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health" [bib_ref] Prebiotics: the concept revisited, Roberfroid [/bib_ref]. A variety of oligosaccharides serve this function and when a prebiotic is combined with a probiotic it is called a symbiotic.
So far as prebiotics are concerned, there has only been one double blind placebo-controlled trial in IBS and this involved the use of a trans-galactooligosaccharide mixture, B-GOS [bib_ref] Clinical trial: the effects of a transgalactooligosaccharide prebiotic on faecal microbiota and..., Silk [/bib_ref]. Compared to placebo, both doses of this prebiotic (3.5g/day and 7g/day) resulted in symptom reduction, as well as specifically stimulating the growth of Bifidobacterium spp.. This is an encouraging observation, but more research is required on dosing and the relative merits of different compounds. With regard to synbiotics, there are many studies of these preparations but their design has not been sufficiently robust to draw firm conclusions [bib_ref] Clinical trial on the efficacy of a new symbiotic formulation, Flortec, in..., Andriulli [/bib_ref] [bib_ref] Prescript-Assist probiotic-prebiotic treatment for irritable bowel syndrome: a methodologically oriented, 2-week, randomized,..., Bittner [/bib_ref] [bib_ref] Prescript-assist probiotic-prebiotic treatment for irritable bowel syndrome: an open-label, partially controlled, 1-year..., Bittner [/bib_ref] [bib_ref] Effect of a symbiotic preparation on the clinical manifestations of irritable bowel..., Colecchia [/bib_ref] [bib_ref] Single-blind follow-up study on the effectiveness of a symbiotic preparation in irritable..., Tsuchiya [/bib_ref]. However, combining a prebiotic and a probiotic could, at least from a theoretical standpoint, increase the 'potency' of such a product.

# D. conclusion
From the results of these studies aimed at modifying the microbiota it is possible to draw two tentative conclusions: Firstly that this approach has therapeutic potential and secondly that if attempting to change the microbiota can improve symptoms, then this supports the view that there might be a microbial imbalance in the first place. However, future trials more explicitly address the question of how symptomatic improvement is achieved. Is it mirrored by a change in the microbiota of the gut or is some other mechanism involved?

## Key points

## Summary of clinical guidance regarding modulation of intestinal microbiome in ibs (fig.9.2)
- There is currently no clinically useful way of identifying whether the microbiota is disturbed in a particular patient with IBS. As a consequence, any approach that aims to alter the gut microbiota has, at present, to be undertaken on an empirical basis .

## - 
Attempts to modify the microbiota should start with the safest and most economical options. Probiotics have a reasonable evidence base and should be tried, for a period of at least one month, at adequate doses before a judgment is made about response to treatment.
Since they do not generally colonise the gut, if effective they will probably have to be administered long term. - Not all probiotic preparations are necessarily of a standard that would be demanded for pharmaceuticals and therefore it is essential to ensure that the source of a particular preparation is completely reliable.

## - 
Probiotics are unlikely to exhibit the potency of pharmacological agents and consequently are likely to be effective in the less severe cases of IBS.

## - 
The utility of testing for SIBO in the setting of IBS remains an area of uncertainty but the lactulose breath test is unreliable and should be abandoned.

## - 
If there is good reason to suspect SIBO then the glucose breath test or jejunal aspirate should be performed.

## - 
Consideration should be given to discontinuing PPIs in those with SIBO.

## - 
There is emerging evidence that non-absorbable antibiotics may have the potential to reduce symptoms in some patients with IBS.

## - 
The non-absorbable antibiotic with the most evidence is rifaxamin with a number needed to treat of 11. However, questions remain about duration of treatment, how long the beneficial effects last, the development of resistance and the long term safety for the population as a whole. Thus antibiotic therapy should be considered only for those failing other treatments.

## A. overview of clinical considerations
Although the science regarding the role of microbiota in FGIDs is fascinating it remains in its infancy;
there is much left to learn. Despite our ignorance, clinicians faced with a patient still have to make decisions. This section aims to help clinicians decide on whether to suggest modulators of intestinal microbiota to benefit patients with FGIDs, particularly IBS to which most of the limited evidence applies.

## B. diet
As described in Chapter 4, diet profoundly alters the microbiota since unabsorbed dietary components provide the nutrients which drive microbial growth. Bloating and diarrhoea have been shown to respond to reducing fibre intake 387 or reducing intake of fructose, fructans and other poorly absorbable but fermentable dietary items, the basis of the "FODMAP diet" [bib_ref] Fructose malabsorption and symptoms of irritable bowel syndrome: guidelines for effective dietary..., Shepherd [/bib_ref]. The great advantage of such treatments is their undoubted safety and prolonged effect [bib_ref] Food intolerance and the irritable bowel syndrome, Nanda [/bib_ref]. Detailed description of how these diets alter gut microbiota are lacking but the change in metabolism of lactulose observed with a low residue diet [bib_ref] Abnormal colonic fermentation in irritable bowel syndrome, King [/bib_ref] is a marker that these diets do alter colonic fermentation, which can be considered a marker of altered microbiome. Systematic exclusion diets may help 458 though they are laborious; it may be more practical to enact targeted exclusion of likely suspects like regularly consumed dairy, wheat, fruit and vegetables. There are few proper randomized, placebo controlled trials because of the difficulty in controlling for the placebo effect. One RCT showed bran aggravated symptoms [bib_ref] Bran supplementation in the treatment of irritable bowel syndrome, Snook [/bib_ref] and hence it would be logical that excluding it would help. Many patients exclude lactose, which in lactose malabsorbers will act as a prebiotic and undoubtedly alter the microbiota.

## C. prebiotics
Lactulose was once widely used as a laxative. It is a potent prebiotic which causes a substantial increase in beta-galactosidase activity and corresponding increased efficiency in metabolising lactulose during chronic dosing [bib_ref] Influence of chronic lactulose ingestion on the colonic metabolism of lactulose in..., Florent [/bib_ref]. More recently studies assessing directly the changes in fecal bacteria have confirmed that lactulose induces a significant increase in faecal Bifidobacterium spp.
counts and b-galactosidase activity [bib_ref] Prospective, randomized, parallel-group trial to evaluate the effects of lactulose and polyethylene..., Bouhnik [/bib_ref]. A novel prebiotic trans-galactooligosaccharide designed to be preferentially metabolised by bifidobacteria has been shown to increase their number in IBS and although the study very small, there was a reduction in bloating and flatulence [bib_ref] Clinical trial: the effects of a transgalactooligosaccharide prebiotic on faecal microbiota and..., Silk [/bib_ref]. However inulin also increases Bifidobacteria but increases flatulence [bib_ref] Prebiotic effect of fruit and vegetable shots containing Jerusalem artichoke inulin: a..., Ramnani [/bib_ref] as most other studies of prebiotics have
shown. This is likely to be a major limitation of prebiotic therapy in IBS.

## D. probiotics
While a meta-analysis has suggested these do have benefit in IBS, the variability in study design and probiotic used suggests that this group of very differing treatments is not well suited to a metaanalytical approach. The studies are often small and poorly designed and since they used unvalidated endpoints are hard to compare with other established treatments. Furthermore there are many different probiotics which vary in their effectiveness and mode of action. Many studies include less than 50 subjects and are obviously underpowered to assess symptoms, which typically need subjects per treatment. A recent systematic review reported that studies with poorer quality scores tended to show larger effects and published data indicates a publication bias, with non-reporting of negative effects in small trials 449 . An alternative approach to manipulating the microbiota would be to alter intestinal transit which can itself alter intestinal microbiota, as previously discussed in Section 4. It is well-established that motility is abnormal in many IBS patients and therapies that address motility improve IBS symptoms.
Thus alosetron slows transit in IBS [bib_ref] Alosetron, a 5-HT3 receptor antagonist, delays colonic transit in patients with irritable..., Houghton [/bib_ref] , while linaclotide 466 , lubiprostone [bib_ref] Effect of a selective chloride channel activator, lubiprostone, on gastrointestinal transit, gastric..., Camilleri [/bib_ref] , and tegaserod 468 all accelerate transit in IBS-C, and all have high quality randomized controlled trials supporting their efficacy in IBS. However only one study using loperamide has examined the impact on the microbiome [bib_ref] Alteration of sulfate and hydrogen metabolism in the human colon by changing..., Lewis [/bib_ref]. Future research should evaluate whether these effective treatments alter the gut microbiota and whether these changes correlate with symptom improvement.

## F. discontinuing proton pump inhibitor (ppi) therapy
Over 40% of reflux patients starting PPI therapy develop bloating after 8 weeks of therapy, and that 1 in 5 meet Rome III criteria for IBS after 6 months of PPI use (without meeting criteria at baseline) [bib_ref] Effects of long-term PPI treatment on producing bowel symptoms and SIBO, Compare [/bib_ref].
Furthermore, data also reveal that return of an abnormal glucose breath test in IBS following rifaximin therapy is independently predicted by use of concurrent PPI therapy [bib_ref] Small intestinal bacterial overgrowth recurrence after antibiotic therapy, Lauritano [/bib_ref]. Thus clinicians should, where possible, consider discontinuing PPI therapy in IBS patients and perhaps replacing with less potent acid suppression such as H 2 blockers or antacids.

## G. antibiotics therapies in ibs
Given the rapid development of antibiotic resistance whenever antibiotics are widely used the use of antibiotics in a condition as ubiquitous as IBS seems problematic, particularly as the condition is chronic and the treatment seems likely to be given repeatedly since the benefit appears to be wearing off by [bib_ref] New insights into the pathogenesis and pathophysiology of irritable bowel syndrome, Ohman [/bib_ref] for empiric treatment was not described by the authors and indeed the evidence discussed above (Chapter 6) suggests that the antibiotic effect was most likely mediated via effects on colonic bacteria rather than those in the small bowel. Although rifaximin was superior to placebo, the number-needed-to-treat (NNT) was 11 which is on par or worse than most other potential treatments. [fig_ref] Table 9 1: [/fig_ref].2. shows the NNT from recent trials in which the methodology is comparable suggesting that at 11 the NNT for rifaximin is at the high end of treatments shown to be better than placebo, while .1. shows the trade-off between effectiveness versus invasiveness/safety of treatments that modulate intestinal microbiota 266, 469-474 .

## Ibs treatment nnt vs. placebo * references
"Placebo without deception" *** 4 [bib_ref] Placebos without deception: a randomized controlled trial in irritable bowel syndrome, Kaptchuk [/bib_ref] Alosetron 8 [bib_ref] Efficacy of alosetron in irritable bowel syndrome: a meta-analysis of randomized controlled..., Cremonini [/bib_ref] Linaclotide ** 8 [bib_ref] Linaclotide improves abdominal pain and bowel habits in a phase IIb study..., Johnston [/bib_ref] Rifaximin 11 [bib_ref] Rifaximin therapy for patients with irritable bowel syndrome without constipation, Pimentel [/bib_ref] Lubiprostone 12 [bib_ref] Clinical trial: lubiprostone in patients with constipation-associated irritable bowel syndrome--results of two..., Drossman [/bib_ref] [bib_ref] Clinical trial: phase 2 study of lubiprostone for irritable bowel syndrome with..., Johanson [/bib_ref] Tegaserod 14 471 ***"Placebo without deception" involves giving a placebo and actively informing the patient that it is an inactive agent. Patients were informed that they received "placebo pills made of an inert substance, like sugar pills, that have been shown in clinical studies to produce significant improvement in IBS symptoms through mind-body self-healing processes." Compared to no treatment, this approach was highly effective in a well-documented randomized controlled trial 474 .

## H. potential risks of gut-directed antibiotic use in clinical practice
Although rifaximin appears to be well tolerated and safe, given its relatively low potency it is important to consider its downside. Rifaximin use can promote rifampin-resistant strains of staphylococci which remained resistant for at least 9 weeks implying that there is no fitness cost to the resistance mutation that emerges [bib_ref] Rifaximin intake leads to emergence of rifampin-resistant staphylococci, Valentin [/bib_ref]. This is potentially problematic, because rifampin is a vital treatment for management of staphyloccal foreign body infections such as prosthetic valve endocarditis or prosthetic joint infections. Although IBS patients are themselves unlikely to develop staphylococci foreign body infections, they could potentially harbour and transmit resistant strains, though further research is needed to reproduce these initial findings and establish the clinical importance of the results. Since host-bacterial interactions is a dynamic process, particularly in diseases states, there is also a need for longitudinal or interventional studies assessing the role of microbiota and diet, the relationship of changes in microbiotia to remission and symptom flare-ups, and stress, infection or therapeutic modulation (e.g. probiotics, prebiotics, antibiotics). The demonstration of a bidirectional brain-gut-microbial axis [bib_ref] The relationship between intestinal microbiota and the central nervous system in normal..., Collins [/bib_ref] and the existence of a systemic immune response to microbial luminal antigens (anti-flagellin antibodies) [bib_ref] Antibodies to flagellin indicate reactivity to bacterial antigens in IBS patients, Schoepfer [/bib_ref] points out that microbial homeostasis may be perturbed beyond the gastrointestinal tract and opens the field to novel avenues which could not be even imaginable only few years ago.
The wide heterogeneity of functional bowel disorders and the inter-individual variability of microbiota profiles suggest that larger sample size studies will be of key importance in the future.
In addition, a recent study demonstrated that the microbiota in humans can be divided into enterotypes [bib_ref] Enterotypes of the human gut microbiome, Arumugam [/bib_ref] and hence, typing patients based on their enterotype could be as important as typing them for other phenotypic and genotypic characteristics in relation to health and disease.
Moreover, microbiota signatures can be developed to contribute to IBS diagnostics and segmentation. When cause-effects have been established then they offer the potential to develop therapeutic avenues. Attention should also be directed to accurate characterization of patient's symptom and, whenever possible, studies should be stratified by factors known to affect intestinal microbiota. As detailed in Chapter 4, this should include primarily the effect of age and diet. In particular, diet has powerful influences on gut microbiota and dietary manipulation is often employed by both patients and clinicians in the attempt to improve symptoms. Researchers should also consider the potential confounding effect of previous or concomitant use of drugs with potential interference on intestinal microbiota including antibiotics, probiotics, laxatives, prokinetcs, proton pump inhibitors and mesalazine.
Currently, there are promising results suggesting that a subgroup of patients with functional bowel disorders may respond favourably to a short course of gut-specific antibiotics. Among the symptoms, bloating and flatulence appear to be especially responsive to non-absorbable antibiotics. However, in order to safely direct these treatment options to the appropriate patients, we need to know more about, predictors of treatment responsiveness, the risk of development of antibiotic resistance, the efficacy and safety of re-treatment schedules and the optimal dosing regimen [bib_ref] Treatment for bacterial overgrowth in the irritable bowel syndrome, Drossman [/bib_ref] [bib_ref] Antibiotic therapy for the irritable bowel syndrome, Tack [/bib_ref]. Further studies should also investigate the mechanism and site of action of non-absorbable antibiotics since amelioration of gas-related symptoms in patients occurred also in patients with no evidence of SIBO [bib_ref] A randomized double-blind placebo-controlled trial of rifaximin in patients with abdominal bloating..., Sharara [/bib_ref]. Probiotics in general seem to have a positive, albeit modest, effect in both children and adults with functional bowel disorders, especially IBS. However, head-to-head comparisons between different probiotic products would be useful and future trials need to be large scale, high-quality and use valid endpoints. It would also be useful if such trials could include an exploration of the possible mechanisms behind symptom improvement.
To conclude, a better definition of the role of intestinal microbiota in the pathogenesis, pathophysiology of functional bowel diseases represents a challenge for the future. Although promising, therapeutic implications will need to be better defined in well conducted large trials.
A strict cooperation of experienced clinical researchers with microbial ecologists should be considered an important factor for the success of these future studies.
[fig] Figure 3 1: Overlap of symptoms defining functional bowel disorders and functional abdominal pain10,14 . [/fig]
[fig] Figure 3 2: Variation in the microbiota composition in the GI tract both with respect to distance from the mouth and form the lumen. [/fig]
[fig] Figure 3 3: Example of bacterial taxonomic classification. [/fig]
[fig] Fig 4. 1: Gut microbiota composition in African children living in rural areas with a polysaccharide-rich diet when compared with Italian city children 45 . (Redrawn from DeFilippo et al 45 ) Adding 48 g of whole grain cereals daily for 3 weeks to the diet in a RCT increased numbers of faecal [/fig]
[fig] Fig 4. 2: Fig 4.2. Gut microbiota and the intrinsic and extrinsic factors that can affect its distribution and composition. A number of host mechanisms participate in gut microbiota modulation, including gastric acid secretion, fluid, anti-commensal sIgA and antimicrobial peptide production, and GI motility. Drugs that block acid secretion and affect GI motility, can indirectly alter the microbiota. Antibiotics, depending on spectrum and dosage, will directly affect microbiota composition inducing dysbiosis. Dietary modifications, including probiotic and fibre supplements will also affect microbiota composition. (MMC: migrating motor complex; PPI: proton pump inhibitor; NSAID: non steroidal anti-inflammatory drugs). [/fig]
[fig] Fig 5. 1: The full circle rRNA approach.g. MetagenomicsAs indicated above 16S rRNA gene-based approaches provide valuable information about the identity, number and diversity of microbial communities. However, further insights into the functional properties of microbes cannot be extrapolated from 16S rRNA and its respective genes. To get insight into the functional potential encoded in the community, metagenomics approaches are needed205 . Metagenomics is defined as the study of genomes recovered from environmental samples which will provide information about the functional capabilities and the phylogenetic distribution of an ecosystem. Thus, instead of looking at one genome with classical genomics approaches, with metagenomics you study the genomes from all microorganisms in an ecosystem simultaneously. Depending on the research question, metagenomic information can be gathered from short insert clone libraries (1-10 Kb), large insert clone libraries (20-100 kb) or direct randomly generated sequences using the novel high throughput sequencing approaches, such as 454 pyrosequencing and Solexa sequencing. Short insert libraries can be used for individual gene-function screening such as identification of specific enzymatic activities206 , while large insert libraries favors [/fig]
[fig] Fig 6. 1: The LHBT measures small intestinal transit rather than SIBO in IBS patients. Upper schematic shows ingestion of test meal with subsequent serial measurement of both H 2 gas, resulting from fermentation of the lactulose by intestinal bacteria, and Tc99 scanning in the cecum. This latter measurement detects when the test meal has reached the cecum. The stylized drawing below shows a representative result from an IBS patient with serial measurements over time. The Tc99 had already reached the cecum in large quantities before the H 2 PPM level has reached the threshold for an abnormal test (seeYu et al. 2011 for details). This demonstrates that the increased H 2 production results from fermentation by colonic bacteria, not by abnormal bacteria small intestine (i.e. SIBO). [/fig]
[fig] Figure 6 2: Multiple factors influence the composition of the intestinal microbiota. [/fig]
[fig] Fig 6. 3: Integrated conceptual framework of the pathophysiology of functional disorders. [/fig]
[fig] Fig 7. 1: Common factors in the pathogenesis of chronic gut disorders. Chronic disorders of the GI tract, including inflammatory bowel disease (IBD), celiac disease (CD) and irritable bowel syndrome (IBS), share three main pathogenetic pathways: genetic predisposition, abnormal host responses and exposure to environmental triggers. In CD, the environmental trigger, gliadin, the toxic protein fraction in gluten, has been identified, as well as the mechanisms of interaction between immune (Tcell) and genetic factors (HLA DQ2/8). Progress has been made in the understanding of IBD pathogenesis. The best characterized genetic mutation associated with IBD (NOD2) may predispose to abnormal host immune responses to the intestinal microbiota. No characteristic genetic mutation has been associated with IBS, but a variety of genes that regulate innate immune function (TNFSF15, IL-6), bacterial recognition (TLR-9) and barrier function (CDH-1) have been detected. These genetic defects may predispose the host to react inappropriately to a variety of environmental triggers such as the intestinal microbiota or specific food antigens such as gluten. [/fig]
[fig] Fig 7. 2: The association between IBS and coeliac disease. IBS symptoms and full-blown CD can coexist. However, milder forms of gluten sensitivity that may or may not belong to the celiac spectrum, can cause IBS-like symptoms. Thus, coexistence of both diseases is possible, but because active CD can cause IBS-like symptoms, this, in itself, rules out "IBS," as the definition of the syndrome requires the absence of organic pathology. Similarly as low-grade inflammation and infectious gastroenteritis, this model proposes mild gluten sensitivity as yet another possible cause of IBS symptoms. [/fig]
[fig] Fig 9. 1: Effectiveness vs. invasiveness/safety of treatments that modulate intestinal microbiota in irritable bowel syndrome (IBS) [/fig]
[fig] Fig 9. 2: Different ways to modulate gut microbiota.10. CONCLUSIONS & RECOMMENDATIONS FOR FUTURE RESEARCH AND DEVELOPMENTIn this review we critically discussed the latest developments in the understanding of the role of intestinal ecosystem and the efficacy of microbiota directed therapies in functional bowel disorders. Several observations suggest that gut bacteria are important in the pathophysiology of functional bowel disorders in some patients. These include: 1) evidence of both qualitative and quantitative changes in intestinal microbiota in IBS; 2) the well-established role enteric infection in the development of IBS symptoms in predisposed individuals and 3) data supporting the evidence that microbiota modulation with probiotics and non-absorbable antibiotics may provide some beneficial effects in subgroups of patients. Nonetheless, as we acknowledge the importance of these recent developments, we also realize that the field is in its infancy and a number of both basic and clinical key issues remain to be addressed in the future.Since the diversity of gut bacteria is under-represented by characterized cultured isolates, the introduction of modern molecular techniques has offered the opportunity to obtain novel information on the phylogenetic and functional properties of microbial ecosystems in health and disease states274 . However, this enormous technological potential has been so far only applied to the field of functional bowel disorders in a limited way. Thus, a deeper phylogenetic characterization of microbiota through high throughput sequencing or phylogenetic microarraying is crucial in future studies. We need to know more about mucosal ecological niches in patients with functional bowel disorders since the vast majority of studies are currently based on faecal samples, which may not representative of the microbiota throughout the intestine. Microbes embedded in the mucous layer likely form a different microbial ecosystem retaining important properties in the regulation expression of host genes (eg, TLR) and epithelial, endocrine and immune physiological functions of the intestine 33, 184 . The controversial SIBO hypothesis in patients with IBS could also take advantage of modern molecular techniques. In fact, currently the SIBO hypothesis is largely based on findings obtained with the lactulose breath test which has poor sensitivity and limited ability to discriminate the detection of ileal as opposed to caecal bacteria 113 The study by Posserud et al. provided no evidence of clear-cut jejunal bacterial overgrowth in patients with IBS compared with controls. However, interestingly enough this study showed sub-threshold increases in cultivable microbes 115 which are worth further characterization with high throughput culture-independent technologies. Despite the need of a deeper characterization of qualitative and quantitative microbiota changes in functional bowel disorders, a phylogenetic approach alone may turn out to be insufficient to elucidate if microbiota changes are cause or consequence of altered bowel physiology. Time has come to move on and direct research to the characterization of functional properties of microbiota in functional bowel disorders. This type of research can take advantage now of a wide range of novel tools including metagenomic, metatranscriptomics, metaproteomics, metabonomic techniques 274 . These studies should be also extended to the identification of the impact of regulatory signals produced by intestinal bacteria to the epithelium, immune system and enteric nerves. In turn, these mechanistic as opposed to descriptive studies could provide novel insights on the role of intestinal bacteria in the pathophysiology of functional bowel disorders. One field of particular interest is related to gut immunity. Microbiota is a driving force for the mucosal immune system and several recent studies have pointed out the presence of low grade immune activation in patients with IBS 13 . It has been hypothesized that low grade inflammation in IBS may occur as a consequence of exaggerated exposure of the immune system to luminal bacterial antigens through a leaky epithelial barrier 475 . Data showing increased expression of TLR recognising pathogen associated molecular patterns in patients with IBS as compared with healthy controls, is in line with this concept and suggest the existence of altered host-microbiota and the innate immune system interactions. Mucosal release of antimicrobial substances such as defensins produced by Paneth cells represent another extremely exciting field of study deserving further attention 476 . [/fig]
[table] Table 5 1: Overview of approaches to study the microbiome. [/table]
[table] Table 6 1: Summary of Studies Culturing Small Bowel Microbiome115,244,[246][247][248][249][250][251][252][253] [/table]
[table] Table 8: 1. Some effects of probiotics  Stimulation of anti-inflammatory and immune responses  Enhancement of epithelial barrier and reduction of bacterial translocation  Inhibition of growth of pathogens such as Salmonella spp.  Inhibition of adhesion of viruses such as rotavirus  Elaboration of active proteins and metabolites with toxin binding, immune modulatory and active bactericidal activity [/table]
[table] Table 9: 1. shows studies with >50 subjects per treatment. Only one small study in mixed IBS reported unfavourable effects with exacerbation of diarrhoea and worsening symptoms even in those with constipation434 . The evidence suggests that other small studies with negative effects have never been published. Few studies have assessed the impact on faecal bacteria and hence shown a direct link between changes in bacteria and symptoms. One study showed a multispecies probiotic containing Lactobacillus spp., Bifidobacterium spp., and Proprionobacterium spp. showed a fall in a phylotype with 94% similarity to Ruminococcus torques and increase in certain Clostridium spp.461 . More studies which demonstrate the effect of probiotics on faecal microbes are needed to see if these changes can be reliably linked to symptom improvement. This would help in determining the true mechanism of action. Several studies have shown a beneficial effect of probiotics on abnormal gut permeability414,462,463 . However although others have linked abnormal permeability to severity of pain464 , the study in children found no relationship between the effect on permeability and the effect on symptoms414 . However gut permeability may be a worthwhile objective biomarker of probiotic effect in future studies.Thus the evidence base is weak and this should be acknowledged when discussing with a patient whether to try probiotics. Safety in the setting of IBS seems good but at least one study found symptoms got worse so the patients should be warned about this and told to discontinue if this happens. At present the strongest evidence is for Bifidobacterium infantis 35624 at a dose of 1 X 10 8 cfu/day443 . The evidence suggests that this should be taken for at least 4 weeks since the benefit did appear greater towards the end of the 4 week trial. Much work needs to be done to define who benefits, what is the best variety of probiotic and what are the best endpoints to determine efficacy. [/table]